Anti-CD40l antibodies and methods for treating CD40l-related diseases or disorders

Abstract

To provide anti-human CD40L antibodies engineered to lack the ability to activate platelets for treating patients having a CD40L-associated disease, pharmaceutical compositions comprising the antibodies, and the use of the antibodies and compositions for treating CD40L-related diseases or disorders.SOLUTION: The present invention provides an isolated antibody that binds to human CD40L, the antibody comprising a light chain and a heavy chain that include variable regions comprising specific amino acid sequences, and an Fc region comprising a specific amino acid sequence, and a method of use thereof.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of U.S. Provisional Patent Application No. 62,111,261, filed February 3, 2015, the entire disclosure of which is incorporated herein by reference.

[0002] Anti-CD40L antibodies, compositions comprising the antibodies, and methods of using them for the treatment of CD40L-associated diseases or disorders.

[0003] Sequence Listing This application contains a sequence listing which has been submitted in electronic, readable form. The electronic sequence listing was newly created on February 2, 2016, is named "384897ST25.txt" and has a size of 32.6 KB. The entire contents of the sequence listing in the electronic "384897ST25.txt" file are incorporated herein by reference in their entirety. [Background technology]

[0004] The interaction of CD40 with its ligand CD40L plays a crucial role in regulating immune responses. Binding of CD40L to CD40 leads to activation of the CD40 pathway, which upregulates costimulatory molecules such as CD80 and CD86. Blockade of the interaction between CD40 and CD40L with monoclonal antibodies has been shown to result in protection from autoimmunity and graft rejection in various preclinical models. Recently, antibodies directed against CD40L have been shown to delay disease onset and extend life expectancy after disease onset in a mouse model of amyotrophic lateral sclerosis (U.S. Patent No. 8,435,514, incorporated herein by reference). In early clinical studies, the humanized anti-CD40L antibody hu5c8 showed efficacy in patients with lupus erythematosus and immune thrombocytopenic purpura. However, the occurrence of thromboembolic events in patients treated with hu5c8 has discouraged further testing. Further in vitro and preclinical animal studies have demonstrated that the interaction of Fc with the Fc receptor FcγRIIa causes platelet activation and aggregation, resulting in thromboembolic events. Various approaches have been taken to reduce or eliminate the interaction of immunoglobulin Fc regions with FcγRIIa, including introducing point mutations in the Fc region to create deglycosylated anti-IC40L IgG1 lacking Fc effector functions. Other approaches use antibodies lacking the Fc region or antibody fragments containing multiple amino acid substitutions in the Fc region. Although the anti-CD40L antibody hu5c8 has shown efficacy in human patients, there are no anti-CD40L antibodies on the market. Thus, there is a need for improved anti-CD40L for administration to humans that does not cause platelet activation or aggregation, yet is stable and binds to CD40L. Summary of the Invention

[0005] The present invention provides anti-CD40L antibodies suitable for use in humans and non-human primates that have an engineered Fc domain to reduce or eliminate the risk of platelet aggregation and associated thromboembolism. In one aspect of the invention, the invention provides antibodies that are humanized versions of the murine anti-human CD40L antibody 5c8. In one embodiment, the antibodies of the invention comprise a human IgG1 consensus framework in which the variable light and variable heavy chains comprise the CDR sequences of 5c8.

[0006] One aspect of the invention is an isolated antibody that binds CD40L and comprises a light chain and a heavy chain, (i) the light chain comprises a light chain variable region comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, and (ii) the heavy chain comprises a variable heavy chain region and an Fc region, a) the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, and b) the Fc region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, wherein the Fc region comprises one or a combination of substitutions selected from the group consisting of C11S, C14S, and P23S. Optionally, the Fc region comprises the additional amino acid substitution C5S.

[0007] Another aspect of the invention is a method for treating a patient having a CD40L-related disease or disorder, comprising administering to the subject a therapeutically effective amount of an antibody according to the invention. One embodiment of the invention is a method for treating a patient having a neurodegenerative or neuromuscular disease or disorder; an inflammatory or immune disease or disorder; or an autoimmune disease, comprising administering to the subject a therapeutically effective amount of an antibody according to the invention. Another embodiment is a method for treating a patient having a CD40L-related disease or disorder, comprising administering to the subject a therapeutically effective amount of an antibody according to the invention, administered in combination with a compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80. [Brief description of the drawings]

[0008] [Figure 1A]The heavy chain amino acid sequence for hu5c8 is shown. Amino acids shown in bold represent amino acids that differ between the heavy chain sequence for 5c8 and the heavy chain sequences for JB5 and JB5-K74R. [Figure 1B] The heavy chain amino acid sequence for JB5 is shown. The amino acids shown in bold represent the amino acids that differ between the heavy chain sequence for 5c8 and the heavy chain sequences for JB5 and JB5-K74R. [Figure 1C] The heavy chain amino acid sequence for JB5-K74R is shown. Amino acids shown in bold represent amino acids that differ between the heavy chain sequence for 5c8 and the heavy chain sequences for JB5 and JB5-K74R. [Figure 2A]

[0036] Figure 1 shows the light chain amino acid sequence for JB5. Amino acids shown in bold represent amino acids that differ between the light chain sequences for 5c8 and JB5-R28K and between the Fc regions for hu5c8 and JB5. [Figure 2B] The light chain amino acid sequence for JB5-R28K is shown. The amino acids shown in bold represent the amino acids that differ between the light chain sequences for 5c8 and JB5-R28K and between the Fc region for hu5c8 and JB5. [Figure 2C]

[0036] Figure 1 shows the Fc region amino acid sequence for hu5c8. Amino acids shown in bold represent amino acids that differ between the light chain sequences for 5c8 and JB5-R28K and between the Fc region for hu5c8 and JB5. [Figure 2D]

[0036] Figure 2 shows the Fc region amino acid sequence for JB5. Amino acids shown in bold represent amino acids that differ between the light chain sequences for 5c8 and JB5-R28K and between the Fc region sequences for hu5c8 and JB5. [Diagram 3] Graph showing relative binding of JB5 antibody (circles, dashed line), hu5c8 antibody (squares-solid line), and control CTLA4-IgG1 (triangles) to human CD40L. [Figure 4]Graph showing binding of hu5c8 antibody to FCGR1A (circles, solid line), FCGR2A (circles, dashed line), FcR3A and FcR3B isoforms of human Fc gamma receptor proteins. [Diagram 5] 1 is a graph showing that the JB5 antibody does not bind to the FCGR1A, FCGR2A, FcR3A or FcR3B isoforms of human Fc gamma receptor proteins. [Figure 6] 1 shows an analytical chromatographic elution profile for the JB5 antibody from a size exclusion column performed at 30° C. [Figure 7] 1 shows an analytical chromatographic elution profile for the hu5c8 antibody from a size exclusion column performed at 30° C. [Figure 8] FIG. 1 is a graph showing binding of the platelet activation marker PAC1 antibody to untreated platelet samples (negative control) as assessed by fluorescence activated cell sorting (FACS). [Figure 9] 1 is a graph showing binding of anti-PAC1 antibodies assessed by FACS. [Figure 10] FIG. 1 is a graph showing binding, assessed by FACS, of anti-PAC1 antibodies to platelets following incubation of the platelets with CD40L. [Figure 11] FIG. 1 is a graph showing binding, assessed by FACS, of anti-PAC1 antibody to platelets following incubation of platelets with immune complexes of CD40L and hu5c8 antibody. [Figure 12] FIG. 1 is a graph showing binding, assessed by FACS, of anti-PAC1 antibodies to platelets after incubation of the platelets with immune complexes of CD40L and JB5 antibodies. [Figure 13] FIG. 13 is a graph showing binding of anti-PAC1 antibodies to platelets, assessed by FACS, following incubation of platelets with immune complexes of CD40L and hu5c8F(ab')2. [Figure 14]1 is a scatter plot graph showing FACS results from platelets from three individuals following incubation of platelets with 20 μM ADP, 5 μg / ml CD40L, immunocomplexes of CD40L and hu5c8, immunocomplexes of CD40L and JB5 antibody, or immunocomplexes of CD40L and hu5c8 F(ab')2. [Figure 15] Provided are the variable light region amino acid sequence of anti-CD40L antibodies JB5 and hu5c8 (SEQ ID NO:1), the variable heavy region amino acid sequence of anti-CD40L antibodies JB5 and hu5c8 (SEQ ID NO:2), the Fc region amino acid sequence of anti-CD40L antibody hu5c8 (SEQ ID NO:3), the Fc region amino acid sequence of anti-CD40L antibody JB5 (SEQ ID NO:4), the variable light region amino acid sequence of anti-CD40L antibody JB5-R28K (SEQ ID NO:5), the variable heavy region amino acid sequence of anti-CD40L antibody JB5-K74R (SEQ ID NO:6), and the light chain amino acid sequence of anti-CD40L antibody JB5 (SEQ ID NO:7). [Figure 16] The light chain synthetic nucleotide sequence (SEQ ID NO:8) encoding the anti-CD40L antibody JB5 is provided, with capital letters representing exons and lower case letters representing intron sequences of the synthetic gene, as is the heavy chain amino acid sequence (SEQ ID NO:9) of the anti-CD40L antibody JB5. [Figure 17] A synthetic nucleic acid sequence (SEQ ID NO:10) encoding the heavy chain of the anti-CD40L antibody JB5 is provided, where capital letters represent exons and lower case letters represent intron sequences of the synthetic gene. [Figure 18] Provided is the amino acid sequence of anti-CD40L antibody JB5-R28K (SEQ ID NO:11), a synthetic nucleic acid sequence encoding the light chain of anti-CD40L antibody JB5-R28K (SEQ ID NO:12), where uppercase letters represent exons and lowercase letters represent intron sequences of the synthetic gene, as well as the heavy chain amino acid sequence of anti-CD40L antibody JB5-K74R (SEQ ID NO:13). [Figure 19] A synthetic nucleic acid sequence (SEQ ID NO:14) encoding the heavy chain of the anti-CD40L antibody JB5-K74R is provided, where capital letters represent exons and lower case letters represent intron sequences of the synthetic gene. [Figure 20]The amino acid sequences of the heavy and light chain CDRs of anti-CD40L antibody JB5 (SEQ ID NOs: 15-20, respectively) and the amino acid sequence of the hu5c8 heavy chain (SEQ ID NO: 21) are provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition Terms such as "comprises," "comprised," "comprising," "contains," and "containing" have the meanings given to them in U.S. Patent Law. These terms are inclusive or open-ended phrases and do not exclude additional, unrecited elements or method steps. Terms such as "consisting essentially of" and "consists essentially of" have the meanings given to them in U.S. Patent Law. These terms permit the inclusion of additional components or steps that do not materially affect the basic and novel characteristics of the invention being claimed. Terms such as "consist of" and "consisting of" have the meanings given to them in U.S. Patent Law and these terms are closed-ended phrases.

[0010] The terms "treat," "treatment," and the like include therapeutic and prophylactic treatment. Therapeutic treatment is treatment of a subject having a symptom or symptoms of the disease, condition, or disorder to be treated. Prophylactic treatment means treatment of a subject having a predisposition to the disease, condition, or disorder who does not exhibit overt signs of the disease, condition, or disorder. Thus, treatment may result in the arrest, partial or complete alleviation or diminution of signs or symptoms of the disease, and specifically includes, without limitation, the prolongation of life.

[0011] "About" indicates that the numerical value presented allows for some slight imprecision (with some approximation to the precision of the value; roughly or reasonably close to the value; approximately). In other cases where the imprecision provided by the term "about" is not understood in the art in its ordinary sense, "about" as used herein represents at least the variation that can arise from ordinary methods of measuring and using such parameters. Moreover, the disclosure of a range includes the disclosure of all values ​​throughout the range and further divided ranges.

[0012] Use of the conjunction "or" is used interchangeably with "at least one of." For example, if a composition includes A or B, then the method must include at least one of A and B, but can also include both A and B. Similarly, a composition that includes "A, B, C, or D" must include at least one of the group A, B, C, and D, but can also include all or any combination of A, B, C, and D.

[0013] Amino acid substitutions are designated by a convention identifying the original amino acid, the position of the amino acid within a specified sequence, and the substituted amino acid; for example, C11S would indicate that the cysteine ​​at position 11 of a polypeptide sequence has been replaced with a serine.

[0014] "5c8" refers to a mouse anti-human antibody that binds to CD40L and is produced by the hybridoma having accession number HB10916 available from the ATCC and described in U.S. Patent No. 5,474,771. "hu5c8" refers to the humanized version of 5c8 whose sequence is disclosed in Karpusas et al., Structure vol. 9, pp321-329, (2001).

[0015] Reference is made herein to the percentage of identity between polypeptide or amino acid sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal sequence alignment of the two sequences. Identity can be measured as "local identity" and "global identity". Local identity refers to the degree of sequence relatedness between polypeptides as determined by matching between strings of sequences. Global identity refers to the degree of sequence relatedness of polypeptides compared to the full length of a reference polypeptide. Unless otherwise specified, identity as used herein refers to global identity. For purposes of this disclosure, the percentage of global identity is calculated using Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) using the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. There are many publicly available software programs that incorporate the Needleman and Wunsch algorithm, for example the GAP program in the GCG software package.

[0016] CD40L is also known as CD154, gp39, T-BAM, 5c8 antigen, or TNF-related activation protein (TRAP).

[0017] Embodiment The present invention provides therapeutic anti-human CD40L antibodies and methods for using the antibodies of the invention to treat patients with CD40L-related diseases or disorders. While various exemplary embodiments of the invention have been provided, the invention should not be limited by the disclosed embodiments but by the claims.

[0018] In one aspect of the invention, the invention provides an antibody that is an engineered version of the anti-CD40L antibody hu5c8 comprising a human IgG1 consensus framework with the variable light and variable heavy chain CDR sequences of hu5c8, with the Fc domain engineered to prevent platelet activation.

[0019] Table 1 provides a description of the SEQ ID NOs referred to in the application. Table 1 TIFF2025081304000002.tif128155

[0020] One embodiment (embodiment A) is an isolated antibody that binds CD40L and comprises a light chain and a heavy chain, wherein the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO:1, and the heavy chain comprises a variable heavy chain region and an Fc region, and the heavy chain variable region has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94% sequence identity to SEQ ID NO:2. or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO:3, and an Fc region comprises an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO:3, wherein said Fc region comprises one or a combination of substitutions selected from the group consisting of C11S, C14S and P23S.

[0021] In another embodiment (embodiment B) is the isolated antibody of embodiment A, wherein the Fc region further comprises the amino acid substitution C5S.

[0022] In a variation of embodiments A and B, the antibody comprises the light chain variable region that does not include any of the substitutions T33W, S26D, or Q27E.

[0023] In another variation of embodiments A and B, the light chain variable region comprises the substitution R28K.

[0024] In some variations of the A and B embodiments, the heavy and light chain CDRs have the sequences listed in Table 2. Table 2 TIFF2025081304000003.tif45155

[0025] In yet another variation of embodiments A and B, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15). In yet other embodiments, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15) and one or both of the amino acid sequences YASNLES (SEQ ID NO: 16) and QHSWEIPPT (SEQ ID NO: 17).

[0026] In some variations of embodiments A and B, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 1. In yet other embodiments, the light chain variable region consists of the amino acids of SEQ ID NO: 1. In some embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 7. In other embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 7. In yet other embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 11. In yet other embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 11. In yet other embodiments, the light chain consists of the amino acid sequence of SEQ ID NO: 11.

[0027] In other variations of embodiments A and B, the antibody comprises a heavy chain variable region that does not comprise any of the substitutions T30H, Y33W, or S54N. In some embodiments of the antibodies of embodiments A and B, the light chain variable region does not comprise any of the substitutions T33W, S26D, or Q27E. In other variations of embodiments A and B, the light chain variable region does not comprise any of the substitutions T33W, S26D, or Q27E, and the heavy chain variable region does not comprise any of the substitutions T30H, Y33W, or S54N.

[0028] In yet another variation of embodiments A and B, the heavy chain variable region comprises the substitution K74R. In one embodiment, the heavy chain variable region comprises one or any combination of the amino acid sequences SYYMY (SEQ ID NO: 18), EINPSNGDTNFNEKFKS (SEQ ID NO: 19), and SDGRNDMDS (SEQ ID NO: 20).

[0029] In another embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the heavy chain variable region consists essentially of the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6. In yet other embodiments, the heavy chain variable region consists essentially of the amino acid sequence of SEQ ID NO: 6. In yet other embodiments, the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 6.

[0030] One embodiment of the present invention is an isolated antibody, wherein the light chain comprises the amino acid sequence of SEQ ID NO:1 and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0031] Another embodiment of the invention is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:7 and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0032] Yet another embodiment is an isolated antibody, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:5 and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0033] Yet another embodiment is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:11 and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0034] Yet another embodiment is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:7 and the heavy chain consists of the amino acid sequence of SEQ ID NO:13.

[0035] Another embodiment is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:11 and the heavy chain consists of the amino acid sequence of SEQ ID NO:13.

[0036] In a preferred embodiment, the antibodies of the invention are stable at 37° C. for at least 12 hours.

[0037] In another aspect, the present disclosure provides a method for treating a patient having a CD40L-related disease or disorder, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. It is contemplated that the antibody of the present invention or a mixture thereof may be administered to a subject as a monotherapy, which means that as used herein, the antibody is the only therapeutic agent directed to the treatment of the underlying disease or disorder for which it is administered to the patient. Monotherapy using the antibody of the present invention does not exclude the administration of other drugs, non-limiting examples of which include muscle relaxants, nonsteroidal anti-inflammatory drugs, analgesics, and antidepressants. Thus, in various embodiments of the present invention, one or a mixture of the antibodies of the present invention is the only therapeutic agent directed to the treatment of the underlying disease or disorder.

[0038] It is also contemplated that the antibodies of the present invention or a mixture thereof can be administered in combination with other therapeutic agents. "In combination with" includes, but is not limited to, administration of the therapeutic agents at different times, with different frequencies, simultaneously, or combined in a single dosage form.

[0039] One embodiment is a method for treating a patient having a neurodegenerative or neuromuscular disease or disorder, including, but not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, or spinocerebellar ataxia, comprising administering to a subject a therapeutically effective amount of an antibody of the invention.

[0040] Another embodiment is a method for treating a subject having amyotrophic lateral sclerosis, comprising administering to the subject a therapeutically effective amount of an antibody of the invention.

[0041] One embodiment of the present invention is a method for treating a subject having an inflammatory or immune disease or disorder, including, but not limited to, colitis, drug-induced lupus nephritis, graft-versus-host disease, transplant rejection and atherosclerosis, comprising administering to the subject a therapeutically effective amount of an antibody of the invention.

[0042] Yet another embodiment is a method for treating a subject having an autoimmune disease, including, but not limited to, systemic lupus erythematosus, type 1 diabetes, myasthenia gravis, inflammatory bowel disease, immune thrombocytopenic purpura, and rheumatoid arthritis, comprising administering to the subject a therapeutically effective amount of an antibody of the invention.

[0043] Yet another embodiment is a method of inhibiting an immune response in a subject comprising administering to the subject a therapeutically effective amount of an antibody of the invention. In one embodiment, the immune response is graft-versus-host disease. In another embodiment, the immune response is transplanted organ rejection.

[0044] In some embodiments, the antibodies of the invention are administered as monotherapy. In one embodiment, antibody JB5 is administered as monotherapy. In another embodiment, antibody JB5-K74R is administered as monotherapy. In yet another embodiment, antibody JB5-R28K is administered as monotherapy. In yet another embodiment, antibody JB5-R28K-K74R is administered as monotherapy.

[0045] In some embodiments of the methods of the present invention, the antibody is administered in combination with another therapeutic agent.

[0046] In some embodiments, the antibodies of the invention are administered in combination with compounds that block the interaction between CD28 and CD86 or between CD28 and CD80.

[0047] In some embodiments, the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is a CTLA4-Ig fusion protein. In one embodiment, the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is abatacept or belatacept or galiximab.

[0048] Pharmaceutical Compositions and Methods of Administration The pharmaceutical composition for use in the method of the present disclosure for treating any of the above-mentioned disorders can be formulated in a conventional manner using one or more physiologically acceptable carriers.Pharmaceutical acceptable carriers are determined in part by the specific composition to be administered and by the specific method used to administer the composition.Therefore, there are a variety of suitable formulations of the compound useful in the method of the present disclosure (see, for example, Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro et al., Eds., Lippincott Williams and Wilkins, 2000).

[0049] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers and preservatives.

[0050] According to the present disclosure, the compound can be administered by any suitable means, which may vary depending on the type of disorder to be treated or the nature of the compound itself. For example, for the antibody of the present invention, the route of administration preferably includes parenteral administration, such as intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferably, parenteral dosage is given by injection, most preferably intravenous, intramuscular or subcutaneous injection. The amount administered depends on various factors, such as clinical symptoms, the weight of the individual, and the presence or absence of other drugs. It should be recognized that the determination of the appropriate dosage form, dosage and route of administration is within the level of ordinary skill in the pharmaceutical and medical arts. EXAMPLES

[0051] The following examples illustrate the methods used to prepare and test the antibodies of the present invention. Suitable modifications and adaptations of the described conditions and parameters normally encountered in the art of molecular biology and immunology will be apparent to those skilled in the art.

[0052] Example 1: Antibody Generation To generate the antibodies of the present invention, nucleic acid sequences encoding the heavy and light chains of the desired antibody were designed to be suitable for expression in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The nucleic acids were then artificially synthesized and ligated into the antibody expression vector BPJPuro using standard molecular biology techniques. BPJPuro is a dual gene mammalian expression vector optimized for selectable and stable immunoglobulin expression in Chinese Hamster Ovary (CHO) cells. This vector is then transfected into CHO cells and stable transfectants are selected.

[0053] Generation of JB5 antibody A nucleic acid (SEQ ID NO: 10) encoding a heavy chain having the amino acid sequence of SEQ ID NO: 9 and a nucleic acid (SEQ ID NO: 8) encoding a light chain having the amino acid sequence of SEQ ID NO: 7 were synthesized and ligated into the antibody expression vector BPJPuro.

[0054] The resulting expression vectors encoding the heavy and light chains were transfected into a CHO line (CHO SA, Cellectis SA, Paris, France) using liposome-mediated transfection. Suitable transfectants were isolated by puromycin selection and subcloned to obtain clonal cell lines. Candidate cell lines were adapted to serum-free suspension culture and screened for IgG production and robust growth. One of the cell lines was selected and named JB5, which was cultivated in a pilot-scale bioreactor and the antibody JB5 was purified from the conditioned medium by sequential enrichment, protein A / G affinity chromatography, and size-exclusion chromatography.

[0055] Example 2: CD40L Binding Assay A three-part sandwich ELISA assay was used to determine the binding kinetics of the JB5 antibody in relation to the parent antibody hu5c8. All washes were performed using three washes of 250 μl PBS. 96-well polystyrene plates were coated with 100 μl / well of JB5 or hu5c8 antibody (2 μg / ml) for 16 h at 4°C. Plates were washed and then blocked with 2% bovine serum albumin / PBS for 1 h at room temperature. Plates were washed and recombinant human CD40L protein (Santa Cruz Biotechnology, Santa Cruz, California, USA) was added to the plates, titrating by two-fold dilutions starting at 2000 ng / ml. After binding and washing, bound CD40L protein was detected with 100 μl of biotinylated goat anti-human CD40L polyclonal antibody (200 ng / ml) and 100 μl of streptavidin-horseradish peroxidase conjugate at 100 ng / ml. Colorimetric detection was performed using the chromagen TMB (3,3',5,5'-tetramethylbenzidine) and spectrophotometric analysis of absorbance at 450 nm. The resulting binding curves (Figure 3) show that JB5 (circles) has very similar CD40L binding to the parent antibody hu5c8 (squares). The control protein CTLA4-IgG1 (triangles), which has the same Fc domain as JB5, did not show any significant binding. The calculated EC50 for hu5c8 and JB5 are 114 and 137 nM, respectively. JB5-R28K and JB5-K74R showed binding similar to that of JB5.

[0056] Example 3: Fc gamma receptor binding assay hu5c8 / human Fc gamma receptor binding assay A solid-phase ELISA binding assay was performed to determine the binding levels of the four human Fc gamma receptor isoforms to the parental hu5c8 antibody. 100 μl / well of hu5c8 antibody (2 μg / ml in phosphate-buffered saline) was added to a 96-well polystyrene plate and incubated at 4° C. for 16 hours. The plate was blocked and recombinant human Fc gamma receptor (FCGR) protein (Santa Cruz Biotechnology, Santa Cruz, California) was titrated by two-fold dilutions at a starting concentration of 5 μg / ml. The following four recombinant FCGR isoforms were tested separately: FCGR1A (CD64), FCGR2A (CD32), FCGR3A (CD16a), FCGR3B (CD16b). After binding and washing, FCGR was detected using the appropriate FCGR isoform-specific mouse monoclonal antibody (1000 ng / ml) and a horseradish peroxidase-conjugated goat anti-mouse IgG detection antibody. Colorimetric detection was performed using the chromagen TMB (3,3',5,5'-tetramethylbenzidine) and absorbance spectrophotometry at 450 nm. The resulting binding curves (Figure 4) demonstrate that the parental antibody binds with high affinity the high affinity FCGR1A (circles, solid line) and FCGR2A (circles, dashed line) receptors expressed on activated platelets. The hu5c8 antibody did not show any binding to the FcR3A or FcR3B isoforms.

[0057] JB5 human Fc gamma receptor binding assay A solid-phase binding assay was used to test binding of human Fc gamma receptors to the mutant JB5 antibodies. 100 μl / well of JB5 antibody (2 μg / ml in phosphate-buffered saline) was coated onto 96-well polystyrene plates for 16 hours. Plates were blocked and recombinant human Fc gamma receptor (FCGR) protein (Santa Cruz Biotechnology, Santa Cruz, California) was titrated by two-fold dilutions at a starting concentration of 5 μg / ml. Four recombinant FCGR isoforms were tested separately: FCGR1A (CD64), FCGR2A (CD32), FCGR3A (CD16a), FCGR3B (CD16b). After binding and washing, FCGR was detected using the appropriate FCGR isoform-specific mouse monoclonal antibody (1000 ng / ml) and a horseradish peroxidase-conjugated goat anti-mouse IgG detection antibody. Colorimetric detection was performed using the chromagen TMB (3,3',5,5'-tetramethylbenzidine) and absorbance spectrophotometry at 450 nm. The resulting binding curves (Figure 5) demonstrate that the JB5 antibody did not bind any of the high affinity FCGR1A or FCGR2A receptors expressed on activated platelets in this assay. Similar to the parental hu5c8 antibody, no binding was observed for FCGR3A or FCGR3B.

[0058] Example 4: Stability of JB5 at 22°C and 37°C Since JB5 lacks three of the disulfide linkages in wild-type IgG1 antibodies, we tested JB5 using size exclusion chromatography to determine whether the antibody was stable, i.e., whether it existed as a tetrameric intact antibody. As a control, hu5c8, which has three disulfide linkages, was used.

[0059] Two experiments were performed comparing JB5 and hu5c8, respectively. In the first experiment, the antibodies were at room temperature (22° C.) before and during chromatography. To simulate in vivo conditions, in the second experiment, the antibodies were incubated in human plasma at 37° C. for 30 min prior to chromatography at 30° C. Twenty micrograms of JB5 or hu5c8 in PBS were injected into a TSK gel G3000SW (7.8 mm×30 cm, 5 μm bead column) (Tosoh Bioscience, King of Prussia, PA) equipped with a pre-column filter TSKgel Guard SW×1 (6.0 mm×4.0 cm, 7 μm bead column). The mobile phase was PBS, the elution rate was 1.0 mL / min, and the absorbance was measured at 280 nm. At both 22° C. and 30° C., JB5 had an observed molecular weight of 183 kDa (FIG. 6) and hu5c8 (FIG. 7) had a MW of 164 kDa, consistent with the antibody being a tetrameric bivalent form. The 19 kDa difference observed between the hu5c8 antibody and JB5 may be due to increased glycosylation of the Fc domain of JB5.

[0060] Example 5: Elimination of platelet activation To determine the effect of JB5 on CD40L immune complex-mediated platelet activation, the antibody was assayed for its ability to induce the platelet cell surface marker protein PAC-1. Whole blood was collected from three healthy volunteers into 3.2% sodium citrate tubes and the first 2 ml was discarded. Platelet-rich plasma was prepared by centrifugation at 120 g for 15 min and platelet counts were determined to be 1×10 5 Recombinant human CD40L (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and test antibodies, hu5c8, JB5, and hu5c8F(ab'), were preincubated at room temperature for 15 min and normalized to cells / ml with phosphate buffered saline. 2Immune complexes were prepared at a 3:1 CD40L:antibody molar ratio (0.6944 nmoles of CD40L:0.2315 nmoles of antibody). The immune complex mixture was diluted in normalized PBS / platelet solution to a final concentration of 5 μg / ml of CD40L and incubated for 30 minutes at 37° C. Negative controls were untreated platelets and CD40L alone. Positive controls for platelet activation were prepared by adding ADP to a final concentration of 20 micromolar in normalized PBS-platelet solution. After 30 minutes of incubation, anti-human PAC-1-FITC conjugated antibody was added to all samples and incubated for 15 minutes. Samples were diluted 1:1 in 2% paraformaldehyde:PBS buffer, allowed to settle on ice for 30 minutes, and centrifuged at 100 g for 5 minutes to pellet the cells. Cells were resuspended in PBS. Fluorescence-activated cell sorting (FACS) was performed on a Guava easyCyte flow cytometer (EMD Millipore, Inc., Billerica, MA, USA). Post-acquisition analysis was performed using Flow Jo software (FlowJo, LLC, Ashland, OR, USA).

[0061] Untreated platelet control samples were used to set negative and positive PAC-1 activation gates (Figure 8). Platelets activated with 20 micromolar ADP had significantly increased PAC-1 cell surface expression (Figure 9). Consistent with published observations (see, e.g., Mirabet, M. et al., Molecular Immunology 45, 937-944 (2008)), CD40L alone was able to activate platelets at low levels (Figure 10). This activation was significantly increased when CD40L was present as an immune complex with the hu5c8 antibody (Figure 11). In contrast, the engineered antibody JB5 complexed with CD40L demonstrated very low levels of platelet activation (Figure 12). This reduction in activation potential of the CD40L:JB5 immune complex is mediated by a loss of FcR interaction, since hu5c8F(ab')2:CD40L immune complexes (Figure 13) also did not activate platelets compared to hu5c8-IgG1:CD40L immune complexes (Figure 11). Figure 14 shows the activation potential of CD40L:JB5 immune complexes in the presence of 20 μM ADP, 5 μg / ml CD40L, immune complexes of CD40L and hu5c8, immune complexes of CD40L and JB5 antibody, or CD40L and hu5c8F(ab') 2 Results from platelets from three individuals following incubation of platelets with immune complexes of JB5, hu5c8F(ab') 2 Platelets did not show any significant platelet activation when compared with CD40L immune complexes (p<0.34) (unpaired t-test, two-tailed; t=1.013, df=4). Furthermore, JB5 immune complexes showed significantly less platelet activation when compared with hu5c8 immune complexes (p<0.005) (unpaired t-test, two-tailed; t=5.586, df=4).

[0062] Although multiple embodiments of the present disclosure have been described, it is apparent that those skilled in the art can modify the basic examples to provide other embodiments that use or incorporate the methods and processes of the present invention. The embodiments and examples are for illustrative purposes and should not be construed as limiting the disclosure; rather, the appended claims define the scope of the invention.

Claims

1. 1. An isolated antibody that binds to CD40L and comprises a light chain and a heavy chain, (i) the light chain comprises a light chain variable region comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1; (ii) the heavy chain comprises a variable heavy chain region and an Fc region; a) the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2; b) the Fc region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3, and the Fc region comprises one or a combination of substitutions selected from the group consisting of C11S, C14S, and P23S; The isolated antibody.

2. The isolated antibody of claim 1 , wherein the Fc region further comprises the amino acid substitution C5S.

3. The isolated antibody of claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:

1.

4. 4. The isolated antibody of claim 1, wherein the light chain variable region does not contain the substitutions T33W, S26D, or Q27E.

5. 5. The isolated antibody of claim 1, wherein the light chain variable region comprises the substitution R28K.

6. 4. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequences ISCRASQRVSSSSTYSYMH (SEQ ID NO: 15), YASNLES (SEQ ID NO: 16), and QHSWEIPPT (SEQ ID NO: 17).

7. The isolated antibody of claim 1 or 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

1.

8. 8. The isolated antibody of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:

2.

9. 9. The isolated antibody of any one of claims 1-8, wherein the heavy chain variable region does not contain the substitutions T30H, Y33W, or S54N.

10. 10. The isolated antibody of claim 1, wherein the heavy chain variable region comprises the substitution K74R.

11. 10. The isolated antibody of any one of claims 1 to 9, wherein the heavy chain variable region comprises the amino acid sequences SYYMY (SEQ ID NO: 18), EINPSNGDTNFNEKFKS (SEQ ID NO: 19), and SDGRNDMDS (SEQ ID NO: 20).

12. The isolated antibody of claim 1, 2 or 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

2.

13. the light chain variable region comprises the amino acid sequence of SEQ ID NO:1; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:2; the Fc region comprises the amino acid sequence of SEQ ID NO:4; 2. The isolated antibody of claim 1.

14. the light chain variable region consists of the amino acid sequence of SEQ ID NO:1; the heavy chain variable region consists of the amino acid sequence of SEQ ID NO:6; The Fc region consists of the amino acid sequence of SEQ ID NO:

4.

2. The isolated antibody of claim 1.

15. the light chain variable region comprises the amino acid sequence of SEQ ID NO:5; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:2; the Fc region comprises the amino acid sequence of SEQ ID NO:4; 2. The isolated antibody of claim 1.

16. the light chain variable region comprises the amino acid sequence of SEQ ID NO:5; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6; The Fc region comprises the amino acid sequence of SEQ ID NO:

4.

2. The isolated antibody of claim 1.

17. 2. The isolated antibody of claim 1, wherein the light chain consists of the amino acid sequence of SEQ ID NO:7 and the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

18. 2. The isolated antibody of claim 1, wherein the light chain consists of the amino acid sequence of SEQ ID NO:7 and the heavy chain consists of the amino acid sequence of SEQ ID NO:

13.

19. 2. The isolated antibody of claim 1, wherein the light chain consists of the amino acid sequence of SEQ ID NO:11 and the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

20. 2. The isolated antibody of claim 1, wherein the light chain consists of the amino acid sequence of SEQ ID NO:11 and the heavy chain consists of the amino acid sequence of SEQ ID NO:

13.

21. 21. The isolated antibody of any one of claims 1 to 20, wherein the antibody is stable at 37°C for a period of at least 12 hours.

22. 22. The isolated antibody of any one of claims 1 to 21, wherein the antibody has been modified to reduce immunogenicity.

23. 23. A method for treating a patient having a CD40L-associated disease or disorder, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 22.

24. 23. A method for treating a patient having a neurodegenerative or neuromuscular disease or disorder; an inflammatory or immune disease or disorder; or an autoimmune disease, comprising administering to a subject a therapeutically effective amount of an antibody described in any one of claims 1 to 22.

25. 25. The method of claim 24, wherein the disease or disorder is an autoimmune disease selected from the group consisting of systemic lupus erythematosus, type 1 diabetes, myasthenia gravis, inflammatory bowel disease, immune thrombocytopenic purpura, and rheumatoid arthritis.

26. 25. The method of claim 24, wherein the disease or disorder is an inflammatory or immune disease or disorder selected from the group consisting of colitis, drug-induced lupus nephritis, graft-versus-host disease, transplant rejection, and atherosclerosis.

27. 25. The method of claim 24, wherein the disease or disorder is a neurodegenerative, neuromuscular disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, or spinocerebellar ataxia.

28. 28. The method of claim 27, wherein the disorder is amyotrophic lateral sclerosis.

29. 23. A method for inhibiting an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody described in any one of claims 1 to 22.

30. 26. The method of claim 25, wherein the immune response is graft-versus-host disease or transplanted organ rejection.

31. 31. The method of any one of claims 23 to 30, wherein the antibody is administered in combination with another therapeutic agent.

32. The method of claim 31 , wherein the antibody is administered in combination with a compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80.

33. The method of claim 32, wherein the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is a CTLA4-Ig fusion protein.

34. 34. The method of claim 33, wherein the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is abatacept or belatacept or galiximab.

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