bispecific symmetrical modified igg4 antibodies in the sequence

专利摘要:
  SYMMETRIC SYMMETRIC BIESPECIFIC ANTIGODS IN SEQUENCE.The present disclosure relates to a symmetrical bispecific antibody of the IgG4 class comprising two heavy chains that each comprise a variable domain, CH1 domain and an articulation region, where, in each heavy chain: cysteine in the CH1 domain which forms a disulfide bond interchanged with a cysteine in a light chain is replaced by another amino acid; and optionally one or more of the amino acids positioned in the upper hinge region is replaced by cysteine, where the sequence of constant region of each heavy chain is similar or identical and the variable region in each heavy chain is different, the formulations comprising the same , the use of each one of the above in the treatment and processes for the preparation of said antibodies and formulations.
公开号:BR112014020627A2
申请号:R112014020627-9
申请日:2013-02-22
公开日:2020-10-27
发明作者:David Paul Humphreys；Shirley Jane Peters
申请人:Ucb Pharma S.A.；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to a bispecific IgG4 antibody that has an altered arrangement of disulfide bonds as compared to a wild type antibody and a method for producing the enhanced antibody. In a further aspect, the present disclosure provides an efficient method for preparing bispecific antibodies.
[0002] [0002] The biopharmaceutical industry that includes recombinant proteins, monoclonal antibodies (mAbs) and drugs based on nucleic acid is growing rapidly. Antibody engineering has resulted in the design and production of antibody fragments or alternative formats. The preferred molecular format together with other aspects such as production yield, protein quality and storage stability are taken into account when selecting an antibody-based protein as a therapeutic agent.
[0003] [0003] The basic structure of all immunoglobulin molecules (lg) comprises two identical heavy chains (HCs) and two identical light chains (LCs) that are coupled by disulfide bonds. Each LC consists of a variable (V1) and constant domain (C)). Based on HC, five major Ig classes are recognized: IgG, IgA, 1gD, IgE and IgM. For IgG, HC consists of a variable domain (Vy4) and three constant domains (Chy1-3). The Ch2 and CH3 domains form the Fc part of the molecule that is responsible for stimulating the effector function and is linked to the Fab fragment (VuV. And CHC,) by a joint region that gives flexibility to the IgG molecule. Two antigen recognition sites are located at the ends of the V, and Vu domains. IgG is further subdivided into 4 different isotypes: I9G1, IgG2, IgG3 and I9G4.
[0004] [0004] Fc-mediated effector functions, that is, antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) are isotype-dependent. Each isotype has evolved to perform a specific function within the body. The I9G1 isotype is currently the most widely used as a therapeutic due to its extended half-life, improved ADCC activation and complement activation. Other isotypes are employed as therapeutic agents as appropriate to the target and desired effect. For example, when target antigens must simply be neutralized and effector functions are less important, alternative isotypes such as IgG2 and IgG4 can be used. Alternatively, IgG with reconstructed Fc / effector function can be considered.
[0005] [0005] —AIgG2 also has minimal associated effector function, but is prone to dimerization that is not fully understood.
[0006] [0006] —AIlgG4 remains a useful isotype due to its relative absence of effective function induction. However, IgG4 also has some inherent practical difficulties, namely its shorter sleep half-life and its ability to undergo “Fab arm swap” (also referred to as dynamic heavy chain swap or heavy chain swap), where the heavy chain and the attached light chain of one antibody are exchanged for the heavy chain and the same light chain attached to another antibody to form another complete antibody composed of two heavy chains and two fixed light chains (van der Neut Kolfschoten et al., 2007 Science 317, 1554 to 1557).
[0007] [0007] Ih alive, Fab arm exchange results in bispecific antibodies that, due to their different variable domains, can co-engage different target antigens. This produces a large percentage of circulating IgG4 that has been observed to be bispecific, but functionally monovalent. (Schuurman, J., Van Ree, R., Perdok, GJ, Van Doorn, HR, Tan, KY, Aalberse, RC, 1999. Normal human immunoglobulin G4 can be bispecific: it has two different antigen-combining sites. Immunology 97 , 693 to 698).
[0008] [0008] Ih vitro, when the I9G4 antibodies are analyzed by non-reducing SDS-PAGE, they were observed to form the so-called "semimolecules" "that each comprise a single pair of covalently associated light / heavy chains caused by the absence of heavy-chain disulfide bonds typically due to the formation of heavy-chain disulfide bonds within the hinge region.The heavy chain of a “semimolecule” may non-covalently associate with the paired heavy chain member of the same , the association being maintained by interactions Ch3: domain Ch3. In the solution, such "semimolecules" are actually observed with the use of methods such as size exclusion chromatography to be completely scaled, which is approximately 150 kDa, but in SDS -Page not reducing are comprised of 75 kD of pairings LC: HC (so-called “semimolecule”).
[0009] [0009] A Ser to Pro mutation at position 241 (numbered according to the Kabat numbering system) in the joint reduces the appearance of these “semimolecules” by non-reducing SDS-PAGE (Angal, S. et al., 1993. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (I9G4) antibody as observed during SDS-PAGE analysis Mol Immunol 30, 105 to 108). In addition, this point mutation does not influence the compact structure of IgG4 thus allowing IgG4 to retain its reduced ability to activate complement.
[0010] [0010] Following the finding of the S241P mutation, additional mutations for IgG4 were investigated in order to understand the interaction between heavy chain in IgG4 antibodies, reduce the effector function of I9gG4 and improve structural stability. In Schuurman et al. (Schuurman, J et al., 2001. The inter-heavy chain disulphide bonds of I9G4 are in equilibrium with intra-heavy chain disulphide bonds. Molecular Immunology 38, 1 to 8), the observed instability of heavy IgG4 disulfide bonds was investigated with the use of IgG4 mutants. In the M1 mutant Cys 131 (numbered according to the EU numbering system or Cys 127 numbered according to the Kabat numbering system), which is involved in the disulfide bond between light / heavy chain (C, -Cu1), it was replaced by serine and it was observed that this mutant resulted in the formation of light chain dimers and heavy chain dimers. In the M2 mutant, cysteine 226 (226 numbered according to the EU numbering system or 239 numbered according to the Kabat numbering system), which is involved in a disulfide bond between heavy chain in the joint, was replaced by serine and it was observed that this mutant had a more stable heavy chain link compared to IgG4 and prevents the formation of a heavy intrachain disulfide link.
[0011] [0011] Mutations in the Ch2 and CH3 domains of I9G4 antibodies have also been investigated in order to reduce the formation of aggregates of I9G4 antibodies. US No. 2008/0063635 Takahashi et al. investigated an IgG4 mutant in which arginine at position 409 (409 numbered according to the EU numbering system or 440 numbered according to the Kabat numbering system) in the Ch43 domain is replaced by lysine, threonine, methionine or leucine in order to inhibit aggregate formation at low pH. Additional mutations in L235, D265, D270, K322, P329 and P331 (L235, D265, D270, K322, P329 and P331 numbered according to the EU numbering system or L248, D278, D283, K341, P348 and P350 numbered according with the Kabat numbering system) are also taught in order to mitigate CDC activity. WOZ2008 / 145142 Van de Winkel et al. reveals stable IgG4 antibodies that have a reduced ability to undergo Fab arm replacement by replacing the arginine residue at position 409, the Phe residue at position 405 or Lys at position 370 (R409, F405 and K370 numbered according to numbering system EU or R440, F436 and K393 numbered according to the Kabat numbering system) even in the absence of the S228P mutation (S228 numbered according to the EU or S241 numbering system according to the Kabat numbering system) in the region articulation.
[0012] [0012] The present invention provides novel mutant antibodies that have advantageous properties that include enhanced biophysical properties compared to wild type antibodies, in particular fragments and I9gG4 of wild type antibodies. In particular, it was surprisingly observed that a change in the location of the cysteine residue in the heavy chain of an IgG4 antibody that forms a disulfide bond with a cysteine in the light chain provides an IgG4 antibody that has improved stability compared to an IgG4 antibody of wild type. It has also been observed that IgG4 antibody mutants are capable of forming bispecific antibodies that have advantageous Fab arm exchange properties.
[0013] [0013] In vitro, the exchange of Fab arms can be promoted by using high concentrations of antibodies and / or by employing chemical stimulants such as glutathione to generate a bispecific format that is stable and suitable for use as a therapeutic agent. This has application in the field of biological pharmaceutical products since bispecific entities can be difficult to express as a single construct. SUMMARY OF THE INVENTION
[0014] [0014] In one aspect, the present invention provides a symmetrical bispecific antibody of the IgG4 class comprising two heavy chains that each comprise a variable region, a Cy1 domain and a hinge region, wherein in each heavy chain: aa cysteine in the Chi domain that forms a disulfide bond interchanged with a cysteine in a light chain is replaced by another amino acid; and b. one or more of the amino acids positioned in the upper joint region are replaced by cysteine,
[0015] [0015] in which a sequence of constant regions of each heavy chain is similar or identical and a variable region in each heavy chain is different.
[0016] [0016] In one embodiment, the cysteine in the Cy1 domain that forms a disulfide bond interchanged with a cysteine in a light chain is cysteine at position 127, numbered according to the Kabat numbering system, for example, as shown in Figure 1b.
[0017] [0017] In one embodiment, cysteine interleaves at position 127, numbered according to the Kabat numbering system, in the Cy1 domain is replaced by another amino acid in one or both of the heavy chains.
[0018] [0018] The one or more amino acids positioned in the upper articulation region that are replaced by cysteine can be selected from 226, 227, 228, 229, 230, 237 and 238, numbered according to the Kabat numbering system, as shown in Figure 1b (underlined amino acids in the upper hinge region). In one embodiment, the one or more amino acids positioned in the upper articulation region that are replaced by cysteine are one or more of the amino acids in the positions selected from 227, 228, 229 and 230, numbered according to the Kabat numbering system, as shown in Figures 1b and 2a.
[0019] [0019] In one embodiment, a cysteine mutation at position 229 reduces the Fab arm switch.
[0020] [0020] In one embodiment, a mutation to cysteine at position 230 in combination with a mutation at position 241 to a non-polar amino acid, for example, selected from proline, alanine, glycine, isoleucine, phenylalanine, tryptophan and valine, is maid.
[0021] [0021] In one embodiment, the Fab arm can be increased by employing a mutation to a polar residue, for example, selected from arginine, aspartic acid, glutamic acid, histidine, lysine, threonine and tyrosine, such as threonine.
[0022] [0022] In one embodiment, a mutation at position 241 is provided for a non-polar amino acid, for example, selected from proline, alanine, glycine, isoleucine, phenylalanine, tryptophan and valine, which is employed.
[0023] [0023] In a further aspect, the present invention also provides a symmetrical bispecific antibody of the IgG4 class comprising two heavy chains each comprising a variable region, a Cy1 domain and a hinge region, wherein in each heavy chain : a. cysteine at position 127, numbered according to the Kabat numbering system, is replaced by another amino acid; and b. cysteine at position 239 or cysteine at position 242, numbered according to the Kabat numbering system, are replaced by another amino acid,
[0024] [0024] in which the constant region sequence of each heavy chain is similar or identical and the variable region in each heavy chain is different.
[0025] [0025] The antibodies provided by the present invention have no effector function and can have advantageous properties compared to a wild-type IgG4 antibody, for example, improved stability, such as improved thermal stability.
[0026] [0026] Although not wishing to be limited by theory, it is a hypothesis that the modified joint in the antibodies in accordance with the present disclosure alleviates the internal tension inherent in the IgG4 molecule and thereby promotes improved stability.
[0027] [0027] Natural heavy chain exchange processes can be promoted in IgG4 antibodies to facilitate the preparation of bispecific IgG4 antibodies in accordance with the present disclosure, for example, in vitro using high concentrations of antibodies and / or using an exchange chemical stimulant such as glutathione.
[0028] [0028] The antibodies of the present disclosure may be beneficial in that they have improved stability on wild-type IgG4 molecules and / or enhanced heavy chain exchange. The antibodies of the present invention can demonstrate reduced heavy chain exchange compared to wild-type IgG4, which provides a bispecific antibody that demonstrates little or no exchange with wild-type IgG4 in vivo due to its reduced propensity to exchange compared to IgG4 and also due to the relatively low concentration of a bispecific antibody in vivo compared to naturally circulating IgG4 antibodies.
[0029] [0029] While not wishing to be bound by theory, it is believed that the exchange between constructs of a similar type than those described in accordance with the present invention is more favorable than the exchange between a construct of the present disclosure and a wild-type IgG4 .
[0030] The bispecific antibodies of the present invention can demonstrate reduced heavy chain exchange at concentrations greater than in vivo concentrations, for example, concentrations of 0.5 mM or less compared to the wild-type IgG4. Although the bispecific antibodies of the invention demonstrate reduced heavy chain exchange compared to wild-type IgG4, they demonstrate a degree of heavy chain exchange compared to I9G1 wt and IgG4 S241P, which is sufficient to create the bispecific antibody from of two antibodies that have different antigen specificities in vitro. The symmetry in the constant region of the constructs advantageously minimizes the internal tension of the antibody in this document and therefore assists stability.
[0031] [0031] Accordingly, the present invention also provides the method of generating a symmetrical bispecific antibody comprising the step of mixing a first IgG4 antibody with a second I9gG4 antibody ex vivo, under conditions conducive to heavy chain exchange, wherein the antigen specificity of variable regions in the first antibody is different from the antigen specificity of variable regions in the second antibody.
[0032] [0032] The method of the present disclosure allows for the efficient preparation of bispecific symmetrical antibodies that employs only sets of routine procedures and stimulating naturally occurring processes. BRIEF DESCRIPTION OF THE FIGURES
[0033] [0033] Figure 1 shows the human Cyl and IgG1 wild type and IgG4 wild type hinge sequences, where the hinge residues are underlined, and the kappa light chain constant sequence.
[0034] [0034] Figure 1b shows:
[0035] [0035] the human kappa light chain constant sequence that indicates the cysteine (underlined) that forms the C, -Cy1 disulfide bond interchain;
[0036] [0036] the human IgG heavy chain N-terminal residues of Cyu1 1, 2, 3 and 4 and the joint region sequences in which the cysteine position (in the upper joint for IgG1 and in Ch41 of the N-terminal for IgG 2, 3 and 4 ) is indicated (underlined) that forms the C, -Cy1 disulfide bond interchain;
[0037] [0037] the human IgD heavy chain N-terminal chi residues and part of the hinge region sequences where the position of cysteine in the N-terminal Ch41 sequence is indicated (underlined) that forms the C disulfide bond , -Ch1 interchanges;
[0038] [0038] C-terminal Cy, N-terminal heavy chain IgM residues and N-terminal Ch2 residues in which the position of cysteine in the N-terminal Ch1 is indicated (underlined) that forms the C disulfide bond -Ch1 interchanges; and
[0039] [0039] —the residues in the upper joint of I9G3 and IgG4, the IgD joint and in the C-terminal Cy1 and the IgM Ch2 where underlined residues indicate the positions where one or more residues can be replaced by cysteine in the antibodies of the present invention .
[0040] [0040] Figure 2a shows the cy1 cysteine residue (C127) which forms the disulfide bond interchanged with a light chain cysteine and the wild-type I9G1 upper and core joint residues of the I9G4 and the positions wherein the mutations were introduced into the IgG4 antibodies of the present invention.
[0041] [0041] Figure 2b shows the cy1 cysteine residue (C127) that forms the disulfide bond interchanges with a cysteine in the light chain and the IgG3 wild-type hinge residues and positions where one or more residues are replaced by cysteine in the IgG3 antibodies of the present invention.
[0042] [0042] Figure 2c shows the cysteine residue Ch1 (C127) that forms the disulfide bond interchanges with a light chain cysteine and selected IgM wild-type Chy1 and Ch2 residues and the positions where one or more residues are replaced by cysteine in the IgM antibodies of the present invention.
[0043] [0043] Figure 2d shows the cy1 cysteine residue (C128) that forms the disulfide bond interchanges with a light chain cysteine and the wild type I | gD joint residues and positions where one or more residues are replaced by cysteine in the IgD antibodies of the present invention.
[0044] [0044] Figure 3a shows the mutations introduced in IgG4 antibodies according to the present invention.
[0045] [0045] Figure 3b shows the positions of the residues in the mutated heavy chain of the I9gG4 antibodies shown in Figure 3a and the predicted disulfide bond that it can form with a cysteine in the light chain (LC) or with another mutated heavy chain (HC). Since cysteine can bind with a cysteine in LC or HC, the underlined chain is the predicted predominant disulfide binding arrangement.
[0046] [0046] Figure 4a shows the mutations introduced in IgG4 antibodies according to the present invention.
[0047] [0047] Figure 4b shows the positions of the cysteine residues in the IgG4 antibodies shown in Figure 4a and the predicted disulfide bond that can form with a cysteine in the light chain (LC) or heavy chain (HC). Since cysteine can bind to a cysteine in LC or HC, the underlined chain is the predominant disulfide binding arrangement envisaged.
[0048] [0048] Figure 5 shows the sequences of the Ch1 and IgG4 antibody hinge region according to the present invention.
[0049] [0049] Figure 6 shows the sequences of Cy41, hinge region, Ch2 and Ch3 of IgG4 antibodies according to the present invention.
[0050] [0050] Figure 7 shows the Western Blot analysis of antibodies according to the present invention with the top gel that shows the results with the use of an Anti-Human Fc Antibody and the bottom gel that shows the results with the use of an Anti-Kappa Antibody.
[0051] [0051] Figure 8 shows the Western Blot analysis of antibodies according to the present invention with the top gel showing the results with the use of an Anti-Human Fc Antibody and the bottom gel showing the results with the use of an Kappa Anti-Human Antibody.
[0052] [0052] Figure 9 shows the Western Blot analysis of antibodies according to the present invention with the top gel that shows the results with the use of an Anti-Human Fc Antibody and the bottom gel that shows the results with the use of an Kappa Anti-Human Antibody.
[0053] [0053] Figure 10 shows the Western Blot analysis of an antibody according to the present invention with the top gel showing the results with the use of an Anti-Human Fc Antibody and the bottom gel showing the results with the use of an Kappa Anti-Human Antibody.
[0054] [0054] Figure 11 shows the results of a Thermoiluor analysis of antibodies of the present invention showing the Fab and Ch2 domain thermostabilities.
[0055] [0055] Figure 12 shows the results of a Thermoiluor analysis of antibodies of the present invention showing the Fab and Cyh2 domain thermostabilities.
[0056] [0056] Figure 13 shows the results of a Thermoiluor analysis of antibodies of the present invention showing the Fab and Ch2 domain thermostabilities.
[0057] [0057] Figure 14 shows the results of a Thermofluor analysis of antibodies of the present invention showing the Fab and Ch2 domain thermostabilities.
[0058] [0058] Figure 15 shows the classification of the Thermostabilities of selected antibodies of the present invention.
[0059] [0059] Figure 16 shows the exchange of heavy chain to wild type Ig9G1, wild type IgG4 and several mutants at two concentrations of GSH and at various time points.
[0060] [0060] Figure 17 shows the heavy chain exchange for wild type IgG4 and several mutants in two concentrations of GSH at various time points.
[0061] [0061] Figure 18 shows the heavy chain exchange for wild type IgG4 and several mutants in various concentrations of GSH at various time points.
[0062] [0062] Figure 19 shows the percentage change of heavy chain exchange for the various mutants in 0.5 mM GSH compared to wild-type IgG4
[0063] [0063] Figure 20 shows the percentage change of heavy chain exchange for several mutants in 5 mM GSH compared to wild-type IgG4.
[0064] [0064] Figure 21 shows the symmetrical arm exchange analysis of IgG4 mutants with alternative residues at position 241. IgG4 WT exchanged more than IgG4 P. The exchange activities of S241G and S241A were similar to each other and significantly smaller and approximately half of I9gG4 WT. S241T switched at levels similar to IgG4 WT.
[0065] [0065] Present invention will now be described in more detail.
[0066] [0066] A symmetrical antibody as used herein is the antibody or antibody fragment in which the heavy chains have a similar or identical sequence in the outer region of the variable regions.
[0067] [0067] Similar to the one used in this document is in the case where the amino acid sequence has 95% identity or higher over the entire analyzed sequence, for example, 96, 97, 98 or 99% identity. The percent identity can be assessed using software known to those skilled in the art.
[0068] [0068] Identical to the employee in this document refers to when there is a sequence identity of 100% through the analyzed sequence, for example, through the entire sequence.
[0069] [0069] In one embodiment, the heavy chain sequences in the antibodies of the present disclosure are covalently linked, for example, via an interchain disulfide bond, for example, a bond that is naturally present in the corresponding wild-type fragment or a bond that has been genetically modified to be present at the desired location in the chains.
[0070] [0070] In one aspect, the antibodies of the present disclosure are characterized by the fact that both heavy chain sequences or fragments have an I9G1-type joint.
[0071] [0071] The core and upper joint of wild type IQG1 has the sequence EPKSCDKTHTCPPCP (SEQ ID No: 224).
[0072] [0072] The core and upper hinge of wild-type IgG4 has the sequence EPKYGPPCPSCP (SEQ ID No: 225).
[0073] [0073] The IgG1 type arniculation as used in this document is intended to refer to the case where one or more, for example, 1 to 5, such as 1, 2 or 3 amino acids are inserted in the IgG4 joint, in particular between EPKYGPP (SEQ ID No: 319) and CPSC and / or one or more of the amino acids YGPP in the IgG4 joint are replaced, for example, to correspond to an amino acid in the I9gG1 joint, in particular G (from YGPP in the IgG4 joint ) is replaced by C or where Y (from YGPP in the I9G4 joint) is replaced by Cou S.
[0074] Accordingly, the present invention also provides a symmetrical bispecific antibody comprising IgG4 heavy chains with an upper, core and lower joint, wherein said upper and core joint in the heavy chain or each heavy chain therein a length of 13 to 17, such as 15 amino acids.
[0075] [0075] In one embodiment, the bispecific symmetrical antibody with an IgG4 heavy first chain has a top hinge and nucleus of 15 amino acids in length.
[0076] [0076] In one embodiment, the upper and core joints of the heavy chains comprise the 12 natural amino acids found in an I9G4 joint and three more amino acids, for example, 3 alanine residues, or 3 glycine residues or a combination thereof.
[0077] [0077] In one embodiment, the upper and core joint in an IgG4 heavy chain of the disclosure consists of an IgG1 type joint, that is, EPKSCDKTHTCPPC SEQ ID No: 25 or a derivative thereof such as: EPKSCDKAAACPPCP SEQIDNo: 26; EPKSCDKGGGCPPCP SFOIDNo: EPKSCDKTHTSPPCP SEQIDNo: 28; EPKSCDKTHTCPPsP —SFQD No: EPKSCDKTHTSPPSP —SEQIDNo: 30; EPKSCDKAAASPPCP SEO o No: EPKSCDKAAACPPSP SEQIDNo: 32; EPKSCDKARASPPsPp —SFQIO No EPKSCDKGGGSPPCP SEQIDNo: 34; EPKSCDKGGGCPPSP SEO D No: EPKSCDKGGGSPPSP SEQID No: 36
[0078] [0078] In one embodiment, the antibody according to the present disclosure comprises an upper joint and nucleus.
[0079] [0079] In one embodiment, the upper joint and the nucleus region are selected from one of the following sequences:
[0080] [0080] In one embodiment, the nucleus hinge region in one or both of the heavy chain sequences or fragments thereof has the sequence CPPCP SEQ ID No: 318.
[0081] [0081] Although not wishing to be bound by theory, it is believed that this sequence is prone to block the dynamic exchange of antibody arms at "in vivo" concentrations, for example, the concentration of less than 0.5 mM of reductant, in particular, reducer concentrations in the order of 5 µM are believed to be physiologically relevant (Zilmer et al., 2005 Drug Design Reviews vol. 2, no. 2, pages 121 to 127, 2005).
[0082] [0082] In one embodiment of the present invention, each bispecific antibody heavy chain has identical core and upper hinge regions selected from the above sequences, and may also have an identical lower hinge region. In an additional embodiment, each bispecific antibody heavy chain has identical Ch1 regions, and can also have identical Ch2 and Ch3 regions. Accordingly, each heavy chain can have identical heavy chain constant region sequences.
[0083] [0083] The "different variable regions" as used in this document are intended to refer to those in which said variable regions have specificity for different antigens. That is, the antigen to which each variable region is specific is a different antigen or a different part of an antigen, for example, a different epitope.
[0084] [0084] “Specific” as used in this document refers to the fact that the binding domains recognized a target antigen with greater affinity and / or avidity than other antigens to which they are not specific (for example, 10, 20, 50, 10 or 1,000 higher). This does not necessarily imply that the specific binding region does not bind any non-target antigens, but that the interaction with the target is such that it can be used to purify the target antigen (to which it is specific) from a complex mixture of antigens, including antigens in the same protein family.
[0085] [0085] In one embodiment, the antibody according to the present disclosure is isolated.
[0086] [0086] Isolated as used in this document is intended to refer to an antibody that is isolated from the human body, for example: prepared by sets of recombinant procedures, purified using a set of procedures such as chromatography and / or in a pharmaceutical formulation.
[0087] [0087] The present invention also provides an expression vector that comprises a sequence encoding the antibodies of the present invention and a host cell that comprises the expression vector.
[0088] [0088] The present invention also provides an antibody as defined above for use in the treatment of a disease or disorder. A method is provided additionally for the treatment of a disease or disorder which comprises administering a therapeutically effective amount of an antibody as defined above.
[0089] [0089] In one embodiment, the antibody according to the present disclosure comprises one or two heavy chain sequences selected independently from a heavy chain sequence disclosed herein.
[0090] [0090] The terms "protein" and "polypeptide" are used interchangeably in this document, unless the context indicates otherwise. "Peptide" is intended to refer to 10 amino acids or less.
[0091] [0091] The terms "polynucleotide" include a gene, DNA, cDNA, RNA, mRNA etc. unless the context indicates otherwise.
[0092] [0092] As used in this document, the term "comprising" in the context of this specification should be interpreted as "including".
[0093] [0093] The term "wild type" in the context of the present invention means an antibody as it can occur in nature or can be isolated from the environment, which does not comprise any genetically modified mutations.
[0094] [0094] The designation for a replacement mutant in this document consists of a letter followed by a number by a letter. The first letter designates the amino acid in the wild type protein. The number refers to the amino acid position at which the amino acid substitution is being made, and the second letter designates the amino acid that is used to replace the wild type amino acid. Residues in the constant and variable domains of antibody are conventionally numbered according to a system designed by Kabat et al. This system is presented in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter “Kabat et al. (Supra)”).
[0095] [0095] Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer additional amino acids or amino acids than in the exact Kabat numbering that corresponds to a shortening of, or insertion into, a structural component, frame or complementarity determining region (CDR), of the domain structure basic variable. The correct Kabat numbering of residues can be determined for a given antibody by aligning homology residues in the antibody sequence with a "standard" numbered Kabat sequence.
[0096] [0096] Alternatively, the numbering of amino acid residues can be performed using the EU index or EU numbering system (also described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
[0097] [0097] An additional numbering system for amino acid residues in antibodies is the IMGT numbering system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 29, 185 to 203 (2005)).
[0098] [0098] The Kabat numbering system is used in this specification unless otherwise indicated that the EU numbering system or the IMGT numbering system is used.
[0099] [0099] Among the four IgG4 isotypes, the intrachain disulfide binding arrangements in the light and heavy chain are similar while the interchain disulfide binding arrangements are unique for each isotype [Reviewed by (Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, MJ, Fodor, S., Kelner, DN ,, Flynn, GC, Liu, YD, Bondarenko, PV, Ricci, MS, Dillon, TM, Balland, A., 2008. Human IgG2 antibodies display disulfide-mediated structural isoforms. J Biol Chem. 283, 16194 to 16205)].
[00100] [00100] As shown in Figure 1b, the hinge region sequences of the four IgG4 isotypes differ. The region of genetic or complete articulation typically consists of residues 226 to 251 (numbering based on the Kabat numbering system). Figure 1b shows the upper, core and lower sections of the articulation regions of the four IgG4 isotypes. For the IgG1 isotype, the upper joint region is residues 226 to 238, the core joint region is residues 239 to 243 and the lower joint region is residues 244 to
[00101] [00101] Figure 1b shows sections of the human IgG light and heavy chain sequences for the IgG isotypes 1 to 4 that indicate the (underlined) cysteine positions that form the C, -Ch1 interchain disulfide bonds. The disulfide bond between Ig-Cy1 of IgG1 is formed between the LC C214 (Kabat numbering system) and C233 (Kabat numbering system) of HC just before the articulation region. In contrast, the disulfide bond Ch1-C, for I9gG2, 3 and 4 is formed between the N terminal of LC C214 and C127 for the intrachain disulfide bond of HC. The sequences of LC and HC surrounding the cysteine residues involved in the formation of C, -Ch1 disulfide bond are shown and aligned in Figure 1b.
[00102] [00102] The present invention investigated how the disulfide bond C, -Ch1 affects the properties of an IgG4 antibody which include the thermostability, structural stability and affinity of the antibody.
[00103] [00103] IgG4 mutants were generated by replacing the cysteine residue in Cu; in position 127 by another amino acid as well as the replacement of one or more of the amino acids in the upper articulation region, preferably the amino acids in the positions selected from 227, 228, 229 and 230, numbered according to the Kabat numbering system, by cysteine. Positions 227, 228, 229 or 230 are in the position that the I9G1 233 cysteine is located or close to it.
[00104] [00104] Each heavy chain can comprise additional mutations that include replacing one or both of the cysteine residues 239 and 242 in the IgG4 hinge region with another amino acid. A mutation to elongate the I9gG4 joint region by three amino acids between positions 238 and 239 to be the same length as the I9gG1 joint was also included in some antibodies. The S241P mutation has also been introduced in some antibodies.
[00105] [00105] The formation of IgG4 semimolecules can be reduced by introducing a Ser to Pro mutation at position 241 (numbered according to the Kabat numbering system) in the joint (Angal, S. et al., 1993. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody. Mol Immunol 30, 105 to 108). In addition, this point mutation did not influence the compact structure of IgG4 thereby allowing IgG4 to retain the reduced ability to activate complement.
[00106] [00106] However, the in vitro exchange of these mutated antibodies can be promoted using high concentrations of antibodies, for example, 1 to 10 mM or more, such as 2, 3, 4, 5, 6, 7, 8, 9 MM. The I9gG4 antibody mutants according to the present invention have been found to have advantageous properties, for example, improved stability.
[00107] [00107] In one embodiment, the I9gG4 antibody mutants according to the present invention exhibit increased thermostability compared to a wild-type IgG4 antibody. It was surprisingly observed that the IgG4 antibody mutants that were mutated to replace the cysteine at position 127 in the Cy41 domain with another amino acid and in which a cysteine was introduced in the heavy chain joint region between positions 227 to 230 showed improved thermostability in compared to a wild-type IgG4 antibody. The mutation to remove the cysteine at position 127 changes the position at which the interchain disulfide bond forms between the heavy chain and the light chain (Cy -Ch1) and forces the light chain to form a disulfide bond with a cysteine that is introduced between positions 227 and 230 in the articulation region of the heavy chain. Consequently, in one embodiment, an I9G4 antibody is provided in which cysteine 127 is replaced by another amino acid and the light chain cysteine is linked via a disulfide bond to a modified cysteine at position 227, 228, 229 or 230.
[00108] [00108] A further enhancement to thermostability was also observed surprisingly by adding three amino acids to the IgG4 articulation region in order to lengthen the I9G4 articulation region.
[00109] [00109] It was surprisingly observed that the IgG4 antibody mutants that were mutated to replace cysteine at position 127 in the Cy1 domain with another amino acid and to replace cysteine at position 239 or position 242 in the heavy chain joint region with another amino acid showed enhanced thermostability compared to a wild type IgG4 antibody.
[00110] [00110] In one embodiment, the antibodies of the present invention show reduced formation of so-called semimolecules, which are formed from a single light chain and a single heavy chain (HL). The antibodies of the present invention that comprise a C239 mutation, but that do not carry a C242 mutation, generally show reduced semimolecule formation. Without being limited by theory, it is believed that this is due to the removal of Cysteine at position 239 which reduces the formation of intrachain disulfide binding in the heavy chain and therefore reduces the number of semimolecules compared to antibodies that do not carry a mutation in C239 or C242. Antibodies that carry a C242 mutation but do not carry a C239 mutation appear to form more semimolecules compared to antibodies that carry a C239 mutation but do not carry a C242 mutation. Without being bound by theory, cysteine at position 239 is believed to be more reactive compared to cysteine at position 242 and is able to form a disulfide bond with a heavy-chain joint cysteine or with light-chain cysteine.
[00111] [00111] The antibodies that carry the mutation in both C239 and C242 form a high proportion of semimolecules due to the lack of inter-disulfide bond formation between two heavy chains. However, antibodies that comprise mutations in both C239 and C242 still have the capacity to form entire antibody molecules due to the binding of heavy chains through non-covalent bonds.
[00112] [00112] The formation of reduced semimolecule is also seen in antibodies that carry the S241P mutation.
[00113] [00113] The antibodies according to the present invention also show comparable affinity to the target antigen in comparison to the wild type I9G4 antibody.
[00114] [00114] Mutations to the heavy chain constant regions of the bispecific antibodies of the present invention are described in more detail below. Methods for replacing amino acids are well known in the art of molecular biology. Such methods include, for example, site-directed mutagenesis using methods such as PCR to exclude and / or replace amino acids or de novo design of synthetic sequences.
[00115] [00115] Figure 2a shows the IgG1 wild-type, IgG4 wild-type hinge residues and the positions at which the mutations were introduced in the antibodies of the present invention. Numbering based on the Kabat numbering system.
[00116] [00116] The antibodies according to the present invention comprise a mutation at position 127 (C127), in which the cysteine residue is replaced by another amino acid, preferably an amino acid that does not contain a thiol group. By replacing swapping or replacing it means that where the cysteine interchain 127 can normally be found in the heavy antibody chain another amino acid is in its place. The C127 mutation can be any mutation suitable for one, two or three of the nucleotides encoding the amino acid at position 127 which changes the amino acid residue of cysteine to another suitable amino acid. Examples of suitable amino acids include serine, threonine, alanine, glycine or any polar amino acid. A particularly preferred amino acid is serine.
[00117] [00117] Substitution of cysteine at position 127 by another amino acid removes cysteine in the Cy1 domain that normally forms a disulfide bond with a cysteine in the light chain in wild-type IgG4. Therefore, in order to form a light chain and heavy chain pairing through an interchain disulfide bond, the light chain must form a disulfide bond with a cysteine that is positioned in the joint region of the heavy chain.
[00118] [00118] In a first aspect of the invention, the antibodies according to the present invention comprise a heavy chain in which one or more of the amino acids at the positions selected from 227, 228, 229 and 230, numbered according to the system of Kabat numbering, are replaced by cysteine. Accordingly, the antibodies according to the present invention can carry one or more of the following mutations: S227C; K228C; Y229C; G230C.
[00119] [00119] In one embodiment, only a residue selected from 227, 228, 229 and 230 is replaced by a cysteine residue.
[00120] [00120] In one embodiment, the antibodies of the present invention carry the Y229C or G230C mutation.
[00121] [00121] The inclusion of a cysteine residue in a selected position from 227, 228, 229 and 230, in the hinge region of the heavy chain provides a new position for an interchain disulfide bond to form between the heavy chain and the light chain. The present inventors have observed that this new interchain disulfide binding arrangement provides IgG4 antibodies that have enhanced thermostability compared to a wild-type IgG4 antibody.
[00122] [00122] Additional mutations can be introduced to the antibodies of that aspect of the present invention. In one embodiment, cysteine at position 239 (C239) and / or cysteine at position 242 (C242), numbered according to the Kabat numbering system, in the heavy chain are replaced by another amino acid, preferably an amino acid that does not contain a thiol group. By exchanging or replacing it means that, where cysteine 239 and / or cysteine 242 can normally be found in the heavy chain antibody, another amino acid is in its place. The C239 and / or C242 mutation can be any suitable mutation for one, two or three of the nucleotides encoding the amino acid that changes the cysteine amino acid residue to another suitable amino acid. Examples of suitable amino acids include serine, threonine, alanine, glycine or any polar amino acid. A particularly preferred amino acid is serine.
[00123] [00123] In one embodiment, the cysteine at position 239 in the heavy chain is replaced by another amino acid and the cysteine at position 242 in the heavy chain is replaced by another amino acid. In this embodiment, substitution of both C239 and C242 removes both cysteine residues in the hinge region of the heavy chain that normally form the heavy interchain disulfide bonds with the corresponding cysteines in another heavy chain. The resulting semimolecules can form entire antibody molecules through non-covalent bonding between two heavy chains.
[00124] [00124] In an alternative embodiment, the cysteine at position 239 in the heavy chain is replaced by another amino acid. In this modality, the cysteine at position 242 is not replaced by another amino acid.
[00125] [00125] In an additional alternative embodiment, the cysteine at position 242 in the heavy chain is replaced by another amino acid. In this modality, the cysteine at position 239 is not replaced by another amino acid.
[00126] [00126] The substitution of C239 or C242, leaves a cysteine in the heavy chain that has the capacity to form a disulfide bond between heavy chain with a cysteine in another heavy chain. Without being limited by theory, it is believed that the replacement of a cysteine in the joint region, particularly the replacement of C239, reduces the formation of an intrachain disulfide bond in the joint region and can therefore reduce the formation of semimolecules. of antibody.
[00127] [00127] In one embodiment of the present invention, where the serine at position 227 is replaced by a cysteine, the antibody preferably does not include mutations at positions C239 and C242. In another embodiment, where the serine at position 227 is replaced by a cysteine, the cysteine at position 239 in the heavy chain is preferably replaced by another amino acid, but the cysteine at position 242 is not replaced by another amino acid.
[00128] [00128] In one embodiment, the antibodies of the present invention comprise an IgG4 heavy chain that is mutated to insert one or more amino acids between amino acids 226 to 243. The number of amino acids entered can be from 1 to 10.1 to 5.1a3 , preferably 1, 2, 3 or 4 amino acids are inserted. Amino acids are preferably inserted between amino acids 238 and 239. Any suitable amino acids can be inserted in the region of articulation, such as alanines, glycines, serines or threonines and combinations thereof. Preferably, three alanines (AAA), three glycines (GGG), three serines (SSS) or three threonines (TTT) are inserted or one threonine, histidine and another threonine (THT). The antibodies of the present invention that comprise an IgG4 heavy chain that has been mutated to insert three amino acids into the hinge region have been shown to have enhanced thermostability.
[00129] [00129] An additional mutation that can be introduced into the antibodies according to the present invention is the S241P mutation. This mutation has previously been shown to reduce the formation of semimolecules (Angal, S. Et al., 1993. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (I9G4) antibody. Mol Immunol 30, 105 to 108). It has been surprisingly found that mutant antibodies of the present invention that comprise the S241P mutation demonstrate some heavy chain exchange in vitro under strong reducing conditions compared to IgG4 P (I9gG4 with S241P). This allows the creation of bispecific antibodies in vitro from mutants of IgG4 antibodies of the present invention. The antibodies according to the present invention can comprise one or more additional mutations in the hinge region. For example, antibodies can additionally comprise one or more of the following mutations S227P, Y229S, P237D and P238K.
[00130] [00130] In one embodiment, the antibody according to the present invention effectively comprises an I9gG1 hinge region of residue 226 to 243 (upper hinge and core hinge). Accordingly, the antibody of the present invention comprises a hinge region where glycine at position 230 is replaced by cysteine, serine at position 227 is replaced by proline, tyrosine at position 229 is replaced by serine, proline at position 237 is replaced by aspartic acid, the proline at position 238 is replaced by lysine, the amino acid sequence threonine-histidine-threonine is inserted between positions 238 and 239 and the serine at position 241 is replaced by proline. These mutations can also be written as S227P, Y229S, G230C, P237D, P238KTHT and S241P, as shown in Figure 2a. It has been observed that the introduction of these additional mutations to the I9gG4 articulation region provides an antibody that has improved thermostability.
[00131] [00131] The antibody according to the present invention preferably has a lower hinge of I9G4 from residue 244 to 251 (APEFLGGP SEQ | D No: 229). Without being limited by theory, it is believed that the IgG4 lower articulation region contributes to the lack of effective function of an Ig9G4 antibody.
[00132] [00132] In a second aspect of the present invention, the symmetrical bispecific antibody is provided in which, in one or both heavy chains, the cysteine interchain at position 127 is replaced by another amino acid, as described above, and the cysteine at position 239 or cysteine at position 242, numbered according to the Kabat numbering system, in the heavy chain are replaced by another amino acid. In this second aspect, none of the residues in positions 227, 228, 229 and 230 is replaced by a cysteine residue.
[00133] [00133] A bispecific symmetrical antibody of the IgG4 class comprising two heavy chains that each comprise a variable domain, Cy1 domain and articulation region, where in each heavy chain, the cysteine interleaves at position 127, numbered according to the Kabat numbering system, is replaced by another amino acid; and cysteine at position 239 and / or cysteine at position 242, numbered according to the Kabat numbering system, are replaced by another amino acid, where the constant region sequence of each heavy chain is similar or identical and the variable region each heavy chain is different.
[00134] [00134] Antibodies according to the second aspect of the present invention have been found to have surprisingly improved thermostability compared to a wild-type IgG4 antibody.
[00135] [00135] In the second aspect of the present invention, the antibody can comprise one or more additional mutations. In one embodiment, the antibody comprises an IgG4 heavy chain that is mutated to insert three amino acids between amino acids 226 to 243, preferably between amino acids 238 and 239, as described above. In an additional embodiment, the antibody comprises the S241P mutation. In an additional embodiment, the antibody may additionally comprise one or more of the following mutations S227P, Y229S, P237D and P238K.
[00136] [00136] Figures 3a and 4a also show the mutations introduced in the IgG4 antibodies according to the present invention. Figures 3b and 4b show the positions of the cysteine residues in the IgG4 antibodies of the present invention and also show the predicted binding of the cysteine to a cysteine in the light chain (LC) or other heavy chain (HC). For the cysteine residues that they present (LC or HC), it is possible that the cysteine is linked to a cysteine in the light chain or in the heavy chain, but where the LC or HC is underlined this is the disulfide bond believed to occur predominantly.
[00137] [00137] In one embodiment, the present invention provides an antibody that comprises two heavy chains that each comprise a variable region, a Cy1 domain and a hinge region, and each heavy chain comprises mutations of an antibody selected from 2, 3, 6, 7, 8, 15, 16, 28, 28P, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 44, 44P, 45, 46, 47 and 48, as shown in Table 1. Accordingly, the present invention provides an antibody comprising two heavy chains that each comprise a variable region, a Cy1 domain and a hinge region and each heavy chain comprises one of the following sequences : SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO : 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO O: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267 and SEQ ID NO: 268.
[00138] [00138] In a preferred embodiment, the antibody of the present invention comprises two heavy chains that each comprise a variable region, a Cy1 domain and a hinge region, and comprise one of the following sequences: SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID
[00139] [00139] In an additional preferred embodiment, the present invention provides an antibody comprising two heavy chains each comprising a variable region, a Cy41 domain, a hinge region, a Ch2 domain and a Cy43 domain and each heavy chain comprises mutations of an antibody selected from 2, 3, 6, 7, 8, 15, 16, 28, 28P, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 44, 44P, 45, 46, 47 and 48, as shown in Table 1. Accordingly, the present invention provides an antibody comprising two heavy chains that each comprise a variable region, a Cun1 domain, a hinge region , a Ch2 domain and a Cy3 domain and each heavy chain comprises one of the following sequences: SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO : 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305 and SEQ ID NO: 306.
[00140] [00140] A particularly preferred antibody of the present invention comprises two heavy chains each comprising a variable region, a Cy1 domain and a hinge region, wherein the heavy chain comprises SEQ ID NO: 36 (antibody 28P), SEQ ID NO: 37 (antibody 44P) or SEQ ID NO: 35 (antibody 48). An additional particularly preferred antibody of the present invention comprises two heavy chains each comprising a variable region, a Cy1 domain, a hinge region, a Ch2 domain and a Cy3 domain in which the heavy chain comprises SEQ ID NO: 62 ( antibody 28P), SEQ ID NO: 63 (antibody 44P) or SEQ ID NO: 61 (antibody 48). Antibodies 28P, 44P and 48 are particularly preferred since they exhibit significantly improved thermostability and additionally exhibit reduced semimolecule formation.
[00141] [00141] Table 1 below lists exemplary antibodies with mutations that have been introduced in comparison to the wild type IgG4 sequence. Table 1 also includes wild-type IgG1 and IgG4 antibodies and control antibodies. Table 1: R z; Domain CH1 and | ... Cn! Number of Heavy Chain Mutations; = Articulation, Ch2 SEQ ID NO: 6 | emscaecas - | as | 28 | 8 C127S, G230C, C239S, C242S 13 C242S 277 315 antibody (Kabat numbering) SEQ ID NO: & CH3 SEQ ID NO:
[00142] [00142] Figures 3a and 4a also show the mutations introduced in IgG4 antibodies according to the present invention. Figures 3b and 4b show the positions of the cysteine residues in the IgG4 antibodies of the present invention and also show the predicted binding of the cysteine to a cysteine in the light chain (LC) or another heavy chain (HC). For the cysteine residues that they present (LC or HC), it is possible that the cysteine is linked to a cysteine in the light chain or in the heavy chain, but where the LC or HC is underlined this is the disulfide bond that is believed to occur predominantly.
[00143] [00143] In a preferred embodiment, the present invention provides an antibody comprising two heavy chains that each comprise a variable region, a Chy1 domain and a hinge region and each heavy chain comprises mutations of an antibody selected from of 2, 3, 6, 7, 8, 15, 16, 28, 28P, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 44, 44P, 45, 46,47 and 48, as shown in Table 1. Accordingly, the present invention provides an antibody comprising two heavy chains that each comprise a variable region, a Cy1 domain and a hinge region and each heavy chain comprises one of the sequences a follow: SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO : 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267 and SEQ ID NO: 268.
[00144] [00144] In a preferred embodiment, the antibody of the present invention comprises two heavy chains that each comprise a variable region, a Cy1 domain and a hinge region, and comprise one of the following sequences: SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267 and SEQ ID NO: 268. More preferably, the antibody of the present invention comprises two heavy chains that each comprise a variable region, a Ch41 domain and a hinge region and each heavy chain comprises one of the following sequences: SEQ ID NO: 243, SEQ ID NO : 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ I D NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO : 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 , SEQ ID NO: 266, SEQ ID NO: 267 and SEQ ID NO: 268.
[00145] [00145] In an additional preferred embodiment, the present invention provides an antibody comprising two heavy chains each comprising a variable region, a Ch41 domain, a hinge region, a Ch2 domain and a Cy43 domain and each heavy chain comprises mutations of an antibody selected from 2, 3, 6, 7, 8, 15, 16, 28, 28P, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 44, 44P, 45, 46, 47 and 48, as shown in Table 1. Accordingly, the present invention provides an antibody comprising two heavy chains that each comprise a variable region, a Cu1 domain, a hinge region , a Ch2 domain and a Ch3 domain and each heavy chain comprises one of the following sequences: SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO : 294, SEQ ID NO: 295, SEQ ID NO: 2 96, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305 and SEQ ID NO: 306.
[00146] [00146] A particularly preferred antibody of the present invention comprises two heavy chains each comprising a variable region, a Cy1 domain and a hinge region, wherein the heavy chain comprises SEQ ID NO: 267 (antibody 28P), SEQ ID NO: 268 (antibody 44P) or SEQ ID NO: 266 (antibody 48). An additional particularly preferred antibody of the present invention comprises two heavy chains each comprising a variable region, a Ch41 domain, a hinge region, a Ch2 domain and a Cy3 domain in which the heavy chain comprises SEQ ID NO: 305 ( antibody 28P), SEQ ID NO: 306 (antibody 44P) or SEQ ID NO: 304 (antibody 48). Antibodies 28P, 44P and 48 are particularly preferred since they exhibit significantly improved thermostability and additionally exhibit reduced semimolecule formation.
[00147] [00147] Antibodies 2,3 and 8 have been shown to form a significant amount of so-called semimolecules (HL). These mutants can form entire antibody molecules (H2L2) in vitro under non-denaturation conditions, but any non-covalent associations between heavy chains and / or between light and heavy chains are removed under non-reducing SDS-PAGE conditions. Although it is often taught to be desirable to reduce the formation of semimolecules, antibodies that have an increased tendency to form semimolecules can be advantageous for certain uses. Antibodies that form stable semimolecules (HL) and little or no whole antibody (H2L2) due to the antibody heavy chain being unable to form a covalent or non-covalent association with another heavy chain are of particular interest. Antibodies that form stable semimolecules can be advantageous for the production of monovalent antibodies. Antibodies that form semimolecules can also provide a useful way to produce a bispecific antibody due to the formation of whole antibodies from semimolecules that have different specificities, wherein the entire antibody is bispecific and monovalent for each antigen. The heavy chains of such a bispecific antibody can be joined non-covalently.
[00148] [00148] Antibody 3 retains C239 in the hinge region, but appears to be unable to form interarticulation heavy chain disulfide bonds, presumably due to the efficient disulfide formation between the C-terminal light chain cysteine and the C239 joint. A comparison of antibodies 2 and 3 shows the extent of the “reach” of the C-terminal cysteine of the light chain, due to the fact that the light chain disulfide binds more efficiently to C239 than to
[00149] [00149] Although the antibodies mutated according to the present invention are described above in relation to the IgG4 isotype, the skilled person will note that the mutations made to the IgG4 antibody can also be applied to other isotypes or classes of antibody that have the same disposition disulfide binding agent than an IgG4 antibody in order to provide an enhanced antibody. Specific examples of antibodies that have the same disulfide binding arrangement as an I9gG4 antibody are I9G3 antibodies, IgM antibodies and 1gD antibodies. As shown in Figure 1b, I9gG3 and IgM have a cysteine at position 127 in the Chy1 domain and IgD has a cysteine at position 128 in the Cit domain that is equivalent to C127 in the Chy1 domain of IgG4 that forms a disulfide bond interchanged with a cysteine in the light chain. Furthermore, it can also be seen from Figure 1b that regions of upper articulation of I9G3 and IgD and the C terminal region of the Cy1 domain and the N terminal region of the Chy2 domain in IgM do not contain a cysteine residue that is equivalent to residues from the upper IgG1 joint region. Accordingly, the present invention additionally provides an IgG3 antibody, an IgD antibody and an IgM antibody in which the cysteine in the Chy1 domain which forms a disulfide bond interchanges with a cysteine in a light chain is replaced by another amino acid and in that one or more amino acids that are in a position structurally analogous to the upper articulation region of I9gG1 or IgG4 are replaced by cysteine.
[00150] Accordingly, the present invention also provides a symmetrical bispecific antibody of the IgG3 class that comprises two heavy chains each comprising a variable region, a Cy1 domain and a hinge region, wherein in each heavy chain: a . the cysteine in the Chi domain that forms a disulfide bond interchanged with a cysteine in a light chain is replaced by another amino acid; and b. one or more of the amino acids positioned in the upper joint region are replaced by cysteine
[00151] [00151] in which the constant region sequence of each heavy chain is similar or identical and the variable region in each heavy chain is different.
[00152] [00152] In a preferred embodiment of the IgG3 antibody of the aspect of the present invention, the cysteine in the Cy1 domain that forms a disulfide bond interchain with a cysteine in a light chain is the cysteine at position 127, numbered according to the system of Kabat numbering, as shown in Figure 1b and 2b.
[00153] [00153] In a preferred embodiment of the IgG3 antibody of the aspect of the present invention, the one or more amino acids positioned in the upper hinge region that can be replaced by cysteine are one or more of the amino acids at the positions selected from 226, 227, 228 229, 230, 232 and 233, numbered according to the Kabat numbering system, as shown in Figures 1b and 2b.
[00154] [00154] The present invention additionally provides a symmetrical bispecific antibody of the class IIM comprising two heavy chains each comprising a variable region, a Cy1 domain and a Ch2 domain, wherein in each heavy chain: aa cysteine in the Cy1 domain which forms a disulfide bond interchanged with a cysteine in a light chain is replaced by another amino acid; and b. one or more of the amino acids positioned in the Chy1 domain or in the Ch2 domain are replaced by cysteine
[00155] [00155] in which the constant region sequence of each heavy chain is similar or identical and the variable region in each heavy chain is different.
[00156] [00156] In a preferred embodiment of the IgM antibody of the aspect of the present invention, the cysteine in the Cy1 domain that forms a disulfide bond interchanged with a cysteine in a light chain is the cysteine at position 127, numbered according to the system of Kabat numbering, as shown in Figures 1b and 2c.
[00157] [00157] In a preferred embodiment of the IgM antibody of the aspect of the present invention, one or more amino acids positioned at the C-terminal end of the Ch1 domain or at the N-terminal end of the Ch2 domain are replaced by cysteine. The position of preferred amino acids at the C-terminal end of the Cy1 domain that can be replaced by cysteine are one or more of the amino acids at the positions selected from 223, 223A, 223B and 223C, numbered according to the Kabat numbering system, as shown in Figures 1b and 2c. The position of preferred amino acids at the N-terminal end of the Ch2 domain that can be replaced by cysteine are one or more of the amino acids at the positions selected from 243G, 243H and 2431, numbered according to the Kabat numbering system, as shown in Figures 1b and 2c. Accordingly, any one or more of amino acids 223 to 243 can be substituted for cysteine.
[00158] [00158] The present invention additionally provides a bispecific symmetrical IgD class antibody comprising two heavy chains each comprising a variable region, a Cy1 domain and a hinge region, wherein in each heavy chain: a. the cysteine in the Chi domain that forms a disulfide bond interchanged with a cysteine in a light chain is replaced by another amino acid; and b. one or more of the amino acids positioned in the joint region is replaced by cysteine
[00159] [00159] in which the constant region sequence of each heavy chain is similar or identical and the variable region in each heavy chain is different.
[00160] [00160] In a preferred embodiment of the IgD antibody of the aspect of the present invention, the cysteine in the Cy1 domain that forms a disulfide bond interchanged with a cysteine in a light chain is the cysteine at position 128 numbered according to the numbering system Kabat, as shown in Figures 1b and 2d.
[00161] [00161] The hinge region of an IgD antibody can be defined as
[00162] [00162] In a preferred embodiment of the IgD antibody of the aspect of the present invention, the one or more amino acids positioned in the hinge region that are replaced by cysteine are one or more of the amino acids at the positions selected from 227, 228, 229, 230, 231, 232 and 233, numbered according to the Kabat numbering system, as shown in Figures 1b and 2d.
[00163] [00163] The Ig9G3, IgD or IgM antibodies provided by the present invention may comprise one or more additional mutations to the hinge region as discussed above with respect to the I9G4 antibody.
[00164] [00164] In this aspect of the present invention, the antibody is preferably of the IgG4 class.
[00165] [00165] The term "antibody" as used herein includes intact antibodies (integer) and functionally active fragments that comprise two heavy chains that each comprise a Vu domain, a CH1 domain and a hinge region. The antibody according to the present invention preferably comprises at least one light chain. Accordingly, the term "antibody" in the present invention encompasses bi, tri or tetravalent antibodies, a dimer of Fab 'and F (ab') fragments, and whole antibody molecules comprising two light and heavy chain pairings.
[00166] [00166] As is well known in the art, a typical Fab 'molecule comprises a pair of heavy and light chains in which the heavy chain comprises a variable region Vu, a constant domain Ch1 and a hinge region and the light chain comprises a variable region V, and a constant domain C ,.
[00167] [00167] In one embodiment, a Fab 'dimer is provided according to the present disclosure, for example, dimerization can be through the joint.
[00168] [00168] In one embodiment, the heavy chain comprises a Ch2 domain and a Ch3 domain and optionally a Ch4 domain. In one embodiment, the antibody comprises two heavy chains, each of which is as defined above in the first or second aspect of the present invention. The antibodies according to the present invention also preferably comprise two light chains, wherein the light chain constant regions are preferably identical. In that embodiment, where the antibody comprises two heavy chains and two light chains, preferably both constant chain heavy chain sequences are identical as defined above by the first or second aspect of the present invention, and both light chain constant region sequences are identical.
[00169] [00169] In a preferred embodiment, the antibody of the present invention is an entire antibody comprising two light chains and two heavy chains, each heavy chain comprising an IgG4 Cut in which the cysteine at position 127, numbered according to the system numbered Kabat, it is replaced by another amino acid, an IgG1 middle and upper joint region, an IgG4 lower joint region, a Ch2 domain and a Ch3 domain.
[00170] [00170] The entire hinge region of an IgG4 antibody typically consists of residues 226 to 251 (numbering based on the Kabat numbering system. However, the hinge region can be shortened or lengthened as required. For example, antibodies according with the first aspect of the present invention, the wild-type amino acid is replaced by a cysteine residue at position 227, 228, 229 or 230, the hinge region can end after the new cysteine residue at position 227, 228, 229 or 230. The antibodies according to the present invention can also comprise one or more additional amino acids positioned at the N-terminus and / or C-terminus of the hinge region. In addition, other characteristics of the hinge can be controlled, such as the distance from the s ) light chain interlinking cysteine (s), the distance between the joint's cysteines and the composition of other amino acids in the joint that may affect the properties joint requirements such as flexibility, for example, glycines can be incorporated into the joint to increase rotational flexibility or prolines can be incorporated to reduce flexibility. Alternatively, combinations of hydrophobic or charged residues can be incorporated into the joint to impart multimerization or purification properties. Other modified hinge regions can be completely synthetic and can be designed to have desired properties such as length, composition and flexibility.
[00171] [00171] The constant region domains, particularly in the Fc domain, when present, employed in the present invention, are preferably IgG4 isotype when antibody effector functions are not required. According to each heavy chain, it preferably comprises an IgG4 Ch2 domain domain and a Cx43 domain, as shown in SEQ ID NO: 64.
[00172] [00172] It will be appreciated that sequence variants of the Fc constant region domains can also be used.
[00173] [00173] In one embodiment, each heavy chain comprises Ig2 Ch2 and C43 domains in which arginine at position 409 (EU numbering) is replaced by lysine, threonine, methionine or leucine in order to inhibit the formation of aggregates in PH low (US No. 2008/0063635 Takahashi et al.) Mutations in L235, D265, D270, K322, P331 and P329 (numbered according to the EU numbering system) are also taught in order to mitigate CDC activity ( US No. 2008/0063635 Takahashi et al.).
[00174] [00174] Each heavy chain can comprise the mutations as taught in WO2008 / 145142 Van de Winkel et al. which reveals stable IgG4 antibodies that have a reduced ability to undergo Fab arm replacement by replacing the arginine residue at position 409, the Phe residue at position 405 or Lys at position 370 (numbered according to the EU numbering).
[00175] [00175] In one embodiment, each heavy chain comprises an IgG4 Ch2 domain and an IgG1 Cy43 domain, as shown in SEQ ID NO: 280
[00176] [00176] In the embodiment of the present invention where the antibody is a mutated IgG3, I | gD or IgM antibody, each heavy chain preferably comprises a Ch2 domain and a Cu3 domain, and optionally a Ch4 domain. In the IgG3 antibody, each heavy chain preferably comprises an IgG3 Ch2 domain and an I9G3 Ch3 domain. In the IgD antibody, each heavy chain preferably comprises the IgD Ch2 domain and a Ch3 domain of
[00177] [00177] In one embodiment, mutations in C127 in the Cy1 domain can be made at equivalent positions in other IgG isotopes (1, 2, 3) or in other classes of antibody. Such mutant antibodies may comprise one or more additional mutations, for example, in the hinge region and / or the Ch2 domain and / or the Ch3 domain. Examples of specific mutations in the articulation region and Ch2 and Cy3 domains are described above in relation to the other aspects of the present invention.
[00178] [00178] Specific examples of the aforementioned mutant antibodies are the IgG4 antibody mutants 4, 5, 5P, 9, 10, 11 and 14 listed in Table 1.
[00179] [00179] Accordingly, the present invention provides an antibody comprising two heavy chains, each heavy chain comprising a Cy1 domain and a hinge region and each heavy chain comprising mutations of an antibody selected from 4, 5, 5P, 9, 10, 11 and 14, as shown in Table 1. Accordingly, the present invention provides an antibody comprising two heavy chains, each heavy chain comprising a Ch41 domain and a hinge region and each heavy chain comprising one of the following strings: SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275 and SEQ ID NO: 276.
[00180] [00180] In an additional embodiment, the present invention provides an antibody comprising two heavy chains, each heavy chain comprising a Cy41 domain, a hinge region, a Ch2 domain and a Cy3 domain and each heavy chain comprising mutations of an antibody selected from 4, 5, 5P, 9, 10, 11 and 14, as shown in Table 1. Accordingly, the present invention provides an antibody comprising two heavy chains, each heavy chain comprising a Ch41 domain , a hinge region, a Ch2 domain and a Ch3 domain and each heavy chain comprises one of the following sequences: SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313 and SEQ ID NO: 316.
[00181] [00181] Antibodies 4 (C127S, C239S and C242S) and 14 (C2398S and C242S) have been shown to form a significant amount of so-called semimolecules (HL). These mutants can form whole antibody molecules (H2L2) in vitro under non-denaturation conditions, but any non-covalent associations between heavy chains and / or between light and heavy chains are removed under non-reducing SDS-PAGE conditions. Although it is often taught to be desirable to reduce the formation of semimolecules, antibodies that have an increased tendency to form semimolecules may be advantageous for certain uses, for example, the in vitro formation of semimolecules can facilitate heavy chain exchange and facilitate preparation bispecific IgG4 antibodies according to the present disclosure.
[00182] [00182] Antibodies 2,3 and 8, as described above, have also been shown to form significant amounts of semimolecules and therefore can also be used in situations where semimolecule formation is desirable.
[00183] [00183] Antibody 3 retains C239 in the hinge region, but appears to be unable to form interarticulation heavy chain disulfide bonds, presumably due to the efficient disulfide formation between the C-terminal light chain cysteine and the C239 joint. A comparison of antibodies 2 and 3 shows the extent of the “reach” of the C-terminal cysteine of the light chain, due to the fact that the light chain disulfide binds more efficiently to C239 than to C242 in the joint region. In addition, antibody 3 has increased stability compared to antibody 2.
[00184] [00184] Antibodies 5 (G230C), 5P (G230C and S241P), 9 (G230C and C239S), 10 (G230C and C242S) and 11 (G230C, C239S and C242S) comprise two cysteines (one cysteine at position 127 in CH1 and 230 in the upper joint) with which the cysteine in the light chain can form a disulfide bond. Accordingly, these antibodies can comprise a disulfide-unbound cysteine in the Cy41 heavy chain or in the hinge region which can be used advantageously for fixing an effector molecule in the disulfide-unbound cysteine. Additional antibody 5 comprises two articulating cysteines in
[00185] [00185] The term "antibody" as used herein includes intact (whole) antibodies and functionally active fragments that comprise two heavy chains that each comprise a Vu domain, a Cy1 domain and a hinge region. The antibody according to the present invention preferably comprises at least one light chain. Accordingly, the term "antibody" in the present invention encompasses bi, tri or tetravalent antibodies, Fab 'and F (ab') fragments, antibody medium molecules or semimolecules comprising a single light and heavy chain pairing and molecules of whole antibody comprising two light chain and heavy chain pairings.
[00186] [00186] As is well known in the art, a typical Fab 'molecule comprises a pair of light and heavy chains where the heavy chain comprises a variable region Vu, a constant domain Ch1 and a hinge region and the light chain comprises a variable region V, and a constant domain C ,.
[00187] [00187] In one embodiment, a Fab 'dimer is provided according to the present disclosure for example, dimerization can be through the joint.
[00188] [00188] In one embodiment, the heavy chain comprises a Ch2 domain and a Ch3 domain and optionally a Ch4 domain. In one embodiment, the antibody comprises two heavy chains, each of which is as defined above in the first or second aspect of the present invention. The antibodies according to the present invention also preferably comprise two light chains. In that embodiment where the antibody comprises two heavy chains, preferably both heavy chain sequences are identical as defined above by the first or second aspect of the present invention.
[00189] [00189] In a preferred embodiment, the antibody of the present invention is an entire antibody comprising two light chains and two heavy chains, each heavy chain comprising an Ig41 Cy41 in which the cysteine at position 127, numbered according to Kabat numbering system, is replaced by another amino acid, an IgG1 middle and upper articulation region, an IgG4 lower articulation region, a Ch2 domain and a Ch3 domain.
[00190] [00190] The complete hinge region of an IgG4 antibody typically consists of residues 226 to 251 (numbering based on the Kabat numbering system). However, the joint region can be shortened or lengthened as required. For example, the antibodies according to the first aspect of the present invention, the wild-type amino acid is replaced by a cysteine residue at position 227, 228, 229 or 230, the hinge region can end after the new cysteine residue at position 227, 228, 229 or 230. The antibodies according to the present invention can also comprise one or more additional amino acids positioned at the N-terminus and / or C-terminus of the hinge region. In addition, other characteristics of the joint can be controlled, such as the distance from the light chain interchain cysteine joint cysteine (s), the distance between the joint cysteines and the composition of other amino acids in the joint that can affect joint properties such as flexibility, for example, glycines can be incorporated into the joint to increase rotational flexibility or prolines can be incorporated to reduce flexibility. Alternatively, combinations of hydrophobic or charged residues can be incorporated into the joint to impart multimerization or purification properties. Other modified hinge regions can be completely synthetic and can be designed to have desired properties such as length, composition and flexibility.
[00191] [00191] The constant region domains, in particular in the Fc domain, when present, employed in the present invention, are preferably of the IgG4 isotype when antibody effector functions are not required. Accordingly, each heavy chain preferably comprises an IgG4 Ch2 domain and a Ch43 domain, as shown in SEQ ID NO: 279.
[00192] [00192] In one embodiment, the antibody is a fragment of monoclonal antibody, completely human, humanized or chimeric. In one embodiment, the antibody is either completely human or humanized.
[00193] [00193] Monoclonal antibodies can be prepared by any method known in the art such as the hybridoma procedure set (Kohler & Milstein, Nature, 1975, 256, 495 to 497), the trioma procedure set, the human B-cell hybridoma procedure (Kozbor et al., Immunology Today, 1983, 4, 72) and the EBV hybridoma procedure set (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, pages 77 to 96, Alan R. Liss, Inc., 1985).
[00194] [00194] Antibodies for use in the invention can also be generated using single lymphocyte antibody methods by cloning and expression of immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies, for example , by the methods described by Babcook, J. et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (15), 7843 to 7848, documents No. * WO 92/02551, WO2004 / 051268 and WO2004 / 106377.
[00195] [00195] Humanized antibodies are antibody molecules of non-human species that have one or more complementarity determining regions (CDRs) of the non-human species and a frame region of a human immunoglobulin molecule that optionally comprises one or more residues of donor of the non-human species (see, for example, document No. US
[00196] [00196] Antibodies for use in the present invention can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al., J. Inmunol. Methods, 1995, 182, 41 to 50; Ames et al., J. Inmunol. Methods, 1995, 184, 177 to 186; Kettleborough et al. Eur. J. Immunol., 1994, 24, 952 to 958; Persic et al., Gene, 1997 187, 9 to 18; and Burton et al., Advances in Immunology, 1994, 57, 191 to 280; in WO 90/02809; in WO 91/10737; in WO 92/01047; in document nº WO
[00197] [00197] Completely human antibodies are those antibodies in which the variable regions and the constant regions (when present) of both light and heavy chains are all of human origin, or substantially identical to sequences of human origin, not necessarily the same antibody. Examples of fully human antibodies may include antibodies produced, for example, by the phage display methods described above and the antibodies produced by mice in which the murine immunoglobulin constant and / or variable region genes have been replaced by human counterparts, by example, as described in general terms in documents No. * EPO5S46073 B1, US 5,545,806, US 5,569,825, US 5,625,126, US
[00198] [00198] The antibody starting material for use in the present invention can be prepared by using sets of recombinant DNA procedures that involve the manipulation and re-expression of DNA encoding the constant and variable region (s) (s) of. Standard molecular biology procedure sets can be used to modify, add or delete amino acids or domains as desired. Any changes to the constant or variable regions are still covered by the terms "variable" and "constant" regions as used in this document.
[00199] [00199] Antibody starting material can be obtained from any species that include, for example, mouse, rat, rabbit, hamster, camel,
[00200] [00200] In one embodiment, the antibody comprises a pair of variable domains that form a binding domain that is a cognate pair. The cognate pair as used in this document is intended to refer to a natural pair of variable domains, that is, isolated from a single antibody or antibody expression cell.
[00201] [00201] The variable domains may have been optimized and / or humanized.
[00202] [00202] The optimized / humanized variable domains derived from a cognate pair will still be considered a cognate pair after optimization / humanization.
[00203] [00203] Consequently, the invention extends to human, humanized or chimeric molecules.
[00204] [00204] In one embodiment, the molecule specifically binds a target antigen. Binding specifically as used in this document is intended to refer to molecules that have a high affinity for a target antigen (to which it is specific) and that binds the antigens to which the dwarf month is specific with a low or much lower affinity ( or none). Affinity measurement methods are known to those skilled in the art and include such assays as BlIAcore'Y,
[00205] [00205] The antibody molecules of the present invention suitably have a high binding affinity, in particular, nanomolar or picomolar. Affinity can be measured using any suitable method known in the art, including BIAcore ™. In one embodiment, the molecule of the present invention has a binding affinity of approximately 100 pM or greater. In one embodiment, the molecule of the present invention has a binding affinity of approximately 50 µM or greater. In one embodiment, the molecule of the present invention has a binding affinity of approximately 40 µM or greater. In one embodiment, the molecule of the present invention has a binding affinity of approximately 30 µM or greater. In one embodiment, the molecule of the present invention is completely human or humanized and has a binding affinity of approximately 100 pM or greater.
[00206] [00206] A derivative of a naturally occurring domain as used in this document is intended to refer to when one, two, three, four or five amino acids in a naturally occurring sequence have been replaced or deleted, for example, to optimize the domain properties such as eliminating undesirable properties, but where the domain characterization feature (s) is / are retained.
[00207] [00207] In one embodiment, the antibody molecules of the present invention comprise one or more albumin-binding peptides. In vivo, the peptide binds albumin, which increases the half-life of the molecule.
[00208] [00208] The albumin-binding peptide can be attached from one or more variable regions, a joint or C-terminus of the molecule or any location that does not interfere with the binding properties of antigen molecules.
[00209] [00209] Examples of albumin-binding peptides are provided in WO No. 2007/106120.
[00210] [00210] It will also be understood by an individual skilled in the art that the antibody can undergo a variety of post-transductional modifications. The type and extent of these modifications often depends on the host cell line used to express the molecule as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diquetopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a basic carboxy-terminal residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705: 129 to 134, 1995).
[00211] [00211] If desired, a molecule for use in the present invention can be conjugated to one or more effector molecule (s). It will be appreciated that the effector molecule can comprise a single effector molecule or two or more such molecules linked in this way to form a single portion that can be attached to the antibody molecule of the present invention. When it is desired to obtain an antibody according to the invention linked to an effector molecule, it can be prepared by sets of standard recombinant DNA or chemical procedures in which the antibody is linked directly or through a coupling agent to the effector molecule. The sets of procedures for conjugating such effector molecules to an antibody are well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., Eds., 1987, pp. 623 to 53 ; Thorpe et al., 1982, Immunol. Rev., 62: 119 to 58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67 to 123). Sets of particular chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO03031581. Alternatively, when the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA procedures, for example, as described in documents No. * WO 86/01533 and EP0392745.
[00212] [00212] The term effector molecule as used in this document includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins, for example, enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, for example, DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporting groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.
[00213] [00213] Examples of effector molecules may include cytotoxins or cytotoxic agents that include any agent that is harmful to (for example, eliminates) cells. Examples include combretastatines, dolastatines, epothilones, staurosporine, maytansinoids, spongistatins, rhizoxin, hayclondrines, roridins, hemiasterline, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, vinoposthine, etoposide, tenoposide, tenoside , daunorubicin, dihydroxy anthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogues or homologues thereof.
[00214] [00214] Effector molecules also include, but are not limited to, antimetabolites (eg, methotrexate, G6-mercaptopurine, G6-thioguanine, cytarabine, S-fluorouracil decarbazine), alkylating agents (eg, meclorethamine, thioepa chlorambucil, melphalan, melphalan (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-platinum cis-dichlorodiamine (11) (DDP)), anthracyclines (eg daunorubicin) and doxorubicin) and doxorubicin (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, anthramycin (AMC), calicheamicins or duocarmicins) and antimitotic agents (eg vincristine and vinblastine).
[00215] [00215] Other effector molecules may include chelated radionuclides such as "In and * ºY, Lu '” ”, Bismuth " , Californium * , Iridium ”** and Tungsten * / Rhenium'! *; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase | inhibitors, taxoids and suramine.
[00216] [00216] Other effector molecules include proteins, peptides and enzymes. The enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, tumor necrosis factor, interferon a, interferon B, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, a thrombotic agent or an antiangiogenic agent, for example, angiostatin or endostatin, or a biological response modifier such as lymphokine, interleukin-1 (IL- 1), interleukin-2 (IL-2), macrophage granulocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other factor growth and immunoglobulins.
[00217] [00217] Other effector molecules may include detectable substances useful, for example, in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography) and metal ions for non-radioactive magnetic. Generally refer to U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use in diagnosis. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansil chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; and suitable radioactive nuclides include Il, 1, Min and ”Tc.
[00218] [00218] In another example, the effector molecule may increase the half-life of the antibody in vivo, and / or reduce the immunogenicity of the antibody and / or improve the delivery of an antibody through an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins or albumin-binding compounds such as those described in WO 05/117984.
[00219] [00219] When the effector molecule is a polymer, it can generally be a synthetic polymer or a naturally occurring polymer, for example, an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalguylene polymer or a polysaccharide branched or non-branched, for example, a homo or heteropolysaccharide.
[00220] [00220] Specific optional substituents that may be present in the synthetic polymers mentioned above include one or more hydroxy, methyl or methoxy groups.
[00221] [00221] Specific examples of synthetic polymers include optionally substituted branched or linear poly (ethylene glycol!), Poly (propylene glycol!) Poly (vinyl alcohol) or derivatives thereof, especially optionally substituted poly (ethylene glycol! 1) such as methoxypoly (ethylene glycol! 1) or derivatives thereof.
[00222] [00222] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
[00223] [00223] The "derivatives" as used herein are intended to include reactive derivatives, for example, selective reactive thiol groups such as maleimides and the like. The reactive group can be linked directly or through a linker segment to the polymer. It will be seen that the residue from such a group will, in some cases, form part of the product as the linking group between the developing antibody and the polymer.
[00224] [00224] The size of the polymer can be varied as desired, but will generally be in an average molecular weight range of 500 Da to 50,000 Da, for example, 5,000 to 40,000 Da such as 20,000 to 40,000 Da. The polymer size can be selected in particular based on the intended use of the product, for example, the ability to locate for certain tissues such as tumors or extend the circulation half-life (for review, see
[00225] [00225] Suitable polymers include a polyalkylene polymer, such as a poly (ethylene glycol!) Or, especially, a methoxy poly (ethylene glycol) or a derivative thereof, and especially with a molecular weight in the range of approximately 15,000 Da to approximately 40,000 Gives.
[00226] [00226] In one example, an antibody for use in the present invention is attached to the poly (ethylene glycol) (PEG) moieties. In a particular example, PEG molecules can be attached via any available amino acid side chain or functional terminal amino acid group located on the antibody, for example, any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids can occur naturally in the antibody or can be made into the antibody using recombinant DNA methods (see, for example, US * No. 5,219,996; US 5,667,425; WO 98/25971). In one example, the molecule of the present invention is a modified antibody in which the modification is the addition, at the C-terminus of the heavy chain thereof, of one or more amino acids to allow attachment of an effector molecule. Multiple sites can be used to attach two or more PEG molecules.
[00227] [00227] In one embodiment, a PEG molecule is linked to a 171 cysteine in the light chain, for example, see document No. WO2008 / 038024 incorporated in this document for reference.
[00228] [00228] Suitable PEG molecules are covalently linked through a thiol group of at least one cysteine residue located on the antibody. Each polymer molecule attached to the modified antibody can be covalently linked to the sulfur atom of a cysteine residue located in the antibody. The covalent bond will generally be a disulfide bond or, in particular, a sulfur-carbon bond. When a thiol group is used as the attachment point for appropriately activated effector molecules, for example, selective thiol derivatives such as maleimides and cysteine derivatives can be used. An activated polymer can be used as the starting material in the preparation of polymer modified antibody as described above. The activated polymer can be any polymer that contains a reactive thiol group such as an α-halocarboxylic acid or ester, for example, iodoacetamide, an imide, for example, maleimide, a vinyl sulfone or a disulfide. Such starting materials can be obtained commercially (for example, from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar, formerly Shearnwater).
[00229] [00229] The present invention also provides isolated DNA that encodes an antibody molecule described herein.
[00230] [00230] In an additional aspect, a vector is provided that comprises said DNA.
[00231] [00231] The general methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made to “Current Protocols in Molecular Biology", 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Manual of Maniatis produced by Cold Spring Harbor Publishing.
[00232] [00232] In an additional aspect, a host cell is provided that comprises said vector and / or DNA.
[00233] Any suitable host cell / vector system can be used for the expression of the DNA sequences encoding the molecule of the present invention. Bacteria, for example, E. coli, and other microbial systems can be used, or for example, mammalian, host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.
[00234] [00234] The present invention also provides a process for the production of an antibody molecule as described in this document which comprises culturing a host cell containing a vector (and / or DNA) of the present invention under conditions suitable for leading to expression of protein from the DNA encoding an antibody molecule of the present invention, and isolating an antibody molecule.
[00235] [00235] For the production of products comprising both heavy and light chains, the cell line can be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used, the vector including the sequences encoding the light and heavy chain polypeptides.
[00236] [00236] The antibody molecules according to the present disclosure are expressed at appropriate levels from host cells that make them conducive to commercial processing.
[00237] [00237] The antibody can be specific for any target antigen. The antigen can be a cell-associated protein, for example, a cell surface protein in cells such as bacterial cells, yeast cells, T cells, endothelial cells or tumor cells, or the same can be a soluble protein. The antigens of interest can also be any medically relevant protein such as those up-regulated proteins during disease or infection, for example, receptors and / or the corresponding ligands. Particular examples of cell surface proteins include adhesion molecules, for example, integrins such as B1 integrins, for example, VLA-4, E-selectin, P selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CDl1a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD40L, CD45, CDW52, CD69, CD134 (OX40), ICOS, BCMP7, CD137, CD27L, CDCP1, CSF1 or
[00238] [00238] Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, II-8, 11-12, 11-13, 11-14, I1L-16 or I1L-17, such as IL17A and / or IL17F, viral antigens, for example, respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins, such as IgE, interferons such as interferon a, interferon B or interferon y, factor tumor necrosis TNF (formerly known as tumor necrosis factor a and referred to herein as TNF or TNFa), tumor necrosis factor B, colony stimulating factors such as G-CSF or GM-CSF and growth factors platelet derivatives such as PDGF-a, and PDGF-fB, WISP-1 and, where appropriate, their receptors. Other antigens include bacterial cell surface antigens, bacterial toxins, viruses such as influenza, EBV, HepA, B and C, bioterrorism agents, radionuclides and heavy metals, and snake and spider poisons and toxins.
[00239] [00239] In one embodiment, a method is provided for generating a bispecific symmetrical antibody comprising the step of mixing a first IgG4 antibody with a second I9G4 antibody ex vivo, under conditions conducive to heavy chain exchange, in which the specificity of antigen of variable regions in the first antibody is different from the antigen specificity of variable regions in the second antibody.
[00240] [00240] In vitro conditions leading to heavy chain exchange include reduction conditions. Suitable reducing agents include GSH, 2-mercaptoethanol, 2-mercaptoethylamine, TBP, TCEP, cysteine-HCl and DTT.
[00241] [00241] Adequate concentrations of reducing agents are in the range of 0.01 to 10 mM such as 0.5 to 5 mM. In addition, the reduction can be achieved with the use of redox buffers, that is, different relative ratios of reduced and oxidized variants of reagents such as, for example: GSH: GSSG and Cys: diCys
[00242] [00242] Suitable conditions that include antibody ratios are in the range of 0.5: 5 to 5:05, such as 1: 1 or 1: 2.
[00243] [00243] Adequate temperature includes 15 to 40 ºC, such as 37 ºC.
[00244] [00244] The reduction conditions can be selected to be between the reducing stability of homodimers and heterodimers.
[00245] [00245] In an alternative embodiment, the antibodies of the disclosure are prepared using a mixed cell culture, for example, -50% exchange occurs. This can yield in the region of 1 to 2 gl of the desired bispecific.
[00246] [00246] In one embodiment, an antibody or fragment obtained or obtainable from a process or method described in this document is provided.
[00247] [00247] In one embodiment, the antibody can be used to functionally alter the activity of the antigen of interest. For example, the antibody can neutralize, antagonize or agonize the activity of said antigen, directly or indirectly.
[00248] [00248] The antibody molecules of the present invention are useful in the treatment and / or prophylaxis of a pathological condition.
[00249] [00249] Accordingly, an antibody is provided according to the present invention for use in treatment, by administering a therapeutically effective amount of it, for example, in a pharmaceutical formulation. In one embodiment, the antibody according to the invention is administered topically to the lungs, for example, by inhalation.
[00250] [00250] The antibodies provided by the present invention are useful in the treatment of diseases or disorders that include inflammatory disorders and diseases, immune disorders and disease, cancers and fibrotic disorders.
[00251] [00251] The term "inflammatory disease" or "disorder" and "immune disease or disorder" includes rheumatoid arthritis, psoriatic arthritis, still disease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerative colitis, SLE (Systemic Lupus Erythematosus) ), asthma, allergic rhinitis, atopic dermatitis, multiple sclerosis, vasculitis, Type | diabetes mellitus, transplantation and graft versus host disease.
[00252] [00252] The term "fibrotic disorder" includes idiopathic pulmonary fibrosis (IPF), systemic sclerosis (or scleroderma), renal fibrosis, diabetic nephropathy, IgA nephropathy, hypertension, end-stage renal disease, peritoneal fibrosis (continuous ambulatory peritoneal dialysis) , liver cirrhosis, aging-related macular degeneration (ARMD), retinopathy, reactive cardiac fibrosis, scarring, keloids, burns, skin ulcers, angioplasty, coronary bypass surgery, arthroplasty and cataract surgery.
[00253] [00253] The term "cancer" includes a new malignant tumor that arises from the epithelium, found on the skin or, more commonly, the lining of organs in the body, for example: sinuses, ovary, prostate, lung, kidney, pancreas, stomach, bladder or intestine. Cancers tend to infiltrate adjacent tissue and spread (metastasize) to distant organs, for example, to bones, liver, lung or brain.
[00254] [00254] The present invention also provides a diagnostic or pharmaceutical composition that comprises an antibody of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier. Accordingly, the use of an antibody of the invention is provided for the manufacture of a medicament. The composition will normally be supplied as part of a sterile pharmaceutical composition that will normally include a pharmaceutically acceptable carrier. A pharmaceutical composition of the present invention can further comprise a pharmaceutically acceptable adjuvant.
[00255] [00255] The present invention also provides a process for the preparation of a diagnostic or pharmaceutical composition which comprises adding and mixing the antibody of the present invention together with one or more of a pharmaceutically acceptable carrier, diluent or excipient.
[00256] [00256] The antibody of the disclosure may be the only active ingredient in the diagnostic or pharmaceutical composition or may be accompanied by other active ingredients that include other antibody ingredients, for example, anti-TNF, anti-IL-16, anti- T cell, anti-| FNy or anti-LPS, or non-antibody ingredients such as xanthines. Other suitable active ingredients include antibodies capable of inducing tolerance, for example, anti-CD3 or anti-CD4 antibodies.
[00257] [00257] In an additional embodiment, the antibody or composition according to the disclosure is used in combination with an additional pharmaceutically active agent, for example, a corticosteroid (such as fluticasone propionate) and / or a beta-2-agonist ( such as salbutamol, salmeterol or formoterol) or inhibitors of cell proliferation and growth (such as rapamycin, cyclophosphamide, methotrexate) or, alternatively, a CD28 and / or CD40 inhibitor. In one embodiment, the inhibitor is a small molecule. In another embodiment, the inhibitor is a target-specific antibody.
[00258] [00258] The pharmaceutical compositions suitably comprise a therapeutically effective amount of the antibody of the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent necessary to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable preventive or therapeutic effect. The therapeutically effective amount may be estimated initially in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates.The animal model can also be used to determine the appropriate concentration range and route of administration. then used to determine useful doses and routes for administration in humans.
[00259] [00259] The therapeutically effective amount needed for a human individual will depend on the severity of the disease state, the individual's overall health, the age, weight and gender of the individual, diet, time and frequency of administration, drug combination (s), reaction sensitivities and tolerance / response to therapy. This amount can be determined by routine experimentation and is within the doctor's judgment. Generally, a therapeutically effective amount will be 0.01 mg / kg to 50 mg / kg, for example, 0.1 mg / kg to 20 mg / kg. The pharmaceutical compositions can conveniently be presented in unit dose forms that contain a predetermined amount of an active agent of the invention per dose.
[00260] [00260] The compositions can be administered individually to a patient or they can be administered in combination (for example, simultaneously, sequentially or separately) with other agents, drugs or hormones.
[00261] [00261] The dose at which an antibody of the present invention is administered depends on the nature of the condition to be treated, for example, the extent of the disease / inflammation present and whether the molecule is being used prophylactically or to treat an existing condition.
[00262] [00262] The frequency of dose will depend on the half-life of the antibody and the duration of its effect. If the antibody has a short half-life (for example, 2 to 10 hours), it may be necessary to provide one or more doses per day. Alternatively, if the antibody has a long half-life (for example, 2 to 15 days), it may be necessary to provide only one dose once a day, once a week, or even once every 1 | or 2 months.
[00263] [00263] The pharmaceutically acceptable carrier itself must not induce the production of antibodies harmful to the individual receiving the composition and must not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
[00264] [00264] Pharmaceutically acceptable salts can be used, for example, mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.
[00265] [00265] Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, can be present in such compositions. Such carriers allow pharmaceutical compositions to be formulated as tablets, pills, pills, capsules, liquids, gels,
[00266] [00266] Suitable forms for administration include forms suitable for parenteral administration, for example, by injection or infusion, for example, by bolus injection or continuous infusion. When the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulation agents, such as suspending, preservative, stabilizing and / or dispersion. Alternatively, the disclosure molecule can be in dry form, for reconstitution before use with an appropriate sterile liquid.
[00267] [00267] Once formulated, the compositions of the invention can be administered directly to the individual. The individuals to be treated can be animals. However, in one or more embodiments, the compositions are adapted for administration to human subjects.
[00268] [00268] Suitably, in formulations according to the present disclosure, the PH of the final formulation is not similar to the value of the isoelectric point of the antibody, for example, if the pH of the formulation is 7 then a pH between 8 to 9 or higher can be appropriate. While not wishing to be bound by theory, it is believed that this can finally provide a final formulation with improved stability, for example, the antibody remains in the solution.
[00269] [00269] The pharmaceutical compositions of this invention can be administered by any number of routes that include, but are not limited to, oral, intravenous, - intramuscular, - intra-arterial, - intramedullary, - intrathecal, intraventricular, transdermal, transcutaneous (e.g. , see document No. WO98 / 20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal. Jet injectors can also be used to administer the pharmaceutical compositions of the invention. Typically, therapeutic compositions can be prepared as injectables, as suspensions or liquid solutions. Solid forms suitable for solution, or suspension, in liquid vehicles before injection can also be prepared.
[00270] [00270] The direct delivery of the compositions will generally be carried out by injection,
[00271] [00271] It will be noted that the active ingredient in the composition will be an antibody. As such, it will be susceptible to degradation in the gastrointestinal tract. Consequently, if the composition is to be administered via a route using the gastrointestinal tract, the composition will need to contain agents that protect the antibody from degradation, but that release the antibody once they have been absorbed from the gastrointestinal tract.
[00272] [00272] An in-depth discussion of pharmaceutically acceptable carriers is available from Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).
[00273] [00273] In one embodiment, the formulation is provided as a formulation for topical administrations that include inhalation.
[00274] [00274] Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free of propellant gases. Inhalable powders according to the disclosure that contain the active substance can consist solely of the substances mentioned above or a mixture of the active substances mentioned above with physiologically acceptable excipient.
[00275] [00275] Such inhalable powders can include monosaccharides (eg, glucose or arabinose), disaccharides (eg, lactose, sucrose, maltose), oligo and polysaccharides (eg, dextrans), polyalcohols (eg, sorbitol, mannitol, xylitol) ), salts (e.g. sodium chloride, calcium carbonate) or mixtures thereof. Mono or disaccharides are used properly, the use of lactose or glucose, particularly, but not exclusively in the form of their hydrates.
[00276] [00276] The particles for deposition in the lung require a particle size of less than 10 microns, such as 1 to 9 microns, for example, from 0.1 to 5 µm,
[00277] [00277] Propellant gases that can be used to prepare inhalable aerosols are known in the art. Suitable propellant gases are selected from hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The propellant gases mentioned above can be used on their own or in mixtures thereof.
[00278] [00278] Particularly suitable propellant gases are derived from halogenated alkane selected from TG 11, TG 12, TG 134a and TG227. Of the hydrocarbons - halogenated - mentioned above - TGi34a - (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.
[00279] [00279] Inhalable aerosols containing propellant gas may also contain other ingredients such as cosolvents, stabilizers, surface active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All of these ingredients are known in the art.
[00280] [00280] Inhalable aerosols containing propellant gas according to the invention can contain up to 5% by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by weight of active ingredient.
[00281] [00281] Alternatively, topical administrations to the lung can also be by administration of a suspension formulation or liquid solution, for example, employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (for example, the nebulizer Pari LC-Jet Plus (R) connected to a Pari Master (R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).
[00282] [00282] The antibody of the invention can be delivered dispersed in a solvent, for example, in the form of a solution or a suspension. It can be suspended in a suitable physiological solution, for example, saline or other pharmacologically acceptable solvent or a buffered solution. Buffered solutions known in the art may contain from 0.05 mg to 0.15 mg of disodium ededate, 8.0 mg to 9.0 mg of NaCl, 0.15 mg to 0.25 mg of polysorbate, 0.25 mg The
[00283] [00283] Therapeutic solution or suspension formulations may also contain one or more excipients. Excipients are well known in the art and include buffers (for example, citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, chloride sodium, liposomes, mannitol, sorbitol, and glycerol. The solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation will generally be provided in a substantially sterile form that employs sterile manufacturing processes.
[00284] [00284] This may include the production and filter sterilization of the buffered solution / solvent used for the formulation, the aseptic suspension of the molecule in the sterile buffered solvent solution, and the distribution of the formulation in sterile receptacles by methods familiar to those skilled individuals common in technique.
[00285] [00285] The nebulizable formulation according to the present disclosure can be supplied, for example, as single dose units (for example, sealed plastic bottles or containers) packaged in foil wrappers. Each vial contains a unit dose in a volume, for example, 2 ml, of solvent / solution buffer.
[00286] [00286] The antibody of the present disclosure is thought to be particularly suitable for nebulized delivery.
[00287] [00287] Understanding in the context of this specification is intended to mean including.
[00288] [00288] Being that technically appropriate modalities of the invention can be combined.
[00289] [00289] The invention will now be described with reference to the following examples, which are merely illustrative and should not be construed in any way as limiting the scope of the present invention. Examples
[00290] [00290] Amino acid mutations were performed using the QuickChange86 Lightening Multisite-Directed Mutagenesis (SDM) kit or the DSM Quickchange & | l kit (obtained from StratageneO) (catalog numbers 210516 and 200521, respectively) and performed according to with the manufacturer's instructions.
[00291] [00291] The mutations were verified by DNA sequencing. IgG4 heavy chains of antibodies 1 to 47 in the following table were produced: Number z; Cy1 and ont domain, from | Kabal Mining | Aicnação | CONES Antibody SEQ ID NO: SEQ ID NO: [e [emscaccas - | 3 | 2 [| 8 | ems.cro0. houses houses | as | 268 and | eocas> rs | am
[00292] [00292] Antibody heavy chain 48 (Sequence ID NO: 266) was generated by
[00293] [00293] All mutant DNA was transfected into CHOK1 cells. The cells (2x10º cells / ml) were resuspended in 1 ml of Earles' Balanced Saline Solution (Sigma) and mixed with 400 µg DNA (200 µg heavy chain Dan and 200 µg kappa light chain DNA). 800 µl aliquots were transferred to 0.4 cm cuvettes (Biorad). For a 500 ml culture, six cuvettes were electroporated under the following parameters: 1 ms, 9.6 Amps; 10 ms, The Amps; 40 ms, 3.2 Amps. The transfected cells were incubated for 24 hours, shaking at 140 rpm in an environment humidified in 5% CO, at 37 ºC and continued from day 2 post-transfection at 32 ºC for 10 to 13 days. On day 4 post-transfection, 1.6 mls 1 M sodium butyrate was added to the culture. Once the cells reached 40% viability or by day 13, the supernatant was harvested. The cultures were centrifuged for 45 minutes at 4,000 rpm. The supernatant was placed through a 0.22 µM Stericup filter (Miliporo) to be purified.
[00294] [00294] The supernatants (200 to 500 ml) were purified using a 5 ml HiTrap MabSelect SuRe Protein column (GE Healthcare, Amersham UK). The samples were prepared by adding 1 / 50º of the volume of 2 M supernatant of Tris-HCI pH 8.5. The samples were loaded onto the column at 1 ml / min. The column was washed with PBS pH 7.4. To elute the samples, 0.1 M sodium citrate, pH 3.4, was passed through the column at 1 ml / min and 0.5 ml fractions were collected. The peak fractions were neutralized by adding 0.125 m! 2 M Tris-HCI pH 8.5 each. UV detection was set at 280 nm. 4, Characterization of purified mutated igG4 antibodies
[00295] [00295] The crude supernatant was centrifuged at 1,200 rpm for 5 minutes and quantified in OCTET. Antibody samples (25 to 30 ng) were prepared by adding the appropriate amounts of antibody, 4x Loading Buffer (Invitrogen) and 2 u! 100 mM NEM. A total volume of 20 ul! was created using dH2O. The samples were then boiled for 3 minutes at 100 ºC and loaded with 4 to 20% Tris-Glycine Gel in 15 1.5 mm wells. The gels were passed through 150 V for 1.5 hours in 1x of Buffer tank. The antibodies were transferred to a nitrocellulose membrane using the iBlot dry transfer system defined to transfer for 8 minutes. The membrane was incubated for 1 h at room temperature (RT) in PBS-TM on a shaking platform, followed by incubation with a rabbit anti-human IgG Fc HRP conjugated antibody (Jackson Immunoscope) or chain HRP conjugated antibody take goat anti-human Kappa (Bethyl) for 1 h, shaking at RT. This was followed by 3 washes of 5 minutes each with PBS-T. The stains were developed using a metal-optimized DAB substrate kit according to the manufacturer's instructions (Pierce).
[00296] [00296] The results of the Western blot analysis are shown in Figures 7, 8, 9 and 10. In Figures 7 to 10, H stands for the heavy chain and, L, the light chain, H2L2 is an entire antibody molecule comprising two heavy chains and two light chains and HL is a semimolecule comprising a heavy chain and a light chain.
[00297] [00297] Figure 7 shows the Western blot analysis for antibodies 15, 16, 6, 7, 8, 17, 18, 19, 5, 5P, 9, 10, 11, 1, 2, 3, 4, 12, 13 and 14. It can be seen from Figure 7 that the antibodies have a good level of H2L2 except for antibodies 4, 8 and 14 which have very little or no H2L2 due to the presence of both C239S and C242S joint mutations . However, antibodies 4, 8 and 14 can form H2L2 by non-covalent bonding between the heavy chains. Mutant 3 also has little H2L2, this mutant retains C239, but is unable to form heavy inter-chain disulfides in the joint, presumably due to the efficient disulfide formation between the C-terminal light chain (LC) cysteine and the joint C239. It can also be seen that antibodies that comprise the C239S mutation, but not C242S (antibodies 2, 6, 9 and 12) show reduced HL formation compared to antibodies that do not comprise C239S or C2428S or antibodies that comprise C242S , but not the C239S. The 5P and 16 antibodies that comprise the S241P mutation also show reduced HL formation. A comparison of mutants 2 and 3 shows the extent of the “reach” of the light chain C terminal cysteine to form a disulfide bond with the heavy chain, it appears that the light chain cysteine binds efficiently to C239 than to C242 in the heavy chain.
[00298] [00298] Figure 8 shows Western blot analysis for antibodies 15, 6, 7, 8, 28, 29, 30, 31, 17, 19, 32, 33, 33, 34, 35, 36, 37, 38 and 39. It can be seen from Figure 8 that the antibodies have a good level of H2L2 except for antibodies 8, 31, 35 and 39 that have very little or no H2L2 due to the presence of C239S and C242S mutations in the joint region and therefore, there are no disulfide bonds formed between two heavy chains. However, antibodies 8, 31, 35 and 39 can form H2L2 by non-covalent bonding between the heavy chains. It can also be seen that antibodies that comprise the C239S mutation, but not C242S (antibodies 6, 29, 33 and 37) show reduced HL formation compared to antibodies that do not comprise C239S or C2428 or antibodies that comprise C2428S, but not C239S. Mutant 15 is able to form a disulfide bond between the light chain and G230C in CH1 and heavy chain disulfides, consequently resulting in a disulfide-bound and fully assembled protein. In addition, a comparison of mutants 15 (C127S G230C), 28 (Ci127S Y229C), 32 (C127S K228C) and 36 (C127S S227C) shows that the position of the cysteine introduced in the upper joint improves the formation of the LC- inter disulfide bond HC. G230 and Y229 are particularly preferred positions for introducing a cysteine. Mutant 28 (C127S Y229C) has a good level of HL and H2 and has,
[00299] [00299] Figure 9 shows the Western blot analysis for antibodies 15, 6, 7, 8, 44, 45, 46, 47, 17 and 19. It can be seen from Figure 9 that the antibodies present a good level of H2L2 except for antibodies 8 and 47 that have very little or no H2L2 due to the presence of C2398S and C2428S mutations in the joint region and, therefore, there are no disulfide bonds formed between two heavy chains. However, antibodies 8 and 47 can form H2L2 by non-covalent bonding between the heavy chains. It can also be seen that antibodies that comprise the C239S mutation, but not C2428S (antibodies 6 and 45) show reduced HL formation compared to antibodies that do not comprise C2398S or C2428 or antibodies that comprise C242S, but not the C239S. In particular, mutant 44 shows that the insertion of three amino acids in the upper joint can also reduce the formation of H and H2 and, consequently, has lower levels of disulfide heterogeneity than the comparable mutant 15.
[00300] [00300] Figure 10 shows the Western blot analysis for antibodies 48, 17, 18 and
[00301] [00301] The thermostabilities of purified mAbs were analyzed in a thermofluor assay using SYPROG Orange to monitor the process of thermal protein unfolding. 5 ul MAb in 1 mg / ml, 5 ul of
[00302] [00302] Figures 11, 12, 13, 14 and 15 show the results of the thermostability analysis of IgG4 Antibody mutants in comparison to IgG1 and IgG4 wild-type antibodies.
[00303] [00303] A comparison of mutant 15 with wild-type IgG4 (mutant 17) shows an increase in Fab Tm due to the altered disulfide disposition. A comparison of mutant 15 and 28 shows the further improvement in Fab Tm for mutant 28 which comprises the Y229C mutation compared to mutant 15 which comprises the G230C mutation. A comparison of mutants 15 and 44 shows that the Fab Tm of a G230C mutant can be increased further by inserting three amino acids into the upper joint. The comparison of mutants 17 and 18 shows that the medium joint mutation S241P does not increase Fab Tm although it significantly reduces the formation of HL. Mutant 48 also has significantly improved Fab Tm when compared to both wild-type IgG4 (mutant 17) and mutant 15.
[00304] [00304] Figure 15 shows the global classification of the thermostabilities of IgG4 mutants selected according to the present invention. All mutants 48, 44, 44P, 46, 45, 6, 29, 30, 28, 28P, 31, 8, 47 and 15 have significantly higher Fab Tm values compared to wild-type IgG4 (mutant 17) and the S241P wild-type IgG4 (mutant 18).
[00305] [00305] A first IGG4 antibody and the potential exchange partner were mixed at a molar ratio of 1: 1 at a total concentration of 100 µg / ml in phosphate buffered saline (PBS) (in mM: 150 NaCl, 10 NaH2PO, pH 7.4). To allow the reduction of disulfide binding, the samples were supplemented with reduced Glutathione (GSH; Sigma) to a final concentration of O, 0.5 or 5 mM. At the beginning of the experiment (t = 0 hour), an aliquot of the mixture was taken, quenched with N-ethylmaleimide (NEM; Sigma) to a final concentration of 10 mM (to inactivate potentially reactive thiol groups) and incubated with the rest of mix for 16 hours at 37 ºC (t = 16 hours). After incubation, the sample of t = 16 hours was quenched as mentioned above. b) Detection and quantification of heavy chain exchange in vitro
[00306] [00306] The presence of functionally active bispecific antibodies was determined using a sandwich MSD assay in which the briskly cooled reaction samples provided in Example 5 a), serially diluted in 1% BSA in PBS (PB) , were pre-incubated with 1 µg / ml of biotinylated antigen 1 (first antibody antigen) in PB for 1 h in shaking TA (200 rpm) before being transferred to pre-blocked streptavidin coated MSD plates ( Meso Scale Diagnostics). After 1 h of incubation in RT with shaking, the wells were washed three times with PBS / 0.1% Tween-20 before being incubated with 1 µg / ml of sulfur-labeled antigen 2 (second antibody antigen in PB). After incubation, the plates were washed as mentioned above and the signs revealed and measured using the manufacturers' reading buffer and the Image Sector 6000 instrument, respectively. The background values obtained from the parallel control reactions in which the biotinylated antigen was replaced by a non-biotinylated alternative, were subtracted from all signals. Duplicate values from at least 3 independent experiments were used in all calculations.
[00307] [00307] The higher the MSD signal, the greater the amount of heavy chain exchange that occurs. The following antibodies were analyzed:
[00308] [00308] Figures 16 to 20 show the results of the quantification of Fab arm change in vitro, in which the y-axis shows the MSD signal, in which the higher the level of the bar, the greater the amount of exchange of Fab's arm.
[00309] [00309] Figure 16 shows the switch from heavy chain to wild type I9G1, wild type IgG4 and IgG4P mutants, 28, 28P, 15, 44, 44P and 48 at various concentrations of GSH and at various time points.
[00310] [00310] Figure 17 shows the heavy chain exchange for wild type IgG4 and mutants 15, 44 and 48 at various concentrations of GSH at various time points. It can be seen that the more the IgG4 joint is mutated to be of the I9G1 type, the more the exchange is reduced, as shown by mutant 48 who has the location exchange.
[00311] [00311] Figure 18 shows the heavy chain exchange for wild type IgG4 and mutants 15 and 28 at various concentrations of GSH at various time points. It can be seen that the mutation at position 229 (mutant 28) reduces the exchange for 4 to 16 hours at both concentrations of reducing agent to a greater extent compared to the mutation at position 230 (mutant 15).
[00312] [00312] Figure 19 shows the percentage change for IgG4P, 15, 28, 44 and 48 mutants in 0.5 mM GSH compared to wild-type IgG4. The exchange can be classified as follows: I9G4 wt> 15 = 44> 48> 28.
[00313] [00313] Figure 20 shows the percentage change for several mutants in 5 mM GSH compared to wild-type IgG4. The exchange can be classified I9G4 wt> 15 = 44> 48> 28.
[00314] [00314] In good agreement with the literature (Labrijn 2011, Lewis 2009, Stubenrauch 2010, Labrijn 2009), it is shown that the S241P mutation in the IgG4 core joint represents a tool to prevent the Fab arm exchange. also that the bispecific mutant antibodies of the present invention may demonstrate less Fab arm exchange than was shown in 0.5 mM GSH, which is 100 times higher than the physiological GSH concentration of 4 to 6 µM plasma (Zilmer et al, 2005. Drug Design Reviews). Accordingly, bispecific antibodies can be raised in vitro by switching Fab arms under reducing conditions, which may then have significantly reduced switching Fab arms in vivo compared to IgG4 wt. Antibody Affinity:
[00315] [00315] The affinity of selected I9G4 mutant antibodies of the present invention to the target soluble cytokine can be measured by BiacoreTV. The assay format is the capture of IgG on an antiFc surface followed by soluble cytokine titration.
[00316] [00316] The term "ki" (s *) refers to the rate of dissociation rate of the antibody-antigen interaction. Said value is also referred to as the kKot value.
[00317] [00317] The term "ka" (Ms), as used in this document, refers to the association rate constant of the antibody-antigen interaction.
[00318] [00318] The term "Kp" (M) or "Kpy" (pM), as used in this document, refers to the dissociation equilibrium constant of the antibody-antigen interaction. Size Exclusion HPLC (SEC) analysis:
[00319] [00319] Approximately 50 µg of purified antibody was passed on HPLC using an S200 column. Abs 1 to 19 were used for the analysis. This result shows that H2L2 associated in a non-covalent manner is formed despite changes to the DSB provisions of a human IgG4 molecule.

权利要求:
Claims (33)
[1]
1. Bispecific symmetrical IgG4 antibody, characterized by the fact that it comprises two heavy chains that each comprise a variable domain, Cy1 domain and an articulation region, in which, in each heavy chain: the cysteine in the Cy1 domain that forms a disulfide bond interchanged with a cysteine in a light chain is replaced by another amino acid; and optionally one or more of the amino acids positioned in the upper hinge region is replaced by cysteine, where the sequence of constant regions of each heavy chain is similar or identical and the variable region in each heavy chain is different.
[2]
2. Symmetrical bispecific antibody, according to claim 1, characterized by the fact that the joint region is an IgG1 type joint.
[3]
3. Symmetrical bispecific antibody according to claim 1 or 2, characterized by the fact that the antibody is isolated.
[4]
4. Symmetrical bispecific antibody according to any one of claims 1 to 3, characterized by the fact that a cysteine interlocks at position 127, numbered according to the Kabat numbering system, in the CH1 domain in one or both heavy chains are replaced by another amino acid.
[5]
5. Symmetrical bispecific antibody according to any of the claims | to 4, characterized by the fact that the one or more amino acids positioned in the upper articulation region of the first heavy chain that are replaced by cysteine are selected from 227, 228, 229 and 230, numbered according to the Kabat numbering system .
[6]
6. Symmetrical bispecific antibody, according to claim 5, characterized by the fact that glycine at position 230, numbered according to the Kabat numbering system, is replaced by cysteine.
[7]
7. Symmetrical bispecific antibody according to claim 5, characterized by the fact that tyrosine at position 229, numbered according to the Kabat numbering system, is replaced by cysteine.
[8]
8. Symmetrical bispecific antibody according to claim 5, characterized by the fact that lysine at position 228, numbered according to the Kabat numbering system, is replaced by cysteine.
[9]
9. Symmetric antibody, as defined in claim 5, characterized by the fact that the serine at position 227, numbered according to the Kabat numbering system, is replaced by cysteine.
[10]
10. Symmetrical bispecific antibody according to any one of the claims | to 9, characterized by the fact that cysteine at position 239 and cysteine at position 242, numbered according to the Kabat numbering system, in one or both heavy chains is / are replaced by another amino acid.
[11]
11. Symmetrical bispecific antibody according to any one of claims 1 to 10, characterized by the fact that the cysteine at position 239, numbered according to the Kabat numbering system, in the first heavy chain is replaced by another amino acid.
[12]
12. Symmetrical bispecific antibody according to any one of claims 1 to 11, characterized by the fact that the cysteine at position 242, numbered according to the Kabat numbering system, in the first heavy chain is replaced by another amino acid.
[13]
13. Bi-specific symmetrical IgG4 antibody, characterized by the fact that it comprises two heavy chains that each comprise a variable domain, Ch1 domain and articulation region, in which, in each heavy chain, the cysteine interleaves at position 127, numbered according to the Kabat numbering system, it is replaced by another amino acid; and cysteine at position 239 and / or cysteine at position 242, numbered according to the Kabat numbering system, is replaced by another amino acid, where the constant region sequence of each heavy chain is similar or identical and the variable region each heavy chain is different.
[14]
14. Symmetrical bispecific antibody according to any one of claims 10 to 13, characterized in that the cysteine at position 239 and / or cysteine at position 242 of a first heavy chain is replaced by an amino acid that does not contain thiol.
[15]
15. Symmetrical bispecific antibody according to claim 14, characterized by the fact that the amino acid that does not contain thiol is serine.
[16]
16. Symmetrical bispecific antibody according to any one of claims 1 to 15, characterized in that the cysteine at position 127 is replaced by an amino acid that does not contain thiol.
[17]
17. Symmetrical bispecific antibody according to claim 16, characterized by the fact that the amino acid that does not contain thiol is serine.
[18]
18. Symmetrical bispecific antibody according to any one of claims 1 to 17, characterized by the fact that one or both of the heavy chains is / are mutated to insert three amino acids between amino acids 226 to 243, numbered according to the system of Kabat numbering.
[19]
19. Symmetrical bispecific antibody according to claim 18, characterized by the fact that one or both heavy chains is / are mutated to insert three amino acids between positions 238 and 239, numbered according to the Kabat numbering system.
[20]
20. Symmetrical bispecific antibody, according to claim 19, characterized by the fact that three alanines are inserted between positions 238 and 239, of the first heavy chain numbered according to the Kabat numbering system.
[21]
21. Symmetrical bispecific antibody according to claim 19, characterized by the fact that a threonine, a histidine and an additional teronin are inserted between positions 238 and 239, of one or both of the heavy chains numbered according to the system numbering system.
[22]
22. Symmetrical bispecific antibody according to any one of claims 1 to 21, characterized by the fact that the serine at position 241, of one or both of the heavy chains numbered according to the Kabat numbering system, is / are replaced (s) for proline.
[23]
23. Symmetrical bispecific antibody according to any one of the claims | to 22, characterized by the fact that in one or both of the heavy chains, glycine at position 230 is replaced by cysteine, serine at position 227 is / are replaced by proline, tyrosine at position 229 is replaced by serine, proline at position 237 is replaced by aspartic acid, proline at position 238 is replaced by lysine, the amino acid sequence threonine-histidine-threonine is inserted between positions 238 and 239 and the serine at position 241 is replaced by proline.
[24]
24. Symmetrical bispecific antibody according to any of the claims | to 23, characterized by the fact that both heavy chains comprise a Ch2 domain and / or a C13 domain.
[25]
25. Symmetrical bispecific antibody according to any of the claims | to 24, characterized by the fact that the region of articulation in each heavy chain is identical.
[26]
26. Symmetrical bispecific antibody according to any one of claims 1 to 25, characterized by the fact that each heavy chain comprises an upper articulation region and the nucleus region of 12 to 17 amino acids in length, for example, 15 amino acids of length.
[27]
27. Symmetrical bispecific antibody according to any one of claims 1 to 26, characterized in that it additionally comprises one or two light chains.
[28]
28. Expression vector, characterized by the fact that it comprises a sequence that encodes an antibody, as defined in any one of claims 1 to 27.
[29]
29. Host cell, characterized by the fact that it comprises a vector, as defined in claim 28.
[30]
30. Antibody according to any one of claims 1 to 27, characterized by the fact that it is for use in the treatment of a disease or disorder.
[31]
31. Method of generating a bispecific symmetric antibody characterized by the fact that it comprises the step of mixing a first IgG4 antibody with a second IgG4 antibody ex vivo, under conditions conducive to heavy chain exchange, in which the antigen specificity of variable regions in the first antibody it is different from the antigen specificity of the variable regions in the second antibody.
[32]
32. Method according to claim 31, characterized in that the conditions conducive to heavy chain exchange are conditions of reduction, for example, in the presence of a suitable buffer.
[33]
33. The method of claim 31 or 32, characterized in that the first IgG4 antibody and the second I9G4 antibody each comprise a Cy1 domain and a hinge region, as defined in any one of claims 1 to 26.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US11059911B2|2021-07-13|Sequence symmetric modified IgG4 bispecific antibodies

---

JP2020090514A|2020-06-11|SEQUENCE ASYMMETRIC MODIFIED IgG4 BISPECIFIC ANTIBODIES

---

US20200157204A1|2020-05-21|Antibodies of the Class IGG4

同族专利:

---

公开号 | 公开日

---

KR20200018706A|2020-02-19|

---

JP6814761B2|2021-01-20|

---

EP2817332B1|2016-07-20|

---

EP2817332A1|2014-12-31|

---

CN104321341B|2018-05-22|

---

EP3156416A1|2017-04-19|

---

AU2018201232A1|2018-03-15|

---

US20150018529A1|2015-01-15|

---

CN104321341A|2015-01-28|

---

KR102077331B1|2020-02-17|

---

JP2015513537A|2015-05-14|

---

US20190202937A1|2019-07-04|

---

IL234102A|2018-06-28|

---

WO2013124450A1|2013-08-29|

---

US11059911B2|2021-07-13|

---

ES2599503T3|2017-02-02|

---

HK1202563A1|2015-10-02|

---

AU2018201232B2|2019-04-18|

---

EA201491569A1|2015-01-30|

---

AU2013224003B2|2017-11-23|

---

MX2014010018A|2014-09-12|

---

CA2864439A1|2013-08-29|

---

AU2013224003A1|2014-09-18|

---

JP6308952B2|2018-04-11|

---

GB201203051D0|2012-04-04|

---

EP3156416B1|2019-11-20|

---

MX356288B|2018-05-22|

---

CN108586615A|2018-09-28|

---

US10221251B2|2019-03-05|

---

CN108586615B|2022-03-08|

---

SG11201404789XA|2014-09-26|

---

JP2018117627A|2018-08-02|

---

IL234102D0|2014-09-30|

---

KR20140127890A|2014-11-04|

---

DK3156416T3|2020-02-24|

---

EA035050B1|2020-04-22|

---

ES2768780T3|2020-06-23|

---

DK2817332T3|2016-11-07|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4741900A|1982-11-16|1988-05-03|Cytogen Corporation|Antibody-metal ion complexes|

---

GB8308235D0|1983-03-25|1983-05-05|Celltech Ltd|Polypeptides|

---

US4816567A|1983-04-08|1989-03-28|Genentech, Inc.|Recombinant immunoglobin preparations|

---

GB8422238D0|1984-09-03|1984-10-10|Neuberger M S|Chimeric proteins|

---

DK336987D0|1987-07-01|1987-07-01|Novo Industri As|immobilization|

---

GB8719042D0|1987-08-12|1987-09-16|Parker D|Conjugate compounds|

---

GB8720833D0|1987-09-04|1987-10-14|Celltech Ltd|Recombinant dna product|

---

US5677425A|1987-09-04|1997-10-14|Celltech Therapeutics Limited|Recombinant antibody|

---

EP1026240A3|1988-09-02|2004-04-07|Dyax Corp.|Generation and selection of recombinant varied binding proteins|

---

US5223409A|1988-09-02|1993-06-29|Protein Engineering Corp.|Directed evolution of novel binding proteins|

---

GB8823869D0|1988-10-12|1988-11-16|Medical Res Council|Production of antibodies|

---

US5530101A|1988-12-28|1996-06-25|Protein Design Labs, Inc.|Humanized immunoglobulins|

---

GB8907617D0|1989-04-05|1989-05-17|Celltech Ltd|Drug delivery system|

---

GB8928874D0|1989-12-21|1990-02-28|Celltech Ltd|Humanised antibodies|

---

AU7247191A|1990-01-11|1991-08-05|Molecular Affinities Corporation|Production of antibodies using gene libraries|

---

AU633698B2|1990-01-12|1993-02-04|Amgen Fremont Inc.|Generation of xenogeneic antibodies|

---

US5780225A|1990-01-12|1998-07-14|Stratagene|Method for generating libaries of antibody genes comprising amplification of diverse antibody DNAs and methods for using these libraries for the production of diverse antigen combining molecules|

---

CA2090126C|1990-08-02|2002-10-22|John W. Schrader|Methods for the production of proteins with a desired function|

---

US5427908A|1990-05-01|1995-06-27|Affymax Technologies N.V.|Recombinant library screening methods|

---

GB9015198D0|1990-07-10|1990-08-29|Brien Caroline J O|Binding substance|

---

US5625126A|1990-08-29|1997-04-29|Genpharm International, Inc.|Transgenic non-human animals for producing heterologous antibodies|

---

US5545806A|1990-08-29|1996-08-13|Genpharm International, Inc.|Ransgenic non-human animals for producing heterologous antibodies|

---

US5661016A|1990-08-29|1997-08-26|Genpharm International Inc.|Transgenic non-human animals capable of producing heterologous antibodies of various isotypes|

---

US5633425A|1990-08-29|1997-05-27|Genpharm International, Inc.|Transgenic non-human animals capable of producing heterologous antibodies|

---

US5770429A|1990-08-29|1998-06-23|Genpharm International, Inc.|Transgenic non-human animals capable of producing heterologous antibodies|

---

CA2089661C|1990-08-29|2007-04-03|Nils Lonberg|Transgenic non-human animals capable of producing heterologous antibodies|

---

US5698426A|1990-09-28|1997-12-16|Ixsys, Incorporated|Surface expression libraries of heteromeric receptors|

---

WO1992009690A2|1990-12-03|1992-06-11|Genentech, Inc.|Enrichment method for variant proteins with altered binding properties|

---

ES2315612T3|1991-04-10|2009-04-01|The Scripps Research Institute|GENOTECAS OF HETERODYMERIC RECEPTORS USING PHAGEMIDS.|

---

GB9112536D0|1991-06-11|1991-07-31|Celltech Ltd|Chemical compounds|

---

PT1024191E|1991-12-02|2008-12-22|Medical Res Council|Production of anti-self antibodies from antibody segment repertoires and displayed on phage|

---

GB9120467D0|1991-09-26|1991-11-06|Celltech Ltd|Anti-hmfg antibodies and process for their production|

---

US5733743A|1992-03-24|1998-03-31|Cambridge Antibody Technology Limited|Methods for producing members of specific binding pairs|

---

AU696293B2|1993-12-08|1998-09-03|Genzyme Corporation|Process for generating specific antibodies|

---

ES2247204T3|1994-01-31|2006-03-01|Trustees Of Boston University|BANKS OF POLYCLONAL ANTIBODIES.|

---

FR2716640B1|1994-02-28|1996-05-03|Procedes Machines Speciales|Device for centering and blocking a workpiece with a view to running it in using an expansion lapper.|

---

US5516637A|1994-06-10|1996-05-14|Dade International Inc.|Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage|

---

JP2978435B2|1996-01-24|1999-11-15|チッソ株式会社|Method for producing acryloxypropyl silane|

---

US5980898A|1996-11-14|1999-11-09|The United States Of America As Represented By The U.S. Army Medical Research & Material Command|Adjuvant for transcutaneous immunization|

---

GB9625640D0|1996-12-10|1997-01-29|Celltech Therapeutics Ltd|Biological products|

---

US20060228364A1|1999-12-24|2006-10-12|Genentech, Inc.|Serum albumin binding peptides for tumor targeting|

---

CN1678634A|2002-06-28|2005-10-05|多曼蒂斯有限公司|Immunoglobulin single variable antigen combination area and its opposite constituent|

---

US6908963B2|2001-10-09|2005-06-21|Nektar Therapeutics Al, Corporation|Thioester polymer derivatives and method of modifying the N-terminus of a polypeptide therewith|

---

CA2507004A1|2002-12-03|2004-06-17|Celltech R & D Limited|Assay for identifying antibody producing cells|

---

GB0312481D0|2003-05-30|2003-07-09|Celltech R&D Ltd|Antibodies|

---

GB0412181D0|2004-06-01|2004-06-30|Celltech R&D Ltd|Biological products|

---

WO2006033386A1|2004-09-22|2006-03-30|Kirin Beer Kabushiki Kaisha|STABILIZED HUMAN IgG4 ANTIBODIES|

---

GB0619291D0|2006-09-29|2006-11-08|Ucb Sa|Altered antibodies|

---

US20100267934A1|2007-05-31|2010-10-21|Genmab A/S|Stable igg4 antibodies|

---

SG193868A1|2007-09-26|2013-10-30|Chugai Pharmaceutical Co Ltd|Modified antibody constant region|

---

EP3153524A1|2008-12-03|2017-04-12|Genmab A/S|Antibody variants having modifications in the constant region|

---

US20120071634A1|2009-03-19|2012-03-22|Chugai Seiyaku Kabushiki Kaisha|Antibody Constant Region Variant|

---

JP5889181B2|2010-03-04|2016-03-22|中外製薬株式会社|Antibody constant region variants|

---

CA2797981C|2010-05-14|2019-04-23|Rinat Neuroscience Corporation|Heterodimeric proteins and methods for producing and purifying them|

---

CA2806252C|2010-07-29|2019-05-14|Xencor, Inc.|Antibodies with modified isoelectric points|

---

GB201014033D0|2010-08-20|2010-10-06|Ucb Pharma Sa|Biological products|

---

PT2794905T|2011-12-20|2020-06-30|Medimmune Llc|Modified polypeptides for bispecific antibody scaffolds|

---

GB201203051D0|2012-02-22|2012-04-04|Ucb Pharma Sa|Biological products|

---

GB201203071D0|2012-02-22|2012-04-04|Ucb Pharma Sa|Biological products|

---

TW201632559A|2015-02-22|2016-09-16|索倫多醫療公司|Antibody therapeutics that bind CD137|

---

US10563966B2|2017-11-13|2020-02-18|Hornady Manufacturing Company|Vibratory powder trickler|WO2006106905A1|2005-03-31|2006-10-12|Chugai Seiyaku Kabushiki Kaisha|Process for production of polypeptide by regulation of assembly|

---

US11046784B2|2006-03-31|2021-06-29|Chugai Seiyaku Kabushiki Kaisha|Methods for controlling blood pharmacokinetics of antibodies|

---

ES2654040T3|2006-03-31|2018-02-12|Chugai Seiyaku Kabushiki Kaisha|Antibody modification method for the purification of bispecific antibodies|

---

PL2202245T3|2007-09-26|2017-02-28|Chugai Seiyaku Kabushiki Kaisha|Method of modifying isoelectric point of antibody via amino acid substitution in cdr|

---

JP5889181B2|2010-03-04|2016-03-22|中外製薬株式会社|Antibody constant region variants|

---

US9334331B2|2010-11-17|2016-05-10|Chugai Seiyaku Kabushiki Kaisha|Bispecific antibodies|

---

GB201203071D0|2012-02-22|2012-04-04|Ucb Pharma Sa|Biological products|

---

GB201203051D0|2012-02-22|2012-04-04|Ucb Pharma Sa|Biological products|

---

KR101895634B1|2013-05-31|2018-09-05|한미약품 주식회사|IgG4 Fc fragment comprising modified hinge region|

---

ES2881306T3|2013-09-27|2021-11-29|Chugai Pharmaceutical Co Ltd|Method for the production of heteromultimers of polypeptides|

---

EP2944962A1|2014-05-14|2015-11-18|UCB Biopharma SPRL|Method for determining antibody specificity|

---

MA40764A|2014-09-26|2017-08-01|Chugai Pharmaceutical Co Ltd|THERAPEUTIC AGENT INDUCING CYTOTOXICITY|

---

TWI691512B|2015-02-20|2020-04-21|日商橘生藥品工業股份有限公司|Fc fusion high affinity IgE receptor alpha chain|

---

EP3279216A4|2015-04-01|2019-06-19|Chugai Seiyaku Kabushiki Kaisha|Method for producing polypeptide hetero-oligomer|

---

US11254744B2|2015-08-07|2022-02-22|Imaginab, Inc.|Antigen binding constructs to target molecules|

---

WO2017159287A1|2016-03-14|2017-09-21|中外製薬株式会社|Cell injury inducing therapeutic drug for use in cancer therapy|

---

EP3433281A1|2016-03-21|2019-01-30|Elstar Therapeutics, Inc.|Multispecific and multifunctional molecules and uses thereof|

---

JP2019532957A|2016-10-06|2019-11-14|グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッドＧｌａｘｏｓｍｉｔｈｋｌｉｎｅ Ｉｎｔｅｌｌｅｃｔｕａｌ Ｐｒｏｐｅｒｔｙ Ｄｅｖｅｌｏｐｍｅｎｔ Ｌｉｍｉｔｅｄ|Antibodies with reduced binding to process impurities|

---

US10995152B2|2016-10-26|2021-05-04|The Board Of Trustees Of The Leland Stanford Junior University|Modified immunoglobulin hinge regions to reduce hemagglutination|

---

KR20190095946A|2016-12-23|2019-08-16|브리스톨-마이어스 스큅 컴퍼니|Therapeutic Immunoglobulin G4 Design for Improved Bioanalysis and Bioprocessing Properties|

---

WO2018147960A1|2017-02-08|2018-08-16|Imaginab, Inc.|Extension sequences for diabodies|

---

US20200291089A1|2017-02-16|2020-09-17|Elstar Therapeutics, Inc.|Multifunctional molecules comprising a trimeric ligand and uses thereof|

---

AR111830A1|2017-05-25|2019-08-21|Squibb Bristol Myers Co|MONOCLONAL ANTI-BODIES ANTAGONISTS AGAINST CD40 AND ITS USES|

---

EP3630836A1|2017-05-31|2020-04-08|Elstar Therapeutics, Inc.|Multispecific molecules that bind to myeloproliferative leukemiaprotein and uses thereof|

---

EP3641802A1|2017-06-22|2020-04-29|INSERM |Methods and pharmaceutical compositions for the treatment of fibrosis with agents capable of inhibiting the activation of mucosal-associated invariant tcells|

---

WO2019009346A1|2017-07-06|2019-01-10|日東紡績株式会社|Anti-human igg4 monoclonal antibody and human igg4 assay reagent using said antibody|

---

WO2019035938A1|2017-08-16|2019-02-21|Elstar Therapeutics, Inc.|Multispecific molecules that bind to bcma and uses thereof|

---

WO2019178362A1|2018-03-14|2019-09-19|Elstar Therapeutics, Inc.|Multifunctional molecules that bind to calreticulin and uses thereof|

---

EP3765516A2|2018-03-14|2021-01-20|Elstar Therapeutics, Inc.|Multifunctional molecules and uses thereof|

---

JP2021521843A|2018-04-25|2021-08-30|プロメテウス バイオサイエンシーズ，インク．|Optimized anti-TL1A antibody|

---

AU2019297451A1|2018-07-03|2021-01-28|Marengo Therapeutics, Inc.|Anti-TCR antibody molecules and uses thereof|

---

AR117091A1|2018-11-19|2021-07-07|Squibb Bristol Myers Co|MONOCLONAL ANTIBODIES ANTAGONISTS AGAINST CD40 AND THEIR USES|

---

AU2020205150A1|2019-01-03|2021-07-22|Assistance Publique-Hôpitaux De Paris |Methods and pharmaceutical compositions for enhancing CD8+ T cell-dependent immune responses in subjects suffering from cancer|

---

EP3927744A1|2019-02-21|2021-12-29|Marengo Therapeutics, Inc.|Multifunctional molecules that bind to t cell related cancer cells and uses thereof|

---

EP3927747A1|2019-02-21|2021-12-29|Marengo Therapeutics, Inc.|Antibody molecules that bind to nkp30 and uses thereof|

---

EP3927746A1|2019-02-21|2021-12-29|Marengo Therapeutics, Inc.|Multifunctional molecules that bind to calreticulin and uses thereof|

---

GB202112702D0|2019-02-21|2021-10-20|Marengo Therapeutics Inc|Multifunctional molecules that bind to t cells and uses thereof to treat autoimmune disorders|

---

AU2020226904A1|2019-02-21|2021-09-16|Marengo Therapeutics, Inc.|Anti-TCR antibody molecules and uses thereof|

---

WO2021009263A1|2019-07-16|2021-01-21|INSERM |Antibodies having specificity for cd38 and uses thereof|

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

---

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. |

---

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

---

2021-07-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

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

优先权:

---

申请号 | 申请日 | 专利标题

---

GBGB1203051.6A|GB201203051D0|2012-02-22|2012-02-22|Biological products|

---

GB1203051.6|2012-02-22|

---

PCT/EP2013/053614|WO2013124450A1|2012-02-22|2013-02-22|Sequence symmetric modified igg4 bispecific antibodies|

---