Company diagnosis for anti-hyaluronan agent therapy and methods of its use

专利摘要:
  MONITORING DIAGNOSIS FOR ANTI-HYALURONAN AGENT THERAPY AND METHODS OF USE OF THE SAME.Diagnostic methods and agents for identifying individuals for the treatment of cancer, with an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan, are provided. Diagnostic agents for detecting and quantifying hyaluronan in a biological sample and monitoring cancer treatment with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, are provided. Combinations and kits for use in the practice of the methods are also provided.
公开号:BR112014009797A2
申请号:R112014009797-6
申请日:2012-10-24
公开日:2020-10-27
发明作者:Ping Jiang；H. Michael Shepard；Lei Huang
申请人:Halozyme, Inc.；
IPC主号:

专利说明:

[0001] [0001] The priority benefit is claimed for US Serial Provisional Application No. 61/628, 187, of October 24, 2011, entitled “Follow-up diagnosis for enzyme therapy that degrades hyaluronan and methods of using it”, for US Provisional Application No. 61/559, 011, of November 11, 2011, entitled “Follow-up diagnosis for enzyme therapy that degrades hyaluronan and methods of using it”, for US Provisional Application No. 61/630, 765 , of December 16, 2011, entitled “Follow-up diagnosis for enzyme therapy that degrades hyaluronan and methods of using it” for US Provisional Application No. 61/714, 700, of October 16, 2012, entitled “Diagnosis follow-up for anti-hyaluronan agent therapy and methods of using it ”. The purpose of each of the applications listed above is incorporated by reference in its entirety.
[0002] [0002] This application is related to US Patent Application No. 13 / 694,071, filed on the same day in the attachment, entitled “Follow-up diagnosis for anti-hyaluronan agent therapy and methods of using it”, which claims priority for US Serial Interim Requirements No. 61/628, 187; 61/559, 011; 61/630, 765 and 61/714, 700.
[0003] [0003] The purpose of the application mentioned above is hereby incorporated by reference in its entirety. INCORPORATION BY LISTING REFERENCE OF SEQUENCE PROVIDED ELECTRONICALLY
[0004] [0004] An electronic version of the Sequence Listing is filed as an attachment, the contents of which are incorporated by reference in their entirety. The electronic file was created on October 24, 2012, is 1827 Kb in size, and is titled 3096segPCl.txt. FIELD OF INVENTION
[0005] [0005] Diagnostic methods and means for identifying people for treating cancer with an enzyme that degrades hyaluronan are provided. Diagnostic agents for the detection and quantification of hyaluronan in a biological sample, and for monitoring cancer treatment, the hyaluronan degrading enzyme are provided. Combinations and kits for use in the practice of the methods are also provided. BACKGROUND
[0006] [0006] Hyaluronan degrading enzymes have been used therapeutically, usually as dispersants and agents in combination with other therapeutic spreading agents. Hyaluronan degrading enzymes can also be used in single agent therapy for the treatment of diseases and disorders associated with hyaluronan. For example, tumors and cancers associated with the accumulation of hyaluronan, and treatment with an enzyme that degrades hyaluronan, inhibits tumor growth and increases vascular perfusion, and improves the delivery of agents - “chemotherapeutic agents for the tumor. There is a need for methods and reagents to improve the treatment of patients who are treated with enzymes that degrade hyaluronan. SUMMARY
[0007] [0007] Here is provided a method for selecting an individual to treat a tumor, with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan. In the method provided, a sample of tissue or body fluid from an individual with a tumor or cancer is contacted with a hyaluronan-binding protein (HABP) that has not been prepared from, or isolated from, animal cartilage. The binding of the hyaluronan-binding protein to the sample is detected, thus determining the amount of hyaluronan in the sample, in which if the amount of hyaluronan in the sample is equal to or greater than a predetermined level, the individual is selected for treatment with an anti-hyaluronan agent, for example, a hyaluronan degrading enzyme.
[0008] [0008] Here is provided a method for selecting an individual for the treatment of a tumor, with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, in which a body fluid of an individual with a tumor or cancer is in contact with a hyaluronan-binding protein (HABP) that has not been prepared from, or isolated from, animal cartilage, and the HABP is bound to the sample by a solid phase with a colorimetric or fluorescent signal, thus determining the amount of hyaluronan in the sample, in which an individual is selected for treatment with an anti-hyaluronan agent, for example, a hyaluronan degrading enzyme, when the predetermined threshold level is High HA. In some examples of the method, the predetermined threshold level is at least or above 0.025 µg HA / ml of sample, 0.030 pg / ml, 0.035 upg / ml, 0.040 pg / ml,
[0009] [0009] Here is provided a method for selecting an individual for the treatment of a tumor, with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, in which a sample of tumor tissue from an individual with a tumor or cancer is in contact with a hyaluronan-binding protein (HABP) that has not been prepared from, or isolated from, animal cartilage, and that the HABP binding to the sample is done by histochemistry, thereby determining the amount of hyaluronan in the sample, in which an individual is selected for treatment with an anti-hyaluronan agent, for example, a hyaluronan degrading enzyme, when the predetermined threshold level is an HA score of at least two +2 ( HA ) Or at least +3 (HA). In some instances, the predetermined threshold level is an HA score of at least +3 (HA **) (high levels). In other examples, the predetermined threshold level is at least one percent of HA positive pixels in the tumor (cells and stroma) for total staining in tumor tissue of at least 10%, 10% to 25% or greater than than 25%. For example, the predetermined threshold level is at least one percent of HA positive pixels in the tumor (cells and stroma) and the total tumor tissue staining of at least 11%, 12%, 13%, 14% , 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.
[0010] [0010] Also provided here is a method for selecting an individual to treat a tumor, with an anti-hyaluronan agent, such as, for example, an enzyme that degrades hyaluronan, in which the individual is then treated with the anti-hyaluronan agent. -hyaluronan, for example, an enzyme that degrades hyaluronan. In some examples, the anti-hyaluronan agent is an enzyme that degrades hyaluronan, which is administered in a dosage amount between about, or between 0.01 npg / kg (of the individual) at 50 vg / kg, 0.01 pg / kg to 20 pg / kg, 0.01 pg / kg to 15 pg / kg, 0.05 vg / kg to 10 ug / kg, 0.75 pg / kg to 7.5 mg / kg or 1.0 vg / kg to 3.0 mg / kg and a frequency of administration twice a week, once a week, once every 14 days, once every 21 days, or once a month. In particular examples of the method, a corticosteroid is administered prior to administration of a hyaluronan-degrading enzyme, or after administration of the hyaluronan-degrading enzyme, usually in an amount sufficient to improve an adverse effect on the individual, from the hyaluronan-degrading enzyme. hyaluronan administered. For example, the amount of corticosteroids is administered between, or about 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 20 mg, 0 , 2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg, 0.4 to 5 mg, 0, 4 to 4 mg, 1 to mg, 1 to 15 mg or 1 to 10 mg.
[0011] [0011] A method for predicting the effectiveness of treating an individual with an anti-hyaluronan agent, for example, a hyaluronan degrading enzyme, is also provided here. In the method provided, a sample of tissue or body fluid from an individual who is, or has been treated with, an anti-hyaluronan agent, for example, a hyaluronan-degrading enzyme, is contacted with a hyaluronan-binding protein (HABP ) that has not been prepared from, or isolated from, animal cartilage, and the binding of the hyaluronan binding protein to the sample is detected,
[0012] [0012] A method for monitoring an individual's treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) is also provided here. In the method provided, a sample of tissue or body fluid from an individual with a tumor or cancer is in contact with a hyaluronan-binding protein (HABP) that has not been prepared from, or isolated from, animal cartilage, and the amount of hyaluronan-binding protein that binds to the sample is detected, thus determining the amount of hyaluronan in the sample, and the level of hyaluronan to be compared with a control or reference sample, to determine, thus, the amount of hyaluronan in the sample in relation to the control or the reference sample, in which the amount of hyaluronan is an indicator of the evolution of treatment.
[0013] [0013] Methods for predicting the effectiveness of treating an individual with an anti-hyaluronan agent (for example a hyaluronan degrading enzyme) and monitoring treatment of an individual with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), in which the treatment is changed based on the determined amount of hyaluronan in the sample, in relation to the control or reference sample, such that if the amount of hyaluronan in the sample is equal to or greater than the value in control or reference sample, treatment is continued or referred by increasing the dose and / or dosage; or if the amount of hyaluronan in the sample is less than the value of the control or reference sample, treatment is continued, reduced, decreasing the dose and / or dose schedule, or terminated. In some instances, the control or reference sample is a sample from a healthy individual, is a baseline sample from the individual, prior to treatment with an anti-hyaluronan agent (eg, hyaluronan-degrading enzyme) or is a sample from an individual, before the last dose of the anti-hyaluronan agent (eg, hyaluronan degrading enzyme). In some instances, the individual has a tumor or cancer, and the sample is a sample of tumor tissue, and detection is performed by histochemistry. In other examples, the individual has a tumor or cancer, and the sample is a body fluid and detection is done by a solid-phase binding assay. In some examples, the solid phase binding assay is a microtiter plate assay and the binding is detected colorimetrically or by fluorescence.
[0014] [0014] In any of the methods provided here, the step of contacting the sample with a HABP can be performed at, or between about pH 5.6 to 6.4. For example, the step of contacting the sample with a HABP is carried out at a pH of about 5.8, 5.9, 6.0, 6.1 or 6.2. In some examples, HABP specifically binds to HA with a binding affinity represented by the dissociation constant (Kd) of at least, or less than 1 x 107 M, 9 x 10º M, 8 x 10º M 7 x10ºM, 6x 10º M, 5 x 10 M, 4 x 10 M, 3 x 10 M, 2 x 10 M, 1 x 10 M, 9 x 10 M, 8 x 10 M, 7 x 10 M, 6 x 10 M, 5 x 10 M , 4 x 10º M, 3 x 10º M, 2 x 10º M, 1 x 10ºM or lower Kd.
[0015] [0015] In any of the methods provided here, HABP can be generated recombinantly or synthetically. In some examples, the HABP contains a connection module. In other examples, the HABP contains two or more connection modules. In other examples, the binding module (or modules) is the only HABP portion of the molecule. Thus, methods are provided here in which HABP contains a binding module selected from CD44, LYVE-1, binding protein / HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin -1, Stabilin -2, CAB61358 and KIAAO5S27. In some examples of the method, HABP contains a portion of a CD44, LYVE-1, binding protein / HAPLNl, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-l, Stabilin -2, CAB61358 and KIAAO5S27, including a connection module or a sufficient portion of a connection module to connect to the HA. In a particular example, HABP is a gene protein stimulated by tumor necrosis factor (TSG-6) or a TSG-6 binding module or a sufficient portion of a TSG-6 binding module to bind to HA. In other examples of the method described here, HABP contains a Gl domain of a type C hyaluronan binding protein, for example, a Gl domain selected from Aggrecan Gl, Versican Gl, Neurocan Gl and Brevican Gl. In particular examples, the Gl domain is the only HABP portion of the molecule.
[0016] [0016] In some examples of the methods provided here, HABP contains the amino acid sequence established in any of SEQ ID NOS: 207, 222, 360, 361, 371-394, 4116-418 and 423-426, or has the amino acid sequence that exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98% , 99% or more of sequence identity for an amino acid sequence defined in any of SEQ ID NOS: 207 360, 361, 371-394, 416-418 and 423-426, and specifically binds to HA, or is a its HA binding domain, or a sufficient portion of it, to specifically bind to HA. In one example, HABP contains a TSG-6 (LM) link module or a sufficient portion of it, which specifically binds to HA. For example, TSG-6-LM contains the amino acid sequence set forth in SEQ ID NO: 207, 360, 417 or 418, or an amino acid sequence comprising at least 65% amino acid sequence identity with the acidic amino acid sequence established in SEQ ID NO: 207, 360, 417 or 418 and specifically binds to HA. In specific examples of the method, HABP contains a binding module set out in SEQ ID NO: 207 or an amino acid sequence comprising at least 65% of the amino acid sequence identity for the amino acid sequence set out in SEQ ID NO: 207 and , specifically, binds to HA. In other examples, HABP contains a connection module that features at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 207, 360, 417 or 418, whereby HABP specifically binds to HA.
[0017] [0017] In some examples of the method provided here, the TSG-6 binding module is modified to reduce or eliminate binding to heparin. For example, heparin binding is reduced at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times or more. In some examples of the method provided here, the TSG-6 linker contains an amino acid substitution at the amino acid position corresponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84, set out in SEQ ID NO: 360 , where a corresponding amino acid residue is identified by aligning a TSG-6-LM set out in SEQ ID NO: 360. For example, the amino acid substitution is in a TSG-6-LM set out in SEQ ID NO: 207, and the amino acid substitution or substitutions are at amino acid residue 21, 35, 42, 55, 57, 73 or 85. The amino acid substitution can be that of a non-basic amino acid residue selected from Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gin (Q), Ala (A), Val (V), He (1), Leu (L), Met (M), Phe (EF), Tyr (Y) and Trp (W). In another example, the TSG-6 linker contains an amino acid substitution corresponding to amino acid substitutions K20A, K34A or K41A in a TSG-6-LM set out in SEQ ID NO: 360, or the substitution by the corresponding residue in another TSG-6-LM. In another example, the TSG-6 linker contains amino acid substitutions corresponding to amino acid substitutions K20A, K34A and K41A in a TSG-6- LM established in SEQ ID NO: 360, or the substitution by the corresponding residue in the other TSG- 6-LM. For example, HABP contains a binding module set out in SEQ ID NO: 361 or 416, or an amino acid sequence comprising at least 65% amino acid sequence identity for the amino acid sequence set out in SEQ ID NO: 361 or 416 , which specifically binds to HA.
[0018] [0018] In some examples of the methods provided here, HABP contains a connection module that features at least 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 361 or 416, whereby HABP specifically binds to HA. In particular examples, HABP contains a connection module established in SEQ ID NO: 361 or SEQ ID NO: 416. In other examples, the connection module is the only part
[0019] [0019] In the methods provided, HABPs, HA-binding domains, binding modules, or portions thereof, can be linked to a multimerization domain that is selected from an immunoglobulin constant (Fc) region, a leucine zipper , complementary hydrophobic regions, complementary hydrophilic regions, compatible protein-protein interaction domains, free thiols that form an intermolecular disulfide bond between two molecules, and a protrusion-in-cavity and a compensating cavity of identical or similar size that form multimers stable.
[0020] [0020] In certain methods provided here, HABP is TSG-6 or a hyaluronan binding region of the same. In some examples of the methods provided here, HABP or TSG-6 has an HA binding affinity of at least 1 x 10º M, 2 x 10º MC, 3 x 10º MU, 4 x 10º MI, 5 x 10º MO, 6 x 10th MC, 7 x 10th MC, 8 x 10th MT, 9 x 10th MI, 1x 10th MT or higher. In other examples, HABP or TSG-6 is conjugated to a detectable portion that is either detectable or detectable. For example, HABP or TSG-6 is biotinylated.
[0021] [0021] In some examples of the methods provided here the sample is a stroma tissue sample, such as a tumor stroma tissue sample. The tissue collected in the methods presented here can be fixed, fresh, frozen or embedded in paraffin. In some instances, the sample is taken from a biopsy of a solid tumor, for example, obtained by needle biopsy, CT guided needle, aspiration biopsy, endoscopic biopsy, bronchoscopic biopsy, bronchial lavage, incisional biopsy, excisional biopsy , punch biopsy, scrape biopsy, skin biopsy, bone marrow biopsy, and Loop Electrosurgical Excision Procedure (LEEP) biopsy. In other examples, the sample is a fluid sample that is blood, serum, urine, sweat, semen, saliva, cerebrospinal fluid, or the lymph sample. In any of the methods provided herein, the sample can be obtained from a mammal. In a special example, the mammal is a human being.
[0022] [0022] In any of the methods provided here, the tumor may be a cancer selected from breast cancer, pancreatic cancer, ovarian cancer,
[0023] [0023] In any of the methods provided here, the anti-hyaluronan agent can be an agent that degrades hyaluronan, or it can be an agent that inhibits hyaluronan synthesis. For example, the anti-hyaluronan agent can be a hyaluronan-degrading enzyme. In another example, the anti-hyaluronan agent is an agent that inhibits the synthesis of hyaluronan. For example, the anti-hyaluronan agent is an agent that inhibits the synthesis of hyaluronan as a sense or antisense nucleic acid molecule against an HA synthase or is a small molecule drug. For example, an anti-hyaluronan agent is 4-methylumbelliferone (MU), or a derivative thereof, or leflunomide or a derivative thereof. Such derivatives include, for example, a 4-methylumbelliferone (MU) derivative, which is 6,7-dihydroxy-4-methyl-coumarin or 5,7-dihydroxy-4-methyl-coumarin.
[0024] [0024] In other examples of methods provided herein, the hyaluronan degrading enzyme is a hyaluronidase. In some instances, the hyaluronan-degrading enzyme is hyaluronidase PH20 or the truncated form thereof for the lack of a C-terminal glycosylphosphatidylinositol (GPI) binding site or a part of the GPI binding site. In specific examples, hyaluronidase PH20 is selected from a human being,
[0025] [0025] In other examples, the anti-hyaluronan agent is a hyaluronan-degrading enzyme that is modified by conjugation with a polymer. The polymer can be a PEG and the anti-hyaluronan agent an enzyme that degrades PEGylated hyaluronan. Thus, in some examples of the methods provided herein, the hyaluronan-degrading enzyme is modified by conjugation with a polymer. For example, the hyaluronan-degrading enzyme is conjugated to a PEG, so the enzyme that degrades hyaluronan is
[0026] [0026] A kit containing a hyaluronan binding agent (HABP) is also provided here to detect the amount of hyaluronan in a sample, where HABP has not been prepared from animal cartilage, and an enzyme that degrades hyaluronan. HABP can be generated recombinantly or synthetically. In some examples, HABP contains a connection module. In other examples, HABP contains two or more connection modules. In some examples, the binding module, or modules, are the only HABP portion of the molecule. For example, HABP contains a binding module selected from CD44, LYVE-l, binding protein / HAPLNI, HAPLN2, HAPLN3, HAPLNA4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-1, Stabilin -2 , CAB61358 and KIAAOS27 or a portion thereof comprising a connection module, or a sufficient portion of a connection module to connect to the HA. In other examples, HABP contains a Gl domain of a type C hyaluronan binding protein, for example, a Gl domain selected from Aggrecan Gl, Versican Gl, Neurocan Gl, and Brevican Gl. In particular examples, the Gl domain is the only HABP portion of the molecule.
[0027] [0027] In some examples, the kit contains a HABP containing the amino acid sequence established in any of SEQ ID NOS: 207, 222, 360, 361, 371-394 and 416-418, and 423-426, or a sequence of amino acids that has at least 65%, 70%, 75%, 80%, 85%, 90%, 91%,
[0028] [0028] In some examples of kits provided here, the TSG-6 binding module is modified to reduce or eliminate binding to heparin. For example, heparin binding is reduced at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times or more. In some examples, the TSG-6 linker contains an amino acid substitution at the amino acid position corresponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84, set out in SEQ ID NO: 360, where the The corresponding amino acid residue is identified by an alignment to TSG-6- LM set out in SEQ ID NO: 360. For example, the amino acid substitution is for a non-basic amino acid residue selected from Asp (D), GIu (E ), Ser (S), Thr (T), Asn (N), Gin (0), Ala (A), Val (V), He (1) Leu (L), Met (M), Phe (F) , Tyr (Y) and Trp (W). Thus, a kit is provided here in which the TSG-6 link module contains an amino acid substitution corresponding to the amino acid substitution K20A, K34A or K41A in a TSG-6- LM set out in SEQ ID NO: 360 or its replacement in corresponding residue in another TSG-6-LM. For example, the TSG-6 linker contains amino acid substitutions corresponding to the amino acid substitutions K20A, K34A and K41A in a TSG-6-LM set out in SEQ ID NO: 360, or the replacement to the corresponding residue in another TSG-6 -LM. Also provided here are kits in which the HABP contains a binding module set out in SEQ ID NO: 361 or 416, or an amino acid sequence comprising at least 65% amino acid sequence identity with the amino acid sequence set out in SEQ ID NO: 361 or 416, which specifically binds to HA. For example, HABP contains a connection module that features at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 361 or 416, whereby HABP specifically binds to HA. In particular examples, the HABP contains a connection module set out in SEQ ID NO: 361 or 416. In other examples, the connection module is the only TSG-6 part of HABP.
[0029] [0029] In some examples of kits provided herein, HABP is a multimer containing a first HA binding domain directly or indirectly linked via a linker to a multimerization domain, and a second HA binding domain directly or indirectly linked through a linker to a multimerization domain. For example, the HA binding domain is a binding module or a Gl domain. The first and second HA binding domains can be the same or different. In a particular example, the first and second HA-binding domains are a TSG-6 binding module, a variant of yours or a sufficient portion of yours, which specifically binds to HA. For example, TSG-6-LM contains an amino acid sequence set out in SEQ ID NO: 207, 360, 361, 416, 417 or 418, or an amino acid sequence that comprises at least 65% amino acid sequence identity for the amino acid sequence set out in SEQ ID NO: 207, 360, 361, 416, 417 or 418, which specifically binds to HA. For example, the connection module features at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 207, 360, 361, 416, 417 or 418, whereby HABP specifically binds to HA. In some methods provided herein, the linker contains an amino acid sequence shown in SEQ ID NO: 207, 360, 361, 416, 417 or 418.
[0030] [0030] In the kits provided, HABPs can be linked to a multimerization domain that is selected from an immunoglobulin constant region (Fc), a leucine closure, complementary hydrophobic regions, complementary hydrophilic regions, compatible protein-protein interaction domains , free thiols that form an intermolecular disulfide bond between two molecules, and a protrusion-in-cavity and a compensating cavity of identical or similar size that form stable multimers. In a particular example, the multimerization domain is an Fc domain or a variant of it that is effective for multimerization. For example, the Fc domain is IgG, IgM or IgE, or the Fc domain has an amino acid sequence shown in SEQ ID NO: 359. In some examples of the methods provided here, HABP is a fusion protein that contains a TSG-6 binding and an immunoglobulin Fc domain. For example, HABP is TSG -6-LM-Fc which has an amino acid sequence set in SEQ ID NO: 212 or 215, or an amino acid sequence that exhibits at least 65% amino acid sequence identity with SEQ ID NO : 212 or 215, and specifically binds to HA, such as an amino acid sequence that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 212 or 215, whereby HABP specifically binds HA. In particular examples, HABP has an amino acid sequence set out in SEQ ID NO: 212 or 215. In any of the methods provided here, HABP can be TSG-6-LM-Fc / nHep that has an amino acid sequence set out in SEQ ID NO: 215 or an amino acid sequence that exhibits at least 65% amino acid sequence identity with SEQ ID NO: 215 and specifically binds to HA, such as an amino acid sequence that exhibits at least 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence set out in SEQ ID NO: 215, whereby HABP specifically binds to HA.
[0031] [0031] In particular examples of kits provided herein, HABP is a hyaluronan or TSG-6 binding region thereof. In some examples of the methods provided here, HABP has a binding affinity, represented by the association constant (Ka) for HA of at least 10º Nº, for example, at least 1 x 10º MN, 2 x 10º MC, 3 x 10º MT, 4 x 10º MO, 5 x 10º MI, 6 x 10º MT, 7 x 10º Mo, 8 x 10º Mo, 9 x 10º Mo, 1x 10º M or superior. For example, HABP has a binding affinity, represented by the dissociation constant (Kd) for HA of at least less than, or less than 1x10 M, 9x10 * M, 8x10 º M, 7x10 ºM, 6x10 MM, 5x10 ºM, 4x10 ºM, 3x10ºM, 2x10M, 1x10 ºM, 9x10 ºM, 8x10 ºM, 7x10 MM, 6x10 ºM, 5x10 ºM, 4x10 ºM, 3x10 ºM, 2x10 M, 1x10 º * M or lower Kd. In other examples, HABP is conjugated to a detectable portion that is either detectably labeled or can be detected. For example, HABP is biotinylated.
[0032] [0032] kits containing a hyaluronan binding agent (HABP) to detect the amount of hyaluronan in a sample, where HABP has not been prepared from animal cartilage, and an anti-hyaluronan agent (for example, example, an enzyme that degrades hyaluronan). Any of the kits provided here may also contain reagents for the detection of HABP. In any example of the kits provided herein, the anti-hyaluronan agent can be any of those described above, or any other part included. For example, the anti-hyaluronan agent can be a hyaluronan-degrading enzyme such as hyaluronidase. For example, the hyaluronan degrading enzyme is hyaluronidase PH20 or the truncated form thereof, for the lack of a C-terminal glycosylphosphatidylinositol (GPI) binding site or a part of the GPI binding site. In some examples, PH20 is selected from a human, monkey, bovine, sheep, rat, mouse, or guinea pig PH20. For example, the hyaluronan-degrading enzyme is a human PH20 hyaluronidase that is neutral-active and N-glycosylated, and is selected from (a) a polypeptide that is a full-length PH20 hyaluronidase or is a truncated form of the C-terminal of PH20, wherein the truncated form includes at least amino acid residues 36-464 of SEQ ID NO: 1, where the full-length PH20 has the amino acid sequence shown in SEQ ID NO: 2; or (b) a hyaluronidase polypeptide comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 983%, 99% or more of sequence identity to the polypeptide, or truncated form of the amino acid sequence shown in SEQ ID NO: 2; or (c) a hyaluronidase polypeptide of (a) or (b) comprising amino acid substitutions, wherein the hyaluronidase polypeptide has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the polypeptide described in SEQ ID NO: 2, Or with their corresponding truncated forms. In particular examples, the hyaluronan-degrading enzyme is a PH20 which comprises a designated rHuPH20 composition. In some examples, the hyaluronan-degrading enzyme is modified by conjugation to a polymer, such as, for example, a PEG, and the enzyme that degrades hyaluronic is PEGylated. Therefore, a kit is provided here in which the hyaluronan degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). Kits are also provided here that also contain a corticosteroid. Any of the kits provided here may also contain a label or package insert for the use of their components.
[0033] [0033] here are provided methods of using a hyaluronan-binding protein (HABP) for selecting an individual to treat a tumor, with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), wherein HABP has not been prepared or isolated from animal cartilage. Also present here are pharmaceutical compositions containing a hyaluronan-binding protein (HABP) for use in selecting an individual for the treatment of a tumor, with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), in that HABP has not been prepared or isolated from animal cartilage.
[0034] [0034] here are provided methods of using a hyaluronan binding protein (HABP) to predict the effectiveness of treating an individual with an anti-hyaluronan agent (e.g., an enzyme that degrades hyaluronan), where HABP does not has been prepared or isolated from animal cartilage. Also present are pharmaceutical compositions that contain a hyaluronan-binding protein (HABP) to predict the effectiveness of treating an individual with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), where HABP has not been prepared or isolated from animal cartilage.
[0035] [0035] In any of the pharmaceutical uses or compositions provided here, HABP may contain a binding module (or modules) or a Gl domain. In some examples, HABP contains a TSG-6 (LM) binding module, a variant of its own , or a sufficient portion thereof, which binds to HA. In a particular example, the TSG-6 binding module is modified to reduce or eliminate binding to heparin. In some examples, HABP contains an amino acid sequence as described in any of SEQ ID NOS: 207, 212, 215, 222, 360, 361, 371-394 and 416-418, and 423-426, or a sequence of amino acids that exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 95%, 95%, 96%, 97%, 98%, 99%, Or more of sequence identity for an amino acid sequence defined in any of SEQ ID NOS: 207, 212, 215, 222, 360, 361,
[0036] [0036] Also provided here is a multimer TSG-6-LM containing a first directly or indirectly linked module via a linker to a multimerization domain, and a second directly or indirectly linked module via a linker to a multimerization domain, in which the first and second polypeptides are not part of the TSG-6 full-length sequence. In some examples, the linker is the only TSG-6 portion of the first polypeptide and the second polypeptide. The first and second connection modules can be the same or different. In some examples, the linker contains an amino acid sequence set out in SEQ ID NO: 207, 360, 417 or 418 or an amino acid sequence that comprises at least 65% amino acid sequence identity set out in SEQ ID NO: 207 , 360, 417 or 418, which specifically binds to HA. For example, the link module displays at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % sequence identity for the amino acid sequence shown in SEQ ID NO: 207, 360, 417 or 418, which specifically binds to HA. In some examples, the linker contains an amino acid sequence shown in SEQ ID NO: 207, 360, 417 or 418.
[0037] [0037] In some examples, the TSG-6 binding module is modified to reduce or eliminate binding to heparin. Binding to heparin is reduced at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times or more. In some examples, the TSG-6 linker contains an amino acid substitution at the amino acid position corresponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84, set out in SEQ ID NO: 360, where a Corresponding amino acid residue is identified by aligning a TSG-6-LM set out in SEQ ID NO: 360. For example, the amino acid substitution is in a TSG-6-LM set out in SEQ ID NO: 207 and the replacement or substitutions amino acid residue is at amino acid residue 21, 35, 42, 55, 57, 73 or 85. The amino acid substitution can be that of a non-basic amino acid residue selected from Asp (D), Glu (E), Ser (S), Thr (7), Asn (N), Gin (0), Ala (A), Val (V), He (1), Leu (L), Met (M), Phe (F), Tyr (Y) and Trp (W). For example, the TSG-6 linker contains an amino acid substitution corresponding to amino acid substitutions K20A, K34A or K41A in a TSG-6-LM set out in SEQ ID NO: 360, or to the substitution by the corresponding residue in another TSG- 6-LM.
[0038] [0038] A TSG-6-LM multimer is also provided here in which the multimerization domain is selected from an immunoglobulin constant region (Fc), a leucine zipper, complementary hydrophobic regions, complementary hydrophilic regions, protein-protein interaction domains compatible proteins, free thiols that form an intermolecular disulfide bond between two molecules, and a protrusion-in-cavity and a compensating cavity of identical or similar size that form stable multimers. In some examples, the multimerization domain is an Fc domain or a variant of it that makes multimerization effective. For example, the Fc domain is IgG, IgM or an IgE. In a particular example, the Fc domain has an amino acid sequence set out in SEQ ID NO:
[0039] [0039] A TSG-6-LM multimer containing a TSG-6 binding module and an immunoglobulin Fc domain is also provided herein. In some examples, the TSG-6-LM multimer contains an amino acid sequence shown in SEQ ID NO: 212 or 215, or an amino acid sequence that exhibits at least 65% amino acid sequence identity for SEQ ID NO: 212 or 215. For example, the TSG-6 multimer contains an amino acid sequence that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 9394 94%, 95%, 96% , 97%, 98%, or 99% sequence identity for the amino acid sequence shown in SEQ ID NO: 212 or 215, and which specifically binds to HA. In particular examples, the TSG-6-LM multimer contains an amino acid sequence shown in SEQ ID NO: 212 or
[0040] [0040] Also provided here are methods for selecting an individual, predicting effectiveness and / or monitoring treatment, using any of the above HABP's to detect HA in vivo by imaging methods. The in vivo imaging method can be magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), computed tomography (CT), axial computed tomography (CAT), electron beam computed tomography (EBCT), high resolution computed tomography (HRCT), hypocycloid tomography, positron emission tomography (PET), scintigraphy, gamma camera, B + detector, a y detector, fluorescence images, low light images, X-rays, and / or bioluminescence image. In such methods, HABP is conjugated, directly or indirectly, to a part that provides a signal or induces a signal that is detectable in vivo. DETAILED DESCRIPTION
[0041] [0041] Outline: A. DEFINITIONS B. HIALURONAN-BINDING PROTEIN AND DIAGNOSIS OF SIDE DISH
[0042] [0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as is commonly understood by a person skilled in the art to which the invention belongs. All patents, patent applications, published applications and publications, GenBank strings, websites and other published materials referred to throughout this description, unless otherwise stated, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms present here, those in this section prevail. whenever reference is made to a URL or other identifiers or addresses, it is understood that such identifiers can change and particular information on the internet can come and go, but the equivalent information is known and can be easily accessed, such as through internet search and / or appropriate databases. References to them show the availability and public disclosure of such information.
[0043] [0043] As used herein, a follow-up diagnosis refers to a diagnostic method and / or reagent that is used to identify individuals sensitive to treatment, with a particular treatment, or to monitor treatment and / or to identify an effective dose for an individual or sub-group or another group of individuals. For the purposes described here, a follow-up diagnosis refers to reagents, such as modified TSG-6 proteins, that are used to detect hyaluronan in a sample. The accompanying diagnosis refers to reagents and also to the test (s) that is / are performed with the reagent.
[0044] [0044] As used herein, hyaluronan (HA, also known as hyaluronic acid or hyaluronate) refers to a naturally occurring polymer of repeated disaccharide units of N-acetylglucosamine and D-glucuronic acid. Hyaluronan is produced by certain tumors.
[0045] [0045] As used herein, "elevated HA", with reference to the amount or level of HA in a tissue or body fluid sample, refers to the degree or extent of HA in the body tissue or fluid sample, in comparison with normal or healthy tissue, or a sample of body fluid. The amount of HA is greater if the amount is at least, or at least about 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times , 30 times, 40 times, 50 times, 60 times, 70 times or higher than the amount or level of HA in a corresponding normal or healthy tissue. It is understood that the amount of HA can be determined and quantified or semi-quantified using methods such as solid-phase or histochemical binding assays. For example, the amount can be based on comparing plasma levels or comparing staining intensity (for example, percentage of positive pixels), as determined by histochemistry. For example, does high HA exist if the HA marker by histochemistry or another method is HA ” and / or if there is an HA staining of more than 25% of the tumor section. For example, there is high HA if there is a strong positive staining ratio (such as brown staining) to the sum of the total colored area that is greater than
[0046] [0046] As used herein, an HA score refers to a semi-quantitative count of HA positivity levels on cell members and in the tumor stroma. The score can be determined by detecting HA in tumor tissues, such as fixed in formalin and tissue embedded in paraffin, by histochemical methods, such as immunohistochemistry or pseudo immunohistochemistry methods, for HA using a HABP. The degree of staining in cells and stroma can be determined visually under a microscope or by available computer algorithm programs and software. For example, images can be analyzed quantitatively using a pixel counting algorithm for HA staining (for example, Aperio Spectrum Software and other "conventional methods that measure or quantify or semi-quantify the degree of staining). A tumor is classified or marked as HA "**" (HA **) under a strong HA stain of more than 25% of the tumor section; as HA "º *" ra * º (HA **) in the strong HA stain between 10 and 25% of the tumor section; and as HA “(HA **) in the strong HA staining of less than 10% of the tumor section. For example, a ratio of strong positive staining (such as brown staining) to the sum of the total colored area can be calculated and marked, if the proportion is greater than 25% of strong positive staining for total staining of tumor tissue is marked as HA, if the proportion is 10-25% of strong positive staining for total staining tumor tissue is marked as HA ”, if the proportion is less than 10% of strong positive staining for c total tumor tissue growth is marked as HA ", and if the ratio of strong positive staining to total staining is 0, the tumor tissue is marked as 0. The method
[0047] [0047] As used herein, a hyaluronan-binding protein (HA-binding protein; HABP) refers to any protein that specifically binds HA to enable the detection of HA. The binding affinity is one that has a constant Ka association that is at least about, that is, at least 10 'M' *. For the accompanying diagnostic methods and products provided here, the HA-binding protein is a synthetic or recombinantly produced protein, not a protein derived from a biological or physiological source, such as cartilage. HA-binding proteins include HA-binding domains, including binding modules that bind to HA and sufficient portions thereof, which specifically bind to HA to allow their detection. Thus, HABPs include any protein that contains a hyaluronan binding region, or a domain or a sufficient portion of it, to specifically bind to HA. Exemplary hyaluronan binding regions are binding modules (binding domains) or Gl domains. A sufficient portion includes at least 10, 20, 30, 40, 50, 60, 70 80, 90, 95 or more contiguous amino acids a connection domain or connection module. HA-binding proteins also include fusion proteins that contain an HA-binding protein, and one or more additional polypeptides, including multimerization domains. Examples of HA-binding proteins include but are not limited to aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, TSG-6 mutants, such as those provided herein, including polypeptides that contain HA-binding domains, and their connection modules that connect to the HA.
[0048] [0048] As used herein, the hyaluronan binding domain or an AH binding domain refers to a region or domain of a HABP polypeptide that specifically binds to hyaluronan with a binding affinity that has a constant of association Ka, which is at least about or is at least 10'M or 10'M or greater, or a dissociation constant Kd, which is less than 10 “M or 10 M or less. Exemplary hyaluronan binding domains include, for example, binding modules (also called binding domains described herein) or Gl domains, or sufficient portions of a binding module or Gl domain that specifically bind to HA.
[0049] [0049] As used herein, the reference to "the only portion of a HABP" is a binding module or Gl domain or grammatical variations thereof, means that the HABP molecule (for example, a TSG-6 binding module ) consists or consists essentially of the ligation module or the Gl domain, but does not include the full length amino acid sequence of the reference HABP. Thus, HABP contains only one hyaluronan binding region, or a sufficient portion of it, to specifically bind to HA. It is understood that HABP can contain additional sequences of non-HABP amino acids, including, but not limited to, sequences that correspond to a detectable moiety or moiety capable of detection, or a multimerization domain.
[0050] [0050] As used herein, modified, with respect to modified HA-binding proteins refers to modifications to alter, normally improve, one or more properties of an HA-binding protein, for detection of diagnostic methods provided here. The modifications include mutations that increase the protein affinity and / or specificity for HA.
[0051] [0051] As used herein, a domain refers to a portion (a sequence of three or more, generally 5 or 7 or more amino acids) of a polypeptide that is structurally and / or functionally distinguishable or definable. For example, a domain includes those that can form a folded structure, independently, within a protein consisting of one or more structural motifs (for example, combinations of alpha helices and / or beta chains linked by circular regions) and / or recognized by virtue of a functional activity, such as kinase activity. A protein can have one, or more, a distinct domain. For example, a domain can be identified, defined or distinguished by the homology of the respective sequence for related family members, such as homology and motifs, which define an extracellular domain. In another example, a domain can be distinguished by its function, such as by enzymatic activity, for example, kinase activity, or an ability to interact with a biomolecule, such as DNA binding, ligand binding and dimerization. A domain can independently have a function or activity, such that the domain independently or fused with another molecule, can exert an activity, such as, for example, proteolytic activity or ligand binding. A domain can be a linear amino acid sequence or a non-linear amino acid sequence of the polypeptide. Many polypeptides contain a plurality of domains.
[0052] [0052] As used herein, a fusion protein refers to a chimeric protein containing two or more portions, of two more proteins or peptides that are directly or indirectly linked via peptide bonds.
[0053] [0053] As used herein, a multimerization domain refers to a sequence of amino acids that promotes the stable interaction of the polypeptide molecule with another polypeptide molecule containing a complementary multimerization domain, which can be the same or a domain different multimerization, to form a stable multimer with the first domain. Generally, a polypeptide is linked directly or indirectly to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, compatible protein-protein interaction domains, such as, but not limited to, an R subunit of PKA and an anchoring domain ( AD), a free thiol that forms an intermolecular disulfide bond between two molecules, and a bulge-in-cavity (for example, “knobs into holes”) and a compensating cavity of identical or similar size, which form stable multimers. The multimerization domain, for example, can be an immunoglobulin constant region. The immunoglobulin sequence can be an immunoglobulin constant domain, such as the Fc domain or portions thereof, from IgG1, IgG2, IgG3 or IgG4, IgA, IgE, IgD and IgM subtypes.
[0054] [0054] As used herein, "knobs into holes" (also referred to here as bulge-in-cavity) refers to certain multimerization domains modified in such a way that the steric interactions between these domains, not only promote stable interaction , but also promote the formation of heterodimers (or multimers) over homodimers (or homomultimers) from a mixture of monomers. This can be achieved, for example, through the construction of protrusions and cavities. small amino acid side chains from the interface of the first polypeptide with larger side chains (for example, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the bulges are optionally created at the interface of a second polypeptide, replacing large side chains of amino acids by minors (for example, alanine or threonine).
[0055] [0055] As used herein, the complementary multimerization domains refer to two or more multimerization domains that interact to form polypeptide multimers with stable bonds for each of these domains. Complementary multimerization domains can be the same domain or a member of a family of domains, such as, for example, Fc regions, leucine zippers, and "knobs" and holes.
[0056] [0056] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide that contains the constant region of an antibody heavy chain, with the exception of the first domain of the immunoglobulin constant region. Thus, Fc refers to the last two immunoglobulin domains of the IgA, Igb, and IgE constant regions, or the last three immunoglobulin domains of the IgE and IgM constant regions. Optionally, an Fc domain can include all or part of the flexible hinge N-terminal for these domains. For IgA and IgM, Fc can include the J chain. For an exemplary IgG Fc domain, it contains immunoglobulin domains Cy2 and Cy3, and, optionally, all or part of the joint between Cyl and Cy2. The boundaries of the Fc region may vary, but usually include at least part of the articulation region. In addition, Fc also includes any allelic or species variant, or any modified form or variant, such as any modified form or variant that changes the link to an FCcR or changes an Fc-mediated effector function. Examples of sequences from other Fc domains, including modified Fc domains are known.
[0057] [0057] As used herein, "Fc chimera" refers to a chimeric polypeptide in which one or more polypeptides are linked, directly or indirectly, to an Fc region or a derivative thereof. Typically, an Fc chimera combines the Fc region of an immunoglobulin with another polypeptide, such as, for example, an ECD polypeptide. Modified Fc polypeptides or derivatives thereof are known to those skilled in the art.
[0058] [0058] As used herein, "multimer", with reference to a hyaluronan-binding protein, refers to a HABP that contains multiple HA-binding sites, for example, at least two, three, or four sites of connection to HA. For example, a HABP multimer refers to a HABP that contains at least two binding modules, each of which is capable of binding to HA. For example, a multimer can be generated, by linking, directly or indirectly, from two or more link modules (for example, the TSG-6 link module). Binding can be facilitated by the use of a multimerization domain, such as an Fc protein.
[0059] [0059] As used herein, an allele variant or an allele variation refers to a polypeptide encoded by a gene that is different from a reference form of a gene (i.e., it is encoded by an allele). Usually, the reference form of the gene encodes a wild-type form and / or a predominant form of a polypeptide, of a population or member, of a unique reference species. Typically, allelic variants, which include variants between some species, normally have at least 80%, 90% or more, of amino acid identity with a wild-type form and / or a predominant form of the same species; the degree of identity depends on the gene, and whether the comparison is made between species or intraspecies. Generally, intraspecific allelic variants have at least about 80%, 85%, 90% or 95% identity or more, with a wild-type form and / or a predominant form, including 96%, 97%, 98% , 99% or more, identity with a wild-type form and / or predominant form of a polypeptide.
[0060] [0060] As used herein, species variants refer to variants of the same polypeptide between two or more species. Generally, interspecies variants have at least about 60%, 70%, 80%, 85%, 90%, or 95% identity or more, with a wild-type form and / or predominant form of other species, including 96% , 97%, 98%, 99% or more, of identity with a wild-type form and / or predominant form of a polypeptide.
[0061] [0061] As used herein, modifications refer to the modification of an amino acid sequence of a polypeptide, or a nucleotide sequence of a nucleic acid molecule, and include deletions, insertions, and substitutions of amino acids and nucleotides, respectively .
[0062] [0062] As used herein, a composition refers to any mixture. It can be a solution, a suspension, liquid, powder, paste, aqueous, non-aqueous solution, or any combination thereof.
[0063] [0063] As used herein, a combination refers to any association between two or more items. The combination may be two or more separate elements, such as two or more sets of compositions, may be a mixture of them, such as a single mixture of two or more items, or any variation. The elements of a combination are generally functionally associated or related. A kit is a package combination that optionally includes instructions for using the combination or its elements.
[0064] [0064] As used herein, normal levels or values can be defined in a variety of ways known to a person skilled in the art. Normally, normal levels refer to the levels of HA expression across a healthy population. Normal levels (or reference levels) are based on measurements from healthy individuals, such as from a specified source (ie blood, serum, tissues, or another source). Often, a normal level will be specified as a "normal range", which typically refers to the range of values on average for 95% of the healthy population. The reference value is used here interchangeably with the normal level, but it can be different from the normal levels, depending on the individuals or the source. Reference levels are usually dependent on normal levels for a given segment of the population. Thus, for the purposes of this invention, a normal or reference level is a predetermined standard or control, by which a test patient can be compared.
[0065] [0065] As used herein, high levels refer to any level of expression or amount of HA above a recited or normal threshold.
[0066] [0066] As used herein, biological sample refers to any sample obtained from a live or viral source, or other source of macromolecules and biomolecules, and includes any type of cell or tissue of an individual, from which , nucleic acid or protein, or another macromolecule can be obtained. The biological sample can be a sample obtained directly from a biological source, or that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissues and organ samples from animals, including samples of biopsied tumors.
[0067] [0067] As used here, detection includes methods that allow the visualization (by eye or equipment) of a protein. A protein can be visualized using an antibody specific to the protein. The detection of a protein can also be facilitated by fusing a protein with a tag, including a tagged epitope or tag.
[0068] [0068] As used herein, a label refers to a detectable compound or composition that is directly or indirectly conjugated to a polypeptide, in order to generate a labeled polypeptide. The marker can be detectable by itself (for example, radioisotope markers or fluorescent markers) or, in the case of an enzyme marker, it can catalyze a chemical change to a substrate compound composition that is detectable. Non-limiting examples of markers include fluorogenic moieties, green fluorescent protein, or luciferase.
[0069] [0069] As used herein, affinity refers to the strength of the interaction between two molecules, such as between a hyaluronan and hyaluronan binding protein. Affinity is often measured by the association equilibrium constant (Ka) or dissociation equilibrium constant (Kd). The binding affinity between the molecules described here, usually has a binding affinity with an association constant (Ka) of at least about 10º 1 / mol, 10º l1 / mol, 10º l1 / mol, 10º l1 / mol or more (usually 10 '1 / mol- 10º 1 / mol or greater). The binding affinity of molecules present here can also be described based on the dissociation constant (Kd) of at least less, or less than, or 10 M, 10 M, 10 M, 10 M, 107 "! M , 10 M or less.
[0070] [0070] As used herein, reference to a sufficient portion of it that binds to HA, means that the binding molecule has a Ka of at least, or at least about 10º to 10º Mº, or a constant of dissociation (Kd) of 1 x 107 or 1 x 10º Mou less than HA.
[0071] [0071] As used herein, specificity (also referred to here as selectively) with respect to two molecules, such as with respect to a hyaluronan and HA-binding protein, refers to a greater affinity that the two molecules exhibit , to each other, in relation to their affinity for other molecules. Thus, a hyaluronan-binding protein (HABP) with a greater specificity for HA means that it binds to other molecules, such as heparin, with a lower affinity than when it binds to HA. Specific binding usually results in selective binding.
[0072] [0072] As used herein, a "Gl domain" refers to a type C HA binding domain of the HA binding protein. The Gl domain contains a module of
[0073] [0073] As used herein, binding modules or binding domains, used interchangeably here, are hyaluronan binding domains that occur in proteins and facilitate HA binding, and which are involved in the assembly of extracellular matrix, cell adhesion and migration. For example, the human TSG-6 binding module contains two alpha helices and two beta antiparallel sheets arranged around a hydrophobic nucleus. This defines the consensus fold for the binding module superfamily, which includes CD44, TSG-6, cartilage binding protein, agrecan and others, as described herein. As used herein, an "Ig module" refers to that portion of the Gl C HABPs type C domain, which is involved in the connection between Type C HABPs. Ig modules of type C hyaluronans interact with one another to form a stable tertiary structure with hyaluronan.
[0074] [0074] As used herein, a "solid phase binding assay" refers to an in vitro assay in which an antigen comes into contact with a ligand, in which an antigen or ligand is attached to a solid support. The solid phase can be a phase in which the components are physically immobilized to a solid support. For example, solid supports include, but are not limited to, a microtiter plate, a membrane (for example, nitrocellulose), a granule, a rod, a thin layer chromatography plate, or other solid support. After antigen-ligand interaction, unwanted or non-specific components can be removed (for example, by washing) and the antigen-ligand complex detected.
[0075] [0075] As used herein, preventing the effectiveness of treatment with an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan, means that the accompanying diagnosis may be an indicator of prognosis for treatment with an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan. For example, based on the results of detecting hyaluronan or another marker with the accompanying diagnosis, it can be determined that an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan, is likely to have some effect in treating individuals.
[0076] [0076] As used herein, prognosis indicator refers to a parameter that indicates the probability of a particular outcome, such that the probability of treatment is effective for a particular disease or individual.
[0077] [0077] As used herein, elevated HA in a sample, refers to an amount of HA in a sample that is increased compared to the level present in a corresponding sample, from a healthy sample or compared to a standard predetermined.
[0078] [0078] As used herein, high levels of hyaluronan refer to the values of hyaluronan, in particular, tissue, body fluid or cells, depending on the disease or condition, as a consequence, or otherwise, observed in the disease . For example, as a consequence of the presence of a tumor rich in hyaluronan, levels of hyaluronan (HA) may be elevated in body fluids, such as blood, urine, saliva and serum, and / or in tumor tissue or a cell. The level can be compared to a standard or other appropriate control, such as a comparable sample from an individual, who does not have the disease associated with HA, such as an individual who does not have a tumor.
[0079] [0079] As used herein, the corresponding waste refers to waste that occurs in aligned locations. Related or variant polypeptides are aligned by any method known to those skilled in the art. Such methods usually maximize combinations, and include methods such as using manual alignments and the numerous alignment programs available (for example, BLASTP) and others known to those skilled in the art. By aligning the polypeptide sequences, one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues, as guides. Corresponding positions can also be based on structural alignments, for example, through computer simulated protein structure alignments. In other cases, the corresponding regions can be identified.
[0080] [0080] As used herein, an anti-hyaluronan agent refers to any agent that modulates the synthesis or degradation of hyaluronan (HA), thereby altering the levels of hyaluronan in a tissue or cell. For the purposes of this invention, anti-hyaluronan agents reduce the levels of hyaluronan in a tissue or cells, compared to the absence of the agent. Such agents include compounds that modulate the expression of the genetic material that encodes HA synthase (SAH), and other enzymes or receptors involved in the metabolism of hyaluronan, or that modulate proteins that synthesize or degrade hyaluronan, including HAS function or activity. . The agents include small molecules, nucleic acids, peptides, proteins or other compounds. For example, anti-hyaluronan agents include, but are not limited to, sense or antisense molecules,
[0081] [0081] As used herein, an enzyme that degrades hyaluronan refers to an enzyme that catalyzes the cleavage of a hyaluronan polymer (also referred to as hyaluronan or HA) into lower molecular weight fragments. Examples of enzymes that degrade hyaluronan are hyaluronidases, and in particular chondroitinases and lyases that have the ability to depolymerize hyaluronan. Exemplary chondroitinases which are enzymes that degrade hyaluronan include, but are not limited to, chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AC lyase (also known as chondroitin sulphate lyase or chondroitin sulphate elimination) and chondroitin C lyase. Chondroitin ABC lyase comprises two enzymes, chondroitin-sulfate-ABC endolysis (EC
[0082] [0082] As used herein, hyaluronidase refers to a class of enzymes that degrades hyaluronan. Hyaluronidases include bacterial hyaluronidases (EC
[0083] [0083] As used herein, "purified bovine testicular hyaluronidase" refers to a purified bovine hyaluronidase from bovine testis extracts (see U.S. Patent Nos. 2,488,564, 2,488,565, 2,806,815,
[0084] [0084] As used herein, "purified sheep testicular hyaluronidase" refers to a purified sheep hyaluronidase from sheep testis extracts (see U.S. Patent Nos. 2,488,564, 2,488,565 and 2,806,815 and Application for PCT International Patent No. WO2005 / 118799). Examples of commercially available purified sheep testicular extract include VitraseO and ovine hyaluronidases, including, but not limited to those available from Sigma Aldrich, Cell Sciences, EMD Chemicals, GenWay Biotech, Inc., Mybiosource.com and Raybiotech, Inc. Also included are hyaluronidases produced recombinantly, such as sheep, but not limited to those generated by expression of a nucleic acid molecule, as described in any of SEQ ID NOs: 66 and 193-194.
[0082] [0082] As used herein, "PH20" refers to a type of hyaluronidase that occurs in sperm and is neutral-active. PH-20 occurs on the surface of the sperm and the acrosome derived from the lysosome, where it is attached to the inner acrosomal membrane. PH20 includes those of any origin, including but not limited to those of human origin, chimpanzees, cynomolgus monkeys, murine, bovine, sheep, guinea pig, rabbit and rat Rhesus monkeys. Exemplary PH20 polypeptides include those of human origin (SEQ ID NO: 1), chimpanzee (SEQ ID NO: 101), Rhesus monkey (SEQ ID NO: 102), cynomolgus monkey (SEQ ID NO: 29), cow (SEQ ID NOS : 11 and 64), mouse (SEQ ID NO: 32), rat (SEQ ID NO: 31), rabbit (SEQ ID NO: 25), sheep (SEQ ID NOS: 27, 63 and 65) and porcupine -Guinea (SEQ ID NO: 30).
[0085] [0085] References for enzymes that degrade hyaluronan include polypeptides from enzymes that degrade precursor polypeptides, and enzymes that degrade mature hyaluronan (such as those in which the signal sequence has been removed), truncated forms of these that have activity, and include allelic variants and variant species, variants encoded by splice variants, and other variants, include polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the precursor polypeptides set out in SEQ ID Nos: 1 and 10-48, 63-65, 67-102, or the mature forms thereof . For example, the reference to the hyaluronan-degrading enzyme also includes the variants of the human PH20 precursor polypeptide set out in SEQ ID NOS: 50-51. Hyaluronan degrading enzymes also include those that contain chemical or post-translational modifications and those that do not contain post-translational or chemical modifications. Such modifications include, but are not limited to, PEGylation, albumination, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. An incomplete PH20 hyaluronidase is any form shortened at the C-terminus thereof, particularly truncated forms that are neutrally active when N-glycosylated.
[0086] [0086] As used herein, a "soluble PH20" refers to any form of PH20 that is soluble under physiological conditions. A soluble PH20 can be identified, for example, by its partition in the aqueous phase of a solution of X-114 Tritono at 37ºC. (Bordier et al., (1981) UU. Biol. Chem., 256: 1604-7). Membrane-anchored PH20, such as lipid-anchored PH20, including GPI-anchored PH20, will partition into the detergent-rich phase, but will partition into the detergent-poor water phase, followed by phospholipase-C treatment. Included among the soluble PH20 are PH20 anchored to the membrane, in which one or more regions associated with PH20 anchoring to the membrane have been removed or modified, in which the soluble form retains hyaluronidase activity. Soluble PH20 also includes recombinant soluble PH20 and those contained in, or purified from, natural sources, such as, for example, from sheep or bovine testis extracts. An example of such soluble PH20 is human soluble PH20.
[0087] [0087] As used herein, soluble human PH20 or sHuPH20 includes PH20 polypeptides in which all or a portion of the glycosylphosphatidylinositol (GPI) anchor sequence is missing at the C-terminal end, such that after expression, the polypeptides are soluble under physiological conditions. Solubility can be determined by any suitable method that demonstrates solubility under physiological conditions. Examples of such methods are the X-1111 Triton O assay, which evaluates the O partitioning in the aqueous phase, and which is described above and in the examples. In addition, a soluble human PH20 polypeptide is, if produced in CHO cells, such as CHO-S cells, a polypeptide that is expressed and secreted into the cell culture medium. Soluble human PH20 polypeptides, however, are not limited to those produced in CHO cells, but can be produced in any cell or by any method, including recombinant expression and polypeptide synthesis. The reference for secretion in CHO cells is defined. Thus, if a polypeptide can be expressed and secreted in CHO cells and is soluble, that is, divisions for the aqueous phase, when extracted with X-114 Tritono, it is a soluble PH20 polypeptide, or it is not so produced. The precursor polypeptides for sHuPH20 polypeptides can include a signal sequence, such as a heterologous or non-heterologous (i.e., native) signal sequence. Examples of precursors are those that include a signal sequence, such as the 35 amino acid signal sequence, at the positions of amino acids 1-35 (see, for example, amino acids 1-35 of SEQ ID NO: 1).
[0088] [0088] As used herein, an "extended soluble PH20" or "esPH20" includes soluble PH20 polypeptides that contain residues up to the signal sequence attached to the GPI anchorage, and one or more contiguous residues of the signal sequence attached to the anchorage to the GPI. GPI, so that esPH20 is soluble under physiological conditions. Solubility under physiological conditions can be determined by any method known to those skilled in the art. For example, it can be evaluated by the X-114 Tritono assay described above and in the examples. In addition, as discussed above, a soluble PH20 is, if produced in CHO cells, such as CHO-S cells, a polypeptide that is expressed and secreted into the cell culture medium. Soluble human PH20 polypeptides, however, are not limited to those produced in CHO cells, but can be produced in any cell or by any method, including recombinant expression and polypeptide synthesis. The reference for secretion in CHO cells is defined. Thus, if a polypeptide can be expressed and secreted in CHO cells and is soluble, that is, divisions for the aqueous phase, when extracted with X-114 TritonO, it is a soluble PH20 polypeptide or it is not so produced. Human soluble esPH20 polypeptides include, in addition to residues 36-490, one or more contiguous amino acids from the 491 amino acid residue position of SEQ ID NO: 1, inclusive, such that the resulting polypeptide is soluble. Exemplary human soluble esPH20 polypeptides are those that have the amino acid residues corresponding to amino acids 36-491, 36-492, 36-493, 36-494, 36-495, 36-496 and 36-497 of SEQ ID NO: 1. Examples of these are those with an amino acid sequence established in any of SEQ ID NOS: 151-154 and 185-
[0089] [0089] As used herein, the reference to "esPH20s" includes —pregnant esPH20 polypeptides and mature esPH20 polypeptides (such as those in which the signal sequence has been removed), incomplete forms of these that have enzymatic activity (retention, at least, 1% 10%, 20%, 30%, 40%, 50% or more of the full length form) and are soluble, and include allelic variants and variant species, variants encoded by splice variants, and other variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or more of sequence identity with the precursor polypeptides set forth in SEQ ID NO: 1 and 3, or their mature forms.
[0090] [0090] As used herein, the reference to "esPH20s" also includes those that contain chemical or post-translational modifications and those that do not contain post-translational or chemical modifications. Such modifications include, but are not limited to PEGylation, albumination,
[0091] [0091] As used herein, "recombinant soluble human PH20 (rHuPH20)" refers to a composition containing soluble forms of human PH20 as expressed and secreted recombinantly in Chinese hamster ovary (CHO) cells. soluble rHuPH20 is encoded by a nucleic acid molecule that includes a signal sequence, and is set out in SEQ ID NO: 49. The nucleic acid encoding soluble rHuPH20 is expressed in CHO cells, which secrete the mature polypeptide. As produced in the culture medium, there is a great heterogeneity at the C-terminal so that the product includes a mixture of species, which can include any one or more amino acids 36-481 and 36- 482 of PH20 (for example, SEQ ID NO : 4 to SEQ ID NO: 9) in varied abundance.
[0092] Similarly, polypeptides and compositions, for other forms of PH20, such as esPH20s expressed recombinantly therefrom, can include a plurality of species whose C-terminal exhibits heterogeneity. For example, recombinantly expressed esPH20 compositions produced by expression of the SEQ ID NO: 151 polypeptide, which encodes an esPH20 that has amino acids 36-497, may include forms with fewer amino acids, such as 36-496, 36- 495.
[0093] [0093] As used herein, an "N-linked portion" refers to an asparagine (N) amino acid residue of a polypeptide that is capable of being glycosylated by post-translational modification of a polypeptide. Examples of N-linked portions of human PH20 include amino acids N82, N166, N235, N254, N368 and N393 of human PH20 described in SEQ ID NO: 1.
[0094] [0094] As used herein, an "N-glycosylated polypeptide" refers to a truncated polypeptide or PH20 form of the same linkage containing oligosaccharides of at least three N-linked amino acid residues, for example, linked portions to N corresponding to amino acid residues N235, N368 and N393 of SEQ ID NO: 1. An N-glycosylated polypeptide can include a polypeptide in which three, four, five and even all N-linked groups are linked to an oligosaccharide. N-linked oligosaccharides can include oligosaccharides, complex, hybrid or sulfated oligosaccharides, or other oligosaccharides and monosaccharides.
[0095] [0095] As used herein, a "partially N-glycosylated polypeptide" refers to a polypeptide that minimally contains an N-acetylglucosamine glycan linked to at least three N-linked moieties. A partially glycosylated polypeptide can include various forms of glycan, including monosaccharides, oligosaccharides, and branched sugar forms, including those formed by treating a polypeptide with EndoH, EndoFl, EndoF2 and / or EndoF3.
[0096] [0096] As used herein, a "deglycosylated PH20 polypeptide" refers to a PH20 polypeptide in which few of all possible glycosylation sites are glycosylated. Deglycosylation can be carried out, for example, by removing glycosylation, avoiding, or modifying the polypeptide to eliminate a glycosylation site. N-glycosylation sites, in particular, are not required for activity, whereas others are.
[0097] [0097] As used herein, "PEGylated" refers to covalent bonding or other stable attachment of polymeric molecules, such as polyethylene glycol (PEGylation PEG portion) to hyaluronan-degrading enzymes, such as hyaluronidases, usually to increase the half - life of the hyaluronan degrading enzyme.
[0098] [0098] As used herein, a "conjugate" refers to a polypeptide linked directly or indirectly to one or more other polypeptides or chemical groups. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods. For example, a conjugate refers to soluble PH20 polypeptides linked directly or indirectly to one or more other polypeptides, or "chemical groups, through which at least one soluble PH20 polypeptide is linked, directly or indirectly, to another polypeptide. or chemical portion, as long as the conjugate retains hyaluronidase activity.
[0099] [0099] As used herein, a "fusion" protein refers to a polypeptide encoded by a nucleic acid sequence that contains a coding sequence for a nucleic acid molecule, and the coding sequence for another acid molecule nucleic, in which the coding sequences are in the same reading frame, such that when the fusion construct is transcribed and translated into a host cell, the protein is produced, containing the two proteins. The two molecules can be adjacent in the construction or separated by a polypeptide linker that contains, 1, 2, 3, or more, but is usually less than 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide.
[0100] [0100] As used herein, a "polymer" refers to any portion of the high molecular weight natural or synthetic weight that is conjugated with, or is stably linked directly or indirectly through a linker, to a polypeptide. Such polymers typically increase the half-life in the serum, and include, but are not limited to, sialic moieties, PEGylation moieties, dextran, sugar and other radicals, such as glycosylation. For example, hyaluronidase, such as a soluble PH20 or rHuPH20, can be conjugated to a polymer.
[0101] [0101] As used herein, a hyaluronidase substrate refers to a substrate (for example, the protein or polysaccharide) that is cleaved and / or depolymerized by a hyaluronidase enzyme. Generally, a hyaluronidase substrate is a glycosaminoglycan. An exemplary hyaluronidase substrate is hyaluronan (HA).
[0102] [0102] As used herein, a disease, disorder or condition associated with hyaluronan, refers to any disease or condition in which hyaluronan levels are elevated as a cause, consequence, or otherwise, seen in the disease or condition. Diseases and conditions associated with hyaluronan are associated with increased expression of hyaluronan in a tissue or cells, increased interstitial fluid pressure, decreased vascular volume and / or increased water content in the tissue. Diseases, disorders or conditions associated with hyaluronan can be treated by administering a composition that contains an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan, such as hyaluronidase, for example, a soluble hyaluronidase, either alone or in combination with, or in addition to another treatment and / or agent. Exemplary diseases and conditions include, but are not limited to, inflammatory diseases and types of cancer rich in hyaluronan. Types of cancer rich in hyaluronan include, for example, tumors, including solid tumors, such as advanced cancers, metastatic cancer, undifferentiated cancer, ovarian cancer, carcinoma in situ (ISC) squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers. They are also examples of diseases and conditions associated with hyaluronan, diseases that are associated with elevated interstitial fluid pressure, such as diseases associated with pressure on the disc, edema, for example, edema caused by organ transplantation, stroke, trauma brain or other injury. Illustrative diseases and conditions associated with hyaluronan include diseases and conditions associated with elevated interstitial fluid pressure, decreased vascular volume and / or increased water content in a tissue, including cancer, disc pressure and edema. In one example, treatment of the disease or disorder associated with hyaluronan includes improvement, reduction, or other beneficial effect on one or more of the interstitial fluid pressure (IFP), decreased vascular volume, and increased water content in the tissue.
[0103] [0103] As used herein, "activity" refers to an activity or functional activities of a polypeptide, or a portion thereof related to a full-length (total) protein. For example, active fragments of a polypeptide can exhibit full-length protein activity. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind or compete with a polypeptide to bind to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide.
[0104] [0104] As used herein, "hyaluronidase activity" refers to the ability to catalyze enzymatic cleavage of hyaluronan. The United States Pharmacopeia (USP) XXIII assay, for hyaluronidase, determines hyaluronidase activity indirectly by measuring the amount of higher molecular weight hyaluronan, Or hyaluronan substrate (HA) remaining after the enzyme, is left to react with HA for 30 min. at 37 ° C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, MD). A Standard Reference Solution can be used in an assay to determine the relative activity, in units, of any hyaluronidase. In vitro assays to determine hyaluronidase activity of hyaluronidases, such as for example PH20, including soluble PH20 and esPH20, are known in the art and described herein. Exemplary assays include the microturbity assay that measures the cleavage of hyaluronan by hyaluronidase indirectly, detecting the insoluble precipitate formed when non-cleaved hyaluronic acid binds to serum albumin; and the biotinylated hyaluronan assay, which measures cleavage of hyaluronan indirectly by detecting the binding of remaining biotinylated hyaluronic acid, not covalently bound to microtiter plate wells, with a streptavidin-horseradish peroxidase conjugate and a chromogenic substrate. Reference standards can be used, for example, to generate a standard curve to determine activity in hyaluronidase units to be tested.
[0105] [0105] As used herein, specific activity refers to the units of activity per mg of protein. The milligrams of hyaluronidase are defined by the absorption of a solution at 280 nm, assuming a molar extinction coefficient of about 1.7 in units of M * cm ".
[0106] [0106] As used herein, "neutral-active" refers to the ability of a PH20 polypeptide to catalyze enzymatic cleavage of hyaluronic acid with a neutral pH (for example, a, or about pH 7.0). Generally, a soluble and neutral-active PH20, for example, an incomplete PH20 of C-terminal or partially glycolated of N, has, or is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000% or more, of activity, compared to the hyaluronidase activity of a corresponding neutral-active PH20, which is not C-terminal incomplete or partially N-glycolated.
[0107] [0107] As used herein, a "signal sequence attached to the GPI anchor", is a C-terminal amino acid sequence that targets in addition to a GPI anchor preformed with the polypeptide within the ER lumen . The signal sequences attached to the GPI anchor are present in the precursor polypeptides of polypeptides anchored by GPI, such as PH20 polypeptides anchored to GPI. The signal sequence attached to the C-terminal GPI anchor typically contains a predominantly hydrophobic region of 8 to 20 amino acids, preceded by a hydrophilic spacer region of 8 to 12 amino acids, immediately downstream of the àw location, or the anchor location. to the GPI. The signal sequences attached to the GPI anchor can be identified using methods well known in the art. These include, but are not limited to, in silico methods and algorithms (see, for example, Udenfriend et al. (1995) Methods Enzymol. 250: 571-582, Eisenhaber et al., (1999) J. Biol. Chem. 292 : 741-758, Fankhauser et al., (2005) Bioinformatics 21: 1846-1852, Omaetxebarria et al., (2007) Proteomics 7: 1951-1960, Pierleoni et al., (2008) BMC Bioinformatics 9: 392), including those that are readily available on bioinformatics sites, such as tools on the ExXPASy Proteomics site (for example, www.expasy.ch/tools/).
[0108] [0108] As used herein, "nucleic acids" include DNA, RNA and their analogs, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a radioactive or fluorescent label, single chain molecules are contemplated. These molecules are usually of such a length that their purpose is statistically unique or of a low number of copies (usually less than 5, usually less than 3), to probe or start a library. Generally, a probe or primer contains at least 14, 16 or 30 contiguous nucleotides in the sequence in a complementary or identical manner to a gene of interest. The probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids in length.
[0109] [0109] As used herein, a peptide refers to a polypeptide that is greater than or equal to two amino acids in length and less than or equal to 40 amino acids in length.
[0110] [0110] As used herein, amino acids that occur in the different amino acid sequences provided here are identified according to known abbreviations, three letters or one letter (Table 1). The nucleotides that occur in the various fragments of nucleic acids are designated with the standard, single-letter designations used routinely in the art.
[0111] [0111] As used herein, an "amino acid" is an organic compound that contains an amine group and a carboxylic acid group. A polypeptide contains two or more amino acids. For the purposes described herein, amino acids include the twenty naturally occurring amino acids, unnatural amino acids and amino acid analogues (i.e., amino acids in which carbon a has a side chain).
[0112] [0112] As used herein, "amino acid residue" refers to an amino acid formed by chemical digestion (hydrolysis) of a polypeptide in its peptide bonds. The amino acid residues described herein are presumably in the "L" isomeric form. Residues in the "D" isomeric form, which are so designated, can be replaced by any L-amino acid residue, provided that the desired functional property is retained by the polypeptide. NH2 refers to the free amine group present at the amino terminal of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. Keeping the standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopting 37 C.F.R. SS1.821-1.822, the abbreviations for amino acid residues are shown in Table 1: TABLE 1 - Correspondence table
[0113] [0113] All amino acid residue sequences, represented here by formulas, have a left-to-right orientation in the conventional direction from the amino terminal to the carboxyl terminal. In addition, the term "amino acid residue" is defined to include the amino acids listed in the Correspondence Table (Table 1) and the modified and unusual amino acids, such as those referred to in 37 C.F.R. S S 1.821 - 1.822, and incorporated herein by reference. In addition, it should be noted that a dash at the beginning or at the end of a sequence of amino acid residues indicates a peptide bond to another sequence of one or more amino acid residues, to an amino-terminal group such as NH, Or a carboxyl-terminal group such as COOH.
[0114] [0114] As used herein, "naturally occurring α-amine acids" are the residues of the respective 20 α-amine acids found in nature, which are incorporated into the protein by specific recognition of the tRNA molecule loaded with its mRNA codon cognate in humans. Non-naturally occurring amino acids thus include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids, and include, but are not limited to, D-stereoisomers of amino acids. Exemplary unnatural amino acids are described herein and are known to those skilled in the art.
[0115] [0115] As used herein, a DNA construct is a single or double-stranded, linear or circular DNA molecule that contains the DNA segments combined and juxtaposed in a way not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
[0116] [0116] As used herein, a DNA segment is a portion of a larger DNA molecule, which has specified attributes. For example, a segment of DNA encoding a specified polypeptide is a portion of a larger DNA molecule, such as a plasmid or plasmid fragment, which from the 5 'to 3' direction, encodes the amino acid sequence of the specified polypeptide .
[0117] [0117] As used herein, the term polynucleotide means a single or double chain polymer of deoxyribonucleotides or ribonucleotides read from the 5 'end to the 3' end. Polynucleotides include DNA and RNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given here in terms of nucleotides (abbreviated as "nt") or base pairs (abbreviated as "bp"). The term nucleotide is used for single-stranded and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote the overall length, and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art, that the two strands of a double-stranded polynucleotide may differ slightly in length, and that their ends can be staggered; thus, all nucleotides in a double-stranded polynucleotide molecule may not be paired. Such unpaired ends - will, in general, be no more than 20 nucleotides in length.
[0118] [0118] As used herein, "similarity" between two proteins or nucleic acids refers to the relationship between the amino acid sequence of the proteins or the nucleotide sequences of the nucleic acids. The similarity can be based on the degree of identity and / or homology of residue sequences and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those skilled in the art. For example, in a method of evaluating sequence similarity, two sequences of amino acids or nucleotides are aligned in a way that provides a maximum level of identity between the sequences. "Identity" refers to the extent to which the amino acid or nucleotide sequences are invariant. The alignment of the amino acid sequences, and some extensive nucleotide sequences, may also consider conservative differences and / or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physical and chemical properties of the waste involved. The alignments can be global (alignment of the sequences compared over the entire length of the sequences and including all residues) or local (alignment of a portion of the sequences, which includes only the most similar region or regions).
[0119] [0119] "Identity" per se has a recognized meaning in the art and can be calculated using published techniques. (See, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer,
[0120] [0120] As used herein, homologous (with respect to nucleic acid and / or amino acid sequences) means about or equal to 25% sequence homology, usually greater than or equal to 25%, 40%, 50% 60% , 70%, 80%, 85%, 90% or 95% sequence homology; the exact percentage can be specified, if necessary. For purposes proposed here, the term "homology" and "identity" are often used interchangeably, unless otherwise specified. In general, to determine the percentage of homology or identity, the strings are aligned so that the highest order correspondence is obtained (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey , 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo, H and Lipton, D., (1988) SIAM J Applied Math 48: 1073). By sequence homology, the number of conserved amino acids is determined by conventional algorithm alignment programs, and can be used with the standard deviation penalties established by each supplier. Substantially homologous nucleic acid molecules could normally hybridize under moderate or high stringency over the entire length of the nucleic acid of interest. Also included are nucleic acid molecules that contain degenerate codons instead of codons from the hybridizing nucleic acid molecule.
[0121] [0121] If any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical "or" homologues "can be determined using computer algorithms known as the“ FASTA ”program, using, for example, the standard parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA85: 2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (Altschul, SF, et al ., Mol Biol 215: 403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, Ee Carrillo, H. and Lipton, D., (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include the DNAStar "MegaAlign" program (Madison, WI) and the "Gap" program at the University of Wisconsin Genetics Computer Group (UWG) (Madison WI). The percentage of homology or identity of proteins and / or nucleic acid molecules can be determined, for example, by comparing the sequence information using a GAP computer program (for example, Needleman et al. (1970) J. Mol. Biol. 48: 443, as reviewed by Smith and Waterman ((1981) Adv. Appl. Math. 2: 482). Briefly, the GAP program defines similarity as the number of aligned symbols (ie nucleotides or amino acids) that are similar, divided by the total number of symbols in the shortest of the two strings.
[0122] [0122] Therefore, as used herein, the term "identity" or "homology" represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least “90% identical to" refers to the 90 to 99.99 percent identity to the polypeptide reference nucleic acid or amino acid sequence. Identity at a 90% or more is indicative of the fact that, assuming for example, a test and reference polypeptide, the length of the 100 amino acids is compared. No more than 10% (that is, 10 out of 100) of the amino acids in the polypeptide of test, differs from that of the reference polypeptide. Similar comparisons can be made between the test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide, or can be grouped into one or more sites of variable length up to the maximum allowed, for example, 10/100 amino acid difference (about 90% identity). Differences are defined as substitutions, insertions or deletions of nucleic acids or amino acids. At the homology or identity level above,
[0123] [0123] As used herein, an aligned sequence refers to the use of homology (similarity and / or identity) to align the corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more strings that are related to 50% or more of identity are aligned. A set of aligned sequences refers to two or more sequences that are aligned at corresponding positions and can include sequences that align derivatives of RNAs, such as ESTs and other cDNAs, aligned with the genomic DNA sequence.
[0124] [0124] As used herein, "primer" refers to a nucleic acid molecule that can function as a starting point for template-directed DNA synthesis under appropriate conditions (for example, in the presence of four different nucleoside triphosphates , and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer, and at an appropriate temperature. It will be appreciated that certain nucleic acid molecules can serve as a "probe" and as a "primer". A primer, however, has a 3 'hydroxyl group by extension. A primer can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reverse transcriptase (RT) -PCR, RNA PCR, LCR, multiplex PCR, PCR panhandle, PCR capture, expression of PCR, RACE 3 'and 5', PCR in situ, PCR per link, and other amplification protocols.
[0125] [0125] As used herein, "primer pair" refers to a set of primers that includes a 5 primer (upstream) that hybridizes to the end complement
[0126] [0126] As used herein, "specifically hybridizes" refers to the heat treatment (annealing), by pairing complementary bases, of a molecule of nucleic acid (for example, an oligonucleotide) to a molecule of target nucleic acid . Those skilled in the art are familiar with in vitro and in vivo parameters that affect specific hybridization, such as the length and composition of the particular molecule. Particularly relevant parameters for in vitro hybridization further include heat treatment and washing temperature, buffer composition and salt concentration. Examples of washing conditions for the removal of non-specifically bound nucleic acid molecules with high stringency are 0.1 x SSPE, 0.1% SDS, 65ºC, and at medium stringency are 0.2 x SSPE, 0.1% SDS , 50 ° C. Equivalent stringency conditions are known in the art. The person skilled in the art can easily adjust the parameters to obtain a specific hybridization of a nucleic acid molecule to a target nucleic acid molecule suitable for a particular application. Complementarily, when referring to two nucleotide sequences, it means that the two nucleotide sequences are able to hybridize, usually with less than 25%, 15% or 5% of negative combination between the opposite nucleotides. If necessary, the complementarity percentage will be specified. Normally, the two molecules are chosen in such a way that they hybridize under stringent conditions.
[0127] [0127] As used herein, substantially identical to a product, means sufficiently similar so that the property of interest is sufficiently stable that the substantially identical product can be used in place of the product.
[0128] [0128] As used herein, it is also understood that the terms "substantially identical" or "similar" vary according to the context, as understood by those skilled in the relevant art.
[0129] [0129] As used herein, an allele variant or allele variation refers to any one, of two or more alternative forms of a gene, that occupy the same “chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Genetic mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. The term "allelic variant" is also used here to refer to a protein encoded by an allelic variant of a gene. Usually, the reference form of the gene encodes a wild-type and / or predominant form of a polypeptide, a population or member of a unique reference species. Typically, allelic variants, which include variants between some species, normally have at least 80%, 90% or more, of amino acid identity with a wild type and / or predominant form of the same species; the degree of identity depends on the gene, and whether the comparison is made between species or intraspecies. Generally, intraspecific allelic variants have at least about 80%, 85%, 90%, 95% or more of identity with a wild form and / or predominant form, including 96% 97%, 98%, 99% or more , identity with a wild form and / or a predominant form of a polypeptide. The reference to an allelic variant here generally refers to variations in proteins between members of the same species.
[0130] [0130] As used herein, "allele", which is used interchangeably here, with "allelic variant" refers to alternative forms of a gene or parts thereof. The alleles occupy the same place or position on homologous chromosomes. When an individual has two identical alleles of a gene, the individual is referred to as being homozygous for that gene or allele. When an individual has two different alleles of a gene, the individual is referred to as being heterozygous for the gene. Alleles of a specific gene can differ from one another in a single nucleotide or several nucleotides, and may include modifications, such as nucleotide substitutions, deletions and insertions. A gene allele can also be a form of a gene that contains the mutation.
[0131] [0131] As used herein, species variants refer to variants of polypeptides between different species, including different species of mammals, such as the rat and man. For example, for PH20, examples of variant species provided here are PH20 of primates, such as, but not limited to humans, chimpanzees and cynomolgus monkeys. Generally, species variants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more, sequence. Matching residues between 2 or more variant species can be determined by comparing and aligning sequences to maximize the number of corresponding nucleotides or residues, for example, such that the identity between the sequences is equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98% or equal to or greater than 99%. The position of interest then determines the number assigned to the reference nucleic acid molecule. The alignment can be carried out manually or visually, in particular, where the sequence identity is greater than 80%.
[0132] [0132] As used herein, a human protein is encoded by a nucleic acid molecule, such as DNA, present in the genome of a human being, including all allelic variants and conservative variations thereof. A variant or modification of a protein is a human protein, whether the modification is based on the wild-type or prominent sequence of a human protein.
[0133] [0133] As used herein, a splice variant refers to a variant produced by differential processing of a primary genomic DNA transcript, which results in more than one type of mRNA.
[0134] [0134] As used herein, the modification is in reference to the modification of an amino acid sequence of a polypeptide, or a nucleotide sequence of a nucleic acid molecule, and includes deletions, insertions and substitutions (for example, substitutions) of amino acids and nucleotides, respectively. Examples of modifications are amino acid substitutions. A polypeptide with amino acid substitutions can have 65%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, sequence identity for a polypeptide that does not contain amino acid substitutions. Amino acid substitutions can be conservative or non-conservative. Generally, any modification of a polypeptide retains activity of the polypeptide. Methods of modifying a polypeptide are routine for those skilled in the art, such as using recombinant DNA methodologies.
[0135] [0135] As used herein, suitable conservative amino acid substitutions are known to those skilled in the art, and can be made generally without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, substitutions of a single amino acid in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th edition, 1987 , The Benjamin / Cummings Pub. Co., P. 224). Such substitutions can be made in accordance with those presented in Table 2 below: TABLE 2 Original waste | Exemplary conservative substitution Ala (A) Gly; Ser arg (8) Asn (N) ve O Gln (9) ERan Gly (6) HS O) TIe (1) Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Read; Tyr Ser (5) Thr (D) Trp (1) Tyr (Y) Trp; Phe TEL
[0136] [0136] Other substitutions are also permissible and can be determined empirically or according to known conservative substitutions.
[0137] [0137] As used herein, the term promoter means a portion of a gene that contains DNA sequences that provide for binding of RNA polymerase and initiation of transcription. Promoter sequences are usually, but not always, found in the 5 'non-coding region of genes.
[0138] [0138] As used herein, the isolated or purified polypeptide or protein, or biologically active portion thereof, is substantially free of cellular material or other contaminating proteins, from the cell or tissue from which the protein is derived, Or substantially free of chemical precursors or other chemical substances when chemically synthesized. The preparations can be determined to be substantially free, if they appear to be free of easily detectable impurities, as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those skilled in the art to evaluate said purity, or sufficient purity, in such a way that further purification that would not be detectable, alters the physical and chemical properties, such as the enzymatic and biological activities of the substance. Methods of purifying the compounds to produce substantially chemically pure compounds are known to those skilled in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such cases, further purification can increase the specific activity of the compound.
[0139] [0139] Thus, reference to a substantially purified polypeptide, such as substantially purified soluble PH20, refers to protein preparations that are substantially free of cellular material, which includes protein preparations, wherein the protein is separated from the components cells of the cells from which it is isolated or recombinantly produced. In one embodiment, the term substantially free of cellular material includes enzyme protein preparations with less than about 30% (by dry weight) of non-enzymatic proteins (also referred to herein as a contaminating protein), generally less than about 20% non-enzymatic proteins, or 10% non-enzymatic proteins, or less than about 5% non-enzymatic proteins. When the enzyme protein is produced recombinantly, it is also substantially free of culture medium, that is, the culture medium represents less than about, or 20%, 10%, or 5% of the volume of the protein preparation. enzyme.
[0140] [0140] As used herein, the term substantially free of chemical precursors or other chemicals includes enzyme protein preparations, in which the protein is separated from chemical precursors, or other chemicals that are involved in protein synthesis. The term includes enzyme protein preparations that have less than about 30% (dry weight), 20%, 10%, 5% or less, of chemical precursors or non-enzymatic chemicals or components.
[0141] [0141] As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or synthetic gene, or a synthetic peptide refers to a nucleic acid molecule or a polypeptide molecule that is produced by recombinant methods and / or chemical synthesis methods.
[0142] [0142] As used herein, production by recombinant means or by recombinant DNA methods, means the use of well-known methods of molecular biology, for the expression of proteins encoded by the cloned DNA.
[0143] [0143] As used herein, vector (or plasmid) refers to distinct elements that are used to introduce a heterologous nucleic acid into cells for both expression and replication. The vectors usually remain episomal, but can be designed to integrate a gene or part of it into a chromosome in the genome. Also included are vectors which are artificial chromosomes, such as artificial yeast chromosomes and artificial mammal chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
[0144] [0144] As used herein, an expression vector includes vectors capable of expressing DNA that is operationally linked to regulatory sequences, such as promoter regions, that are capable of effecting the expression of these DNA fragments. Such additional segments may include promoter and termination sequences and, optionally, may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to recombinant DNA or construction DNA, such as a plasmid, phage, recombinant virus or other vector that, after introduction into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those of skill in the art, and include those that are replicable in eukaryotic cells and / or prokaryotic cells, and those that remain episomal, or those that integrate into the host cell genome.
[0145] [0145] As used herein, vector also includes "virus vectors" or "viral vectors". Viral vectors are viruses that are operationally linked to exogenous genes to transfer (as vehicles or transporters, the exogenous genes in cells.
[0146] [0146] As used herein, "operationally" or "operationally linked" when referring to DNA segments, means that the segments are arranged so that they work together for their intended purposes, for example, transcription begins downstream of the promoter, and upstream of any transcribed sequences.The promoter is generally the domain to which the transcriptional machinery binds to initiate transcription, and proceeds through the coding segment to the terminator.
[0147] [0147] As used herein, the term "evaluate" is intended to include quantitative and qualitative determination, in the sense of obtaining an absolute value for the activity of a protein, such as an enzyme, or a domain thereof, present in the sample , and also obtain an index, ratio, percentage, visual or other value indicative of the level of activity. The assessment can be direct or indirect. For example, the chemical species actually detected do not, of course, need to be the enzymatically cleaved product itself, but it can, for example, be a derivative of you or some other substance. For example, the detection of a cleavage product can be a detectable portion, such as a fluorescent portion.
[0148] [0148] As used herein, biological activity refers to the in vivo activities of a compound, or physiological responses that result after the in vivo administration of a compound, composition or other mixture. The biological activity, therefore, encompasses the therapeutic effects and the pharmaceutical activity of such compounds, compositions and mixtures. Biological activities can be observed in in vitro systems designed to test or use such activities. Thus, for the purposes described here, a biological activity of a hyaluronidase enzyme is its degradation of hyaluronic acid.
[0149] [0149] As used herein, equivalent, when referring to two nucleic acid sequences, means that the two sequences in question encode the same sequence of equivalent amino acids or proteins. When equivalent is used in reference to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence, with substitutions only for amino acids that do not substantially alter the activity, or function of the protein, or the peptide. When equivalent refers to a property, the property need not be present to the same extent (for example, two peptides can exhibit different rates of the same type of enzyme activity), but the activities are generally substantially the same.
[0150] [0150] As used herein, "modular" and "modulation" or "alter" refer to a change in an activity of a molecule, such as a protein. Examples of activities include, but are not limited to, biological activities, such as signal transduction. Modulation can include an increase in activity (that is, an increase in regulation or agonist activity), a decrease in activity (that is, infra-regulation or inhibition) or any other change in an activity (such as a change in periodicity, frequency , duration, kinetics or other parameter). Modulation can be context dependent, and is usually compared to a designated state, for example, the wild-type protein, the protein in a constitutive state, or the protein expressed in a designated cell type or condition.
[0151] [0151] As used herein, a composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous solution, or any combination thereof.
[0152] [0152] As used herein, a combination refers to any association between two or more items. The combination may be two or more separate elements, such as two compositions or two sets, may be a mixture of them, such as a single mixture of two or more items, or any variation. The elements of a combination are generally functionally associated or related. For example, a combination can be a combination of the compositions provided herein.
[0153] [0153] As used herein, a kit refers to a combination of components, such as a combination of the compositions of the invention and another item for the purposes, including, but not limited to reconstitution, activation and delivery instruments / devices , administration, diagnosis and evaluation of a biological activity or property. Kits optionally include instructions for use.
[0154] [0154] As used herein, "disease or disorder" refers to a pathological condition in an organism, resulting from the cause or condition, including, but not limited to infections, conditions, genetic diseases, and characterized by identifiable acquired symptoms. Diseases and disorders of interest present here are the diseases and disorders associated with hyaluronan.
[0155] [0155] As used herein, "treatment" of an individual with a disease or condition means that the individual's symptoms are partially or totally relieved, or remain static after treatment. Thus, treatment includes prophylaxis, therapy and / or cure. Prophylaxis refers to the prevention of a potential disease and / or the prevention of a worsening of symptoms or progression of a disease.
[0156] [0156] As used herein, a pharmaceutically effective agent includes any therapeutic agent or bioactive agents, including, but not limited to, for example, chemotherapeutic agents, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.
[0157] [0157] As used herein, treatment means any way in which, the symptoms of a condition, disorder or disease, or other indication, are improved or otherwise beneficially altered.
[0158] [0158] As used herein, therapeutic effect means an effect resulting from the treatment of an individual that normally changes, improves, the symptoms of a disease or condition, or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound that results in a therapeutic effect, after administration to an individual.
[0159] [0159] As used herein, the term "individual" refers to an animal, including a mammal, such as a human.
[0160] [0160] As used herein, a "patient" refers to a human being, who presents symptoms of a disease or disorder.
[0161] [0161] As used herein, an "individual" can be a subject.
[0162] [0162] As used herein, on the same means within an amount that a person skilled in the art considers to be the same, or to be within an acceptable error range. For example, normally, for pharmaceutical compositions, an amount within at least 1%, 2%, 3%, 4%, 5% or 10% is considered to be approximately the same. Such amount may vary, depending on the tolerance of variation of the composition, in particular, by individuals.
[0163] [0163] As used herein, dosage regimen refers to the amount of the agent, for example, the composition containing a hyaluronan-degrading enzyme, for example, a soluble hyaluronidase or other agent administered, and the frequency of administration. The dosage regimen is a function of the disease or condition being treated, and so can vary.
[0164] [0164] As used herein, the frequency of administration refers to the time between successive treatment administrations. For example, the frequency can be days, weeks or months. For example, the frequency can be more than once a week, for example, twice a week, three times a week, four times a week, five times a week, six times a week, OR daily. Frequency can also be one, two, three or four weeks. The particular frequency is a function of the particular disease or treated condition. Generally, the frequency is more than once a week, and it is usually, twice a week.
[0165] [0165] As used herein, an "administration cycle" refers to the repeated administration schedule of the dosage regimen of administration of the enzyme and / or a second agent that is repeated over successive administrations. For example, an exemplary administration cycle is a 28-day cycle with administration twice a week for three weeks, followed by a week of discontinued dosing.
[0166] [0166] As used herein, when referring to the dosage based on the individual's mg / kg, an average human being is considered to have a mass of about 70 kg -75 kg, such as 70 kg.
[0167] [0167] As used herein, amelioration of symptoms of a particular disease or treatment disorder, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to any reduction, permanent or temporary, lasting or transient, of symptoms or, adverse effects of a condition, such as, for example, reduction of adverse effects associated with, or occurring. after administration of an enzyme that degrades hyaluronan, such as PEGylated hyaluronidase.
[0168] [0168] As used herein, prevention or prophylaxis refers to reducing the risk of developing a disease or condition.
[0169] [0169] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to the amount of an agent, compound, material, or composition, which contains a compound that is at least sufficient to produce a therapeutic effect. Therefore, it is the amount needed to prevent, cure, improve, interrupt or partially arrest a symptom of a disease or disorder.
[0170] [0170] As used herein, unit dose form refers to physically distinct units suitable for human and animal individuals, and individually packaged as is known in the art.
[0171] [0171] As used herein, a single dosage formulation refers to a formulation as a single dose.
[0172] [0172] As used herein, formulation for direct administration means that the composition does not require further dilution for administration.
[0173] [0173] As used herein, an "article of manufacture" is a product that is made and sold. As used throughout this application, the term is intended to encompass anti-hyaluronan agents, for example, the enzyme that degrades hyaluronan, such as hyaluronidase, and second agent compositions contained in packaging articles.
[0174] [0174] As used herein, fluid refers to any composition that can flow. Fluids include compositions that are in the form of semi-solids, ointments, solutions, aqueous mixtures, gels, lotions, creams and other compositions.
[0175] [0175] As used herein, a cell extract or lysate refers to a preparation or fraction that is made from a lysed or interrupted cell.
[0176] [0176] As used herein, animal includes any animal, such as, but not limited to primates, including humans, gorillas and monkeys; rodents, such as mice and rats; birds, such as chickens; ruminants, such as goats, cows, deer, sheep; pigs and other animals. Non-human animals do not include human beings, such as the contemplated animal. Hyaluronidases are provided here from any animal, vegetable, prokaryotic and fungal source. Most hyaluronidases are of animal origin, including the origin of mammals. Usually hyaluronidases are of human origin.
[0177] [0177] As used herein, anticancer treatments include the administration of drugs and other agents for the treatment of cancer, as well as treatment protocols, such as surgery and radiation therapy. Anti-cancer treatments include the administration of anti-cancer agents.
[0178] [0178] As used herein, an anti-cancer agent refers to any agents or compounds used in anti-cancer treatment. These include any agents, when used alone or in combination with other compounds, that can alleviate, reduce, improve, prevent, or place, or maintain, a state of remission of clinical symptoms, or diagnostic markers associated with tumors and cancer, and can be used in combinations and compositions provided herein. Exemplary anti-cancer agents include, but are not limited to, degrading enzymes, such as hyaluronan, enzymes that degrade the PEGylated hyaluronan provided herein, used alone or in combination with other anti-cancer agents, such as chemotherapeutic agents, polypeptides, antibodies, peptides, molecules small or gene therapy vectors, viruses or DNA.
[0179] [0179] As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it may be from a volunteer normal, unaffected by the condition of interest. A control can also be an internal control.
[0180] [0180] As used herein, the singular forms "one", "one" and "o (a)" include plural referents unless the context clearly indicates otherwise. Thus, for example, the reference to a compound that comprises or contains an "extracellular domain" includes compounds with one or a plurality of extracellular domains.
[0181] [0181] As used herein, ranges and quantities can be expressed as "about" a particular value or range, which also includes the exact value. So, "about 5 bases" means "about 5 bases" and also "5 bases". Generally "about" includes an amount that is expected to be within the experimental error.
[0182] [0182] As used herein, "optional" or "optionally" means that the event or circumstance described subsequently occurs or not, and that the description includes cases in which the said event or circumstance occurs, and examples where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.
[0183] [0183] As used herein, the abbreviations for any protecting groups, amino acids and other compounds are, unless otherwise indicated, according to their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Nomenclature Biochemistry (see, (1972) Biochem. 11: 17226). B. HYALURONAN-BINDING PROTEIN AND DIAGNOSIS FOLLOW-UP
[0184] [0184] Sensitive and specific methods are provided here to detect and closely monitor levels of hyaluronan (HA) associated with the disease, particularly in the extracellular matrix (ECM) of tumor tissues. The follow-up diagnostic methods provided here are based on the finding that the accumulation of AH correlates specifically with, and predicts, aggressive disease, in particular, with respect to cancer. In addition, the follow-up diagnostic method provided here is also based on the finding that HA specifically provides information on prognosis selection and superior treatment, compared to other markers involved in the metabolic pathway of HA associated with diseases and conditions associated with hyaluronan , such as hyaluronidase or hyaluronidase synthases. Thus, the The method provided here uses improved hyaluronan-binding protein reagents (HABP) that have specificity, high affinity and low variability, for the specific and sensitive detection of AH. Enhanced HABP reagents are also provided here.
[0185] [0185] In one example, the improved HABPs provided here, like any described in Section C, can be a follow-up diagnosis for the selection of patients with HA associated diseases, for example HA associated tumors, by treatment with an agent anti-hyaluronan, or an enzyme that degrades hyaluronan, like any established in Section E (for example, a hyaluronidase or hyaluronidase modified as PEGylated PH20, ie PEGPH20). In such an example, the method is useful for the classification of patients for the selection of therapies, such as cancer therapy, and in particular, it refers to the measurement of AH levels that correlate with the responsiveness to therapy with an anti-hyaluronan agent, for example, therapy with an enzyme that degrades hyaluronan, such as therapy by PEGPH20 for the treatment of patients with advanced tumors.
[0186] [0186] In another example, the improved HABPs provided here, such as any described in Section C, can also be used in methods of monitoring the effectiveness or responsiveness to treatment with an anti-hyaluronan agent or hyaluronan-degrading enzyme , such as any defined in Section E (for example, a hyaluronidase or modified hyaluronidase, such as PH20
[0187] [0187] Combinations and kits containing an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan (for example, any provided hereinafter in Section E) and an improved HABP (for example, any one provided here, below, in Section C), and optionally, other accompanying reagents, for use in the selection, monitoring and / or treatment of diseases and conditions associated with HA, especially cancer.
[0188] [0188] Hyaluronan (HA; also called hyaluronan or hyaluronate) is a linear glycosaminoglycan polymer (GAG) that contains disaccharide subunits of N-acetyl-glucosamine and D-glucuronic acid repeated through GlcUA-B1,3-GleNAC-Bl1 bonds , 4. Hyaluronan is synthesized by a class of hyaluronan synthases, HAS1, HAS2 and HAS3. These enzymes act by elongating hyaluronan by adding glucuronic acid and No. acetylglucosamine to the nascent polysaccharide, since it is extruded through the cell. In addition to HA synthases, HA levels are normally maintained by their catabolism by hyaluronidases, specifically the modified enzyme hyaluronidase 1l1 (Hyall). The dynamic transformation of HA is balanced by biosynthesis and catabolism, to maintain a constant concentration in normal tissue.
[0189] [0189] HA is a component of the extracellular matrix (ECM). It is ubiquitously distributed in tissues and located in extracellular and pericellular matrices, as well as inside cells. HA has a wide variety of biological functions such as contribution to tissue homeostasis and biomechanics, cell proliferation, immune adhesion and activation, and cell migration during dynamic cellular processes. These processes are mediated by the interaction of HA with HA-binding proteins, such as TSG-6, versican, inter-alpha-trypsin inhibitor, CD44, endothelial HA receptor (LYVE-1-1) and RHAMM .
[0190] [0190] Hyaluronan accumulation is associated with various conditions of malignant and autoimmune diseases (Járvelãinen H, et al. (2009) Pharmacol Rev 61: 198-223; Whatcott CJ, et al. (2011) Cancer Discovery 1: 291-296). For example, certain diseases are associated with the expression and / or production of hyaluronan, including inflammatory diseases and cancers. HA is linked to a variety of biological processes involved in the progression of such diseases (for example, see Itano et al. (2008) Semin Cancer Bioll18 (4): 268-274; Tammi et al. (2008) Semin Cancer Biol 18 ( 4): 288-295).
[0191] [0191] In particular, HA is a component of the tumor matrix and is present in many solid tumors. The accumulation of HA within a tumor focus prevents cell-cell contact, promotes epithelial-mesenchymal transitions, is involved with the p53 tumor suppressor pathway through its RHAMM and CD44 receptors, and recruits tumor-associated macrophages (Itano et al. (2008) Cancer Sci 99: 1720-1725; Camenisch et al. (2000) J Clin Invest 106: 349-
[0192] [0192] HA accumulation has been correlated with the expression of the HAS gene and / or the expression of the HYAL gene (Kosaki et al. (1999) Cancer Res. 59: 1141-1145; Liu et al. (2001) Cancer Res 61: 5207-5214; Wang et al. (2008) PLoS 3: 3032; Nykopp et al. (2010) BMC Cancer 10: 512). Studies in the art in different ways have shown that HA, SAH or Hyall can be used as indicators of cancer prognosis. In addition, studies have suggested that selective Hyall inhibition, such as by means of antisense methods, or by hyaluronan synthesis by SAH, such as by the use of 4-methylumbelliferone, are methods of treating tumors (Yoshihara et al (2004) JJ. Biol. Chem. 279: 33281-33289). In addition, hyaluronidase, such as PH20 as discussed below, has also been used to treat diseases and conditions associated with hyaluronan (see for example, Thompson et al. (2010) Mol. Cancer. Ther 9: 3052-303064 ).
[0193] [0193] As shown in the Examples, it is now noted here that the phenotype of an HA cell, and in particular the formation of a pericellular tumor matrix,
[0194] [0194] It is also found here, that the amount or extent of HA accumulation measured, also correlates with the responsiveness to treatment with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, such as PH20 . Anti-hyaluronan agents, such as enzymes that degrade hyaluronan, such as PH20, exhibit useful properties for the single agent or a combined therapy of diseases and conditions that exhibit the accumulation of hyaluronan (hyaluronic acid, HA). Such disease conditions and / or disorders associated with hyaluronan, include cancers and inflammatory diseases. Cancers rich in hyaluronan include, but are not limited to, tumors, including solid tumors, for example, types of cancer in advanced stage, metastatic cancer, undifferentiated cancer, ovarian cancer, carcinoma in situ (I1SC) squamous cell carcinoma (SCC), cancer prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers.
[0195] [0195] For example, enzymes that degrade HA, such as hyaluronidase, for example PH20, have been demonstrating the removal of HA from tumors, resulting in reduced tumor volume, reduced IFP, slowed down tumor cell proliferation, and the improved efficacy of chemotherapy drugs and coadministered biological agents, allowing for increased tumor penetration (see, for example, Published Application US No. 20100003238 and Published PCT Application Published No. WO 2009/128917; Thompson et al. (2010 ) Mol. Cancer, Ther 9: 3052-3064).
[0196] [0196] The ability of a hyaluronidase, such as PH20, to degrade HA to serve as a therapeutic agent for diseases and disorders associated with hyaluronan, can be exploited, by modification, to increase systemic half-life. The increase in half-life allows not only the removal of HA, but also, due to its continuous presence in the plasma and its ability to degrade HA, it reduces or decreases the degree of HA regeneration within diseased tissues, such as the tumor . Thus, maintaining plasma enzyme levels can remove HA, such as the HA tumor, and also neutralize HA resynthesis. PEGylation is an established technology used to increase the half-life of therapeutic proteins in the body,
[0197] [0197] It is found here that the growth inhibitory activity of an anti-hyaluronan agent, and in particular an enzyme that degrades hyaluronan, for example, a hyaluronidase, such as PH20 or PEGPH20, in tumor cells is correlated with the degree of HA levels. As shown in the Examples, tumors can be characterized in phenotypic groups (for example, HA ”, HA” , HA ”) based on the amount of HA expression in the tumor. Elevated tumor-associated HA (HA) resulted in accelerated tumor growth in animal models, and increased tumor inhibition by a hyaluronan-degrading enzyme (eg PEGPH20). For example, tumor growth inhibition associated with an HA ”phenotype was 97%, whereas it was only 44% and 16% for HA phenotypes” or HA ”of the tumor, respectively. The data indicate that the continued growth of some tumors depends on the density and amount of HA in the tumor microenvironment, and that the depletion of HA from a tumor rich in HA (for example, HA ”**) has a more pronounced on the growth of the tumor, than the depletion of HA from a moderate or poor tumor of HA (for example, HA * º, HA “!) or a deficient tumor of HA. Thus, as shown here, the degree of HA accumulation in tumor tissues, measured using a HABP, is predictive of the degree of tumor growth inhibition in vivo mediated by an anti-hyaluronan agent (for example, PEGPH20).
[0198] [0198] Based on the results presented above and here in the Examples, the HA biomarker, detected using a HABP, was specifically correlated with the response to an anti-hyaluronan treatment, for example, a treatment with enzymes that degrade hyaluronan (for example PEGPH20). Thus, here is provided a method of using a HABP for prognosis and for preventing the degree of sensitivity and, therefore, the responsiveness, to an anti-hyalruonan agent, for example, an enzyme that degrades hyaluronan (for example, a hyaluronidase or modified hyaluronidase, such as PEGylated PH20, i.e. PEGPH20).
[0199] [0199] By value as a reagent, the sensitivity and specificity of a HABP is desired, as well as reproducibility due to low variability. For example, the detection and measurement of HA in tissues is limited, using existing reagents. Currently, the method used to detect or measure HA in tissues through immunohistological staining depends mainly on proteins or HA-binding domains derived from animal cartilage tissue. These include HABP purified from bovine nasal cartilage proteoglycan by extraction with 4 M guanidine-HCl, and then by affinity chromatography, using resin coupled to HA. The resulting HA of animal origin is composed of two main components: Gl Aggrecan domain and binding module. Due to the variation from batch to batch, as well as different modifications used in the method to prepare HABP, there is variability in the art, in terms of differences in the HABP staining patterns and staining profiles, making comparisons between studies difficult. Thus, due to their existence as a heterogeneous mixture of components and of any validated procedure for their production, alternative HABP proteins are provided here for use in accompanying diagnostics for the prognosis of the disease and prevention of treatment effectiveness in conjunction with a anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan).
[0200] [0200] Thus, the HABP reagent for use in the methods provided herein, includes any HABP other than a HABP purified from animal cartilage, such as, for example, purified from nasal bone cartilage, as used in the art, employing a method as described by E-Laurent et al. (1985) Ann. Rheum. Dis. 44: 83-88) or its modified method. Exemplary HABPs for use in the methods described herein are presented in section C. Such proteins include, for example, HABPs containing binding domains of one or more HA, including one or more binding modules for binding to HA. In some examples, HABP contains an HA binding domain (for example, a binding module) of aggrecan, versican, neurocan, brevican, phosphacan HAPLNl, HAPLN2, HAPLN3, HAPLN4, TSG-6, Stabilin- 1, Stabilin - 2, CAB61358 and KIAAO5S27. In some examples, HABP contains a Gl aggrecan domain, Gl versican domain, Gl neurocan domain, Gl brevican domain, or a Gl phosphacan domain. In some examples, HABP contains a Gl domain of aggrecan, versican, neurocan, brevican, or phosphacan and a binding protein selected from HAPLN-1, HAPLN-2, HAPLN-3, or HAPLN-4. In some examples, HABP contains a TSG-6, Stabilin-1, Stabilin-2, CAB61358, or KIAAOS27 connection module.
[0201] [0201] In some examples, HABP is a modified HABP, such as, for example, a modified protein aggrecan, versican, neurocan, brevican, phosphacan, HAPLN-1, HAPLN-2, HAPLN-3, HAPLN-A4, Stabilin -1l, Stabilin-2, CAB61358, KIAAON5S27, or TSG-6, such as, for example, TSG-6-LM-Fc. In some examples, HABP is a modified HABP that is modified to improve its binding to HA, such as, for example, TSG-6-LM-FcAHep.
[0202] [0202] In particular, the HABP provided here 1) can be produced recombinantly in an expression system, such as a mammalian expression system; 2) it has better biophysical properties, such as stability and / or solubility; 3) it can be purified by simple purification methods, such as by a step of affinity purification methods; 4) it is capable of being detected by procedures compatible with the binding assays, and, in particular, immunohistochemistry or ELISA methods; 5) can be expressed in multimeric form (for example, through dimerization) to show an increase or a high affinity for HA; and / or 6) exhibits specificity for HA, compared to other GAGs.
[0203] [0203] In one example, HABPs which are HA proteins of individual modules, which can be produced recombinantly in expression systems, are provided for use in the methods herein. In particular, HABP reagents containing a binding module are provided herein. For example, the HABPs provided here are of the HABP class A type containing only the binding module (LM) or a sufficient portion of it to bind to the hyaluronan. Examples of such HABPs are the genes stimulated by tumor necrosis factor (TSG) -6-LM (binding module established in SEQ ID NO: 360), Stabilin-1-LM or Stabilin-2-LM (binding module established in SEQ ID no: 371 or 372, respectively), CAB61358-LM (connection module established in SEQ ID NO: 373) or KIAAON5S27-LM (connection module established in SEQ ID NO: 374).
[0204] [0204] In another example, HABPs that are directly or indirectly linked to a multimerization domain are provided for use in the methods presented here. HA binding domains, such as a binding module, from HABPs, can be directly or indirectly linked, such as covalently linked, non-covalently linked, or chemically linked, to form multimers of two or more HA binding domains . The HA-binding domains can be the same or different. In particular, the HA binding domain is a binding domain or module. Thus, multimers can be formed by dimerizing two or more binding domains. In one example, the multimers can be linked through disulfide bonds formed between cysteine residues in different HA-binding domains. For example, a multimerization domain can include a portion of an immunoglobulin molecule, such as a portion of an immunoglobulin constant region (Fc). In another example, multimers can include an HA-binding domain together through covalent or non-covalent interactions with the peptide moieties fused to the polypeptide. Such peptides can be peptide ligands (spacers), or peptides that have the property of promoting multimerization. In the additional example, multimers can be formed between two polypeptides through chemical bonding, for example, using heterobifunctional linkers. A description of multimerization domains is provided below. Examples of a HABP multimer is a binding module (LM) fused to an Fc. For example, a HABP reagent for use in the methods described herein is TSG-6-LM-Fc.
[0205] [0205] In another example, they are provided for use in the methods present here HABP which are modified, for example, by substitution of amino acids, to present increased specificity for hyaluronan in comparison with other GAGs. For example, a mutant TSG-6-LM containing amino acid substitution (s) is provided in amino acid residues 20, 34, 41, 54, 56, 72 and / or 84, and in particular, in amino acid residues 20 , 34, 41, and / or 54 (corresponding to the amino acid residues set forth in SEQ ID NO: 206). The amino acid substitution can be that of any other amino acid residue, and it is usually a non-basic amino acid residue. For example, amino acid substitution can be for Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (1), Leu (L) Met (M), Phe (F), Tyr (Y) or Trp (W). The substitution or substitutions of amino acids confer decreased binding to heparin. The bond can be reduced at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times or more, compared to TSG-6-LM binding to heparin that does not contain amino acid substitution. Examples of a TSG-6 LM mutant for use as a reagent in the process provided herein is K20A / K34A / K41A. Thus, for example, heparin binding is reduced in such a way that the specificity for hyaluronan is increased. The TSG-6-LM mutant can be conjugated directly or indirectly to a multimerization domain to generate multimers. An example of a reagent for use in the methods herein is TSG-6-LM (K20A / K34A / K41A) -Fc.
[0206] [0206] Either reagent can be used alone or in combination, in a follow-up diagnostic method. For example, in a sandwich ELISA or competitive ELISA, two or more of the above reagents can be used. As described here below, any of the HABPs provided herein can be coupled directly or indirectly to a unit that is capable of detection. In some examples, HABPs that bind to HA, for example, in a tumor sample, can be detected using a secondary reagent, such as an antibody that binds to HABP. In some examples, HABPs are modified to allow detection of binding to HA. For example, HABPs can be conjugated to a detectable molecule, which allows both direct detection and secondary detection agents, such as antibodies that bind to modified HABPs and are coupled to detectable proteins, such as fluorescent probes or enzymes. detectable substances, such as horseradish peroxidase.
[0207] [0207] The HABPs provided herein can be used individually or in combination, in methods of prognosis or diagnostic monitoring, which use binding assays on various biological samples from patients with a disease or condition associated with hyaluronan, or at risk or suspected having a disease or condition associated with hyaluronan. For example, HABPs can be used in trials in patients with a solid tumor, or at risk of developing a solid tumor or other type of cancer. In particular examples, TSG-6-LM, TSG-6-
[0208] [0208] In the exemplary methods provided herein, diagnostic and prognostic methods are follow-up methods for therapy with an anti-hyaluronan agent, such as a hyaluronan-degrading enzyme, for example, a hyaluronidase or modified hyaluronidase, such as PH20 or PEGPH20. Detection of AH can inform treatment selection, initiation, dose customization or termination, and therefore can serve to individualize treatment with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan.
[0209] [0209] For example, a follow-up diagnostic method for HABP can be used to determine whether an individual who is predisposed to a disease or condition associated with hyaluronan (eg cancer), or who suffers from a disease or condition associated with hyaluronan (eg cancer) will benefit, or is expected to be responsive to treatment with an anti-hyaluronan agent, such as a hyaluronan-degrading enzyme. In the method, the level of HA expression from samples of individuals predisposed or known to have a disease or condition associated with hyaluronan (eg cancer) can be determined, and the level of HA expression in samples of individuals in comparison with predetermined levels of HA that classify responsiveness of an anti-hyaluronan agent, for example, the hyaluronan-degrading enzyme. It is within the level of a person skilled in the art to determine the threshold level of HA to classify the response to treatment with an enzyme that degrades hyaluronan. For example, does it appear here that there is a significant correlation between the accumulation of elevated HA, and the inhibition of tumor growth, in which the tumor growth inhibition response is correlated with an HA phenotype ” as quantified by immunohistochemistry of tumor tissues. Thus, in the follow-up diagnostic method provided here, a tumor sample is evaluated for HA levels, using a HABP reagent provided here by immunohistochemistry methods or other adaptive methods, for scoring. If the HA phenotype is HA ”, as determined by methods known to a person skilled in the art and described herein, then the individual is selected as a candidate for treatment with an enzyme that degrades hyaluronan, such as —hyaluronidase or modified hyaluronidase (eg PH20 or PEGPH20). Similar quantification and classification methods can be used by assessing HA in body fluids, such as blood or plasma. Dosages and regimens of a hyaluronan-degrading enzyme, such as PH20 or PEGPH2O0, for treatment, are provided here.
[0210] [0210] The HABP reagents provided herein can detect HA using any binding assay known to a person skilled in the art, including, but not limited to, the enzyme immunoassay (ELISA) or any other similar immunoassay, including a sandwich ELISA or competitive ELISA; immunohistochemistry (IHC); flow cytometry, or western blot. The binding assay can be performed on samples obtained from a patient's body fluid, cells or tissue sample of any kind, including plasma, urine, tumor tissues or suspected tumor (including fresh, frozen, and fixed or embedded in paraffin), lymph node tissue or bone marrow.
[0211] [0211] Once the amount of HA in the sample is determined, the value can be compared with a threshold or control level. For example, if the amount of HA is determined to be high in the sample, the individual is selected as a candidate for tumor therapy. Examples of methods for stratifying tumor samples or samples of body fluids for diagnosis, prognosis or selection of individuals for treatment are provided here.
[0212] [0212] In one example, a diagnostic method uses a sample of tumor tissue, tumor cells or a body fluid containing proteins, from a patient. In the method, the presence and level of HA expression can be determined using a HABP, for example, TSG-6-LM, TSG-6-LM-Fc or its variant or mutant, as provided herein. The level of HA expression is determined and / or scored, and compared with predetermined HA phenotypes associated with the disease. As described below, these predetermined values can be determined by comparing or knowing the levels of HA in a corresponding normal sample, as determined by the same detection assay and using the same HABP reagent. It is within the level of a person skilled in the art to determine the threshold level for the diagnosis of diseases, depending on the particular disease, the assay to be used for the detection of AH and / or the HABP detection reagent being used. For example, in body fluids such as plasma, HA levels greater than 0.02 pg / ml, 0.03 pg / ml, 0.04 po / ml, 0.05 uvg / ml, 0.06 pg / ml or higher are correlated to the presence of a tumor or cancer. In another example, in tumor tissue immunohistochemistry methods, an HA score or HA ” may be a determinant of the disease. If the level is indicative of disease, then the patient is diagnosed with a tumor.
[0213] [0213] In another example, a prognostic method uses a sample of tumor tissue, tumor cells or a body fluid containing proteins from a patient. In the method, the presence and level of HA expression can be determined using a HABP, for example, TSG-6-LM, TSG-6-LM-Fc or its variant or mutant, as provided herein. The level of HA expression is determined and / or scored, and compared to predetermined HA phenotypes associated with the disease. As described below, these predetermined values can be determined by comparing or knowing the levels of HA in a corresponding normal sample, or samples from sick patients as determined by the same detection assay, and using the same HABP reagent. It is within the skill of a person skilled in the art to determine threshold levels for disease prognosis, depending on the particular disease, the assay to be used for the detection of AH and / or the HABP detection reagent used. The level of HA expression indicates the expected course of disease progression in the patient. For example, high levels of AH, assessed by immunohistochemistry methods using a quantitative scoring scheme (for example, AH) correlated to the existence of malignancy in a variety of types of cancer. In another example, HA levels in body fluids, such as plasma greater than 0.06 mg / mL of HA, are also associated with the stage of advanced disease.
[0214] [0214] In another example of follow-up diagnostic methods, the level of HA expression in samples from individuals previously treated with an enzyme that degrades hyaluronan can be monitored to determine whether an individual administered with the agent achieved an effective level of drug in the blood in order to optimize the dosage or schedule.
[0215] [0215] The following sections describe exemplary HABP reagents and assays for conducting HA detection methods for use in diagnostic and prognostic methods, and in particular, as a company for therapy with an anti-hyaluronan agent, for example. example, an enzyme that degrades hyaluronan. Also described are anti-hyaluronan agents, which include enzyme agents that degrades hyaluronan, for use in the treatment of diseases and disorders associated with hyaluronan, and kits and combinations of HABP reagents, with said agents (for example, enzymes that degrade hyaluronan) . Any of the above methods can be performed using any of the HABP reagents described, and the assay detection methods, alone or in conjunction with therapy with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan).
[0216] [0216] The methods provided here are directed to the quantitative or semi-quantitative measurement of hyaluronan, in a sample, such as a tumor or fluid sample from an individual who has a tumor, or suspected of having a tumor, using a hyaluronan binding protein (HABP). As described here, tumors that express high levels of hyaluronan are responsive to treatment with an anti-hyaluronan agent (for example,
[0217] [0217] The accompanying HABP diagnostics provided here can be used in conjunction with therapy with an anti-hyaluronan agent, such as therapy with enzymes that degrade hyaluronan, or any described in Section E, to select or identify patients expected to be responsive to treatment and / or to monitor treatment and its effectiveness, thus providing a better treatment scheme for diseases or conditions “associated with hyaluronan. For example, the HABP follow-up diagnosis provided here can be used to select and / or monitor individuals or patients who have a tumor or cancer. In addition, accompanying HABP diagnostics can also be used in other diagnostic and prognostic methods, for diseases or conditions associated with hyaluronan, such as tumors or cancers.
[0218] [0218] Hyaluronan binding proteins are provided here for use in the methods provided here for the detection and quantification of hyaluronan in a sample. Hyaluronan binding proteins can contain full-length HABP polypeptides, or parts of them containing HA binding domains from HABPs, or parts of them sufficient to bind to HA. Typically, HABPs or portions thereof that contain an HA binding domain or sufficient portion thereof, that bind HA, or variants or multimers thereof, exhibit a binding affinity with a dissociation constant (Kd) of, at least less than, or less than, or 1 x 10 ”M and, in general, at least less than, or less than, or 9x10 * M, 8x10 * M, 7x10 * M, 6x10 MM, 5x10 ºM, 4x10 ºM, 3x10 ºM, 2x10 ºM, 1x10 ºM, 9x10 ºM, 8x10 MM, 7x10 ºM, 6x10ºM, 5x10ºM, 4x10M, 3x10M, 2x10M, 1x10º M or lower Kd. As discussed here, the displayed binding affinity is generally displayed under conditions that allow the ideal to be reached, or close to the ideal of binding to hyaluronan. In one example, pH conditions can affect the binding. For example, here as a follow-up diagnosis, binding assays using a TSG-6 reagent, including TSG-6-LM or enough portions of it to bind HA, its variants and multimers thereof, are generally conducted at a pH of about 5.8 to 6.4, such as about, or pH 6.0.
[0219] [0219] Hyaluronan-binding proteins are of two types: hyaluronan-binding proteins that have an HA-binding domain, which contains one or two binding modules, and proteins that have an HA-binding domain that is not a connection module. In particular examples, the accompanying diagnostics provided here are derived from HABP-binding molecules that have only a single binding domain that confers binding on HA, which can simplify expression, production and purification methods.
[0220] [0220] The HABPs provided herein can be derived from known HABPs or can be generated synthetically. In some examples, HABPs can be generated synthetically, based on conserved residues from the HA-binding domains of known HABPs. The HABPs provided herein can also be derived from HABPs generated from HA-binding protein screening methods, such as phage display or affinity-based screening methods.
[0221] [0221] HABPs, including the HA-binding domains of HABPs, or portions thereof, which are sufficient to bind to HA, provided herein, can be modified to improve one or more properties of the HABPs for use in the methods provided herein . For example, the respective HABPs, or HA-binding fragments, provided herein, can be modified to increase protein expression in mammalian expression systems, improve biophysical properties, such as stability and solubility, improve purification and protein detection, to increase the specificity of HA and / or increase affinity to HA, as long as they retain their ability to bind to HA. For example, an HABP or HA binding fragment thereof, provided herein for use in the methods, can be modified to increase its specificity to hyaluronan compared to other glycosaminoglycans. In another example, a HABP or HA binding fragment thereof, provided here for use in the methods, can be linked directly or indirectly to a multimerization domain, to increase the number of HA binding sites in the molecule and therefore increase affinity for binding to HA.
[0222] [0222] In addition, for use as a follow-up diagnosis referred to here, any of the HABPs, or parts of them (for example, link modules or sufficient portions of them to link to the HA) can be modified to facilitate detection. For example, follow-up diagnoses are modified by conjugating, directly or indirectly, biotin, a fluorescent fraction, a radioactive marker or another detectable marker.
[0223] [0223] A description of exemplary HABPs for use as diagnostic follow-up agents described herein, and modifications thereof, is provided below.
[0224] [0224] Here are provided as accompanying diagnostic reagents for use in the methods presented here, HA binding proteins (HABP) or portions thereof, which contain at least one binding module or binding domain, and, in general, by at least two or more connection modules. In some examples, the HABP contains a Gl domain that contains two link modules. The connection to the HA is mediated through the connection module. Binding modules, also called tandem repeats of proteoglycans, are about 100 amino acids (aa) in length, with four cysteines that are disulfide bound in the Cysl-Cys4 and Cys2-Cysôi pattern. The three-dimensional structure of the connection modules consists of two alpha helices and two triple-stranded anti-parallel beta sheets.
[0225] [0225] There are three categories of proteins that contain the binding module: a domain-type A protein that contains a single binding module; Type B domain proteins, which contain a single binding module spanning a -N and C-terminal flank region; and C-type proteins that have an extended structure called the G1 domain, which contains an n-terminal Ig type V domain, followed by an adjacent pair of two binding modules. Modeling and comparison studies have demonstrated a high degree of resolution and conservation of certain amino acids between proteins that contain binding modules that correlate with interaction with HA (Blundell et al. (2005) J. Biol. Chem., 280: 18189- 18201). For example, central HA-binding amino acid residue corresponding to Tyr59 and Tyr78, numbered with reference to TSG-6-LM set out in SEQ ID NO: 360 are conserved between HABPs that contain module-binding through identical amino acids or conservative (for example, hydrophobic residues with a flat and wide front or aromatics, which can also accumulate against a GlcNAc ring, for example, Phe, His, Leu or Val) in the corresponding position, based on alignment with TSG-6 -LM (for example, described in SEQ ID NO: 360). In addition, basic residues in the positions corresponding to positions 11 and 81 presented in SEQ ID NO: 360, are also found in other connection modules, as determined by the alignment.
[0226] [0226] HA binding proteins containing binding modules for use in the methods provided herein include, but are not limited to, TSG-6 (for example, established in SEQ ID NO: 206 as the precursor and SEQ ID NO0: 222 as the mature protein without a signal sequence; or LM established in SEQ ID NO: 207, 360, 417 or 418, which represents various lengths of the LM as reported in the literature), stabilin-l (for example, established in SEQ ID NO : 223 or the mature form thereof; or LM set out in SEQ ID NO: 371), stabilin-2 (for example, set out in SEQ ID NO: 224 or the mature form thereof; or LM set out in SEQ ID NO: 372 ), CD44 (for example, established in SEQ ID NO: 227 or the mature form thereof; or LM established in SEQ ID NO: 375), LYVE-1 (for example, established in SEQ ID NO: 228 or the mature form or the connection module established in SEQ ID
[0227] [0227] In particular, in the examples presented here, the HABP used in the methods described herein contains at least one connection module, and in some cases, contains at least two or at least three connection modules. The HABP can be a complete HABP containing a connection module. For example, the accompanying diagnostic reagent, for use in the method of this invention, may contain an amino acid sequence as described in any of SEQ ID NOS: 206, and 223-236, the mature form of which, or a sequence of amino acids that display at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % sequence identity with a sequence shown in any of SEQ ID NOS: 206, and 223-236. For example, the HABP for use as a follow-up diagnosis here may be full-length TSG-6, having the amino acid sequence set out in SEQ ID NO: 222, or an amino acid sequence that displays at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, with a sequence presented in SEQ ID NO: 222.
[0228] [0228] In other examples, the accompanying diagnostic reagent, for use in the methods described here, contains only the binding module or a sufficient portion of a binding module to bind to the HA derived from a full-length HABP shown in any of SEQ ID NOS: 206 and 223-236, or the mature form thereof (without the signal sequence). In some examples, the HABP that contains a link module or modules, is not the complete sequence of an HABP established in any of SEQ ID NOS: 206, and 223-236, or the mature form thereof (without the sequence of signal). It is understood that the portion of a linker or HABP is generally a contiguous sequence of amino acids that is generally at least 50 amino acids in length, 60, 70, 80, 90, 100, 200, 300 or more amino acids. In some instances, the binding module or modules are the only HABP portion of the accompanying diagnostic binding molecule. For example, the accompanying diagnostic reagent, for use in the method described here, contains only part of a full-length HABP, and has an amino acid sequence defined in any of SEQ ID NOS: 207, 360, 361, 371 -394 and 416 -418, or an amino acid sequence that displays at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% sequence identity with a sequence shown in any of SEQ ID NOS: 207, 360, 361 371-394 and 416-418.
[0229] [0229] In the examples presented here, the accompanying diagnostic reagent for use in the methods described herein contains a Gl domain or sufficient portion thereof, to specifically bind to HA. The HABP containing the Gl domain can be derived from a full-length HABP presented in any of SEQ ID NOS: 233-236, or a mature form thereof. In some examples, the HABP containing the Gl domain is not the complete sequence of HABPs established in any of SEQ ID NOS: 233-236 or mature form thereof. It is understood that the portion of a HABP that contains a Gl domain is generally a contiguous sequence of amino acids that is generally at least 100 amino acids in length, such as 150, 200, 250, 300, 400 or more amino acids. In some examples, the Gl domain is the only HABP portion of the accompanying diagnostic binding molecule. For example, the accompanying diagnostic reagent for use in the method described here contains only a portion of a full-length HABP, and has a Gl domain with an amino acid sequence as described in any of SEQ ID NOS: 423-426, Or an amino acid sequence that displays at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence shown in any of SEQ ID NOS: 423-426.
[0230] [0230] In some examples, the monitoring diagnosis may contain more than one connection module, such as two or three connection modules. The connection modules can be from the same or different from the HABP. Follow-up diagnostics can contain linkage modules that are linked directly or indirectly to form a single polypeptide. In other examples, the accompanying diagnosis may contain binding modules that are established as a separate polypeptide that is chemically linked, such as through a disulfide bond. An example of a HABP fragment provided, for use in the methods described herein, is the TSG-6 binding domain (TSG-6-ME), or a portion thereof, sufficient to bind to HA.
[0231] [0231] In some examples, HABP is a multimer containing two or more binding modules that are linked directly or indirectly through a multimerization domain, to effect the formation of dimer or trimer molecules, and the generation of multiple sites. connection to HA. For example, a follow-up diagnosis for use in the methods described here is one that is generated by the expression of a nucleic acid molecule that codes for the binding module shown in any of SEQ ID NOS: 207, 360, 361, 371- 39 and 416-418, or an amino acid sequence that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, with a sequence shown in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 linked directly or indirectly to a nucleic acid which encodes a multimerization domain, such as an Fc part of an immunoglobulin. Thus, the resulting HABP multimer or LM multimer contains a first polypeptide defined in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418, or an amino acid sequence that exhibits at least 65%, 70% , 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence displayed in any one of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418, linked directly or indirectly to a multimerization domain; and a second polypeptide as described in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418, or an amino acid sequence that exhibits at least 65%, 70%, 75%, 80% , 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence presented in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418, directly or indirectly linked to a multimerization domain. The sequence of the binding module in the first and second polypeptides can be the same or different. An example of a HABP multimer provided for use in the methods described herein is a multimer containing two polypeptide chains, each of which contains TSG-6-LM, its variant, or a portion thereof sufficient to bind directly or indirectly bound HA to a multimerization domain, which performs multimerization. For example, a TSG-6-LM: Fc molecule is provided here for use in the methods (see for example, SEQ ID NO: 212 or 215).
[0232] [0232] A description of exemplary HABPs containing binding domains, including structure and function description, is provided below. Any of the HABPsS or portions thereof, described as a fragment containing only one binding domain, or sufficient portion of it to bind to HA, can be used as a follow-up diagnostic reagent in the methods described herein.
[0233] [0233] HABPs that are members of the type A subgroup, which contain a unique binding module that binds to hyaluronan, are provided here for use in the methods described here. Type A HABPs bind to HA with a minimum chain length of six sugars, hexasaccharide (HA), or greater. Members of the type A subgroup, which can be used as a follow-up diagnosis in the methods provided herein include, but are not limited to, TSG-6, Stabilin-1, Stabilin-2, CAB61358 and KIAAO5S27, connecting modules thereof, or sufficient parts of a connection module connected to the HA.
[0234] [0234] An example of a type A subgroup HABP is provided for use as an accompanying diagnostic reagent in the methods provided here, TSG-6, or a binding module thereof, a sufficient portion of a binding module for connect to HA, its variants or its multimers. Gene 6 that stimulates tumor necrosis factor (TSG-6, alpha-induced tumor necrosis factor 6 protein, TNFAIP6; SEQ ID NO: 206) is a secreted - 35 kD glycoprotein composed of a single CUB C domain -terminal and N-terminal connection module. TSG-6 expression is induced in different cell types by inflammatory mediators, including cytokines and growth factors. Through its binding module, TSG-6 is a potent inhibitor of polymorphonuclear leukocyte migration. TSG-6 forms a stable complex with the serine protease inhibitor Inter-alpha-inhibitor (IalI) and potentiates Ial's antiplasmin activity. TSG-6 is also important for the formation and remodeling of HA-rich pericellular linings and extracellular matrices.
[0235] [0235] The human TSG-6 transcript (SEQ ID NO: 205) is normally translated to form a 277 amino acid precursor of peptides (SEQ ID NO: 206) that contains a 17 amino acid signal sequence at the N-terminus. The mature TSG-6 (established in SEQ ID NO: 222), therefore, is a 260 amino acid protein containing amino acids 18-277 of SEQ ID NO: 206 (Lee et al. (1992) J Cell Biol 116: 545- 557). TSG-6 consists of two main domains, the connection module and the CUB domain. The TSG-6 binding module is referred to in the literature to be located at amino acids 35-129, 36-128, 36-129 or 36-132 of SEQ ID NO: 206 (presented as SEQ ID NOS: 207, 360, 417 or 418, respectively). It is understood that the reference to the locus of a domain can vary according to several amino acids, due to differences in alignments. Thus, for the present purposes, a TSG-6-LM is established in any one of SEQ ID NOS: 207, 360, 417 or 418, or that varies from such sequence, by one, two or three amino acids. The CUB domain is located at amino acids 135-246 of SEQ ID NO: 206. Human TSG-6 has two potential N-linked glycans to residues N118 and N258 of
[0236] [0236] The TSG-6 connection module (SEQ ID NO: 360) has a relatively small dimension and a well-characterized structure. The structure of the three-dimensional TSG-6 binding domain was determined and was found to have the same fold as other known binding modules, with two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic nucleus (Kohda et al. ( 1996) Cell 86: 767-775). In addition, the interaction of the TSG-6 and HA binding module has been studied revealing that the aromatic rings of Tyrl2, Tyr59, Phe70, Tyr78, Trp88 or the basic residues Lysll, Lys72, Asp77, Arg 81, and Glu86 of the domain TSG-6 (SEQ ID NO: 360) are important for binding to HA (see, for example, Kahmann et al. (2000) Structure 15: 763-774; Mahoney et al. (2001) J Biol Chem 276: 22764-22771; Kohda et al. (1996) Cell, 88: 767-775; Blundell et al. (2003) 3 Biol Chem 278: 49261-49270; Lesley et al. (2004) J Biol Chem 279: 25745-25754 ; Blundell et al. (2005) J Biol Chem 280: 18189-18201). Structural studies also show that there is only a single HA binding site contained in the binding module, which is located at a region of the molecule based on the structural map of the Lysll, Tyrl2, Tyr59, Phe70 and Tyr7 / 78 residues, more directly involved in binding to HA (see, for example Mahoney et al. (2001) J Biol Chem 276: 22764-22771).
[0237] [0237] The TSG-6 binding module displays binding activity to various glycosaminoglycans. For example, studies have revealed the binding of the HA-binding module, chondroitin-4-sulfate (C4S), G1 domain of the proteoglycan aggrecan, heparin and the iliac bicunin chain (see, for example, Milner et al. ( 2003) Journal of Cell Science, 116: 1863-1873; Mahoney et al. (2005) Journal of Biological Chemistry, 280: 27044-27055). The binding of TSG-6 to heparin and HA is mediated by a distinct binding site on the TSG-6 LM. The residues involved in TSG-6-LM binding to HA are Lysll, Tyrl2, Tyr59, Phe70 and Tyr'78, in which the Kl1Q, Y12F, Y59F, F70V and Y78F mutants have between and 100 times less AH binding affinity, compared to the wild type; the residues in the TSG-6-LM involved in heparin binding are Lys20, Lys34, Lys41l, Lys54, Arg56 and Arg84, whereby mutants K20A, K34A, K41A and K54A exhibit weakened heparin binding properties; and residues involved in TSG-6-LM binding to bicunin overlap but are not identical to the HA binding site (Mahoney et al. (2005) Journal of Biological Chemistry, 280: 27044-27055).
[0238] [0238] The binding of TSG-6 to hyaluronan is pH dependent, with the binding activity shown at an acidic pH of about 5.6-6.4, such as, or about 5.8 to 6, 0.
[0239] [0239] TSG-6 polypeptides, HA-binding domains thereof, for example, TSG-6 binding modules, or fragments thereof sufficient to bind to HA, provided herein for use as a follow-up diagnosis in the methods of this invention, can include any of SEQ ID NOS: 206, 207, 222, 360, 417 or 418, or their variants, such as variants that exhibit at least 65%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of SEQ ID NOS: 206, 207, 222, 360, 417 or
[0240] [0240] Variants of TSG-6 or HA-binding fragments thereof, for use in the methods provided, include variants with an amino acid modification that is an amino acid substitution (substitution), deletion or insertion. Exemplary modifications are amino acid substitutions, such as an amino acid substitution in any of the amino acid residues 4, 6, 8, 13, 20, 29, 34, 41, 45, 54, 67, 72 or 96, corresponding to the residues in TSG-6 set forth in SEQ ID NO: 360, 417 or 418. The amino acid substitution can be that of any other amino acid residue. Examples of amino acid substitutions of TSG-6 polypeptides or HA-binding fragments thereof, provided herein, for use as a company diagnostic reagent in the methods provided herein, include modified TSG-6 polypeptides or their HA-binding fragments, which contain at least one amino acid substitution that corresponds to H4K, EHA4S, E6A, E6K, R8A, K13A, K20A, H29K, K34A, K41A, H45S, K54A, N67L, N67S, K72A, H96K, K34A / K54A or K20A / K34A or K20A / K34A / K41A corresponding to the residues in TSG-6 established in SEQ ID NO: 360, 417 or 418 (see, for example, Mahoney et al. (2005) J Biol Chem 280: 27044-27055, Blundell et al. (2007) J Biol Chem 282: 12976-12988, Lesley et al. (2004) J Biol Chem279: 25745-25754, Kahmann et al. (2000) Structure 15: 763-774). It is understood that important residues or otherwise necessary for the binding of TSG-6 to HA, as described above, or any known to one skilled in the art, are generally invariable and cannot be altered. Thus, for example, amino acid residues 11, 12, 59, 70, 78 and 81 of SEQ ID NO: 360 in the TSG-6 binding module are generally invariant and are not altered. Furthermore, it is understood that amino acid modifications that result in improper folding or disturbance of the binding module folding are generally invariable. Thus, for example, a TSG-6 modification, provided for use in the methods described herein, will not contain any one or more of the amino acid modifications H4S, H29A, H45A, H45K, R56A, D77A, R84A and D89A of SEQ ID NO: 360 (Mahoney et al. (2005) J Biol Chem 280: 27044-27055, Blundell et al. (2007) J Biol Chem 282: 12976-12988, Lesley et al. (2004) J Biol Chem 279: 25745-25754) .
[0241] [0241] In particular, the modification, for example, the amino acid substitution or substitutions, are those that confer a change, such as an improvement, in the activity in relation to a TSG-6 that does not contain the modification. These variants include those that contain amino acid modifications that improve the binding affinity of TSG-6 to HA, increase the specificity of TSG-6 for HA, and / or increase the solubility of TSG-6. For example, provided here for use in the methods described here are variants of TSG-6, HA-binding domains, or portions thereof, sufficient to bind HA, which increase the specificity of TSG-6 for HA, decreasing the binding of TSG-6 to other glycosaminoglycans, including heparin, chondroitin-4-sulfate, heparan sulfate and dermatan sulfate.
[0242] [0242] Example of a TSG-6 polypeptide provided herein for use in the methods described herein is a TSG-6 polypeptide that contains at least one HA binding domain, for example, a TSG-6 binding module. Thus, a TSG-6 connection module, or a variant thereof, is provided here for use in the methods provided. An example of such a reagent is a polypeptide that has an amino acid sequence set out in SEQ ID NO: 207, 360, 361, 416, 417 or 418, or has an amino acid sequence that exhibits at least 65%, 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, 99% or more of sequence identity with any of SEQ ID NOS : 207, 360, 361, 416, 417 or 418. For example, the TSG-6 binding module can be modified to change its specificity, affinity or solubility, while retaining its ability to bind to HA.
[0243] [0243] In yet another example, the affinity of the TSG-6 binding module is increased by dimerization or multimerization, such as, for example, by merging a multimerization domain, such as an Fc domain (see Section C3 below) . Thus, the TSG-6 binding module can be modified to produce a multimer containing two or more binding modules that are directly or indirectly linked through a multimerization domain to effect the formation of dimer or trimer molecules, and to generate multiple HA-binding sites. For example, a follow-up diagnosis, for use in the methods described here, is one that is generated by the expression of a nucleic acid molecule that codes for the binding module presented in any of the
[0244] [0244] In another example, the TSG-6 link module is linked to an Fc domain to increase its solubility (see Section C3 below).
[0245] [0245] An example of a HABP of the type A subgroup, provided for use as a follow-up diagnostic reagent in the methods provided here, Stabilin-1 or Stabilin-2, or a connection module thereof, a sufficient portion of a connection module to connect to the HA, variants of the same or multimers thereof. Stabilin-l1 (also called STABl, CLEVER-l, KIAAO246, FEEL-1, FEX-l and FELE-l; SEQ ID NO0: 223) and Stabilin-2 (also called STAB2, FEEL-2, CD type FELL2 precursor -44, DKFZp434E0321, FEX2, and the hyaluronan receptor for endocytosis / HARE; SEQ ID NO: 224) are members of type I transmembranes, from a family of homologs of the facycline-type hyaluronan (HA) receptors. Both contain seven facycline-like adhesion domains, multiple EGF-like replicates, and HA-linked binding modules. Both (Stabilin-1 and Stabilin-2) are expressed in the sinusoidal endothelium and macrophages, although each is functionally distinct. Stabilin-1 is involved in two intracellular traffic pathways: receptor-mediated endocytosis and recycling; and transiting between the endosomal compartment and the trans-Golgi network (TGN). Stabilin-2 acts as a scavenger receptor for HA and AGE-modified proteins.
[0246] [0246] The precursor sequence to Stabilin-l is described in SEQ ID NO: 223. The Stabilin-l1 binding module is located at 2208-2300 of SEQ ID NO: 223, and is described in SEQ ID NO: 371. The precursor sequence for Stabilin-2 is shown in SEQ ID NO: 224, and the Stabilin-2 binding module is located at amino acids 2198-2290 of SEQ ID NO: 224, and is described in SEQ ID NO: 372.
[0247] [0247] polypeptides Stabilin-l or Stabilin-2, HA binding domains therefor, for example, modules
[0248] [0248] Also provided here for use as a follow-up diagnosis in the methods described herein is a Stabilin-1-LM or Stabilin-1-LM multimer which exhibits an increased affinity for HA. For example, a follow-up diagnosis, for use in the methods described here, is one that is generated by the expression of a nucleic acid molecule that codes for the binding module shown in any of SEQ ID NOS: 371 or 372, or a nucleic acid encoding a binding module that has an amino acid sequence that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence presented in any of SEQ ID NOS: 371 or 372, linked directly or indirectly to a nucleic acid encoding a multimerization domain, such as an Fc portion of an immunoglobulin. Thus, the resulting LM multimer contains a first polypeptide defined in any of SEQ ID NOS: 371 or 372, or an amino acid sequence that exhibits at least 65%, 70% 75%, 80%, 84%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, with a Sequence displayed in any of SEQ ID NOS: 371 or 372, linked directly or indirectly to a multimerization domain; and a second polypeptide as described in any of SEQ ID NOS: 371 or 372, or an amino acid sequence that exhibits at least 65%, 70%, 75%, 80% 84%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence presented in any of SEQ ID NOS: 371 or 372, directly or indirectly linked to a domain multimerization.
[0249] [0249] HABPs that are members of the type B subgroup with an HA binding domain, which contain a unique binding module with the N- extensions, are provided here as a follow-up diagnostic reagent for use in the methods described herein. and C-terminal, which bind to hyaluronan. Unlike the HA-binding domain of the Type A / TSG-6 subgroup, the sequences that flank the binding domains are essential for the structural integrity of the type B domain, and are necessary for HA-binding. Members of the Type B subgroup of HABPs for use in the methods provided herein include, but are not limited to, CD44 and LYVE-l1, or HA-binding fragments thereof.
[0250] [0250] A Type B subgroup HABP provided for use in the methods described herein is CD44, HA binding domains of CD44, or portions thereof sufficient to bind to HA.
[0251] [0251] variants are also provided herein, for use in the methods provided, including allelic variants, species variants and other variants that contain an amino acid modification, provided that the variants retain their ability to bind to HA. Variant species of CD44 for use in the methods provided herein include, but are not limited to mouse (SEQ ID NO: 259), rat (SEQ ID NO: 260), bovine (SEQ ID NO: 261), dog (SEQ ID NO : 262), horse (SEQ ID NO: 263), hamster (SEQ ID NO: 264), baboon (SEQ ID NO: 265) and golden hamster (SEQ ID NO: 266). Variants of CD44, or acceptable HA binding fragments, for use in the methods provided, include variants that have an amino acid change, and that exhibit a change, such as an improvement, with respect to a CD44 activity that does not contain the modification. These variants include those that contain amino acid modifications that improve the affinity of CD44 binding to HA, increase the specificity of CD44 to HA, and / or increase the solubility of CD44.
[0252] [0252] A HABP of the type B subgroup, which is LYVE-1, LYVE-1 HA binding domains, or sufficient portions to bind HA, is provided herein for use in the methods described herein. Lymphatic vessel endothelial hyaluronan (HA) receptor (LYVE-l, also called CRSBP-1, HAR, and XLKDl; SEQ ID NO: 228) is a type of 60-kDa transmembrane glycoprotein that is expressed on both lumen surfaces and ablumenal of the lymphatic endothelium, as well as in the hepatic arterial sinusoidal endothelium. LYVE-l1 participates in the internalization of HA for the degradation and transport of HA, from tissues to the lumen of the lymphatic vessels. The location of HA directed to Lyve-l for lymphatic surfaces, also affects aspects of “tumor metastases or immune system response. The LYVE-1l binding module is located at amino acids 40-129 of SEQ ID NO: 228 and is described in SEQ ID NO: 376. Thus, provided for use in the methods described here, they are fragments of LYVE-l1 that retain the ability to bind to HA, for example, a fragment of LYVE-1 that contains a binding domain, and the N- and C-terminal domains that flank, or a portion thereof sufficient to effect binding to HA.
[0253] [0253] are also provided herein for use in the methods provided, variants, including allelic variants, species variants, and other variants that contain an amino acid modification, as long as the variants retain their ability to bind to HA. Variant species of LYVE-1 include, but are not limited to, mouse (SEQ ID NO: 267) and bovine (SEQ ID NO: 268). Variants of LYVE-l, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to a LYVE-l activity that does not contains the modification. These variants include those that contain amino acid modifications that improve the binding affinity of LYVE-1 to HA, increase the specificity of LYVE -1 for HA, and / or increase the solubility of LYVE-1.
[0254] [0254] Provided herein for use as a follow-up diagnostic reagent in the methods described herein are HABPs that are members of the type C subgroup, having an HA-binding domain that contains an immunoglobulin (Ig) domain, which can mediate binding between the binding protein and other type C HA binding proteins, and two binding modules, both of which are necessary for binding to HA. The Ig domain and two binding modules collectively make up the Gl domain of Type C HABPs. Members of the type C subgroup of HABPs for use in the methods provided herein include, but are not limited to, HAPLN1 / binding protein, HAPLN2, HAPLN3, HAPLNA4, aggrecan, versican, brevican, neurocan, and phosphacan, or HA-binding fragments thereof.
[0255] [0255] The hyaluronan and proteoglycan-binding protein (HAPLN) family is composed of four secreted proteoglycans that bind to HA, and contain an Ig-type C2 domain and two binding domains.
[0256] [0256] A HABP of the type C subgroup provided here for use in the methods is HAPLNlI, the HA-binding domains of HAPLNI, or sufficient portions to bind to the HA. Hyaluronan and proteoglycan binding protein 1 (HAPLN1, also called CRTL1 and binding protein; SEQ ID NO: 229) contributes to the stability and flexibility of the extracellular matrix, through HA stabilization interactions with chondroitin sulfate proteoglycans . HALPN1 contains two binding modules (amino acids 159-253 and amino acids 260-350 of SEQ ID NO: 229) that bind to HA and an Ig module (amino acids 53- 160 of SEQ ID NO: 229) that binds to the module Ig from the Gl domain of aggrecan. HAPLNl] stabilizes HA associations with aggrecan, forming a ternary complex, containing “a linear HA skeleton with aggrecan and HAPLN 1 perpendicularly linked. Aggrecan and HAPLN1] are positioned parallel to each other, while HA runs between the two HAPLN1 connection modules and the two aggrecan connection modules. The complex creates a gelatinous substance with resistance to deformation. HAPLNI also stabilizes the interaction of HA with other chondroitin sulfate proteoglycans, such as versican, neurocan, and brevican, which also have Gl domains containing an Ig module and two binding modules, similar to aggrecan.
[0257] [0257] The HAPLN1 Gl domain] contains the Ig domain and the two link modules. The Ig domain of the G1 domain of HAPLN] 1 is located at amino acids 53-160 of SEQ ID NO: 229. The binding modules of the HAPLNI Gl domain are located at amino acids 159-253 and 259-350 of SEQ ID NO: 229 and are shown in SEQ ID NOS: 377 and 378. Thus, fragments of HAPLN1I are provided for use in the methods described herein that retain the ability to bind to HA, for example, a fragment of HAPLN1 that contains the Gl domain, or enough of it to bind to HA. For example, provided for use in the methods described herein, it is a HA-binding fragment from HAPLNI that contains at least the two binding modules.
[0258] [0258] Normally, for use as a diagnosis for the detection of HA, HAPLN 1 is supplied in combination with another HA-binding protein that contains the HA-binding region, such as, for example, the Gl domain of another Type C HABP, such as aggrecan, versican, brevican, neurocan or phosphacan.
[0259] [0259] Variants, including allelic variants, species variants and other variants that contain an amino acid modification are also provided for use in the methods provided, provided that the variants retain their ability to bind to HA.
[0260] [0260] HAPLN1 variant species] include, but are not limited to cattle (SEQ ID NO: 269 and 273),
[0261] [0261] Provided herein for use in the methods described herein is a HABP of the type C subgroup that is HAPLN2, HA-binding domains of HAPLN2, or portions thereof sufficient to bind to HA. Hyaluronan-binding protein 2 and proteoglycan (HAPLN2; SEQ ID NO: 230), also known as brain-binding protein 1, is predominantly expressed in the brain. The HAPLN2 Gl domain contains the Ig domain and the two link modules. The Ig domain of the HAPLN2 Gl domain is located at amino acids 49-149 of SEQ ID NO: 230. The binding modules of the HAPLN2 Gl domain are located at amino acids 148-241 and 247-337 of SEQ ID NO: 230 and are presented in SEQ ID NOS: 379 and 380.
[0262] [0262] Thus, provided for use in the methods described here, are fragments of HAPLN2 that retain the ability to bind to HA, for example, a fragment of HAPLN2 that contains the Gl domain or a sufficient portion of it to bind to HA THERE IS. For example, a HA-binding fragment of HAPLN2 that contains at least the two binding modules is provided for use in the methods described herein. Typically, for use as a diagnosis for the detection of HA, HAPLN2 is supplied in combination with another HA-binding protein that contains the HA-binding region, such as, for example, the Gl domain of another HABP-type C, such as aggrecan, versican, brevican, neurocan or phosphacan.
[0263] [0263] variants are also provided herein for use in the methods provided, including allelic variants, species variants, and other variants that contain an amino acid modification, provided that the variants retain their ability to bind to HA. Variant species of HAPLN2 include, but are not limited to, mouse (SEQ ID NO: 276), rat (SEQ ID NO: 277) and bovine (SEQ ID NO: 278). Variants of HAPLN2, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to a HAPLN2 activity that does not contain the change . These variants include those that contain amino acid modifications that improve the binding affinity of HAPLN2 for HA, increase the specificity of HAPLNI for HA, and / or increase the solubility of HAPLN2.
[0264] [0264] A type C subgroup HABP provided for use in the methods described herein is HAPLN3, HA-binding domains of HAPLN3, or portions thereof sufficient to bind to HA. Hyaluronan-binding protein 3 and proteoglycan, (HAPLN3; SEQ ID NO: 231), functions in hyaluronan acid binding and cell adhesion. HAPLN3 is regulated in breast cancer and, therefore, may be related to the development of cancer and metastasis. The Gl domain contains the Ig domain and the two connection modules. The Ig domain of the HAPLN3 Gl domain is located at amino acids 62-167 of SEQ ID NO: 231. The binding modules of the G1 domain of HAPLN3 are located at amino acids 166-260 and 266-357 of SEQ ID NO: 231, and are presented in SEQ ID NOS: 381 and
[0265] [0265] Thus, fragments of HAPLN3 that retain the ability to bind to HA are provided for use in the methods described herein, for example, a fragment of HAPLN3 that contains the G1 domain or a sufficient portion of it to bind to HA. For example, a HA-binding fragment of HAPLN3 is provided for use in the methods described herein that contains at least the two binding modules. Typically, for use as a diagnosis for the detection of HA, HAPLN3 is supplied in combination with another HA-binding protein that contains the HA-binding region, such as, for example, the Gl domain of another Type C HABP. , such as aggrecan, versican, brevican, neurocan or phosphacan.
[0266] [0266] Variants, including allelic variants, species variants, and other variants that contain an amino acid modification are also provided for use in the methods described herein, provided that the variants retain their ability to bind to HA. Variant species of HAPLN3 include, but are not limited to, mouse (SEQ ID NO: 279), rat (SEQ ID NO: 280) and bovine (SEQ ID NO: 281). Variants of HAPLN3, or HA-binding fragments thereof, for use in the methods provided include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to a HAPLN3 activity that does not contain the change. These variants include those that contain amino acid modifications that improve the binding affinity of HAPLN3 for HA, increase the specificity of HAPLN3 for HA, and / or increase the solubility of HAPLN3.
[0267] [0267] A type C subgroup HABP, which is HAPLNA4, HA-binding domains of HAPLN4, or sufficient portions to bind to HA is provided for use in the methods described herein. Hyaluronan and proteoglycan-binding protein 4, (HAPLN4; SEQ ID NO: 232), also known as brain-binding protein 2, is predominantly expressed in the brain. HAPLN4 participates in the development of the perineuronal matrix. Human and mouse HAPLN4 share 91% amino acid sequence identity. The HAPLN4 Gl domain contains the Ig domain and the two link modules. The Ig domain of the HAPLN4 Gl domain is located at amino acids 60-164 of SEQ ID NO:
[0268] [0268] Thus, fragments of HAPLN4 that retain the ability to bind to HA are provided for use in the methods described herein, for example, a fragment of HAPLN4 that contains the Gl domain or a portion thereof sufficient to effect binding to the HA. THERE IS. For example, a HA-binding fragment of HAPLN4 is provided for use in the methods described herein that contains at least the two binding modules. Typically, for use as a diagnosis for the detection of HA, HAPLN4 is supplied in combination with another HA-binding protein that contains the HA-binding region, such as, for example, the Gl domain of another type C HABP. , such as aggrecan, versican, brevican, neurocan or phosphacan.
[0269] [0269] HAPLN4 variants, including allelic variants, species variants, and other variants that contain an amino acid modification, are also provided for use in the methods provided herein, provided that the variants retain their ability to bind to HA. Variant species of HAPLN4 include, but are not limited to, mouse (SEQ ID NO: 282), bovine (SEQ ID NO: 283) and rat (SEQ ID NO: 284). Variants of HAPLN4, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change, and that exhibit a change, such as an improvement, in relation to a HAPLN4 activity that does not contain the change . These variants include those that contain amino acid modifications that improve the binding affinity of HAPLN4 to HA, increase the specificity of HAPLNA4 to HA, and / or increase the solubility of HAPLNA4.
[0270] [0270] A type C subgroup HABP that is aggrecan, the HA binding domains of aggrecan, or portions thereof sufficient to bind HA are provided for use in the methods described herein. Aggrecan (SEQ ID NO: 233) belongs to the chondroitin sulfate (CS) proteoglycan family, which also includes versican, brevican, neurocan, and phosphacan. Each aggrecan molecule contains approximately 100 and 30 side chains of keratin sulfate and glycosaminoglycans (GAG), respectively. Agrecam associates non-covalently with hyaluronan through the binding modules and an Ig domain at its N-terminal end. It is the most abundant of proteoglycans in the cartilage, and contributes to the load-bearing capacity of this tissue.
[0271] [0271] The Gl domain of aggrecan is located at amino acids 45-352 of SEQ ID NO: 233. The Ig domain of Gl domain of aggrecan is located at amino acids 45-154 of SEQ ID NO: 233 and is described in SEQ ID NO : 423. Aggrecan Gl domain binding modules are located at amino acids 153-247 and 254-349 of SEQ ID NO: 233, and are presented in SEQ ID NOS: 385 and 386. Link modules 3 and 4 are shown in SEQ ID NOS: 387 and 388. In this way, fragments of aggrecan that retain the ability to bind to HA are provided for use in the methods described herein, for example, an aggrecan fragment that contains the Gl domain or a portion enough of it to connect to the HA.
[0272] [0272] aggrecan variants are also provided herein for use in the methods provided, including allelic variants, species variants and other variants that contain an amino acid modification, as long as the variants retain their ability to bind to HA. Variant species of aggrecan include, but are not limited to, pork (SEQ ID NO: 285), chicken (SEQ ID NO: 286), mouse (SEQ ID NO: 287), bovine (SEQ ID NO: 288), dog ( SEQ ID NO: 289), rat (SEQ ID NO: 290) and rabbit (SEQ ID NO: 291). Variants of aggrecan, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to an aggrecan activity that contains the change. These variants include those that contain amino acid modifications that improve aggrecan binding affinity for HA, increase aggrecan specificity for HA, and / or increase aggrecan solubility.
[0273] [0273] A Type C subgroup HABP that is brevican, brevican HA binding domains, or portions thereof, sufficient to bind HA is provided for use in the methods described herein. Brevican (SEQ ID NO: 234) is a member of the 160 kDa aggrecan / versican proteoglycan family of matrix proteins. It is derived from the brain and serves as a link between hyaluronan and other matrix molecules, such as tenacins and fibulins. The Gl domain of brevican is located at amino acids 51- 356 of SEQ ID NO: 234, and is described in SEQ ID NO: 424. The Ig domain of the Gl domain of brevican is located at amino acids 51-158 of SEQ ID NO: 234 The binding modules for the brevican G1 domain are located at amino acids 157-251 and 258-353 of SEQ ID NO: 234, and are shown in SEQ ID NOS: 389 and 390. Thus, they are provided for use in the methods described herein, fragments of brevican that retain the ability to bind to HA, for example, a fragment of brevican that contains the Gl domain or a sufficient portion of it to effect binding to HA. For example, a brevican HA binding fragment is provided for use in the methods described herein, which contains at least the two binding modules.
[0274] [0274] Brevican variants are also provided for use in the methods provided herein, including allelic variants, species variants, and other variants that contain an amino acid modification, as long as the variants retain their ability to bind to HA. Variant brevican species include, but are not limited to, rat (SEQ ID NO: 292), mouse (SEQ ID NO: 293), bovine (SEQ ID NO: 294) and cat (SEQ ID NO: 295). The brevican variants, or HA-binding fragments thereof, for use in the methods provided include variants that have an amino acid change, and that exhibit a change, such as an improvement, from a brevican activity that does not contain the modification. These variants include those that contain amino acid modifications that improve the brevity binding affinity for HA, increase the brevican specificity for HA, and / or increase the brevican solubility.
[0275] [0275] A Type C subgroup HABP that is versican, versican HA binding domains, or portions thereof, sufficient to bind HA is provided for use in the methods described herein. Versican (SEQ ID NO: 235) is a large extracellular matrix proteoglycan, which is present in a variety of tissues. It plays important structural roles, forming loose hydrated matrices, during development and illness. It also interacts directly or indirectly with cells to regulate these physiological processes, such as cell adhesion, survival, proliferation, and motility. The versican Gl domain is located at amino acids 38-349 of SEQ ID NO: 235 and is described in SEQ ID NO: 425. The Ig domain of the versican Gl domain is located at amino acids 38- 151 of SEQ ID NO: 235. The binding modules of the versican Gl domain are located at amino acids 150-244 and 251-346 of SEQ ID NO: 235, and are presented in SEQ ID NOS: 391 and 392. Thus, they are provided for use in the methods described herein, fragments of versican that retain the ability to bind to HA, for example, a fragment of versican that contains the Gl domain or a sufficient portion of it, to effect binding to HA. For example, a versican HA binding fragment is provided for use in the methods described herein that contains at least the two binding modules.
[0276] [0276] Also provided for use in the methods provided herein, are versican variants, including allelic variants, species variants, and other variants that contain an amino acid modification,
[0277] [0277] A type C subgroup HABP that is neurocan, neurocan HA binding domains, or portions thereof, sufficient to bind HA is provided for use in the methods described herein. Neurocan, also known as CSPG3 and 1Dl (SEQ ID NO: 236), is a secreted chondroitin sulfate proteoglycan, which is mainly expressed in the central nervous system. Human neurocan is expected to be cleaved following Met635, resulting in N-terminal (Neurocan-130) and C-terminal (Neurocan-C) fragments. Neurocan and Neurocan-C are produced by astrocytes and accumulate in the matrix surrounding the bundles of axons, and neuronal cell bodies. Neurocan-130 is found mainly in the cytoplasm of glial cells. Neurocan inhibits neuronal adhesion and growth of neurites through interactions with a variety of matrices and transmembrane molecules. The neurocan Gl domain is located at amino acids 53-359 of SEQ ID NO: 236 and is described in SEQ ID NO: 426. The Ig domain of the neurocan Gl domain is located at amino acids 53-161 of SEQ ID NO: 236. The neurocan Gl domain binding modules are located at amino acids 160-254 and 261-356 of SEQ ID NO: 236, and are shown in SEQ ID NOS: 393 and 394. Thus, provided for use in the methods described herein, are fragments of neurocan that retain the ability to bind to HA, for example, a fragment of neurocan that contains the G1 domain or a sufficient portion of it to effect binding to HA. For example, provided for use in the methods described herein, it is a neurocan HA-binding fragment that contains at least the two binding modules.
[0278] [0278] Also provided herein for use in the methods provided are variants, including allelic variants, species variants, and other variants that contain an amino acid modification, as long as the variants retain their ability to bind to HA. Variant species of neurocan include, but are not limited to, mouse (SEQ ID NO: 301), rat (SEQ ID NO: 302) and chimpanzee (SEQ ID NO: 303). Neurocan variants, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to a neurocan activity that does not contain the change . These variants include those that contain amino acid modifications that improve neurocan HA binding affinity, increase neurocan specificity for HA, and / or increase neurocan solubility.
[0279] [0279] A type C subgroup HABP which is phosphacan, phosphacan HA binding domains, or parts thereof sufficient to bind HA is provided for use in the method provided herein. Phosphacan (SEQ ID NO: 340), a chondroitin sulfate proteoglycan isolated from the rat brain, which binds to neurons and neural cell adhesion molecules, and modulates cell interactions, and other nerve tissue development processes through heterophilic binding to the cell surface and extracellular matrix molecules, and by competition with transmembrane phosphatase ligands. Phosphacan has 76% identity with the extracellular portion of a human receptor tyrosine phosphatase protein (zeta / beta RPTP) and represents a variant of mRNA splicing of the largest transmembrane protein.
[0280] [0280] Variants, including allelic variants, species variants, and other variants that contain an amino acid modification are also provided for use in the methods described herein, provided that the variants retain their ability to bind to HA. Variant species of phosphacan include, but are not limited to, rat phosphacan (SEQ ID NO: 237). Phosphacan variants, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to a phosphacan activity that does not contain the change . These variants include those that contain amino acid modifications that improve phosphacan binding affinity for HA, increase phosphacan specificity for HA, and / or increase phosphacan solubility.
[0281] [0281] In some examples, provided for use in the methods described herein, are HA binding proteins that do not contain binding modules. HA-binding proteins without binding modules for use in the methods provided herein include, but are not limited to, HABP1 / CIQBP, lailine, RHAMM, IaI, CDC37, PHBP, SPACR, SPACRCAN, CD38, IHABP4 and PEP-l, or HA-binding fragments thereof.
[0282] [0282] A hyaluronan binding protein 1, HABPl binding domains or portions thereof, are provided for use in the methods described herein to bind HA. Hyaluronan-binding protein 1 (HABP1; SEQ ID NO: 240), also known as C1IqBP / C1aR and p32, is a ubiquitous acid glycoprotein that functions in spermatogenesis, and as a receptor for pro-inflammatory molecules. HABPl binds to extracellular hyaluronan, vitronectin, a component of the cla complement, HMW kininogen, and bacterial and viral proteins. Intracellular HABP1l binds to molecules containing the Claq globular domain, multiple isoforms of PKC, mitochondrial HRK, adrenergic receptors and GABA-A, the ASF / SF2 splicing factor mRNA, and the CBF transcription factor.
[0283] [0283] Also provided for use in the methods described herein are variants, including allelic variants, species variants and other variants, which contain an amino acid modification, provided that the variants retain their ability to bind to HA. Variants of HABP1, or HA-binding fragments thereof, for use in the methods provided, include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to a HABP1l activity that does not contain the change . These variants include those that contain amino acid modifications that improve the binding affinity of HABPl to HA, increase the specificity of HABPl to HA, and / or increase the solubility of HABP1.
[0284] [0284] A lailine, HA binding domains of lailine, or portions thereof sufficient to bind HA are provided for use in the methods described herein. Lailin (SEQ ID NOS: 238 and 239) is the transmembrane protein with homology to type C lecithins, and is named after the amino acid six L-A-Y-I-L-I, in its transmembrane segment. Lailin binds specifically to hyaluronan and is found in the extracellular matrix of most animal tissues and body fluids. And therefore, they are able to modulate cellular behavior and functions during tissue remodeling, development, homeostasis and disease.
[0285] [0285] Also provided for use in the methods described herein are variants, including allelic variants, species variants, and other variants that contain an amino acid modification, as long as the variants retain their ability to bind to HA. Variant species of lailin include, but are not limited to, mouse (SEQ ID NO: 304), Chinese hamster (SEQ ID NO: 305) and rat (SEQ ID NO: 306). Lailin variants, or HA-binding fragments thereof, for use in the methods provided, include variants which have an amino acid modification, and which exhibit a change, such as an improvement, in relation to a lailin activity that does not contain the modification. These variants include those that contain amino acid modifications that improve the binding affinity of lailin to HA, increase the specificity of lailin to HA, and / or increase the solubility of lailin.
[0286] [0286] An RHAMM, RHAMM domains, or their portions sufficient to bind to HA is provided for use in the methods described here. The receptor for HA-mediated motility (RHAMM, SEQ ID NO: 242) is a membrane-associated protein, the size of which ranges from - 59 to 80 kDa. RHAMM is expressed in most cell types and functions to mediate cell adhesion and motility in response to binding to HA. Variants, including allelic variants, species variants, and other variants that contain an amino acid modification are also provided herein for use in the methods described herein, provided that the variants retain their ability to bind to HA. Variant species of RHAMM include, but are not limited to, mouse (SEQ ID NO: 307) and rat (SEQ ID NO: 308). RHAMM variants, or HA-binding fragments thereof, for use in the methods provided include variants that have an amino acid change and that exhibit a change, such as an improvement, in relation to an RHAMM activity that does not contain the change. These variants include those that contain amino acid modifications that improve the binding affinity of RHAMM for HA, increase the specificity of RHAMM for HA, and / or increase the solubility of HABP1.
[0287] [0287] Other HA-binding HABPs, some of which contain hyaluronan binding domains that can be used in the methods provided herein include, but are not limited to, TIoaI (SEQ ID NOS: 243-245), CDC37 (SEQ ID NO: 250), PHBP (SEQ ID NO: 251), SPACR (SEQ ID NO: 246), SPACERCAN (SEQ ID NO: 247), CD38 (SEQ ID NO: 248), IHABP4 (SEQ ID NO: 249) and PEP-l1 (SEQ ID NO: 241), or HA-binding domains, or parts thereof sufficient to bind to HA.
[0288] [0288] Modified HABPs are provided here to improve one or more properties of HABPs for use in the methods provided herein. Such properties include modifications that increase protein expression in mammalian expression systems, improve biophysical properties, such as stability and solubility, improve protein purification and detect and / or increase affinity for HA, via dimerization of HA. fusion protein.
[0289] [0289] HABPs intended for use in the methods described here can be linked directly or indirectly to a multimerization domain. The presence of a multimerization domain can generate multimers of the HA-binding domains of HABPs, or even to increase the HA-binding sites of a molecule. This can result in an increased affinity of HABP for HA. For example, the affinity of a HABP multimer can be increased by 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, compared to a HABP polypeptide that does not contain a multimerization domain. The affinity of a HABP multimer for HA, when represented as the dissociation constant (Kd), is generally at least less than or less than or 1x10 º M to 1x10 * º M, such as, at least , less than, or less than, Or 9x10 ºM, 8x10 ºM, 7x10 M, 6x10 ºM, 5x10 M, 4x10 M, 3x10ºM, 2x10ºM, 1x10ºM, 9x10ºM, 8x10 * ºM, TxX10 * º M, 6x10 ºº M, 5x10 ºM, 4x10ººM, 3x10 ºM, 2x10ººM, 1x10 * º M or lower Kd.
[0290] [0290] Multimers are provided herein which include an HA binding domain or sufficient portion thereof to bind the HA of a first HABP and an HA binding domain or sufficient portion thereof to bind the HA of a second HABP , in which theThe first and second HA-binding domains are directly or indirectly linked via a linker to a multimerization domain. The first and second HA-binding domains can be from the same HABP or a different HABP. For example, if the HA-binding domain is the same, then homodimers or homotrimers can be generated. If the HA-binding domain is different, heterodimers or heterotrimers can be generated. For example, HA-binding domains, such as an Or O module binding domain, of HABPs, can be covalently linked, non-covalently linked, or chemically linked, to form multimers of two or more HA binding domains. The connection modules can be connected to form higher dimers, trimers or multimers. In some cases, multimers can be formed by dimerizing two or more HABP polypeptides, each containing an HA-binding domain.
[0291] [0291] Any part of a HABP, including an HA-binding domain, can be used as a multimer partner. For example, any of the HABPs described above, or those presented in any of SEQ ID NOS: 206-207, 222-340, 407-414 or any portion of an HABP, including an HA-binding domain, for example, a binding domain or module and its variants, including the HA binding domains established in any of SEQ ID NOS: 341 and 371-394 can be used to generate chimeric HABP polypeptides, wherein all or part of the polypeptide HABP is connected to a multimerization domain. Usually, at least one, but sometimes the two HABP portions are wholly or part of an HABP sufficient to connect to the HA connected to a multimerization domain. Examples of HABPs, or portions thereof, for use as multimerization partners are described here above and are presented in any of SEQ ID NOS: 206-207, 222-341, 371-394, 407-414, 416-418 or 423 -426. In some instances, at least one of the multimer partners is all or part of the HABP including the HA-binding domain. An example of a multimeric HABP polypeptide is a multimer formed between the HA binding domain (for example, binding domain or binding module), or part of it, from aggrecan, versican, neurocan, brevican, phosphacan, HAPLN1, HAPLN2, HAPLN3, HAPLN4 Stabilin-1, Stabilin-2, CAB61358, KIAAON5S27 or TSG- protein
[0292] [0292] The multimerization between two HABP polypeptides can be spontaneous, or it can occur due to the forced attachment of two or more polypeptides. In one example, multimers can be linked by disulfide bonds formed between cysteine residues in different HABP polypeptides or domains, or sufficient portions thereof, which bind to HA. In another example, multimers can include a HABP polypeptide or domain, or sufficient part of it to bind to HA, joined through covalent or non-covalent interactions to portions of fused peptides for each polypeptide. Such peptides can be peptide ligands (spacers) or, for example, peptides that have the property of promoting multimerization. In a further example, multimers can be formed between two polypeptides through chemical bonding, for example, using heterobifunctional linkers.
[0293] [0293] Peptide linkers can be used to produce HABP polypeptide multimers, such as, for example, a multimer in which at least one multimerization partner contains an HA binding domain (e.g., a binding domain or module) ). In one example, the peptide linkers can be fused to the C-terminal end of a first polypeptide and the N-terminal end of a second polypeptide. This structure can be repeated multiple times, such that at least one, preferably two, three, four, or more polypeptides are linked to each other by means of peptide linkers, at their respective ends. For example, a multimer polypeptide can have a sequence of Z, -X-Z ,, where 2; and Z ;, are each a sequence of all or part of a HABP including an HA-binding domain, and where X is a sequence of a binding peptide. In some cases, 72.1 and / or Z7 is the entirety of a HABP including an HA-binding domain. In other cases, 2; and / or Z, is part of an HABP including an HA-binding domain. 72, and Z7, are the same or are different. In another example, the polypeptide has a sequence of 2Z; -X-Z2 (-X-2), where '"n" is an integer, that is, generally, 1 or 2.
[0294] [0294] Normally, the peptide linker is of sufficient length to allow one or both of the HA binding domains to bind to a hyaluroran substrate, or to allow interaction between the HA binding domains (for example, interaction of two Ig modules from the Gl binding domains to Type C HABPs).
[0295] [0295] Binding portions are described, for example, in Huston et al. (1988) PNAS 85: 5879-5883, Whitlow et al. (1993) Protein Engineering 6: 989-995, and Newton et al., (1996) Biochemistry 35: 545-553. Other suitable peptide linkers include any of those described in U.S. Patent No. 4751180 or 4935233, which are incorporated herein by reference. A polynucleotide that encodes a desired peptide linker can be inserted between, and in the same reading frame, as a polynucleotide that encodes all or part of a
[0296] [0296] The attachment of one HABP polypeptide to another HABP polypeptide to create a heteromultimeric fusion polypeptide can be direct or indirect. For example, the bonding of two or more HABP polypeptides can be achieved by chemical bonding or facilitated by heterobifunctional linkers, such as any known in the art, or provided herein.
[0297] [0297] Numerous heterobifunctional cross-linking reagents that are used to form covalent bonds between amine groups and thiol groups, and to introduce thiol groups into proteins, are known to those skilled in the art (see, for example, the PIERCE CATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describes the preparation, and the use of such reagents, and provides a commercial source of such reagents, see also, for example, Cumber et al. (1992) Bioconjugate Chem. 3: 397- 401; Thorpe et al. (1987) Cancer Res. 47: 5924-5931; Gordon et al. (1987) Proc. Natl. Acad. Sci. 84: 308-312; Walden et al. (1986) J. Mol. Cell. Immunol. 2: 191-197; Carlsson et al. (1978) Biochem. J. 173: 7723-737; Mahan et al. (1987) Anal. Biochem. 162: 163-170; Wawryznaczak et al. (1992 ) Br. J. Cancer 66: 361-366; Fattom et al. (1992) Infection & Immun. 60: 584-589). These reagents can be used to form covalent bonds between the N-terminal portion of one HABP polypeptide, including an HA binding domain and the C-terminal portion of the other HABP polypeptide, including an HA binding domain, or between each of those portions and a binder.
[0298] [0298] The interaction of two or more HABP polypeptides can be facilitated by linking them, either directly or indirectly, to any portion or other polypeptide that are themselves capable of interacting to form a stable structure. For example, separate HABP encoded polypeptide chains can be joined by multimerization, where the polypeptide multimerization is mediated by a multimerization domain. Typically, the multimerization domain provides the formation of a stable protein-protein interaction between a first HABP polypeptide and a second HABP polypeptide. HABP polypeptides include, for example, a link (directly or indirectly) of a nucleic acid that encodes an HA binding domain (for example, a domain or binding module) from an HABP with a nucleic acid that encodes a multimerization domain. Typically, at least one multimerization partner is a nucleic acid that encodes the entire part of a HABP including an HA-binding domain, directly or indirectly linked to a multimerization domain, thus forming a chimeric molecule. Homo or heteromultimeric polypeptides can be generated from co-expression of separate HABP polypeptides. The first and second HABP polypeptide can be the same or different.
[0299] [0299] Generally, a multimerization domain includes anyone capable of forming a stable protein-protein interaction. The multimerization domains can interact through an immunoglobulin sequence (for example, Fc domain; See, for example, International Patents Pub. No. WO 93/10151 and WO 2005/063816; and No. 2006/0024298; No. 5457035 in the USA ), leucine zipper (e.g., nuclear transformation proteins, fos and jun or the c-myc proto-oncogene, or general Nitrogen control (GCN4)), a hydrophobic region, a hydrophilic region, or free thiol, which forms an intermolecular disulfide bridge between the chimeric molecules of a homo- or heteromultimer. In addition, a multimerization domain can include an amino acid sequence that comprises a protuberance complementary to an amino acid sequence that comprises an orifice, as described, for example, in the U.S. Patent. At the.
[0300] [0300] A HABP polypeptide, such as, for example, any provided herein, including any HA binding domain (e.g., a binding domain or module) of an HABP, can be joined anywhere, but usually through from its N- or C-terminal, to the N- or C- terminal of a multimerization domain, to form a chimeric polypeptide, the connection can be direct or indirect by means of a linker. In addition, the chimeric polypeptide can be a fusion protein, or it can be formed by chemical bonding, such as through covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide that contains a multimerization domain, the nucleic acid that encodes all or part of a HABP that includes an HA-binding domain can be operably linked to the nucleic acid that encodes the sequence of the domain of multimerization, directly or indirectly, or, optionally, through a binding domain. Typically, the construct encodes a chimeric protein in which the C-terminal of the HABP polypeptide is joined to the n-terminal of the multimerization domain. In some cases, the construct may encode a chimeric protein in which the N-terminal of the HABP polypeptide is joined to the N- or C-terminal end of the multimerization domain.
[0301] [0301] A polypeptide multimer contains two chimeric proteins created by linking, directly or indirectly, two of the same, or different HABP polypeptides directly or indirectly, to a multimerization domain. In some instances, where the The multimerization domain is a polypeptide, a fusion gene that encodes the chimeric polypeptide of the HABP multimerization domain, is inserted into an appropriate expression vector. The resulting chimeric proteins from the resulting HABP multimerization domain can be expressed in host cells transformed with the recombinant expression vector, and left to pool in multimers, in which the multimerization domains interact to form multivalent polypeptides. Chemical ligation of multimerization domains for HABP polypeptides can be performed using heterobifunctional linkers, as discussed above.
[0302] [0302] The resulting chimeric polypeptides, and multimers formed from them, can be purified by any suitable method, such as, for example, by affinity chromatography on protein A or protein G columns. When two nucleic acid molecules that encoding different chimeric HABP polypeptides are transformed into cells, the formation of homo- and heterodimers will occur. Conditions for expression can be adjusted so that the formation of heterodimers is favored over the formation of homodimers.
[0303] [0303] Domains of multimerization include those comprising a free thiol moiety capable of reacting to form an intermolecular disulfide bond, with a multimerization domain of an additional amino acid sequence. For example, a multimerization domain can include a portion of an immunoglobulin molecule, such as, from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IoM and IgE. Such a portion is usually an immunoglobulin constant (Fc) region. Fusion protein preparations containing polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) have been described, see, for example, Ashkenazi et al. (1991) PNAS 88: 10535; Byrn et al. (1990) Nature, 344: 677; and Hollenbaugh and Aruffo, (1992) “Construction of
[0304] [0304] antibodies bind to specific antigens and contain two identical heavy chains and two identical light chains, covalently linked by disulfide bonds. Both heavy and light chains have variable regions that bind to the antigen, and constant regions (c). In each chain, a domain (V) has a variable amino acid sequence, depending on the specificity of the molecule's antibody. The other domain (C) has a more or less constant sequence common among molecules of the same class. Domains are numbered sequentially from the amino-terminal end. For example, the IgG light chain is composed of two immunoglobulin domains with N- to C-terminal bonds, in the order V, -Crn, referring to the variable domain of the light chain and constant domain of the light chain, respectively. The IgG heavy chain is composed of four immunoglobulin domains linked from the N- to the C-terminal of the order of Vi Crl-Cr2-Cx3, referring to the variable heavy domain, contain a constant heavy domain 1, constant heavy domain 2, and constant heavy domain 3. The resulting antibody molecule is a four-chain molecule in which each heavy chain is linked to a light chain by a disulfide bond, and the two heavy chains are linked to each other via disulfide bonds. The binding of heavy chains is mediated by a flexible zone of the heavy chain, known as an articulation region. Fragments of antibody molecules can be generated, such as, for example, through enzymatic cleavage. For example, after papain protease cleavage, a dimer of the heavy chain constant regions, the Fc domain, is cleaved from the two Fab regions (i.e., the portions containing the variable regions).
[0305] [0305] In humans, there are five antibody isotypes classified based on their heavy chains designated as delta (3), gamma (y), mu (p), alpha and (a) and epsilon (c), giving rise to antibody classes IgD, IgG, IgM, IgA and IgE, respectively. The IgA and IgG classes contain the subclasses IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4. The sequence differences between the immunoglobulin heavy chains cause the fact that the various isotypes differ in, for example, the number of C domains, the presence of a joint region, and the number and location of the interchain disulfide bridges. For example, the IgM and IgE heavy chains contain an additional C domain (C4), which replaces the hinge region. The Fc regions of IgG, IgD, IgA pair with each other through their Cy3, Cô3, and Ca3i domains, whereas the Fc regions of IgM and IgE dimerize through their Cyp4 and C £ e4 domains. IgM and IgA form multimeric structures with ten and four antigen-binding sites, respectively.
[0306] [0306] Chimeric HABP immunoglobulin polypeptides provided herein include a full-length immunoglobulin polypeptide. Alternatively, the immunoglobulin polypeptide is less than the full length, that is, it contains a heavy chain, light chain, Fab, Fabo, Fv, or Fc. In one example, the chimeric HABP immunoglobulin polypeptides are assembled as monomers or hetero- or homomultimers, and particularly as dimers or tetramers. Chains or basic units of different structures can be used to assemble the monomers and hetero- and homo-multimers. For example, a HABP polypeptide can be fused all or part of an immunoglobulin molecule, including all or part of a CH, Cr, Vu, or V domain, of an immunoglobulin molecule (such as, for example, U.S. Patent At the.
[0307] [0307] Normally, the immunoglobulin portion of a chimeric HABP protein includes the heavy chain of an immunoglobulin polypeptide, more generally, the heavy chain constant domains. Examples of sequences of regions constant in the human IgG subtype heavy chain are shown in SEQ ID NOS: 355 (IgGl1), SEQ ID NO: 356 (IgG2), SEQ ID NO: 357 (IgG3), and SEQ ID NO : 358 (IgG4). For example, for the exemplary heavy chain constant region described in SEQ ID NO: 355, the Cxl domain corresponds to amino acids 1-98, the hinge region corresponds to amino acids 99-110, the Cx2 domain corresponds to amino acids 111-223, and the Cr; x3 domain corresponds to amino acids 224-330.
[0308] [0308] In one example, an immunoglobulin polypeptide chimeric protein can include the Fc region of an immunoglobulin polypeptide. Usually, such a fusion retains at least one functionally active joint, Cy, 2 and C1i3 domains of the constant region of an immunoglobulin heavy chain. For example, an IgGl full-length Fc sequence includes amino acids 99- 330 of the sequence shown in SEQ ID NO: 355. An exemplary Fc sequence for hIgGl is set out in SEQ ID NO: 359, and contains almost the entire sequence linkage corresponding to amino acids 100-110 of SEQ ID NO: 355, and the complete sequence for the Cr62 and Cx3 domain, as shown in SEQ ID NO: 355. Another exemplary Fc polypeptide is the Fc polypeptide shown in SEQ ID NO:
[0309] [0309] In addition to hIgG1l Fc, other Fc regions can also be included in the chimeric HABP polypeptides provided herein. For example, when effector functions mediated by Fe / FeyR interactions are to be minimized, fusion with IgG isotypes that barely recruit complement or effector cells from, such as, for example, IgG2 or IgG4 Fc, are contemplated. In addition, Fc fusions may contain immunoglobulin sequences that are substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including, but not limited to IgG (including the human subclasses IgG1l, 19G2, I19gG3, or I19G4) , IgA (including human subclasses IgAl and IgA2), antibody classes IgD, IgE, and IgM. In addition, the linkers can be used to covalently link to another Fc polypeptide to generate an Fc chimera.
[0310] [0310] Modified Fc domains are also contemplated for use in chimeras with HABP polypeptides. In some examples, the Fc region is modified in such a way that it changes in the connection to an FcR, therefore, it should result in an effected function (that is, more or less) altered than the effector function of an Fc region of a chain heavy dose of wild-type immunoglobulin. Thus, a modified Fc domain may have altered affinity, including, but not limited to, increase or decrease or no affinity for the Fc receptor. For example, the different IgG subclasses have different affinities for FcyR5, with IgGl and IgG3, usually having a substantially better bond than the Ig6G2 and IgGi receptors. In addition, different FcyR5 can mediate different effector functions. FcyRl, FcyRIIa / c, and FcyRIIIa are positive regulators of activation triggered in the immune complex, characterized by having an intracellular domain that has a tyrosine-based immunoreceptor activation motive (ITAM). FcyRIIb, however, has a tyrosine-based immunoreceptor inhibition motive (ITIM) and is therefore an inhibitor. In some cases, a HABP polypeptide Fc chimeric protein provided herein can be modified to increase binding to the complement protein Clq. In addition, an Fc can be modified to alter its binding to the FcRn, thereby improving the pharmacokinetics of a chimeric HABP-Fc polypeptide. Thus, altering the affinity of an Fc region for a receptor is able to modulate the effector functions and / or pharmacokinetic properties associated with the Fc domain. Modified Fc domains are known to a person skilled in the art and described in the literature, see for example, U.S. Patent No. 5,457,035; US Patent Publication No. 2006/0024298; and International Patent Publication No. WO 2005/063816 for exemplary modifications.
[0311] [0311] Typically, a polypeptide multimer is a dimer of two chimeric proteins created by linking, directly or indirectly, two of the same or different HABP polypeptides to an Fc polypeptide. In some examples, a fusion gene that encodes the chimeric protein HABP-Fc is inserted into an appropriate expression vector. The resulting HABP-Fc chimeric proteins can be expressed in host cells transformed with the recombinant expression vector, and left to assemble as antibody molecules, in which interchain disulfide bonds are formed between the Fc moieties, to produce bivalent HABP polypeptides.
[0312] [0312] The resulting chimeric polypeptides containing Fc portions and multimers formed from them, can be easily purified by affinity chromatography on protein A or protein G columns.
[0313] [0313] HABP chimeric polypeptides that contain the Fc regions can also be modified to include a label with metal chelates or another epitope. The labeled domain can be used for rapid purification by metal chelate chromatography, and / or by antibodies, to allow the detection of immunoprecipitation western blots, or activity depletion / block in bioassays.
[0314] [0314] Exemplary HABP-Fc chimeric polypeptides include the TSG-6 binding module fusion protein (TSG-6-LM) and Fc. A TSG-6-LM-Fc example is described in SEQ ID NO: 212, and encoded by a nucleotide sequence described in SEQ ID NO: 211. In addition, HABP-Fc molecules, including, for example, exemplary TSG-6-Fc molecules, may optionally contain an epitope marker, or an expression and secretion signal. For example, the exemplary chimeric polypeptide TSG-6-LM-Fc presented as SEQ ID NO: 212 contains a human immunoglobulin light chain leader kappa (x) signal peptide sequence (SEQ ID NO: 210), a fragment Fc of a human IgGl heavy chain (SEQ ID NO: 204) and a human TSG-6 binding module (SEQ ID NO: 207). The cDNA sequence encoding the chimeric TSG-6-LM-Fc polypeptide is described in SEQ ID NO: 211. The DNA encoding the heavy chain regions of human IgGl and the human TSG-6 binding module is connected with a 6 bp Agel restriction enzyme cleavage site and a 12 bp sequence, GACAAAACTCAC (SEQ ID NO: 208), which encodes four additional amino acids (DKTH; SEQ ID NO: 209).
[0315] [0315] Another method of preparing HABP polypeptide multimers for use in the methods provided herein involves the use of a leucine zipper domain. Leucine zippers are peptides that promote multimerization of the proteins in which they are found. Typically, leucine zipper is a term used referring to a repetitive hepta pattern containing 4-5 leucine residues present as a conserved domain in various proteins. Leucine zips fold like short, parallel coiled spirals, and are believed to be responsible for the oligomerization of the proteins of which they form a domain. The dimer formed by a leucine closure domain is stabilized by the repetition of seven, designated (abcdefg) n
[0316] [0316] Exemplary leucine zippers, for use as multimerization domains described herein, are derived from either of the two nuclear transformation proteins, fos and jun, which have leucine zipper domains, or the proto-oncogene product murine, c-myc. The leucine zipper domain is necessary for biological activity (DNA binding) in these proteins. The products of the fos and jun nuclear oncogenes contain leucine closure domains that preferably form a heterodimer (0 'Shea et al. (1989) Science, 245: 646; Turner and Tijian (1989) Science, 243: 1689). For example, the leucine closing domains of human transcription factors c-jun and c-fos have been shown to form stable heterodimers with a 1: 1 stoichiometry (see, for example, Busch and Sassone-Corsi (1990) Trends Genetics, 6: 36-40; Gentz et al., (1989) Science, 243: 1695-1699). Although the formation of Jun-Jun homodimers has also been shown, they are about 1000 times less stable than jun-fos heterodimers.
[0317] [0317] Thus normally a HABP polypeptide multimer provided herein is generated using a combination. Generally, the leucine zipper domain of both c-jun and c-fos is fused to the C-terminal structure of a HABP of a polypeptide by genetic engineering of fusion genes. Examples of the amino acid sequences of c-jun and c-fos leucine zippers are shown in SEQ ID NOS: 362 and 363, respectively. In some cases, a sequence of a leucine zipper may be modified, such as by adding a cysteine residue, to allow the formation of disulfide bridges, or the addition of a tyrosine residue at the C-terminal part to facilitate measurement of the peptide concentration. Such exemplary amino acid sequences encoded by a modified c-jun and c-fos leucine zipper are shown in SEQ ID NOS: 362 and 363, respectively. In addition, the binding of a HABP polypeptide to a leucine zipper may be direct or may employ a flexible binding domain, such as, for example, an IgG hinge region, or other small amino acid polypeptide binding agents such as glycine , serine, threonine, or alanine, in various lengths and combinations. In some cases, separation of a leucine zipper from the C-terminus of an encoded polypeptide can be effected by fusion with a sequence encoding protease cleavage sites, such as, for example, a thrombin cleavage site. In addition, chimeric proteins can be labeled, such as, for example, by a 6XHistag, to allow rapid purification by means of metal chelate chromatography and / or by epitopes, to which antibodies are available, such as, for example, example, myc tag, to allow detection in western blots, immunoprecipitation, or activity depletion / block bioassays.
[0318] [0318] Another exemplary leucine zipper domain for use as a multimerization domain is derived from a nuclear protein that functions as an activator for the transcription of a family of genes involved in general nitrogen control metabolism (GCN4) in S. cerevisiae.
[0319] [0319] Example of another type of multimerization domain, for use in modifying a HABP provided for use in the methods described here, is one in which multimerization is facilitated by protein-protein interactions between the different subunit polypeptides. Examples of such a multimerization domain are derived from the CAMP-dependent protein kinase mechanism (PKA), with its kinase A anchoring protein (AKAP) anchoring domain (AKAP). Thus, a heteromultimeric HABP polypeptide can be generated by linking (directly or indirectly) a nucleic acid that encodes a HABP polypeptide, such as an HA-binding domain of a HABP polypeptide, with a nucleic acid that encodes a sequence of subunits PKA R (i.e., SEQ ID NO: 367). This results in a homodimeric molecule, due to the spontaneous formation of a dimer by the R subunit. In parallel, another HABP fusion polypeptide can be generated by binding a nucleic acid that encodes another HABP polypeptide to a nucleic acid sequence that encodes an AKAP AD sequence (i.e., SEQ ID NO: 368). After co-expressing the two components, such as the color transfection of the chimeric HABP components in host cells, the dimeric R subunit then provides a coupling site for binding to the AD sequence, which results in a heteromultimeric molecule. This binding event can be further stabilized by covalent bonds, such as, for example, disulfide bridges. In some examples, a flexible binding residue can be fused between the nucleic acid encoding the HABP polypeptide and the multimerization domain. In another example, the fusion of a nucleic acid, which encodes a polypeptide
[0320] [0320] Other domains of multimerization that can be used for multimerization of a supplied HABP, for use in the methods presented herein, are known to those skilled in the art, and are those that facilitate the protein-protein interaction of two or more polypeptides, which are generated and expressed separately as HABP mergers. Examples of other multimerization domains that can be used to provide protein-protein interactions between two chimeric polypeptides include, but are not limited to, the barnase-barstar module (see, for example, Deyev et al., (2003) Nat. Biotechnol. 21: 1486-1492); use of certain protein domains (see, for example, Terskikh et al., (1997) Proc Natl Acad Sci USA 94: 1663-1668 and Muller et al., (1998) FEBS Lett. 422: 259-264); use of specific peptide motifs (see, for example, Kruif et al., (1996) JJ. Biol. Chem. 271: 7630-7634 and Muller et al., (1998) FEBS Lett. 432: 45-49); and the use of disulfide bridges for increased stability (by Kruif et al., (1996) JU. Biol. Chem. 271: 7630-7634 and Schmiedl et al., (2000) Protein Eng. 13: 725-734) .
[0321] [0321] In another example, HABPs are provided for use in the present methods, which are modified, for example, by substitution of amino acids, to expose a greater specificity for hyaluronan compared to other GAGs. For example, a mutant TSG-6-LM containing the substitution of amino acid (s) is provided here at amino acid residue 20, 34, 41, 54, 56, 72 and / or 84, and in particular at the amino acid residue 20, 34, 41, and / or 54 (corresponding to the amino acid residues set forth in SEQ ID NO: 360). The amino acid substitution can be that of any other amino acid residue, and is usually a non-basic amino acid residue. For example, amino acid substitution can be for Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (0), Ala (A), Val (V), Ile (1), Leu (L), Met (M), Phe (F), Tyr (Y) or Trp (W). The substitution or substitutions of amino acids confer decreased binding to heparin. The bond can be reduced at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times 100 times or more, compared to TSG-6-LM binding to heparin that does not contain amino acid substitution. Examples of a TSG-6-LM mutant for use as a reagent in the process provided herein is K20A / K34A / K41A. Thus, for example, heparin binding is reduced in such a way that the specificity for hyaluronan is increased. The TSG-6-LM mutant can be directly or indirectly conjugated to a multimerization domain to generate multimers. For example, an example of a reagent for use in the methods described herein is TSG-6-LM (K20A / K34A / K41A) -Fc.
[0322] [0322] For use in the diagnostic methods provided herein, HA-binding proteins can be modified to contain a detectable protein or a portion to facilitate detection.
[0323] [0323] HA-binding proteins for use in the diagnostic methods provided herein can be modified by, conjugation to detectable moieties including, but not limited to, peptides, radioactive labels, fluorescent molecules, chemiluminescent molecules, bioluminescent molecules, Fc domains, biotin, enzymes that catalyze a detectable reaction or catalyze the formation of a detectable product, and proteins that bind to a detectable compound. Detectable moieties, including proteins and compounds, or moieties that facilitate detection are known to a person skilled in the art. Detectable portions can be used to facilitate the detection and / or purification of HABP.
[0324] [0324] In one example, the HA-binding protein is modified by conjugation to a detectable protein or a protein that induces a detectable signal. The detectable protein or protein that induces a detectable signal can be selected from a luciferase, a fluorescent protein, a bioluminescent protein, a receptor protein or vehicle that binds to, and / or carries a contrast agent, chromophore, compound or linker that can be detected. For example, the detectable protein or protein that induces a detectable signal is a green fluorescent protein (GFP) or a red fluorescent protein (RFP).
[0325] [0325] The detectable markers can be used in any of the diagnostic methods provided here.
[0326] [0326] Examples of such proteins are enzymes that can catalyze a detectable reaction or catalyze the formation of a detectable product, such as, for example, luciferases, such as a beetle luciferase, a Renilla luciferase, a firefly luciferase or beta-glucuronidase (GusA). Examples of such proteins are also proteins that emit a detectable signal, including fluorescent proteins, such as green fluorescent protein (GFP) or a red fluorescent protein (RFP). A variety of DNA sequences that encode proteins that emit a detectable signal or that can catalyze a detectable reaction, such as fluorescent or luminescent proteins, are known and can be used in the methods provided herein. Examples of genes coding for light-emitting proteins include, for example, the Vibrio harveyi bacterial luciferase genes (Belas et al., (1982) Science 218: 791-793), Vibrio fischerii bacterial luciferase (Foran and Brown, (1988) Nucleic acids Res. 16: 177), firefly luciferase (de Wet et al., (1987) Mol. Biol. 7: 725-737), Aequorea victoria aequorin (Prasher et al., (1987) Biochem 26: 1326-1332), Renilla luciferase from Renilla renformis (Lorenz et al, (1991) Proc Natl Acad Sci USA 88: 4438-4442) and green fluorescent protein from Aequorea victoria (Prasher et al., (1987) Gene 111 : 229-233). The luxA and luxB genes for bacterial luciferase can be combined to produce the fusion gene (Fab2), which can be expressed to produce a fully functional luciferase protein (Escher et al., (1989) PNAS 86: 6528-6532).
[0327] [0327] Exemplary detectable proteins that can be conjugated to HA-binding proteins, for use in the diagnostic methods provided herein, also include proteins that can bind a contrast agent, chromophore, or a compound or linker, which can be detected , such as a transferrin receptor or a ferritin; and reporter proteins, such as E. coli B-galactosidase, B-glucuronidase, xanthine-guanine phosphoribosyltransferase (gpt), and alkaline phosphatase. Also examples of detectable proteins, proteins that can specifically bind a detectable compound, including, but not limited to, receptors, metal-binding proteins (e.g., siderophages, ferritins, transferrin receptors), ligand-binding proteins, and antibodies .
[0328] [0328] HABP can also be conjugated to a protein or peptide tag. In one example, the HA-binding protein is conjugated to an Fc domain.
[0329] [0329] the detectable markers can be coupled or conjugated to a HABP by recombinant methods or by chemical methods. For example, conjugation can be carried out by binding the protein, directly or indirectly, to a linker such as a peptide linker or a chemical linker. The linkers can be polypeptide sequences, such as polyglycine sequences between about 5 and 200 amino acids. Proline residues can be incorporated into a linker polypeptide to prevent the formation of significant secondary structural elements, i.e. helix a / sheet B, by the linker. An example of a flexible linker is a polypeptide that includes a glycine chain with a proline intermediate. In other examples, a chemical linker is used to synthetically link or recombinantly produce subsequences of the linkage and labeling domain. Such flexible connecting elements are known to persons skilled in the art. For example, poly (ethylene glycol) binders are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide bonds, sulfhydryl bonds, or heterofunctional bonds.
[0330] [0330] An HA-binding protein suitable for use as a diagnostic agent can be selected based on one or more desired properties or activities, including, but not limited to, specificity or affinity for HA, solubility, peptide stability, homogeneity, ease of expression and purification, minimum batch of variations for batch variations in the expressed peptide, and low sample variability in binding to HA and detection. In some instances, a single polypeptide diagnostic agent is included in more than one diagnosis with multiple polypeptide components. For example, a binding module that binds to HA in the absence of a complete binding protein. The ability of a HABP provided herein to bind to hyaluronan can be assessed by methods well known in the art, including, but not limited to ELISA-based assays, competitive binding assays with HA, heparin and other glycosaminoglycans, such as chondroitin sulfates A or C, heparan sulfates or dermatan sulfates. Examples of assays to assess HA-binding activity are provided here in section D, and in the Examples. D. TESTS AND CLASSIFICATION
[0331] [0331] The methods provided herein are based on assaying the expression or levels of hyaluronan (HA) in a sample or samples, such as a tissue sample or body fluid sample. The methods here are based on binding methods using an accompanying diagnosis of hyaluronan-binding protein (HABP, such as TSG-6-LM, multimer or variant) for the evaluation, determination, quantification and / or otherwise , specifically, detection of hyaluronan expression or levels in a sample. The assays can be carried out in vitro or in vivo. By comparison with a control or reference sample, or classifications based on a predetermined level, these values can be used for the diagnosis or prognosis of a disease or condition associated with hyaluronan, to predict the responsiveness of an individual who has a disease or condition associated with hyaluronan for an enzyme therapy that degrades hyaluronan, and / or to control or predict the effectiveness of treatment for an individual who has a disease or condition associated with hyaluronan, who has been treated with an enzyme therapy that degrades hyaluronan. For example, as described herein, HA levels and measurement have been found to be specifically associated with responsiveness to treatment with an enzyme that degrades hyaluronan, such as a hyaluronidase or modified hyaluronidase (e.g., PEGylated hyaluronidase as PEGPH20) .
[0332] [0332] In any of the above examples, the diseases or conditions associated with hyaluronan are those diseases and conditions where the levels of hyaluronan are elevated as a cause or effect, or otherwise, seen in the disease or condition. Exemplary diseases or conditions associated with hyaluronan include, but are not limited to, those associated with elevated interstitial fluid pressure, cancer and, in particular, cancer rich in hyaluronan, edema, pressure on the disc, inflammatory disease, and others diseases associated with hyaluronan. In some cases, diseases and conditions associated with hyaluronan are associated with increased pressure in the interstitial fluid, decreased vascular volume and / or increased water content in the tissue, such as a tumor. In particular, diseases and conditions associated with hyaluronan, include, but are not limited to, types of cancer rich in hyaluronan, for example, tumors, including solid tumors such as end-stage cancers, metastatic cancers, undifferentiated cancers, cancer of the ovary, carcinoma in situ (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers.
[0333] [0333] In one example, based on hyaluronan levels or expression, a patient or individual may be selected for treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan). For example, a sample from an individual can be contacted with a follow-up diagnosis of hyaluronan-binding protein (HABP), such as TSG-6-LM, a multimer or a variant thereof, and the HABP binding to the sample can be detected to determine the amount of hyaluronan in the sample. Based on the predetermined selection or classification criteria, as described herein, a patient can be diagnosed with a disease or condition associated with hyaluronan, and therefore selected for the treatment of the disease or condition. In addition, based on the predetermined selection or classification criteria, khaki described, the present methods can be used for the individual's prognosis. Depending on the course of the disease or condition, the dose, treatment schedule and / or dosing regimen of the therapeutic agent (for example, an enzyme that degrades hyaluronan) can be optimized and adjusted accordingly. In particular examples described herein, based on the predetermined selection or classification criteria, as described herein, a patient or individual may be selected for treatment, which is expected to be responsive to treatment with an anti-hyaluronan agent, for example , an enzyme that degrades hyaluronan, such as a modified hyaluronidase or hyaluronidase (for example, a PEGylated hyaluronidase such as PEGPH20). Thus, the method can be used to predict the effectiveness of treatment by an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan.
[0334] [0334] In the examples of methods described herein, the effectiveness of treatment by an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, can be determined by monitoring the expression or levels of hyaluronan, over the course of treatment. Thus, the method is a method of post-treatment of the condition and / or resolution of the monitored disease, the information of which can be used to alter the course of treatment of an individual who depends on the individualized state information. For example, a sample from an individual can be contacted with a follow-up diagnosis of hyaluronan-binding protein (HABP), for example, TSG-6-LM, a multimer or a variant thereof, and the binding of HABP to the sample can be detected in order to determine the amount of hyaluronan in the sample. The expression or level of hyaluronan in the sample can be compared with a control or reference sample in order to assess differences in levels or expression of hyaluronan. For example, elevated or accumulated levels of hyaluronan in a sick individual, compared to a healthy or normal individual, are indicative of a disease or condition associated with hyaluronan (eg, tumor or cancer) and the extent of expression or levels of hyaluronan correlates with the aggressiveness of the disease. In such methods, the control or reference sample is a sample from a healthy individual, it is a baseline sample from the individual, prior to treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan)
[0335] [0335] It is within the skill of a person skilled in the art to assess, quantify, determine and / or detect the levels of hyaluronan in a sample using a follow-up diagnosis of HABP, such as TSG-6-LM, multimer (eg TSG-6LM-Fc) or a variant thereof, as described herein. The assays include in vitro or in vivo assays. Examples of binding assays that can be used to assess, determine, quantify and / or otherwise detect specifically the expression of hyaluronan or levels in a sample include, but are not limited to, solid phase binding assays (eg, assay immunoenzymatic (ELISA)), radioimmunoassay (RIA), immunoradiometric assay, fluorescence assay, chemiluminescent assay, bioluminescent assay, western blot and histochemical methods such as immunohistochemistry (IHC) or pseudo immunohistochemistry, using a non-binding agent -antibody. Solid-phase binding assay methods, such as ELISA methods, for example, the assay can be in sandwich format or competitive inhibition format. In other examples, in vivo imaging methods can be used.
[0336] [0336] The methods for assessing the accumulation of hyaluronan are based on the ability of a follow-up diagnosis HABP to bind to HA, in a sample, for example, a tissue or cell sample, such that the amount of the accompanying diagnosis of HABP that binds, correlates with the amount of HA in the sample. Any accompanying HABP diagnostics provided herein can be used to detect AH, using tissue staining methods known to a person skilled in the art, including, but not limited to cytochemical or histochemical methods, such as immunohistochemistry (IHC) or histochemistry, using a non-antibody agent (for example, pseudo immunohistochemistry). Such histochemical methods allow the quantitative or semi-quantitative detection of the amount of HABP that binds to HA in a sample, such as a sample of tumor tissue. In such methods, a tissue sample can be contacted with a HABP reagent provided herein, and in particular one that is detectably labeled or capable of detection, under conditions that allow binding to the HA associated with the tissue or cell.
[0337] [0337] A sample for use in the methods provided here, as determined by histochemistry can be any biological sample, which can be analyzed for your HA levels, such as a tissue or cell sample. For example, a tissue sample can be a solid tissue, including a fresh, frozen and / or preserved organ, or tissue sample or biopsy or aspirate, or cells. In some instances, the tissue sample is tissue or cells obtained from a solid tumor, such as primary and metastatic tumors, including but not limited to, breast, colon, rectum, lung, stomach,
[0338] [0338] When the tumor is a solid tumor, the isolation of tumor cells is usually achieved by surgical biopsy. Biopsy techniques that can be used to harvest an individual's tumor cells include, but are not limited to, needle biopsy, cT-guided needle, aspiration biopsy, endoscopic biopsy, bronchoscopic biopsy, bronchial lavage, incisional biopsy, excisional biopsy , punch biopsy, scrape biopsy, skin biopsy, bone marrow biopsy, and Loop Eletrosurgical Excision Procedure (LEEP) biopsy. Typically, a sterile, non-necrotic biopsy sample is obtained with more than 100 mg, but which can be less, such as less than 100 mg, 50 mg or less, 10 mg or less, or 5 mg or less; or more, such as more than 100 mg, 200 mg or more, or 500 mg or more, 1 g or more, 2 g or more, 3 g or more, 4 g or more, or 5 g or more. The size of the sample to be extracted for dosing may depend on a number of factors including, but not limited to, the number of tests to be performed, the health of the tissue sample, the type of cancer, and the condition of the individual. The tumor tissue is placed in a sterile container, such as a sterile tube or culture plate, and can optionally be immersed in an appropriate medium.
[0339] [0339] The tissue obtained from the patient after the biopsy is often fixed, usually by formalin (formaldehyde) or glutaraldehyde, for example, or by immersion in alcohol. For histochemical methods, the tumor sample can be processed using known techniques, such as dehydration and incorporation of the tumor tissue into a paraffin wax or other solid supports known to those skilled in the art (see Plenat et al., (2001) Ann Pathol January 21 (1): 29-47), which cuts the tissue into sections suitable for staining, and processing the sections for staining, according to the selected histochemical staining method, including removal of supports solids for the incorporation of organic solvents, for example, and rehydration of preserved tissue. Thus, samples for use in the methods described herein may contain compounds that are not naturally present in a tissue or cell sample, including, for example, preservatives, anticoagulants, buffers, fixatives, nutrients and antibiotics.
[0340] [0340] In exemplary methods for selecting an object for treatment with an enzyme that degrades hyaluronan, the harvesting of tumor tissue is usually performed before treatment of the individual with an enzyme that degrades hyaluronan. In exemplary therapy methods of monitoring a tumor with an enzyme that degrades hyaluronan, harvesting the tumor tissue from the individual can be performed before, during or after that individual has received one or more treatments with a degrading enzyme hyaluronan.
[0341] [0341] The assays for use in the methods provided here are those in which the HA present in the sample is detected through histochemistry or immunohistochemistry. Histochemistry (HC) is a staining method, based on enzymatic reactions using a binding partner, such as an antibody (for example, monoclonal or polyclonal antibodies) or another binding partner, to detect specific cells or proteins, such as tissue antigens, or biomarkers, for example, HA. For example, histochemistry assays, for use in the methods described herein, include those in which a HABP is used as a binding partner to detect HA associated with cells or tissues. Typically, histochemistry protocols include detection systems that form the presence of visible markers, either for the human eye or an automated scanning system, for qualitative or quantitative analyzes. In a direct HC assay, binding is determined directly after binding of the binding partner (eg, first antibody) to the tissue or biomarkers, due to the use of a labeled reagent. In an indirect HC assay, a secondary antibody or second binding partner is needed to detect the binding of the first binding partner, since it is not labeled.
[0342] [0342] In such methods, in general, a slide tissue sample is labeled with a labeled binding reagent (e.g. labeled HABP) using common histochemical techniques. Thus, in the exemplary HC methods provided here, the accompanying HABP diagnostics are modified to contain a detectable group (as described in section 3C above). In some examples, the accompanying diagnoses of HABP are conjugated to small molecules, for example, biotin, which are detected through a labeled binding partner, or antibody. In some examples, the IHC method is based on staining with a HABP protein that is detected by enzymatic staining with horseradish peroxidase. For example, HABP can be biotinylated and detected with avidin or straptavidin conjugated to the detectable protein, such as horseradish peroxidase (see Example 6 below). In other examples, HABP tracking diagnostics are conjugated to detectable proteins that allow direct detection, such as, for example, HABP tracking diagnostics conjugated to a fluorescent protein, protein or bioluminescent enzyme. Various enzymatic staining methods are known in the art for detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens, such as DAB, AEC or Fast Red. In other examples, the accompanying HABP diagnostics are conjugated with peptides or proteins that can be detected by a labeled binding partner or antibody.
[0343] [0343] IN other examples, HA is detected by HC methods using a HABP follow-up diagnosis provided here, in which HABP follow-up diagnostics are detected by labeled secondary reagents, such as labeled antibodies that recognize one or more HABP epitopes, domains binding HABP, or HA binding fragments. In other examples,
[0344] [0344] The resulting marked specimens are each photographed using a system for viewing the detectable signal and acquiring an image, such as a digital color image. Methods for acquiring images are well known to a person skilled in the art. For example, once the sample has been colored, any optical or non-optical imaging device can be used to detect the stain or mark of biomarkers, such as vertical or inverted optical microscopes, confocal scanning microscopy, cameras, microscope scanning or tunneling, probe microscopes and infrared image detectors. In some instances, the image may be captured digitally. The images obtained can then be used to determine quantitatively or semi-quantitatively, the amount of HA in the sample. Various systems for processing, digitizing and automatic sample analysis, suitable for use with immunohistochemistry, are available in the art. Such systems “can include automatic staining and microscopic scanning, computerized image analysis, serial section comparison (to control variation in sample orientation and size), digital report generation, and sample archiving and tracking (such as slides, which tissue cuts are placed). Cellular imaging systems are commercially available, in which conventional light microscopes are combined with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostaining samples. See, for example, the CAS-200 system (Becton, Dickinson & Co.). In particular, the detection can be done manually or by means of image processing techniques that involve computer processors and software. When using software, for example, images can be configured, calibrated, standardized and / or validated based on factors including, for example, the quality of the color or the intensity of the color, using procedures known to a person skilled in the art (see for example, U.S. patent published No. US20100136549).
[0345] [0345] The image can be quantitatively or semi-quantitatively analyzed and classified based on the color intensity of the sample. Quantitative or semi-quantitative histochemistry, refers to the method of scanning and scoring samples that have been subjected to histochemistry, to identify and quantify the presence of a specified biomarker, such as an antigen or other proteins (for example, HA). Quantitative or semi-quantitative methods can employ imaging software to detect color density or amount of color, or methods of detecting color by the human eye, in which a trained operator sorts the results numerically. For example, images can be analyzed quantitatively using a pixel counting algorithm (for example Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUAO platform), and other standard methods that measure or quantify or semi-quantify the degree of coloring; see , for example U.S. Patent No. 8,023,714; U.S. Patent No. 7,257,268; U.S. Patent No.
[0346] [0346] Using histochemistry, such as immunohistochemistry or pseudo histochemistry methods, the amount of HA detected is quantified and given as a positive HA score and / or pixels. For example, the amount of HA detected in the sample can be quantified as a percentage of positive HA pixels. In some examples, the amount of HA present in a sample is quantified as the percentage of the colored area, for example, the percentage of HA positive pixels. For example, a sample can have at least, or about, at least, or about O, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of positive HA pixels compared to the total staining area.
[0347] [0347] In some examples, the sample is given a score which is a numerical representation of the intensity or amount of the sample's histochemical staining, and represents the amount of target biomarker (for example, HA) present in the sample. The values of optical density or percentage area can be assigned a score on a scale, for example, on an integer scale, for example, 0-10, 0-5, or 0-3. In particular examples, the amount of hyaluronan in which a sample is rated on a scale of 0-3, for example, 0, HA ", HA", and HA ". The amount of HA present is relative to the percentage of HA pixels , that is, low percentages of HA pixels indicate a low level of HA, while high percentages of HA pixels indicate high levels of HA. Scores can be correlated with percentages of positive HA pixels, such that the percentage of The painted surface is scored as 0, HA, HA ”, and HA * , representing no coloring, less than 10% coloring, 10-25% coloring or more than 25% coloring, respectively. the ratio (for example, strong pixel staining to total colored area) is greater than 25%, the tumor tissue is marked, as HA + 3, if the proportion is 10-25% strong positive staining to total staining, tumor tissue is scored as HA + 2, if the proportion is less than 10% of strong positive staining for staining total, the tumor tissue is scored, as HA + l1, and if the ratio of strong positive staining to total staining is O, the tumor tissue is scored as 0. A score of O or HA + l indicates low levels of HA in the sample analyzed, while an HA score of +2 or +3 indicates high levels of HA in the tested samples.
[0348] [0348] Methods for assessing hyaluronan accumulation are based on the ability of a HABP follow-up diagnosis to bind to HA in a sample, such that the amount of the HABP follow-up diagnosis that binds to HA , is correlated with the amount of HA in the sample. In particular, solid phase bonding tests can be used. Examples of binding assays that can be used to assess, determine, quantify and / or otherwise specifically, detect expression, or levels of hyaluronan in a sample include, but are not limited to, the immunoassay (ELISA), radioimmunoassay (RIA ), immunoradiometric assay, fluorescence assay, chemiluminescent assay, bioluminescent assay. For example, a follow-up diagnosis of HABP provided herein can detect HA using any binding assay known to a person skilled in the art, including but not limited to the immunoassay assay (ELISA), or any other similar immunoassay, including a sandwich ELISA or competitive. Examples of methods provided herein include ELISA-based methods for the quantitative or semi-quantitative detection of the amount of HABP that binds HA to a sample, such as a tumor tissue sample or fluid sample, from an individual who has a tumor or is suspected of having a tumor. Solid phase binding assays can be used when HA is detected in a body fluid.
[0349] [0349] As described here, patients who exhibit high levels of hyaluronan production in tumor tissue also have high levels of hyaluronan in the blood. Thus, the methods provided herein encompass methods of predicting the individual's responsiveness to treatment with an enzyme that degrades hyaluronan, the choice for treating individuals with an enzyme that degrades hyaluronan, or to monitor treatment with an enzyme that degrades hyaluronan, include the assessment of the accumulation of hyaluronan in a fluid sample, from a patient with a tumor, or from a patient suspected of having a tumor.
[0350] [0350] Fluid samples for analysis of HA production in a disease associated with HA, such as cancer, include but are not limited to serum, urine, plasma, cerebrospinal fluid, and lymph. The individual may have or be suspected of having cancer, such as primary and metastatic tumors, in the breast, colon, rectum, lung, stomach, ovary, cervix, uterus, testicles, bladder, prostate, thyroid, lung cancer. In particular examples, cancer is advanced cancer, metastatic cancer, undifferentiated cancer, ovarian cancer, carcinoma in situ (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-cell lung cancer -small, breast cancer, colon cancer.
[0351] [0351] In exemplary methods to predict an individual's responsiveness to treatment with an enzyme that degrades hyaluronan, or to select individuals being treated with an enzyme that degrades hyaluronan, collecting a fluid sample from an individual is generally performed before treatment of the individual with an enzyme that degrades hyaluronan. In exemplary therapy methods of monitoring a tumor with an enzyme that degrades hyaluronan, the collection of the fluid sample from an individual can be performed before, during or after that individual has received one or more treatments with a degrading enzyme hyaluronan. The collection of the fluid sample can also be performed before, during, or after the respective individual has undergone one or more cycles of anti-cancer therapy, such as radiation and / or chemotherapy.
[0352] [0352] The fluid sample can then be assessed for the presence or amount of HA, using a solid phase binding assay. Solid phase binding assays can detect a substrate (for example, HA) in a fluid sample by binding the substrate to a binding agent that is attached or immobilized on a solid surface. A substrate-specific antibody or binding protein (for example, a HABP provided herein), coupled to the detectable marker (for example, an enzyme), is applied and left to bind to the substrate. The presence of the antibody or binding protein is then detected and quantified. Detection and quantification methods include, but are not limited to, colorimetric, fluorescent, luminescent or radioactive methods. The choice of the detection method depends on the detectable marker used. In some examples, a colorimetric reaction using the enzyme bound to the antibody. For example, enzymes commonly used in this method include horseradish peroxidase and alkaline phosphatase. The amount of substrate present in the sample is proportional to the amount of color produced. A substrate pattern is generally used to improve quantitative accuracy. The concentration of HA in a sample can be calculated by interpolating the data for the standard curve. The amount of HA can be expressed as a sample concentration of fluid.
[0353] [0353] In an exemplary method, a HABP reagent that is generally unmarked is first immobilized on a solid support (for example, coated in the wells of a microtiter plate), followed by incubation with a fluid sample containing HA (for example , serum or plasma) to capture HA. After washing the fluid sample with an appropriate buffer, binding to HA can be detected. In some examples to detect binding to HA, a second HABP which is the same or different from immobilized HABP, and which is labeled (labeled HABP), such as a biotinylated HABP, is used to bind to HA on the plate. After removal of the unbound labeled HABP, the labeled bound HABP is detected using a detection reagent. For example, biotin can be detected using an avidin detection reagent. In some examples, the HABP attached to the board is different from the HABP used for detection. In other examples, the HABP attached to the board and the HABP for detection are the same. In other examples, to detect binding to HA, it is detected by adding HABP and subsequently adding an anti-HABP antibody. For example, for the detection of TSG-6 or TSG-6-LM, a monoclonal antibody from the anti-TSG-6 binding module can be used, such as the antibodies designated A38 and Q75 (see, Lesley et al. (2002 ) J Biol Chem 277: 26600-26608). Anti-HABP antibodies can be labeled for detection Or they can be detected with a secondary antibody that binds to the first antibody. In still other examples to detect binding to HA, said is directly detected with an anti-HA antibody. Anti-HA antibodies are well known to a person skilled in the art, and include, for example, a polyclonal sheep anti-hyaluronan antibody (for example, Abcam t * 53842 and À 93321).
[0354] [0354] In some examples here, the amount of HA is detected by in vivo imaging methods. In such methods, HABP, such as a TSG-6-LM, a multimer or a variant thereof (for example, TSG-6LLM-Fc), is conjugated to a detectable portion or a fraction that is capable of detection by a Image. Exemplary imaging methods include, but are not limited to,
[0355] [0355] In particular, HABP, such as TSG-6-LM, multimer or a variant thereof (for example, TSG-6LLM-Fc), is marked or conjugated to a unit that provides a signal or induces a signal that is detectable in vivo when worked on, such as, but not limited to, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET), scintigraphy, gamma camera, a B + detector, a y detector, fluorescence image and bioluminescence image. illustrative monitoring / imaging methods include any of a variety of magnetic resonance methods, such as magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS), and also include any of a variety of tomographic methods including tomography computed tomography (CT), computed axial tomography (CAT), electron beam computed tomography (EBCT), high resolution computed tomography (HRCT), hypocycloidal tomography, positron emission tomography (PET), gamma rays (after the annihilation of a positron and a PETscan electron), single photon emission computed tomography (SPECT), spiral computed tomography and ultrasonic tomography. Other exemplary imaging methods include low-light images, X-rays, ultrasound signal, fluorescence absorption and bioluminescence. In addition, proteins can be labeled with light emitters or other electromagnetic spectrum emitters, such as fluorescent compounds or molecules. Detection can be carried out by detecting emitted light or other electromagnetic radiation emitted.
[0356] [0356] detectable markers include reagents with directly detectable elements (for example radiolabeled) and reagents with indirectly detectable elements (for example, a reaction product). Section C.3.c also describes discoverable markers. Examples of detectable markers include radioisotopes, bioluminescent compounds, chemiluminescent compounds, fluorescent compounds, metal chelates and enzymes. A detectable marker can be incorporated into a HABP by recombinant or chemical methods.
[0357] [0357] Markers suitable for X-ray imaging are known in the art, and include, for example, bismuth (III), gold (III), lanthanum (III) or lead (II); a radioactive ion, such as 67Copper, 67Gallium, 68Gallium, l11INdium, 113Indium, 123Iodine, 125Iodine, 131Iodine, 197Mercury, 203Mercury, l86Rhenium, 188Rhenium, 97Rubidium, 103Rubidium, 99Tetetium or 90Yttrium; a nuclear magnetic resonance isotope, such as cobalt (IT), copper (II), chromium (III), Dysprosium (III), Erbium (III), Gadolinium (III), Holmium (III), iron (II ), Iron (III),
[0358] [0358] Contrast agents are used for magnetic resonance imaging. Examples of contrast agents include iron, gold, gadolinium and gallium. Markers "suitable for magnetic resonance imaging are known in the art, and include, for example, fluorine, gadolinium chelates, metals and metal oxides, such as, for example, iron, gallium, gold, gadolinium, magnesium, compounds marked “H,“ F, ºC, and “N. The use of chelates in contrast agents is known in the art. Markers suitable for tomography imaging methods are known in the art, and include, for example, Bremissores, such as "o, * N," Vo or "" Cu or (b) yY-emitters, such as "ºI. Other radionuclides examples that can be used, for example, as markers for PET include “co,“ Ga, “Ga,“ Cu (II), Sou (II), Tc, Ni, Fe and ** F. The reagent, such as TSG -6 Or its FC portion may be conjugated to a marker and / or the appropriate protein may include a radioactive marker in its constituent molecules.
[0359] [0359] A list with examples of radionuclides useful for the imaging methods provided here includes, for example, carbon, “Fluorine,“ Pearbono, UPNitrogen, "Nitrogen,“ Poxygen, “Fluorine,“ Fluorine, “sodium, * Phosphate, “Potassium, chromium, Iron,“ Iron, Cobalt, “Cobalt, copper,“ Gallium, “ºGallium," Selenium, “krypton, CRubidium, PEStronium,” PEstrontium, Pytrium, * Technetium, 1st Palladium, ÓRuthenium, “Indian,“ Lutetium , “Iodine,“ Iodine, “Iodine," PxXxenon, Vesium, 1 Ssamário, * PGadolínio, * Dysprosium,! 6HÓI1mio, ““ Itérbio, “Leutium, ** Rhenium, * ºRenio,“ * Iridium,
[0360] [0360] Fluorescent markers can also be used. These include fluorescent proteins, fluorescent probes or fluorescent substrate. For example, fluorescent proteins can include, but are not limited to, fluorescent proteins, such as green fluorescent protein (GFP), or their counterparts or RFP; fluorescent dyes (for example, fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Greene O, rhodamine and its derivatives (for example, Texas red and tetramethyl rhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, Alexa FluorO, Li-CORO, CyDyesO or DyLightoe Fluors); tdTomato, mCherry, mPlum, Neptune, TagRFP, mKate2, TurboRFP and TurboFP635 (Katushka). The fluorescent reagent can be chosen based on the user's desired excitation and emission spectra. Fluorescent substrates can also be used, and result in fluorescent cleavage products.
[0361] [0361] In vivo imaging methods can be used for the diagnosis of tumors or cancers associated with HA. This technique allows the diagnosis, without the use of biopsy. In vivo imaging methods based on the extent or degree of attachment of a HABP to a tumor, can also be used for the prognosis of cancer patients. In vivo imaging methods can also be used to detect metastatic cancers in other parts of the body or circulating tumor cells (CTC). It is within the level of a person skilled in the art to determine the basic levels of hyaluronan in other tissues, in addition to tumors.
[0362] [0362] Once the amount of hyaluonan in the sample is determined, the value can be compared with a threshold or control level. The control or threshold level is usually a predetermined threshold level, or quantity that is indicative of a disease or condition associated with hyaluronan (for example, a tumor or cancer). Such a level or quantity can be determined empirically by one skilled in the art. It is understood that the predetermined specific selection or classification criteria, for the methods described here, are dependent on the particular assay that is used to detect hyaluronan and the particular sample to be tested. It is within the skill of a person skilled in the art to determine whether an assay is compatible with the test of a particular sample. Generally, in vitro solid phase assays are used to test samples of body fluids. Solid-phase assays, such as histochemistry or immunohistochemistry are generally used to test tissue samples. It is also understood that, in methods that involve comparisons with a predetermined level or quantity, Either for a control or a reference sample, that references are made with the same type of sample and using the same assay and HABP reagent (including the same detectable portion and detection method).
[0363] [0363] For example, the predetermined threshold level can be determined based on the level or quantity of the marker in a sample or control reference, such as the average or median level, or quantity of the marker in a population of individuals, so to assess differences in levels or expression. In one example, the predetermined threshold level may represent the average or median level, or the amount of hyaluronan in a sample from a healthy individual, or from an individual known to have a disease or condition associated with hyaluronan (for example, a tumor or cancer). In one embodiment, the level or predetermined amount of hyaluronan in a normal tissue or body fluid sample is the average level or amount seen in normal samples (for example, all normal samples analyzed). In another embodiment, the level or amount of hyaluronan from a normal tissue or body fluid sample is the average value for the level or quantity seen in normal samples. The predetermined threshold level can also be based on the level or amount of hyaluronan in a cell line or other control sample (for example, tumor cell line). As described below, these predetermined values can be determined by comparing or knowing the levels of HA in a corresponding normal sample, as determined by the same detection assay and using the same HABP reagent.
[0364] [0364] The sample or control reference can be another tissue, cells or body fluid, such as a tissue, cell or body fluid, for example, a tissue, cell or body fluid that is analogous to the sample to be tested, but isolated from a different individual. The control or reference individual can be a normal individual or population of individuals (that is, who does not have a disease or condition), an individual who has a disease, but does not have the type of disease Or condition that the tested individual has or you are suspected of having, for example, an individual who does not have a disease or condition associated with hyaluronan (for example, a tumor or cancer), or an analogous tissue from another individual who has a similar disease or condition, but whose disease does not it is so severe and / or expressed relatively less hyaluronan. For example, when the cell, tissue or fluid to be tested is an individual or a population of individuals who have a type of cancer, the level or quantity of the marker can be compared to the level or quantity of the marker in a tissue, cells or fluid of an individual who has less severe cancer, such as early, differentiated or other cancer. In another example, a reference sample or control is a fluid, tissue, extract (eg, cell or nuclear extract), nucleic acid or peptide preparation, cell line, biopsy, standard sample or other, with a known quantity or quantity relative to hyaluronan, such as a sample, for example, a tumor cell line, known to express relatively low levels of HA, such as exemplary tumor cell lines that express low levels of HA, for example, HCT 116 cell line , the HT29 cell line, NCI H460 cell line, DUl45 cell line, Capan-l cell line, and tumors from tumor models generated using such cell lines.
[0365] [0365] In any method described here, the level (or levels) of hyaluronan in samples from individuals known or suspected of suffering from a disease or condition associated with hyaluronan (eg cancer) can be determined at the same time as determining the level (or levels) of hyaluronan in reference or normal tissues. Alternatively, the levels of hyaluronan in samples from individuals known or suspected of suffering from a disease or condition associated with hyaluronan (for example, cancer can be compared with the level (or levels) of hyaluronan previously determined in normal tissue or body fluid. , the level of hyaluronan in normal or healthy samples or other reference samples used in any detection, comparison, determination, or evaluation may be a level or quantity determined before any detection, determination or evaluation of the level or quantity of hyaluronan, in a sample from a human patient.
[0366] [0366] The level or amount of hyaluronan is determined and / or scored, and compared with the predetermined phenotypes of hyaluronan associated with the disease. It is within the level of a person skilled in the art to determine the threshold level for the diagnosis of diseases, depending on the particular disease, the assay to be used for the detection of the hyaluronan and / or the HABP detection reagent being used. It is within the skill of a person skilled in the art to determine the threshold level of hyaluronan to classify the response to treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan). Examples of methods for stratifying tumor samples or samples of body fluids for diagnosis, prognosis or selection of individuals for treatment are provided here.
[0367] [0367] It is understood that the particular change, for example, increase or decrease in hyaluronan is dependent on the assay used. In an ELISA assay, an increase or decrease in absorbance at a particular wavelength or the amount of protein (for example, as determined by means of a standard curve) can be expressed relative to a control. In a PCR assay, such as RT-PCR, it can be compared to the control of expression levels (for example, expressed as change of times) using methods known to those skilled in the art, such as the use of standards.
[0368] [0368] In the particular examples of the methods described herein, an individual is selected as a candidate for therapy with an anti-hyaluronan agent, if the amount of hyaluronan is determined to be high in the sample. For example, elevated or accumulated levels of hyaluronan in a sick individual, compared to a healthy or normal individual, are indicative of a disease or condition associated with hyaluronan (for example, tumor or cancer). Hyaluoronan can be raised to 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more. Thus, in examples of the methods described here, when the amount of hyaluronan in a sample from an individual is to be tested, the detection of the marker can determine whether the amount of HA for the sample (for example, cancer cells, tissue or fluid) of the individual is high, compared to a predetermined level or quantity, or control sample. In one example, the individual is determined to have a disease or condition associated with hyalruonan, if the amount of HA in the tissue, cells or fluid is increased to, or about 0.5 times, |] times, 2 times, 3 times , 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times, or more, compared to the level or quantity predetermined, or value, or control sample.
[0369] [0369] An individual can be selected as a candidate for therapy with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) based on the level or amount of hyaluronan in a sample (for example, a body fluid or other fluid) of the individual. HA greater than 0.010 pg / ml, 0.015 pg / ml, and generally greater than 0.02 pg / ml, 0.03 pg / ml, 0.04 pg / ml, 0.05 vg / ml, 0 , 06 pg / ml or higher, correlates with the presence of a tumor or cancer. Using such methods, in the exemplary methods provided herein, an individual can be selected for treatment with an anti-hyaluronan agent (e.g., enzyme that degrades hyaluronan), if the concentration of HA in the fluid sample, such as a serum sample , contains HA levels greater than 0.010 ug / ml, 0.015 pg / ml, and generally greater than 0.02 ug / ml, 0.03 pg / ml, 0.04 pg / ml, 0.05 pg / mL, 0.06 vg / mL or greater.
[0370] [0370] An individual can be selected as a candidate for therapy with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) based on the level or amount of hyaluronan in a cell or tissue sample. In one example, if the level is indicative of disease, then the patient is diagnosed with a disease or condition associated with hyaluronan. For example, using tumor tissue immunohistochemistry methods an HA score or HAº * can be a determinant of the disease. For example, a percentage of HA staining over the total tumor area greater than 10%, such as 10 to 25%, or greater than 25% is indicative of disease. In the methods described here, an individual is selected for treatment with an anti-hyaluronic agent (for example, an enzyme that degrades hyaluronan), if the sample's scale score is an HA sample or HA ”. For example, a higher score, for example, HA, indicates that the individual has a tumor rich in HA, indicative of the presence of a tumor that would benefit from treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluonan ) and thus is a candidate for therapy with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan). In other examples, an individual may be selected for treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) based on the percentage of staining, for example, if the degree of staining of HA is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of the total staining area, and generally at least 25% or more.
[0371] [0371] The effectiveness of treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), or responsiveness to treatment, can also be monitored by comparing an individual's level or amount of hyaluronan over time. Changes in the level or amount of hyaluronan can be used to optimize the dosage or schedule of treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan). In the method, the level of expression in HA samples, in particular, assessed in tumor tissues (for example, by means of immunohistochemistry or similar method), from treated individuals, is compared with a predetermined level of expression of THERE IS. For the purpose of monitoring treatment after administration of a hyaluronan-degrading enzyme, in particular, one with a prolonged half-life (for example, PEGPH20), the sample that is controlled is not a body fluid, where systemic levels of enzyme may be present.
[0372] [0372] For monitoring treatment purposes, the predetermined level of HA can be from a normal or healthy individual, a baseline HA value prior to treatment, the previously measured HA level, in the same individual in one previous time after treatment, or a classification or stratification of AH levels known to be associated with disease progression or regression.
[0373] [0373] In monitoring methods and methods of determining treatment effectiveness, particular therapy can be changed during the course of treatment to maximize individual response. The dosage and treatment schedule can be modified in response to changes in levels. Combination therapy with other anti-cancer agents can also be used in such treatment methods. It is within the skill of the attending physician to determine the exact course of treatment. For example, the treatment can be modified, such that the amount of dosage, schedule (for example, the frequency of administration), or oThe regimen is adjusted accordingly, such as discontinued, decreased or made less frequent, or in combination with a second treatment for the disease or condition. On the other hand, if the level of hyaluronan is above a comparative reference or control sample, it is likely that the patient will not respond to treatment. In such cases, the nature and particular type of anti-hyaluronan agent (e.g., the hyaluronan-degrading enzyme) or combination therapy may be modified or altered. In other cases, the dosage, quantity, schedule and / or regimen can be adjusted accordingly, such as increased or made more frequent. It is included in the treatment level, the doctor to determine the exact course of the respective treatment.
[0374] [0374] For the purpose of monitoring the effectiveness of treatment, the predetermined levels or amounts of hyaluronan can be determined empirically, where theO level or quantity indicates that the treatment is working. These predetermined values can be determined by comparing or knowing the levels of HA in a corresponding normal sample, or samples from patients with the disease as determined by the same detection assay and using the same HABP reagent. For example, high levels of HA, as assessed by immunohistochemistry methods using a quantitative scoring scheme (eg, HA ”) or the percentage of tumor staining for hyaluronan greater than 25% is correlated with existence of malignancy in a variety of cancers, and indicates that the patient is not responding to treatment. In another example, HA levels in body fluids such as plasma greater than 0.015 ug / ml, and generally greater than 0.02 ug / ml, such as 0.03 ug / ml, 0.04 pg / mL, 0.05 pg / ml or 0.06 pg / ml HA, are associated with advanced disease. On the other hand, is it likely that an individual is responding to treatment, if the sample mean scale is less than HA ” or HA ”, or if the percentage of HA staining is less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less. An individual is likely to respond to treatment if the level of HA in body fluids such as plasma is less than 0.03 µg / mL, 0.02 µg / mL, 0.01 µg / mL or less.
[0375] [0375] In the methods described herein, the comparison of a predetermined level or levels of a control or reference sample can be determined by any method known to those skilled in the art. For example, comparing a level of hyaluronan with a reference,
[0376] [0376] The methods provided herein include methods of treating a tumor-bearing individual with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, in which the individual was selected for treatment based on the level of HA detected in the tumor. Treatment methods include methods for assessing the effects of treatment with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, such as the effectiveness of a treatment, such as, for example, tumor inhibition or regression, or side effects of treatment, such as, for example, musculoskeletal side effects. Therapies combined with one or more additional anti-cancer agents, or an agent for the treatment of one or more side effects of therapy with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) are also provided.
[0377] [0377] Anti-hyaluronan agents include agents that inhibit hyaluronan synthesis or degrade hyaluronan. anti-hyaluronan agents, such as hyaluronan-degrading enzymes, can be used to treat a disease or condition associated with hyaluronan, including tumors and cancers, or inflammatory diseases or conditions. For example, the accumulation of HA, such as by altered hyaluronan metabolism, the distribution and function associated with arthritis, immune and inflammatory diseases, pulmonary and vascular diseases, and cancer (Morohashi et al. (2006) Biochem. Biophys. Comm., 345: 1454-1459). Such diseases can be treated by inhibiting the synthesis of HA or degraders of HA (see, for example Morohashi 2006; U.S. application published No. 20100003238 and PCT published International Application No. WO 2009/128917). In some instances, such treatments that decrease the levels of hyaluronan in cells and tissues may be associated with adverse side effects, such as musculoskeletal side effects. Thus, treatment with an anti-hyaluronan agent may further include treatment with a corticosteroid to improve or reduce these side effects.
[0378] [0378] HA can be synthesized by three enzymes, which are the products of three related mammalian genes, identified as HA synthases, designated as has-1, has-2 and has-3. Different cell types express different SAH enzymes, and the expression of SAH mRNAs is correlated with HA biosynthesis. The silencing of SAH genes in tumor cells is known to inhibit the growth of tumors and metastases. An anti-hyaluronan agent includes any agent that inhibits, reduces or down-regulates the expression or level of an HA synthase. Such agents are known to a person skilled in the art or can be identified.
[0379] [0379] For example, downregulation of a SAH can be achieved by providing the oligonucleotides that specifically hybridize, or otherwise, interact with one or more nucleic acid molecules that encode a SAH. For example, anti-hyaluronan agents that inhibit hyaluronan synthesis include sense or antisense molecules against a has gene.
[0380] [0380] Another example of an anti-hyaluronan agent that is an inhibitor of HA synthesis is 4-methylumbelliferone (4-MU; 7-hydroxy-4-methylcoumarin) or a derivative thereof. 4-MU works by reducing the precursor UDP-GlIcUA, which is necessary for the synthesis of HA. For example, in mammalian cells, HA is synthesized by SAH using UDP-glucuronic acid (UGA) and UDP-N-acetyl-D-glucosamine precursors. 4-MU interferes with the process by which UGA is generated, thus depleting the intracellular accumulation of UGA, and resulting in the inhibition of HA synthesis. 4-MU is known to have anti-tumor activity (see for example, Lokeshwar et al. (2010) Cancer Res., 70: 2613-23; Nakazawa et al. (2006) Cancer Chemother. Pharmacol., 57: 165- 170; Morohashi et al. (2006) Biochem. Biophys. Res. Comm., 345-1454-1459). Oral administration of 4-MU with 600 mg / kg / d reduces metastases by 64% in the Bl6 melanoma model (Yoshihara et al. (2005) FEBS Lett., 579: 2722-6). The 4-MU structure is shown below. In addition, 4-MU derivatives exhibit anti-cancer activity, in particular 6.7 dihydroxy coumarin-4-methyl-coumarin and 5,7-dihydroxy-4-methyl (see, for example 4-Methylumbelliferone (4 -MU; C10oHgO3) HO. OO
[0381] [0381] Other examples of anti-hyaluronan agents are tyrosine kinase inhibitors, such as leflunomide (Arava), or genistein or erbstatin. Leflunomide is also an inhibitor of pyrimidine synthesis. Leflunomide is a drug known to treat rheumatoid arthritis (RA), and is also effective in treating allograft rejection as well as xenografts. HA is known to contribute directly or indirectly to RA (see, for example, Stuhlmeier (2005) J Immunol., 174: 7376- 7382). Tyrosine kinase inhibitors inhibit expression of the hasl gene (Stuhlmeier 2005).
[0382] [0382] In one example, leflunomide or its derivatives, in general, are available in the form of tablets “containing 1-100 mg of active drug, for example, 1, 5, 10, 20, 30, 40, 50, 60 , 70, 80, 90 or 100 mg of drug. For the treatment of a disease and conditions associated with hyaluronan, for example, rheumatoid arthritis, or a tumor or cancer, it is administered at 10 to 500 mg per day, usually 100 mg per day. Dosing can be continued as needed for the treatment of the disease or conditions, or it can be decreased or reduced to successively lower doses. For example, for the treatment of rheumatoid arthritis, leflunomide can be administered at an initial loading dose of 100 mg daily for three days and then administered at a constant dose of 20 mg / day.
[0383] [0383] Hyaluronan is an essential component of the extracellular matrix and one of the main constituents of the interstitial barrier. By catalyzing the hydrolysis of hyaluronan, enzymes that degrade hyaluronan decrease the viscosity of hyaluronan, thereby increasing tissue permeability and the rate of absorption of fluids administered parenterally. As such, enzymes that degrade hyaluronan, such as hyaluronidases, have been used, for example, as dispersing or spreading agents, together with other agents, drugs and proteins to improve their dispersion and delivery.
[0384] [0384] Hyaluronan-degrading enzymes act to degrade it by cleaving hyaluronan polymers, which are composed of repeating units of disaccharides, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GleNAC), linked between itself through alternating glycosidic bonds B-l1 - 4 and B-1 - 3. Hyaluronan chains can reach about 25,000 repetitions of disaccharide or more, in length, and hyaluronan polymers can vary in size from about 5000 to 20,000 .000 Da, in vivo. Therefore, enzymes that degrade hyaluronan, for the uses and methods provided, include any enzyme that has the ability to catalyze the cleavage of a hyaluronan polymer or disaccharide chain. In some examples, the enzyme that degrades hyaluronic acid cleaves the glycosidic bond fB-1> 4 in the hyaluronan chain or polymer. In other examples, the enzyme that degrades hyaluronic acid catalyzes the cleavage of the glycosidic bond B-1 »3 of the hyaluronan chain or polymer.
[0385] [0385] Thus, enzymes that degrade hyaluronan, such as hyaluronidases, are a family of enzymes that degrade hyaluronic acid, which is an essential component of the extracellular matrix, and one of the main constituents of the interstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, one of the main constituents of the interstitial barrier, hyaluronan degrading enzymes decrease the viscosity of hyaluronic acid, thus increasing tissue permeability. As such, hyaluronan degrading enzymes, such as hyaluronidases, have been used, for example, as a spreading or dispersing agent in conjunction with other agents, drugs and proteins to improve their dispersion and delivery. Hyaluronan degrading enzymes are also used as an adjuvant to increase the absorption and dispersion of other injected drugs, by hypodermoclysis (subcutaneous fluid administration), and as an adjuvant in subcutaneous urography to improve the resorption of radiopaque agents. Hyaluronan-degrading enzymes, such as hyaluronidase, can be used in applications of ophthalmic procedures, for example, sub-Tenon block and peribulbar in local anesthesia before ophthalmic surgery. Hyaluronidase can also be used in other therapeutic and cosmetic uses, for example, by promoting akinesia in cosmetic surgery, such as blepharoplasty and facial lifting.
[0386] [0386] Various forms of hyaluronan-degrading enzymes, including hyaluronidases, have been prepared and approved for therapeutic use in individuals, including humans. The compositions and methods provided can be used, by means of these and other therapeutic uses, for the treatment of diseases and conditions associated with hyaluronan. For example, animal hyaluronidase preparations include Vitrase (ISTA Pharmaceuticals), a purified sheep testicular hyaluronidase, Amphadase (Amphastar "Pharmaceuticals), bovine testicular hyaluronidase and Hydase (Prima Pharm Inc.), a bovine testicular hyaluronidase. It is understood that any hyaluronidase preparation of animal origin can be used in the methods and uses provided herein (see, for example, US Patents. Nos. 2,488,564
[0387] [0387] Examples of enzymes that degrade hyaluronan in the compositions and methods provided here,
[0388] [0388] As described below, enzymes that degrade hyaluronan exist in membrane-bound or soluble forms that are secreted from cells. For the purposes of this invention, enzymes that degrade soluble hyaluronan are provided for use in the methods, uses, compositions or combinations described herein. Thus, when hyaluronan degrading enzymes include a glycosylphosphatidylinositol (GPI) anchor and / or are otherwise membrane-anchored or insoluble, such hyaluronan degrading enzymes are provided here in soluble form, by truncation or suppression of the GPI anchor, to process the secreted and soluble enzyme. Thus, enzymes that degrade hyaluronan include incomplete, for example, incomplete variants to remove all or a portion of a GPI anchor. Hyaluronan degradation enzymes provided herein also include alleles or species variants, or other variants, of an enzyme that degrades soluble hyaluronan. For example, hyaluronan-degrading enzymes may contain one or more variations in their primary sequence, such as amino acid substitutions, additions and / or deletions. A variant of a hyaluronan-degrading enzyme generally exhibits at least or about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity, relative to the hyaluronan degrading enzyme that does not contain the variation. Any variation may be included in the hyaluronan-degrading enzyme for the purposes described herein, provided that the enzyme retains hyaluronidase activity, such as at least or about 5%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of the activity of a degrading enzyme of hyaluronan that does not contain the variation (as measured in in vitro and / or in vivo assays well known in the art, and described herein).
[0389] [0389] Where the methods and uses provided herein describe the use of a soluble hyaluronidase, according to any degrading enzyme of hyaluronan, generally a degrading enzyme of soluble hyaluronan, can be used. It is understood that any hyaluronidase can be used in the methods and uses provided herein (see, for example, U.S. Patent. No.
[0390] [0390] Hyaluronidases are members of a large family of hyaluronan degrading enzymes. There are three general classes of hyaluronidases: mammals, bacterial hyaluronidases and leeches hyaluronidases, other parasites and crustaceans. Such enzymes can be used in compositions, combinations and methods provided herein.
[0391] [0391] Mammalian type hyaluronidases (EC
[0392] [0392] Mammalian hyaluronidases can be subdivided into those that are neutrally active, predominantly found in extracts of testicles and active acid, predominantly found in organs such as the liver. Exemplary neutral-active hyaluronidases include PH20, including but not limited to PH20 derived from different species, such as sheep (SEQ ID NOS: 27, 63 and 65), cattle (SEQ ID NO: 11 and 64) and humans (SEQ ID NO: 1). Human PH20 (also known as PH20 sperm surface protein or SPAM 1), is generally linked to the plasma membrane by means of a glycosylphosphatidylinositol (GPI) anchor. It is naturally involved in sperm-egg adhesion and aids in penetrating the cumulus cell layer through hyaluronan digestion through the sperm.
[0393] [0393] In addition to human PH20 (also called SPAM1), five similar hyaluronidase genes have been identified in the human genome, HYAL1, HYAL2, HYAL3, HYALA4 and HYALPl. HYALP1] is a pseudo-gene and HYAL3 (SEQ ID NO: 38) has not been shown to have enzymatic activity for any known substrates. HYALA (precursor polypeptide established in SEQ ID NO: 39) is a chondroitinase and exhibits little activity compared to hyaluronan. HYAL1 (polypeptide precursor established in SEQ ID NO: 36) is the prototypical acid-active enzyme and PH20 (precursor polypeptide established in SEQ ID NO: 1) is the prototypical neutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 and HYAL2 (precursor polypeptide set out in SEQ ID NO: 37) do not generally have catalytic activity at neutral pH (i.e., pH 7). For example, HYAL1I has little in vitro catalytic activity over pH 4.5 (Frost et al. (1997) Anal. Biochem. 251: 263-269). HYAL2 is an acid-active enzyme with very low specific activity in vitro. Hyaluronidase enzymes can also be characterized by those that are generally linked to the plasma membrane by means of an anchor of glycosylphosphatidylinositol (GPI) such as human HYAL2 and human PH20 ((Danilkovitch-Miagkova et al. (2003) Proc Natl Acad Sci USA 100 (8): 4580-5), and those that are generally soluble, such as human HYAL1 (Frost et al. (1997) Biochem Biophys Res Commun. 236 (1): 10-5).
[0394] [0394] PH20, like other mammalian hyaluronidases, is an endo-B-1-N-acetyl-hexosaminidase, which hydrolyzes the glycosidic bond B1 - 4 of hyaluronan in various lengths of oligosaccharides, such as tetrasaccharides and hexassaccharides. It has both hydrolytic and transglycosidase activity, and can degrade hyaluronic acid and chondroitin sulfates, such as C4-S and C6-S. PH20 is naturally involved in sperm-egg adhesion and assists sperm penetration of the cumulus cell layer by digestion of hyaluronan. PH20 is located on the surface of the sperm and lysosome-derived acrosome, where it is attached to the inner acrosomal membrane. PH20 of the plasma membrane has hyaluronidase activity only at neutral pH, while PH20 of the internal acrosomal membrane has activity under both neutral and acidic pH. In addition to being a hyaluronidase, PH20 is reported to be a receptor for HA-induced cell signaling, and a receptor for the pellucid zone around the oocyte.
[0395] [0395] Exemplary PH20 proteins include, but are not limited to, human PH20 polypeptides (precursor polypeptide set out in SEQ ID NO: 1, mature polypeptide as explained in SEQ ID NO: 2), chimpanzee (SEQ ID NO: 101), Rhesus monkey (SEQ ID NO: 102) bovine (SEQ ID NOS: 11 and 64), rabbit (SEQ ID NO: 25), sheep PH20 (SEQ ID NOS: 27, 63 and 65), Cynomolgus monkey (SEQ ID NO: 29) , guinea pig (SEQ ID NO: 30), rat (SEQ ID NO: 31) and mouse (SEQ ID NO: 32).
[0396] [0396] Bovine PH20 is a 553 amino acid polypeptide precursor (SEQ ID NO: 11). The alignment of bovine PH20 with human PH20 shows only weak homology with several existing gaps, from amino acid 470 to the respective carboxy, due to the absence of a GPI anchor with the bovine polypeptide (see, for example, Frost Gl ( 2007) Expert Opin. Drug, Deliv. 4: 427-440). In fact, clean GPI anchors are not foreseen in many other PH20 species other than humans. Thus, sheep and bovine PH20 polypeptides naturally exist as soluble forms. Although there is a bovine PH20 very sensitively bound to the plasma membrane, it is not anchored by means of a sensitive phospholipase anchorage (Lalancette et al. (2001) Biol Reprod. 65 (2): 628-36). This unique characteristic of bovine hyaluronidase has allowed the use of the enzyme hyaluronidase from bovine testicles as a soluble extract for clinical use (WydaseO, HyalaseO).
[0397] [0397] Human PH20 mRNA transcription is normally translated to generate a 509 amino acid precursor polypeptide (SEQ ID NO: 1) that contains a 35 amino acid signal sequence at the N-terminus (amino acid residue positions 1-35) and a 19 amino acid glycosylphosphatidylinositol anchor signal sequence (GPI) at the C-terminus (positions of amino acid residues 491-509). Mature PH20, therefore, is a 474 amino acid polypeptide established in SEQ ID NO: 2. Following the transport of the precursor polypeptide to the ER and removal of the signal peptide, the C-terminal GPI-linked signal peptide is cleaved to facilitate covalent attachment of a GPI anchor to the newly formed C-terminal amino acid at the amino acid position , which corresponds to the 490 position of the precursor polypeptide established in SEQ ID NO: 1. Thus, a mature polypeptide anchored to GPI of amino acid 474 with an amino acid sequence established in SEQ ID NO: 2, is produced.
[0398] [0398] Human PH20 exhibits hyaluronidase activity at neutral and acidic pH. In one aspect, human PH20 is the prototypical neutral-active hyaluronidase that is generally blocked to the plasma membrane by means of a GPI anchor. In another aspect, PH20 is expressed on the inner acrosomal membrane where it has hyaluronidase activity at neutral and acidic pH. PH20 contains two catalytic sites in distinct regions of the polypeptide: the peptide 1 and 3 regions (Chem et al. (2001) Matrix Biology20: 515-525). The evidence indicates that Peptide region 1 of PH20, which corresponds to amino acid positions 107-137 of the mature polypeptide as explained in SEQ ID NO: 2, and positions 142-172 of the precursor polypeptide established in SEQ ID NO: 1, are necessary for enzymatic activity, at neutral pH. The amino acids at positions 111 and 113 (which correspond to the mature PH20 polypeptide established in SEQ ID NO: 2), within this region, are reported to be important for activity, such as mutagenesis by results of amino acid substitution in PH20 polypeptides with 3% of hyaluronidase activity, or detectable hyaluronidase activity, respectively, compared to PH20 (Arming et al., (1997) Eur. J. Biochem. 247: 810-814).
[0399] [0399] The peptide region 3, which corresponds to the amino acid positions 242-262 of the mature polypeptide as explained in SEQ ID NO: 2, and the positions 277-297 of the precursor polypeptide set out in SEQ ID NO: 1, are reported as important for enzyme activity at acidic pH. Within this region, the amino acids at positions 249 and 252 of the mature PH20 polypeptide are referred to as essential for activity, as mutagenesis of the results in a polypeptide essentially devoid of activity (Arming et al., (1997) Eur. J. Biochem. 247 : 810-814).
[0400] [0400] In addition to the catalytic sites, PH20 also contains a hyaluronan binding site. Experimental evidence indicates that this site is located in the Peptide 2 region, which corresponds to amino acid positions 205-235 of the precursor polypeptide set out in SEQ ID NO: 1 and positions 170-200 of the mature polypeptide set out in SEQ ID NO: 2. This region is highly conserved among hyaluronidases, and is similar to the heparin-binding motif. Mutation of the arginine residue at position 176 (which corresponds to the mature PH20 polypeptide set out in SEQ ID NO: 2) to a glycine, results in a polypeptide with only about 1% of the hyaluronidase activity of the wild type polypeptide (Arming et al., (1997) Eur. J. Biochem. 247: 810-814).
[0401] [0401] There are seven potential glycosylation sites, including the N-linked glycosylation sites, on human PH20 at N82, Nl66, N235, N254, N368, N393, S490 of the polypeptide exemplified in SEQ ID NO: 1. Because amino acids 36 -464 of SEQ ID NO: 1 are reported to contain the minimally active human PH20 hyaluronidase domain, the S-490 glycosylation site is not required for proper hyaluronidase activity. There are six disulfide bonds in human PH20. Two disulfide bonds between cysteine residues C60 and C351 and between C224 and C238 of the polypeptide exemplified in SEQ ID NO: 1 (corresponding to residues C25 and C316, and C189 and C203 of the mature polypeptide as explained in SEQ ID NO: 2 , respectively). Another four disulfide bonds are formed between the cysteine residues, C376 and C387; between C381 and C435; between C437 and C443; and between C458 and C464 of the polypeptide exemplified in SEQ ID NO: 1 (corresponding to residues C341 and C352; between C346 and C400; between C402 and C408, and between C423 and C429 of the mature polypeptide as explained in SEQ ID NO: 2, respectively).
[0402] [0402] Bacterial hyaluronidases (EC 4.2.2.1 or
[0403] [0403] Examples of bacterial hyaluronidases for use in the compositions, combinations and methods provided include, but are not limited to, enzymes that degrade hyaluronan in microorganisms, including the strains of Arthrobacter, Bdellovibrio, Clostridium, Micrococcus, Streptococcus, Peptococcus, Propionibacterium , and Streptomyces. Particular examples of such strains and enzymes include, but are not limited to, Arthrobacter sp. (strain FB24 (SEQ ID NO: 67)), Bdellovibrio bacteriovorus (SEQ ID NO: 68), Propionibacterium acnes (SEQ ID NO: 69), Streptococcus agalactiae ((SEQ ID NO: 70); 18RS21 (SEQ ID NO: 71) ); serotype Ia (SEQ ID NO: 72); serotype III (SEQ ID NO: 73)), Staphylococcus aureus (COL strain (SEQ ID NO: 74); MRSA252 strain (SEQ ID NOS: 75 and 76); MSSA476 strain (SEQ ID NO: 77); NCTC strain 8325 (SEQ ID NO: 78); bovine strain RF122 (SEQ ID NOS: 79 and 80); strain USA300 (SEQ ID NO: 81)), Streptococcus pneumoniae ((SEQ ID NO : 82); ATCC strain BAA-255 / R6 (SEQ ID NO: 83); serotype 2, strain D39 / NCTC 7466 (SEQ ID NO: 84)), Streptococcus pyogenes (serotype Ml (SEQ ID NO: 85); serotype M2, strain MGAS 10270 (SEQ ID NO: 86); serotype M4, strain MGAS 10750 (SEQ ID NO: 87); serotype M6 (SEQ ID NO: 88); serotype Ml12, strain MGAS2096 (SEQ ID NOS: 89 and 90 ); serotype M12, strain MGAS9429 (SEQ ID NO: 91); serotype M28 (SEQ ID NO: 92)), Streptococcus suis (SEQ ID NOS: 93-95); Vibrio fischeri (strain ATCC 700601 / ES114 (SEQ ID NO: 96)), and the hyaluronidase enzyme Streptomyces hyaluronolyticus, which is specific for hyaluronic acid and does not cleave chondroitin or chondroitin sulfate (Ohya, T. and Kaneko, Y (1970) Biochim, Biophys, Acta 198: 607). Leeches hyaluronidase, other parasites, and crustaceans (EC 3.2.1.36) are endo-b-glucuronidases that generate end products of tetra- and hexassaccharides. These enzymes catalyze the hydrolysis of 1-3 bonds between B-D-glucuronate and N-acetyl-D-glucosamine residues in hyaluronate. Examples of leeches hyaluronidases include, but are not limited to, Hirudimidae hyaluronidase (eg, Hirudo medicinalis), Erpobdellidae (eg, Nephelopsis obscura and Erpobdella punctata), Glossiphoniidae (eg, Desserobdella picta, Helobdata, Placobdella ornata and Theromyzon sp.) And Haemopidae (Haemopis marmorata) (Hovingh et al. (1999) Comp Biochem Physiol B Biochem Mol. Biol. 124 (3): 319-26). An exemplary hyaluronidase from bacteria that has the same mechanism of action as leech hyaluronidase is that originated from cyanobacteria, Synechococcus sp. (strain RCC307, SEQ ID NO: 97).
[0404] [0404] In addition to the hyaluronidase family, Other degrading enzymes of hyaluronan can be used in the compositions, combinations and methods provided. For example, enzymes, which include chondroitinases and lyases, which have the ability to cleave hyaluronan, can be used. Exemplary chondroitinases that can degrade hyaluronan include, but are not limited to, chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AC lyase (also known as chondroitin sulphate lyase or chondroitin sulphate elimination) and chondroitin C lyase. methods for producing and purifying such enzymes for use in the compositions, combinations and methods provided herein, are known in the art (for example, U.S. Patent No. 6,054,569; Yamagata, et al. (1968) J. Biol. Chem. 243 (7): 1523-1535; Yang et al. (1985) JJ. Biol. Chem. 160 (30): 1849-1857).
[0405] [0405] Chondroitin ABC lyase contains two enzymes, chondroitin-sulfate-ABC endolysis (EC 4.2.2.20) and chondroitin-sulfate-ABC exolysis (EC 4.2.2.21) (Hamai et al. (1997) J Biol. Chem. 272 ( 14): 9123-30), which degrade a variety of glycosaminoglycans like chondroitin sulfate and dermatan sulfate. Chondroitin sulphate, chondroitin-proteoglycan sulphate and dermatan sulphate are the preferred substrates for chondroitin-sulphate-ABC endolase, but the enzyme can also act on hyaluronan at a lower rate. Chondroitin-sulphate-ABC endolyses a variety of chondroitin-sulphate and dermatan-sulphate glycosaminoglycans, producing a mixture of different sized AM4-unsaturated oligosaccharides, which are ultimately degraded to AM4-unsaturated tetra- and disaccharides. Chondroitin-sulfate-ABC exoliase has the same substrate specificity, but removes disaccharide residues from the non-reducing ends of both polymeric chondroitin sulfates and their oligosaccharide fragments produced by chondroitin-sulfate-ABC endoliase (Hamai, A. et al. (1997). J. Biol. Chem. 272: 9123-9130). Exemplary chondroitin-sulfate-ABC endoliases and chondroitin-sulfate-ABC exoliases include, but are not limited to those of Proteus vulgaris and Pedobacter heparinus (Proteus vulgaris chondroitin-sulfate-ABC endoliase is established in SEQ ID NO: 98 (Sato et al. ( 1994) Appl. Microbiol. Biotechnol. 41 (1): 39—46).
[0406] [0406] Chondroitin AC lyase (CE 4.2.2.5) is active in chondroitin sulfates A and C, chondroitin and hyaluronan, but is not active in dermatan sulfate (chondroitin sulfate B). Examples of bacterial chondroitinase AC enzymes include, but are not limited to, Pedobacter heparinus and Victivallis vadensis, set out in SEQ ID NOS: 99 and 100 respectively, and Arthrobacter aurescens (Tkalec et al. (2000) Applied and Environmental Microbiology 66 ( 1): 29-35; Ernst et al. (1995) Critical Reviews in Biochemistry and Molecular Biology 30 (5): 387-444).
[0407] [0407] Chondroitinase Cc cleaves chondroitin sulfate Cc producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-68S). It also cleaves hyaluronic acid producing unsulfated unsaturated disaccharide (delta Di-OS). Examples of chondroitinase C enzymes from bacteria include, but are not limited to, Streptococcus and Flavobacterium (Hibi et al. (1989) FEMS-Microbiol-Lett. 48 (2): 121-4; Michelacci et al. (1976) J. Biol. Chem. 251: 1154-8; Tsuda et al. (1999) Eur. J. Biochem. 262: 127-133).
[0408] [0408] Enzymes that degrade soluble hyaluronan, including soluble hyaluronidases, are provided in the compositions, combinations, uses and methods described herein. “Soluble hyaluronan degrading enzymes include any enzyme that degrades hyaluronan that is secreted from cells (for example CHO cells) upon expression, and that exists in soluble form. These enzymes include, but are not limited to, soluble hyaluronidases, including non-human soluble hyaluronidases, including non-human animal soluble hyaluronidase, bacterial soluble hyaluronidase and human hyaluronidase, Hyall, bovine and sheep PH20, their allelic variants and other variants thereof. For example, included among enzymes that degrade soluble hyaluronan are enzymes that degrade hyaluronan that have been modified to be soluble. For example, hyaluronan degrading enzymes that contain a GPI anchor can be solubilized by truncating and removing all or part of the GPI anchor. In one example, the human PH20 hyaluronidase, which is normally anchored to the membrane by means of a GPI anchor, can be made soluble by truncation and removal of the whole, or part of the GPI anchor at the C-terminal.
[0409] [0409] Enzymes that degrade soluble hyaluronan also include neutral-active and acid-active hyaluronidases. Depending on factors such as, but not limited to, the desired level of enzyme activity after administration and / or the site of administration, neutral-active and acid-active hyaluronidases can be selected. In a particular example, the hyaluronan-degrading enzyme for use in the compositions, combinations and methods described herein is a soluble neutral-active hyaluronidase.
[0410] [0410] Examples of a soluble hyaluronidase is PH20, from any species, such as any established in any of SEQ ID NOS: 1, 2, 11, 25 27, 29-32, 63-65 and 101-102 , or its truncated forms, missing all or a portion of the GPI anchor at the C-terminal end, while hyaluronidase is soluble (secreted by expression) and retains hyaluronidase activity. Also included among soluble hyaluronidases are allelic variants or other variants of any of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, or their truncated forms. Allelic variants and other variants are known to a person skilled in the art, and include polypeptides with 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of SEQ ID NO: 1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, or their truncated forms. Amino acid variants include conservative and non-conservative mutations. It is understood that residues that are important or otherwise required for hyaluronidase activity, as described above, or any known to those skilled in the art, are generally invariable and cannot be altered. These include, for example, waste from active sites. Thus, for example, amino acid residues 111, 113 and 176 (corresponding to residues in the mature PH20 polypeptide described in SEQ ID NO: 2) of a human PH20 polypeptide, or its soluble form, are generally invariable and are not altered. Other residues that confer glycosylation and disulfide bond formation necessary for proper folding can also be invariant.
[0411] [0411] In some cases, the soluble hyaluronan degrading enzyme is normally anchored to GPI (such as, for example, human PH20) and is made soluble by truncation at the C-terminus. Such truncation can remove the entire sequence of the GPI anchor binding signal, or it can remove only some of said sequences. The resulting polypeptide, however, is soluble. In cases where the soluble hyaluronan degrading enzyme maintains a part of the GPI anchor-binding signal sequence, 1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the anchor-binding signal sequence of GPI can be retained, as long as the polypeptide is soluble. Polypeptides containing one or more amino acids from the GPI anchor are termed as enzymes that degrade prolonged soluble hyaluronan. One skilled in the art can determine whether a polypeptide is GPI-anchored using methods well known in the art. Such methods include, but are not limited to, the use of known algorithms to predict the presence and location of the GPI anchor-binding signal sequence and location w, and the performance of solubility analyzes before and after digestion with specific phospholipase from phosphatidylinositol C (PI-PLC) or D (PI-PLD).
[0412] [0412] Prolonged soluble hyaluronan degrading enzymes can be produced by making C - terminal truncations of any naturally occurring hyaluronan degrading enzyme to GPI, so that the resulting polypeptide is soluble and contains one or more amino acid residues from the signal sequence. link to GPI anchor (see, for example, published U.S. Pat. Application No. US20100143457). Examples of extended soluble hyaluronan degrading enzymes that are truncated at the C-terminus, but retain a portion of the GPI anchor binding signal sequence include, but are not limited to, the extended soluble PH20 polypeptide (esPH20) of primate origin, such as , for example, human and chimpanzee esPH20 polypeptides. For example, esPH20 polypeptides can be made by C-terminal truncation of any of the mature polypeptides Or precursors exposed in SEQ ID NOS: 1, 2 or 101, or allelic or another variation thereof, including its active fragment, in which the The resulting polypeptide is soluble and retains one or more amino acid residues from the GPI anchor binding signal sequence. Allelic variants and other variants are known to a person skilled in the art, and include polypeptides with 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, or more of identity sequence of any of SEQ ID NO: 1 or 2. The esPH20 polypeptides provided herein can be truncated at the C-terminus by
[0413] [0413] Normally, for use in the compositions, combinations and methods of this invention, an enzyme that degrades soluble human hilauronan, such as a soluble human PH20, is used. Although enzymes that degrade hilauronan, such as PH20, from other animals can be used, such preparations are potentially immunogenic, since they are proteins of animal origin. For example, a significant proportion of patients demonstrate prior sensitization secondary to the food eaten, and since these are animal proteins, all patients have a risk of later sensitization. Thus, non-human preparations may not be suitable for chronic use. If non-human preparations are desired, it is contemplated that these polypeptides can be prepared to have reduced immunogenicity. Such modifications are within the level of a person skilled in the art and may include, for example, the removal and / or replacement of one or more antigenic epitopes on the molecule.
[0414] [0414] Hyaluronan degrading enzymes, including hyaluronidases (for example, PH20), used in the methods described herein can be produced recombinantly, or can be purified or partially purified from natural sources, such as, for example, from of testis extracts. Methods for producing recombinant proteins, including enzymes that degrade recombinant hyaluronan, are provided here, elsewhere, and are well known in the art.
[0415] [0415] Examples of a soluble hyaluronidase is soluble human PH20, recombinant soluble forms of human PH20 have been produced and can be used in the compositions, combinations and methods described herein. The production of such soluble forms of PH20 described in U.S. Published U.S. Patent Requirements US20040268425; US20050260186, US20060104968, US20100143457 and PCT International Application No. WO2009111066. For example, soluble PH20 polypeptides include truncated C-terminal variant polypeptides, which include an amino acid sequence in SEQ ID NO: 1, or are at least 91%, 92%, 93%, 94%, 95%, 95 %, 97%, 98% sequence identity with an amino acid sequence included in SEQ ID NO: 1, retain hyaluronidase activity and are soluble. Included among these —polypeptides are soluble PH20 polypeptides that lack all or a portion of the GPI anchor binding signal sequence.
[0416] [0416] Also included are extended soluble PH20 polypeptides (esPH20) that contain at least one GPI anchor amino acid. Thus, instead of having a GPI anchor covalently attached to the C-terminal of the protein in the ER and being anchored to the extracellular sheet of the plasma membrane, these polypeptides are secreted and soluble. PH20 truncated C-terminal polypeptides can be truncated at the C-terminal by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids, compared to the wild-type full-length polypeptide, such as a wild-type full-length polypeptide with a displayed sequence in SEQ ID NOS: 1 or 2, or allelic variants or species variants, or other variants thereof.
[0417] [0417] For example, water-soluble forms include, but are not limited to, truncated polypeptides at the human PH20 C-terminal described in SEQ ID NO: ll with a C-terminal amino acid residue of 467, 468, 469, 470 , 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495 , 496, 497, 498, 499 or 500 of the amino acid sequence shown in SEQ ID NO: 1, or polypeptides that exhibit at least 85% identity with it. Soluble forms of human PH20 generally include those containing amino acids 36-464 shown in SEQ ID NO: 1. For example, when expressed in mammalian cells, the N-terminal 35 amino acid signal sequence is cleaved during processing, and the mature form of the protein is secreted. Thus, mature soluble polypeptides contain amino acids 36 to 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO:
[0418] [0418] For example, examples of truncated C-terminal PH20 polypeptides, which exhibit hyaluronidase activity, which are secreted from cells and are soluble, include any of the mature forms of truncated human PH20 shown in Table 3, or variants thereof , which exhibit hyaluronidase activity. For example, PH20 or its truncated form contains the amino acid sequence established in any of SEQ ID NOS: 4- 9, 47, 48, 150-170 and 183-189, or an amino acid sequence that exhibits at least 85% of sequence identity with any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189. For example, the PH20 polypeptide can have at least 85%, 86%, 87%, 88%, 89%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of SEQ ID NOS: 4- 9, 47, 48, 150-170 and 1183-189.
[0419] [0419] Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure polypeptide retention activity, since glycosylation is important for catalytic activity, and the stability of hyaluronidase . Such cells include, for example, Chinese hamster ovary (CHO) cells (for example, DG44 CHO cells).
[0420] [0420] Recombinant soluble forms of human PH20 have been generated and can be used in the compositions, combinations and methods provided herein. The generation of such soluble forms of recombinant human PH20 is described, for example, in U.S. Patent Application Published No. US20040268425; US20050260186; US20060104968; US20100143457; and International PCT No. WO2009111066. Examples of such polypeptides are those generated by expression of a nucleic acid molecule that encodes amino acids 1-482 (set out in SEQ ID NO: 3). Such an exemplary nucleic acid molecule is described in SEQ ID NO: 49. Post-translational processing removes the 35 amino acid signal sequence, leaving a soluble recombinant human PH20 of 447 amino acids (SEQ ID NO: 4). As produced in the culture medium, there is heterogeneity at the C-terminal such that the product, designated rHuPH20, includes a mixture of species, which can include any one or more of SEQ ID NOS. 4-9 in huge abundance. Normally, rHuPH20 is produced in cells that facilitate the correct N-glycosylation to retain activity, such as CHO cells (for example, DG44 CHO cells).
[0421] [0421] Glycosylation, including N- and O- linked glycosylation, of some hyaluronan-degrading enzymes, including hyaluronidases, may be important for their catalytic activity and stability. Although changing the type of glycan that modifies a glycoprotein can have dramatic effects on a protein's antigenicity, structural fold, solubility, and stability, most enzymes are not thought to require glycosylation for 6Optimal enzyme activity. For some hyaluronidases, removal of N-glycosylation can result in almost complete inactivation of hyaluronidase activity. Thus, for such hyaluronidases, the presence of N-linked glycans is important for the generation of an active enzyme.
[0422] [0422] N-linked oligosaccharides fall into several “main” types (oligomannose, complex, hybrid, sulfated), all of which have (Man) 3-GlcNACc- GlcNAc-colors linked through the Asn residue amide nitrogen in -Asn-Xaa- Tre / Ser- sequences (where Xaa is not Pro). Glycosylation at one site - Asn-Xaa-Cys-has been reported for clotting protein C. In some cases, an enzyme that degrades hyaluronic acid, such as hyaluronidase, can contain both N-glycosidic and O-glycosidic bonds. For example, PH20 has O-linked oligosaccharides, as well as N- linked oligosaccharides. There are seven potential N-linked glycosylation sites in N82, Nl66, N235, N254, N368, N393, N490 of the human PH20 exemplified in SEQ ID NO: 1. Amino acid residues N82, N166 and N254 are occupied by complex type glycans while amino acid residues N368 and N393 are occupied by high mannose glycans. The amino acid residue N235 is occupied by about 80% of high glycans like mannose and 20% of complex type of glycans. As noted above, N-linked glycosylation in N490 is not necessary for hyaluronidase activity.
[0423] [0423] In some examples, hyaluronan degrading enzymes for use in the compositions, combinations and / or methods provided are glycosylated at one or all glycosylation sites. For example, for human PH20, or a soluble form thereof, 2, 3, 4, 5, or 6 of the N - glycosylation sites corresponding to amino acids N82, Nl66, N235, N254, N368, N393 and SEQ ID NO: 1 are glycosylated. In some examples, hyaluronan degrading enzymes are glycosylated at one or more native glycosylation sites. In other examples, hyaluronan degrading enzymes are modified at one or more non-native glycosylation sites to confer glycosylation of the polypeptide to one or more additional sites. In such examples, the attachment of additional sugar moieties can improve the pharmacokinetic properties of the molecule, such as improved half-life and / or improved activity.
[0424] [0424] In other examples, hyaluronan degrading enzymes for use in the compositions, combinations and / or methods provided herein are partially deglycosylated (or N-partially glycosylated polypeptides). For example, soluble partially deglycosylated PH20 polypeptides that retain all or part of the hyaluronidase activity of a fully glycosylated hyaluronidase can be used in the compositions and / or methods provided herein. Examples of partially deglycosylated hyalurodinases include soluble forms of partially deglycosylated PH20 polypeptides of any kind, as defined in any of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63, 65, and 101-102 , or their allelic variants, truncated variants, or other variants thereof. Such variants are known to a person skilled in the art, and include polypeptides with 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or more of sequence identity with any of the SEQ ID NOS: 1, 2, 11, 25 27, 29-32, 63, 65 and 101-102, or truncated forms. The partially deglycosylated hyaluronidases provided herein also include hybrid, fusion and chimeric partially deglycosylated hyaluronidases and partially deglycosylated hyaluronidase conjugates.
[0425] [0425] Glycosidases or glycosyl hydrolases are enzymes that catalyze the hydrolysis of the glycosidic bond to generate two smaller sugars. The main types of N-glycans in vertebrates include high mannan glycans, hybrid glycans and complex glycans. There are several glycosidases that result in partial protein deglycosylation, including: EndoFl, which cleaves high mannose and hybrid type glycans; EndoF2, which cleaves biternary complex type glycans; EndoF3, which cleaves biternary complex and still branched glycans; and EndoH, which cleaves high-mannose and hybrid-type glycans. Treatment of a hyaluronan-degrading enzyme, such as soluble hyaluronidase, such as a
[0426] [0426] Enzymes that degrade partially deglycosylated hyaluronan, such as soluble partially deglycosylated hyaluronidases, can be produced through digestion with one or more glycosidases, usually a glycosidase that does not remove all N-glycans, but can only partially deglycosylate the protein. For example, treatment of PH20 (for example, a recombinant PH20 called rHuPH20) with one or all of the glycosidases (for example, EndoFl, EndoF2 and / or EndoF3) above results in partial deglycosylation. These partially deglycosylated PH20 polypeptides can exhibit an enzyme activity of hyaluronidase that is comparable to fully glycosylated polypeptides. In contrast, treatment of PH20 with PNGaseF, a glycosidase that cleaves all N-glycans, results in the complete removal of all N-glycans and thus renders PH20 enzymatically inactive. Thus, although all N-linked glycosylation sites (such as, for example, amino acids N82, N1l66, N235, N254, N368, N393 of human PH20, exemplified in SEQ ID NO: 1) can be glycosylated, treatment with one or more glycosidases can process the degree of reduced glycosylation compared to a hyaluronidase that is not digested with one or more glycosidases.
[0427] [0427] Partially deglycosylated hyaluronan degrading enzymes, including partially deglycosylated soluble PH20 polypeptides, can have 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the glycosylation level of a polypeptide fully glycosylated. In one example, 1, 2, 3, 4, 5 or 6 of the N-glycosylation sites corresponding to amino acids N82, N166, N235, N254, N368, N393 and SEQ ID NO: 1 are partially deglycosylated, such that they no longer contain high mannose glycans or complex type glycans, but they contain at least one N-acetylglucosamine portion. In some examples, 1, 2 or 3 of the N-glycosylation sites corresponding to amino acids N82, N166 and N254 of SEQ ID NO: 1 are deglycosylated, that is, they do not contain a portion of sugar. In other examples, 3, 4, 5, or 6 of the N-glycosylation sites corresponding to amino acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 1 are glycosylated. Glycosylated amino acid residues minimally contain an N-acetylglucosamine portion. Typically, partially deglycosylated degrading hyaluronan enzymes, including partially deglycosylated soluble PH20 polypeptides, exhibit hyaluronidase activity which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400% 500%, 1000% or more of the hyaluronidase activity exhibited by the fully glycosylated polypeptide.
[0428] [0428] In one example, the intended compositions and combinations contain enzymes that degrade hyaluronan, in particular soluble hyaluronidases, which have been modified by conjugation with one or more polymeric molecules (polymer), usually to increase the half-life of the degrading enzyme in hyaluronan, for example, to promote the prolonged / sustained effects of treatment on an individual.
[0429] [0429] Covalent stable fixation or other stable fixation (conjugation) of polymeric molecules, such as polyethylene glycol (PEGylation portion (PEG)), for hyaluronan degrading enzymes, such as hyaluronidases, confer beneficial properties to the degrading enzyme-polymer composition resulting hyaluronan. Such properties include improved biocompatibility, protein span (and enzyme activity), half-life in blood, cells and / or other tissues within an individual, effective shielding of protein from proteases and hydrolysis, improvement improved biodistribution, pharmacokinetics and / or pharmacodynamics, and greater solubility in water.
[0430] [0430] Thus, in the special examples presented here, the hyaluronan-degrading enzyme is conjugated to a polymer. Examples of polymers are such as polyols (i.e., poly-OH), polyamines (i.e. poly-NH2) and polycarboxyl acids (i.e. poly-COOH), and other heteropolymers ie polymers that comprise one or more different groups of coupling, for example, a hydroxyl group and amine groups. Examples of suitable polymer molecules include polymer molecules selected from polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polypropylene glycols (PEG), methoxypolyethylene glycol (MPEG) glycols and polypropylene glycols, PEG-glycidyl ethers (Epox -PEG) PEG-oxycarbonylimidazole (CDI-PEG) branched polyethylene glycols (PEGS), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D, L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene acid anhydride co-maleic, dextrans, including carboxymethyl-dextran, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, carboxyethylcellulose hydroxyethylcellulose and hydroxypropylcellulose, chitosan hydrolysates, amides, such as hydroxyethyl-amides and glycosides-amides and its derivatives, quar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, hydrolyzed alginic acid and bio - polymers.
[0431] [0431] In particular, the polymer is a polyethylene glycol (PEG). Polymeric molecules suitable for attachment to hyaluronan-degrading enzymes, including hyaluronidases, include, but are not limited to, polyethylene glycol (PEG) and PEG derivatives such as methoxy-polyethylene glycols (MPEG), PEG-glycidyl ethers (Epox-PEG) ), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, and polyethylene oxide (PEO) (see for example, Roberts et al., Advanced Drug Delivery Review (2002) 54: 459-476; Harris and Zalipsky, S ( eds.) "Poly (ethylene glycol), Chemistry and Biological Applications" ACS Symposium Series 680, 1997; Mehvar et al., JJ. Pharm. Pharmaceut. Sci., 3 (1): 125-136, 2000; Harris, ( 2003) Nature Reviews Drug Discovery 2: 214-221; and Tsubery, (2004) 3. Biol. Chem. 279 (37): 38118-24). The polymeric molecule can be of a molecular weight that normally ranges between about 3 kDa and about 60 kDa. In some embodiments, the polymeric molecule that is conjugated to a protein, such as rHuPH20, has a molecular weight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 kDa.
[0432] [0432] Various methods of modifying polypeptides by covalently linking (conjugating) a PEG or PEG derivative (i.e., "PEGylation") are known in the art (see, for example, US 2006/0104968; US Pat. No. 5,672,662; US Pat. No. 6,737,505; and US 2004/0235734). Such techniques are described here elsewhere.
[0433] [0433] Pharmaceutical compositions of anti-hyaluronan agents, for example, an enzyme that degrades hyaluronan or its modified form (e.g., PEGylated hyaluronan degrading enzymes, such as pegylated hyaluronidases), are provided for use in the treatment methods provided. Also here are pharmaceutical compositions that contain a second agent that is used to treat a disease or disorder associated with a disease or condition associated with hyaluronan, such as cancer. Examples of such agents include, but are not limited to, anti-cancer agents that include drugs, polypeptides, nucleic acids, antibodies, peptides, small molecules, gene therapy vectors, viruses and other therapeutic agents. Anti-hyaluronan agents, for example, an enzyme that degrades hyaluronan or its modified form (for example, PEGylated hyaluronan degrading enzymes, such as PEGylated hyaluronidases or PEGPH20), can be co-formulated or co-administered with pharmaceutical formulations of such second agents, to improve their delivery to the desired locations or tissues within the body, associated with excess or accumulation of hyaluronic acid.
[0434] [0434] Pharmaceutically acceptable compositions are prepared, in view of approvals to a regulatory agency or other agency, in accordance with pharmacopoeia generally recognized for use in animals and humans. The compounds can be formulated in any pharmaceutical preparations suitable for any oral and intravenous administration, such as solutions, suspensions, powders or sustained release formulations. Typically, compounds are formulated in pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126. The formulation must be suitable for the mode of administration.
[0435] [0435] In one example, the pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions. If supplied in liquid form, the pharmaceutical compositions can be supplied as a concentrate preparation to be diluted to a therapeutically effective concentration, prior to use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). In another example, pharmaceutical preparations can be presented in the form of lyophilisate for reconstitution with water or another suitable vehicle before use.
[0436] [0436] Pharmaceutical compositions may include vehicles such as a diluent, adjuvant, excipient or vehicle with which the compositions (e.g., corticosteroid or anti-hyaluronan agent, such as PEGylated hyaluronan degrading enzymes) are administered. Examples of suitable pharmaceutical carriers are described in "Remington Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound or agent, generally, in purified form or in partially purified form, together with a suitable amount of vehicle in order to provide the form for appropriate administration to the patient. Such pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soy oil, mineral oil, and sesame oil. Water is a typical vehicle. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The compositions may contain, together with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum arabic, gelatin, glucose, molasses, polyvinylpyrrolidine, celluloses and their derivatives, povidone, crospovidones and other binding agents known to those skilled in the art. suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, lime, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skimmed milk, glycerol, propylene, glycol, water and ethanol. For example, suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. The composition, if desired, can also contain other minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other agents of this type, such as, for example, acetate. sodium, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
[0437] [0437] Pharmaceutically acceptable vehicles used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents,
[0438] [0438] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Preparations for intraprostatic administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready for use combination with a vehicle immediately before use, sterile emulsions. The solutions can be aqueous or non-aqueous.
[0439] [0439] Normally, the dose of an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, is one that also achieves a therapeutic effect in the treatment of a disease or condition associated with hyaluronan, such as cancer. Thus, compositions of an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, are included in an amount sufficient to exert a therapeutically useful effect. The composition containing the active agent can include a pharmaceutically acceptable carrier. Compositions of an anti-hyaluronan agent can also include a second therapeutic agent.
[0440] [0440] Therapeutically effective concentrations of an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, can be determined empirically by testing the compounds in known systems in vitro and in vivo, such as the assays provided herein. For example, the concentration of an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan or its modified form (for example, an enzyme that degrades PEGylated hyaluronan, such as PEGylated hyaluronidase) depends on the rates of absorption, inactivation and excretion, of the characteristics physical-chemical, dosing schedule, and amount administered, as well as other factors known to those skilled in the art. For example, it is understood that the precise dosage and duration of treatment are a function of the tissue to be treated, the disease or condition to be treated, the route of administration, the patient or individual and the anti-hyaluronan agent in particular, and can be determined empirically using known test protocols or by extrapolation, from in vivo or in vitro test data and / or can be determined from known dosage regimens of the particular agent. The amount of an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan or its modified form (for example, an enzyme that degrades PEGylated hyaluronan, such as a PEGylated hyaluronidase), to be administered for the treatment of a disease or condition, for example a disease or condition associated with hyaluronan, such as a tumor rich in HA, can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be used to help identify optimal dosing ranges. The exact dosage, which can be determined empirically, may depend on the particular enzyme, the route of administration, the type of disease to be treated and the severity of the disease.
[0441] [0441] For example, methods of using agents —anti-hyaluronan, such as hyaluronan-degrading enzymes, or their modified forms (eg, PEGylated forms) for the treatment of diseases and conditions associated with hyaluronan are well known in the art (see for example, US Patent Application Pub. No. 20100003238 and International Published PCT Application No. WO 2009/128917). Thus, the dosages of an anti-hyaluronan agent, such as a hyaluronan-degrading enzyme, for example, a hyaluronidase, can be chosen based on conventional dosing regimes for that agent, according to a particular route of administration.
[0442] [0442] Examples of effective amounts of an anti-hyaluronan agent for the treatment of a disease or condition associated with hyaluronan is a dose ranging from 0.01 ug to 100 pg per kg of body weight. For example, an effective amount of an anti-hyaluronan agent is a dose ranging from 0.01 pg to 100 mg per kg of body weight, such as 0.01 ug to 1 mg per kg of body weight, 1 pg to 100 ug per kg of body weight, from 1 ug to 10 ug per kg of body weight or from 0.01 mg to 100 mg per kg of body weight. For example, effective amounts include at least or about, at least, or about 0.01 µg or, 0.05, 0.1,
[0443] [0443] For example, agents and treatments for the treatment of diseases and conditions associated with hyaluronan, such as anti-cancer agents, are well known in the art (see for example, US Application No. 20100003238 and Published International Application PCT No. WO 2009/128917). Thus, the dosages of a hyaluronan-degrading enzyme, for example, a hyaluronidase, or other agents of a second composition can be chosen based on conventional dosing regimes for that agent, according to a particular route of administration.
[0444] [0444] Examples of effective amounts of a hyaluronan-degrading enzyme are aa doses ranging from 0.01 µg to 100 g per kg of body weight. For example, an effective amount of a hyaluronan-degrading enzyme is a dose ranging from 0.01 µg to 100 mg per kg of body weight, such as from 0.01 µg to 1 mg per kg of body weight, from 1 µg to 100 µg per kg of body weight, from 1 µg to 10 µg per kg of body weight or from 0.01 mg to 100 mg per kg of body weight. For example, effective amounts include at least or about, at least, or about 0.01 µg or, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500 , 600, 700, 800, 900 or 1000 mg / kg of body weight. Other examples of effective amounts include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g / kg of body weight. For example, an enzyme that degrades hyaluronan, for example, a hyaluronidase (for example, a PEGylated hyaluronidase such as PEGPH20), can be administered at or about 0.1 pg / kg to 1 mg / kg, for example 0.5 ug / kg at 100 ug / kg, 0.75 pg / kg at 15 pg / kg, 0.75 ng9 / kg at 7.5 pg / kg or 1.0 p9g / kg at 3.0 g / kg. In other examples, an enzyme that degrades hyaluronan, for example a hyaluornidase (for example, a PEGylated hyaluronidase such as PEGPH20), can be administered at or 1 mg / kg to 500 mg / kg, for example, 100 mg / kg to 400 mg / kg, such as 200 mg / kg. Generally, the compositions contain 0.5 mg to 100 grams of a hyaluronan degrading enzyme, for example, 20 µg to 1 mg, such as 100 µg to 0.5 mg, or can contain from 1 mg to 1 gram, such as 5 mg to 500 mg.
[0445] [0445] The dose or compositions can be for single dose administration or for multiple dose administration. The dose or composition can be administered in a single administration once, several times a week, twice a week, every 15 days, 16 days, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, or 30 days, once a month, several times a year or annually. In other examples, the dose or composition can be divided and administered once, several times a week, twice a week, every 15 days, 16 days, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30 days, once a month, several times a year or annually. Hyaluronan-degrading enzyme compositions can be formulated as liquid compositions or can be lyophilized. The compositions can also be formulated as a tablet or capsule.
[0446] [0446] The description of dosages and dosage regimens of a sample of enzymes that degrade hyaluronan conjugated to a polymer (for example, linked to PEG) is provided below for use in the methods described herein. Hyaluronan-degrading enzymes can be used alone in therapy with a single agent or in combination with other agents, for use in the treatment of a disease or condition associated with HA, such as cancer. As discussed elsewhere elsewhere, in “specific examples of the methods and uses herein, the agents can be administered in combination with a corticosteroid, in order to mitigate a side effect associated with the treatment of an anti-hyaluronan agent.
[0447] [0447] A hyaluronan-degrading enzyme, such as an enzyme that degrades PEGylated hyaluronan (eg, a hyaluronidase), can be administered systemically, for example, intravenously (IV), intramuscularly, or by any other systemic route . In particular examples, lower doses may be given locally. For example, local administration of a hyaluronan-degrading enzyme, such as an enzyme that degrades PEGylated hyaluronan, for example, a PEGylated hyaluronidase (for example PH20) includes intratumoral administration, arterial injection (for example, hepatic artery), administration intraperitoneal, intravesical administration, and other local pathways used for cancer therapy, which can increase local action at a lower absolute dose.
[0448] [0448] The exemplary dosage range is, or is, about 0.3 Units / kg to 320,000 units / kg, such as Units / kg of 320,000 Units / kg of PEGylated hyaluronidase, or a functionally equivalent amount of another enzyme that degrades PEGylated hyaluronan. It is understood here that a unit of activity is normalized to a standard activity, for example, an activity as measured in a microturbity assay that tests for hyaluronidase activity. PEGylated soluble hyaluronidase may exhibit less activity per mg of total protein, that is, it has a lower specific activity, compared to a soluble native hyaluronidase not so conjugated. For example, an exemplary rHuPH20 preparation has a specific activity of 120,000 Units / mg, while a pegylated form of rHuPH20 exhibits a specific activity of, or about 32,000 units / mg. Typically, a pegylated form of a hyaluronan-degrading enzyme, such as, for example, hyaluronidase rHuPH20, has a specific activity in the range of between or about 18,000 and at, or about 45,000 U / mg. In one example, the hyaluronan degrading enzyme PEG can be supplied as a stock solution, for example, at 3.5 mg / ml to 112,000 U / ml (- 32,000 U / mg), with a molar ratio of PEG to protein between 5: 1 and 9: 1, for example, 7: 1, or it can be provided in a less concentrated form. For the purposes of this invention, dosages may refer to Units.
[0449] [0449] For example, a hyaluronan-degrading enzyme PEG, such as hyaluronidase, for example PEGPH20, can be administered intravenously twice a week, once a week or once every 21 days. Normally, the hyaluronan-degrading enzyme PEG is administered twice a week. the administration cycle can be for a defined period, usually for 3 weeks or 4 weeks. The administration cycle can be repeated in a dosage regimen for more than one month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more. Generally, the administration cycle is repeated at the discretion of an attending physician and may depend on factors such as the remission of the disease or condition, the severity of the disease or condition, adverse events and other factors. In other examples, in subsequent cycles of administration, the hyaluronan-degrading enzyme may be administered less frequently. For example, in a first cycle, the hyaluronan-degrading enzyme is administered twice a week for four weeks, and in subsequent cycles of administration, the hyaluronan-degrading enzyme is administered once a week, or once every two weeks, once every 3 weeks (for example, once every 21 days) or once every four weeks. As described herein, the dose or dosing regimen of corticosteroids is dependent on the dosing regimen of hyaluronan degrading enzyme.
[0450] [0450] Although dosages may vary depending on the disease and the patient, the hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase, is generally administered in an amount that is or is about, in the range of 0.01 ug / kg , such as 0.0005 mg / kg (0.5 pg / kg9) to 10 mg / kg (320,000 U / kg), for example, 0.02 mg / kg to 1.5 mg / kg, for example, 0 , 05 mg / kg. PEGylated hyaluronidase can be administered, for example, at a dosage of, at or about 0.0005 mg / kg (of the individual), 0.0006 mg / kg, 0.0007 mg / kg, 0.0008 mg / kg , 0.0009 mg / kg, 0.001 mg / kg, 0.0016 mg / kg, 0.002 mg / kg, 0.003 ma / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.016 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0 , 25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0, 6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg , 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg,
[0451] [0451] A hyaluronan degrading enzyme, such as PEGylated hyaluronidase (for example PH20), provided herein, for example, PEGPH20, can be administered at, or about 1 Unit / kg to 800,000 Units / kg, such as 10 to 800,000 Units / kg, 10 to 750,000 Units / kg, 10 to 700,000 Units / kg, 10 to 650,000 Units / kg, 10 to 600,000 Units / kg, 10 to 550,000 Units / kg, 10 to 500,000 Units / kg, 10 to 450,000 Units / kg, 10 to 400,000 Units / kg, 10 to 350,000 Units / kg, 10 to 320,000 Units / kg, 10 to 300,000 Units / kg, 10 to 280,000 Units / kg, 10 to 260,000 Units / kg, 10 to 240,000 Units / kg, 10 to 220,000 Units / kg, 10 to 200,000 Units / kg, 10 to 180,000 Units / kg, 10 to 160,000 Units / kg, 10 to 140,000 Units / kg, 10 to 120,000 Units / kg, 10 to 100,000 Units / kg, 10 to 80,000 Units / kg, 10 to 70,000 Units / kg, 10 to 60,000 Units / kg, 10 to 50,000 Units / kg, 10 to 40,000 Units / kg, 10 to 30,000 Units / kg, 10 to 20,000 Units / kg , 10 to 15,000 Units / kg, 10 to 12,800 Units / kg, 10 to 10,000 Units / kg, 10 to 9,000 Units / kg, 10 to 8,000 Units / kg, 10 to 7,000 Units / kg, 10 to 6,000 Units / kg, 10 to 5,000
[0452] [0452] Generally, when the specific activity of PEGylated hyaluronidase is, or is, about 18,000 U / mg to 45,000 U / mg, generally at or about 18,000 U / mg to 45,000 U / mg, usually at or about 1 Unit / kg (U / kg), 2 U / kg, 3 U / kg, 4 U / kg, 5 U / kg, 6 U / kg, 7 U / kg, 8 U / kg, 8
[0453] [0453] In some respects, the PEGylated hyaluronan degrading enzyme is formulated and dosed to maintain at least 3 U / ml of PEGylated hyaluronidase in plasma (see for example U.S. Patent Application No. US20100003238 and US Patent Application USA published No. WO2009128917). For example, PEGylated soluble hyaluronidase is formulated for systemic administration of an amount sufficient to maintain at least about 3 U / mL, in plasma, in general, 3 U / mL-12 U / ml or more, for example , from at least, or over, or at a level 4 U / mL, 5 U / mL, 6 U / mL, 7 U / mL, 8 U / mL, 9 U / mL, U / mL, 11 U / ml, 12 U / ml, 13 U / ml, 14 U / ml, 15 U / ml, 16 U / ml, 17 U / ml, 18 U / ml, 19 U / ml, 20 U / ml, 25 U / mL, 30 U / mL, U / mL, 40 U / mL, 45 U / mL, 50 U / ML or more. Generally, for the purposes described herein, at least 3 U / mL of hyaluronidase in plasma, or about 0.02 mg / kg (from the subject), 0.03 mg / kg, 0.04 mg / kg, 0, 05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.30 mg / kg, 0.35 mg / kg, 0.40 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg : kg, 0.6 mg / kg, 0.7 mg / kg,
[0454] [0454] It is within the level of a person skilled in the art, to determine the amounts of degrading enzyme of PEGylated hyaluronic acid, for example, PEG PH20, to maintain at least 3 U / mL of hyaluronidase in the blood. The level of hyaluronidase in the blood can be monitored over time to ensure that a sufficient amount of hyaluronidase is present in the blood. Any assay known to a person skilled in the art to measure hyaluronidase in plasma can be performed. For example, a microturbity or enzyme assay, described in the Examples presented here, can be performed on proteins in plasma. It is understood that plasma usually contains hyaluronidase enzymes. Such plasma hyaluronidase enzymes normally have acidic pH activity (U.S. Pat. No. 7,105,330). Thus, before treatment with a modified enzyme, plasma levels of hyaluronidase should be determined and used as a baseline. Subsequent measurements of plasma levels of hyaluronidase after treatment can be compared to levels before treatments. Alternatively, the assay can be performed under pH conditions that suppress endogenous lysosomal hyaluronidase activity in the plasma, which normally has activity at acidic pH. Thus, where modified soluble hyaluronidase is active at a neutral pH (for example human PH20), only the level of modified neutral-active soluble hyaluronidase is measured.
[0455] [0455] In other examples, the PEGylated hyaluronan degrading enzyme is formulated and administered at a lower dose, which is found here for therapeutic purposes, to treat a disease or conditions associated with hyaluronan, in the absence of a detectable level of hyaluronidase maintained in the blood. For example, PEGylated soluble hyaluronidase is administered in an amount that is less than 20 pg / kg, for example 0.01 ug / kg to 15 pg / kg, 0.05 ug / kg to 10 pg / kg, 0.75 pg / kg at 7.5 vg / kg or 1.0 pg / kg at 3.0 pg / kg, such as, or about 0.01 µg / kg (of the individual), 0.02 pg / kg, 0.03 upg / kg, 0.04 vg / kg, 0.05 ug / kg, 1.0 pg / kg, 1.5 npg / kg, 2.0 np9g / kg, 2.5 p9 / kg, 3 .0 pg / kg, 3.5 pg / kg, 4.0 p9 / k9, 4.5 pg / kg, 5.0 p9I / kg, 5.5 pg / kg, 6.0 pg / kg, 7, 0 np9 / kg9, 7.5 npg / kg, 8.0 vo / kg, 9.0 pg / kg, 10.0 pog / kg, 12.5 p9g / kg or 15 pupg / kg. Generally, when the specific activity of hyaluronidase is modified or is from about 20,000 U / mg to 60,000 U / mg, usually at or about 35,000 U / mg, 200 Units to 50,000 (U) are administered, such as 200 U, 300 U; 400 U; 500 U; 600 U; 700 U; 800 U; 900 U; 1,000 U; 1250 U; 1500 U; 2000 U; 3000 U; 4000 U; 5,000 U; 6,000 U; 7,000 U; 8,000 U; 9,000 U; 10,000 U; 20,000 U; 30,000 U; 40,000 U; or 50,000 U is administered. To maintain these levels, administration can be done daily, several times a week, twice a week, weekly or monthly.
[0456] [0456] Normally, the volumes of injections or infusions of PEGylated hyaluronidase contemplated herein are from, or about 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or more. The PEGylated hyaluronan degrading enzyme, such as a PEGylated hyaluronidase can be supplied as a stock solution or about 50 U / ml, 100 U / ml, 150 U / mML, 200 U / ml, 400 U / mML or 500 U / ml (or a functionally equivalent amount) or can be supplied in a more concentrated form, for example at, or about 1000 U / ml, 2000 U / ml, 3000 U / ml, 4000 U / ml, 5000 U / ml, 6000 U / ml, 7000 U / ml, 8000 U / ml, 9000 U / ml, 10,000 U / ml, 11,000 U / ml, 12,000 U / ml, or 12,800 U / ml, for direct use or by dilution, at a effective concentration before use. The volume of enzyme that degrades PEGylated hyaluronan, such as PEGylated hyaluronidase, administered is a function of the required dosage, but can be varied depending on the concentration of a hyaluronan-degrading enzyme, such as an available stock formulation of soluble hyaluronidase. For example, it is contemplated here, that the enzyme that degrades PEGylated hyaluronan, such as PEGylated hyaluronidase, is not administered in volumes greater than about 50 ml, and is usually administered in a volume of 5-30 ml, usually in one volume that is not greater than about 10 mL. The PEGylated hyaluronan degrading enzyme, such as a PEGylated hyaluronidase, can be supplied as a liquid or lyophilized formulation. Lyophilized formulations are ideal for storing large doses of PEGylated hyaluronan degrading enzyme units.
[0457] [0457] Anti-hyaluronan agents, such as enzymes that degrade hyaluronan, or its modified form (for example, an enzyme that degrades PEGylated hyaluronan or PEGylated hyaluronidase such as PEGPH20) can be administered in combination therapy, for example,
[0458] [0458] A preparation of a second agent or agents, or treatment or treatments can be administered at once, or can be divided into a smaller number of doses to be administered at intervals of time. The selected treatment preparations / agents can be administered in one or more doses over the course of a treatment period, for example, over several hours, days, weeks or months. In some cases, continuous administration is useful. It is understood that the precise dosage and course of administration depend on the indication and tolerability of the patient. Generally, dosage regimens for second agents / treatments described herein are known to a person skilled in the art.
[0459] [0459] For example, an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan or its modified form conjugated to a polymer (for example, an enzyme that degrades PEGylated hyaluronan, such as PEGylated hyaluronidase), is administered with a second agent or treatment, or for the treatment of the disease or condition.
[0460] [0460] For example, an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, can be administered in conjunction with anti-cancer agents (see, for example, US Publication No. US2010-0003238). The anticancer agent (s) or treatment (s) for use in combination with a hyaluronan degrading enzyme includes, but is not limited to, surgery, radiation, medications, chemotherapy, polypeptides, antibodies, peptides, molecules small or gene therapy vectors, viruses or DNA.
[0461] [0461] In other examples, the treatment methods provided herein include methods of administering one or more anti-hyaluronan agents for therapy, in addition to a hyaluronan-degrading enzyme.
[0462] [0462] In some instances, a corticosteroid can be administered to improve the side effects or adverse effects of a hyaluronan-degrading enzyme in combination therapy (U.S. Patent Application published No. 13 / 135,817). In some instances, glucocorticoids are selected from cortisone, dexamethasones, hydrocortisones, methylprednisolones, prednisolones and prednisones. In a particular example, the glucocorticoid is dexamethasone. Generally, corticosteroids are administered orally, although any method of corticosteroid administration is contemplated. Normally, the glucocorticoid is administered in an amount between, or about 0.4 and 20 mg, for example, at or about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg
[0463] [0463] Polypeptides of a hyaluronan binding protein, for use in the "provided compositions and methods, or a hyaluronan degrading enzyme, for example, a soluble hyaluronidase, for the treatment presented here, can be obtained by methods well known in the art. art, for protein purification and expression of recombinant proteins. Any method known to those skilled in the art for the identification of nucleic acids that encode the desired genes, can be used. Any method available in the art can be used to obtain a hyaluronan binding protein that encodes a full-length genomic DNA or CcDNA clone (ie, covering the entire coding region) or a hyaluronidase, such as, from a cell or tissue source. Hyaluronan-binding proteins or modified or variant hyaluronidases, can be manipulated from a wild-type polypeptide, such as by site-directed mutagenesis.
[0464] [0464] Polypeptides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include nucleic acid amplification and library screening, including antibody-based nucleic acid hybridization screening and activity-based screening.
[0465] [0465] Methods for amplifying nucleic acids can be used to isolate nucleic acid molecules encoding a desired polypeptide, including, for example, polymerase chain reaction (PCR) methods. A nucleic acid containing the material can be used as a starting material, from which a nucleic acid molecule encoding the desired polypeptide can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (eg, blood, serum, saliva), samples from healthy and / or sick individuals can be used in amplification methods. Nucleic acid libraries can also be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on expressed sequences from which a desired polypeptide is generated. Primers can be designed based on the translation of a polypeptide amino acid sequence. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.
[0466] [0466] Additional nucleotide sequences can be linked to a nucleic acid molecule encoding the polypeptide, including ligand sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, an expression vector protein, or a vector designed to amplify the DNA sequences that encode the protein's nucleus. In addition, additional nucleotide sequences that specify the functional DNA elements can be operationally linked to a nucleic acid molecule that encodes polypeptide. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to facilitate protein secretion. Such sequences are known to those skilled in the art. Additional nucleotide residue sequences, such as base sequences that specify protein binding regions, can also be linked to nucleic acid molecules that encode the enzyme. Such regions include, but are not limited to, residue sequences that facilitate or encode proteins that facilitate the absorption of an enzyme in specific target cells, or otherwise alter the pharmacokinetics of a synthetic gene product. For example, enzymes can be linked to PEG moieties.
[0467] [0467] In addition, labels or other portions can be added, for example, to aid in the detection or affinity purification of the polypeptide. For example, additional sequences of nucleotide residues, such as base sequences that specify a marker epitope or other detectable marker can also be linked to enzyme-encoding nucleic acid molecules. Examples of such sequences include nucleic acid sequences that encode a His tag (for example, 6exHis, HHHHHH; SEQ ID NO: 54) or Flag tag (DYKDDDDK; SEQ ID NO: 55).
[0468] [0468] The identified and isolated nucleic acids can then be inserted into a suitable cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, modified plasmids or viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PpCMVA4, pBR322 or pUC derived plasmid, or Bluescript vector (Stratagene, La Jolla, CA). Other expression vectors include the expression vector HZ24 exemplified here. Insertion into a cloning vector can, for example, be performed by ligating the DNA fragment into a cloning vector that has complementary cohesive terminals. Insertion can be performed using TOPO cloning vectors (Invitrogen, Carlsbad, CA). If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be modified enzymatically. Alternatively, any desired location can be produced by linking nucleotide sequences (ligands) to the DNA terminals; these linked ligands may contain specific chemically synthesized oligonucleotides that encode restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and protein gene can be modified by homopolymeric tails. Recombinant molecules can be introduced into host cells through, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated.
[0469] [0469] In specific modalities, the transformation of host cells with recombinant DNA molecules that incorporate the isolated protein gene, cDNA, or a synthesized DNA sequence allows the generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by culture transformers, isolating the recombinant DNA molecules, from the transformers and, when necessary, recovering the inserted gene from the isolated recombinant DNA.
[0470] [0470] For recombinant expression of one or more of the desired proteins, such as any hyaluronan binding protein or hyaluronan degrading enzyme described herein, the nucleic acid that contains all or a portion of the nucleotide sequence encoding the protein, it can be inserted into an appropriate expression vector, that is, a vector that contains the necessary elements for the transcription and translation of the coding sequence of the inserted proteins. the necessary transcription and translation signals can also be provided by the native promoter for the enzyme genes, and / or their flanking regions.
[0471] [0471] Vectors are also provided that contain a nucleic acid that encodes the enzyme. The cells containing the vectors are also provided. The cells include eukaryotic and prokaryotic cells, and the vectors are suitable for any use in them.
[0472] [0472] Prokaryotic and eukaryotic cells, including endothelial cells containing the vectors, are provided. Such cells include bacterial cells, yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells. The cells are used to produce a protein therefrom, growing the cells described above under conditions such that the encoded protein is expressed by the cell, and by recovering the expressed protein. For the purposes described herein, for example, the enzyme can be secreted into the medium.
[0473] [0473] Vectors are provided that contain a nucleotide sequence that encodes the hyaluronan-degrading enzyme polypeptide, in some instances, a soluble hyaluronidase polypeptide, along with the native or heterologous signal sequence, as well as several of its copies. The vectors can be selected for the expression of the enzyme protein in the cell, or in such a way that the enzyme protein is expressed as a secreted protein.
[0474] [0474] A variety of host-vector systems can be used to express the protein's coding sequence. These include, but are not limited to, mammalian cell systems infected with viruses (for example, vaccinia virus, adenovirus and other viruses); insect cell systems infected with viruses (for example, baculovirus); microorganisms such as yeast vectors containing yeast; or bacteria transformed with bacteriophage, DNA, DNA plasmid, or DNA cosmid. The elements of expression of vectors vary in their strengths and specificities. Depending on the host-vector system used, any one of a number of appropriate translation and transcription elements may be used.
[0475] [0475] Any methods known to those skilled in the art for inserting DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene containing appropriate translational / transcriptional control signals, and protein coding sequences. These methods can include in vitro recombinant DNA and synthesis techniques and in vivo recombinants (genetic recombination). The expression of nucleic acid sequences encoding proteins, or domains, derivatives, fragments or their homologues, can be regulated by a second nucleic acid sequence, so that the genes or fragments thereof are expressed in a host transformed with the recombinant DNA molecule. For example, protein expression can be controlled by any promoter / stimulator known in the art. In a specific embodiment, the promoter is not native to the gene for a desired protein. Promoters that can be used include, but are not limited to, the SV40 early promoter (Bernoist and Chambon, Nature
[0476] [0476] In a specific embodiment, a vector that is used contains a promoter operationally linked to nucleic acids that encode a desired protein, or a domain, fragment, derivative or homologue thereof, one or more origins of replication and, optionally, a or more selectable markers (for example, an antibiotic resistance gene). Examples of plasmid vectors for the transformation of E. coli cells include, for example, the expression vectors Pp0EOE (available from Qiagen, Valencia, CA, see also the literature published by Qiagen describing the system). Vectors may have a T5 phage promoter (recognized by E. coli, RNA polymerase) and a double lac operator repression module to provide highly regulated, high-level expression of recombinant proteins in E. coli, a binding site to the synthetic ribosome (RBS II) for efficient translation, a 6XHis tag coding sequence, TO and Tl transcription terminators, ColEl origin or replication, and a beta-lactamase gene to confer resistance to ampicillin. The poQE vectors allow the placement of a 6xHis tag on either the N- or C-terminal of the recombinant protein. Such plasmids include POE 32, poE 30, and poE 31, which provide multiple cloning sites for all three reading frames, and to provide expression of 6xHis labeled proteins at the n-terminus. Other examples of plasmid vectors for the transformation of E. coli cells include, for example, pET expression vectors (see, U.S. Patent No.
[0477] [0477] Examples of a vector for mammalian cell expression is the HZ24 expression vector. The HZ24 expression vector was derived from the backbone of the pCI vector (Promega). It contains DNA encoding the beta-lactamase resistance gene (AMpR), an origin of F1 replication, a cytomegalovirus (CMV) enhancer / early promoter region, and an SV40 late polyadenylation signal (SV40). The expression vector also has an internal ribosome entry site (IRES) of the ECMV virus (Clontech) and the mouse dihydrofolate reductase gene.
[0478] [0478] Hyaluronan-binding proteins and enzyme polypeptides that degrade hyaluronan, including soluble hyaluronidase polypeptides, can be produced by any method known to those skilled in the art, including in vivo and in vitro methods. Desired proteins can be expressed in any appropriate organism to produce the necessary amounts and forms of proteins, such as, for example, those necessary for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms, such as E. coli, yeasts, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts may differ in their levels of protein production, as well as the types of post-translational modifications that are present in the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need for methods for purification.
[0479] [0479] Many expression vectors are available and known to those skilled in the art, and can be used for the expression of proteins. The choice of the expression vector will be influenced by the choice of the host's expression system. In general, expression vectors can include transcription promoters and, optionally, enhancers, translation and transcription signals, and translation termination signals. The expression vectors that are used for the stable transformation usually have a selectable marker that allows the selection and maintenance of the transformed cells. In some cases, a source of replication can be used to amplify the number of copies of the vector.
[0480] [0480] Hyaluronan-binding proteins and hyaluronan-degrading enzyme polypeptides, such as soluble hyaluronidase polypeptides, can also be used, or expressed as fusion proteins. For example, a fusion enzyme can be generated to add functionality to an enzyme. Examples of enzyme fusion proteins include, but are not limited to, fusions of a signal sequence, a tag, such as for location, for example, a His6 tag or myc tag, or a tag for purification, for example , a TSG fusion, and a sequence to direct protein secretion and / or membrane association.
[0481] [0481] Prokaryotes, in particular E. coli, provide a system for the production of large amounts of proteins. E. coli transformation is a simple and rapid technique well known to those skilled in the art. E. coli expression vectors can contain inducible promoters, such promoters are useful for inducing high levels of protein expression, and for the expression of proteins that exhibit some toxicity to host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature-regulated APL promoter.
[0482] [0482] Proteins, such as any provided herein, can be expressed in the cytoplasmic environment of E. coli.
[0483] [0483] yeast cells such as Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are well-known hosts of yeast expression, which can be used for the production of proteins, as any described herein. Yeast can be transformed with replicating episomal vectors or by stable chromosomal integration by homologous recombination. Usually, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7 and GAL5 and metallothionein promoters, such as the CUPl, AOX1l or other Pichia promoter, or another yeast promoter. Expression vectors generally include a selectable marker, such as LEU2, TRP1l, HIS3, and URA3, for selecting and maintaining transformed DNA. Proteins expressed in yeast are often soluble. Coexpression with chaperonins such as Bip and protein disulfide isomerase can improve levels of expression and solubility. In addition, proteins expressed in yeast can be directed to secretion using secretion signal peptide fusions, such as the Saccharomyces cerevisae yeast combination factor secretion signal and fusions with yeast cell surface proteins, such as such as the Aga2p combination adhesion receptor or Arxula adeninivorans glucoamylase. A protease cleavage site, such as for the Kex-2 protease, can be manipulated to remove the fused sequences of expressed polypeptides as they exit the secretion pathway. Yeast is also capable of glycosylation in Asn-X-Ser / Thr motifs.
[0484] [0484] Insect cells, in particular, using baculovirus expression, are useful for the expression of polypeptides, including hyaluronan-binding proteins and hyaluronan-degrading enzyme polypeptides, such as soluble hyaluronidase polypeptides. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a restrictive host variety that improves safety, and reduces regulatory concerns for eukaryotic expression. Typical expression vectors use a promoter for high-level expression, such as the baculovirus polyhedrin promoter. Baculovirus systems commonly used include baculoviruses such as the nuclear polyhedrosis virus Autographa californica (ACNPV), and the nuclear polyhedrosis virus Bombyx mori (BMNPV) and an insect cell line, such as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta ( A7S) and Danaus plexippus (DpNl). For high-level expression, the nucleotide sequence of the molecule to be expressed, is fused immediately downstream of the virus polyhedrin initiation codon. Mammalian secretion signals are accurately transformed into insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DPN 1) produce proteins with glycosylation patterns similar to mammalian cell systems.
[0485] [0485] An alternative expression system in insect cells is the use of stable transformed cells. Cell lines, such as Schneider 2 (52) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes albopictus) can be used for expression. The Drosophila metallothionein promoter can be used to induce high levels of expression in the presence of induction by heavy metals with cadmium or copper. Expression vectors are normally maintained by the use of selectable markers, such as neomycin and hygromycin.
[0486] [0486] Mammalian expression systems can be used to express proteins, including hyaluronan binding proteins and hyaluronan degrading enzyme polypeptides, such as soluble hyaluronidase polypeptides. Expression constructs can be transferred to mammalian cells through viral infection, such as adenovirus or through direct DNA transfer, such as liposomes, calcium phosphate, DEAE-dextran, and by physical means, such as electroporation and microinjection. Expression vectors for mammalian cells typically include a mRNA cap site, a TATA box, a translation initiation sequence (Kozak consensus sequence) and polyadenylation elements. IRES elements can also be added to allow bicistronic expression with another gene, such as a selectable marker. Such vectors generally include transcriptional promoter-enhancers for high-level expression, for example, the SV40 promoter-enhancer, human cytomegalovirus (CMV), and the long terminal repeat of the Rous sarcoma virus (RSV). These enhancer-promoters are active in many types of cells. Promoter and enhancer regions of tissues and cells can also be used for expression. Exemplary promoter / enhancer regions include, but are not limited to those of genes, such as elastase, insulin, immunoglobulin, mammary tumor virus, albumin, alpha-fetoprotein, alpha-1-antitrypsin, beta globin, basic protein myelin, light chain 2 and gonadotropic myosin that releases control of the hormonal gene. selectable markers can be used to select and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select only cells that express the DHFR gene. Fusion with cell surface signaling molecules, such as TCR-C and FckxRI-y can direct the expression of proteins in an active state on the cell surface.
[0487] [0487] Many cell lines are available for expression in mammals, including mouse, rat, human, monkey, chicken and hamster cells. Examples of cell lines include, but are not limited to CHO, Balb / 3T3, HeLa, MT2, mouse NSO (non-secretory) and other myeloma, hybridoma and heterohibridoma cell lines, lymphocytes, fibroblasts, Sp2 / 0, COS, NIH3T3, HEK293, 293S, 2B8 and HKB. Cell lines are also available adapted for serum-free media, which facilitates the purification of secreted proteins from the cell culture medium. Examples include CHO-S cells (Invitrogen, Carlsbad, CA., cat $ 11619-012) and the serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84: 332-342). The cell lines that are also available are adapted to grow in special media optimized for maximum expression. For example, DG44 CHO cells are adapted to grow in suspension culture in a chemically defined medium free from animal products.
[0488] [0488] Cells from transgenic plants and plants can be used to express proteins, such as any described here. Expression constructs are normally transferred to plants through direct DNA transfer, such as bombardment of microprojectiles and PEG-mediated transfer to protoplasts, and with Agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcription termination elements and translation control elements. expression vectors and transformation techniques are generally divided between dicotyledonous hosts, such as Arabidopsis and tobacco, and monocotyledon hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter, and the ubiquitin and UBQO3 gene promoters. Selectable markers, such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate the selection and maintenance of transformed cells. The transformed plant cells can be maintained in culture as cells, aggregated (callus tissue) or regenerated in whole plants. Transgenic plant cells can also include algae modified to produce hyaluronidase polypeptides. Because plants have more different patterns of glycosylation than mammalian cells,
[0489] [0489] methods for purifying polypeptides, including hyaluronan-binding proteins and hyaluronan-degrading enzyme polypeptides, (for example, “soluble hyaluronidase polypeptides) or other proteins, from host cells, will depend on the host cells chosen and expression systems. For secreted molecules, proteins are usually purified from the culture medium after removal of the cells. For intracellular expression, cells can be lysed and proteins purified from the extract. When transgenic organisms, such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material, to make an extract of lysed cells. In addition, the production of transgenic animals can include the production of polypeptides in milk or eggs, which can be collected, and if necessary, proteins can be extracted and further purified using standard methods in the art.
[0490] [0490] Proteins can be purified using protein purification techniques known in the art, including, but not limited to, SDS -PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, and ion exchange chromatography , such as anion exchange. Affinity purification techniques can also be used to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind to hyaluronan-binding proteins, or hyaluronidase enzymes, can be used in affinity purification. Expression constructs can also be modified to add an affinity marker for a protein, such as a myc epitope, TSG or His6 fusion and purified affinity with myc antibody, glutathione resin and Ni-resin, respectively. Purity can be assessed by any method known in the art, including gel electrophoresis and staining, and spectrophotometric techniques. Purified rHuPH20 compositions, as provided herein, typically have a specific activity of at least 70,000 to 100,000 units / mg, for example, about 120,000 units / mg. The specific activity may vary according to the modification, such as with a polymer.
[0491] [0491] Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine, mainly because PEG is a biocompatible, non-toxic water-soluble polymer that is normally non-immunogenic (Zhao and Harris, ACS Symposium Series680: 458 - 72, 1997). In the area of drug delivery, PEG derivatives have been widely used in covalent binding (i.e., "PEGylation") for proteins, to reduce immunogenicity, proteolysis and renal clearance, and to improve solubility (Zalipsky, Adv. Drug Del Rev. 16: 157-82, 1995). Similarly, PEG has been linked to low molecular weight, relatively hydrophobic drugs, to improve solubility, reduce toxicity and change biodistribution. Normally, PEGylated drugs are injected as solutions.
[0492] [0492] A closely related application is the synthesis of networked degradable PEG networks or formulations for use in drug administration, since much of the same chemistry used in the design of soluble degradable drug vehicles can also be used in the creation of degradable gels (Sawhney et al., Macromolecules 26: 581-87, 1993). It is also known that intermacromolecular complexes can be formed by mixing solutions of two complementary polymers. Such complexes are generally stabilized by electrostatic interactions (polyanion-polycation) and / or hydrogen bonds (polyacid-polybase) between the involved polymers, and / or by hydrophobic interactions between the polymers in the surrounding aqueous medium (Krupers et al., Eur. Polym J. 32: 785-790, 1996). For example, the mixture of solutions of polyacrylic acid (PAAC) and polyethylene oxide (PEO), under the appropriate conditions, results in the formation of complexes based mainly on hydrogen bonding. The dissociation of these complexes under physiological conditions has been used for the delivery of free (ie, non-pegylated) drugs. In addition, complementary polymer complexes have been formed from both homopolymers and copolymers.
[0493] [0493] Numerous PEGylation reagents have been described in the art. Such reagents include, but are not limited to, activated PEG N-hydroxysuccinimidyl (NHS), mPEG succinimidyl, mMPEG,; - N-hydroxysuccinimide, MPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mMPEG succinimidyl butane acid, mMPEG succinimyl butaneate succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, pEG-nitrophenyl carbonate PEG, mPEG-benzotriazole carbonate, PEG, MPEG butial; branched butyraldehyde, mPEG acetyl, MPEG piperidone, mPEG methyl ketone, mPEG “no binder”
[0494] [0494] In one example, polyethylene glycol has a molecular weight ranging from about 3 kDa to about 50 kD, and usually from about 5 kDa to about 30 kD. Covalent binding of PEG to the drug (known as "PEGylation") can be accomplished by known chemical synthesis techniques. For example, protein PEGylation can be accomplished by reacting the NHS-activated PEG with the protein under suitable reaction conditions.
[0495] [0495] While numerous reactions have been described for PEGylation, those that are most commonly applied confer directionality, use mild reaction conditions, and do not require extensive downstream treatment to remove toxic catalysts or by-products. For example, monomethoxy PEG (MPEG) has only one reactive hydroxyl terminal, and thus, its use limits some of the heterogeneity of the resulting PEG-protein product mixture. Activation of the hydroxyl group at the end of the polymer opposite the methoxy terminal group is generally necessary to perform an efficient protein PEGylation, in order to make the derivatized PEG more susceptible to nucleophilic attack. The nucleophilic attack is usually the epsilon-amine group of a lysyl residue, but other amines can also react (for example, N-terminal alpha-amine or histidine ring amines) if local conditions are favorable. More targeted binding is possible in proteins that contain a single lysine or cysteine. The last residue can be targeted by PEG - maleimide for the specific modification of the thiol. Alternatively, PEG-hydrazide can react with an enzyme that degrades oxidized hyaluronan with periodate, and is reduced in the presence of NaCNBH ;. More specifically, PEGylated CMP sugars can react with an enzyme that degrades hyaluronan in the presence of appropriate glycosyl transferases. One technique is the "PEGylation" technique, in which a number of polymeric molecules are coupled to the polypeptide in question. When using this technique, the immune system has difficulties in recognizing the epitopes on the surface of the polypeptide responsible for the formation of antibodies, thus reducing the immune response. For polypeptides introduced directly into the circulatory system of the human body to give a specific physiological effect (i.e. pharmaceuticals) the typical potential immune response is an IgG and / or IgM response, whereas polypeptides that are inhaled through the respiratory system ( for example, industrial polypeptide) can potentially cause an IgE response (that is, an allergic response). One of the theories that explain the reduction of the immune response is that the protective epitope (s) of the polymer molecule (s) on the surface of the polypeptide responsible for the immune response, leads to the formation of antibodies. Another theory, or at least a partial factor, is that the heavier the conjugate, the lower the immune response is obtained.
[0496] [0496] Normally, to produce the PEGylated hyaluronan degradation enzymes provided herein, including the PEGylated hyaluronidases, portions of PEG are conjugated, through covalent bonding, to the polypeptides. PEGylation techniques include, but are not limited to, specialized binders and coupling chemicals (see, for example, Roberts, Adv. Drug Deliv. Rev. 54: 459-476, 2002), linking multiple portions of PEG to a single conjugation site (such as through the use of branched PEG, see, for example, Guiotto et al., Bioorg. Med. Chem. Lett. 12: 177-180, 2002), site-specific PEGylation and / or mono-PEGylation (see, for example, Chapman et al., Nature Biotech. 17: 780-783, 1999), and site-directed enzymatic PEGylation (see, for example Sato, Adv. Drug Deliv. Rev., 54: 487-504, 2002). Methods and techniques described in the art can produce proteins with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, of 10 PEG or PEG derivatives linked to a single protein molecule (see, for example, example, USA 2006/0104968).
[0497] [0497] As an exemplary illustration method for making PEGylated hyaluronan degrading enzymes, such as PEGylated hyaluronidases, PEG aldehydes, succinimides and carbonates, each being applied each conjugating portions of PEG, PEG succinimidil normally, to rHuPH20. For example, rHuPH20 was conjugated to exemplary succinimidyl reagents - “monoPEG (MPEG), including mPEG-Succinimidyl Propionates (mMPEG-SPA), MPEG-Succinimidyl Butanoates (mPEG-SBA), and (“ branched ”PEGS for binding) mMPEG2-N - hydroxylsuccinimide. These PEGylated succinimidyl esters contain carbon structures of different lengths between the PEG group and the activated cross-linking agent, and a single PEG or branched group. These differences can be used, for example, to provide different reaction kinetics, and to potentially restrict sites available for PEG binding to rHuPH20 during the conjugation process.
[0498] [0498] Succinimidil PEGS (as above), which comprise either straight or branched PEGs, can be conjugated to rHuPH20. PEG can be used to generate reproducible rHuPH20s containing molecules that have, on average, between about 3-6 or 3-6 molecules of PEG per hyaluronidase. Such PEGylated rHuPH20 compositions can be easily purified to obtain compositions that have specific activities of about 25,000 or 30,000 units / mg of protein, hyaluronidase activity, and are substantially free of non-PEGylated rHuPH20 (less than 5% non-PEGylated) .
[0499] [0499] Using various PEG reagents, exemplary versions of hyaluronan-degrading enzymes, in particular soluble recombinant human hyaluronidase (eg, rHuPH20), can be prepared, for example, using mPEG-SBA (30 kD), mPEG-SMB (30 kD), and branched versions based on mPEG2-NHS (40kD) and mPEG2-NHS (60 kD). PEGylated versions of rHuPH20 were generated using NHS chemicals, as well as carbonates, and aldehydes, using each of the following reagents: mMPEG2-NHS-40K branched, mMPEG-NHS-10K branched, mPEG-NHS-20K branched, mPEG2-NHS-60K branched; mPEG-SBA-5K, mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB-20K, mPEG-SMB-30K; mPEG-butyraldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin. PEGylated hyaluronidases were also prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including PEGylated hyaluronidases with Dowpharma PEG-nitrophenyl carbonate (30 kDa) and PEG propionaldehyde (30 kDa).
[0500] [0500] In one example, PEGylation includes the conjugation of mPEG-SBA, for example, mPEG-SBA-30K (having a molecular weight of about 30 kDa) or other succinimidyl esters derived from butaneic acid PEG, to a hyaluronidase soluble. Succinimidyl esters of PEG butanoic acid derivatives such as mPEG-SBA-30K readily pair with protein amino groups. For example, the covalent conjugation of m-PEG-SBA-30K and rHuPH20 (which is about 60 kDa in size) provides stable amide bonds between rHuPH20 and mPEG, as shown in Scheme 1, below.
[0501] [0501] Usually, mPEG-SBA-30K or another PEG is added to the hyaluronan-degrading enzyme, in some cases, a hyaluronidase, in a 10: 1 PEG: polypeptide molar ratio in a suitable buffer, for example, 130 mM NaCl / 10 mM HEPES at pH 6.8 or 70 mM phosphate buffer, pH 7, followed by sterilization, for example, sterile filtration, and continuous conjugation, for example, with stirring, overnight at 4ºC in a cold room. In one example, the enzyme that degrades conjugated PEG hyaluronan is concentrated and has the buffer exchanged.
[0502] [0502] Other methods of succinimidyl esters for coupling PEG butanoic acid derivatives, such as mPEG -SBA-30K are known in the art (see for example, U.S. Patent No. 5,672,662; U.S. Patent No. 6,737,505; and 2004/0235734). For example, a polypeptide, such as an enzyme that degrades hyaluronan (for example, a hyaluronidase), can be coupled to an activated PEG derivative of NHS by reaction in a borate buffer (0.1 M, pH 8.0) for one hour at 4ºC. The resulting PEGylated protein can be purified by ultrafiltration. Alternatively, PEGylation of a bovine alkaline phosphatase can be achieved by mixing the phosphatase with mPEG-SBA in a buffer containing 0.2 M sodium phosphate and 0.5 M NaCl (pH 7.5) at 4ºC for 30 minutes. Unreacted PEG can be removed by ultrafiltration. Another method reacts polypeptide with mPEG-SBA in deionized water, so that triethylamine is added to increase the pH to 7.2-9. The resulting mixture is stirred at room temperature for several hours to complete the PEGylation.
[0503] [0503] methods for the PEGylation of polypeptides that degrade hyaluronan, including, for example, animal hyaluronidase and bacterial hyaluronan degrading enzymes, are known to a person skilled in the art. See, for example, European Patent No. EP 0400472, which describes the pegylation of bovine testes hyalurorase and chondroitin ABC-lyase. In addition, US Publication No. 2006014968 describes the PEGylation of a human hyaluronidase derived from human PH20. For example, the PEGylated hyaluronan degrading enzyme generally contains at least three portions of PEG per molecule. For example, the hyaluronan degrading enzyme may have a PEG to molar ratio of the protein 5: 1-9: 1, for example, 7: 1. G. METHODS OF EVALUATING ACTIVITIES AND MONITORING THE EFFECTS OF ANTI-HYALURONAN AGENTS
[0504] [0504] Anti-hyaluronan agents, for example, enzymes that degrade hyaluronan, such as a modified hyaluronidase or hyaluronidase (for example PH20 or PEGPH20) act as therapeutic agents, either alone, or in combination with secondary agents, such as chemotherapeutic agents, for the treatment of diseases and conditions associated with hyaluronan, in specific cancers (see, for example, USA 2010/0003238 and WOO09 / 128, 917). In addition, as described elsewhere here, therapy with an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, may be accompanied by treatment with a corticosteroid to minimize systemic side effects, for example, musculoskeletal, of PEGylated hyaluronidase. Follow-up diagnostics of HABP, such as TSG-6-LM or TSG-6-LM: Fc or its variants, provided herein, can be used in conjunction with therapy with an anti-hyaluronan agent, for example, a therapy of hyaluronan-degrading enzyme, used to treat diseases or disorders associated with hyaluronan, such as cancer, to control the responsiveness and effectiveness of treatment with the agent (for example, an enzyme that degrades hyaluronan). In addition, auxiliary or supplementary methods can also be used to assess the effects of anti-hyaluronan agents (eg, enzymes that degrade hyaluronan) from treatment, alone or in combination with corticosteroids. It is within the level of a person skilled in the art, to assess the improvement of side effects by corticosteroids, as well as studies of efficacy, tolerability and pharmacokinetics of anti-hyaluronan agent therapy, including hyaluronan-degrading enzyme therapy. This section provides a description of auxiliary or supplementary methods that can be used to assess the efficacy, responsiveness, tolerance and / or pharmacokinetics of a hyaluronan degrading enzyme therapy.
[0505] [0505] In vivo assays can be used to evaluate the effectiveness of corticosteroids in improving or eliminating musculoskeletal side effects that can be caused by an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, such as hyaluronidase or modified hyaluronidase, to expose a longer systemic half-life (for example PH20 or PEGPH20). Side effects that can be assessed include, for example, muscle and joint pain, stiffness in the upper and lower limbs, cramps, myositis, muscle pain and tenderness throughout the body, weakness, fatigue and / or a decrease in range of movement of knee and elbow joints. Trials to assess side effects may include animal models in which the animal can be observed for reduced movement, changes in behavior or posture, radiological findings, histopathological changes and other notable clinical observations. Other trials may include clinical trials in humans, in which patients may be asked about symptoms, assessed by physical examination, imaging (for example, MRI or PET) or by radiological evaluation. The improvement of a side effect caused by the administration of an anti-hyaluronan agent (for example, a hyaluronan-degrading enzyme agent) is seen when the side effect is improved, eliminated, reduced or decreased in the presence of the corticosteroid, compared to your absence.
[0506] [0506] In these examples, the dose of anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) and / or corticosteroids, can be varied to identify the optimal or minimum dose necessary to obtain the activity, improving side effects . Such studies are at the level of an expert in the art. In addition, the dosage regimen can be varied. For example, studies can be performed using a monthly, fortnightly, hyaluronan-degrading enzyme dosing schedule once a week, twice a week, three times a week, four times a week or more. In addition, the corticosteroid can be administered before, concurrently and / or subsequent to the administration of the anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan).
[0507] [0507] For example, in vivo animal models can be used to assess the ability of corticosteroids, such as dexamethasone, to improve or eliminate the side effects associated with the administration of the anti-hyaluronan agent (for example, the hyaluronan-degrading enzyme ). Animal models may include non-human primates, such as cynomolgus monkeys and rhesus monkeys, beagle dogs, for example, or any other animal that has adverse side effects, in response to treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan, such as PEGylated hyaluronidase, for example PEGPH20). Animal models can be treated with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) in the presence or absence of observed or measured corticosteroid and musculoskeletal effects.
[0508] [0508] For example, animals, such as cynomolgus monkeys, beagles or other similar animal models, capable of observable or quantifiable musculoskeletal events, can be treated with the hyaluronan degrading enzyme in the presence or absence of corticosteroids. In one example, a group of animals, for example, cynomolgus monkeys or beagles, are administered with an anti-hyaluronan agent, such as an enzyme that degrades hyaluronan alone, for example, a PEGylated hyaluronidase, such as by intravenous administration. For example, administration can be twice a week. Treatment may continue until changes in the range of motion of joints in the legs are seen in the knee and elbow, or stiffness or reduced mobility is observed. Then, another group of animals can be treated with the anti-hyaluronan agent (for example, the hyaluronan-degrading enzyme) and administered corticosteroids, such as by administering oral doses of dexamethasone or another corticosteroid, given on the same day. the anti-hyaluronan agent (eg, enzyme that degrades hyaluronan) was administered. Groups of animals can be compared, for example, through physical examination of the range of motion of the joints, or other reduced mobility,
[0509] [0509] In another example, the efficacy of corticosteroids, such as dexamethasone, in improving or eliminating adverse side effects associated with the administration of anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) can be evaluated in patients “ humans with solid tumors. For example, human patients can be dosed to examine the ability of corticosteroids to improve and / or eliminate adverse events mediated by the anti-hyaluronan agent (for example, mediated by enzymes that degrade hyaluronan), including, but not limited to any or more of the following characteristics: muscle and joint pain, stiffness of the upper and lower limbs, cramps, myositis, muscle pain and tenderness throughout the body, weakness, fatigue. Patients can be treated with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) with or without co-treatment with a corticosteroid, such as dexamethasone. During and after administration of an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), the side effects of both treatment groups can be assessed. A doctor can determine the severity of symptoms through physical examination of the individual, including, for example, the patient's complaints, vital signs, changes in body weight, 12-lead ECG, echocardiogram, clinical chemistry, or imaging (MRI, PET or radiological evaluation). The severity of symptoms can be quantified using the NCI common terminology criteria for the Adverse Event classification system (CTCAE). CTCAE is a descriptive terminology used for reporting adverse events (AE). The rating scale (severity) is provided for each AE term. CTCAE displays grades 1 to 5, with clinical descriptions of severity for each adverse event based on the following general guidance: Grade 1 (mild AE); Grade 2 (moderate AE); Grade 3 (severe AE); Grade 4 (life-threatening or disabling AE); and Grade 5 (AE-related death). The ability of a corticosteroid to improve adverse side effects associated with the administration of an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) can be measured by observing a reduction in severity or rating on the CTCAE scale by one or more side effects in individuals treated with the anti-hyaluronan agent (for example, the hyaluronan-degrading enzyme) and corticosteroids, compared to patients treated with the same anti-hyaluronan agent (for example, the hyaluronan-degrading enzyme) alone, or that is, the severity of side effects is reduced from grade 3 to grade 1 or grade 2.
[0510] [0510] In another example, human patients can be dosed to assess the tolerability of a dose escalation of an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) and to assess dose-limiting toxicity, as measured by severity side effects. In one example, the maximum tolerated dose of an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) that can be tolerated in the presence of an improving agent, such as a corticosteroid, can be determined. Treatment regimens may include dose escalation, in which each patient receives a higher dose of hyaluronan-degrading enzyme, at the same dose level as corticosteroids. Patients can be monitored to determine adverse effects for the highest dose of hyaluronan-degrading enzyme that can be administered with a corticosteroid, before side effects are not tolerated. Tolerability can be measured based on the severity of symptoms emerging during and after treatment. Doses of an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) can be scaled up until the adverse effects reach a predetermined level, for example, grade 3. Dosing regimens can also include a reduction in the amount of corticosteroids administered to examine the need to continue with the corticosteroid, and the possibility of acclimatization to the anti-hyaluronan agent in relation to the resulting side effects.
[0511] [0511] As described herein, the measurement and level of HA phenotypes is a biomarker that is associated with, and correlates with, the effectiveness and activity of an anti-hyaluronan agent (for example, the hyaluronan-degrading enzyme) . For example, for cancer patients with tumors, such as advanced solid tumors, associated with the tumor and reduced stroma, it is a biomarker of the activity of an administered enzyme that degrades hyaluronan. A HABP binding assay to detect HA present in tissue (eg, tumor biopsy) or body fluids (eg, plasma), as described here, elsewhere, can be performed to assess and monitor the therapeutic effect of a anti-hyaluronan (for example hyaluronan degrading enzyme).
[0512] [0512] In addition, the assays can be performed separately or in conjunction with the HABP assays described, used here, to detect HA in tissues (eg tumor biopsy) or body fluids (eg plasma) to assess better effects of an anti-hyaluronan agent (eg, an enzyme that degrades hyaluronan) on hyaluronic acid inhibition or degradation activity. In particular, for the treatment of a disease or conditions associated with hyaluronan, such as cancer, clinical measures or biomarkers associated with the activity of an anti-hyalruonan agent, for example, an enzyme activity that degrades hyaluronan include, but not are limited to, reduced metabolic activity of tumors, increased apparent diffusion and improved tumor perfusion and / or increased HA catabolites. Additional tests to measure such biomarkers may include, but are not limited to, measurements of hyaluronan catabolites in blood or urine, measurements of hyaluronidase activity in plasma, or measurements of interstitial fluid pressure, vascular volume or water content in tumors. It is within the skill of a person skilled in the art to perform such tests.
[0513] [0513] These tests can be performed on animal models treated with a hyaluronan degrading enzyme or on human patients. For example, animal models of diseases, disorders or conditions associated with hyaluronan can be used to assess the in vivo efficacy of administration of an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, such as hyaluronidase or modified hyaluronidase to display the increased half-life (for example, PH20 or PEGPH20). Another agent, such as a chemotherapeutic agent, can also be included in the assessment of activity. Examples of diseases associated with hyaluronic acid, for which an appropriate animal model can be used, include solid tumors, for example, types of cancer in advanced stage, metastatic cancer, undifferentiated cancer, ovarian cancer, carcinoma in situ (ISC ), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers. They are also examples of diseases and disorders associated with hyaluronan, inflammatory diseases, pressure on the disc, cancer and edema, for example, edema caused by organ transplantation, stroke, brain trauma or other injury.
[0514] [0514] Animal models may include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys and rhesus monkeys. In some instances, immunodeficient mice, such as hairless or SCID mice, are transplanted with a tumor cell line from a cancer associated with hyaluronan, to establish an animal model of the respective cancer. Examples of cancer cell lines associated with hyaluronan include, but are not limited to, PC3 prostate carcinoma cells, BxPC-3 pancreatic adenocarcinoma cells, MDA-MB-231 breast carcinoma, MCF-7 breast tumor cells , BT474 breast tumor cells, Tramp-C2 prostate tumor cells, and Mat-LyLu prostate cancer cells, and other cell lines described here, which are associated, for example,
[0515] [0515] In other examples, dogs, such as beagle dogs, can be treated with an anti-hyaluronan agent in the presence or absence of a corticosteroid, such as dexamethasone. Tissues such as skin or skeletal muscle tissue are biopsied and stained for hyaluronan and evaluated visually. The tissues of animals treated with an anti-hyaluronan agent alone, are then compared to the tissues of animals treated with the anti-hyaluronan and corticosteroid to measure the effect of corticosteroid on anti-hyaluronan activity.
[0516] [0516] Assays for the activity of an anti-hyaluronan agent, such as an enzyme activity that degrades hyaluronan, can also be performed in humans. For example, assays to measure a biomarker associated with anti-hyaluronan activity (for example, an enzyme that degrades hyaluronan) can be performed on human subjects known or suspected of having a disease or condition associated with hyaluronan (for example, cancer), and has been treated with an enzyme that degrades hyaluronan (eg PEGPH20).
[0517] [0517] The activity of an enzyme that degrades hyaluronan can be assessed using methods well known in the art. For example, the USP XXII hyaluronidase assay indirectly determines activity by measuring the amount of hyaluronic acid or hyaluronan that is not degraded, the substrate (HA) that remains after the enzyme can react with HA for 30 min. at 37 ° C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, MD). A hyaluronidase (USP) reference standard or National Formulary (NF) Standard Hyaluronidase solution can be used in an assay to determine the activity, in units, of any hyaluronidase. In one example, activity is measured using a micro turbidity assay. This is based on the formation of an insoluble precipitate, when hyaluronic acid binds to serum albumin. Activity is measured by incubating hyaluronidase or a sample containing hyaluronidase, for example, in blood or plasma, with sodium hyaluronate (hyaluronic acid) for a defined period of time (for example, 10 minutes) and then , precipitating undigested sodium hyaluronate with the addition of acidified albumin serum. The resulting sample turbidity was measured at 640 nm after an additional development period. The decrease in turbidity resulting from hyaluronidase activity on the sodium hyaluronate substrate is a measure of the enzyme activity of hyaluronidase.
[0518] [0518] In another example, hyaluronidase activity is measured using a microtiter assay, in which residual biotinylated hyaluronic acid is measured followed by incubation with hyaluronidase, or a sample containing hyaluronidase, for example, in blood or plasma (see for example, Frost and Stem (1997) Anal. Biochem. 251: 263-269, U.S. Patent Publication No. 20050260186). The free carboxyl groups in the hyaluronic acid glucuronic acid residues are biotinylated, and the biotinylated hyaluronic acid substrate is covalently coupled to a microtiter plate. After incubation with hyaluronidase, the residual biotinylated hyaluronic acid substrate is detected by an avidin-peroxidase reaction and, in comparison with the next reaction obtained with hyaluronidase patterns of known activity. Other assays for measuring hyaluronidase activity are also known in the art and can be used in the methods described herein (see, for example Delpech et al., (1995) Anal. Biochem. 229: 35-41; Takahashi et al., ( 2003) Anal. Biochem. 322: 257-263).
[0519] [0519] The ability of an active hyaluronan-degrading enzyme, such as a soluble modified hyaluronidase (eg, bound to PEG PH20) to act as a propagating or diffusing agent, for example, for chemotherapeutic agents, can also be assessed. For example, trypan blue dye can be injected, such as subcutaneously or intradermally, with or without an enzyme that degrades hyaluronan in the lateral skin on each side of hairless mice. The dye area is then measured, for example, with a microcalibrator, to determine the ability of the hyaluronan-degrading enzyme to act as a spreading agent (see, for example, U.S. Published Application No. 20060104968).
[0520] [0520] In another example, blood and urine can be collected at different time points during the patient's treatment and tested for hyaluronan catabolites. The presence of catabolites is indicative of hyaluronan degradation and is therefore a measure of hyaluronidase activity. The plasma enzyme can also be evaluated and measured over time after administration. For example, HA catabolites, which are degradation products of HA-disaccharides, can be evaluated using high performance liquid chromatography (HPLC) to separate and measure the areas of the saccharide peaks. Example 15 exemplifies this test.
[0521] [0521] The reduction in the metabolic activity of the tumor is associated with the activity of the anti-hyaluronan agent (eg, hyaluronic acid degrading enzyme). The metabolic activity of the tumor can be assessed using standard procedures known in the art. For example, positron emission tomography [18F] - fluorodeoxyglucose (FDG-PET) can be used. PET is a non-invasive diagnosis that produces images and quantitative parameters of perfusion, cell viability, proliferation and / or metabolic activity of tissues. The images resulting from the use of different biological substances (for example, sugars, amino acids, metabolic precursors, hormones) marked with positron-emitting radioisotopes. For example, FDG is a glucose analog and is absorbed by living cells through the first phases of the normal glucose pathway. In cancers, there is an increase in glycolytic activity resulting in the capture of FDG in the cancer cell. A decrease in FDG entrapment correlates with a decrease in tumor metabolic activity and anti-tumorigenic activity. Guidelines for PET imaging are known to a person skilled in the art, and should be followed by any physician or treatment technician.
[0522] [0522] Additional methods of assessing the activity of an anti-hyaluronan agent (eg, the hyaluronan-degrading enzyme) include assays that assess the diffusion of water in tissues. As discussed here at another time, tissues that generally accumulate hyaluronan, have a higher pressure of interstitial fluid than normal tissue, due to the concomitant accumulation of water. Thus, tissues that accumulate HA, such as tumors, have high interstitial fluid pressure, which can be measured by various methods known in the art. For example, diffusion magnetic resonance, such as ADC MRI or DCE MRI, can be used. Water diffusion can be assessed by these procedures, and is directly related to the presence of tissues rich in hyaluronic acid, such as solid tumors (see, for example, Chenevert et al. (1997) Clinical Cancer Research, 3: 1457-1466) . For example, tumors that accumulate hyaluronan have a noticeable increase in ADC MRI or DCE MRI because of an increase in perfusion. Such assays can be performed in the presence and absence of a hyaluronan degrading enzyme, and the results have been compared. Diffusion measurement method is a useful measure to assess cellular changes after these therapies.
[0523] [0523] The activity of an anti-hyaluronan agent (for example, enzymes that degrade hyaluronan) is associated with reduced tumor size and / or volume. The size and volume of the tumor can be monitored based on techniques known to a person skilled in the art. For example, the size and volume of the tumor can be monitored by radiography, ultrasound, necropsy, by using calipers, by microcCT or by * F-FDG-PET. The size of the tumor can also be assessed visually. In particular examples, the size of the tumor (diameter) is directly measured using calipers.
[0524] [0524] In other examples, tumor volume can be measured using an average of tumor diameter measurements (D) obtained by ultrasound assessments or calipers. For example, tumor volume can be determined using VisualSonics Vevo 770 high-resolution ultrasound or other similar ultrasound. The volume can be determined from the formula V = D "xn / 6 (for the diameter measured using calipers) or V = Dº xdxn / 6 (for the diameter measured using ultrasound, where d is the depth or thickness) For example, gauge measurements can be made of the length of the tumor (1) and the width (w) and the volume of the tumor calculated as length x width X 0.52. In another example, microCT scans can be used to measure the volume of the tumor (see for example, Huang et al. (2009) PNAS, 106: 3426-3430). As an example, rats can be injected with 74% injection of Optiray Pharmacy ioversol contrast medium (eg 741 mg of loversol / mL), anesthetized rats, and computed tomography performed using a MICROCAT 1A scanner or similar scanner (for example, IMTEK) (40 kV, 600 VA, 196 rotation steps, total angle or rotation = 196) The images can be reconstructed using the software (for example, software program RVA3; ImTeK). can be determined using the available software (for example, Amira 3.l software; Mercury Computer Systems). The volume or size of the tumor can also be determined based on the size or weight of a tumor.
[0525] [0525] The percentage of tumor growth inhibition can be calculated based on volume, using the equation% TGI = [1- (TnhW-To) - (Cn-Co)] x100%, where "T,", é The average tumor volume for the treatment group on day "n" after the last dose of hyaluronan-degrading enzyme; "TÁ" is the average tumor volume, in the treatment group on day O, before treatment; "Cn" is the average tumor volume for the control group corresponding to day "n"; and "CY" is the average tumor volume in the control group on day O, before treatment. The statistical analysis of the tumor volume can be determined.
[0526] [0526] pharmacokinetic or pharmacodynamic studies can be performed using animal models or can be performed during studies with patients to assess the pharmacokinetic properties of an anti-hyaluronan agent, for example, a degrading enzyme of hyaluronan, such as hyaluronidase or modified hyaluronidase (e.g., PEGPH20). Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys and rhesus monkeys. In some cases, pharmacokinetic or pharmacodynamic studies are performed using healthy animals. In other examples, studies are conducted using animal models of a disease for which hyaluronan therapy is considered, for example, animal models of any disease or disorder associated with hyaluronan, for example, a tumor model.
[0527] [0527] The pharmacokinetic properties of an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan, such as modified hyaluronidase) can be assessed by measuring parameters such as maximum concentration (peak) (Cmax), peak time (that is, when the maximum concentration occurs; Tmax), the minimum concentration (that is, the minimum concentration between doses; Cmin), the elimination half-life (T1, / 2) and the area under the curve (this ie, the area under the curve generated by time plotted versus concentration; AUC), after administration. The absolute bioavailability of the agent or enzyme (for example, a hyaluronidase) can be determined by comparing the area under the curve after subcutaneous delivery (AUCsc) with the intravenous delivery followed by AUC (AUCiv). The absolute bioavailability (F), can be calculated using the formula: F = ([AUC] sc * dosesc) / ([AUC] 14x dose ;,). A variety of doses and different dosing frequency can be administered in pharmacokinetic studies to assess the effect of increasing or decreasing the concentration of an anti-hyaluronan agent, for example, an enzyme that degrades hyaluronan, such as hyaluronidase or modified hyaluronidase (e.g. PH20 PEGylated) in the dose. H. KITS AND MANUFACTURING ITEMS
[0528] [0528] Provided here are kits for use in selecting patients for treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan), to predict the effectiveness of treatment with an anti-hyaluronan agent (for example an enzyme that degrades hyaluronan), to determine the prognosis of a patient with a disease associated with HA, or to monitor the effectiveness of treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) for the treatment of diseases associated with HA , in particular cancer. The kits provided here contain a HABP reagent provided here, for the detection and quantification of hyaluronan in a sample and, optionally, the reagents for carrying out the methods. For example, kits may additionally contain reagents for tissue collection, tissue preparation and processing, and reagents for quantifying the amount of HA in a sample, such as, but not limited to, detection reagents, such as antibodies, buffers, substrates for enzymatic staining, chromatography or other materials, such as slides, containers, microtiter plates and, optionally, instructions for carrying out the methods. Those skilled in the art will recognize many other possible containers and plates, and reagents that can be used to come into contact with different materials. Kits can also contain control samples representing tissues with different levels of HA, or colored reference samples, for the HA content for comparison and classification of test samples. The diagnosis of supplied HABP can be provided in a lyophilized or other, stable formulation of the diagnostic agent. In some examples, the kit includes a device, such as an automated cell imaging system (ACIS) fluorometer, luminometer or spectrophotometer for detecting the assay.
[0529] [0529] Also provided are combinations of a HABP reagent provided here, including improving the HABP reagents provided here, and an enzyme that degrades hyaluronic acid. As described herein, HABPs can be used as complementary diagnostic agents for treatment with a hyaluronan-degrading enzyme. Such combinations can optionally be packaged in kits for use in patient selection for treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) and treatment of such patients with the anti-hyaluronan agent (for example). example, an enzyme that degrades hyaluronan), to predict the effectiveness of treatment with an anti-hyaluronan agent (for example, an enzyme that degrades hyaluronan) in a patient and treatment of such patients with the anti-hyaluronan agent (for example enzyme that degrades hyaluronan), for determining the prognosis of a patient with a disease associated with HA, and treating such patients with anti-hyaluronan agent (eg, enzyme that degrades hyaluronan), or to monitor the effectiveness of a patient's treatment with an anti-hyaluronan enzyme (for example, an enzyme that degrades hyaluronan) for the treatment of diseases associated with HA, in particular cancer, and treatment of such patients with the anti-hyaluronan agent (eg enzyme that degrades hyaluronan) based on the effectiveness of the treatment.
[0530] [0530] The kits provided herein may also include reagents for detecting the expression of one or more additional proteins or encoding RNA in the sample, such as, for example, one or more additional cancer markers, such as, for example, but not limited to, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) CAl25, CAl9 -9, prostate specific antigen (PSA), human chorionic gonadotropin (HCG), HER2 / neu antigen, CA27.29, CYFRA 21-2 , LASA-P, CAl5-3, TPA, S-100 and CA-125. IT. EXAMPLES
[0531] [0531] The following examples are included for illustrative purposes only, and are not intended to limit the scope of the invention.
[0532] [0532] Chinese hamster ovary (CHO) cells were transfected with plasmid HZ24 (set out in SEQ ID NO: 52). The HZ24 plasmid vector for the expression of soluble rHuPH20 contains a pCI vector structure (Promega), the DNA encoding amino acids 1-482 of human PH20 hyaluronidase (SEQ ID NO: 49), an internal ribosomal entry site (IRES) of the ECMV virus (Clontech), and the rat dihydrofolate reductase (DHFR) gene. The pCI backbone vector also includes the DNA encoding the beta-lactamase resistance (AmpR) gene, the source of replication, a cytomegalovirus (CMV) immediate early enhancer / promoter region, a chimeric intron, and a signal of late polyadenylation of SV40 (SV40). The DNA encoding the soluble rHuPH20 construct contains an Nhel site and a Kozak consensus sequence before the DNA encoding methionine at the position of amino acid 1 of the native human PH20 35 amino acid signal sequence, and a stop codon in the sequence of the tyrosine-encoding DNA corresponding to the amino acid position 482 of the human PH20 hyaluronidase described in SEQ ID NO: 1), followed by a BamHI restriction site. The construct pCI-PH20-IRES-DHFR SV40pa (HZ24), therefore, results in a single species of mRNA directed by the CMV promoter that encodes amino acids 1- 482 of human PH20 reductase (set out in SEQ ID NO: 3 and amino acids 1-186 of rat dihydrofolate reductase (set out in SEQ ID NO: 53 separated by the internal ribosome entry site (IRES).
[0533] [0533] Non-transfected CHO cells are grown in GIBCO CD-CHO modified GIBCO media for DHFR (-) cells, supplemented with 4 mM glutamine and 18 ml / L of Plurionic F68 / L (Gibco), seeded at 0.5 x 106 cells / ml in a shaker vessel in preparation for transfection. The cells were grown at 37ºC in 5% CO; in a humidified incubator, shaking at 120 rpm. Exponentially, the growth of untransfected CHO cells was tested for viability before transfection.
[0534] [0534] Sixty million viable cells from the non-transfected CHO cell culture were pelleted and resuspended at a density of 2 x 10 th cells in 0.7 ml of 2 x transfection buffer ((2xHeBS: 40 mM Hepes, pH 7.0 , 274 mM NaCl, 10 mM KCl, 1.4 mM NaszHPOs, 12 mM dextrose) For each aliquot of resuspended cells, 0.09 mL (250 pg) of the linear HZ24 plasmid (linearized by digestion overnight with Cla I ( New England Biolabs) was added, and the cell / DNA solutions were transferred to 0.4 cm BTX (Gentronics) electroporation cuvettes at room temperature, a negative electroporation control was performed without the plasmid DNA mixed with the cells. cell / plasmid mixtures were electroporated with a 330 V and 960 npF capacitor discharge or at 350 V and 960 yupF.
[0535] [0535] The cells were removed from the cuvettes after electroporation and transferred to 5 ml of modified CD-CHO media for DHFR (-) cells, supplemented with 4 mM glutamine and 18 ml / L of Plurionic F68 / L (Gibco), and left to grow in a 6-well tissue culture plate, without selection for 2 days at 37ºC, in 5% CO, in a humidified incubator.
[0536] [0536] Two days after electroporation, 0.5 ml of tissue culture medium was removed from each well and tested for the presence of hyaluronidase activity, using the microturbity assay described in Example 3. Cells that express the highest levels high levels of hyaluronidase activity were collected from well-counted tissue culture and diluted to 1 x 10º to 2 x 10º of viable cells per mL. A 0.1 ml aliquot of the cell suspension was transferred to each well of five, 96-well round-bottom tissue culture plates. One hundred microliters of CD-CHO medium (GIBCO) containing 4 mM GlutaMAX * -1 supplement (GIBCO ”", Invitrogen Corporation) and, without hypoxanthine and thymidine supplements, were added to the wells containing the cells (final volume of 0, 2 mL).
[0537] [0537] Ten clones were identified from the 5 plates grown without methotrexate. Six of these HZ24 clones were expanded in culture and transferred to shake flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1El1, and 4D10 were plated in 96-well round-bottom tissue culture plates using a two-dimensional infinite dilution strategy, in which cells were diluted 1: 2 below the plate, and 1: 3 by the plate, from 5000 cells on the upper left side of the well. Diluted clones were cultured in a background of 500 non-transfected DG44 CHO cells per well, to provide necessary growth factors for the first days in culture. Ten plates were made per subclone, with 5 plates containing 50 nM methotrexate and 5 plates without methotrexate.
[0538] [0538] Clone 3D3 produced 24 visual subclones (13 from treatment without methotrexate, and 11 from treatment with 50 nM methotrexate). Significant hyaluronidase activity was measured in the supernatants of 8 of the 24 sub-clones (> 50 units / ml), and these 8 subclones were expanded into T-25 tissue culture flasks. Clones isolated from the methotrexate treatment protocol were expanded in the presence of 50 nM methotrexate. The 3D35M clone was expanded by 500 nM methotrexate in shaken flasks, and gave rise to clones producing more than 1000 units / ml of hyaluronidase activity (3D35M clone; or Genl 3D35M). A main cell bank (MCB) of 3D35M cells was then prepared.
[0539] [0539] The Genl 3D35M cell line described in Example 1A was adapted to higher levels of methotrexate, to produce generation 2 (Gen2) clones. 3D35M cells were seeded from cultures containing methotrexate, established in CD CHO medium containing 4 mM GlutaMAX-1 "and 1.0 µM methotrexate. The cells were adapted to a higher level of methotrexate, by growth, and passed 9 times for a period of 46 days at 37ºC, 7% humidified CO incubator. The population of amplified cells was cloned by limiting dilution in 96-well tissue culture plates containing medium with 2.0 µM methotrexate. 4 weeks, clones were identified and 3E10B clones selected for expansion. 3E10B cells were cultured in CD CHO medium containing 4 mM GlutaMAX-1 "and 2.0 µM methotrexate, for 20 passages. A main cell bank (MCB), of the cell line 3E110O0B was created and frozen, and used for further studies.
[0540] [0540] Cell line amplification continued by culturing 3E10B cells in CD CHO medium containing 4 mM GlutaMAX-1 "" º and 4.0 µM methotrexate. After the 12th pass, the cells were frozen in flasks as a research cell bank (RCB). One vial of the RCB was thawed and grown in a medium containing 8.0 µM methotrexate. After 5 days, the methotrexate concentration in the medium was increased to 16.0 µM, then 20.0 µM, after 18 days. Eighth pass cells in medium containing 20.0 µM methotrexate were cloned by limiting dilution in 96-well tissue culture plates containing CD CHO medium with 4 mM GlutaMAX-1 "” and 20.0 pM methotrexate. The clones were identified 5 to 6 weeks later, and clone 2B2 was selected for expansion in medium containing 20.0 pM methotrexate, after the 11th pass, 2B2 cells were frozen in flasks as a research cell bank (RCB).
[0541] [0541] The resulting 2B2 cells are deficient in dihydrofolate reductase (dhfr-) from DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20). Soluble PH20 is present in 2B2 cells in a copy number of about 206 copies / cell. Southern blot analysis of 2B2 genomic DNA from Spe I-, Xba I- and BamH I / Hind III digested, using a rHuPH20 specific probe, revealed the following restriction digest profile: a large hybridization band of - 7 , 7 kb and four smaller hybridization bands (- 13.9, - 6.6, - 5.7, and - 4.6 kb) with Spe I digested DNA; a large hybridization band of - 5.0 kb and two smaller hybridization bands (- 13.9 and - 6.5 kb) with XbaIl digested DNA; and a single - 1.4 kb hybridization band, observed using 2B2 DNA digested with BamH I / Hind III. Analysis of the mRNA transcript sequence indicated that the derived CDNA (SEQ ID NO: 56) was identical to the reference sequence (SEQ ID NO: 49), except for a base pair difference at position 1131, which was observed as being a thymidine (T) instead of the expected cytosine (C). This is a silent mutation, with no effect on the amino acid sequence. Example 2 Production and Purification of rHuPH20 A. Production of Soluble rHuPH20 Gen2 in a 300 L Bioreactor Cell Culture
[0542] [0542] A vial of HZ24-2B2 cells (Example 1B) was thawed and expanded from shaking vials through 36 vials in CD-CHO medium (Invitrogen, Carlsbady, CA) supplemented with 20 pnM of methotrexate and GlutaMAX- 1 "(Invitrogen). Briefly, a flask of cells was thawed in a 37ºC water bath, the medium was added, and the cells were centrifuged. The cells were resuspended in a 125 ml flask with 20 ml of medium fresh, and placed in a bath at 37ºC, 7% CO incubator. The cells were expanded to 40 mL in a 125 mL shaken flask.
[0543] [0543] A 400 L reactor was sterilized and 230 mL of CD-CHO medium was added. Before use, the reactor was checked for contamination. Approximately 30L of cells were transferred from the 36L spinner flasks to the 400L bioreactor (Braun) at an inoculation density of 4.0 x 10º viable cells per ml, and a total volume of 260 L. The parameters were adjusted for temperature 37ºC; Speed impeller 40-55 RPM; Container pressure: 3 psi; Air Sprayer 0.5-1.5 L / min .; Air Overlap: 3 L / min. The reactor was sampled daily for cell counting, pH checking, medium analysis, protein production and retention. In addition, during the run, nutrient feeds were added. In 120 hours (day 5), 10.4 L of t1 feed medium (4xCD-CHO + 33 9 / L Glucose + 160 mL / L Glutamax-17 + 83 mL / L Yeastolate + 33 mg / L rHulnsulin) were added. In 168 hours (day 7), 10.8 L of t2 feed (2xCD-CHO + 33 g / L Glycoser80 mL / L Glutamax-l1 "+167 mL / L Yeastolate + 0.92 g / L sodium butyrate) was added, and the culture temperature was changed to 36.5ºC. At 216 hours (day 9), 10.8 L of feed * 3 (1 x CD-CHO + 50 g / L Glucose + 50 mL / L Glutamax-1 "+ 250 mL / L Yeastolate + 1.80 g / L sodium butyrate) was added, and the culture temperature was changed to 36ºC. At 264 hours (day 11), 10.8 L of feed $ 4 (1 x CD-CHO + 33 g / L Glucose + 33 mL / L Glutamax-1 "+250 mL / L Yeastolate + 0.92 g / L of sodium butyrate) was added, and the culture temperature was changed to 35.5 ° C. The addition of feed media was observed to dramatically increase the production of soluble rHuPH20 in the final stage of production. The reactor was harvested at 14 or 15 days , or when cell viability dropped below 40%. The process resulted in a final productivity of 17,000 units per ml, with a maximum cell density of 12 million cells / ml. At harvest, the culture was sampled for mycoplasma, loading biological, endotoxin and viral in vivo and in vitro, Transmission Electron Microscopy (TEM) and enzymatic activity.
[0544] [0544] The culture was pumped by a peristaltic pump through four modules of the Millistak filtration system (Millipore) in parallel, each containing a layer of diatomaceous earth graded to 4-8 µm and a layer of graduated diatomaceous earth to 1.4-1.1 pm, followed by a cellulose membrane, then through a second single Millistak filtration system (Millipore), containing a layer of diatomaceous earth graded to 0.4-0.11 pm and a layer of diatomaceous earth graded to <0.1 µm, followed by a cellulose membrane, and then through a 0.22 μm final filter of sterile flexible bag for a single use with a capacity of 350 L. The collected cell culture fluid was supplemented with 10 mM EDTA and 10 mM Tris at a pH of 7.5. The culture was concentrated 10 x with a tangential flow filtration apparatus (TFF), using four Sartoslice TFF polyether sulfone (PES) filters with a 30 kDa cut-off molecular weight (MWCO) (Sartorius), followed by an exchange of the 10 x 10 mM Tris buffer, 20 mM Na2SO4, pH 7.5 for a 0.22 pm final filter, for a 50 L sterile storage bag.
[0545] [0545] The concentrated diafiltered crop was inactivated by viruses. Before viral inactivation, a solution of 10% Triton X-100, 3% tri (n-butyl) phosphate (TNBP) was prepared. The concentrated diafiltered crop was exposed to 1% Triton X-100, 0.3% TNBP for 1 hour in a 36 L reaction glass container, immediately before purification on the OQ column.
[0546] [0546] An ion exchange column (9 L resin, H = 29 cm, D = 20 cm) of Q-Sepharose (Pharmacia) was prepared. Washed samples were collected for pH determination, conductivity test and endotoxins (LAL). The column was equilibrated with 5 volumes of 10 mM Tris, 20 mM NaszSOa, pH 7.5. After viral inactivation, the concentrated diafiltered crop (Example 2A) was loaded onto the Q column at a flow rate of 100 cm / h. The column was washed with 5 volumes of 10 mM Tris, 20 mM Na2 SO4, pH 7.5 and 10 mM Hepes, 50 mM NaCl, pH 7.0 of the column. The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH 7.0 for a final 0.22 µm filter in a sterile bag. The fluid sample was tested for biological load, protein concentration and hyaluronidase activity.
[0547] [0547] Phenyl-Sepharose hydrophobic interaction chromatography (Pharmacia) was performed next. A Phenyl-Sepharose (PS) column (19-21 resin L, H = 29 cm, D = 30 cm) was prepared. The wash was collected and sampled for pH, conductivity and endotoxins (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate, CaCl, 0.1 mM, pH 7.0. The protein eluate from the Q-Sepharose column was supplemented with 2M ammonium sulfate, 1M potassium phosphate, and CaCl stock solutions, to obtain final concentrations of 5 mM, 0.5 M and 0, 1 mM, respectively. The protein was loaded onto the PS column, at a flow rate of 100 cm / h, and flow through the column collected. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl ,, pH 7.0 at 100 cm / h, and the wash was added to the flow through it. Combined with washing the column, the flow was passed through a final 0.22 µm filter into a sterile bag. The past flow was sampled for microbial load, protein concentration and enzyme activity.
[0548] [0548] An aminophenyl boronate column (ProMedics) was prepared. The wash was collected and sampled for pH, conductivity and endotoxins (LAL assay). The column was equilibrated with 5 volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The past PS stream containing purified protein was loaded onto the aminophenyl boronate column at a flow rate of 100 cm / h. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20 mM bicin, 0.5 M ammonium sulfate, pH 9.0. The column was washed with mM of bicin, 100 mM sodium chloride, pH 9.0. The protein was eluted with 50 mM Hepes buffer, 100 mM NaCl, pH 6.9, and passed through a sterile filter to a sterile bag. The eluted sample was tested for biological load, protein concentration and enzymatic activity.
[0549] [0549] The hydroxyapatite (HAP) column (BioRad was prepared. The wash was collected and tested for pH, conductivity and endotoxins (LAL assay). The column was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, CaCl,; 0.1 mM, pH 7.0 The purified aminophenyl boronate protein was supplemented with final concentrations of 5 mM potassium phosphate and CaCl; 0.1 mM, and loaded onto the HAP column at a rate flow rate of 100 in / h The column was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, CaCl; at 0.1 mM. The column was then washed with 10 m potassium phosphate. mM, pH 7, 100 mM NaCl, 0.1 mM CaCl The protein was eluted with 70 mM potassium phosphate, pH 7.0, and passed through a 0.22 µm sterile filter into a bag The eluted sample was tested for biological load, protein concentration and enzymatic activity.
[0550] [0550] The purified PAH protein was then passed through a virus removal filter. The sterile Viosart filter (Sartorius) was prepared, first, by washing with 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filtered buffer was sampled for pH and conductivity. The purified PAH protein was pumped through a peristaltic pump, through the 20nM virus removal filter. The protein filtered in 70 mM potassium phosphate, pH 7.0, was passed through a final 0.22 pm filter into a sterile bag. The viral filtered sample was tested for protein concentration, enzyme activity, oligosaccharides,
[0551] [0551] Hyaluronidase activity of soluble rHuPH20 in cell culture samples such as plasma, purification fractions and purified solutions was determined using a turbidimetry assay, which is based on the formation of an insoluble precipitate, when hyaluronic acid binds to serum albumin, or a biotinylated hyaluronic acid substrate assay, which measures the amount of enzymatically active rHuPH20 or PEGPH20, by digesting the non-covalently bound biotinylated hyaluronic acid (b-HA) substrate to microtiter plates multi-well plastic.
[0552] [0552] Soluble rHuPH20 hyaluronidase activity is measured by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a certain period of time (10 minutes) and then precipitating undigested sodium hyaluronate, with the addition of acidified serum albumin. The resulting sample turbidity was measured at 640 nm after a 30 minute development period. The decrease in turbidity resulting from the enzyme activity on the sodium hyaluronate substrate is a measure of the hyaluronidase activity of soluble rHuPH20. The method is performed using a calibration curve generated with dilutions of a standard working reference soluble rHuPH20 assay, and measurements of the sample activity are made in relation to this calibration curve.
[0553] [0553] Sample dilutions were prepared in enzyme diluting solutions. The enzyme diluent solution was prepared by dissolving 33.0 t + 0.05 mg of hydrolyzed gelatin in 25.0 ml of 50 mM PIPES reaction buffer (140 mM NaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of sterile water for injection (SWFI), and dilution of 0.2 mL of solution in 25% Buminate, for mixing and centrifugation for 30 seconds. This was carried out within 2 hours of use and stored on ice until needed. The samples were diluted to about 1 to 2 U / ml. Generally, the maximum dilution per step does not exceed 1: 100, and the initial sample size for the first dilution was not less than 20 pL. The minimum sample volumes required to perform the test were as follows: In the Samples process, FPLC fractions: 80 ul; tissue culture supernatants: 1 ml; Concentrated material: 80 1nL; Final or purified step material: 80 pl. Dilutions were made in triplicate in a 96-well low protein binding plate, and 30 µl of each dilution was transferred to Optilux black / light-bottom plates (BD Biosciences).
[0554] [0554] Dilutions of the known soluble rHuPH20 with a concentration of 2.5 U / ml were prepared in enzyme diluent solution, to generate a standard curve, and the Optilux plate was added in triplicate. Dilutions included O U / mL, 0.25 U / mL, 0.5 U / mL, 1.0 U / mL, 1.5 U / mL, 2.0 U / mL, and 2.5 U / mL. “Null reagent” wells that contained 60 1 “npL of diluent enzyme solution were included in the plate as a negative control. The plate was then covered and heated in a heating block for minutes at 37ºC. The cap was removed and the plate was shaken for 10 seconds. After stirring, the plate was returned to the heating block, and the MULTIDROP 384 liquid handling device was started with 0.25 mg / ml of a hot sodium hyaluronate solution
[0555] [0555] MULTIDROP 384 was prepared to stop the reaction by preparing the machine with working solution and changing the volume setting to 240 pl. (25 mL of serum stock solution [1 volume of Horse Serum (Sigma) was diluted with nine volumes of 500 mM acetate buffer solution, and the pH was adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM of acetate buffer). The plate was removed from the heating block and placed over the MULTIDROP 384, and 240 upL of working serum solutions were distributed to the wells. The plate was removed and shaken in a plate reader for 10 seconds. After a further 15 minutes, the turbidity of the samples was measured at 640 nm, and the hyaluronidase activity (in U / mL) of each sample was determined by adjustment with the standard curve.
[0556] [0556] The specific activity (units / mg) was calculated by dividing the hyaluronidase activity (U / mL) by the protein concentration (mg / mL).
[0557] [0557] The biotinylated hyaluronic acid assay measures the amount of enzymatically active rHuPH20 or PEGPH20 in biological samples by digesting a non-covalently high-molecular weight biotinylated hyaluronic acid (b-HA) substrate (- 1.2 megadaltons) attached to plastic multi-well microtiter plates. The rHuPH20 or PEGPH20 in standards and samples are left to incubate on a plate coated with b-HA at 37ºC. After a series of washes, remaining uncleaved / bound b-HA is treated with the horseradish peroxidase streptavidin conjugate (SA-HRP). The reaction between immobilized SA-HRP and the chromogenic substrate, 3.3 ', 5.5'-tetramethylbenzidine (TMB), produces a blue colored solution. After stopping the acid reaction, the formation of the yellow soluble reaction product is determined by reading the absorbance at 450 nm, using a microtiter plate spectrophotometer. The decrease in absorbance at 450 nm resulting from enzyme activity on the biotinylated hyaluronic acid (b-HA) substrate is a measure of the soluble rHuPH20 hyaluronidase activity. The method is performed using a calibration curve generated with dilutions of rHuPH20 or soluble PEGPH20 reference standard, and measurements of the sample activity are made in relation to this calibration curve.
[0558] [0558] The sample dilutions and the calibrator were prepared in assay diluent. The Assay Diluent was prepared by adding 1% v / v plasma banks (from appropriate species) to 0.1% (w / v) BSA in HEPES, pH 7.4. Everything was prepared daily and stored at 2 to 8ºC. Depending on the types of species, as well as the anticipated level of hyaluronidase, single or multiple dilutions were prepared to ensure at least that a sample dilution would fall within the range of the calibration curve. To guide the dilution selection of the test sample (s), the known information about the dose of hyaluronidase administered, route of administration, approximate plasma volume of the species, and the point in time were used to calculate the levels of hyaluronidase activity. .
[0559] [0559] In this example, rHuPH20 was pegylated by the reaction of the enzyme with linear methoxy poly (ethylene glycol) butanoic acid N-hydroxysuccinimidyl ester (mMPEG-SBA-30K). A. Preparation of mPEG-SBA-30K
[0560] [0560] In order to generate PEGPH20, rHuPH20 (which is approximately 60 kDa in size) was covalently conjugated to a linear methoxy poly (ethylene glycol) butanoic acid (MPEG-SBA-30K) N-hydroxysuccinimidyl ester molecular weight of approximately 30 kDa. The structure of mPEG-SBA is shown below, where n * 681. o entratonal- The n mMPEG-SBA IS
[0561] [0561] The methods used to prepare the mMPEG-SBA-30K that was used for PEGylated rHuPH20 are described, for example, in U.S. Patent No. 5672662. Briefly, mPEG-SBA-30K is made according to the following procedure .
[0562] [0562] A solution of ethyl malonate (2 equivalents) dissolved in dioxane is added dropwise to sodium hydride (2 equivalents) and toluene, under an atmosphere of nitrogen. Methane sulfonate mPEG (1 equivalent, MW 30 kDa, Shearwater) is dissolved in toluene and added to the above mixture. The resulting mixture is refluxed for approximately 18 hours. The reaction mixture is concentrated to half its original volume, extracted with 10% aqueous NaCl solution, extracted with 1% aqueous hydrochloric acid, and the aqueous extracts are combined. The collected aqueous phases are extracted with dichloromethane (3x), and the organic layer is dried over magnesium sulfate, filtered and evaporated to dryness. The resulting residue is dissolved in 1N sodium hydroxide, containing sodium chloride, and the mixture is stirred for 1 hour. The pH of the mixture is adjusted to about 3 by adding 6N hydrochloric acid. The mixture is extracted with dichloromethane (2 x).
[0563] [0563] The organic layer is dried over magnesium sulfate, filtered, concentrated, and placed over cold diethyl ether. The precipitate is collected by filtration and dried in vacuo. The resulting compound is dissolved in dioxane and refluxed for 8 hours, and then concentrated to dryness. The resulting residue is dissolved in water and extracted with dichloromethane (2 x), dried over magnesium sulfate, and the solution is concentrated by rotary evaporation and then poured into cold diethyl ether. The precipitate is collected by filtration and dried in vacuo. The resulting compound (1 equivalent) is dissolved in dichloromethane, and N-hydroxysuccinimide (2.1 equivalents) is added. The solution is cooled to 0 ° C and a solution of dicyclohexylcarbodiimide (2.1 equivalents in dichloromethane is added dropwise. The solution is stirred at room temperature for approximately 18 hours. The reaction mixture is filtered, concentrated and precipitated in diethyl ether The precipitate is collected by filtration and dried in vacuo to obtain the mPEG -SBA-30K powder which is then frozen at <-15 ° C.
[0564] [0564] To make PEGPH2O0, mMPEG-SBA-30K was coupled to the amine group (s) of rHuPH20 by covalent conjugation, providing stable amide bonds between rHuPH20 and mPEG, as shown below, where n * 681.
[0565] [0565] Prior to conjugation, the purified bulk rHuPH20 protein prepared in Example 2B was concentrated to 10 mg / mL using 10 KkDa polyethersulfone (PES) tangential flow filtration (PES) cassettes (Sartorius) with an area of 0.2 m filtration, and buffer exchanged against 70 mM potassium phosphate at pH 7.2. The concentrated protein was then stored at 2 to 8 ° C until use.
[0566] [0566] To combine rHuPH20, mMPEG-SBA-30K (Nektar) was thawed at room temperature, in the dark, for no more than 2 hours. Depending on the lot size, a 3 "sterile stir bar was placed in a 1 or 3 liter Erlenmeyer flask, and a rHuPH20 protein from exchanged buffer was added. Five grams of dry mPEG-SBA-30K powder per gram of rHuPH20 (10: 1 mPEG-SBA-30K molar ratio: rHuPH20) was added to the flask under a vacuum cover, and the mixture was mixed for 10 minutes, or until the mMPEG-SBA-30K was completely dissolved. agitation rate was created in such a way that centrifugation without foaming occurred.
[0567] [0567] The solution was then filtered under a class 100 cover by pumping the solution, through a peristaltic pump through a 0.22 mM polystyrene, cellulose acetate filter capsule (Corning 50 mL tubetop filter) to a new 1 or 3 liter Erlenmeyer flask, containing a 3 "sterile stir bar. The volume of the PEGPH20 reaction mixture was determined by mass (1 g / mL density) and 0.22 µm of a filter used for filtration was examined in a post-use integrity test.
[0568] [0568] The mixture was then placed on a stirring plate at 2 to 8ºC, and mixed for 20 + 1 hours in the dark. The agitation rate was again set in such a way that centrifugation occurred without foaming. The entire Erlenmeyer flask was wrapped in aluminum foil to protect the solution from light. After mixing, the reaction was stopped by adding 1 M glycine to a final concentration of 25 mM. The samples were removed from the vessel to test the pH and conductivity. The pH and conductivity were then adjusted by adding a solution of 5 mM Tris Base (5.65 L / L) and 5 mM Tris, 10 mM NaCl, pH 8.0 (13.35 L / L ) to proceed with Q Sepharose purification.
[0569] [0569] A QFF Sepharose ion exchange column (GE Healthcare) (Length = 21.5-24.0 cm, diameter = 20 cm) was prepared by equilibration with 5 column volumes (36 L) of 5 mM Tris, 10 mM NaCl, pH 8.0. The conjugate product was loaded onto the QFF column at a flow rate of 95 cm / h. The column was then washed with 11 L of equilibration buffer (5 mM Tris, 10 mM NaCl, pH 8.0) at a flow rate of 95 cm / h, followed by a wash with 25 L of equilibration buffer at a flow rate of 268 centimeters / hr. The protein product was then eluted with 5 mM Tris, 130 mM NaCl, pH 8.0 at a flow rate of 268 cm / h. The resulting purified PEGPH20 was concentrated to 3.5 mg / mL, using a 30 kDa polyethersulfone (PES) tangential flow filtration (TFF) filter cassette (Sartorius) with a 0.2 m filtration area, and buffer switched against 10 mM histidine, 130 mM NaCl, pH 6.5. The resulting material was tested for enzyme activity as described in Example 3. The rHuPH20 material PEGylated to a concentration of 3.5 mg / mL (final enzymatic activity of 140,000 U / mL) was filled, in 3 mL volumes, in 5 ml glass bottles with a siliconized bromobutyl rubber stopper and a frozen aluminum flip-off seal (frozen overnight in a freezer at -20ºC, and then placed at -80ºC for longer periods of storage). PEGylated rHuHP20 contained about 4.5 moles of PEG per mole of rHuPH20.
[0570] [0570] The PEGylated material rHuPH20 (PEGPH20) was analyzed by gel electrophoresis. Three batches of PEGPH20 made as described in Example 4A above, revealed an identical, unreacted, multiple-banded pattern representing PEG and various species of unreacted PEGPH20 conjugates, and migrated at different distances. Based on the comparison with the migration of a molecular weight marker, the bands representing the species ranged from about 90 kDa to 300 kDa, with three dark bands migrating above the 240 kDa marker. These data indicated that PEGPH20, generated by covalent conjugation of MPEG-SBA-30K, contained a heterogeneous mixture of PEGPH20 species, probably including mono-, di- and tri-PEGylated proteins. The lack of a visible band at 60 KDa suggests that all proteins that reacted with PEG, and that did not detect native rHuPH20, were present in the mixture. Example 5 Competence of tumor cells to form pericellular matrix and Relationship with the content of hyaluronan tumor cells (HA), Hyaluronan synthase levels (SAH) and hyaluronidase expression (Hyal) A. Comparison of hyaluronan acid content in tumor cells (AH), Expression of HAS1, 2, 3 and Hyal l1 and 2, and formation of the pericellular matrix
[0571] [0571] In this Example, the amount of endogenous HA synthesis enzymes, hyaluronan synthase (HAS) 1, 2, and 3, hyaluronidase (Hyal) 1 and (Hyal) 2, and the amount of hyaluronan accumulation (HA) , in tumor cells was compared with the presentation of each correlated with the formation of the pericellular matrix by the tumor cells.
[0572] [0572] Ten tumor cell lines of different tissue origin (eg, prostate, breast, ovary, pancreas and lung) and species origin (eg, human, rat and mouse) were examined in the study. The following cell lines were obtained from the American Type Culture Collection (ATCC): 4T1l mouse breast tumor (ATCC CRL-2539), human prostate adenocarcinoma PC-3 (ATCC CRL-1435), human pancreatic adenocarcinoma BxPC- 3 (ATCC CRL-1687), MDA MB 231 breast adenocarcinoma (ATCC HTB-26), Matilylu mouse malignant prostate carcinoma (ATCC JHU -92), human pancreatic adenocarcinoma AsPc -1 (ATCC CRL -1682), human prostate carcinoma DU-145 (ATCC HTB-81), and human pancreatic carcinoma MIA PaCa 2 (ATCC CRL -1420). ATCC cell lines were grown in recommended culture medium containing 10% FBS at 37ºC in a humidified incubator provided with 5% CO0 / 95% air. MDA-MB-23l-Luc cells (Cat. No. D3H2LN), which express the North American firefly luciferase gene, were purchased from Caliper Life Sciences Inc., and cultured in RPMI medium containing 10% FBS.
[0573] [0573] Cell lines DU-l145 / HAS2 and MDA-MB-231-Luc / HAS2 were generated by transducing cell lines DU-l45 and MDA-MB-23l-Luc with a retrovirus encoding hyaluronan synthase 2 ( HAS2) (SEQ ID NO: 195). To generate the HAS2 retroviruses, the His6 N-terminal hHAS2 cDNA (SEQ ID NO: 196) was inserted into the AvrII and NotI sites of the pLXRN vector (SEQ ID NO: 197; Clontech, Cat No. 631512), which includes the neomycin resistance, to create pLXRN-hHAS2 (SEQ ID NO: 201). The plasmid pLXRN-hHAS2 was then co-transfected with the envelope vector pVSV-L (SEQ ID NO: 198 Clontech, part of Cat. No. 631530) in GP-293 cells, using the Lipofectamine 2000 reagent (Life Technologies ). A DU-1445 Mock cell line was also generated by co-transfection of the empty plasmid pLXRN and the envelope vector pVSV-G.
[0574] [0574] The virus titer was determined by the quantitative PCR method (retro-X "qRT- PCR Titration Kit; Clontech, catalog No. 631453), using the following primers (Clontech Catalog No. * K1060-E): pLXSN 5 'primer (1398-1420):
[0575] [0575] To establish HAS2 expression cell lines, cancer cells confluent in 70% DU-145 and MDA MB 231 Luc, were incubated with a 60: 1 to 6: 1 ratio of retroviruses in DMEM (Mediatech) containing 10% of FBS for 72 hours. The cultures were maintained in a selective medium containing 200 µg / ml of G418. Cancer cells expressing stable HAS2 were generated after 2 weeks of conditional media selection of G418.
[0576] [0576] An assay based on hyaluronan-binding protein (HABP) was used to determine the amount of hyaluronan produced by tumor cells. Assays based on HABP are preferable to chemical methods, for the measurement of HA as a tumor microenvironment biomarker (TME) because HABP preferably detects HA composed of at least 15 (n-acetyl glucose - glucuronic acid) disaccharides, which they are competent to bind HA-binding proteins (see, for example, Haserodt S, et al. (2011) Glycobiology 21: 175-183).
[0577] [0577] Tumor cells were seeded to 1 x 10º cells in 75 cm flasks ” and incubated for 24 hours. Tissue culture supernatants were harvested for HA quantitation using an enzyme-linked HABP sandwich assay (R & D Systems, Catalog No. DY3614), which uses recombinant human aggrecan as an HA capture and detection reagent (domains G1-IGD-G2 recombinant human aggrecan, Val20-Gly676 Adhesion No. NP-037359 (SEQ ID NO: 202), with the 10-HIS tag C-terminal, R & D Systems, Catalog No. 1220-PG). The test for the detection of HA was performed according to the manufacturer's instructions. Briefly, the assay plates were coated with recombinant human aggrecan, and samples (i.e., tissue culture supernatants) containing HA were added to the plate (three independent replicates of each cell line were tested). The plates were washed and bound HA was detected using recombinant human biotinylated aggrecan. After removing the unbound probe, streptavidin conjugated to horseradish peroxidase (HRP) was added as a secondary detection reagent. After washing the plate, the bound HRP was detected by incubation with the 1: 1 substrate solution of H, 0.;) / Tetramethylbenzidine (R & D Systems) and quantified by optical density detection at 450 nm, using a microplate reader SpectraMax M3 Multi-Mode (Molecular Devices, CA). The concentration of HA in the culture medium for each type of tumor cell was expressed as the average concentration of HA (ng / mL) in culture medium (Table 5).
[0578] [0578] The RNA was extracted from cell sediment using an RNeasy Mini Kit (Qiagen GmbH) according to the manufacturer's instructions. The extracted RNA was then quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). Quantitative real-time PCR (aRT-PCR) using specific primers for the gene was used to quantify the levels of mMRNA for each hyaluronan and hyaluronidase synthase. QaRT-PCR primers were purchased from Bio Applied Technologies Joint, Inc., (San Diego,
[0579] [0579] For the PCR reactions, the samples were mixed with a mixture of iQ SYBR Green master (Bio-Rad) and the pairs of primers assigned to each gene. The PCR reactions were performed in a Bio-Rad Chromo 4 qPCR device. The synthesis of the first strand was carried out under the following conditions: 42ºC for 2 minutes for the DNA elimination reaction, 42ºC for minutes for reverse transcription, and 3 minutes at 95ºC during reverse transcriptase inactivation. For amplification, 3 minutes of initial denaturation at 95ºC, 45 cycles of 15 seconds of denaturation and annealing extension 1 minute at 58ºC were used. The CT value of gene expression from each sample was calculated through normalization with the internal maintenance GAPDH gene and the relative values were plotted. Table 5 lists the CT values for each tumor cell type for each of the assay genes.
[0580] [0580] Monolayer cultures of the ten cell lines were cultured and tested for the formation of the pericellular matrix facilitated by aggrecan. To view the pericellular arrays of HA mediated by agracane in vitro, particle exclusion assays were used as previously described in Thompson C B, et al. (2010) Mol Cancer Ther 9: 3052-3064, with some modifications. Briefly, cells were seeded at 1 x 105 cells per well in a six-well plate for 24 hours, and then treated with cell culture media alone or media containing 1000 U / ml of rHuPH20 at 37 ° C for 1 hour. Pretreatment with rHuPH20 inhibits the formation of the pericellular matrix; thus, it was used as a negative control for the formation of the pericellular matrix, for each cell type. The cells were then incubated with 0.5 mg / ml of bovine aggrecan (Sigma-Aldrich), at 37 ° C for 1 hour. Subsequently, the media was removed and replaced with 10º / ml 2% suspension of mouse red blood cells fixed with glutaraldehyde (RBC) isolated from Balb / c mice (Taconic, Hudson, NY), in PBS, pH 7.4. The particles were left to stand for 15 minutes. The cultures were then photographed with a phase contrast microscope, coupled with a camera scanner and an imaging program (Diagnostic Instruments). The particle exclusion area and cell area were measured using the SPOT Advance program (Diagnostic Instruments, Inc., Sterling Heights, MI). The area of the pericellular matrix was calculated as the area of the matrix minus the cell area, and expressed as a (Table 5).
[0581] [0581] The concentration of HA in conditioned medium, as determined by the HABP-based detection assay, correlated with the area of the aggrecan-mediated pericellular matrix formed by tumor cells in monolayer culture (Table 5, P <0.0029 ). In addition, cell lines that were modified to express hyaluronan synthase 2 (HAS2), DU-145 / HAS2 and MDA-MB-231 / HAS2, showed increased HA production, and increased formation of pericellular matrix in vitro , compared to the respective parental cell lines. In contrast, no correlation was found between pericellular matrix formation and relative levels of HAS1, 2 or 3 mRNA expression, or Hyal 1 or 2. These results indicate that the direct measurement of HA associated with the tumor cell specifically predicts a predictor for the formation of the pericellular matrix. TABLE 5. Quantification of HA production, formation of pericellular matrix, expression of SAH and Hyal in PME HA tumor cell lines in MRNA HAS mMRNA isoform ”isoform CV lines Hyaluronidase cell HAS1 HAS2 HAS3 Hyall Hyal2 of tumor MDA-MB- -
[0582] [0582] In this example, the concentration of hyaluronan in different tumor cell lines was assessed by immunohistochemical analysis and compared with the ability of PEGPH20 to inhibit tumor growth from xenograft tumors generated from tumor cell lines, and growth of tumor colonies from HA-rich tumor cell lineage.
[0583] [0583] Fourteen xenograft tumors derived from the tumor cell line were generated from the following tumor cell lines: DU-145 human prostate carcinoma (simulated ATCC HTB - 81 transfected with empty plasmid pLXRN, see Example 5.A .1), DU-1145 HAS2 (see Example 5.A.1), human breast adenocarcinoma MDA MB 231 (ATCC HTB-26), MDA-MB-231 NY Luc (D3H2IN, Caliper Life Sciences), MDA MB 231 Luc / HAS2 (see “Example
[0584] [0584] Atypical hairless (Taconic) nude / nude (NCR) or Balb / c (Harlan) or Balb / c six to eight week old mice (Shanghai Laboratory Animal Center, CAS (SLACCAS), see Example 7) housed in micro-isolators, in a controlled environment room on a 12-hour light / 12-hour dark cycle, and received sterile food and water ad libitun. All animal studies were performed according to approved IACUC protocols.
[0585] [0585] For the generation of tumors, the mice were inoculated with peritibial tumor cells (intramuscular injection adjacent to the periosteum of the right tibia), subcutaneously (sc, from the right rear leg), or in the breast fat pad according to Table 6, below.
[0586] [0586] For peritibial tumors, tumor volumes were determined using high-resolution VisualSonics Vevo 770 ultrasound. For subcutaneous tumors and breast fat pad, tumor volumes were calculated by measuring the gauge of the length (L) and width (W) of the solid tumor masses. The tumor volume (VT) was calculated as: (L x w) / 2. The animals were selected for treatment with PEGPH20 when tumor volumes reached - 400-500 mm. The animals were randomized into treatment and control groups (no 26 rats / group).
[0587] [0587] Treatment with PEGPH20 and analysis of tumor growth inhibition (TGI) were performed as described (Thompson et al.). The mice were treated with vehicle (10 mM histidine, pH 6.5, 130 mM NaCl) or PEGPH20 for 2-3 weeks, according to the schedule shown in Table 7. Tumor volumes were measured twice a week. When the tumor size exceeded 2,000 mr, the animals were removed from the study and humanely euthanized. PEGPH20 PEGPH20 Dose models Quant. by | Freq.de No. GT Number of (mg / kg) 0.1mL | dosage Doses (x day) | animals dose (iv) two 0.7% Dul45 Mock 15 10,000 U times 6 (das) 8 p / week two MDA MB 231 4.5 3,000 U times 6 0% (dl7) 5 p / week two MDA MB - veres D / 23% oo: week MDA two a3: MB 23 4.5 -3,000 U | times p / 5 to 10 Luc / HAS2 week (o; '6 two 18%, AsPc- 4.5 73,000 U | times p 4 (11) 9 week two 248 MIA PaCa 2 4.5 -3,000 U | times p (915) 9 week two 61º 4Q1 9 - oU times for 21º 5 4Q1 3.9 3,000 U | times p 6 (day) 5 week two 155 BxPC3 5 - oU times for From 7 BxPC3 4.5 3,000 U zes p 4 (of) week
[0588] [0588] The TGI for each tumor model was calculated based on the volume of the final day of the study, as shown in Table 7. The percentage of Tumor Growth Inhibition (TGI) for each respective tumor model was calculated using the following equation: STGI = [1- (Th-To) = (Cn-Co)] x100% where "Tn" is the average tumor volume for the treatment group (the animals that received PHylated rHuPH20) on day "n", after the last dose of PEGylated rHuPH20; "T, W" is the average tumor volume, from the treatment group on day O, before treatment; "Cr," is the average tumor volume for the control group corresponding to day "n"; and "Cy" is the average tumor volume in the control group on day 0, before treatment. The statistical analysis of tumor volumes between the control and treatment groups was performed using a one-way ANOVA test with a P value of P <0.05 defined as statistically significant.
[0589] [0589] At the end of the tumor growth inhibition study, each of the fourteen xenograft tumors generated were analyzed for HA content by histochemistry, using protein bound to biotinylated hyaluronic acid (B-HABP) as a probe for detection and digital quantification of HA.
[0590] [0590] the tumor tissues were harvested, fixed in a 10% solution of neutral buffered formalin (NBF), embedded in paraffin and cut into 5 mM sections. For histochemical analysis, the cuts were dewaxed and rehydrated. Endogenous peroxidases were blocked with a peroxide block solution (Invitrogen, CA, USA) for 2 minutes. Non-specific staining was blocked using 2% BSA in 2% normal goat serum PBS for 1 hour at room temperature (RT) before incubation with 2.5 pg / ml biotinylated protein bound to HA (B-HABP, Catalog No. 400763, Seikagaku, Tokyo, Japan) for 1 hour at 37 ° C., To confirm the specificity of the staining, a subset of sections was pretreated with rHuPH20 (1000 U / mL) in PIPES buffer (25 mM PIPES, NaCl at 70 mM, 0.1% BSA, pH 5.5) at 37 ° C for 1 h before the addition of B-HABP. After washing to remove the primary reagent, the samples were incubated with a solution of streptavidin horseradish peroxidase (BD Pharmingen, Catalog No. 550946) for 30 minutes at RT and detected with 3, 3'-diaminobenzidine (DAB; Dako, No. Catalog K3467). The sections were then contrasted in Gill's hematoxylin (Vector Labs, Catalog No. H-3401), dehydrated and mounted in Cytoseal 60 medium (American Mastertech).
[0591] [0591] An Aperio T2 ScanScope (Aperio) was used to generate high resolution images of the tissue cuts. The images were analyzed quantitatively with Aperio Spectrum software, using a brown pixel counting algorithm (HA) counter. The tissue nucleus in sections with less than 10% of tumor cells or more than 50% of necrotic tissue was excluded from the evaluation. PC3 xenograft tumor tissues (HA ”*) were used as a positive control. A proportion of the strong positive colored area (brown) to the sum of the total colored area was calculated and scored +3, +2, +1 or 0, when the proportion was more than 25%, 10-25%, less than 10, or O, respectively.
[0592] [0592] Spearman's correlation coefficient was used to assess the relationship between HA expression and response to PEGPH treatment20
[0593] [0593] The results shown in Table 8, compare the measured level of HA in tissue sections taken from the xenograft tumor, and the percentage of tumor growth inhibition (TGI) by PEGPH20. The results are from tumors treated with at least 1 mg / kg of PEGPH20. Doses above 1 mg / kg did not increase inhibition of tumor growth. In the PC-3 (HA * ) And BxPC3 (HA ) Animal models, no significant increase in efficacy was observed with doses greater than pg / kg and 100 pg / kg, respectively.
[0594] [0594] Despite the diversity of tumor cell types (human, murine and rat), there was a significant correlation (P <0.001, Spearman r = 8, n = 14) between the increase in the intensity of HA staining B-HABP-mediated and PEGPH20 in vivo anti-tumor activity (Table 8) TABLE 8: Intensity of HA staining in the tumor and corresponding PEGPH20-mediated growth inhibition Models Tumor Type TGI positive pixels (source) HA ( %) (%) [DU145S Mock ——Jca, Prostate (AU [MAB 23 [ca mama Do [in PA
[0595] [0595] The effect of increasing HA production in a tumor cell on increasing the sensitivity of tumors to treatment with PEGPH20 was further analyzed in tumor xenografts expressing exogenous hyaluronan synthase 2 (SAH). As shown in Example 5, the production of HA by the DU-1445 tumor cell line could be increased by transducing the cells with a gene encoding HAS2, which led to increased formation of the pericellular matrix in vitro. In addition, DUl42-HAS2 showed increased HA staining and increased tumor inhibition by PEGPH20, in the xenograft models previously described. In this example, the effectiveness of treatment with PEGPG20 over time was compared in the DU-145 xenograft against DU-145-HAS2.
[0596] [0596] Rat xenograft models were prepared as described above in Example 6A. Briefly, the mice were inoculated with both the DU-1445 / vector controls and the DU-145 / HAS2 cells, as shown in Table 6. When the tumors reached approximately 500 mm ”in size, the mice were divided into groups of treatment (n = 8) and treated only with vehicle or PEGPH20. For the treatment of PEGPH20, the mice were injected through the tail vein at a dose of 4.5 mg / kg, twice a week, for 3 weeks. The tumor volume was monitored by caliper measurement, as described above. Xenograft tumors were analyzed for HA content by histochemistry using protein bound to biotinylated hyaluronic acid (B-HABP), as described above, 24 hours after the last treatment with PEGPH20.
[0597] [0597] The DU-145 prostate tumor xenograft with HAS2 overexpression grew more aggressively in hairless mice than the parental cell line transfected with an empty vector (DU-l45 Mock), similar to previous reports (Table 7 ) (Thompson et al. (2010)). PEGPH20 inhibited tumor growth in DU-145-HAS2 tumors (TGI = 50%, P <0.001, n = 8), but not in DU-1445 vector control tumors (TGI = 0.7%, P <0 , 05, n = 8). In addition, B-HABP histochemical staining of PEGPH20 treated tumors showed a removal of HA in tumor samples, compared to control tumors. These data suggest that the accumulation of HA in ECM facilitates the development of the tumor, and this increase in the accumulation of HA associated with the tumor is related to the anti-tumor activity of PEGPH20.
[0598] [0598] In this experiment, the dose-dependent effect of PEGPH20 in inhibiting tumor growth of HA-rich tumors was examined. Rat xenograft models were prepared as described above in Example 6A. Briefly, the mice were inoculated with human pancreatic adenocarcinoma BxPC-3 (ATCC CRL - 1687) or with human prostate adenocarcinoma PC-3 (ATCC CRL-1435) according to Table 6. When did the tumors reach approximately 500 mm in size, the mice were divided into treatment groups (n = 10) and treated with vehicle alone or PEGPH20. For the PEGPH20 treatment, the mice were injected systemically into the tail vein at a dose of 0.01, 0.1, 1, 4.5 and 15 mg / kg (350, 3500, 35000, 157500 and 500000 L / kg, respectively) twice a week for 2 weeks. The tumor volume was monitored by caliper measurement, as described above.
[0599] [0599] The maximum effective dose of PEGPH20 has been found to be below 1 mg / kg. Significant tumor inhibition was observed at all doses of PEGPH20 in the PC-3 xenograft model (P <0.001 for 0.1, 1, 4.5, 15 mg / kg dose, P <0.01 for 0.01 mg / kg, compared to O vehicle) and for all doses greater than 0.01, in the BxPC-3 xenograft model (P <0.001 for 0.1, 1, 4.5, 15 mg / kg doses compared with the vehicle). There was no significant increase in efficacy at doses greater than 0.01 pg / kg (PC3, HA ) Or 0.1 pg / kg (BxPC3, HA ").
[0600] [0600] To determine whether PEGPH20 can inhibit the anchorage-independent growth and proliferation of cells rich in prostatic tumor hyaluronan (PC3) in vitro, a three-dimensional clonogenic assay was performed on the cells. PC3 cells, at approximately 80% confluence, were trypsinized, harvested, and washed once in complete medium. Cell density was adjusted for cells 8 x cells 10 / ml and suspended in MatrigelO (BD Biosciences, San Jose, CA) on ice. 0.025 ml of this cell / Matrigel O mixture was seeded in a 48 well cell culture plate that had been pre-coated with MatrigelO in 0.1 ml per well, and solidified at 37 ° C for 1 hour. For continuous exposure, for 17 days, to control the API buffer and various concentrations of PEGPH20, 0.6 mL / well of API buffer containing complete medium, 1, 3, 10 and 100 U / mL of PEGPH20 were added to the top the appropriate well. The wells were incubated at 37ºC, in a humidified atmosphere with 5% CO, in the air, for 17 days, fresh treatment medium, including the appropriate concentration of the enzyme, when appropriate, was replaced every 34 days during the period of 17 days.
[0601] [0601] On the 17th, colony growth was assessed by imaging with a Nikon Eclipse TE2000U inverted microscope attached to an Insight FireWire digital camera (Diagnostic Instruments, Michigan). The number of colonies and diameter of each colony in uM was measured using the ImageJ software (open source software, a publicly available program for viewing and analyzing images, for calculating the area and pixel value) and coupled calibration function ( colony volumes were calculated using colony diameter and using the formula: 4/3 nrº.
[0602] [0602] The average volume of well colonies for each condition was determined and the effects of PEGPH20 on colony volume evaluated by comparing the average volume of colonies in the control sample buffer (API (active pharmaceutical ingredient) (10 mM Hepes and 130 mM NaCl, pH 7.0), without enzyme) for samples that were incubated in the presence of PEGylated rHuPH20. Inhibitory ratios were calculated using the formula: (Average volume of control-average volume of treaties) / (average volume of control) * 100.
[0603] [0603] PEGylated rHuPH20 induced a dose-dependent inhibition of growth, as evidenced by the lower colony volume compared to the control. Based on proportions of inhibition calculated according to the above formula, cultures were incubated in the presence of
[0604] [0604] The ICs, of PEGPH20 in colony volume reduction, determined using the Graphpad PrismO 4 program (GraphPad Software, Inc., La Jolla, CA), was approximately 1.67 U / mL. The average number of colonies was 10.17 t + 1.56 per well in vehicle-treated (control) cultures, and 11.50 + 0.89 per well in cultures treated with 100 U / mL PEGPH20. The difference in the number of colonies was not significant between the control and the 100 U / mML cultures (n = 6, p> 0.05). These results indicate that PEGPH20 can inhibit the proliferation and / or survival of cancer cells rich in hyaluronan.
[0605] [0605] In an independent experiment, PC3 cells were seeded on the reconstituted basement membrane (matrigel) as described above, and continuously exposed to the vehicle or 0.1, 1, 10, 100, and 1000 U / ml PEGPH20 for 19 days . The images were then digitally captured, and the colony volume was assessed using the ImageJ program. The colony volume inhibition compared to the control was 22, 45, 63, 73 and 74%, respectively, (P <0.01 for 1 U / ml, P <0.001 for 10 U / ml and above in relation to to the vehicle, n = 3).
[0606] [0606] Previous work has shown that elevated HA accumulation occurs in non-small cell lung cancer (NSCLC) (Hernández JIJ R, et al. (1995) Int 3 Biol Markers 10: 149-155 and Pirinen R, et (2001) Int 3 Cancer 95: 12-17). In contrast, cell lines of NSCLC derivatives exhibit low levels, suggesting that NSCLC cell lines lose HA expression during in vitro passage. Thus, the expression of HA in biopsies of primary tumors was examined.
[0607] [0607] A tissue microarray panel (TMA) of 190 NSCLC biopsies (USA Biomax, Inc.) was examined for histotype and HA accumulation. The HA content was determined by histochemical staining of B - HABP, as described in Example 6. Samples were scored as 3, 2, 1 or O, when the proportion of the area of the strong positive stain (brown) to the sum of the total of colored area was more than 25%, 10-25%, less than 10% or 0, respectively. In this panel, cell types of adenocarcinoma (ADC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC) were observed at frequencies of 32%, 51% and 3%, respectively, classified based on the pathology diagnosis provided by US Biomax (Table 9). Other unidentified subtypes correspond to about 11% of the 190 samples examined.
[0608] [0608] Analysis of the accumulation of HA associated with a tumor showed that all histotypes have subsets of cells that express the HA phenotype with high HA ”, with an overall rate of about 27% (Table 9). In particular, 40% of SCC cases that exhibited the HA phenotype ”, while 11% of ADC cases and 33% of LCC cases, were scored as HA '*. 34% of SCC cases exhibited the HA “phenotype, while 48% of ADC cases and 50% of LCC cases were scored as HA” P.
[0609] [0609] In order to prospectively test the relationship between HA overexpression and the PEGPH20-mediated HA depletion NSCLC antitumor response, tumor explants from human NSCLC patients with different degrees of HA accumulation were selected and evaluated for to the ability to respond to PEGPH20 treatment in a xenograft tumor model. Primary explants characterized by HA accumulation were used for this study, as the explant models contain a more representative sample of the genetic diversity of the tumors intact, and must maintain aspects of the native tumor stroma.
[0610] [0610] Tumor biopsies were obtained from sixteen patients with NSCLC, and maintained a low number of passages subcutaneously in hairless mice (Crown Bioscience, Beijing, China). NSCLC tumor explants were screened for HA accumulation in explant tissues from passages 1-4, and were assigned to an HA phenotype (i.e., 1, 2 or 3) by histochemical staining of B-HABP , as described above. Three squamous cell explants (SCC) were prospectively selected for xenograft transplantation, representing the HA * (LUM697), HA * (LUM330) and HA ”(LUMBS8).
[0611] [0611] When the tumors sown for the selected tumor explants reached 500-700 mm3 in size, the mice were sacrificed, and the tumors were extracted and chopped into 3x3x3 mm fragments. A fragment of each tumor was implanted subcutaneously in the rear right flank of a female mouse without Balb / c (n = 10 for each group), as shown in Table 6. Tumor volumes were determined by calibrated measurements of the largest longitudinal diameter (length (L)) and the largest transverse diameter (width (W)) and estimated by calculating (L x W) / 2. When did the average tumor size reach 500 mm (range 300-600 mm ), the animals were randomly divided into two groups. For therapy, animals were treated with vehicle or PEGPH20 at 4.5 mg / kg, twice a week for five doses, as shown in Table 7, above. The percentage of tumor growth inhibition (TGI%) and statistical analysis were performed as described in Example 6.
[0612] [0612] The HA phenotype order ranking (ie, 1, 2 or 3) as determined by histochemistry was found to predict the degree of inhibition of tumor growth by PEGPH20 (Table 9). For example, the percentage inhibition of growth was 97% for LUM697 (HA), 44% for LUM330 (HA + 2), and 16% for LUM858 (HA + 1). In addition, tumor regression was observed in the LUM697 (HA) tumor explant group, but not in the LUM330 (HA ) And LUM858 (HA) groups: 4 out of 10 animals with LUM697 (HA) tumors had decreased tumor compared to pre-therapy.
[0613] [0613] To test whether depletion of HA has antiproliferative effects on tumor cells in vivo, tumor xenografts PC-3 (HA +3) treated with PEGPH20 were examined for levels of DNA synthesis.
[0614] [0614] Atypical nude / nu (NCR) mice from six to eight weeks of age were intraperitoneally implanted with PC-3 tumor cells, as described in Example 6 (cells 1 x 10 in 0.05 ml per mouse ). The tumor volume was monitored by caliper measurement. When the tumors reached + 400 mmº, the mice were treated with either vehicle or PEGPH20 (1 mg / kg (35,000 U / kg) or 4.5 mg / kg (157,500 U / kg); about 700 U / dose or 3150 U / dose based on 20 g of body weight of the rat, at 100 pl, by injection into the tail vein, twice a week, for two weeks.The mice were administered 10 mg / kg 24 hours before the end of the study. of BrdU (0.2 mL) (Invitrogen, Cat. t 00-0103) intraperitoneally, the tumors were excised from the mice, fixed in 10% buffered formalin and embedded in paraffin. The tissues were cut into 5 mM sections , and cell proliferation was assessed after staining with an anti-BrdU antibody (BrdU Staining Kit; Invitrogen, Cati $ 93-3943) according to the manufacturer's instructions.
[0615] [0615] Animals treated with PEGPH20 were compared to animals treated with vehicle. A 58.3% reduction in synthetically active nuclei was observed in tumors treated with PEGPH20, compared to tumors treated with the vehicle (the percentage of BrdU positive nucleus was reduced from 4.8% to 2%). This result resembles the observed xenograft inhibition of prostate growth PC3 (HA!) Or pancreatic BxPC3 (HA *) as a result of treatment with PEGPH20 (- 50% TGI in doses of 1 mg per kg or more) (see Example 6 ).
[0616] [0616] Previous studies have shown that Treatment of HA tumors ”with PEGPH20 has a dramatic effect on tumor interstitial fluid pressure (IFP), and therefore the fluid pressure differential between the tumor and the external environment ( see Thompson et al. (2010)). Physical changes in the TME can have an impacting gene expression (Shieh AC (2011) Ann Biomed Eng 39: 1379-1389). In order to test whether HA removal has an impact on the volume or expression of TME proteins, such as murine collagen I (Collal), murine collagen VíCol5al), and tenascin C (TNC), which are found in remodeling the active matrix, were examined.
[0617] [0617] Tumor tissues with adjacent skin from PC-3 tumors generated in Example 8A were fixed in 10% neutral buffered formalin for 48 hours, processed using a tissue processor (Tissue-TEKVIP,
[0618] [0618] NCR nude / nude mice bearing PC3 tumors were produced and treated with vehicle or PEGPH20 as described in Example 6A. The animals were sacrificed 8 and 48 hours after treatment with vehicle or PEGPH20. Tumor tissues were excised under sterile conditions and quickly frozen in liquid nitrogen. The total RNA was isolated from frozen tissue, in accordance with Asuragen's usual operating procedures. The purity and quantity of the total RNA samples were determined by absorbance readings at 260 and 280 nm, using a NanoDrop ND-1000 UV spectrophotometer. The integrity of the total RNA was qualified by Agilent Bioanalyzer 2100 microfluidic electrophoresis. The samples for mRNA profile studies were processed by Asuragen, Inc. using Affymetrix Mouse 430 Arrays
[0619] [0619] Collal specific reduction in PC-3 tumors was observed after depletion of HA by treatment with PEGPH20. An 80% reduction compared to Collal staining for vehicle-treated tumors was observed (P <0.05 t test). Collal staining on the skin of mice treated with PEGPH20, however, remained stable. In addition, decreased levels of murine (stromal) mRNAs for Collal, Col5al, and TNC were observed as measured by mRNA expression array analysis. TNC ARNM was influenced more significantly (66% reduction) followed by Collal (53% reduction) and Col5al (45% reduction). These results suggest that HA depletion resulted in significant changes in protein expression within the TME.
[0620] [0620] A fusion protein, TSG-6-LM-Fc, containing the TSG-6 binding module and the IgG Fc domain was generated. A mutant TSG-6-LM-Fc / AHep fusion protein in which the heparin binding region of the TSG-6 binding module has been mutated has also been generated.
[0621] [0621] The de novo DNA synthesis (GenScript, NJ) was used to generate the nucleic acid encoding the TSG-6-LM-Fc fusion protein. The nucleic acid contains a DNA encoding a human immunoglobulin light chain leader kappa (x) signal peptide sequence (SEQ ID NO: 210), a 669 bp long cDNA fragment, of human IgGl heavy chain ( Gl No. 5,031,409; SEQ ID NO: 203, which codes for the peptide sequence shown in SEQ ID NO: 204) and a 285 bp long cDNA fragment from the human TSG-6 binding module region ( SEQ ID NO: 216, which codes for the peptide sequence shown in SEQ ID NO: 207, which corresponds to amino acid positions 35 to 129 of the TSG-6 pre-protein, Gl No. 315139000, set out in SEQ ID NO: 205 (mRNA) and SEQ ID NO: 206 (protein)). The human IgGl heavy chain and regions of the human TSG-6 binding module were linked with an Agel 6 bp restriction enzyme cleavage site, and a 12 bp sequence, GACAAAACTCAC (SEQ ID NO: 208) which encodes four amino acids additional (DKTH; SEQ ID NO: 209), originally published as part of the IgGl Fc sequence ((Nucleic Acids Research, 1982, Vol. 10, p4047). Two unique restriction enzyme cleavage sites, Nhel at the 5 'end and BamHI at the 3 'end, were synthesized to flank the fusion protein sequence.The synthesized fragment has a sequence shown in SEQ ID NO: 217. The fragment was codon optimized for enhanced protein expression, and synthesized by synthesis of new DNA The optimized codon fragment has a sequence set out in SEQ ID NO: 211. The protein sequence for the TSG-6-LM-Fc fusion protein is described in SEQ ID NO: 212.
[0622] [0622] the synthesized optimized codon fragment was inserted through Nhel and BamHI cleavage sites into the bicistronic mammalian expression vector pHZ24 IRES (SEQ ID NO: 52) using well-known DNA recombination procedures (by restriction enzymes and binding reagents obtained from New England
[0623] [0623] In order to improve the specificity of binding to (HA), to reduce the binding to other GAG chains, a construct that encodes a mutant fusion protein, TSG-6-LM-Fc / AHep, which contains 3 mutations from lysine to alanine at amino acid positions 55, 69, 76 of the constructed TSG-6 linker. The mutations reduce the heparin-binding activity of the TSG-6 binding module, while not affecting the HA-binding activity (see Mahoney DJ et al. (2005) J Biol. Chem. 280: 27044-27055), which reports 10 times less heparin-binding activity for the triple mutant; K20A / K34A / K41A at the heparin binding site). TSG-6-LM-Fc / AHep was generated by mutagenesis of the nucleic acid fragment encoding the fusion proteinTSG-6-LM-Fc and insertion into the pHZ24 IRES vector, to generate pHZ24-TSG-6-LM-Fc / AHep (SEQ ID NO: 218). The sequence of the TSG-6-LM-Fc / AHep fragment is set out in SEQ ID NO: 214, which encodes the TSG-6-LM-Fc / AHep fusion protein set out in SEQ ID NO: 215.
[0624] [0624] FreeStyle CHO-S suspension cells (Invitrogen) were used for expression of the TSG-6-LM-Fc and TSG-6-LM-Fc / NHep fusion proteins. The FreeStyle CHO-S suspension cell line was maintained in CHO-S CD (Invitrogen) culture medium prior to transfection. For the preparation of cells for transfection and recombinant protein expression, Freestyle CHO-S cells were cultured in Freestyle expression medium (Invitrogen) supplemented with 8 mM L-glutamine in shaking flasks at 37ºC., In a humid atmosphere of CO; 8% in air, on an orbital shaking platform at 125 rpm of rotation, with loose bottle caps to allow aeration.
[0625] [0625] Transient transfection of cells in suspension was performed according to the manufacturer's instructions. Briefly, the cells were divided at a density of 6 x 10º / ml 24 hours before transfection, and transfected using lipid FreeStyle Max with a DNA / lipid ratio in the ratio of 1: 1. After 96 hours post-transfection, cells were harvested at 4,000 g for 20 min., And supernatants were harvested. An analysis of the temporal evolution of the protein expression level during post-transient transfection revealed that the protein expression level reached a plateau after 96 hours after transfection. Thus, the recombinant protein was harvested 96 hours post-transfection.
[0626] [0626] The TSG-6-LM-Fc and TSG-6 LM-Fc / AHep fusion proteins expressed in the collected supernatants were affinity purified by protein A resins (Bio-r Rad, Hercules, CA) according to manufacturer's instructions. Briefly, the collected supernatants were adjusted to pH 7.4, 0.15 M NaCl with 1 M Tris-HCl, pH 7.4 (Teknova Catalog No. T1074) and 5 M NaCl (Sigma) and diluted 3 times with buffer, before being loaded onto a protein A column. The eluted product was immediately neutralized with 1M Tris-HCl, pH 8.5, and dialyzed against a balanced phosphate solution (PBS, 137 mM NaCl, 2 KCl , 7 mM, NazHPOs, 8 mM, KH> PO, 1.46 mM, and pH 7.4) at 4ºC, and stored at -20ºC. The yield of purified proteins from the supernatants by means of a single Protein A affinity column step was between 3 to 5 mg / liter.
[0627] [0627] The purity, size and identity of the purified fusion protein was determined by SDS-PAGE 4-20% gradient gel under conditions of reduction and non-reduction, and Western Blot analysis. 60 ng of purified protein was used in the analysis. The size of the purified fusion proteins was about 40 kDa under reducing conditions, and about 80 kDa under non-reducing conditions, indicating that the expressed proteins form homodimers through disulfide bonds in the IgG Fc hinge region. The purity of the protein samples was greater than 95%. The purified proteins were stable in PBS for at least one month at 4 ° C, without any visible degradation, or loss of binding activity.
[0628] [0628] The identity of the TSG-6 binding module in TSG-6-LM -Fc and TSG-6-LM-Fc / AHep was evaluated by Western blot with goat anti-human IgG TSG-6 antibodies (R & D Systems, Inc., Minneapolis, MN) followed by rabbit anti-goat IgG-HRP antibody (EMD, San Diego, CA). Recombinant human full-length TSG-6 protein (R & D Systems, Inc., Minneapolis, MN) was used as a positive control. The pattern of proteins detected by Western blot analysis under reducing and non-reducing conditions was the same as the SDS-PAGE analysis, except for a small amount of upper bands observed under the non-reducing condition, probably representing tetramers of recombinant proteins with based on its molecular weight size.
[0629] [0629] The identity of the Fc portion in the purified recombinant proteins was confirmed by Western blot analysis with rabbit anti-human IgGFc HRP (Jackson ImmunoResearch, West Grove, PA). The pattern of detected proteins was the same as for the SDS-PAGE and anti-TSG-6 analyzes, which indicates that the purified proteins contain both the TSG-6 binding module (LM) as well as hIgGFc.
[0630] [0630] To analyze whether proteins were glycosylated, the purified proteins were treated with PNGase F glycosidase (0.5 units per ng of protein), which removes N-linked oligosaccharides from proteins, and analyzed by SDS-PAGE and Western blot. A difference of 5 kDa in molecular weight of proteins was observed between before and after treatment with PNGase F, which indicates that the expressed proteins were glycosylated.
[0631] [0631] Two formats were used to test the binding of both TSG-6-LM-Fc and its mutant for HA and heparin. In one format, the connection of TSG-6-LM-Fc and TSG-6 LM-Fc / NHep to immobilized HA or heparin on a microplate was used. In the second format, the binding of biotinylated HA and heparin to immobilize the recombinant TSG-6-LM-Fc and TSG-6-LM-Fc / AHep proteins in a microplate was used.
[0632] [0632] Fusion homodimers of the TSG-6-LM Fc and wild-type mutant were tested for their binding activities to HA and heparin, using microplates coated with heparin E and also HA. Briefly, hyaluronan with an average MW of about 1000 kDa (Lifecore, Chaska, MN) or heparin with an average MW of 15 kDa (Calbiochem, San Diego, CA), at a concentration of 100 pg / ml in sodium carbonate buffer of 0 , 5 M, pH 9.6, were poured into 96 well plates, in duplicate, with 100 pnl / well, and incubated at 4ºC overnight. The plates were blocked with 1% BSA in PBS to reduce non-specific binding. The purified TSG-6-LM-Fc and TSG-6-LM-Fc / AHep protein samples were diluted to give a range of concentrations from 0.31 to 40 ng / ml for binding to the HA coated plate, 0, 78 to 100 ng / ml for binding to heparin coated plate. For each sample, 100 µl per well in duplicate was added to microplates and incubated at room temperature for 1 hour. The plates were washed with 0.05% Tween 20 PBS, 5 times, to remove unbound protein. TSG-6-LM-Fc and TSG-6-LM-Fc / AHep bound to AH and heparin were detected with rabbit anti-human IgG Fc-HRP antibody (Jackson ImmunoResearch, West Grove, Pa.), Followed by substrate of TMB (3.3 ', 5.5'-tetramethylbenzidine) (KPL, Gaithersburg, MD). The samples were incubated for 60 minutes with rabbit anti-human IgG Fc-HRP antibody. After washing, HRP binding was detected with a TMB solution for 10-15 minutes of development time, followed by the addition of phosphoric acid reagent to stop color development. Absorbance was measured at DO450 using a Spectra M3 spectrophotometer from Molecular Devices.
[0633] [0633] Both TSG-6 -LM -Fc and TSG-6-LM-Fc / AHep exhibited the same binding activity on the HA coated plate; and their HA-binding activity titration curves were almost overlapping, indicating that the two expressed HA-bound proteins, with high affinity, based on EC50 values, from HA-binding titration curves. The triple mutation at the heparin binding site has no effect on HA binding. In contrast, the binding of the two proteins to the heparin-coated plate showed a significant difference. The wild type of TSG-6-LM-Fc bound to heparin, albeit with relatively low binding activity, compared to its binding to HA, which may be due to the difference in size of the two GAG chains coated on the plates. The TSG-6-LM-Fc mutant protein exhibited about 10% of heparin binding activity compared to wild type, which was consistent with the result reported for the triple mutant TSG-6-LM (Mahoney DJ et al. (2005) B. Binding of biotinylated HA and Heparin to recombinant immobilized TSG-6 -LM -Fc and TSG-6-LM-Fc / AHep
[0634] [0634] The wild-type GAG binding properties of TGS6 -LM -Fc and TSG-6-LM-Fc / AHep were further examined by coating the microplates with recombinant proteins and assessing their binding to biotinylated HA and heparin.
[0635] [0635] For the preparation of the microplates, TSG-6-LM-Fc and TSG-6-LM-Fc / AHep at a concentration of 2 µg / ml in 1 x PBS buffer were distributed in 96-well plates, in duplicate, with 100 nl / well, and incubated at 4ºC overnight. The plates were blocked with 1% BSA in PBS to reduce non-specific binding.
[0636] [0636] The purified TSG-6-LM-Fc and TSG-6-LM-Fc / AHep protein samples were diluted to give a concentration range of 0.31 - 40 ng / ml for binding to the HA coated plate 0.78 to 100 ng / ml for binding to the heparin-coated plate. 100 pl per well of each sample in duplicate, was added to the microplate and incubated at room temperature for 1 hour. TSG-6-LM-Fc and TSG-6-LM-Fc / NHep linked to HA and heparin were detected with anti-human IgG Fc-HRP (Jackson ImmunoResearch, West Grove, Pa.), Followed by TMB substrate (3 , 3 ', 5,5'-tetramethylbenzidine) (KPL, Gaithersburg, MD).
[0637] [0637] By biotinylation of HA, the carboxyl groups in HA were used for conjugation through chemical hydrazide. Briefly, biotin-hydrazide was dissolved in DMSO at a concentration of 25 mM, and added at a volume ratio of 6: 100 in a HA solution, containing 1000 kDa or 150 kDa of molecular weight of HA (Lifecore Biomedical, LLC Chaska, MN) at 1 mg / ml in 0.1 M MES, pH 5.0. 1-Ethyl- 3- [3-dimethylaminepropyl] carbodiimide hydrochloride (ECD) and sulfo-N-hydroxysuccinimide (sulfo-NHS) were added to the conjugation reaction at a concentration of 40 µM and 850 µM, respectively, to mediate conjugation biotin-hydrazide and HA. The reaction was maintained at 4 ° C overnight, with stirring. The excess amount of chemicals was removed from biotinylated HA by dialysis. Biotinylated heparin was purchased from EMD, San Diego (Catalog No. 375054).
[0638] [0638] Hyaluronan or biotinylated heparin were diluted in PBS with a concentration range of 0.78 ng / ml to 100 ng / ml, 100 p1pl / well dispensed, and incubated at room temperature for 1 hour. The plates were washed with 0.05% Tween 20 PBS, 5 times to remove unbound protein. Bound biotinylated hyaluronan and heparin were detected with anti-streptavidin-HRP (Jackson ImmunoResearch, West Grove, PA), followed by TMB substrate (3.3 ', 5.5'-tetramethylbenzidine) (KPL, Gaithersburg, Md. ) as described above. Absorbance was measured at OD450.
[0639] [0639] The binding results observed were similar for the binding assay performed in Example 10A, which used immobilized HA and heparin and free TSG-6-LM-Fc and TSG-6-LM-Fc / AHep. There was no difference in binding activity of TSG-6-LM-Fc and TSG-6-LM-Fc / AHep immobilized for biotinylated HA, or in the binding titration curves between TSG-6-LM-Fc and TSG-6 -LM-Fc / AHep, and a significant reduction in the binding of the mutant TSG-6-LM-Fc / AHep to biotinylated heparin, compared to that of the wild-type protein, was also observed. Therefore, the HA and heparin binding properties of wild type TSG-6-LM - Fc and its mutant can be evaluated, both in the recombinant protein coated and GAG coated formats; and both formats revealed similar connection patterns.
[0640] [0640] The HA-binding affinity of TSG-6 -LM -Fc was measured using Bio-Layer Interferometry (BLI) technology using the Octet Qke instrument (ForteBio, Menlo Park, CA). The recombinant full-length TSG-6 protein (R & D Systems, Inc., Minneapolis, MN) was used as a control. Briefly, biotinylated HA with an average molecular weight of 150 kDa was immobilized in streptavidin-coated biosensors for 240 seconds. TSG-6-LM-Fc and TSG-6- LM-Fc / AHep were then associated with immobilized HA for 180 seconds, in different concentrations in PBS at pH 6.0 or pH 7.4, followed by dissociation of proteins bound in PBS at pH 6.0 or pH 7.4 for 240 seconds. The results of the binding kinetics were analyzed with the software provided by the manufacturer. The results for the calculated binding affinity are shown in Table
[0641] [0641] The HA and heparin GAG binding sites of the TSG-6 binding module are located in different regions of the binding module. In order to determine whether the two binding sites would interfere with each other during interaction with the TSG-6 binding module or in the presence of other GAG chains, a competitive inhibition assay was performed to assess the binding of HA or heparin in the presence of other GAG chains.
[0642] [0642] 96-well microplates coated with HA and heparin were prepared as described in Example 10A. TSG-6-LM-Fc and TSG-6-LM-Fc / AHep, at a concentration of 40 ng / ml for HA coated plates, and 100 ng / ml for heparin coated plates, were preincubated with four different GAG chains: HA (Lifecore Biomedical, LLC Chaska, MN), chondroitin sulfate A (EMD, San Diego, California, Catalog No. 230687) chondroitin sulfate C (EMD, San Diego, California, Catalog No. 2307 ) and heparin sulfate (EMD, San Diego, California, Catalog No. 375095) in three different concentrations (0.11, 0.33, 1.0 ug / ml) or without GAG chain as a control, at room temperature for 10 minutes. The samples were then distributed (100 pl) in duplicate to the 96-well microplates coated with HA and heparin, and incubated at room temperature for 1 hour. The plates were washed with 0.05% Tween 20 PBS, 5 times, to remove unbound protein. TSG-6-LM-Fc and TSG-6-LM-Fc / AHep were detected with anti-human IgG Fc-HRP (Jackson ImmunoResearch, West Grove, PA), followed by
[0643] [0643] For the HA coated plate, both TSG-6 -LM -Fc and TSG-6-LM-Fc / AHep revealed similar competitive inhibition patterns. The binding of TSG-6-LM-Fc to immobilized HA was effectively inhibited by preincubating the same amount of protein with different doses of free HA (about 68%, 85%, and 93% inhibition for doses 0, 11, 0.33, 1.0 pg / ml doses, respectively), but was not affected by pre-incubation with different doses of free heparin or free chondroitin sulfate C. Some inhibition of TSG-6-LM-Fc and TSG-6-LM-Fc / AHep was observed for pre-incubation with chondroitin sulfate A, although it was less than HA (about 23%, 43%, and 63% of inhibition for doses 0.11, 0.33, 1.0 pg / ml doses). Thus, approximately 10 times more chondroitin sulfate A was needed for inhibition. (In independent experiments up to 30 times the amount of chondroitin sulfate A, was necessary for inhibition in relation to HA). Because TSG-6-LM-Fc and TSG-6-LM-Fc / AHep have shown similar inhibition with pre-incubation with chondroitin sulfate A, the HA-binding site in the TSG-6 module is likely to be responsible for binding to chondroitin sulfate A.
[0644] [0644] For heparin coated plates, the binding of TSG-6-LM-Fc to heparin was efficiently inhibited, not only through pre-incubation with heparin, but also through pre-incubation with both HA and sulfate chondroitin A. These data show that the binding of TSG-6-Fc-LM to HA can block its heparin binding activity. As expected, the TSG-6-LM-Fc / AHep mutant does not bind to heparin and, consequently, exhibited readings close to those for both control and pre-incubation samples.
[0645] [0645] This study demonstrates that the binding of the TSG-6 binding module to HA is not affected by the presence of free heparin or a preformed TSG-6-heparin complex, while its binding to heparin is significantly inhibited by the presence of free HA or TSG-6 preformed HA. Based on these observations it is concluded that TSG-6-LM binds to HA and heparin simultaneously, or the binding of TSG-6-LM to HA is stronger than its binding to heparin. The formation of the HA and TSG-6-LM complex can cause changes in the conformation of the protein or other modalities of the protein that are not favorable for its binding to heparin.
[0646] [0646] In this example, the specificity and binding activity of TSG-6-LM-Fc, TSG-6-LM-Fc / AHep and the binding protein (HABP) for HA, heparin, and other GAGs were compared. For this experiment, biotinylated HA-binding proteins, TSG-6-LM-Fc and TSG-6-LM-Fc / AHep HA were generated and compared to the commercially available biotinylated HA-binding protein (HABP) (Seikagaku, Tokyo , Japan) for its binding activity on coated GAG chain plates.
[0647] [0647] A random labeling approach was used to conjugate biotin to residues containing primary amine (Lys) in the protein directly, without pre-incubation with free HA, in order to protect the HA binding sites. By biotinylation of TSG-6-LM-Fc and TSG-6-LM-
[0648] [0648] For comparison, TSG-6-LM-Fc and TSG-6-LM-Fc / AHep proteins were also biotinylated using the targeted labeling method, which combines the biotin units for sugar chains on the proteins, for example oxidation of the polysaccharide chain of the protein, using NaIO, followed by biotin-hydrazide. Briefly, 1 ml of protein at a concentration of 1 mg / ml in 0.1 M phosphate buffer, pH 7.2, was oxidized for the first time by sodium periodate (NaIO04) to a final concentration of 5 mg / ml, at 4ºC for 30 minutes. The reaction converts the two adjacent primary hydroxyl groups into sugars for the corresponding reactive aldehyde groups. The oxidized protein was dialyzed against 0.1 M phosphate buffer, pH 7.2. The dialysed protein was then mixed with 50 mM hydrazide-biotin prepared in DMSO, volume ratio of
[0649] [0649] After conjugation and removal of free biotin, the HA binding activity of biotin-TSG-6 -LM - Fc and biotin-TSG-6-LM-Fc / AHep was tested together with corresponding unlabeled proteins for examine whether the labeling would cause a reduction in HA binding activity, using the binding assay, as described in Example 10A, using HA coated plates. No difference was found in HA-binding activity between unlabeled proteins vs. labeled.
[0650] [0650] For the preparation of coated microplates GAG, HA, heparin, chondroitin sulfate A, or chondroitin sulfate C, at a concentration of 100 pg / ml in 0.5 M sodium carbonate buffer, 100 pL were dispensed / well, in duplicate 96-well plates, and incubated at 4ºC overnight. The plates were blocked with 1% BSA in PBS to reduce non-specific binding. The three biotinylated proteins, TSG-6-LM-Fc, TSG-6-LM-Fc / NHep and biotinylated HABP were diluted to concentrations of 0.05 to 100 ng / ml to bind to HA, chondroitin sulfate coated plates A and chondroitin sulfate C, and 0.23 to 500 ng / ml for binding to heparin coated plates. Diluted protein samples were dispensed into the plates, 100 µl / well, in duplicate, and incubated at room temperature for 1 hour. Proteins bound to GAG-coated plates were detected with streptavidin-HRP (Jackson ImmunoResearch, West Grove,
[0651] [0651] All three biotinylated GAG binding proteins showed strong HA binding activity on the HA coated plate. At a protein concentration of 11.1 ng / ml, which represents a dilution less than the maximum binding concentrations (ie 33.3 ng / ml and 100 ng / ml) of HA, the binding of TSG-6-LM -Fc and TSG-6-LM-Fc / AHep biotinylated for HA, was approximately 14 times on the background, and the B-HABP binding to HA was approximately 9 times on the background.
[0652] [0652] Biotinylated HABP and TSG-6-LM-Fc / NHep exhibited little binding activity against the wild-type biotinylated plate.-TSG-6-LM-Fc also showed negative heparin binding activity, which suggests that the NHS-PEG4-biotin random labeling approach caused a loss of heparin-binding activity. When TSG-6-LM-Fc was biotinylated by the targeted labeling approach, as described above, heparin binding was restored and the protein exhibited heparin-binding activity similar to unlabeled TSG-6-LM-Fc. Thus, the biotin modification of lysines at the heparin site of TSG-6-LM-Fc could abolish its heparin binding activity.
[0653] [0653] All three proteins showed no binding activity to the chondroitin sulfate coated plate, but showed strong binding to the chondroitin sulfate coated plate A. TSG-6 -LM -Fc and biotin-TSG-6-LM Biotinylated-Fc / AHep have been observed to exhibit sometimes greater binding activity than biotin-HABP. At a protein concentration of 11.1 ng / ml, the binding of biotinylated TSG-6-LM-Fc and TSG-6-LM-Fc / AHep to the
[0654] [0654] The concentration of hyaluronan was determined in clinical samples of human plasma, using a sandwich binding assay. Plasma samples were obtained from 19 patients with solid tumors and various types of advanced stage tumors who were enrolled in a clinical study (Phase 1-101 and Phase 1-102, see table 11) by assessing the PEGPH20 dosage escalated in patients, in the presence or absence of dexamethasone. In addition, plasma samples were also obtained from twenty (20) normal patients (obtained from BioReclamation, Hicksville, NY). Before treatment with PEGPH20, baseline HA levels were determined as follows.
[0655] [0655] 96-well Immulon 4HBX flat-bottom microtiter plates (Immulon / Thermo; Catalog N * 3855) were coated with a recombinant human aggrecan
[0656] [0656] Before incubating the plate with the sample, plasma samples and a standard curve were prepared. Briefly, the plasma test samples were obtained and stored at <€ 60ºC, until analysis. Immediately before analysis, the test samples were thawed on wet ice and quickly mixed by centrifugation immediately before dilution. Then, several serial dilutions of test plasma sample dilutions were prepared to ensure that at least one sample dilution was within the range of the calibration curve, by diluting with Reagent Diluent (PBS Tween solution -20 to 5%, prepared by adding 6.5 ml of Tween-20 (Sigma; Catalog No. P7949) to 123.5 ml of phosphate buffered saline (PBS; Cellgro; Catalog No. 21-
[0657] [0657] For the standard curve, a stock of hyaluronan (132 kD), 1,800 ng / ml; R & D Systems, Catalog No. 842.164) was diluted by serial dilution in reagent diluent (5% Tween 20 in PBS) to final concentrations of 500 ng / ml, 167 ng / ml, 55.6 ng / ml, 18.5 ng / ml, 6.2 ng / ml, 2.1 ng / ml, and 0.68 ng / ml. A well containing blank reagent diluent was also included in the standard.
[0658] [0658] Then, at the end of the blocking step, each well was washed five (5) times with 1 x “PBST (1 x PBS, 0.05% Tween 20) wash buffer, using the ELX405Select CW plate washer. The test samples, controls and standard curve were added to the coated plate and blocked by adding 100 pL of each, in triplicate, to the wells of the plate. The plate was covered with an adhesive plate sealer and incubated at room temperature for approximately 2 hours. After incubation, each well was washed five (5) times with 1 x PBST wash buffer (1 x PBS, 0.05% Tween 20), using the ELXx405Select CW plate washer.
[0659] [0659] To detect the binding of HA to the coated rHu- aggrecan, a biotinylated rHuAggrecan detection reagent (72 pg / ml, R & D Systems, Catalog No. 842163) was added to the plate.
[0660] [0660] Based on the DO450 nm value, the concentration of intact hyaluronan for each sample was determined by interpolation from the standard curve. The results were multiplied by the sample dilution factor. The data were presented as the average of all values within the limits of quantification of the calibration curve, in ng / ml. The results are shown in Tables 11 and 12. The results show that the mean plasma HA in healthy humans was 0.015 ug / ml, whereas in phase 1 individuals, it was 0.06 ug / ml. This represented a statistically significant difference with p <0.0001. TABLE 11. HA in plasma of individuals with tumors Result. Individual Tumor Type Sex (ng / mL FE gar e Es 9 o [E 12 61 T lung cancer 348.3 non-small cell 1
[0661] [0661] Histochemical detection of AH was obtained from a pre-biopsy tumor specimen and a post-treatment metastatic liver biopsy sample, from a 4-week biopsy sample from the patient dosed with 1.6 u1pg / kg PEGPH20 + dexamethasone. The pre-dose biopsy (pre-biopsy) was an archived sample obtained in 2007 (3.5 years prior to treatment with PEGPH20). The post-treatment biopsy sample was obtained 3 days after the last dose (8th dose) in a treatment regimen of PEGPH20 plus dexamethasone, from a female colon cancer patient with liver metastases. Specifically, the patient's post-treatment biopsy was obtained after a PEGPH20 treatment cycle at 1.6 pg / kg of a weekly program, twice for the administration cycle, with co-treatment with dexamethasone. The treatment cycle was defined as a period of 28 days, with PEGPH20 administered intravenously (IV) and dexamethasone administered orally. On each dosing day, a pre-medication regimen of 4 mg dexamethasone was administered orally one hour before PEGPH20, followed by a second dose of 4 mg dexamethasone 8 to 12 hours after dosing PEGPH20.
[0662] [0662] Briefly, tumor biopsies were fixed in normal buffered formaldehyde (NBF) and 5 µm section slices, and stained using a biotin-labeled hyaluronan-binding protein (HABP-bio) (Seikagaku, Japan). After washing to remove the primary reagent, a labeled secondary reagent was used. The cores were counterstained using a DAPI reagent (4 ', 6-diamidine-2-phenylindole). The micrographs were captured using an inverted Nikon Eclipse TEZ000U fluorescence microscope, coupled to an Insight FireWire digital camera (Diagnostic Instruments, Michigan) or ZEISS overhead scope (Carl Zeiss, Inc.), which has the same imaging system.
[0663] [0663] Histochemical staining of samples with biotinylated HA-binding protein demonstrated a decrease in pericellular and stromal HA levels after a PEGPH20 treatment cycle. The results are summarized in Table 13. The H score represents the relative intensity of pericellular and stromal AH. The data demonstrate the ability of PEGPH20 to degrade HA associated with a tumor, as demonstrated by a reduction in HA staining on the tumor biopsy after treatment.
[0664] [0664] This Example describes a method for determining the HA-disaccharide content in plasma as a measure of HA catabolites, which are the degradation products after the enzymatic activity of PEGPH20. The method uses the hydrolysis of HA with Chondroitinase ABC to release the HA-disaccharides, derivatize them with 2-amine acridone (AMAC) and analyze them on a reverse phase HPLC with fluorescence detection. Quantification of HA-disaccharides is performed by comparison with HA-disaccharide standards. This assay was used to measure the enzymatic activity of PEGPH20, by monitoring the concentrations of hyaluronan catabolites in the plasma of patients who were selected in patient programs after treatment with PEGPH20.
[0665] [0665] In the method, a standard working solution was generated. First, a diluted stock solution (DSS) was generated from a HA-disaccharide (SS) stock solution. The HA SS disaccharide was generated by adding 1 ml of water to a vial of HA-Disac (V-labs, Cat. No. C3209) containing 2 mg of lyophilized powder to obtain a uniform suspension. To generate diluted stock solutions, 5 pL of the SS solution was diluted with 125 npL of water to produce a DSS1 solution (containing 200 pmols / unl HA-Disac; 200 nmol / ml HA-Disac). Five serial dilutions in water were made to generate DSS2 (containing 40 pmols / pyl HA-Disac; 40 nmol / ml HA-Disac) and then DSS3 (containing 8 pmols / ul HA-Disac; 8 nmols / ml HA-Disac). Then, the standard working solutions were generated as set out in 25% human serum albumin (SAH) (ABO Pharmaceuticals, Cat. No. 1,500,233) or normal mouse plasma (Bioreclamation, Cat. No. MSEPLEDTA2- BALB-M), as established in Tables 14 and
[0666] [0666] The sample was then hydrolyzed. Said sample (e.g., plasma) was prepared using about 100 pg of protein in a polypropylene tube and adjusting the volume to 340 pl with water. A blank matrix was also prepared using dilution buffer (1.59 g HEPES, 5.07 g NaCl, 1800 ml water, pH 7.0) equivalent to the sample volume, and the volume was adjusted to 340 pl . The hydrolysis of samples and a blank matrix were performed by adding 60 pl of TFA to the sample tube and the crude tubular matrix, and the contents were mixed and incubated at 100ºC for 4 hours. the vials were allowed to cool to room temperature. They were evaporated until a vac speed was used. Then, 300 ml of water was added to each tube, and centrifuged to resuspend the samples.
[0667] [0667] For the derivation of hydrolyzed samples, raw and working samples, 45 pÀpL of each sample (sample, blank, or working sample) was evaporated until it dried in a Speed Vac. Then, 10 pl of SAS were added to the dry sample, blank and working standard. Then, 50 µl of ABA / NaCNBH3 labeling solution was added. the tubes were shaken and spun briefly. Then 440 µl of the mobile phase A was added and the tubes were mixed well. Mobile phase A was prepared as follows: 132 ml of 1 M ammonia acetate buffer (Sigma, Cat. A7330) was added to a 1 liter volumetric flask, and water was added to fill the flask. After derivation, nominal loads in columns per 20 pl of injection for working standards are as defined in Table 16.
[0668] [0668] The HPLC column was equilibrated at a flow rate of 1.0 mL / min. with the initial settings of the mobile phase, as described in Table 20. The system was left to balance until the baseline was constant. HPLC analysis was performed with the device parameters, as described in Table 17.
[0669] [0669] The sequence for the sample analysis was as follows: WSS5 (1 injection) for conditioned column / balance / detector gain; water injection (1 injection); WSS3 (3 injections); WSS1l (1 injection); WSS2 (1 injection); WSS4 (1 injection); WSS5 (1 injection); Water (1 injection); Blank Matrix (1 injection); Sample 1 (1 injection); Sample 2 (1 injection); WSS3 (3 injections); Water (1 injection). The system was considered adequate when there was separation of acceptable quality; the signal for the noise ratio for the shortest peak of the monosaccharide in the WSSl sample was equal to or greater than 10; the relative standard deviation (RSD) of the peak areas, for each monosaccharide pattern, for the six injections of WSS3, was equal to or less than 4%; the correlation coefficient (r) was 0.99 (r was measured using the software to plot the peak area of each work pattern against the load on the column (expressed as pmol) using the first three injections with the standard norm WSS3, and calculating the slope, intercept and correlation coefficient for work patterns, using a linear least squares regression model); peak areas for peaks corresponding to monosaccharides were not more than 2% of the peak area measured by WSS5; and the peak areas for the peaks corresponding to the monosaccharides in the water injection, were not more than 0.5% of the peak areas measured for WSS5.
[0670] [0670] The mean corrected peak area for each monosaccharide in each sample preparation was determined. Valley-to-valley integration was used for the GalN peak. To determine this, the linear curves generated from the working patterns were used to calculate the amount of each monosaccharide loaded for each sample preparation. For each type of monosaccharide, the average molar ratio of monosaccharides per protein molecule for each sample was calculated. Then, for each sample, the total sum of the average molar proportions of all five monosaccharides was determined. The calculations were performed based on the following: the molecular weight (MW) of non-glycosylated hyaluronidase protein is 51106 g / mol; the total volume of each sample was 500 pl; the sample dilution factor is 0.15; the volume of each injection is 20 pl; and the conversion factor from mg to pg is 10º. The calculations were performed as follows, for each monosaccharide:
[0671] [0671] The amount of monosaccharides for each preparation was calculated using the following equation: Monosaccharide (pmol) = Peak area-Intercept slope
[0672] [0672] The number of monosaccharides per protein molecule was calculated using the following formula: Monosaccharide to protein ratio = Monosaccharide (pmol) x MW x 500pl 0.1 mg x 10º x 20p1l x 0.15
[0673] [0673] The results for each sample were reported as the ratio of monosaccharides per protein to each monosaccharide, along with the sum of the five proportions of monosaccharides.
[0674] [0674] The disaccharide assay described above was used to measure AH and its catabolites, from patients involved in phase I clinical studies, who received IV doses of PEGPH20, in doses ranging from 0.5 uvg / kg to 50 pg / kg over a cycle of dosing regimen with or without dexamethasone. Plasma concentrations of HA before dosing with PEGPH20 were usually less than 1 µg / ml or below the level of quantification (0.5 pg / ml) for all patients in the study.
[0675] [0675] Plasma collected from a patient who received a single dose of 50 upg / kg of PEGPH20 was assessed over time after treatment. The results show that the plasma concentrations of hyaluronan increased significantly. In this patient, while PEGPH20 concentrations decreased with a terminal half-life of 2 days, high concentrations of HA catabolites accumulated more slowly, and persisted for up to 2 weeks of post-PEGPH20 treatment, with an observed maximum plasma concentration of HA around 200 hours post-dose of PEGPH20.
[0676] [0676] Plasma was also collected from 12 additional patients starting 24 hours after the initial dose of PEGPH20, or were treated with 0.5 pg / kg PEGPH20 twice a week (1 patient), 0.5 ug / kg every 21 days (3 patients), 0.75 upg / kg every 21 days (4 patients), 1.0 upg / kg every 21 days (3 patients), or 1.5 1ug / kg every 21 days (1 patient). The results show that after administration of single or multiple doses of PEGPH20, which ranged from 0.5 u1pg / kg to 1.5 vg / kg, catabolic HA levels increased in a dose-dependent manner over the course of a week.
[0677] [0677] These results are consistent with the expected mechanism of activity of PEGPH20, and support the role of HA as a biomarker for PEGPH20 pharmacodynamics.
[0678] [0678] Diffusion weighted MRI was performed using a single spin-echo pulse sequence to estimate pixel-by-pixel values for apparent diffusion coefficient. Dynamic contrast enhanced the magnetic resonance imaging (DCE-MRI) included during infusion with a contrast agent. Calibration was performed using a two-part simulator containing an air chamber and an ice / water mixture. The examinations were performed in the pre-treatment and in the post-treatment.
[0679] [0679] Apparent Diffusion Coefficient Magnetic Resonance (ADC-MRI) measures the volume of water that has passed through the cell membrane based on a calculation derived from pre- and post-treatment scans. ADC-MRI scans were completed for a total of 10 out of 14 patients in a phase I clinical trial, which evaluates treatment with PEGPH20 without dexamethasone premedication, and in 4 patients in a phase I clinical trial , which assesses PEGPH20 treatment with dexamethasone premedication.
[0680] [0680] The analysis of the images obtained from each patient was performed by an Imaging Endpoints radiologist (Scottsdale, AZ), and the quantitative ADC estimates were computed for the tissues in each patient.
[0681] [0681] Dynamic Contrast-enhanced MRI measures blood flow that indicates a change in tumor vascularization. The exams were completed in 4 patients, in a phase I of a clinical study, which evaluates the treatment with PEGPH20 with a premedication with dexamethasone. The analysis of images obtained from each patient was performed by a radiologist at Imaging Endpoints (Scottsdale, Arizona), and quantitative estimates of the volume transfer coefficient (Ktrans), blood volume (Vp) and fraction of extracellular volume (Ve) were computed for tissues in each patient. A summary of the DCE-MRI results associated with the tumor regions is shown in Table 19. Significant increases in the Ktrans parameter were observed in the two patients who were checked on the day of dosing with PEGPH20. The increase in Ktrans within the dosing hours is consistent with preclinical data showing that PEGPH20 causes vascular decompression and increased blood flow (Thompson et al. (2010) Mol. Cancer. Ther., 9: 3052-64. DCE Tumor Modification - Dose and Frequency MRI scan days from Post-dose baseline 7 amstasone; 2x / week DL exametasone; Increase in ktrans, Ve, Vp (8
[0682] [0682] DCE-MRI imaging was also performed on a patient with a pancreatic tumor enrolled in a phase I clinical study receiving 3.0 ug / kg + dexamethasone / wk over a 28-day administration cycle. Pre-dose and post-dose images were obtained as follows: 8 hours (Day 1), 24 hours (day 2) and 3 days after the fourth weekly dose of PEGPH20 in cycle 1 (end of cycle 1) . The results are shown in Table 20, and show that PEGPH20 increases the tumor Ktrans measured by serial DCE-MRI. TABLE 20. DCE-MRI results of a patient receiving 3.0 ug / kg + dexamethasone / per week uu A A jm 1
[0683] [0683] Positron emission tomography (PET) using FDG, a glucose analog, was used to obtain metabolic tissue activity, in terms of regional glucose absorption. FDG-PET was performed on a patient with metastatic rectal carcinoma with lung metastasis in a phase I clinical study, receiving 3.0 nug / kg + dexamethasone; 2 * / per week, in a 28-day administration cycle. Pre-dose images were obtained 8 hours after the dose, 24 hours after the dose, and at the end of the cycle (1 day after the eighth dose). The standardized FDG uptake value (SUV) was determined using standardized methods. The results are shown in Table 21, and showed that the patient exhibited a decrease in the metabolic activity of the tumor after treatment with PEGPH20 of reference pulmonary metastases. TABLE 21. FDG-PET results of a patient receiving 3.0 pg / kg dexamethasone 2x / per week Localization | 8h E 24h [n8n | Dia26 / 0 24h the anatomic of (SUV | Line (SUV) | To (SUV) | for day | Baseline |) Base 24h 26 Base for (SUV) for 8 h | Day 26 Upper lobe segment 12.9 | 9.4 -27% -15% 8 [E —38% lower left Base of left lung 11.2 [9.1 -19% 7.1 | -22% 6.7 6% 40% Upper right lobe. In the Region 6.8 4.5 34% 3.9 13% 4 + 3% 41% perihilar dir. Lower lobe 8.1 5.4 | -33% 5.2 to 4. 10% 42% dir.
[0684] [0684] The results show that the various modalities of tumor imaging can be used to demonstrate and monitor the activity of PEGPH20 in the tumor tissue.
[0685] [0685] Control mice and mice bearing a hyaluronAN-rich tumor were administered with TSG-6-LM-Fc / NHep labeled with DyLight 755 Fluor labeling reagent (TSG-6-LM-Fc / AHep ”" "**), and mice were photographed to assess tumor binding and distribution of TSG-6-LM-Fc / AHep "" **, specificity was also assessed by comparing staining and distribution to an IgerrTo control, for the generation of tumor-bearing mice peritibial BxPC3, mice were inoculated with human pancreatic adenocarcinoma BxPC-3 tumor cells (ATCC CRL-1687), subcutaneously (sc, right hind paw) at 1 x 10 'cells / 0.1 ml. For the generation of HA * Dul45-Has2 and HA-DU145 tumor-bearing mice, the mice were inoculated with both Dul45-Has2 cells (produced as described above) and Dul45 cells, in a peritibial manner (intramuscular injection adjacent to the periosteum of the right tibia on both sides) at 5x10º / 0.05 mL.
[0686] [0686] TSG-6-LM-Fc / aAHep ”W" ** was generated by fluorescence, by labeling TSG-6-LM-Fc / AHep (generated as described in Example 9) with DyLight 755, using ammo reactive dye kit Thermo Scientific DyLight 755 (Catalog No. 84538; Thermo Scientific, Rockford, IL), according to the manufacturer's protocol.
[0687] [0687] Mice with a peritibial tumor HA BxPC3, about 18 to 20 mm in diameter, were injected intravenously with 5 µg or 10 µg of TSG-6-LM-Fc / AHepD "" **. In a group of mice, said mice were pretreated with intravenous administration of PEGPH20 at 4.5 mg / kg, three (3) hours before administration of TSG-6-LM-Fc / AHep ”" **,
[0688] [0688] A full fluorescent body imaging system (IVIS Lumina XR, Caliper Life Sciences Mountain View, CA) was used to track fluorescence in the animal. The selective excitation of DyLight755 was performed using a D / 745 nm long-pass filter, and the emitted fluorescence was collected through a D800 nm long-pass filter. The three groups of mice (not injected, TSG-6-LM-Fc / AHep "" "** and PEGPH20 + TSG-6-LM-Fc / AHep" "" **) were photographed at various time points after TSG- 6-LM- Fc / aHep Too (1 hour, 4 hours, day 1, day 2, day 3, day 4 day 5 and day 6). For imaging, uninjected control mice were also evaluated. Fluorescent images were captured with a super-cooled, highly sensitive digital camera. Fluorescent images were subsequently analyzed with Living Image (Caliper Life Sciences, Mountain View, CA).
[0689] [0689] The results show that at 1 hour and 4 hours after injection, TSG-6-LM-Fc / AHep "" "** was detected as circulating in the bloodstream, and was also detected as a start of tumor attachment Tumor binding was dose-dependent, with increased staining intensity seen with the 10 mg dose, and less tumor binding was detected by imaging in mice treated with PEGPH20, at all doses and time intervals. later moments after the injection (for example, a day or two days), binding to the liver was also detected, although this was lower in the mice injected with the 1 µg low dose TSG-6-LM-Fc / AHep ” "" **. TSG-6-LM- Fc / AHep "" "** reached maximum levels between days 1 and 2, as assessed by image analysis. In mice treated with low dosage, TSG-6-LM-Fc / AHep "" "** was eliminated on the third day after injection. TSG-6-LM-Fc / AHep S still circulated in mice treated with high doses 5 days after injection, and all links to the tumor were decreased 6 days after injection.
[0690] [0690] In summary, in vivo imaging results show that TSG-6-LM-Fc / AHep "" "** binding was dose dependent and peaked 1-2 days after injection. , removal of HA by PEGPH20 resulted in less binding of TSG-6-LM-Fc / AHep "" "**. The TSG-6-LM-Fc / AHep link "" "** was cleared from the tumor 6 days after injection.
[0691] [0691] Mice with HA * tumor DU1l45-HAS2 and HA -DUl45 were injected intravenously with 5 µg TSG-6-LM-Fc / AHep ”T *, The rats were photographed daily after the injection of TSG-6-LM-Fc / AHepDL755. Although a low level of background staining of the HA-DUl45 tumor was detected, there was much more TSG-6-LM-Fc / AHep "" ** linkage to HA-rich Dul45-has2 assessed by imaging results. The binding was peak on day 1-2, as determined by the intensity of the staining. Thus, the results show that the more HA is present in the tumor, the more TSG-6-LM-Fc / AHep ”" "** binds to the tumor.
[0692] [0692] The specificity of TSG-6-LM-Fc / AHepDL755 for HA-rich tumors was further evaluated by comparing the binding of TSG-6-LM-Fc / AHep ”" "** or IgG” "" ** to mice with HA peritibial tumor ” BxPC3. Mice with HA + + BxPC3 peritibial tumor were injected intravenously with 5 µg TSG-6-LM-Fc / AHep ”" **, or with 5 ng IgE. The mice were photographed daily after injection. showed little or no detectable staining of IgGDL755 to the tumor and thus a greater binding of TSG-6-LM-Fc / AHep ”" *** to the PC3 tumor than IgE ”.
[0693] [0693] Since the modifications will be apparent to those skilled in the art, it is intended that this invention be limited only by the scope of the appended claims.

权利要求:
Claims (47)
[1]
1. TSG-6 binding module multimer (LM) characterized by the fact that it comprises: a first polypeptide containing a TSG-6-LM linked directly or indirectly through a linker to a multimerization domain; and a second polypeptide containing a TSG-6-LM linked directly or indirectly, via a linker to a multimerization domain, where: the first and second multimerization domains interact to form a multimer containing two or more TSG binding modules -6; the first and second polypeptides do not comprise the TSG-6 full length sequence; and the TSG-6-LM multimer has a binding affinity for hyaluronan (HA) with an association constant of at least 10 'M.
[2]
2. TSG-6-LM multimer according to claim 1, characterized by the fact that the binding module is the only TSG-6 portion of the first polypeptide and the second polypeptide.
[3]
The TSG-6-LM multimer according to either of Claims 1 and 2, characterized in that the first and the second connection modules are the same or different.
[4]
4. TSG-6-LM multimer according to any one of claims 1, 2 or 3, characterized by the fact that TSG-6-LM comprises the amino acid residue sequence established in SEQ ID NO: 207, 360, 417 or 418, or a sequence of amino acid residues comprising at least 85% amino acid sequence identity with the amino acid sequence set out in
SEQ ID NO: 207, 360, 417 or 418, which specifically binds to HA.
[5]
5. TSG-6-LM multimer according to any one of claims 1, 2, 3 or 4, characterized in that the TSG-6-LM is modified to reduce or eliminate binding to heparin.
[6]
6. TSG-6-LM multimer according to claim 5, characterized by the fact that TSG-6-LM comprises an amino acid substitution at the amino acid position corresponding to amino acid residue 20, 34, 41, 54, 56 , 72 or 84 set out in SEQ ID NO: 360, where a corresponding amino acid residue is identified by aligning a TSG-6-LM set out in SEQ ID NO: 360.
[7]
7. TSG-6-LM multimer, according to claim 6, characterized by the fact that the amino acid substitution is in a non-basic amino acid residue selected from Asp (D), Glu (E), Ser (S) , Thr (T), Asn (N), Gln (0), Ala (A), Val (V), Ile (1), Leu (L) Met (M), Phe (F), Tyr (Y) and Trp (W).
[8]
The TSG-6-LM multimer of any one of claims 5, 6 or 7, characterized in that the TSG-6-LM comprises an amino acid substitution corresponding to one or more amino acid substitutions K20A, K34A or K41A in a TSG-6-LM established in SEQ ID no: 360, or the substitution in the corresponding residue in the other TSG-6-LM.
[9]
9. TSG-6-LM multimer according to any one of claims 5, 6, 7 or 8, characterized in that the TSG-6-LM comprises the amino acid sequence set out in SEQ ID NO: 361 or 416, or an amino acid sequence comprising at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 361 or 416, which specifically binds to HA.
[10]
The TSG-6-LM multimer of any one of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized by the fact that the multimerization domain is selected from an immunoglobulin constant region ( Fc), a leucine zipper, complementary hydrophobic regions, complementary hydrophilic regions, compatible protein-protein interaction domains, free thiols that form an intermolecular disulfide bond between two molecules, and a protrusion-in-cavity and a clearing cavity identical or similar size that form stable multimers.
[11]
11. TSG-6-LM multimer, of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, characterized by the fact that the multimerization domain is a constant region of immunoglobulin ( Fc) or a variant thereof, which performs multimerization.
[12]
TSG-6-LM multimer according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, characterized in that the first and second polypeptides comprise, each, a TSG-6-LM and an immunoglobulin Fc domain.
[13]
13. TSG-6-LM multimer according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, characterized in that it comprises the established amino acid sequence as amino acids 24-349 in SEQ ID NO: 212 or 215, or an amino acid sequence that has at least 85% amino acid sequence identity with amino acids 21-349 of SEQ ID NO: 212 or
215.
[14]
The TSG-6-Lm multimer according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12 or 13, characterized by the fact that it comprises an amino acid sequence encoded by the nucleotide sequence established in any of SEQ ID NOS: 211, 214 or 217, or a nucleotide sequence that exhibits at least 85% sequence identity with the nucleotide sequence established in any of SEQ ID NOS: 211, 214 or 217.
[15]
15. TSG-6-LM multimer according to claim 11, characterized by the fact that the multimerization domain comprises the sequence of amino acid residues established in SEQ ID NO: 359 or a sequence of amino acid residues having 85% of sequence identity with it.
[16]
16. Nucleic acid molecule encoding the TSG-6-LM multimer according to any one of claims 1 to 15, characterized in that: the nucleic acid molecule encodes a fusion polypeptide containing a directly or indirectly linked TSG-6-LM through a linker to a multimerization domain; and the multimerization domain is a polypeptide that interacts with itself to form a stable protein-protein interaction, so the encoded protein forms a multimer containing at least two TSG- binding modules
6.
[17]
17. Nucleic acid molecule according to claim 16, characterized by the fact that the nucleotide sequence established in any of SEQ ID NOS: 211, 214 or 217, or a nucleotide sequence that exhibits at least 85% of sequence identity with the nucleotide sequence established in any of SEQ ID NOS: 211, 214 or 217.
[18]
18. Vector characterized in that it comprises the nucleic acid molecule according to any of claims 16 or 17.
[19]
19. Isolated cell or cell culture characterized by the fact that it comprises the nucleic acid molecule according to any of claims 16 or 17.
[20]
20. Method for producing a TSG-6-LM multimer, characterized in that it comprises: introducing the nucleic acid molecule according to any of claims 16 or 17 into a cell; culturing the cell under conditions under which the fusion polypeptide is expressed by the cell; and optionally, recovering the TSG-6-LM multimer.
[21]
21. Kit or combination characterized in that it comprises: a first composition comprising a TSG-6-LM multimer according to any one of claims 11 to 5; and a second composition comprising an anti-hyaluronan agent; and optionally, reagent for detecting the TSG-6-LM multimer.
[22]
22. Kit or combination according to claim 21, characterized in that the anti-hyaluronan agent is a degrading enzyme of hyaluronan or is an agent that inhibits hyaluronan synthesis.
[23]
23. Kit or combination according to claim 22, characterized in that the anti-hyaluronan agent is a hyaluronic acid degrading enzyme which is a hyaluronidase.
[24]
24. Kit or combination according to claim 23, characterized in that the hyaluronan-degrading enzyme is a PH20 hyaluronidase or the truncated form thereof without a C-terminal glycosylphosphatidylinositol (GPI) binding site, or a part of the GPI connection location.
[25]
25. Kit or combination, according to claim 24, characterized by the fact that PH20 or its truncated form comprises the amino acid sequence established in any of SEQ ID NOS: 4 to 9, 47, 48, 150 to 170 and 183 at 189, or an amino acid sequence that exhibits at least 85% sequence identity with any of SEQ ID NOS: 4 to 9, 47, 48, 150 to 170 and 183 to 189.
[26]
26. Kit or combination according to any one of claims 20, 21, 22, 23, 24 or 25, characterized in that the anti-hyaluronan agent is a hyaluronan-degrading enzyme that is modified by conjugation with a polymer.
[27]
27. Kit or combination, according to claim 26, characterized by the fact that the polymer is PEG and the hyaluronic acid degrading enzyme is PEGylated.
[28]
28. Method for selecting an individual for the treatment of a tumor, with an anti-hyaluronan agent, characterized by the fact that it comprises: contacting a sample of tissue or body fluid previously obtained from an individual who has a tumor or cancer, with a TSG-6-LM multimer according to any one of claims 1 to 15; and detect the binding of the TSG-6-LM multimer to the sample, thus determining the amount of hyaluronan in the sample, in which if the amount of hyaluronan in the sample is equal to or greater than a predetermined threshold, the individual is selected for treatment with an anti-hyaluronan agent.
[29]
29. Method according to claim 28, characterized by the fact that the predetermined threshold level is high HA.
[30]
30. Method according to either of claims 28 or 29, characterized in that the predetermined threshold level is at least or greater than 0.025 ug HA / ml of sample, 0.030 upg / ml, 0.035 upg / ml , 0.040 µg / ml, 0.045 µg / ml, 0.050 µg / ml, 0.055 mg / ml, 0.060 µg / ml, 0.065 µg / ml, 0.070 µg / ml, 0.08 µg / ml, 0.09 vg / ml, 0.1 vg / ml, 0.2 pg / ml, 0.3 pg / ml or higher; or the sample is a sample of tumor tissue and the predetermined threshold is an HA score of at least +2 (HA ) or at least +3 (HA); or the sample is a sample of tumor tissue and the predetermined threshold level is at least one percent of HA positive pixels in the tumor (cells and stroma) for total staining in the tumor tissue of at least 10%, 10 % to 25% or greater than 25%.
[31]
31. Method according to claim 30, characterized by the fact that the predetermined threshold level is an HA score of at least +3 (HA) (high levels).
[32]
32. Method for predicting the effectiveness of treating an individual with an anti-hyaluronan agent characterized by the fact that it comprises: contacting a sample of tissue or body fluid from an individual who is or has been treated with an anti-hyaluronan agent with a multimer TSG-6-LM according to any one of claims 1 to 15; detect the binding of the TSG-6-LM multimer to the sample, thereby determining the amount of hyaluronan in the sample, in which the detection of a decrease in hyaluronan compared to previous treatment with the anti-hyaluronan agent or before the previous dose of anti-hyaluronan agent, indicates that the treatment is effective.
[33]
33. The method of claim 28, 29, 30, 31 or 32, characterized in that the anti-hyaluronan agent is a hyaluronan degrading enzyme or is an agent that inhibits hyaluronan synthesis.
[34]
34. The method of claim 33, characterized by the fact that the anti-hyaluronan agent is a hyaluronan degrading enzyme which is a hyaluronidase.
[35]
35. Method according to any of claims 28, 29, 30, 31, 32 or 33, characterized in that the anti-hyaluronan agent is a soluble PH20 hyaluronidase or a Cr-terminal truncated form of a human PH20 hyaluronidase without all or a portion of the GPI binding site.
[36]
36. Method according to claim 35, characterized in that the anti-hyaluronan agent is a hyaluronan degrading enzyme which is a PH20 hyaluronidase or a truncated form thereof without all or a portion of the GPI binding site.
[37]
37. Method according to claim 36, characterized by the fact that PH20 or its truncated form comprises the amino acid sequence established in any of SEQ ID NOS: 4 to 9, 47, 48, 150 to 170 and 183 to 189 , or an amino acid sequence that exhibits at least 85% sequence identity with any of SEQ ID NOS: 4 to 9, 47, 48, 150 to 170 and 183 to 189.
[38]
38. Method according to any one of claims 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, characterized in that the anti-hyaluronan agent is a hyaluronan-degrading enzyme that is modified by conjugation with a polymer.
[39]
39. Method according to claim 38, characterized in that the polymer is PEG and the hyaluronic acid degrading enzyme is PEGylated.
[40]
40. Method according to any one of claims 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, characterized by the fact that the individual from whom the sample is obtained has a tumor or cancer.
[41]
41. Method of diagnosing a disease Or condition associated with hyaluronan characterized by the fact that it comprises: contacting a sample of tissue or body fluid previously obtained from an individual with a TSG-6-LM according to any of claims 1 to 14; and detect the binding of TSG-6-LM, thus determining the amount of hyaluronan in the sample, in which the individual is diagnosed with a disease or condition associated with hyaluronan, if the amount of hyaluronan is above a predetermined level or is greater than hyaluronan level of a reference sample.
[42]
42. Method according to claim 41, characterized by the fact that the disease or condition associated with hyaluronan is a tumor or cancer.
[43]
43. Method according to either of claims 40 or 41, characterized in that the reference or predetermined level is the average level of hyaluronan present in healthy or normal tissue, or fluid samples from a population of individuals of control.
[44]
44, Method according to any one of claims 40, 41, 42 or 43, characterized by the fact that:
the predetermined threshold level is greater than 0.015 µg HA / ml of sample; or is the sample a tumor or tissue sample and the predetermined threshold level is an HA score of HA * ', HA ” or HA ”.
[45]
45. Method according to any one of claims 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44, characterized by the fact that : the sample is a sample of stromal tissue; or the sample is a fluid sample that is a sample of blood, serum, urine, sweat, semen, saliva, cerebrospinal fluid, or lymph.
[46]
46. Method according to any one of claims 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45, characterized by the fact that the sample is a sample of stromal tissue from a tumor.
[47]
47. Method according to claim 46, characterized by the fact that the tumor is a cancer selected from breast cancer, pancreatic cancer, ovarian cancer, colon cancer, lung cancer, non-cell lung cancer small, carcinoma in situ (ISC), squamous cell carcinoma (SCC), thyroid cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, brain cancer, bladder cancer, stomach cancer, hepatoma, melanoma, glioma , retinoblastoma, mesothelioma, myeloma, lymphoma and leukemia.

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法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-06-15| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-06-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-17| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 9A ANUIDADE. |

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2021-10-13| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161628187P| true| 2011-10-24|2011-10-24|

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US61/628,187|2011-10-24|

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US201161559011P| true| 2011-11-11|2011-11-11|

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US61/559,011|2011-11-11|

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US201161630765P| true| 2011-12-16|2011-12-16|

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US61/630,765|2011-12-16|

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US201261714700P| true| 2012-10-16|2012-10-16|

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US61/714,700|2012-10-16|

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PCT/US2012/061743|WO2013063155A2|2011-10-24|2012-10-24|Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof|

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