ANTI-CLUSTER OF DIFFERENTIATION LIGAND 40 (CD40L) ANTIBODIES AND METHODS FOR TREATING DISEASES OR DISORDERS RELATED TO THE CLUSTER OF DIFFERENTIATION LIGAND 40 (CD40L)

Abstract

Genomipulated human anti-CD40L antibodies to lack the ability to activate platelets and methods for treating patients with a human CD40L-associated disease.

Description

Anti-Cluster of Differentiation 40 (CD40L) Ligand Antibodies and Methods for Treating Diseases or Disorders RELATED TO CUMULUS DIFFERENTIATION LIGAND 40 (CD40L) ccacnn / zznz / E / YiAi Field of Invention Anti-CD40L antibodies, compositions comprising the antibodies and a method for using them for the treatment of CD40L-related diseases or disorders. Background of the Invention The interaction of CD40 with its ligand CD40L plays a critical role in regulating immune responses. The binding of CD40L to CD40 triggers activation of the CD40 pathway, which increases costimulatory molecules such as CD80 and CD86. Blocking the interaction between CD40 and CD40L using monoclonal antibodies has been shown to protect against autoimmunity and graft rejection in several preclinical models. Recently, in a mouse model of amyotrophic lateral sclerosis, an antibody targeting CD40L was shown to delay disease onset and prolong survival after disease onset. (U.S. Patent No. 8,435,514, incorporated herein by reference.) In early clinical studies, the Ref. 333990, the humanized anti-CD40L antibody hu5c8, exhibited efficacy in patients with lupus and in patients with immune thrombocytopenic purpura. However, further studies were discontinued due to incidents of thromboembolism in patients treated with hu5c8. Furthermore, in vitro and preclinical animal studies established that the interaction of iron (Fe) with the iron receptor FcyRIIa led to platelet activation and aggregation, resulting in thromboembolic events. Several strategies have been pursued to reduce or eliminate the interaction of the Fe region of the immunoglobulin with FcyRIIa, including the introduction of a point mutation in the Fe region to produce an aglycosylated anti-CD40L IgG1 lacking the Fe effector function. Other strategies utilize antibody fragments lacking the Fe region or antibodies containing multiple amino acid substitutions in the Fe region.Although the anti-CD40L antibody hu5c8 has shown efficacy in human patients, no anti-CD40L antibodies are currently commercially available. Therefore, there is a need for improved anti-CD40L antibodies for human administration that do not cause platelet activation or aggregation, are stable, and bind to CD40L. Brief Description of the Invention The present invention provides anti-CD40L antibodies, suitable for use in humans and non-human primates, having an Fe domain that has been genome-manipulated to reduce or eliminate platelet aggregation and the concomitant risk of thromboembolism. In one aspect of the invention, the present invention provides antibodies that are humanized versions of the human 5c8 anti-CD40L mouse antibody. In one embodiment, an antibody of the present invention comprises a human IgG1 consensus framework wherein the variable light chain and variable heavy chain comprise the 5c8 CDR sequences. One aspect of the present invention is an isolated antibody that binds to CD40L and comprises a light chain and a heavy chain, wherein (i) the light chain comprises a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID No. 1; (ii) the heavy chain comprises a heavy chain variable region and an Fe region, wherein (a) the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID No. 2; and (b) the Fe region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID No. 3, wherein the Fe region comprises one or a combination of substitutions selected from the group consisting of CHS, C14S, and P23S. Optionally, the Fe region comprises an additional C5S amino acid substitution. ccacnn / zznz / E / YiAi Another aspect of the present invention is a method for treating a subject with a CD40L-associated disease or disorder, comprising administering to the subject a therapeutically effective amount of an antibody according to the invention. One embodiment of the present invention is a method for treating a subject with 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 subject with a CD40L-associated disease or disorder, comprising administering to the subject a therapeutically effective amount of an antibody according to the invention in combination with a compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80. Brief Description of the Figures Figures IA, IB, and IC show the heavy chain amino acid sequences for hu5c8 (FIG. 1A), JB5 (FIG. IB), and JB5-K74R (FIG. 1C). Amino acids shown in bold indicate those that differ between the heavy chain sequences for 5c8 and the heavy chain sequences for JB5 and JB5-K74R. Figures 2A–2D show the light chain amino acid sequence for JB5 (FIG. 2A), the ccacnn / zznz / E / YiAi light chain amino acid sequence for JB5-R28K (FIG. 2B), the Fe region amino acid sequence for hu5c8 (FIG. 2C), and the Fe region amino acid sequence for JB5 (FIG. 2D). Amino acids shown in bold indicate those that differ between the light chain sequences for 5c8 and JB5-R28K and between the Fe regions for hu5c8 and JB5. Figure 3 is a graph showing the relative binding to human CD40L of antibody JB5 (circles, dotted line), antibody hu5c8 (squares-solid line) and the CTLA4-IgGl control (triangles) Figure 4 is a graph showing the binding of the hu5c8 antibody to the FCGR1A (circle, solid line), FCGR2A (circle, dashed line), FCR3A, and FCR3B isoforms of the human Fe gamma receptor protein. Figure 5 is a graph showing that the JB5 antibody does not bind to the FCGR1A, FCGR2A, FCR3A, or FCR3B isoforms of the human Fe gamma receptor protein. Figure 6 shows the analytical chromatography elution profile for antibody JB5 run at 30 °C from a size exclusion column. Figure 7 shows the analytical chromatography elution profile for the antibody hu5c8 run at 30 °C from a size exclusion column. Figure 8 is a graph showing the binding of the ccacnn / zznz / E / YiAi PACI antibody platelet activation marker to untreated platelet samples (negative control), as assessed by fluorescence activated cell sorting (FACS). Figure 9 is a graph showing the binding, as assessed by FACS, of an anti-PACl antibody. Figure 10 is a graph showing the binding, as assessed by FACS, of an anti-PACl antibody to platelets after incubation of the platelets with CD40L. Figure 11 is a graph showing the binding, as assessed by FACS, of an anti-PACl antibody to platelets with a CD40L immune complex and the hu5c8 antibody. Figure 12 is a graph showing the binding, as assessed by FACS, of an anti-PACl antibody to platelets with an immune complex of CD40L and the JB5 antibody. Figure 13 is a graph showing the binding, as assessed by FACS, of an anti-PACl antibody to platelets with an immune complex of CD40L and the F(ab')2 of hu5c8. Figure 14 is a scatter plot graph showing the FACS results of platelets from three people after incubation of platelets with 20μM ccacnn / zznz / E / YiAi of ADP, 5 pg / ml of CD40L, the CD40L immune complex and hu5c8, the CD40L immune complex and JB5 antibody, or the CD40L immune complex with hu5c8 F(ab')2. Figure 15 provides 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 Fe region amino acid sequence of anti-CD40L antibody hu5c8 (SEQ ID No.:3), the Fe 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 provides the synthetic light chain nucleotide sequence encoding the anti-CD40L antibody JB5 (SEQ ID No.: 8), with uppercase letters representing exons and lowercase letters representing intron sequences of the synthetic gene, and also provides the heavy chain amino acid sequence of the anti-CD40L antibody JB5 (SEQ ID No.: 9). Figure 17 provides a synthetic nucleic acid sequence that encodes for the heavy chain of the anti-CD40L antibody JB5 (SEQ ID No.: 10), the uppercase letters represent the exons and the lowercase letters represent the intron sequences of the synthetic gene. Figure 18 provides the amino acid sequence of the anti-CD40L antibody JB5-R28K (SEQ ID No.: 11), a synthetic nucleic acid sequence encoding the light chain of the anti-CD40L antibody JB5-R28K (SEQ ID No.: 12), uppercase letters representing exons and lowercase letters representing intron sequences of the synthetic gene, and also provides the heavy chain amino acid sequence of the anti-CD40L antibody JB5K74R (SEQ ID No.: 13). Figure 19 provides a synthetic nucleic acid sequence encoding the anti-CD40L antibody heavy chain JB5-K74R (SEQ ID No.: 14), with uppercase letters representing exons and lowercase letters representing intron sequences of the synthetic gene. Figure 20 provides the amino acid sequences of the heavy and light chain CDRs of the anti-CD40L antibody JB5 (SEQ ID Nos. 15-20, respectively) and the amino acid sequence of the heavy chain of hu5C8 (SEQ ID No. 21). Detailed Description of the Invention DEFINITIONS Terms such as “comprises,” “comprising,” “comprising,” “containing,” “containing,” and the like have the meanings ascribed to them in U.S. patent law; these terms are inclusive or indefinite and do not exclude additional or unenumerated method elements or steps. Terms such as “essentially consisting of” and “essentially consisting of” have the meanings ascribed to them in U.S. patent law; these terms permit the inclusion of additional ingredients or steps that do not materially affect the novel and basic features of the claimed invention. The terms “consists of” and “consisting of” have the meanings ascribed to them in U.S. patent law; these terms are defined. The terms "treat," "treatment," and similar terms include both therapeutic and prophylactic treatment. Therapeutic treatment refers to the treatment of a subject who has signs or symptoms of the disease, condition, or disorder being treated. Prophylactic treatment refers to the treatment of a subject who is predisposed to the disease, condition, or disorder but does not exhibit clear signs of it. Therefore, treatment may result in the disappearance, partial or total relief, or reduction of the signs or symptoms of the disease, and specifically includes, without limitation, the prolongation of survival. "Around" indicates that the stated numerical value allows for a slight degree of imprecision (in some strategies, to the point of exactness; approximately or reasonably close to the value; close). If the imprecision provided by "around" is not otherwise understood in the art with this common meaning, then "around," as used herein, indicates at least the variations that may arise from customary methods of measurement and the use of parameters. Furthermore, the description of intervals includes the description of all values ​​and also the subdivisions within the whole range. The conjunction "or" is used interchangeably with "at least one of". For example, when a composition comprises A or B, the method must comprise at least one of A and B, but it can also comprise A and B. Likewise, a composition comprising "A, B, C or D" must comprise at least one of the group A, B, C and D, but it can also comprise all or any combination of A, B, C and D. Amino acid substitutions are denoted by the convention in which the original amino acid, the position of the amino acid in the specified sequence, and the replacing amino acid are identified; for example, CHS would indicate that the cysteine ​​at position 11 of the polypeptide sequence is replaced with a serine. “5c8” refers to the anti-human mouse antibody that binds to CD40L and is produced by the hybridoma available from ATCC under accession number HB10916 and described in U.S. Patent No. 5,474,771. “hu5c8” refers to the humanized version of 5c8, the sequence of which is described in Karpusas, et al., Structure vol. 9, pp. 321–329, (2001). The description refers to the percentage of identity between the polypeptide and amino acid sequences. The percentage of identity between the two sequences is a function of the number of identical positions they share, taking into account the number and length of gaps that must be introduced for optimal alignment of the two sequences. Identity can be measured as "local identity" or "global identity." Local identity refers to the degree of sequence relationship between the polypeptides, as determined by the match between strands of the sequences. Global identity refers to the degree of sequence relationship of a polypeptide compared to the full length of the reference polypeptide. Unless otherwise specified, as used herein, identity means global identity.For the purposes of this description, the percentages for the global identity ccacnn / zznz / E / YiAi are calculated using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:444-453) which uses the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frame-shift gap penalty of 5. There are many publicly available computer programs that incorporate the Needleman and Wunsch algorithm, for example, the GAP program in the GCG software package. CD40L is also known as CD154, gp39, T-BAM, 5c8 antigen, or TNF-related activation protein (TRAP). MODALITIES The present invention provides therapeutic human anti-CD40L antibodies and methods for using the antibodies of the invention to treat patients with a CD40L-associated disease or disorder. Several exemplary embodiments of the present invention are provided; however, the invention shall be limited by the claims and not by the embodiments described. In one aspect of the invention, the present invention provides antibodies that are modified versions of the anti-CD40L antibody hu5c8 comprising a human IgG1 consensus framework having the variable light chain and variable heavy chain CDR sequences of hu5c8 with a ccacnn / zznz / E / YiAi domain Iron modified to avoid platelet activation. Table 1 provides a description of the SEQ ID N.s proteins mentioned in the application. ccacnn / zznz / E / YiAi Table 1 SEQ ID No.: Sequence Description 1 Amino acid sequence of light chain variable region (hu5c8 and JB5) 2 Amino acid sequence of heavy chain variable region (hu5c8 and JB5) 3 Amino acid sequence of Fe region (hu5c8) 4 Amino acid sequence of Fe region of JB5 5 Amino acid sequence of light chain variable region of JB5-R28K 6 Amino acid sequence of heavy chain variable region of JB5-K74R 7 Amino acid sequence of light chain of JB5 8 Nucleic acid sequence of light chain of JB5 9 Amino acid sequence of heavy chain of JB5 10 Nucleic acid sequence of heavy chain of JB5 11 Amino acid sequence of light chain of JB5-R28K 12 Nucleic acid sequence of synthetic light chain gene of JB5-R28K 13 Amino acid sequence of JB5-K74R heavy chain 14 Nucleic acid sequence of synthetic gene of JB5-K74R heavy chain 15 CDR-1 of the variable light chain amino acid sequence of JB5 16 CDR-2of the variable light chain amino acid sequence of JB5 17 CDR-3 of the variable light chain amino acid sequence of JB5 18 CDR-1 of the variable heavy chain amino acid sequence of JB5 19 CDR-2 of the variable heavy chain amino acid sequence of JB5 20 CDR-3 of the variable heavy chain amino acid sequence of JB5 21 Heavy chain amino acid sequence of Hu5c8 One modality (Modality A) is an isolated antibody that binds to 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 with SEQ ID No. 1, and the heavy chain comprises a heavy chain variable region and an Fe region, wherein the heavy chain variable 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 with SEQ ID No. 1.°:2 and the Fe 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 with SEQ ID No. 3, wherein the Fe region comprises one or a combination of substitutions selected from the group consisting of CHS, C14S, and P23S. Another modality (modity B) is an isolated antibody according to modality A, wherein the Fe region also comprises the C5S amino acid substitution. ccacnn / zznz / E / YiAi In variations of modes A and B, the antibody comprises a variable light chain region that does not include any of the T33W, S26D, and Q27E substitutions. In other variations of modes A and B, the antibody comprises a light chain variable region comprising the R28K substitution. In some variations of modalities A and B, the heavy and light chain CDRs have the sequences indicated in Table 2 ccacnn / zznz / E / YiAi Table 2 ISCRASQRVSSSTYSYMH light chain CDRl (SEQ ID NO:15) YASNLES light chain CDR2 (SEQ ID NO:16) QHSWEIPPT light chain CDR3 (SEQ ID NO:17) SYYMY heavy chain CDR1 (SEQ ID NO:18) EINPSNGDTNFNEKFKS heavy chain CDR2 (SEQ ID NO:19) Heavy Chain CDR3 SDGRNDMDS (SEQ ID NO:20) In another additional variation of modes A and B, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID No.: 15). In other additional modes, 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). In some variations of modes A and B, the variable region of the light chain comprises the amino acid sequence of SEQ ID No. 1. In other additional modes, the variable region of the light chain consists of the amino acid of SEQ ID No. 1. In some modes, the light chain consists essentially of the amino acid sequence of SEQ ID No. 7. In other modes, the light chain consists of the amino acid sequence of SEQ ID No. 7. In other additional modes, the light chain comprises the amino acid sequence of SEQ ID No. 11. In other additional modes, the light chain consists essentially of the amino acid sequence of SEQ ID No. 11. In other additional modes, the light chain consists of the amino acid sequence of SEQ ID No. 11. In other variations of modes A and B, the antibody comprises a variable heavy chain region that does not include any of the T30H, Y33W, and S54N substitutions. In some variations of the antibodies of modes A and B, the variable light chain region does not include any of the T33W, S26D, and Q27E substitutions. In other variations of modes A and B, the variable light chain region does not include any of the T33W, S26D, and Q27E substitutions, and the variable heavy chain region does not include any of the T30H, Y33W, or S54N substitutions. In other additional variations of modalities A and B, the heavy chain variable region comprises the K74R substitution. 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). In one embodiment, the variable heavy chain region comprises the amino acid sequence of SEQ ID No. 2. In another further embodiment, the variable heavy chain region essentially consists of the amino acid sequence of SEQ ID No. 2. In another further embodiment, the variable heavy chain region consists of the amino acid sequence of SEQ ID No. 2. In some embodiments, the variable heavy chain region comprises the amino acid sequence of SEQ ID No. 6. In other further embodiments, the variable heavy chain region essentially consists of the amino acid sequence of SEQ ID No. 6. In other further embodiments, the variable heavy chain region consists of the amino acid sequence of SEQ ID No. 6. One embodiment of the present invention is an isolated antibody, wherein the light chain comprises the amino acid sequence of SEQ ID No. 9 and the heavy chain consists of the amino acid sequence of SEQ ID No. 9. Another embodiment of the present 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. Another additional modality 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. Another additional modality 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. Another additional modality 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. Another modality is an isolated antibody, where 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. In preferred embodiments, the antibody of the present invention is stable at 37 °C for a period of at least 12 hours. In another aspect, the present description provides methods for treating subjects with a CD40L-associated disease or disorder comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. It is contemplated that an antibody of the invention, or mixtures thereof, may be administered to the subject as monotherapy, which, as used herein, means that the antibody is the sole therapeutic agent administered to the patient for the treatment of the underlying disease or disorder. Monotherapy with the use of an antibody of the invention does not preclude the administration of other drugs; non-limiting examples of these include muscle relaxants, nonsteroidal anti-inflammatory drugs, pain medications, and antidepressants.Therefore, in various embodiments of the invention, one or a mixture of the antibodies of the invention is the sole therapeutic agent directed at treating the underlying disease or disorder. It is also contemplated that the antibodies of the invention, or mixtures thereof, may 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, at different frequencies, simultaneously, or combined in a unit-dose form. A modality is a method for treating a subject with a neurodegenerative or neuromuscular disease or disorder comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. Neurodegenerative or neuromuscular diseases or disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, and spinocerebellar ataxia. Another modality is a method for treating a subject with amyotrophic lateral sclerosis comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. Another embodiment of the present invention is a method for treating a subject with an inflammatory or immune disease or disorder comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. Inflammatory or immune diseases or disorders include, but are not limited to, colitis, drug-induced lupus nephritis, graft-versus-host disease, transplant rejection, and atherosclerosis. Another additional modality is a method for treating a subject with an autoimmune disease comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. Autoimmune diseases include, but are not limited to, systemic lupus erythematosus, type 1 diabetes, myasthenia gravis, inflammatory bowel disease, immune thrombocytopenic purpura, and rheumatoid arthritis. Another embodiment is a method for inhibiting an immune response in a subject comprising administering to the subject a therapeutically effective amount of an antibody of the present invention. In one embodiment, the immune response is graft-versus-host disease. In another embodiment, the immune response is organ transplant rejection. In some embodiments, an antibody of the present invention is administered as monotherapy. In one embodiment, the antibody is JB5 and is administered as monotherapy. In one embodiment, the antibody is JB5-K74R and is administered as monotherapy. In another embodiment, the antibody is JB5-R28K and is administered as monotherapy. In one embodiment, the antibody is JB5-R28K-K74R and is administered as monotherapy. In some embodiments of the methods according to the present invention, the antibody is administered in combination with another therapeutic agent. In some embodiments, the antibody of the present invention is administered in combination with a compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80. In some formulations, the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is a CTLA4-Ig fusion protein. In one formulation, the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is abatacept, belatacept, or galiximab. Pharmaceutical compositions and methods of administration To treat any of the preceding disorders, pharmaceutical compositions can be formulated for use in accordance with the methods described herein, using one or more physiologically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular compositions administered, as well as by the particular method used to administer the composition. Consequently, there is a wide variety of suitable formulations of the compounds useful in the methods described herein (see, for example, Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro et al. Eds., Lippincott Williams and Wilkins, 2000). Suitable formulations for parenteral administration include sterile aqueous and non-aqueous isotonic injectable solutions that may contain antioxidants, buffers, bacteriostatics, and solutes that convert the formulation to isotonic with the blood of the desired recipient; and sterile aqueous and non-aqueous suspensions that may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. According to the present description, the compounds can be administered by any suitable means, which may vary depending on the type of disorder being treated and the nature of the compound itself. For example, for the antibodies of the present invention, the routes of administration preferably include parenteral, e.g., intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous. Preferably, the parenteral dosage is administered by injection, more preferably intravenous, intramuscular, or subcutaneous injection. The amount to be administered will depend on a variety of factors such as the clinical symptoms, the individual's weight, and whether other drugs are being administered. It should be appreciated that determining the appropriate dosage forms, dosage amounts, and routes of administration is within the usual level of expertise in pharmaceutical and medical techniques. Examples The following examples illustrate the methods used to produce and evaluate the antibodies of the invention. Appropriate modifications and adaptations of the described conditions and parameters, which are normally found in molecular biology and immunology techniques, will be evident to a person skilled in the art. Example 1: Antibody production In order to produce the antibodies of the invention, the 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 lyqed into the BPJPuro antibody expression vector using standard molecular biology techniques. BPJPuro is a dual-gene mammalian expression vector optimized for the selectable and stable expression of immunoglobulins in Chinese hamster ovary (CHO) cells. The vector was then transfected into the CHO cells, and stable transectants were selected. JB5 antibody production 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 to the BPJPuro antibody expression vector. The resulting expression vector encoding for the heavy and light chains was transfected into the CHO line (CHO SA, Cellectis SA, Paris, France) using liposome-mediated transfection. Stable transfectants were isolated by puromycin selection and subcloned to provide 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 designated JB5, cultured in a pilot-scale bioreactor, and the JB5 antibody was purified from the conditioned medium by sequential concentration, protein A / G affinity chromatography, and size exclusion chromatography. Example 2: CD40L Binding Test A three-part sandwich ELISA was used to determine the binding kinetics of JB5 antibody to the progenitor antibody hu5c8. All washes were performed using three 250 µA washes of PBS. A 96-well polystyrene plate was coated with 100 µA / well of either JB5 or hu5c8 antibody (2 pg / ml) for 16 hours at 4 °C. The plate was washed and then blocked with 2% bovine serum albumin / PBS for 1 hour at room temperature. The plate was washed again, and recombinant human CD40L protein (Santa Cruz Biotechnology, Santa Cruz, California, USA) was added to the plate, adjusted by a 2-fold dilution starting at 2000 ng / ml. After binding and washing, the bound CD40L protein was detected using 100 μA of biotinylated goat polyclonal anti-human CD40L antibody (200 ng / ml) and 100 μA of radish streptavidin-peroxidase conjugate at 100 ng / ml.Colorimetric detection was performed using the chromagen TMB (3,3',5,5'-tetramethylbenzidine) and spectrophotometric absorption analysis at 450 nm. The resulting binding curves (Figure 3) show that JB5 (circle) has a binding to CD40L very similar to that of the progenitor antibody hu5c8 (square). The control protein CTLA4-IgGl (triangle), which has the same Fe domain as JB5, did not exhibit significant binding. The calculated EC50 for hu5c8 and JB5 is 114 and 137 nM, respectively. JB5-R28K and JB5-K74R exhibited binding similar to that of JB5. Example 3: Fe gamma receptor binding assays Human Fe gamma receptor binding assay / hu5c8 A solid-phase ELISA binding assay was performed to determine the binding level of four human Fe gamma receptor isoforms to the progenitor antibody hu5c8. 100 μA / well of hu5c8 antibody (2 pg / ml in phosphate-buffered saline) was added to the wells of a 96-well polystyrene plate and incubated for 16 hours at 4°C. The plate was then blocked, and human Fe gamma receptor (FCGR) proteins (Santa Cruz Biotechnology, Santa Cruz, California) were titrated by 2x dilution from a starting concentration of 5 pg / ml. Four recombinant FCGR isoforms were evaluated separately as follows: FCGR1A (CD64), FCGR2A (CD32), FCGR3A (CD16a), and FCGR3B (CD16b). After binding and washing, FCGR was detected using a murine monoclonal antibody specific for the appropriate FCGR isoform (1000 ng / ml) and a goat anti-mouse IgG detector antibody conjugated to radish peroxidase.Colorimetric detection was performed using the TMB (3,3', 5,5'-tetramethylbenzidine) chromagen, and spectrophotometric absorption analysis was performed at 450 nm. The resulting binding curves (Figure 4) demonstrate that the progenitor antibody hu5c8 binds with high affinity to the FCGR1A (circled, solid line) and FCGR2A (circled, dashed line) receptors expressed on activated platelets. The hu5c8 antibody did not exhibit binding to the FCR3A and FCR3B isoforms. Human Fe Gamma Receptor and JB5 Binding Assays: A solid-phase binding assay was used to evaluate the binding of human Fe gamma receptor isoforms to the mutant antibody JB5. A 96-well polystyrene plate was coated with 100 μA / well of JB5 (2 pg / ml in phosphate-buffered saline) for 16 hours. The plate was blocked, and human Fe gamma receptor (FCGR) proteins (Santa Cruz Biotechnology, Santa Cruz, California) were titrated with a 2x dilution to a starting concentration of 5 pg / ml. Four recombinant FCGR isoforms were evaluated separately as follows: FCGR1A (CD64), FCGR2A (CD32), FCGR3A (CD16a), and FCGR3B (CD16b). After binding and washing, FCGR was detected using a murine monoclonal antibody specific for the appropriate FCGR isoform (1000 ng / ml) and a goat anti-mouse IgG detector antibody conjugated to radish peroxidase.Colorimetric detection was performed using the TMB chromagen (3,3',5,5'-tetramethylbenzidine) and spectrophotometric absorption analysis at 450 nm. The resulting binding curves (Figure 5) demonstrate that the JB5 antibody does not bind to either the FCGR1A or the high-affinity FCGR2A receptor, expressed on activated platelets, in this assay. As with the progenitor antibody hu5c8, no binding to FCGR3A or FCGR3B was observed. Example 4: Stability of JB5 at 22 °C and 37 °C Because JB5 lacks three of the disulfide bonds of natural IgG1 antibodies, JB5 was evaluated using size exclusion chromatography to determine if the antibody was stable, i.e., if it existed as a fully intact, tetrameric antibody. Hu5c8, which has all three disulfide bonds, was used as a control. Two experiments were conducted, in each comparing JB5 with hu5c8. 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 minutes before chromatography at 30 °C. Twenty micrograms of JB5 or hu5c8 were injected into PBS on a TSK gel G3000SW (7.8 mm x 30 cm, 5 pm bead column) equipped with a TSKgel Guard SW xl pre-column filter (6.0 mm x 4.0 cm, 7 pm bead column) (Tosoh Bioscience, King of Prussia, PI). The mobile phase was PBS and the elution rate was 1.0 mL / minute and the absorbance was measured at 280 nm. At 22 °C and 30 °C JB5 had an observed molecular weight of 183 kDa (Figure 6) and hu5c8 (Figure 7) had an MW of 164 kDa consistent with the divalent, tetrameric form of the antibody.The observed difference of 19 kDa between the antibody hu5c8 and JB5 may be due to the increased glycosylation of the Fe domain of JB5. Example 5: Elimination of platelet activation To determine the effect of JB5 on CD40L immune complex-mediated platelet activation, the antibody was assayed to evaluate its ability to induce the platelet cell surface marker protein PAC-1. Whole blood was drawn from three healthy volunteers into 3.2% sodium citrate tubes, discarding the first 2 ml. Platelet-rich plasma was prepared by centrifugation for 15 minutes at 120 g. Platelet counts were normalized with phosphate-buffered saline to 1905 cells / ml. Recombinant human CD40L immune complexes (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the test antibodies hu5c8, JB5, and hu5c8 F(ab')2 were prepared at a CD40L:antibody molar ratio of 3:1 (0.6944 nmol). CD40L:0.2315 nmol antibody) by pre-incubation at room temperature for 15 minutes. The immune complex mixture was diluted to a final concentration of 5 pg / ml CD40L in platelet solution / PBS-normalized and incubated at 37 °C for 30 minutes. Negative controls were untreated platelets and CD40L alone. The positive platelet activation control was prepared by adding ADP to a final concentration of 20 micromolar in platelet solution normalized with PBS. After 30 minutes of incubation, the human anti-PAC-I-FITC conjugated antibody was added to all samples and incubated for 15 minutes. Samples were diluted 1:1 in 2% paraformaldehyde:PBS buffer, fixed on ice for 30 minutes, and centrifuged at 100 g for 5 minutes to granulate the cells. The 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 FlowJo software ccacnn / zznz / E / YiAi (FlowJo, LLC, Ashland, OR, USA). A control sample of untreated platelets was used to fix the PAC-1 positive and negative activation cuts (Figure 8). Platelets activated with 20 micromolar ADP exhibited a significant increase in PAC-1 cell surface expression (Figure 9). Consistent with published observations, see, for example, Mirabet, M., et al., Molecular Immunology 45, 937-944 (2008), CD40L alone was able to activate platelets at a low level (Figure 10). This activation increased significantly when CD40L was present with the hu5c8 antibody as an immune complex (Figure 11). In contrast, the genome-manipulated antibody JB5 complexed with CD40L showed very low levels of platelet activation (Figure 12).This reduction in the potency activation of a CD40L:JB5 immune complex is mediated by the loss of interaction with FcR because the hu5c8:CD40L F(ab')2 immune complex (Figure 13) also did not activate platelets compared to the hu5c8IgGl:CD40L immune complex (Figure 11). Figure 14 shows the platelet activation results from three individuals after incubation of platelets with 20 pM ADP, 5 pg / ml CD40L, the CD40L and hu5c8 immune complex, the CD40L and JB5 antibody immune complex, or the CD40L with hu5c8 F(ab')2 immune complex. The JB5 immune complex ccacnn / zznz / E / YiAi did not exhibit significant platelet activation compared to the CD40L immune complex with F(ab')2 of hu5c8 platelets (p<0.34 (unpaired 2-tailed T test; t=1.013, df=4).Furthermore, the JB5 immune complex exhibited significantly less platelet activation compared to the hu5c8 immune complex (p<0.005 (unpaired 2-tailed T test; t=5.586, df=4). Although several embodiments of the present invention are described, it will be evident that a person skilled in the art can alter the basic examples to provide other embodiments that use or encompass the methods and processes of the present invention. The embodiments and examples are for illustrative purposes only and should not be construed as limiting the description; rather, the appended claims define the scope of the present invention. It is hereby stated that, as of this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.

Claims

1. An isolated antibody that binds to CD40L for use in the treatment of a CD40L-associated disease or disorder: wherein the disease or disorder is selected from systemic lupus erythematosus, type 1 diabetes, myasthenia gravis, inflammatory bowel disease, immune thrombocytopenic purpura, rheumatoid arthritis, colitis, drug-induced lupus nephritis, organ transplant rejection, graft-versus-host disease, transplant rejection, atherosclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, or spinocerebellar ataxia; wherein the isolated antibody comprises a light chain and a heavy chain; wherein the light chain 1 comprises a light chain variable region that shares at least 95% sequence identity with SEQ ID NO: 1;wherein the light chain comprises each of a light chain of CDR1, a light chain of CDR2, and a light chain of CDR3; wherein the light chain of CDR1 has a sequence consisting of SEQ ID NO:15; wherein the light chain of CDR2 has a sequence consisting of SEQ ID NO:16; and wherein the light chain of CDR3 has a sequence consisting of SEQ ID NO:17; wherein the heavy chain comprises a variable region of heavy chain that shares at least 95% sequence identity with SEQ ID NO:2, wherein the heavy chain comprises each of a heavy chain of CDR1, a heavy chain of CDR2, and a heavy chain of CDR3; wherein the heavy chain of CDR1 has a sequence consisting of SEQ ID NO:18; wherein the heavy chain of CDR2 has a sequence consisting of SEQ ID NO:19; and wherein the heavy chain of CDR3 has a sequence consisting of SEQ ID NO:

20.

2. The antibody for use according to claim 1, wherein the light chain variable region consists of the amino acid sequence SEQ ID NO: 1 . ccacnn / zznz / E / YiAi 3. The antibody for use in accordance with claim 1 or 2, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO:

2.

4. The antibody for use in accordance with any of claims 1 to 3, wherein the antibody light chain shares at least 95% sequence identity with SEQ ID NO:

7.

5. The antibody for use in accordance with any of claims 1 to 4, wherein the antibody heavy chain shares at least 95% sequence identity with SEQ ID NO:

9.

6. The antibody for use in accordance with any of claims 1 to 5, wherein the disease or disorder is amyotrophic lateral sclerosis, drug-induced lupus nephritis, systemic lupus erythematosus, organ transplant rejection, graft-versus-host disease, type 1 diabetes, transplant rejection, immune thrombocytopenic purpura, and rheumatoid arthritis.

7. The antibody for use in accordance with any of claims 1 to 5, wherein the disease or disorder is transplant rejection and / or type 1 diabetes.

8. The antibody for use in accordance with any of claims 1 to 5, wherein the disease or disorder is amyotrophic lateral sclerosis.

9. The antibody for use according to any of claims 1 to 5, wherein the drug is used in combination with a second drug 5 that blocks the interaction between CD28 and CD86 or between CD28 and CD80, wherein the second drug is a CTLA4-Ig fusion protein.

10. The antibody for use according to any of claims 1 to 5, wherein the drug is used in combination with a second drug that blocks the interaction between CD28 and CD86 or between CD28 and CD80, wherein the second drug is abatacept, belatacept, or galiximab.