cd40l antagonizing antibody polypeptides

专利摘要:
antibody polypeptides that antagonize cd40l, their use, nucleic acid, vector, host cell, and pharmaceutical composition. The present invention relates to antibody polypeptides which specifically bind to human cd40l. antibody polypeptides do not activate platelets. antibody polypeptides are useful in treating diseases that involve activation of cd40l, such as graft-related diseases and autoimmune diseases. antibody polypeptides can be domain antibodies (dabs) that comprise a single vh or vk domain. the half-life of antibody polypeptides can be increased by modifying the antibody polypeptides to be dual specific reagents that can also bind to human serum albumin (hsa) or other antigen. the present invention also relates to a nucleic acid encoding an antibody polypeptide, a vector comprising said nucleic acid, a host cell comprising said vector, a pharmaceutical composition comprising an antibody polypeptide, and the use of a polypeptide of antibody for the preparation of a medicine for the treatment of a patient who has or is at risk of having an immune disease.
公开号:BR112014009069A2
申请号:R112014009069-6
申请日:2012-10-12
公开日:2020-10-27
发明作者:Steven G. Nadler；Olga Ignatovich；Philip DREW；James K. Tamura；Laura Price；Robert P. Rehfuss；Suzanne J. Suchard；Anish SURI；James William BRYSON；Aaron YAMNIUK；Steven Grant
申请人:Bristol-Myers Squibb Company；Domantis Limited；
IPC主号:

专利说明:

[005] [005] The crucial role of CD40-CD40L interactions in immune and inflammatory responses has made them a promising target for the treatment of pathological immuno-inflammatory processes. Blocking CD40-CD40L interactions through monoclonal antibodies (mAbs) for specific CD40L successfully prevents allograft rejection in primates and treats autoimmune diseases and atherosclerosis in animal models. Montgomery et al., Transplantation 74: 1365-1369 (2002).
[006] [006] In humans, two different anti-CD40L mAb clones have been used in clinical trials for the treatment of different autoimmune diseases. Maribel et al., Mol. Immunol. 45: 937-44 (2008). Monoclonal antibodies, however, can exhibit a surprisingly high incidence of thromboembolic complications (ET), such as atherothrombotic central nervous system events, myocardial infarction, pulmonary embolism and deep vein thrombosis. For example, the usefulness of the anti-CD40L mAb clone hu5c8 (anti-CD40L mAb, Biogen) is limited by a surprisingly high incidence of TE complications. TE by these antibodies is believed to result from the formation of higher-order immunological complexes (CIs) of the mAbs with CD40L attached to the membrane in platelets or spills.
[007] [007] Anti-CD40L antibody antagonists that are less likely to cause platelet aggregation and thus cause thromboembolism are still needed in a clinical setting. New antibody polypeptides that specifically bind to human CD40L are provided. Antibody polypeptides advantageously do not cause platelet aggregation. Antibody polypeptides are useful in the treatment of diseases that involve CDA40L activation, including autoimmune diseases, transplant rejection and allergic responses. The antibody polypeptides comprise a variable domain. Examples of antibody polypeptides are in the form of a domain antibody (dAb) that contains a single variable domain. Alternatively, dAbs can be bispecific reagents that comprise a second variable domain that can bind with another antigen, such as human serum albumin (HSA), for example.
[008] [008] An antibody polypeptide is provided that comprises a first variable domain that specifically binds to human CD40L, wherein the first variable domain comprises the amino acid sequence of one of the selected variable domains of the BMS2h lineage. An isolated antibody polypeptide is additionally provided that comprises a first variable domain that specifically binds to human CD40L, wherein CD40L comprises the amino acid sequence of SEQ ID NO: 1, wherein the sequence of a-
[009] [009] An antibody polypeptide selected from the BMS2h-572 lineage group is also provided, wherein the amino acid sequence of the first variable domain further comprises: (a) a CDR1 region that has a Trp-X sequence, -Leu-Met-Gly (SEQ ID NO: 2), where X, is Glu or Gln; (b) a region of CDR2 that has a sequence Gly-lle-Glu-Gly-Pro-Gly-Asp-Val-Thr-Tyr-Tyr-Ala- —Asp-Ser-Val-Lys-Gly (SEQ ID NO: 3); and (c) a CDR3 region that has a Lys-X, -Y, - Z> -Ser-Asp-Tyr sequence (SEQ ID NO: 4), where X, is Asp or Glu, Y, is Ala or Ser and Z> is Lys, Asn or Arg. Also provided is the antibody polypeptide, in which the amino acid sequence of the first variable domain further comprises: (a) an FR1 region that has a Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly sequence -Gly-Gly-Leu-Val-GlIn-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala- Ala-Ser-Gly-Phe-Thr-Phe-Asn (SEQ ID NO: 5 ); (b) an FR2 region that has a Trp-X, -Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu- Glu-Trp-Val-Ser sequence (SEQ ID NO: 6), where X ; is Ala or Val; (c) an FR3 region that has an Arg-Thr-Phe-lle-Ser-Arg-Asp- Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-GlIn-Met-Asn-Ser sequence -Leu-Arg-Ala-Glu- Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Val-Lys-Val-Gly (SEQ ID NO: 7); and (d) an FR4 region that has an Arg-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser sequence (SEQ ID NO: 8). Alternatively, the first variable domain of the antibody polypeptide can comprise the amino acid sequence of BMS2h-572-633;
[0010] [0010] An antibody polypeptide selected from the BMS2h-719 lineage group is also provided, which comprises a first variable domain with the following consensus sequence: Glu-Val-Gln-Leu-Leu-Glu-Ser -Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly-Gly-Ser-Leu- Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-X, -Y, -Tyr-Glu-Met-Z, - Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-Ser-lle-Ser- Ser-Asp-Gly- Ser-Phe-Thr-Tyr-Tyr-Ala-A, -Ser-Val-Lys-Gly-Arg-Phe-Thr- Ile-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr -Leu-Gln-Met-Asn-Ser- Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Ala-B, -Pro-Phe-Thr-Glu-
[0011] [0011] An antibody polypeptide selected from the BMS2h-503 lineage group is also provided, which comprises a first variable domain with the following consensus sequence: Asp-lle-Gln-Met-Thr-Gln-Ser -Pro-Ser-Ser-Leu-Ser-Ala-Ser-Val-Gly-Asp-Arg- Val-Thr-Ile-Thr-Cys-Arg-Ala-Ser-His-X, -lle-GIn-Arg- Tyr-Leu-Ser-Trp- Tyr-Gln-Gln-Lys-Pro-Gly-Lys-Ala-Pro-Lys-Leu-Leu-lle-Leu-Trp-Gly-Ser- Gln-Leu-Gln-Ser- Gly-Val-Pro-Ser-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Gly- Thr-Asp-Phe-Thr-Leu-Thr-lle-Ser-Ser-Leu-Gln-Pro-Glu- Asp-Phe-Ala- Thr-Tyr-Tyr-Cys-Gly-Gln-Trp-Trp-Ala-Pro-Pro-Gln-Thr-Phe-Gly-Gln- Gly-Thr-Lys-Val-Glu-lle- Lys-Arg (SEQ ID NO: 10), where X, is His or Asp.
[0012] [0012] An antibody polypeptide selected from the BMS2h-116 lineage group is also provided, comprising a first variable domain with the following consensus sequence: Asp-lle-Gln-Met-Thr-Gln-Ser -Pro-Ser-Ser-Leu-Ser-Ala-X, -Val-Gly-Asp-Arg-Val- Thr-lle-Thr-Cys-Arg-Ala-Ser-Gln-Pro-lle-Gly-Pro- Asp-Leu-Leu-Trp-Tyr- GIn-Gln-Lys-Pro-Gly-Lys-Ala-Pro-Lys-Leu-Leu-lle-Tyr-GlIn-Thr-Ser-lle- Leu-Arg-Ser- Gly-Val-Pro-Ser-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Glu-Thr- Asp-Phe-Thr-Leu-Thr-lle-Ser-Asn-Leu-Gln-Pro-Glu- Asp-Y-Ala-Z, -Tyr- Tyr-Cys-GlIn-Gln-Tyr-Trp-Ala-Phe-Pro-Val-Thr-Phe-Gly-A, -Gly-Thr-Lys- Val-Val- lle-Lys-Arg (SEQ ID NO: 11), where X, is Ser or Tyr; Y; is Leu ouPhe; Z, is ThrouLys; eA, is Lys, Arg or Gln.
[0013] [0013] An antibody polypeptide is also provided that comprises a first variable domain that specifically binds to human CD40L, wherein the antibody polypeptide is an antibody domain (dAb). The antibody polypeptide can be a fusion polypeptide comprising the first variable domain and a domain
[0014] [0014] An antibody polypeptide is also provided that comprises a first variable domain that specifically binds to human CD40L, wherein the antibody polypeptide further comprises a second variable domain that specifically binds to a second antigen, wherein the second antigen it is an antigen other than human CD40L. The second antigen can be a differentiation cluster (CD) molecule or a Class II Histo-Compatibility Complex (MHC) molecule. Alternatively, the second antigen may be serum albumin (SA).
[0015] [0015] Also provided is a nucleic acid that encodes any of the antibody polypeptides provided here. It is also considered a vector that comprises nucleic acid. An isolated host cell can comprise such a vector.
[0016] [0016] A pharmaceutical composition is provided which comprises a therapeutically effective amount of the antibody polypeptide provided herein and a pharmaceutically acceptable carrier. The pharmaceutical composition can further comprise an immunosuppressive and immune-modulating and / or anti-inflammatory agent.
[0017] [0017] A method of treating an immune disease is provided in a patient in need of such treatment which comprises administering to the patient a therapeutically effective amount of the pharmaceutical composition described herein. An example of a method delivers the pharmaceutical composition in combination with an immunosuppressive / immunomodulatory and / or anti-inflammatory agent. The immune disease can be an autoimmune disease or a graft-related disease. Alternatively, the immune disease is a graft-related disease. In addition, graft-related disease may include rejection of solid organ, tissue and / or cell transplantation. Alternatively, graft-related disease is graft versus host disease (GVHD). The graft-related disease may still be an acute transplant rejection. Alternatively, the graft-related disease may be a chronic transplant rejection.
[0018] [0018] The method of treating a graft-related disease is also provided, in which the pharmaceutical composition is administered with a mutant CTLA4 molecule. The mutant CTLA4 molecule can be L104EA29Y-lg (belatacept).
[0019] [0019] A method of treating an immune disease is also provided in a patient in need of such treatment that includes administering to the patient a therapeutically effective amount of the pharmaceutical composition provided here, in which the immune disease is selected from the group consisting of Ad-dison disease, allergies, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune mumps, colitis, coronary heart disease, coronary heart disease Crohn's, diabetes, including Type 1 and / or Type 2 diabetes, epididymitis, glomerulonephritis, Graves' disease, Guil- lain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug products, systemic lupus erythematosus, male infertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, arthritis rheumatoid, sarcoidosis
[0020] [0020] A use of an isolated antibody polypeptide disclosed here is also provided for the preparation of a drug for the treatment of a patient, in which the patient has or is at risk of having an immune disease. Additionally, a use of an isolated antibody polypeptide disclosed herein is provided for the preparation of a medicament for the relief of at least one symptom of an immune disease in a patient who needs it.
[0021] [0021] An isolated antibody polypeptide is additionally provided here which comprises a first variable domain, wherein said antibody polypeptide specifically binds to human CD40L, where CD40L comprises the amino acid sequence of SEQIDNO: 1 , in which the antibody polypeptide competes with the binding of BMS2h-572-633 and in which the antibody polypeptide inhibits the binding of CD40L to CD40 with an EC50 of 100pM up to 100nM. In one aspect, the first variable domain comprises the amino acid sequence of one of the antibody polypeptides selected from the lineage group consisting of BMS2h-572, BMS2h-719, BMS2h-503 and BMS2h-116. In another aspect, the first variable domain comprises an amino acid sequence at least 95% identical to BMS2h-572-6, BMS2h-572-608, BMS2h-572-614, BMS2h-572-619, BMS2h-572-633, BMS2h -572-634, BMS2h-572-635, BMS2h-719-2, BMS2h-719-202, BMS2h-719-203, BMS2h-719-213, BMS2h-719-214,
[0022] [0022] FIG. 1A describes the domain antibody comprising a V variable domain, BMS2h-572-633 fused to a modified IgG1 Abatacept Fc cause.
[0023] [0023] FIG. 1B shows the amino acid sequence (SEQ ID NO: 1355) of the BMS2h-572-633 variable domain. The Fc fusion protein is a molecular weight dimer of 77,984 Daltons, with each polypeptide chain consisting of 354 amino acids. The variable domain is fused by a ligand with the mutated Fc construct of human I9G1, in which three cysteine residues are replaced by serine and proline is replaced by a serine residue.
[0024] [0024] FIG. 2 describes a working model for the CD40-40L pathway. The upper panel shows the initial stages of a meeting between a T cell and an APC. The initial encounter controlled by the T cell receptor (TCR) commitment of the pMHC complex (signal 1) coupled with an initial CD28-CD80 interaction (signal 2) is sufficient for expression on the cell surface of trimeric CD40L (bottom panel). The commitment of CD40 to CD40L results in several biological responses outlined in the gray box.
[0025] [0025] FIG. 3 provides sequences (SEQ ID NOS 1356-1361, respectively, in order of appearance) of several Fc domains. Binding regions are shown in the boxes.
[0026] [0026] FIG. 4 shows examples of various antibodies with Fc-formatted domain (SEQ ID NOS 1362-1365, respectively, in order of appearance). Linking regions are indicated by the boxes.
[0027] [0027] FIG. 5 describes SPR sensorgram data for the binding of 12.5-0.39nM BMS-986004 (2: 1 dilution series) with biot-IZ-hCD40L captured on a strepta-vidine SPR sensor chip at 25ºC . The colored lines show the data from the double reference sensorgram and the black lines show the Langmuir adjustment of 1: 1 Langmuir to the data, with an apparent Kd value influenced by the avidity of 0.11 nM.
[0028] [0028] FIG. 6 shows ITC data for titrations of 19 µM IZ-hCD40L within 2 µM BMS-986004 (in black) or 18 µM BMS-986004 within 2 µM IZ-hCD40L (in blue). The molar ratio (apparent stoichiometry) is defined per mole of | Z-hCD40L trimer and per mole of divalent BMS-986004 Fc dimer. The molar ratio values obtained in the form of equivalence points on the abscissa suggest that more than one mole of BMS-986004 can be separated per mole of IZ-hCD40L trimer; however, an exact structural model for the complex cannot be determined from the ITC data alone. The squares represent the integrated heat of the connection data and the solid lines represent the best fit to a "2-site model".
[0029] [0029] FIG. 7 shows the in vivo efficacy of the mouse dAb-Fc CDA40L representative (KLH-induced antibody response).
[0030] [0030] FIG. 8 demonstrates that mouse BMS-2m-126-24-Fc dAb and antibody MR-1 inhibit TNBS-induced colitis in mice.
[0031] [0031] FIG. 9 shows that BMS-2m-126-24-Fc and CTLA4-Ig work synergistically to prolong the survival of cardiac allografts.
[0032] [0032] FIG. 10 provides a hypothetical model for platelet aggregation mediated by anti-CD40 monoclonal antibody.
[0033] [0033] FIG. 11A shows the plasma concentration vs. the time profile of BMS-986004 after IV dosing of 11 mg / kg in monkeys.
[0034] [0034] FIG. 11B demonstrates plasma concentration vs. time profiles of BMS-986003 after dosing | V of 2 mg / kg in monkeys.
[0035] [0035] FIG. 12 shows plasma concentrations vs. the time profiles of BMS-986003 (after dosing SC at 0.2, 2 and 20 mg / kg in monkeys) and 5c8 IgG1 (after dosing IV at 20 mg / kg in monkeys).
[0036] [0036] FIG. 13 shows plasma concentrations vs. the time profiles of BMS-2m-126-24-CT after 1 mg / kg dosage IVe SC and 10 mg / kg dosage SC to mice.
[0037] [0037] FIG. 14 demonstrates the PK / PD modeling of plasma exposures to BMS-986003 and 5c8-lgG1 and anti-KLH antibody response (IgG titers).
[0038] [0038] FIG. 15 shows the PK / PD modeling of plasma exposures to BMS-986004 and RO ex vivo on peripheral blood mononuclear cells (PBMC).
[0039] [0039] FIG. 16 shows that IV.3 blocks platelet-mediated 5c8 / sCD40L IC activation in human blood.
[0040] [0040] FIG. 17 shows the effect of Fc variants on platelet activation in human blood.
[0041] [0041] FIG. 18 demonstrates the activation of platelets with 5c8-CT / sCDA40L IC in the blood of human donors genotyped in relation to the FcegRlla polymorphism.
[0042] [0042] FIG. 19 diagram the activation of platelets by various antibodies in the blood of human donors.
[0043] [0043] FIG. 20 shows the levels of platelet activation by various antibodies, including BMS-986003, in transgenic mice that express hFcgRlla.
[0044] [0044] Figures 21, 22, 23 and 24 show ClushalW2 alignments of domain antibody polypeptides representative of lines BMS2h-572, BMS2h-719, BMS2h-503 and BMS2h-116, respectively. FIG. 21 shows SEQ ID NOS 243, 251, 257, 262 and 274-276, respectively, in order of appearance, FIG. 22 shows SEQ ID NOS 352, 354-355 and 357-361, respectively, in the order of appearance, FIG. 23 shows SEQ ID NOS 1087-1088, respectively, in order of appearance and FIG. 24 shows SEQ ID NOS 970-971 and 974, respectively, in order of appearance.
[0045] [0045] FIG. 25 shows data from the SPR sensorgram for binding experiments using dAbs BMS2h-503-1, BMS2h-572-6, BMS2h-719-17 monovalent and fragment of monovalent Fab of 5c8, in which the indicated molecules compete with each other by binding with CDA40L (biotinylated IZ-hCD40L).
[0046] [0046] FIG. 26 shows data from the SPR sensorgram for experiments that test the binding of BMS2h-572-619-CT-long, BMS2h-572-633-CT-long, BMS2h-572-6-CT-long, BMS2h- 719- 202-CT-long, = BMS2h-572-608-CT-long, - BMS2h-572-634-CT-long, BMS2h-572-614-CT-long, BMS2h-572-635-CT-long and 5c8-CT-long to a CDA40L monomer (3 upper panels) or a CD40L trimer (3 lower panels). DETAILED DESCRIPTION
[0047] [0047] Antibody polypeptides are provided that specifically bind to human CD40L. Antibody polypeptides do not activate platelets, and antibody polypeptides are useful in treating diseases that involve CD40L activation, such as graft-related diseases and autoimmune diseases. Antibody polypeptides can be selected using a primary selection that uses cell binding assays, followed by one or more rounds of error-prone or affinity maturation directed to degenerate oligonucleotides. As a result, a genus of antibody polypeptides is provided that specifically bind to CD40L.
[0048] [0048] A "lineage" is a set of related antibody polypeptides that have been prepared starting from a common precursor through error-prone affinity maturation or targeting degenerate oligonucleotides, which is disclosed in the following examples and expects them to connect to the CD40L. The nomenclature of the antibody polypeptides is used to designate the various strains. The nomenclature "BMS2h-572," for example, refers to antibody polypeptides of lineage 572, which were produced against human CDA40L. The "BMS2h-572 lineage" antibody polypeptides include BMS2h-572-1 through BMS2h-572-19, BMS2h-572-21 through BMS2h-572-24, BMS2h-572-601 through BMS2h-572-627 and BMS2h- 572-630 through BMS2h-572-635.
[0049] [0049] Consequently, in one aspect, an antibody polypeptide comprises a variable domain that specifically binds to human CD40L, where the antibody polypeptide competes with the binding of any of the domain antibodies (dAbs) listed in TABLE 1 or TABLE 3. For example, the antibody polypeptide can compete with selected dAb from the 2h strain. The dAb can also be selected from a strain selected from the group consisting of BMS2h-116, BMS2h-503, BMS2h-572 and BMS2h-719, such as dAb BMS2h-572-633, BMS2h-572-608 or
[0050] [0050] The antibody polypeptides can be an antibody domain that contains a single variable domain. The antibody polypeptides can also comprise additional domains, such as an Fc domain. For example, the antibody polypeptide may comprise a second variable domain that specifically binds to human serum albumin (HSA). Such dual specific antibody polypeptides may have a longer half-life, for example.
[0051] [0051] As used herein, "specific binding" refers to the binding of an antigen by an antibody polypeptide with a dissociation constant (K4g) of approximately 1 µM or less which is measured, for example, by the resonance of surface plasmon (SPR). Suitable test systems include the BlAcore surface plasmon resonance system "" and the BlAcore kinetic evaluation software "" (for example, version 2.1). The affinity or Ka for the specific binding interaction can be approximately 1 µM or less, approximately 500 nM or less or approximately 300 nM or less.
[0052] [0052] The term "approximately" will be understood by ordinary experts in the art and will vary to some extent on the context in which it is used. It generally covers approximately a range of values that are plus / minus 10% of a stated value.
[0053] [0053] According to this detailed description, the following abbreviations and definitions apply. It should be noted that as used here, the forms in the singular "one", "one", "o" and "a" include
[0054] [0054] Antibody polypeptides that bind to human CD40L are provided. CD40L is also known as CD154, gp39, TNF-related activation protein (TRAP), 5c8 antigen or T-BAM. Structural information relevant to human CD40L can be found, for example, at UniProt Accession Number P29965. "Human CD40L" refers to CD40L which comprises the following amino acid sequence: 10 20 30 40 50 60 MIETYNOTSP RSAATGLPIS MKIFMYLLTV FLITOMIGSA LFAVYLHRRL
[0055] [0055] CD40L was also sequenced in Sus scrofa, Mus musculus, Canis familiaris, Bos ffini, Macaca mulatta, Aotus tivirgatus, Callithrix jacchus, Cercocebus torquatus atys, Macaca nemestrina,
[0056] [0056] The binding of the present antibody polypeptides to CD40L antagonizes CD40L activity. "CD40L activities" include, but are not limited to, co-stimulation and activation of an APC in association with stimulation of the T cell receptor by MHC molecules on the APC, secretion of all immunoglobulin isotypes in the presence cytokines, stimulation of B cell proliferation, cytokine production, antibody class change and affinity maturation. For example, patients with X-linked hyper-IgM syndrome express functional CD40 on their B cells, but their activated T cells have a defective CD40L protein, resulting in their inability to activate B cells and induce immunoglobulin subtype exchange. Aruffo et al., Cell 72: 291-300 (1993).
[0057] [0057] CD40L activities can be mediated through interaction with other molecules. "CD40L activities" include the functional interaction between CD40L and the following molecules: CD40 (CD40L receptor), integrin a5B1 and allbB3. For example, CD40L binds to its receptor, CD40, which is expressed on a variety of APCs, such as B cells, macrophages and dendritic cells, as well as on stromal cells, vascular endothelial cells and platelets.
[0058] [0058] As used here, the terms "activate", "active" and "activated (a)" refer to an increase in a measurable CD40L activity provided by at least 10% over a reference , for example, at least 10%, 25%, 50%, 75% or even 100% or more. CD40L activity is "antagonized" if the activity is reduced by at least 10% and in an example of modality, at least 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95%, 97% or even 100% (that is, without detectable activity), in relation to the absence of the antagonist. For example, an antibody polypeptide can antagonize some or all of CD40L activity. In one mode,
[0059] [0059] The antibody polypeptides comprise a variable domain. In one embodiment, the antibody polypeptides are in the form of a dAb that contains a single variable domain. The antibody polypeptides can be full-length anti-CD40L immunoglobulin molecules that comprise two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In this embodiment, the amino terminal portion of each chain includes a variable domain (V, or Vy4) of approximately 100-120 amino acids. The complementarity determining regions (CDRs) contained therein are primarily responsible for the recognition of antigens, although structural residues may play a role in binding epitopes. The carboxy-terminal "half" of each heavy chain defines a constant region (Fc) primarily responsible for the effector function.
[0060] [0060] Antibody polypeptides can also be "fragments" that comprise a portion of the full-length anti-CD40L immunoglobulin molecule that comprises a variable domain that specifically binds to CD40L. Thus, the term "antibody polypeptides" includes an antigen-binding heavy chain, a light chain, a heavy chain-light chain dimer, a Fab fragment, an F (ab ') fragment, a fragment Fv, a single-chain Fv (scFv) and a dAb, for example. The term "antibody polypeptides" thus includes polypeptides produced through recombinant engineering and expression, as well as monoclonal antibodies produced
[0061] [0061] Light chains are classified as kappa (K) or lambda (A) and are characterized by a particular constant region, C, as is known in the art. Heavy chains are classified as y, u, q, à or e and define the isotype of an antibody as IgG, IgM, IgA, IgD or IgE, respectively. The heavy chain constant region is comprised of three domains (CH1, CH2 and CH3) for IgG, IgD and IgA; and four domains (CH1, CH2, CH3 and CH4) for IgM and IgE.
[0062] [0062] Each light chain variable domain (V,) and each heavy chain variable domain (V4) is composed of three CDRs and four structural regions (FRs), arranged from the amino terminal to the carboxy terminal in the order below : FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDRs are referred to as "LCDRI1, LCDR 2 and LCDR3" and the three heavy chain CDRs are referred to as "HCDR1, HCDR2 and HCDR3".
[0063] [0063] As used herein, the term "Fc domain" refers to the constant region antibody sequences that comprise the constant domains CH2 and CH3 that are delimited according to Kabat et al., Sequences of Immunological Interest, 5th ed., US Dept. Health & Human Services, Washington, D.C. (1991). The Fc domain can be derived from an IgG1 or IgG4 Fc region, for example.
[0064] [0064] A variable domain can be merged with an Fc domain. Examples of various antibodies with Fc-formatted domain and their potency are given in TABLE 6. FIG. 3 provides the strings for the various Fc domains provided here. Binding regions are shown in the boxes. As used in TABLE 6, "Fc" indicates that the dAb is fused to a short human IgG1 Fc. "CT Long Fc", also called CT-L2, refers to the CTLA4 Fc. The underlined S represents point cysteine-to-serine mutations made to eliminate disulfides in the Fc hinge. "CT Short", also called deCT-S1, is short of CT Long in 7 amino acids. "N297Q Long Fc", also referred to as N297Q-LA4, is the Fc domain of human IgG1 with an N297Q mutation made to eliminate the N-linked carbohydrate in Fc. "N297Q Short Fc", also called N297Q-S3, is shorter than N297Q Long Fc by 7 amino acids and is a human IgG1 with an N297Q point mutation made to eliminate the N-linked carbohydrate in the Fc domain. "CT-Fc SP5" is CT Long Fc, where SP5 refers to the octeonectin signal peptide used for secretion from the mammalian expression host. The cleavage site is indicated by "". FIG. 4 further provides examples of various Fc domain formations.
[0065] [0065] When a variable domain is fused to an Fc domain, the carboxyl terminal of the variable domain (a V, or Vu domain, including dAbs) can be linked or fused to the amino terminal of the F2 CH2 domain. Alternatively, the carboxyl terminus of the variable domain can be linked or fused to the amino terminus of a CH1 domain, which itself is fused to the CH2 domain of Fc. The protein can comprise the hinge region between the CH1 and CH2 domains as a whole or in part.
[0066] [0066] CDRs contain most of the residues that form specific interactions with the antigen. In one embodiment, the variable domain of an antibody polypeptide comprises regions of CDR1, CDR 2 and CDR3 that have the same amino acid sequence as the regions of CDR1, CDR2 and CDR3 of one of the dAbs listed in TABLE 1 or in TABLE 3 or that each one differs from the regions of CDRI1, CDR 2 and CDR3 in one, two or three amino acids. For example,
[0067] [0067] A "domain antibody" (dAb) comprises a single variable domain (V, or Vu) domain that is capable of binding specifically and monovalently with an antigen, such as CD40L. For example, a dAb may have a Vu structure, characteristic of a camelid dAb. A "V4 domain" as used here is understood to include a Vu structure. In another embodiment, the Vu "domains (including all characteristics and the combination of features presented as modalities here) are non-V4n domains. The dAbs can form homo- or heterodimers in solution. Although not limited by any particular theory, we believe It is known that the dAbs disclosed here do not cause platelet aggregation, because antibodies containing mutated Fc constructs do not bind to FeyRlla (also known as CD32a) on the platelet surface and do not activate the platelets.
[0068] [0068] As used herein, the term "variable domain" refers to immunoglobulin variable domains defined by Kabat et al., Sequences of Immunological Interest, 5th ed., U.S. Dept. Health & Human Services, Washington, D.C. (1991). The numbering and positioning of the CDR amino acid residues within the variable domains is in accordance with the well-known Kabat numbering convention.
[0069] [0069] The term "human", when applied to antibody polypeptides, means that the antibody polypeptide has a sequence, for example, structural regions and / or CH domains, derived from a human immunoglobulin. A sequence is "derived from" a sequence that encodes human immunoglobulin when the sequence
[0070] [0070] Antibody polypeptides can be administered to human patients while enormously avoiding the antibody immune response often elicited by the administration of antibodies from other species, for example, mice. For example, murine antibodies can be "humanized" by grafting murine CDRs into a human variable domain RF, according to procedures well known in the art. The human antibodies that are disclosed here, however, can be produced without the need for genetic manipulation of a murine antibody sequence.
[0071] [0071] The variable domains may comprise one or more RFs with the same amino acid sequence as a corresponding structural region encoded by a gene segment of human germline antibody. For example, a domain antibody may comprise the Vu DP47, DP45 or DP38 germline gene segments, the WV.DPK9 germline gene segment, the J 'JH4b segment or the J. J.1 segment.
[0072] [0072] Changes in the antibody polypeptide sequences can be made while maintaining the ability to specifically bind to CD40L. Specifically, antibody polypeptides (for example, a dAb) can comprise a variant variable domain that maintains the function of specifically binding to CD40L like dAb BMS2h-572-633. In one embodiment, the variant variable domain can compete with BMS2h-572-633 for specific binding to CD40L. The error-prone affinity maturation, which is disclosed in the examples below, provides an example of a method for the production and identification of antibody polypeptides with variant sequences that specifically bind to CD40L.
[0073] [0073] For example, a variant variable domain can differ from one of the variable domains listed in TABLE 1 and TABLE 3 by up to 10 amino acids or any intermediate integral value, where the variant variable domain specifically binds to CD40L. Alternatively, the variant variable domain can have at least 90% sequence identity (for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) to a sequence listed in this Sequence Listing. Non-identical amino acid residues or amino acids that differ between two sequences can represent amino acid substitutions, additions or deletions. Residues that differ between two sequences appear in the form of non-identical positions, when the two sequences are aligned by any appropriate amino acid sequence alignment algorithm, such as BLAST.
[0074] [0074] In one embodiment, amino acid substitutions can be made in individual FR regions, so that an RF comprises 1, 2, 3, 4 or 5 amino acid differences in relation to the amino acid sequence of the Corresponding FR encoded by a gene segment of human germline antibody. In another embodiment, the variant variable domain can contain one or two amino acid substitutions in a CDR. In other modalities, amino acid substitutions in the FR and CDR regions can be combined. The representative variable domains that specifically bind to CD40L are listed in TABLE 1 and
[0075] [0075] Information regarding the boundaries of the V, or V "domains of heavy and light chain genes can be used to design PCR primers to amplify the variable domain from a heavy chain coding sequence or cloned light encoding an antibody polypeptide known to bind to CD40L The amplified variable domain can be inserted into a suitable expression vector, for example, pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133 -4137) and expressed, alone or in the form of a fusion with another polypeptide sequence, using techniques well known in the art.Based on the disclosed amino acid and polynucleotide sequences, the fusion protein can be produced and purified using just common expertise in any suitable mammalian host cell line, such as CHO, 293, COS, NSO and the like, followed by purification using one or a combination of methods, including chromatography. protein A affinity imaging, ion exchange, reverse phase techniques or similar.
[0076] [0076] In one aspect, the antibody polypeptide is a "dual specific" antibody polypeptide comprising a first variable domain that specifically binds to human CD40L. The dual specific antibody polypeptides comprise a second variable domain that specifically binds to a second antigen that is not human CD40L.
[0077] [0077] In another embodiment, the second antigen can be a cell surface molecule of an immune effector cell or a soluble molecule such as a cytokine, for example. The binding of the dual specificity antibody polypeptide could be used to antagonize CD40L and antagonize a biological activity of the second antigen. The cell surface molecules of effective cells
[0078] [0078] In one embodiment, the antibody polypeptides of a specific dual linker can be linked by an "amino acid linker" or a "linker". For example, one dAb can be fused to the N-terminus of an amino acid linker and another dAb can be fused to the C-terminus of the linker. While amino acid linkers can be of any length and consist of any combination of amino acids, the length of the linker can be relatively short (for example, five or less amino acids) to reduce interactions between linked domains. The amino acid composition of the linker can also be adjusted to reduce the number of amino acids with bulky side chains or amino acids that are likely to introduce secondary structure. Suitable amino acid linkers include, but are not limited to, those up to 3, 4, 5, 6, 7, 10, 15, 20 or 25 amino acids in length. Representative amino acid ligand sequences include (GGGGS), (SEQ ID NO: 12), where n can be any integer between 1 and 5. Other suitable ligand sequences can be selected from the group consisting of AS, AST, TVAAPS (SEQ ID NO: 13), TVA and ASTSGPS (SEQ ID NO: 14).
[0079] [0079] Binding of the second antigen can increase the in vivo half-life of the antibody polypeptide. For example, the second variable domain of the dual specific antibody polypeptide can specifically bind to serum albumin (SA), for example, human serum albumin (HSA). The antibody polypeptide formatted to bind to | it may have an in vivo t-a ("alpha half-life") or t-B ("beta half-life") half-life over the same unformatted antibody polypeptide. Half-lives t-a and t-B measure how quickly a substance is distributed within and eliminated from the body. The connection to | it can be performed by fusing the antibody polypeptide with a second variable domain capable of specifically binding to |, for example. Anti-albumin antibodies in human serum are well known in the art. See, for example, Abcam & O, Human Serum Albumin Antibodies ab10241, ab2406 and ab8940, available on the Internet at the hypertext transfer protocol www.abcam.com/index.html or GenWay, ALB antibody, available on the Internet in the hypertext transfer protocol www.genwaybio.com. The variable domains that specifically link to | they can be obtained starting from any of these antibodies then fused to an antibody polypeptide of the disclosure using recombinant techniques that are well known in the art.
[0080] [0080] Alternatively, binding of the antibody polypeptide to | can be performed by directly fusing the antibody polypeptide sequence with a sequence that encodes | using techniques well known to the person skilled in the art. The strings that encode | they can be obtained by PCR using primers derived from the cDNA sequence available in GenBank Accession No. NMOO00477, for example.
[0081] [0081] In one embodiment, the half-life of the domain antibody composition linked to | is increased by 10% or more. In another embodiment, the half-life of the domain-bound antibody composition | is in the range of 0.25 hour to 6 hours. In another embodiment, the half-life has the domain-bound antibody composition | is increased by 10% or more. In another embodiment, the half-life has the domain-bound antibody composition | is in the range of 12 to 48 hours.
[0082] [0082] In another embodiment, an antibody polypeptide can be formatted to increase its half-life in vivo through PEGUuUlation. In one embodiment, the PEG is covalently linked. In another way, PEG is linked to the antibody polypeptide in a cysteine or lysine residue. In yet another embodiment, the PEG-bound antibody polypeptide has a hydrodynamic size of at least 24 kD. In yet another modality, the total PEG size is 20 to 60 kD, inclusive. In yet another embodiment, the PEG-bound domain antibody has a hydrodynamic size of at least 200 kD.
[0083] [0083] PEGylation can be achieved using various PEG binding groups including, but not limited to, active N-hydroxylsuccinimide ester, succinimidyl propionate, maleimide, vinyl sulfone or thiol. A PEG polymer can be linked to an antibody polypeptide at a predetermined position or can be randomly linked to the domain antibody molecule. PEGylation can also be mediated through a peptide linker linked to a domain antibody. That is, the PEG group can be linked to a peptide linker fused to an antibody polypeptide, where the linker provides the site (for example, a free cysteine or lysine) for PEG binding. Antibody PEGylation methods are well known in the art, as disclosed in Chapman, et al., "PEGylated antibodies and antibody fragments for improved therapy: a review", Adv. Drug Deliv. Rev. 54 (4): 531-45 (2002), for example.
[0084] [0084] Antibody polypeptides can also be designed to form a dimer, trimer, tetramer or other multimer. Antibody polypeptides, for example, dAbs, can be linked to form a multimer by various methods known in the art, including, but not limited to, expression of monomers in the form of a fusion protein, attachment of two or more monomers via a peptide linker between monomers or by chemically joining monomers after translation, with each other directly or via a linker through disulfide bonds or through connection to a di-, tri- or multivalent (for example, a multi-arm PEG). In one embodiment, the multimer can bind to a single CD40 molecule.
[0085] [0085] A pharmaceutical composition comprises a therapeutically effective amount of one or more antibody polypeptides and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers that increase the half-life or effectiveness of the fusion protein. The compositions can be formulated to provide rapid, controlled or delayed release of the active ingredient (s) after administration. Suitable pharmaceutical compositions and processes for preparing them are well known in the art. See, for example, Remington, THE SCIENCE AND PRACTICE OF PHARMACY, A. Gennaro et al., Eds., 21st ed., Mack Publishing Co. (2005).
[0086] [0086] The pharmaceutical composition can also comprise
[0087] [0087] As used herein, a "patient" means an animal, for example, a mammal, including humans. The patient can be diagnosed with an immune disease. "Treatment" or "treating" or "treating" refers to the process that involves relieving the progression or severity of a symptom, disorder, health condition, or disease. An "immune disease" refers to any disease associated with the development of an immune reaction in an individual, including a cellular and / or humoral immune reaction. Examples of immune diseases include, but are not limited to, graft-related disease, inflammation, allergy and autoimmune disease. Autoimmune disease can be selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, diabetes, psoriasis, scleroderma, atherosclerosis, inflammatory bowel disease and ulcerative colitis.
[0088] [0088] Diseases that can be treated by administering the pharmaceutical composition can be selected from the group consisting of Addison's disease, allergies, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune mumps, colitis, coronary heart disease, Crohn's disease, diabetes, including Type 1 and / or Type 2 diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug products (eg, Factor VIl in hemophiliacs), systemic lupus erythematosus, male infertility, multiple sclerosis, myasthenia severe, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroid, transplant rejection and vasculitis and. Autoimmune-mediated health states include, but are not limited to, health states where the affected tissue is the primary target and in some cases, the secondary target. Such health conditions include, but are not limited to, AIDS, atopic allergy, bronchial asthma, eczema, leprosy, schizophrenia, inherited depression, tissue and organ transplantation, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus phenomenon, anaphylaxis, alcohol dependence and drug addiction.
[0089] [0089] The preferred indications for the administration of the present pharmaceutical compositions are, for example, immune thrombocytopenic purpura, systemic sclerosis, myasthenia gravis, allograft rejection and graft-versus-host disease.
[0090] [0090] The pharmaceutical composition can be administered alone or in combination therapy, (ie, simultaneously or sequentially) with an immunosuppressive / immunomodulatory and / or anti-inflammatory agent. Different immune diseases may require the use of specific auxiliary compounds useful for the treatment of immune diseases, which can be determined on a patient-to-patient basis. For example, the pharmaceutical composition can be administered in combination with one or more suitable adjuvants, for example, cytokines (IL-10 and I1L-13, for example) or other immune stimulators, for example, chemokines, antigens and associated peptides
[0091] [0091] For example, the disclosed pharmaceutical composition can be co-administered, concomitantly or sequentially, with a cytotoxic T lymphocyte antigen 4 (CTLAA4) mutant, such as L104EA29Y-lg (belatacept). CTLA4 binds to CD80 (B7-1) and CD86 (B7-2) with greater avidity than CD28 and is transiently expressed on T cells after their activation, when this interrupts the interaction between CD28 and CD80 / 86. Oosterwegel et al., Curr. Opin. Immunol. 11: 294-300 (1999). This creates a negative feedback signal for T cell activation.
[0092] [0092] CTLAA mutant molecules, including L1I04EA29Y-lg, have greater avidity of binding to CD80 / 86 compared to wild-type CTLA4. The intervention of the CD28-CD80 / 86 pathway by L104EA29Y-lg has been successfully adopted, for example, to treat graft-related diseases in transplant models with non-human primates, alone or in combination with other immunosuppressive agents. Larsen et al., Amer. J. Transplant. 5: 443 (2005). U.S. Patent Application number 2010/0166774 describes the structure of L1I04EA29Y-lg, the methods of producing it and a formulation comprising a CTLAA4 molecule; and the patent application is incorporated here by reference. U.S. Patents No.
[0093] [0093] Any suitable method or route can be used to administer the antibody polypeptide or pharmaceutical composition. Administration routes include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration. A therapeutically effective dose of the antibody polypeptide (s) administered
[0094] [0094] The ability of the antibody polypeptides of the disclosure to antagonize CD40L can be tested in one of the several in vitro or in vivo model systems available. Suitable human, animal and cell model systems are described below. Additional cell assay systems are described in the examples.
[0095] [0095] The potential role of CD40-CD40L in the pathogenesis of ITP is reported by Patel et al., British J. Haematology 141: 545-548 (2008). Antiplatelet autoantibodies in patients with ITP bind to circulating platelets and accelerate their destruction. The primary mechanism by which anti-CD40L antibodies are believed to increase platelet count in ITP is by blocking activation based on T-cells of autoreactive B cells that produce anti-platelet antibodies. Anti-CD40L antibodies can also block the expression of CD40L on platelets, thus preventing the self-presentation of platelet glycoprotein antigens to macrophages. In addition, anti-CD40L mAbs inhibit direct interactions between platelet CD40L and other cells, such as cells
[0096] [0096] Patel et al. Demonstrated the efficacy of two humanized monoclonal anti-CD40L antibodies, hu5c8 and IDEC-131, in 46 human patients with chronic ITP refractory to conventional therapies. Patients had a general response rate of 24%, characterized by higher platelet counts. This demonstrated the potential function of CD40-CD40L in the pathogenesis of ITP.
[0097] [0097] The expression of glomerular and tubular CD40 is remarkably regulated further in proliferative nephritis. Several studies have reported CD40L hyperexpression by T cells and high soluble sSCD40L concentrations in human lupus. Kimura et al., Therapeutic Apheriss and Dialysis 9: 64-68 (2005); Vakkalanka et al., Arthritis & Rheumatism 42: 871-881 (1999).
[0098] [0098] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of several autoantibodies and the hyperactivity of B. Grammer et al., J. Clin. Invest. 112: 1506-1520 (2003) report the results of the treatment of patients with SLE with the humanized anti-CD40L mAb 5c8 (BG9588). See also Huang et al., Arthritis & Rheumatism 46: 1554-1562 (2002). Grammer et al. Report that peripheral CD19 + B cells were examined before and after treatment with anti-CD40L mAb. Before treatment, patients with SLE manifested activated B cells that expressed CD40L, CD69, CD38, CD5 and CD27. Activated B cells disappeared from the periphery during and after treatment. Before treatment, patients with active SLE had secreting CD3g cells "" "a" º | H circulating that were not found in individuals.
[0099] [0099] Proliferative lupus glomerulonephritis is a prolonged autoimmune disease with an increasing and decreasing course, characterized by a higher level of anti-dsDNA antibodies, decreased serum C3 concentrations and hematuria. Boumpas et al., Arthritis & Rheumatism 48: 719-727 (2003) report results from an open phase II multicenter study that evaluates the toxicity and efficacy of BG9688, a humanized anti-cCD40L monoclonal antibody, in patients with glomerulonephritis of proliferative lupus. Although the study had to be terminated prematurely because of thromboembolic events occurring in patients on various protocols of BG9588, a short course of treatment with anti-CD40L antibody in patients with proliferative lupus nephritis reduced anti-dsDNA antibodies, increasing the C3 concentrations and reduced hematuria, suggesting that the drug has an immunomodulatory function.
[00100] [00100] Crohn's disease (CD) and ulcerative colitis (UC) are IBDs that are characterized by leukocytic infiltrates in the inflamed intestinal mucosa, which consists primarily of activated CD25 + cells, B cells and macrophages. Ludwiczek et al., Int. J. Colorectal Dis. 18: 142-147 (2003) reported that in CD patients, the levels in the sCD40L plasma were significantly higher than in healthy individuals. In addition, patients with DC with fistulas and / or abscesses had significantly higher levels of SCD40L than patients with uncomplicated DC. It has also been reported that the CDA40-CD40L pathway contributes to the proinflammatory function of intestinal epithelial cells in IBD. Borcherding et al., Am. J. Pathol. 176: 1816-1827 (2010). CD patients also have an increased level of systemic thromboembolism, and the hyperactive state of the platelets of such patients is likely to result from the increased release of sSCD40L as a consequence of its higher endogenous CD40L content. Menchen et al., Gut 58: 920-928 (2009); See also Danese et al., Gut 52: 1435-1441 (2003).
[00101] [00101] Kasran et al., Aliment. Pharmacol. The R. 22: 111-122 (2005) investigated the use of a chimeric human anti-CD40 mMAb ch5D12 to treat Crohn's disease. The mAb was administered to 18 patients with moderate to severe CD in a phase | / lia study with an open dose increase of a single dose. Of the 18 patients, 13 (or 72%) experienced a favorable response to the antibody infusion and 4 patients (or 22%) experienced a remission. Treatment with anti-CD40 mAb reduced the activity of microscopic disease and the intensity of the infiltrate of cells of the lamina propria and the mAb was well tolerated.
[00102] [00102] Rheumatoid arthritis is a systemic autoimmune disease with intra-articular inflammation as a dominant characteristic that affects up to 1% of the population. The disease can be clinically subdivided by the presence or absence of autoantibodies (antibodies to the cyclic citrullinated peptide (CCP) or rheumatoid factor (RF), both of which are highly correlated with each other. Ray-chaudhuri et al., Nature Genetics 40: 1216-1223 (2008) reported that they conducted a meta-analysis of two published studies of the extended genome association (GWA) totaling 3,393 cases and 12,462 controls, in order to identify the risk loci of RA in European populations. genotyped 31 main classification short nucleotide polymorphisms (SNPs) not previously associated with RA in an independent replication of 3,929 post-RA autoantibody cases and 5,807 combined controls from eight separate collections. - mum in the locus of the CDA40 gene, which implied a central function for the CD40 signaling pathway in the pathogenesis of RA.The strong association of the CD40 gene c with susceptibility to RA was robustly repeated in another study in a large UK cohort of 3,962 RA patients. Orozso et al., Ann. Rheum. Dis. 69: 813-816 (2010).
[00103] [00103] An important function of CD40L has also been discovered in the pathogenesis of juvenile idiopathic arthritis (JIA). Prahalad et al., Pediatric Rheumatology 6: 1-8 (2008). IABA is a heterogeneous group of arthropathies of unknown etiology. SSCD40L was found to be significantly elevated in the serum of children with JIA, along with some cytokines. Logistic regression analysis suggested that sCD40L, as well as IL-6 and TNFa, were positively associated with JIA. sSCD40L was elevated in all JIA subtypes, with the highest levels among the most severe subtypes. These results implied sCD40L as a potential biomarker for the treatment and monitoring of patients with JIA.
[00104] [00104] It has also been shown that activated T cells from patients with psoriatic arthritis (PSA) and particularly those with active disease, have a significantly increased expression.
[00105] [00105] Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by fibrous and vascular changes in the skin and internal visceral organs. In a study involving 52 Japanese patients with SSc, serum SCD40L levels were elevated when compared to healthy controls. Komura and others, J. Reumatol. 31: 514-519 (2004). In addition, sSCD40L levels in patients with SSc were higher in patients with systemic lupus erythematosus (SLE) who had elevated sSCD40L levels compared to controls and sSCD40L levels correlated positively with peptide levels. reactive C in patients with SSc. It has also been reported that blocking CD40L with anti-CD40L antibody in cultured T and B cells from patients with SSc inhibited the production of anti-topoisomerase antibody | Kuwana et al., J. Immunol. 155: 2703-2714 (1995). These results suggest that inhibition of CD40-CD40L interactions may be potential therapeutic targets in SSc as well as SLE therapy.
[00106] [00106] Several studies have suggested a function of the CD40-CD40L signaling pathway during atherogenesis. Mach and others demonstrated that in mice, treatment with monoclonal anti-CD40L antibody limited atherosclerosis in mice that lacked a low-density lipoprotein receptor that was fed a high-cholesterol diet for 12 weeks. Nature 394: 200-203 (1998). The antibody reduced the size of atherosclerotic lesions of the aorta by 59% and its lipid content by 79%. Additionally
[00107] [00107] Treatment with anti-CD40L antibody from mice deficient in relation to the low-density lipoprotein receptor during the second half of a 26-week regimen of a high cholesterol diet did not regress, but significantly reduced the additional progression of atherosclerotic lesions established within the aortic arch and particularly the thoracic and abdominal aorta, when compared to the control treatment. Schonbeck et al., Proc. Natl. Acad. Sci. 97: 7458-7463 (2000). In addition, treatment with anti-CD40L altered the atheroma composition in ways believed to favor plaque stability, for example, reduced relative content of macrophages and lipids, as well as increased relative content of smooth muscle and collagen cells. These studies provided support for the importance of the CD40-CDA40L signaling pathway in atherosclerosis and its complications, such as coronary artery disease.
[00108] [00108] Targeting the CD40-CD40L pathway has long been of great interest for preventing rejection of solid organ transplants (SOT), particularly in view of the promising data from several transplant studies published in primates not human. It has been shown that the expression CD40L reduction on CD4 + T lymphocytes activated ex vivo correlates with excellent renal allograft function. Lederer et al., / Nt. Arch. Allergy Immunol. 133: 276-284 (2004). In addition, several studies have shown that anti-CD40L mAbs can both prevent and reverse acute allograft rejection in primates. For example, Kirke others, Proc. Natl. Acad. Sci. USA 94: 8789-8794 (1997)
[00109] [00109] In pediatric human patients who were undergoing acute graft rejection after liver or small intestine transplantation, a correlation was observed between the expression of CD40L on CD8 + T cells and the risk of transplant rejection. Ashokkumar et al., Amer. J. Transplantation 9: 179-191 (2009) and Ashokkumar et al., Surgery 146: 166-173 (2009). Similarly, in adult patients who were undergoing allograft rejection after liver or kidney transplantation, histological analysis demonstrated an association between CD40L expression and acute or chronic rejection. Bartlett and others
[00110] [00110] Several studies support targeting CD40L more than CD40 to achieve better transplant efficiency. For example, graft survival is longer and more durable when CD40L is selectively blocked, compared to CD40. Gilson et al., J. Immunol. 183: 1625-35 (2009). In addition, recent data suggest that blocking CD40L may increase the induction of Tregs and / or suppressor cells to promote graft survival. Garcia et al, J. Clin. Inv. 120: 2486-96 (2010). Also, blocking CD40L, but not CD40, demonstrated the induction of longer-lived immune tolerance resulting in indefinite graft survival, particularly when combined with blocking the B7 pathway. Kenyon et al., Proc. Natl. Acad. Sci. USA 96: 8132-8137 (1999); Kawai et al., Amer. J. Transplantation 4: 1391-1398 (2004); Preston et al., Amer. J. Transplantation 5: 1032-1041 (2005); Adams and others, J. Immunol. 174: 542-50 (2005). The synergy of blocking the CD40-40L and B7-CD28 pathways in improving graft survival is especially important, because it presents the domain antibodies currently disclosed as a natural choice for the combination with belatacept (CTLA4-Ig) for SOT.
[00111] [00111] Chronic and acute graft-versus-host disease (cCGVHD and aGVHD) are complications that can occur after a stem cell or bone marrow transplant in which the transplanted donor cells attack the transplant recipient's body. Acute GVHD in humans occurs within approximately 60 days post-transplant and results in damage to the skin, liver and intestines through the action of cytolytic lymphocytes. Chronic GVHD occurs later and is a systemic autoimmune disease that primarily affects the skin, resulting in polyclonal activation of B cells and the overproduction of Ig and autoantibodies.
[00112] [00112] CD40L-CD40 interactions appear to be critical in the development of both CGVHD and aGVHD. Durie et al., J. Clin. Invest 94: 1333-1338 (1994). In an in vivo mouse model, the anti-CD40L antibodies blocked the following phenomena associated with cG-VHD: splenomegaly, production of polyclonal Ig in vitro, elevated serum IgE levels and anti-DNA autoantibodies in serum and generation cytotoxic anti-host T cells. Antibody production remained inhibited for extended periods of time after the end of administration of the anti-CD40L antibody. In mice with aGVHD, which is associated with the induction of a deep antiallogenic cytotoxic T cell (CTL) response, treatment with anti-CDA40L prevented the generation of H-2b-derived CTL. The results of the study suggest that CD40L-CD40 interactions are critical in GVHD and that CD40L can be a valuable ligand for targeting immunotherapeutic agents to control GVHD.
[00113] [00113] Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent autoimmune disorders caused by autoantibodies against nicotinic acetylcholine receptors (AChR) at the neuromuscular junction of the muscle skeletal. The function of CD40-CD40L in EAMG was shown in mice knocked out compared to CD40L (CD40L - / -). Shi et al., Eur. J. Immunol. 28: 3587-3593 (1998). The mice not cautious about CD40L were completely resistant to EAMG induction and had decreased Th1 and Th2 responses as well as severely impaired T cell-dependent AChR reactive responses.
[00114] [00114] It has also been demonstrated that blocking the signaling of
[00115] [00115] CD40-CD40L interactions on T cells and antigen presentations are important for adaptive immune responses, such as proliferation of B cells, production of immunoglobulin (lg), regulation for more than co-stimulating activity (CD80, CD86 ), production of cytokines and exchange of lg classes. The receptor and the ligand are also expressed on platelets (off-target cell population), where CD40 is constitutively found on platelets, while CD40L is expressed on activated platelets and cleaves sCD40L (> 90% circulating sCD40L are derived from the platelets). Feroni et al., Curr. Med. Chem. 14: 2170-2180 (2007). At least three anti-CD40L monoclonal antibodies (mAb) caused TE in clinical and / or non-clinical studies conducted in non-human primates (NHP). husc8 (BG9588) caused ET in several clinical tests (lupuse kidney transplantation). Boumpas et al., Arthritis & Rheumatism 48: 719-727 (2003). IDEC131 caused ET in a patient in a Crohn's disease test, leading to the completion of the tests in progress at that time. Sidiropoulus & Boumpas, Lupus 13: 391-397 (2004). Both hu5c8 and ABI 1793 (which binds to CD40 in an epitope other than 5c8) caused TE / thrombosis in trans-
[00116] [00116] One hypothesis is that the TE associated with the administration of these antibodies is mediated by the platelet crosslinking mediated by the anti-CD40Lmab-CD40L immune complex (CI), facilitated by the binding of the CI with FcegRlla, a receptor IgG Fc, causing activation and aggregation (FIG. 10). It is therefore expected that blocking the interaction of the IgG Fc cluster with FegRlla will alleviate platelet cross-linking and thrombosis. Approaches and methods developed to assess the risk for TE / thrombosis are described in the Examples below. EXAMPLES
[00117] [00117] TABLE 1 lists representative anti-CD40L VH domain amino acid sequences useful for the disclosed antibody polypeptides. TABLE 2 discloses the representative nucleic acids encoding the VH domain sequences listed in TABLE
[00118] [00118] The following example describes the generation of the 2h line of variable domains of human anti-CD40L, called BMS2h-2 to BMS2h-785. After recombinant expression of a repertoire of variable immunoglobulin domains isolated on the fire surface, selection is carried out by contacting the phage repertoire with immobilized target antigen, washing to remove unbound phage and propagating the phage switched on. This process is often referred to as "mining". This is applicable to the verification of isolated immunoglobulin variable domains, as well as other antibody fragments that can be expressed on a display library, for example, scFv, Fab and Fab ”. Alternatively, the phage can be pre-selected for the expression of folded limb variants properly by "mining" against an immobilized generic ligand (for example, protein A or protein L) that is only bound by folded limbs. This has the advantage of reducing the proportion of non-functional members, thereby increasing the proportion of members that are likely to bind to a target antigen. Pre-selection with generic ligands is taught in WO 99/20749, for example. The verification of antibody libraries on phages is generally described, for example, by Harrison et al., Meth. Enzymol. 267: 83-109 (1996).
[00119] [00119] The verification is commonly performed using purified antigen immobilized on a solid support, for example, plastic tubes or wells or on a chromatography matrix, for example, Sepharose "" (Pharmacia). The verification or selection can also be performed on complex antigens, such as the cell surface (Marks et al., BioTechnology 11: 1145 (1993); Kruif and others
[00120] [00120] Three rounds of selection using decreasing concentrations of antigen (500 nM in round 1; 50 nM in round 2; 50 nM or 5 nM in round 3 depending on the production of the used library) were carried out in parallel against the triple mutant of biotinylated human CD40L monomer (1.2 mol biotin / mol CD40L) (T211E, S222Y, H224K, [108-261] Construction tf7) supplied by Bristol-Myers Squibb. The phage from the Domantis 4G and 6G DAb libraries were combined into groups a) through g) indicated below before the start of the selections:
[00121] [00121] 4GVHCDR3 lengths between 7 - 10 amino acids.
[00122] [00122] 4GVHCDR3 lengths between 11 - 15 amino acids.
[00123] [00123] 4GVHCDR3 lengths between 7 - 15 amino acids.
[00124] [00124] 4GVK
[00125] [00125] 6GVH CDR3 lengths between 7-9
[00126] [00126] 6GVHCDR3 lengths between 10-15
[00127] [00127] 6GVK
[00128] [00128] Each round of selection involved adding the desired concentration of biotinylated CD40L to a mixture of 200 µl of phage (from one of the previously listed naive library groups or subsequent selection of the production phage) and 1000 µl of 2% MPBS (Phosphate-Buffered Saline Solution) containing 2% (w / v) Marvel [Premier Foods, UK] J) and incubating at room temperature for 1 hour using the seesaw mix. The biotinylated antigen phage complex was then captured by adding 100 μL of Dynabeads M-280 Streptavidin [Invitrogen, UK] resuspended for 5 minutes with a seesaw mixer at room temperature. The Dynabeads were then recovered using a KingFisher magnetic separator [Thermo Fisher Scientific, UK] and washed 7 x 1 mL of PBST (PBS containing 0.1% (v / v) polyoxyethylene sorbitan 20 monolaurate [Sigma-Aldrich, UK ]) followed by 1 x 1 mL of PBS. The bound phage retained on the washed Dynabe-ads was eluted by incubating with 500 μL of trypsin-PBS (50 μl of 10 mg / ml of trypsin [Sigma-Aldrich, UK] dissolved in 50 mM of Tris- HCl pH 7.4, 1 mM CaCl, added to 450 µl PBS). The solution containing the phages was recovered and 250 μl were used to infect 1.75 ml of E. coli TG1 in a logarithmic growth phase (at a DOgo, 0.4) for 30 minutes at 37ºC.
[00129] [00129] ELISAs of monoclonal phage were performed after selection rounds 2 and 3. All washes were performed using 3 washes of 250 μl of PBST followed by 3 washes of 250 μl of PBS. The plates were coated throughout night at 4ºC with 50 µl / well of 1 µg / mL NeutrAvidin [Thermo Scientific, UK] in 0.2 M carbonate-bicarbonate buffer, pH 9.4. The plates were washed and then blocked with 2% MPBS for 1 hour at room temperature. The plates were then washed and incubated with 50 µl / well of -> - 1.0 µgm of triple mutant of biotinylated CDA40L monomer in 2% MPBS. The plates were washed and 25 µl / well of phage supernatants were added to an equivalent volume of 2% MPBS and incubated for 1 hour at room temperature. The plates were washed and the bound phage detected with 50 µl / well of anti-M13-HRP conjugate [GE Healthcare, UK] diluted 1: 5000 in 2% MPBS and incubated for 1 hour at room temperature. The plates were washed and the ELISA developed using 50 µl / well of Sure-Blue 1-Component TMB MicroWell Peroxidase solution [KPL Inc, USA]. The colorimetric reaction was stopped by adding an equivalent volume of 1M HCl and the ELISA plate was read at 450 nm. The specific phage was identified by comparison with wells coated with NeutrAvidin, but without a triple biotinylated CD40L monomer mutant. Recovery of dAb genes from Plasmid MidiPrep pDOM4:
[00130] [00130] The dAb V genes from the products of round 3 below were recovered through digestion with pDOM4 phage vector DNA restriction enzyme:
[00131] [00131] 4G VH CDR3 lengths between 7 - 10 amino acids (50 nM antigen concentration).
[00132] [00132] 4G VH CDR3 lengths between 11 - 15 amino acids (50 nM antigen concentration).
[00133] [00133] 4G VH CDR3 lengths between 11 - 15 amino acids (5 nM antigen concentration).
[00134] [00134] 4G VH CDR3 lengths between 7 - 15 amino acids (50nM antigen concentration) ..
[00135] [00135] 4G VK (50 nM antigen concentration).
[00136] [00136] 4GVK (5nM antigen concentration).
[00137] [00137] 6G VH CDR3 lengths between 7-9 (50 nM antigen concentration).
[00138] [00138] 6GVHCDR3 lengths between 10-15 (5 nM antigen concentration).
[00139] [00139] Approximately 20 µg of MidiPrep [Qiagen, UK] DNA was digested with Sa / le Notl as follows: 20 µl of DNA (1 µg / uL) was mixed with 1.5 µl of Sa / l (20 U / uL) [NEB, UK] and 3ul Notl (10 U / uL) [NEB, UK], 41 / L Buffer 3 [NEB, UK], 0.4 uL BSA (10 mg / mL) [NEB, UK] and tissue culture grade water [Sigma, UK] added up to 40 ul. The samples were incubated for 5 hours at 37ºC in an air incubator after which the digested dAb genes were isolated by running the dilution mixture on a 2% agarose gel [E-gel, Invitrogen, UK] , the appropriate DNA strands were cut and cleaned using a PCR purification kit [Qiagen, UK]. The purified V genes were ligated into a pDOMS expression vector with double digestion with Sall and Notl. Soluble daAb ELISA:
[00140] [00140] The dAbs that bind have been identified as follows.
[00141] [00141] Unique dAbs were identified by sequencing the DNA of ELISA positive clones. The unique dAbs were expressed as follows in bottles with 250 ml baffles, to which it was added:
[00142] [00142] 50mL of Terrific Broth [Sigma-Aldrich, UK].
[00143] [00143] 100 µg / ml carbenicillin [Sigma-Aldrich, UK].
[00144] [00144] 1got of defoamer A204 [Sigma-Aldrich, UK].
[00145] [00145] “Novagen Overnight Express Autoinduction Kit [Novagen, UK].
[00146] [00146] A bacterial scrape from a fresh confluent 9 cm diameter agar plate or from a glycerol stock of the desired dAb clone was used to inoculate the Broth Terri fi c, then the vial was sealed with Milliwrap PTFE membrane [ Milpore, UK] and incubated for 48 h, shaking at 250 rpm at 30ºC. The bacterial culture throughout the night was clarified by centrifugation and the HV or VK dAb purified using Streamline Protein A [GE Healthcare, UK] or Protein L agarose [generated on site] respectively.
[00147] [00147] dAbs inhibitors were initially identified by checking for purified dAb in a CD40L receptor-sphere binding (RBA) assay. Sphero polystyrene streptavidin spheres (0.5% w / v, 6.7 µm in diameter) [Saxon, Europe] were prepared and washed according to the manufacturer's instructions. The beads were then pelleted at 11,600 g for 1 minute, the supernatant discarded and the beads resuspended in 1 ml of PBS using a vortex. The washing step was repeated two more times, the supernatant was discarded and the spheres were resuspended in 1 ml (0.5 mg / ml) of biotinylated human IZ-CD40L in PBS and incubated overnight at room temperature. environment with rotating seesaw movement. After incubation, the beads were pelleted and washed three times with 1 ml of PBS as before and then resuspended in 0.5 ml of PBS containing 0.1% bovine serum albumin (BSA). The antigen-coated beads were then diluted 1:10 in PBS containing 0.1% BSA before use. The reagents for the RBA assay were added as follows to duplicate wells on an FMAT plate with a transparent bottom with black on the sides of 384 wells [Applied Biosystems, UK]:
[00148] [00148] 12.5 µL of dAb protein or buffer control. The initial concentration of dAb titration was typically 10 µM (final concentration) which was diluted 1: 3.3 (ie, 30 µl of sample added to 70 µl of PBS containing 0.1% BSA ) to produce an 8-point titration effect curve.
[00149] [00149] 12.5uUL CD40-Fc [rovided by Bristol-Myers Squibb, USA; batch CY24Feb06-1] at 0.2 ug / mL (for a final concentration of
[00150] [00150] 12.5 µL of human mouse antidcFc mix [Sigma-Aldrich, UK] at 2 µg / mL (for a final concentration of 0.5 pg / mL) and Alexa Fluor 647 goat anti-mouse [Invitrogen, UK] a1ugml (to a final concentration of 0.25 µg / mL) diluted in PBS containing 0.1% BSA.
[00151] [00151] 12.5 μl of spheres coated with IZ-CD40L described previously were added in the center of the well so that they did not spread to the edge of the well.
[00152] [00152] After adding the reagents to the 384-well plate, it was incubated at room temperature for 6 hours in the dark and then read on an AB8200 FMAT system [Applied Biosystems, UK]. Example 2 Selection of dAb for Clone BMS2h-572
[00153] [00153] Three rounds of selection using decreasing concentrations of antigen (300 nM in round 1; 30 nM in round 2; 3 NM in round 3) were performed in parallel against biotinylated human leucine-CD40L zipper (IZ-hCD40L) (1.42 mol of biotin / mol of trimer) supplied by Bristol-Myers Squibb. The phage from the dAb naive 4G and 6G Domantis libraries were combined into groups a) to h) indicated below before starting the selections:
[00154] [00154] 4GVH CDR3 lengths between 7 - 9 amino acids.
[00155] [00155] 4GVHCDR3 lengths between 10 - 12 amino acids.
[00156] [00156] 4GVHCDR3 lengths between 13 - 15 amino acids.
[00157] [00157] 4GVK
[00158] [00158] 6GVHCDR3 lengths between 7-9
[00159] [00159] 6G VH CDR3 lengths between 10-12
[00160] [00160] 6G VH CDR3 lengths between 13-15
[00161] [00161] 6GVK
[00162] [00162] Each round of selection involved adding the desired concentration of biotinylated CD40L to a phage mixture (from one of the previously listed naive library groups or subsequent selection production phage) in 1000 µl of 2% MPBS (So - Phosphate-Buffered Saline solution containing 2% (w / v) of Marvel [Premier Foods, UK]) and incubation at room temperature for 1 hour using the seesaw mix. The biotinylated antigen phage complex was then captured by adding 100 μl of resuspended Dynabeads M-280 Streptavidin [Invitrogen, UK] (rounds 1 and 3) or 50 μl of M-280 tosylactivated Dynabeads (Invitrogen) that were coupled with NeutrAvidin [Thermo Fisher Scienti fi c, UK] (round 2) and incubated for 5 minutes with a seesaw mix at room temperature. The Dynabeads were then recovered using a KingFisher magnetic separator [Thermo Fisher Scientific, UK] and washed 7 x 1 mL of PBST (PBS containing 0.1% (v / v) 20 polyoxyethylene sorbitan monolaurate [Sig- ma-Aldrich , UKJ) followed by 1 x 1 ml PBS. The bound phage retained on the washed Dynabeads were eluted by incubation with 500 µL of trypsin-PBS (50 µl of 10 mg / ml of trypsin [Sigma-Aldrich, UK] dissolved in 50 mM Tris-HCI pH 7.4, 1 mM CaCl, added to 450 µl PBS). The phage-containing solution was recovered and 250 μl was used to infect 1.75 mL of E. coli TG1 in a logarithmic growth phase (at a DOo of 0.4) for 30 minutes at 37ºC. The culture infected with E. coli TG1 phage was centrifuged at 11,600 g in a microcentrifuge for 1 minute and the resulting cell pellet was resuspended in 1 ml of 2xTY (16 g of Tryptone, 10 g of Yeast Extract and 5 g NaCl in 1 liter, autoclaved for 15 minutes at 121ºC) and plated on a 9 cm Petri dish containing TYE medium supplemented with 15 pug / mL tetracycline.
[00163] [00163] Monoclonal phage ELISAs were performed after selection rounds 2 and 3. All washes were performed using 3 washes of 250 μl of PBST followed by 3 washes of 250 μl of PBS. The plates were coated throughout night at 4 ° C with 50 µl / well of 1 µg / mL of IZ-hCD40L in PBS. The plates were washed and then blocked with 2% MPBS for 1 hour at room temperature. The plates were washed and 25 µl / well of phage supernatants was added to an equivalent volume of 2% MPBS and incubated for 1 hour at room temperature. The plates were washed and bound phage was detected with 50 µl / well of anti-M13-HRP conjugate [GE Healthcare, UK] diluted 1: 5000 in 2% MPBS and incubated for 1 hour at room temperature. The plates were washed and the ELISA was developed using 50 µl / well of Sure-Blue 1-Component TMB MicroWell Peroxidase solution [KPL Inc, USA]. Area-
[00164] The dAb V genes from the products of rounds 2 and 3 were recovered by digesting with restriction enzymes Sall and Notl from the pDOMA4 phage vector and ligated into a pDOMSB expression vector double-digested with Sa / l and Notl. Soluble dAb ELISA:
[00165] [00165] The binding dAbs have been identified as follows. Ninety-six individual colonies containing cloned dAb V genes within the soluble dAb expression vector pDOMS5 were selected from each production in 200 μL of Terrific Broth containing OEx nEx Autoinduction medium [Novagen, UK] and incubated throughout overnight at 37º C with agitation at 250 rpm in Costar 96 Well Cell Culture Clusters [Corning Incorporated, USA] sealed with a gas-permeable adhesive plastic strip. The cultures were centrifuged to pellet the supernatant cells and analyzed by ELISA with antigen binding to dAbs that bound to IZ-hCD40L. 96 wells MaxiSorp 96 [Nunc, USA] were coated overnight at 4 ° C with 50 µl / well of 1 µg / ml of IZ-hCD40L in PBS. All washes were performed as described for the phage ELISA. The plates were blocked for 1 hour at room temperature with 200 µl of PBS containing 1% Tween 20. The ELISA plate was washed and the culture supernatant containing dAb was clarified by centrifugation at 1,800 g for 10 min at 4ºC, then added to the ELISA plate (30 uL / well) to which an equivalent volume of PBST was added. The plates were incubated for 1 hour at room temperature.
[001668] [001668] The dAbs BMS2h-503, BMS2h-719 and BMS2h-572 were submitted to error-prone affinity maturation to generate the lines BMS2h-503, BMS2h-719 and BMS2h-572, respectively. This was performed using random mutagenesis in which on average 3.6 amino acid changes were introduced by dAb. The phage libraries (mean size of 6x10º) were selected using biotinylated monomeric and trimeric human CD40L with alternating streptavidin / neutralravidine antigen bead capture (as described). Three rounds of selections using decreasing antigen concentrations (100 nM in round 1; 10 nM in round 2; 1 NM in round 3) were performed. Sequencing was used to monitor diversity after each round of selection. The selection productions (round 2 selected in the trimer from CD40L to BMS2h-572;
[00167] [00167] The dAbs BMS2h-572-6, BMS2h-503-1 and BMS2h-719-2 were cloned into the vector pPDOM38 containing Fc tail derived from human I9G1 to create DMS0502, DMS0500 and DMSO0501, respectively.
[00168] [00168] OdAbBMS2h-572-6 was submitted to the affinity maturation
[00169] [00169] Library 1 - 5 wastes in the diversified CDR1
[00170] [00170] Library 2 - 6 residues in the CDR 2 diversified
[00171] [00171] Library3-13 wastes in the diversified CDR2
[00172] [00172] Library 4 - 7 wastes in the diversified CDR3
[00173] [00173] In each library, diversification was performed using nnS codons where n maintained a large fraction of the original base (85%) and separation from the rest between the equimolar amounts of the remaining three bases (5% each) and S represents G or C. The phage libraries (mean size 8x10º) were selected using biotinylated monomeric and trimeric human CD40L with alternating antigen streptavidin / neutravidine spheres (as described). Libraries 2 and 3 were brought together during the selection process. Three rounds of selections using decreasing antigen concentrations (50 nM in round 1; 5 nM in round 2; 1 NM in round 3 with a 200-fold excess of the competitor - non-biotinylated CD40L trimer). Sequencing was used to monitor diversity after each round of selection. The selection productions (rounds 2 and 3) were subcloned into the soluble expression vector pDOM13 (without C terminal tag) (as described) and verified in the form of supernatants of monoclonal bacterial microculture by BlAcore in relation to the improved rate dissociation compared to that of parental clones on CD40L both monomeric and trimeric. The enhanced variants identified had the DNA sequenced and the unique dAbs were expressed, purified and then analyzed using the BMS2h RBA beads as well as assays controlled by cellular CD40L (as described). As a result, the dAbs BMS2h-572-608, BMS2h-572-614 and BMS2h-572-619 have been identified.
[00174] [00174] Sequence analysis revealed that all amino acid differences between BMS2h-572-608 and the parental dAb BMS2h-572-6 were located on CDR1 and the differences between BMS2h-572-614 and parental dAb BMS2h-572-6 were located on CDR3. Both matured dAbs shared CDR2 with the parental dAb BMS2h- 572-6. This created an opportunity to build a combination mutant that had CDR1 of BMS2h-572-608 and CDR3 of BMS2h-572-
[00175] [00175] OdAb BMS2h-572-633 was cloned into the vector pDOM38 containing Fc tail derived from human IgG1 to create DMS0507. The construct was transiently expressed in HEK293 cells and the protein was purified using Protein A. The purified Fc fusion was analyzed by Biacore for binding to monomeric and trimeric CD40L as well as in various cell assays (as described). Example4 Cellular CD40L Activity Assays
[00176] [00176] The anti-human CD40L dAbs have been functionally analyzed for their ability to antagonize CD40L activities. The CD40L activities tested were cell proliferation and cytokine production through hCD40L-controlled activation of primary monocyte-derived dendritic cells (DCs). Unless otherwise noted, all assays were performed in RPMI medium supplemented with 10% fetal calf serum (FCS). The results of several assays, described in detail below, are shown in TABLE 5 and TABLE 6. Proliferation of primary human B cells controlled by soluble IZ-hCDA40L:
[00177] [00177] 1x10 th human amygdala B cells were incubated with 0.6 µg / ml of IZ-hCD40L along with variable titration of dAb or mMAb in a final volume of 200 µL / well in a rounded 96-well bottom plate. The plates were incubated at 37ºC for 72 hours after which thymidine (º H; 0.5 pci / well) was added for 6 hours. B cell proliferation was quantified based on the incorporation of thymidine. All tests, unless otherwise stated, were performed in RPMI medium supplemented with 10% fetal calf serum (FCS). Proliferation of primary human B cells controlled by CHO-hCDA40L:
[00178] [00178] CHO cells were transfected with human CD40L to break a stable cell line expressing high levels of CD40L on the cell surface. CHO-CDA40L cells were irradiated at 10,000 Rads before incubation with human B cells. 1x10º human B amygdala cells were incubated with 1x10º CHO-CDA40L cells (1: 100 ratio of CHO-CD40L: human B cells) together with the variable titration of dAb or mAb in a final volume of 200uL / well in a 96-well rounded bottom plate. The plates were incubated at 37ºC for 72 hours after which thymidine ( H; 0.5 pci / well) was added for 6 hours. B cell proliferation was quantified based on the incorporation of thymidine. All tests, unless otherwise stated, were performed
[00179] [00179] T cells were isolated from human peripheral blood mononuclear cells (PBMCs) and enriched using goat red blood cell (SRBC) affinity. The enriched human T cells were cultured with PM-LCLs (B cell line transformed with EBV; irradiated at 10,000 Rads) in a ratio of 5: 1 (T: LCL) for 6 days at 37ºC to generate a cell population Allogeneic tees. On day 6, the expanded T cells were isolated and irradiated at 3000 Rads and then cultured (5x10º T cells / well) with primary human amygdala B cells (1x10º B cells / well) in a ratio of 1 : 2 in a 96-well flat-bottom plate coated with anti-CD3 mAb (OKT3). Variable titers of dAbs / mAbs were added to each well; the final volume in each well was 200 µL. The test plates were incubated at 37ºC for 3 days. The proliferation of human B cells was determined by adding thymidine (º * H; 0.5 pci / well) to the cultures over the past 18 hours. All tests, unless otherwise stated, were performed in RPMI medium supplemented with 10% fetal calf serum (FCS). In some cases, the supernatant was collected and measured for the presence of IL-6. CHO-hCD40L controlled activation of dendritic cells (DCs) derived from primary human monocytes:
[00180] [00180] Human PBMCs were enriched in relation to monocytes through the elimination of T cells via SRBC restoration. The monocyte-enriched PBMCs were grown with 10 ng / ml GM-CSF and 5 ng / ml IL-4 in 6-well plates for six days at 37ºC. The cultured plates were replenished with fresh medium (with GM-CSF and IL-4) on days 2 and 5. Immature DCs were used in the cell assays on day 6. 8x10º immature DCs were cultured with 4x10º CHO-hCD40L cells (irradiated 10,000 Rads) along with variable titers of dAbs / mAbs on a 96-well flat-bottom plate.
[3] [3] | : Ss = + + "ne. O:: |) o +": | : So + | o | =) + H GS à:: o o + + + + o o - - 3 3 & e AR sl Ss are à | à + "o F S ê FT = | o 2 & S $ z 7" = 8 e & F | : = | | o + H "2 e 3 | u à +" o e 2 is Ss + SIS a À ++ cs o + 8 ee Ss ss) 3 GS 8 d + n <& $ and o 5 Sã S | Ss S o ã a Ss:: Ss à + + gs | S $ 2 4 to + H Ss | ] | : e 2 É + - ss: | ::::: - o o S | + o | z: ê: - + "e Ss SS:: õ: - S s 8 o SS 8 2 3 à a: 8 2 à É + Sl = | ê 3 z & à +" 8 = = go: 2 2 o: | : | : the S | e + "s S 3 S K ú e o - | S 2 | Z +" S 7 3 to 2 8 + + Y Ss 3 & 2 É + "+" S SS: S | Z + s: 3 à & ++:: | - angel 3 + "Ss F S ê ED e 2 2 3:: =) and à ê 3 3 o - Ss 8 2 ê à:: à 8 + gs; | S ê à | É + +" = 1 S | à 1 + Za: ê 2 ê n + "- | Ss oO ole </ 22/22 ES &, L q Sel Ss PE ST Se 5 | à Sl a çççE 8 2 3 2) tz 25 2nd 2a 8s & s 7 o -; SS SS only 28 g) 2 If sl 3 m ES SS are Io Sound E. Tx ol K ES O SS 5 of SE: 8 ES 6º Sound of Ss SE 32 o le RS SsS2 Ss Ss Om ES 32) 8th Ss sz) SS 53) 53 SS IF If Ss AS s Ss If) ss in 1st action NL so Ns a Ss ê 2 3 ê Fra 53 E Ss S and Ss & | S a Ss Ss a
[00181] [00181] BMS-986004 is a dimeric fusion protein, composed of a modified IgG1 Fc fragment linked to the C-terminus of dAb BMS2h-572-633. Surface plasmon resonance (SPR) was used to characterize the kinetics and affinity of BMS-986004 or the binding of the monovalent component domain antibody BMS2h-572-633 to CD40L. The values of BMS-986004 were compared with those for the reference antibodies 5c8-l9G1 and 5c8-CT and FAB of the monovalent component of 5c8. The SPR experiments used an hcD40L construct containing an N-terminal isoleucine zipper motif (IZ-hCD40L) that facilitates the specific assembly of the CD40L molecule in the native trimeric form. A biotinylated version of IZ-hCD40L (biot-IZ-hCD40L) with equivalent binding activity was also used for some SPR experiments.
[00182] The monovalent domain antibody BMS2h-572-633 binds biot-IZ-hCD40L with a Kd of 7.8 nM, compared to an affinity of 5.4 nM for the monovalent FAB fragment of 5ce8, TABLE 7. Due to the fact that BMS-986004 is bivalent and the IZ-hCD40L target is trivalent, SPR binding data is influenced regardless of whether the CD40L target is on the surface of a chip or in solution. To estimate binding affinity influenced by avidity, the SPR data for the binding of BMS-986004 to a biot-IZ-hCD40L surface was adjusted for a 1: 1 Langmuir model, suggesting a dissociation constant of less than 1 nM, TABLE 7. Similar results were obtained for 5c8-IgG1 and 5c8-CT. TABLE 7 Kinetics and affinity values of IZ-hCD40L that are determined
[00183] [00183] The value is influenced by the greed due to the bivalence of the analyte.
[00184] [00184] AFIG.5 shows SPR sensorgram data for the 12.5-0.39 nM connection of BMS-986004 (2: 1 dilution series) with biot-IZ-hCD40L captured on a sensor chip SPR with strepavidin at 25 ° C. The colored lines show the sensor data with double reference and the black lines show the 1: 1 Langmuir adjustment to the data, with an apparent Kd value influenced by the avidity of 0.11 nM.
[00185] [00185] The affinity and thermodynamics of the binding of BMS-986004 to CD40L were also characterized in solution using isothermal titration calorimetry (ITC) at temperatures ranging from 15 37ºC. These data suggested the presence of several thermodynamically different binding modes (FIG. 6) with Kd values for different modes beyond the high affinity detection limit (Kd <2 nM) (TABLE 8), consistent with the data of SPR. The affinity of the 5c8 FAB monovalent FAB fragment for IZ-hCD40L which is determined by ITC (3.5 nM) was also consistent with the value determined by SPR.
[00186] [00186] The Fc domain of BMS-986004 (called "CT") was engined starting from a wild-type IgG1 Fc domain to maintain the ability to bind to FCRn, but to interrupt the binding to Fcy. To confirm that the engineered molecule has the binding profile of the desired Fc receptor, the binding affinities of BMS-986004 for human FcRn and human Fcy receptors CD64 (FcyRI)) CD32a (FcyRlla),) CD32b / c (FeyRllb / c), CD16a (FeyRllla),) CD16b (FeyRlllb) were measured using SPR, compared to 5c8-lgG1 and 5c8-CT. For these experiments, BMS-986004 was captured through the domain antibody domains on a biot-IZ-hCD40L sensor surface and the soluble Fc receptor proteins were tested for binding as an exposed Fc domain . Similarly, 5c8-l9G1 and 5c8-CT were captured on a biot-IZ-hCD40L surface through the FAB domains, with binding to the soluble FcR.
[00187] [00187] BMS-986004 bound to FcºRn with Kd of 670 nM at pH 6.0 which is the relevant pH for binding within the endosome, TABLE 9. However, binding was significantly reduced (Kd> 5000 nM) in Neutral pH suggesting the efficient release of FCcRn under these conditions. BMS-986004 bound to CD64 with a Kd of 0.6 nM and had a statistically weak affinity for CD32a, CD32b / c, CD16a and CD16b (Kd> 3000 nM). Both 5c8-lgG1 and 5c8-CT had an affinity for FcRn similar to that of BMS-986004. 5c8-CT, which has the Fc "CT" region identical to that of BMS-986004, also had FcyR-like properties similar to those of BMS-986004, while 5c8-lgG1, which has a wild-type IgG1 Fc domain, bound more strongly to the FcyRs, TABLE 9. TABLE 9 Affinity to the Fc receptor that is determined using SPR (Biacore). BMS Kd- 5c8-lgG1 Kd 5c8-CT Kd 986004 (nM) (nM) (nM) [FR and 6 are 720 hFeRn [DR TI | es - 5 | 6:05 |
[00188] [00188] Connection of CD32a to 5c8-lg9G1 was biphasic. Kd was estimated as —-10 ”M based on the steady state adjustment to the dominant link wind. This Kd is in the KD range reported in the literature for the binding of CD32a to IgG1. Example 6 Cell Based / n-Vitro Assays
[00189] [00189] The potency of BMS-986004 has been evaluated in several primary immune cell assays to ensure robust potency across different cell types. Primary human B cell proliferation assays were conducted in two ways, which are described in detail in Example 4 above: (1) the recombinant CD40L trimer was used to control B cell proliferation; and (2) CHO cells expressing CD40L on the membrane (CHO-CD40L) were used to induce B cell proliferation. The usefulness of CHO-CDA40L cells was particularly important to ensure that signals from CD40L bound to membrane were inhibited equally well when compared to the soluble CD40L trimer. CHO-CD40L cells were also used to control the activation of differentiated primary human DCs from the monocyte culture derived from PBMCs in the presence of GM-CSF and
[00190] [00190] A receptor occupation method was developed to measure the involvement of the CD40L target by BMS-986003 in whole blood samples from cynomolgus monkeys and, subsequently, in BMS-986004 in human whole blood samples. BMS-986003 is a dAb that shares the same amino acid sequence as BMS-986004, except for a non-native glycine residue at its amino terminus.
[00191] [00191] Occupancy is measured on CD4 + T cells by flow cytometry using an anti-CD40L mMAb that competes for binding to CD40L with BMS-986003 / BMS-986004 and cross-reacts with human and cynomolgus monkey CD40L . In the presence of bound dAb, the anti-CD40L detection mAb is blocked from binding to CD40L in a concentration-dependent manner, providing a measure of target occupation. Whereas basal CD40L is expressed at low levels on resting T cells in the blood
[00192] [00192] The target binding potency in whole blood for BMS-986003 and BMS-986004 is closely correlated between the human being and the cynomolgus monkey. ECso values for BMS-986003 and BMS-986004 are also similar when linked to baseline CD40L and induced by PHA. In addition, these values are comparable to those obtained in human cell-based in vitro assays (see TABLE 10). Based on the measured ECo values, complete saturation of the target in peripheral blood should be achieved in concentrations <10nM.
[00193] [00193] To support the preclinical PK / PD profile of BMS-986003 and BMS-986004, RO was evaluated both in the study with KLH in cynomolgus monkey (immunization with lockhole lobe hemocyanin) with BMS -986003 and in the IV binding study with BMS-986004. Additional details of these findings can be found in the Examples below. Example 8 In vivo pharmacology
[00194] [00194] To show the effectiveness of a CD40L dAb in mouse disease models, a 2m126-24 mouse CD40L dAb was formatted with mouse IgG1 Fc with the D265A point mutation to further decrease the effector function of Fc. This 2m126-24-Fc mouse substitute dAb shows potency comparable to that of BMS-986004 and MR-1, a hamster mouse anti-CD40L antibody (TABLE 12). TABLE 12 Comparison of the In vitro EC50 (nM) Assay Power - EC50 (nM) Assay of the EC50 Assay (nM) of Ps-rm Ta O DA with CHO-CD4OL Human Being Sc8 8.0 3.0 20+ 20 BMS-986004 5.0 +0.5 1.0 +0.5 1.9 + 0.6 Mouse Í 2m126-24-Fc 4.7 +0.9 0.4 +0.06 0.5 + 0.2 MR-1 (mAb) 1.7 +04 0.6 +0.2 0.6 +0.3 KLH-Induced Antibody Response Inhibition by Mouse CDA40L dAb
[00195] [00195] Females of BALB / c mice received intraperitoneal injection (ip) of 250 ug of KLH on day 0. The mice were dosed subcutaneously (sc) with MR-1 or BMS-2m- 126-24 -Fc at the doses indicated on day -1 and day 6. Blood was collected and serum was analyzed for anti-KLH IgM on day 7 and IgG on day 14 by ELISA. The serum from BALB / c mice collected on day 14 after immunization with KLH was pooled and used as a positive comparator and the data are expressed as a proportion of the test serum titer in relation to the BALB serum titer. / c grouped. As shown in FIG. 7, BMS-2m-126-24-Fc de-
[00196] [00196] Males of SJL / J mice received intrathecal administration of 2.5 mg of Trinitrobenzene sulfonic acid (TNBS) in 50% EtOH through a catheter inserted 4 cm away from the anus. The mice were dosed once s.c. with MR-1 or BMS-2m-126-24-Fc at the doses indicated 4 hours before TNBS injection. FIG. 8 shows the changes in mean body weight and the percentage of survival in groups of mice treated with PBS / IgG or varying dose levels of MR-1 or dAb. Abatacept was used as a positive control (20 mg / kg, i.p. on alternate days). A typical profile of TNBS-induced colitis was shown in the IgG control group: loss of body weight, with maximum on day 3-4; colitis-related death occurring on day 3 and later; and mice that survived showing signs of recovery after day 4. Treatment with the CDA40L dAb or antibody (both tested at 2, 8 and 20 mg / kg) caused dose-dependent inhibition of body weight loss and the increase in the survival rate; both compounds at 8 mg / kg produced a degree of efficacy that is comparable to that of Abatacept at 20 mg / kg. In conclusion, the mouse CD40L dAb BMS-2m-126-24-Fc demonstrated efficacy comparable to that of anti-CD40L antibody MR-1 in a model of acute TNBS-induced colitis. Synergistic effect between CTLA4 Ig and CD40L dAb in mice - "Heart-to-Ear" Transplantation Model in Mice
[00197] [00197] Heart grafts from neonatal C57BI / 6 mice (48-72 h) were implanted into the subcutaneous pouch created in the ear auricles of BALB / c mice. The mice were treated with CTLA4-Ig (ip 2x / (week), BMS-2m126-24-Fc (sc 1X / week) or a combination of both at the indicated doses, with the first dosage starting the day before transplantation. The rejection time was defined by the absence of cardiac contractility for three consecutive days, which is evaluated daily by the allograft electrocardiogram (ECG) device, as expected, without any treatment, the C57BL / 6 mice that received the heart from Neonatal BALB / c rejected the graft immediately afterwards, with an average survival time (MST) of 12 days, monotherapy with 3.20 mg / kg dAb or 25 mg / kg CTLA4-Ig had no or little impact on prolongation of allograft survival (MST: 12, 15 and 13 days respectively). However, in groups treated with a combination of 20 mg / kg dAb and 25 mg / kg CTLA4-lg, graft survival was significantly prolonged exhibiting 35-day MST (FIG. 9). These data provided fundamentals the logical for the combination of CD40L dAb with belatacept in human kidney transplant patients. Studies of future transplants in non-human primates will further define the level of disease and assess the potential effect on induction of tolerance with CD40L dAb BMS-986004. Example9
[00198] [00198] Several in vivo studies have been conducted to characterize the PK and PD of BMS-986004, BMS-986003 and a mouse CD40L-Fc dAb substitute BMS-2m-126-24-CT, in the non-clinical setting co. The main findings are summarized below.
[00199] [00199] Bioanalytical methods based on the enzyme-linked immunosorbent assay (ELISA) were developed to support PK studies, acute and chronic efficacy studies in mice and exploratory PK / PD studies using cyano-molgus monkeys . In all cases, whole blood was obtained and plasma was prepared in the presence of EDTA, the samples were then subjected to ELISA analysis.
[00200] [00200] BMS-986004 plasma concentrations were measured with an ELISA assay that used the human CD40L antigen to capture the analyte from the test samples. The test samples were thawed at 4ºC, mixed well and diluted 1: 100 in the assay diluent composed of 1x PBS, 0.05% Tween-20 and 1% BSA (PTB). Subsequent sample dilutions were made using 1% normal monkey plasma / PTB as a diluent. This allowed the test analyte to be analyzed at various dilutions (10º - 10%) while the sample matrix was maintained at 1%.
[00201] [00201] Recombinant human trimeric CDA40L was obtained from Protein Structure and Science (PSS), LVL and was attached to 96-well plates at a final concentration of 2 µg / mL. Test samples, quality control (QC) samples and standards were detected with affinity-purified rabbit heavy chain domain (Vh) polyclonal antibody (Covance Research Products, Denver, PA ) diluted to a concentration of 0.25 µg / ml in
[00202] [00202] The performance of the QC samples, measured by deviating the calculated concentration from its nominal value, indicated that the reference material was stable in pure monkey plasma at concentrations of 30 - 1000 ng / mL when stored at -70º C for more than 2 months.
[00203] [00203] The CD40L-specific dAb of mouse BMS-2m-126-24-CT was measured in mouse plasma samples to provide exposure data in support of several acute and chronic efficacy studies as well as in PK assessment.
[00204] [00204] Although the assay format for mouse dAbs was quite similar to that for human dAbs in Monkey samples, there were some differences. The mouse plasma matrix was diluted 1:10 (10%) in the assay diluent and all subsequent dilutions of the test samples were made using 10% mouse matrix. Similarly, all standards and QCs were also incubated on ELISA plates in 10% of mouse plasma. The concentration of BMS-2m-126-24-CT in the test samples from the mice was measured using mouse CDA40L to capture the analyte. Since the mouse dAb has a Vk structure, all test samples, COQs, and standards were detected with affinity purified rabbit kappa polyclonal antibody (Vk) (Covance Research Products, Denver, PA ) diluted to a concentration of 0.5 µg / ml in PTB. The rest of the assay and the analysis procedure were similar to the procedure for the analysis of human CD40L dAbs. The approval criteria for retro-calculated concentrations of standards and QCs were also similar to those for human CD40L dAbs. The quantitative range of BMS-2m-126-24-CT that is determined from the standard curve was 12.5 to 600 ng / mL in the pure sample matrix. Non-Clinical Pharmacokinetics
[00205] [00205] TABLE 13 summarizes the PK parameters for BMS-986004, BMS-986003 and BMS-2m-126-24-CT in non-clinical animal species. TABLE 13 Single-dose PK parameters (mean + SD) from two species of non-clinical animals Species | dAb Rota Dose | Cmax | Tmax | AUCO-inf | T1 / 2 CLTp Vss F Eee STE TES) EEE and es eos e
[00206] [00206] BMS-986004 and BMS-986003 exhibited comparable PK profiles in monkeys (FIG. 11A and FIG. 11B). After | V administration, plasma concentrations of BMS-986004 and BMS -986003 exhibited a biexponential decline of up to 504 and 408 h, respectively. Accelerated elimination was observed thereafter in 50% of monkeys registered in both studies. Testing for immunogenicity of plasma samples collected 38 d after treatment with BMS-986004 suggested that all monkeys developed anti-drug antibody (ADA); and that monkeys with higher ADA levels required faster elimination. Although no immunogenicity test has been conducted for the PK IV study with BMS-986003, a similar level of immunogenicity was observed in monkeys after subcutaneous dosing with BMS-986003 in the PK / PD study, suggesting that both the proteins were immunogenic in monkeys. The terminal half-life (T1 / 2) of 124 and 106 h for BMS-986004 and BMS-986003 was therefore determined using the exposures collected up to two weeks (336 h) only. The volume of distribution at steady state (Vss) of BMS-986004 and BMS-986003 was 0.098 and 0.074 L / kg, respectively. The values are greater than the volume of the plasma (0.06 L / kg) but less than the volume of the extracellular fluid (0.2 L / kg), suggesting that the proteins reside largely in the extracellular space. The elimination of total body plasma (CLTp) of BMS-986004 and BMS-986003 was 0.59 and 0.65 mL / h / kg, respectively.
[00207] [00207] The PK parameters of BMS-986004 in monkeys were compared with those of abatacept, a similarly sized protein (78.5 vs 78-kDa BMS-986004, based on the amino acid sequence), with the same modified human IgG1 Fc format. As expected, the parameters of BMS-986004 were almost identical to those of abatacept (CLTp of 0.6 mL / h / kg, Vss of 0.087 L / kg, T1 / 2 of 5d), suggesting that PK in humans of BMS- 986004 and a-batacept should probably be similar.
[00208] [00208] The absorption of BMS-986003 after subcutaneous administration (SC) was evaluated in the PK / PD study in monkeys. Monkeys received BMS-986003 in the form of single subcutaneous doses of O (vehicle control), 0.2, 2 and 20 mg / kg, 24 h before immunization with keyhole keyhole limpet hemocyanin (KLH) ), a T cell dependent antigen. After dosing, BMS-986003 was slowly absorbed, with a Tmax ranging from 6-96 h (FIG. 12). The exposure of BMS-986003 appeared to be less than dose-proportional across all dose levels. With a dose ratio of 1: 10: 100, the average proportions of Cmax and AUCO-inf were 1:12:80 and 1: 7: 44, respectively. With exposure after dose IV (2 mg / kg) as a reference and assuming linear PK after dosing | V, the SC biological availability of BMS-986003 was 88%, 74% and 44% in 0.2, 2 and 20 mg / kg, respectively. Terminal T1 / 2 was fused by the immunogenicity observed with most monkeys in 2 to 5 weeks after dosing. Therefore, T1 / 2 was estimated to be 85, 66 and 105 h at 0.2, 2 and 20 mg / kg, respectively.
[00209] [00209] The PK of 5c8-lg9G1, a human anti-CD40L monoclonal antibody used as a positive control in the PK / PD study, was evaluated after IV administration at 20 mg / kg (FIG. 12). 5c8-l9G1 exhibited plasma exposures 10 times higher and T1 / 2 4 times longer when compared to BMS-986003 provided SC in the same dose (TABLE 13).
[00210] [00210] The PK of the mouse substitute dAb-Fc fusion protein, BMS-2m-126-24-CT, was evaluated in mice after a single IV and SC administration (TABLE 13). After a single IV (1 mg / kg), plasma concentrations followed a monoexponential decline with a 101 h terminal T1 / 2 (FIG. 13). The CLTp was 1.85 ml / h / kg; and Vss was 0.26 L / kg, indicating distributon extracellular distribution. After single SC doses of 1 and 10 mg / kg, BMS-2m-126-24-CT was slowly absorbed with a Tmax of 24 h. Systemic exposures increased in a dose-proportional manner. With a 1:10 dose ratio, Cmax and AUCO-inf increased by 1:11. The terminal T1 / 2 was 100 and 120 hem 1 and 10 mg / kg, respectively. The proportion of dose-adjusted exposure (AUCO-inf) after SC and IV administration was greater than 1, suggesting complete absorption after SC administration. Pharmacokinetic / Pharmacodynamic Modeling
[00211] [00211] APD of BMS-986003 was measured in the form of suppression of the anti-KLH antibody response in the PK / PD study. BMS-986003 suppressed 70% of the antibody response to KLH (% of response su- a- AUEC 1008h IgG titers Sua all * 100 + AUVECO1OOShtcriptsdelgG group with vehicle) | primida =) at the highest dose of 20 mg / kg. Marginal suppression (15%) and none of the antibody response occurred at 2 and 0.2 mg / kg. In comparison, 5c8-1I9G1 exhibited plasma exposures 10 times higher and T1 / 2 4 times longer than BMS-986003 at the same dose level (20 mg / kg). As a result, 5c8-l9G1 suppressed 97% of the anti-
[00212] [00212] For 5c8-lg9G1, a model of two compartments with central elimination was used. The anti-KLH antibody response, expressed as the mean value of IgG titers, was modeled using a 6-compartment signal transduction model. The KLH kinetics in the body was assumed to be a 1-compartment model. The inhibition of IgG production by BMS-986003 and 5c8-IgG1 has been described using an Imax model, with a maximum inhibition equal to 100%. As shown in FIG. 14, the curves fitted to the model were able to describe the profile of both PK and PD. The plasma IC50 of BMS-986003 and 5c8-lgG1 for suppression of KLH-induced IgG production was estimated to be 74 + 14 and 60 + 18 nM, respectively. These results demonstrated that the potency of these two molecules was comparable in vivo.
[00213] [00213] The CD40L receptor (RO) occupation of BMS-986004 was measured in the PK IV study. After IV administration of 11 mg / kg, the RO of BMS-986004 on peripheral blood mononuclear cells (PBMC) was time and concentration dependent. PK / PD modeling was performed to estimate an EC50 of RO. Plasma concentrations were modeled using a modified two-compartment model with an additional ADA-mediated elimination constant introduced 504 h after
[00214] [00214] The objectives of this study were 1) to determine the tolerance capacity of BMS-986003, including its potential immunogenicity, when supplied subcutaneously in the form of a single dose to monkeys; 2) evaluate their PD (for example, inhibition of antibody response to T cell-dependent antigen) and PK profiles; 3) evaluate the BMS-986003 receptor occupation and peripheral T cell counts after subcutaneous dosing; and 4) assist in the selection of doses for studies of kidney transplantation and first dosage in humans.
[00215] [00215] BMS-986003 was administered s.c. to the posterior chest in the form of single doses of O (vehicle control), 0.2, 2 or 20 mg / kg to groups of 2 cynomolgus monkeys by sex. Two additional monkeys / sex received a single 20 mg / kg intravenous dose of 5c8-I9G1, a monoclonal antibody to human CD40L that was used as a positive control in this study. All doses were administered in 2 mL / kg in the vehicle (PBS; pH 7.2). To assess the effects on the T cell-dependent antibody response, animals were immunized approximately 24 hours after dosing with the test article or immediately after dosing the positive control with 10 mg KLH by intramuscular injection (quadri-
[00216] [00216] At doses <20 mg / kg, BMS-986003 was slowly absorbed (Tmax = 6-96 h) and Cmax and AUCtot values increased less than dose-proportional across all groups doses and there were no evident gender differences. The estimated T1 / 2 values ranged from 69-104 h over all doses. BMS-986003 was substantially immunogenic; all monkeys developed a positive anti-drug antibody (ADA) response during the 6-week post-dose period. At 0.2 and 2 mg / kg, the group's average total ADA response had its maximum on day 22 at the group's average end point titers (EPT) of 4203 and 6469, respectively. At 20 mg / kg, the ADA response, while positive, was somewhat delayed and partially suppressed, consistent with the target's pharmacology, peaking at day 36 in an average group EPT of 1828. Additional characterization of antibodies demonstrated most of the binding to the dAb portion (other than Fc) of the molecule and it was shown that these antibodies block the binding of BMS-986003 to CD40L in 2 different immunological assay formats suggesting that the ADA was neutralizer. In addition, the formation of ADA appeared to accelerate the elimination of BMS-986003 in several monkeys.
[00217] [00217] The average PK parameters for BMS-986003 are shown in TABLE 14. TABLE 14
[00218] [00218] The molecular weight used for the conversion was 78,104 Da for BMS-986003, 150000 Da for mAb 5c8-IgG1.
[00219] [00219] ND = not determined; AUCextra for male individuals was above 20%, therefore T1 / 2 was not reported.
[00220] [00220] There were no clinical observations or effects related to BMS-986003 or 5c8-lgG1 on body weights or parameters of clinical pathology except 1 male subject treated with 5c8-lg9G1 who had red blood cells (0.74 x control), hemoglobin (0.73x pre-dose) and hematocrit (0.75x pre-dose) reduced on day 8 and 3 of 4 monkeys receiving 5c8-IgG1 had reduced lymphocytes (0.53x to 0.65x pre -dose) on day 8, suggestive of lymphocyte elimination.
[00221] [00221] CD40L receptor occupation was generally time and dose dependent and more sustained after administration of mg / kg of BMS-986003, consistent with higher and more sustained exposures at this dose and PD activity. For BMS-986003, maximum mean receptor occupation on peripheral blood mononuclear cells (PBMC) was achieved in 24 h (97%), 6 h (99%) or 48 20 h (99%) post-dose, decreasing to <90% occupancy in 240, 360 or 696 h and to <50% occupancy in 360, 696 or 1032 h, at 0.2, 2 or 20 mg / kg, respectively. In comparison, for 5c8-lgG1 at 20 mg / kg, the maximum mean receptor occupation on PBMCs was reached in 48 hours (> 100%) and was sustained at> 97% during the student (1032 hours until the 44th day).
[00222] [00222] “BMS-986003 suppressed the antibody response to KLH only at the high dose of 20 mg / kg. On days 8-30 at 20 mg / kg, there was a suppression of 69 to 83% of the mean antibody response of the geometrical group to KLH, compared to the control group, with a maximum suppression of 83% occurring on day 16 There was no suppression of the antibody response in 0.2 or 2 mg / kg of BMS-986003. These data demonstrate that BMS-986003 at a sustained receptor occupation of> 90% for at least 1 month and at sustained plasma concentrations above -10 ug / mL until day 11 is able to inhibit a T cell-dependent antibody response in cynomolgus macaques. For the positive control antibody, 5c8-lg9G1, a 74-97% suppression of the mean geometric group antibody response to KLH occurred on days 8-30, with a maximum suppression of 97% on day 16 which was generally sustained until the 30th.
[00223] [00223] No changes related to BMS-986003 biologically relevant in absolute numbers of B cells (CD45 +, CD20 +, CD3-), total T cells (CD45 +, CD3 +), T helper cells (CD45 +, CD3 +, CD4 +, CDB8- ), cytotoxic T cells (CD45 +, CD3 +, CD4-, CD8 +) or natural killer cells (CD45 +, CD3-, CD16 +) occurred during the study, which confirmed the absence of any Fc-effecting function. However, on day 8, 3 of 4 monkeys treated with 20 mg / kg of 5c8-lgG1 had populations of T lymphocytes (0.53x-0.66x pre-dose), both T helper cells (0.64x to 0, 77x pre-dose) and cytotoxic (0.40x to 0.61x pre-dose) reduced, which suggested elimination.
[00224] [00224] In conclusion, BMS-986003 administered in the form of single SC doses of 0.2, 2 or 20 mg / kg (AUC <14195 ug * h / mL) was well tolerated in cynomolgus monkeys with no related adverse effects to the drug. The positive control, 5c8-l9G1, at a dose of 20 mg / kg, resulted in complete sustained inhibition of the antibody response to KLH and sustained receptor occupation of approximately 100% over 30 days post-dose. Smooth elimination of T cells was also seen on day 8 in monkeys that received 5c8-I9G1 (0.40x to 0.77x pre-dose), which was not seen with BMS-
[00225] [00225] A hypothesis has been raised that the TE associated with the administration of anti-CD40L monoclonal antibodies is mediated by the platelet crosslinking mediated by the anti-CD40L immune complex (IC) mAb-CD40L, facilitated by the binding of the IC with FegRlila, a IgG Fc receptor, causing activation and aggregation (FIG. 10). It is experienced, therefore, that blocking the interaction of the Fc delgG cluster with FcegRlla decreases platelet cross-linking and
[00226] [00226] Several in vitro assays were conducted to test the hypothesis that platelets are activated by IC CD40L mab / sCD40L in a FcgRlla-dependent manner. The 5c8-Ig9G1 positive control was used to validate the assays before testing BMS-986003 and BMS-
[00227] [00227] Studies have been conducted with the blocking antibody of FcecoRIla IV3 to test whether platelet activation by IC 5c8 / sCD40L was truly mediated by FCcgRIIA. Human donor blood was preincubated with 0.5 µg / µl of the FcgRila IV.3 blocking antibody prior to dilution and incubation with detection antibodies as previously described. Adenosine diphosphate (ADP), a platelet activator through a different mechanism
[00228] [00228] A requirement for potential candidate molecules was the absence of binding to FcgRlla to prevent potential platelet activation. Several 5c8 constructs containing different IgG1 (eg 5c8-CT and N297Q) or IgG4 (eg 5c8-S228P) mutations have been expressed and verified against Fc tails that did not activate platelets using molar ratios different from SCD40L in relation to mAbs. Wild type and major mutated constructs activated platelets except for 5c8-CT and 5c8-N297Q (FIG. 17). The absence of Fc (5c8-Fab2) also did not activate platelets, further confirming that platelet activation by IC is mediated by Fc. The CT tail was chosen to format dAb candidates BMS-986003 and BMS-986004. Effect of the FcgRlla polymorphism on platelet activation
[00229] [00229] The gene for FcgRlla is variable at codon 131, resulting in His-Arg polymorphism (CAT / CGT). The distribution of the genotype in approximately 100 individuals with an approximately equal distribution of Caucasians and African Americans was A / A (homozygous for His; 14%), A / G (heterozygous for His / Arg; 60%) and G / G (homozygous for Arginine; 26%) for Caucasian Americans and A / A (30%), A / G (51%) and G / G (19%) for African Americans. Reilly et al., Clin. Diagn. Lab. Immunol. 1: 640-644 (1994). Fc-dependent platelet aggregation was observed in samples from R131 individuals when treated with anti-CD9 in the form of Fc mIgG2 or mIgG1, whereas platelets from H131 individuals aggregated only with anti-CD9 as the mIgG2 format ; this suggests that Fc-dependent aggregation with an IgG1 mAb could potentially segregate a patient population into those who respond little and much, which was previously reported with this polymorphism. Tomiyama et al., Blood 80: 2261-2268 (1992).
[00230] [00230] The experiments previously described using 5c8, supported the selection of the CT tail as the best format for BMS-986004 (also called BMS2h-572-633-CT-L2). The blood obtained from 6 donors was treated with 5c8-IgG1, 5c8-CT, F (ab); and BMS-986004. Platelets were activated by 5c8-lgG1, but not by any of the other constructs, including BMS-986004 (FIG. 19), suggesting that this dAb has no or low risk of causing platelet and TE activation in clinical studies. BMS-986003: Activation of platelets in the blood of mice expressing hFecgRlla
[00231] [00231] To further confirm that platelet activation by anti-CD40L antibodies was mediated by the FcgRlla receptor, the blood of transgenic mice expressing the human receptor (genotype R131) was treated with 5c8-lgG1, 5c8-lgG2a, dAb -IgG1, 5c8-CT and BMS-986003 (also called BMS-2h572-633-CT). Platelets were specifically activated by 5c8-lg9G1, 5c8-lgG2a and IC dAb-IlgG1 / sCD40L in blood from mice expressing hFcegRlla, but not in wild-type littermates. 5c8-CT and BMS-986003 did not activate platelets, further confirming a low risk of TE with the antibodies disclosed here (FIG. 20). Example 12 Epitope Binding Experiments
[00232] [00232] AFIG.25 shows SPR sensorgram data for experiments designed to test whether or not the modern dAb molecules BMS2h-503-1, BMS2h-572-6, BMS2h-719-17 and the fragment all monovalent anti-CD40L Fab 5c8 compete with each other for binding to CD40L. The experiments were performed using biotinylated CD40L (biot-IZ-hCD40L) that was captured on the surface of a sensor chip with streptavidin. The tests involved sequential injection of a specified molecule (phase "a"), immediately followed by injection of the same molecule in the presence of a second specified molecule (phase "b"), followed by dissociation (phase " ç"). The competition for binding is identified in the form of a reduction (blocking) of the binding signal for the second molecule in the presence of the first, with the blocking level being governed by the association and dissociation kinetics of each molecule. For each pair of molecules tested, it was shown that the binding of the second molecule was reduced when the first molecule was present. These results suggest that BMS2h-503-1, BMS2h-572-6, BMS2h-719-17 and 5c8Fab compete with each other by binding to biot-IZ-hCD40L.
[00233] [00233] FIG. 26 shows SPR sensorgram data for the binding of the indicated dAb-CT-long and 5c8-CT-long molecules to the human CD40L monomer (triple mutant CDA40L (T211E, S222Y, H224K, [108-261])) or to the CD40L trimer (IZ-hCD40L). Ab-CT-ong and 5c8-CT-long molecules were captured through their "CT-long" Fc domain on the surface of an immobilized human IgG anti-Fc antibody sensor chip (Biacore, GE Healthca - re). The data in the top 3 panels show that the human CD40L monomer specifically binds to BMS2h-719-202-CT-long and 5c8-CT-long, but does not bind to any of the indicated dAb-CT-long molecules containing dAbs of the BMS2h-572-6 strain. In contrast, the bottom 3 panels show that the CD40L trimer (IZ-hCD40L) binds strongly to all tested dAb-CT-long molecules of the BMS2h-572-6 strain, as well as to BMS2h-719-202-CT - far to 5c8-CT-long. These results suggest that the BMS2h-572-6xx-CT-long lineage molecules are specific to an epitope that is present only on the CD40L trimer and is not present on the monomeric human CD40L, whereas BMS2h-719-202 -CT-long and 5c8-CT-long bind to an epitope that is present on both the CD40L monomer and trimer.
[00234] [00234] Although the present modalities have been described in detail with reference to the previous examples, it is understood that several modifications can be made without departing from the spirit of these modalities and would be easily known to the person skilled in the art.

权利要求:
Claims (16)
[1]
1. Isolated antibody polypeptide, characterized by the fact that it comprises a first variable domain that specifically binds to human CD40L, where CD40L comprises the amino acid sequence of SEQ ID NO: 1, in which the sequence of amino acids of the first variable domain comprises BMS2h-572-633 (SEQ ID NO: 274) or differs from the amino acid sequence of BMS2h-572- 633 (SEQ ID NO: 274) by up to 5 amino acids, and in which said polypeptide antibody antibody is a domain antibody (dAb).
[2]
2. Antibody polypeptide according to claim 1, characterized by the fact that the amino acid sequence of the first variable domain comprises: (a) a region of CDR1 that has a sequence Trp-X, - Leu-Met-Gly (SEQ ID NO: 2), where X, is Glu or Gln; (b) a CDR2 region that has a Gly-lle-Glu-Gly-Pro-Gly-Asp-Val-Thr-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly sequence (SEQ ID NO: 3 ) and (c) a CDR3 region that has a sequence Lys-X, - Y2-Z2-Ser-Asp-Tyr (SEQ ID NO: 4), where X, is Asp or Glu, Y, is Ala or Ser and Z, is Lys, Asn or Arg.
[3]
Antibody polypeptide according to claim 2, characterized in that the amino acid sequence of the first variable domain comprises: (a) a region of FR1 that has a sequence Glu-Val-Gln-Leu-Leu- Glu-Ser-Gly-Gly-Gly-Leu-Val-GlIn-Pro-Gly-Gly-Ser-Leu- Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-Asn ( SEQ ID NO: 5); (b) an FR2 region that has a Trp-X sequence, - Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser (SEQ ID NO: 6), where X , is Ala or Val; (c) an FR3 region that has an Arg-Thr-
Phe-lle-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn- Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val- Tyr-Tyr-Cys-Val-Lys-Val-Gly (SEQ ID NO: 7) and (d) an FR4 region that has an Arg-Gly-GIn-Gly-Thr-Leu-Val-Thr-Val- sequence Being-Being (SEQ ID NO: 8).
[4]
Antibody polypeptide according to claim 1, characterized in that the first variable domain comprises the amino acid sequence of BMS2h-572-633 (SEQ ID NO: 274).
[5]
Antibody polypeptide according to claim 1, characterized in that the antibody polypeptide is SEQ ID NO: 1355.
[6]
An antibody polypeptide according to any one of claims 1 to 4, characterized in that the antibody polypeptide is a fusion polypeptide comprising the first variable domain and an Fc domain.
[7]
Antibody polypeptide according to claim 6, characterized in that the fusion polypeptide comprises an amino acid sequence selected from the group consisting of a CT-Long domain (SEQ ID NO: 1357), a CT-short domain ( SEQ ID NO: 1358), an N297Q Long Fc domain (SEQ ID NO: 1359) and an N297Q Short Fc domain (SEQ ID NO: 1360).
[8]
Antibody polypeptide according to any one of claims 1 to 5, characterized in that the antibody polypeptide further comprises a second variable domain that specifically binds to a second antigen, wherein the second antigen is an antigen other than human CD40L.
[9]
9. Nucleic acid, characterized by the fact that it encodes the antibody polypeptide as defined in any one of claims 1 to 8.
[10]
10. Vector, characterized by the fact that it comprises the nucleic acid as defined in claim 9.
[11]
11. Isolated host cell, characterized by the fact that it comprises the vector as defined in claim 10.
[12]
Pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the antibody polypeptide as defined in any of claims 1 to 8, optionally an immunosuppressive / immunomodulatory and / or anti-inflammatory agent, and a pharmaceutically acceptable carrier .
[13]
13. Use of an isolated antibody polypeptide as defined in any of claims 1 to 8, and optionally in combination with an immunosuppressive / immunomodulatory and / or anti-inflammatory agent, characterized by the fact that it is for the preparation of a medicine for the treatment of a patient, in which the patient has or is at risk of having an immune disease, in which the immune disease is selected from the group consisting of Addison's disease, allergies, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, colitis, coronary heart disease, Crohn's disease, diabetes, including Type 1 and / or Type 2 diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug products, systemic lupus erythematosus , male infertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, transplant rejection, vasculitis, AIDS, atopic allergy , bronchial asthma, eczema, leprosy, schizophrenia, inherited depression, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction
cardio, stroke, autism, epilepsy, Arthus phenomenon, anaphylaxis, alcohol dependence and drug addiction.
[14]
14. Use according to claim 13, characterized by the fact that the immune disease is a transplant rejection, and that the transplant rejection is a graft-related disease, and in which the isolated antibody polypeptide is optionally coadmitted. administered with a mutant CTLA4 molecule.
[15]
15. Use according to claim 13, characterized by the fact that the immune disease is idiopathic thrombocytopenic purpura.
[16]
16. Invention, in any form of its embodiments or in any applicable category of claim, for example, product or process or use encompassed by the material initially described, revealed or illustrated in the patent application.

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

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

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

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

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

优先权:

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