Treatment method for amyotrophic lateral sclerosis

Abstract

Methods and kits for treating neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, and spinocerebellar ataxia are provided herein. Also provided are methods for predicting or measuring a response to treatment by measuring biomarker levels in a sample, and methods for modulating biomarker levels.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 365,458, filed May 27, 2022, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application is filed electronically with an accompanying sequence listing. The sequence listing is provided as a file entitled "ELDN.010WO.xml", created on April 20, 2023, having a size of 27,094 bytes. The electronic form of the sequence listing information is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] Background The present disclosure relates to methods of treating neurodegenerative diseases such as amyotrophic lateral sclerosis. Methods of predicting and / or measuring a response to treatment, such as measuring the levels of one or more biomarkers, are also provided. Embodiments also relate to methods of modulating the levels of one or more biomarkers.

[0004] Description of Related Art Amyotrophic lateral sclerosis (ALS), also sometimes called Lou Gehrig's disease, is a progressive, fatal neurodegenerative disease characterized by muscle fiber atrophy resulting from the degeneration of motor neurons in the spinal cord and brain. Approximately 30,000 people in the United States are affected by ALS, and only about 10% of cases are classified as familial ALS. In a subset of familial patients with mutations in the metabolic enzyme superoxide dismutase 1 (SOD1), the pathological progression may be due to an unknown gain of function associated with the mutant form of the enzyme (SOD1 - dependent) (Rosen, 1993). However, in the majority of ALS cases, the SOD1 gene does not contain mutations, the activity of the SOD1 enzyme is normal, and the mechanism of disease pathology is unknown (SOD1 - independent). Thus, the remaining 90% of ALS cases are classified as sporadic cases in which the genetic element or pathogen has not been fully characterized.

[0005] Since the cause of the sporadic disease form is unknown, researchers have embarked on a gene transfer strategy to create an experimental model of the disease. Identification of the role of the SOD1 gene has led to the generation of transgenic rodent models of ALS. Human SOD1 G93A The transgenic mouse line carrying 23 copies of the transgene (the "G93A mouse") is the most widely used mouse model of ALS and is recognized as the standard model for ALS treatment research (the "G93A mouse") (see Tu PH et. al. (1996) Proc Natl Acad Sci U S A 93:3155-3160 and Gurney ME (1997) J Neuro Sci 152 Suppl 1: S67-S73).

[0006] ALS is characterized by the loss of motor neurons in the spinal cord that results in muscle atrophy, but the disease is also manifested by changes in axonal transport, protein aggregation, excitotoxicity, astrocyte increase, mitochondrial dysfunction, microglial activation, and synaptic remodeling. The activation of microglia, the increase in astrocytes, and the presence of infiltrating inflammatory cells from the periphery are well described. There is accumulation of IgG immunoreactive deposits in the spinal cord of ALS patients and infiltration of lymphocytes, dendritic cells, monocytes, and macrophages into the spinal cord in ALS. The role of infiltrating immune cells is poorly understood, but recent studies have suggested that the infiltrating T cell population is neuroprotective and not cytotoxic. ALS has an immune component mediated by the activation of microglia and astrocytes, but is not considered an autoimmune disease. Different from diseases such as rheumatoid arthritis or systemic lupus erythematosus, for which the involvement of specific immunoregulatory pathways (e.g., co-stimulatory pathways) has been described, the involvement of such pathways has not been described for ALS. Summary of the Invention Problems to be Solved by the Invention

[0007] Currently, physicians have limited options for the treatment of ALS. At present, riluzole is the only drug approved by the FDA for the treatment of ALS. In clinical trials, riluzole has shown only a modest benefit in a slight increase in survival time. Therefore, an effective treatment for ALS is urgently needed.

Means for Solving the Problems

[0008] Disclosed herein is a method for predicting the response of a subject having a disease or disorder to treatment with a compound against CD40L or CD40. In some embodiments, the method comprises obtaining a sample from the subject; measuring the concentration of at least one biomarker present in the sample, wherein the at least one biomarker is NFL, MMP9, MMP3, IL6, IL18, IgA, TNFR2, TNFa, IL2ra, Factor VIII, CRP, SAP, MCP1, En-Raged, MIG, vWF, IgE, IL8, IP10, C3, and MMP2, and the concentration of the at least one biomarker predicts that the subject may be responsive to treatment of the disease or disorder with a compound against CD40L or CD40. In some embodiments, the disease or disorder is an inflammation, a neurodegenerative disease, or a neuromuscular disease. In some embodiments, the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia. In some embodiments, the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the subject is a mammal and / or a human. In some embodiments, the compound blocks the interaction between CD40 and CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is tegoprubart. In some embodiments, the compound is MR1 or 5c8.

[0009] Methods of treating a subject having a disease or disorder are also disclosed herein. In some embodiments, the method comprises administering a compound against CD40L or CD40 in a therapeutically effective dose, and by administering the therapeutically effective dose, the compound in the plasma of the subject is maintained at a concentration of 5-100 μg / mL; preferably, at a dose of 30-60 μg / mL. In some embodiments, the disease or disorder is an inflammation, a neurodegenerative disease, or a neuromuscular disease. In some embodiments, the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia. In some embodiments, the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the subject is a mammal and / or a human. In some embodiments, the compound blocks the interaction of CD40 and CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is tebgobart. In some embodiments, the compound is MR1 or 5c8. In some embodiments, the compound is administered at least once every two weeks. In some embodiments, the compound is administered for a period of at least 12 weeks. In some embodiments, the compound is administered at a dose of 200-100 mg / kg; preferably, at a dose of 1-8 mg / kg; most preferably, at a dose of 2-4 mg / kg. In some embodiments, the method further comprises administering a second pharmaceutically effective compound. In some embodiments, the second compound blocks the interaction of CD28 and CD86 or the interaction of CD28 and CD80. In some embodiments, the second compound targets at least one of the biomarkers selected from the group consisting of interleukins, cytokines, and inflammation-inducing markers.In some embodiments, the second compound targets at least one of the biomarkers selected from the group consisting of NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrinogen, IgE, ICAM, factor VIII, Ena-78, Pal1, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, and MMP2. In some embodiments, the second compound is a CTLA4-Ig fusion protein, abatacept, belatacept, or galiximab. In some embodiments, the compound is administered orally, parenterally, or topically. In some embodiments, the compound is administered parenterally. In some embodiments, the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection. In some embodiments, 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 immune response, graft rejection, and atherosclerosis. In some embodiments, 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, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease. In some embodiments, the compound targeting CD40L or CD40 comprises (1) a CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; (2) a CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; and (3) a CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; for the heavy chain variable region (V H ) and (4) a CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; (5) a CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; and (6) a CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20; for the light chain variable region (V L) Also included is an antibody. In some embodiments, the compound that targets CD40L or CD40 has a heavy chain variable region (V H ) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4; and a light chain variable region (V L ) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8.

[0010] Methods for modulating the concentration of at least one biomarker in a subject are also disclosed herein. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound against CD40L or CD40, and the at least one biomarker is NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrinogen, IgE, ICAM, factor VIII, Ena-78, Pal1, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10, or MMP2. In some embodiments, the concentration of at least one biomarker increases after administration of the compound. In some embodiments, the concentration of at least one biomarker decreases after administration of the compound. In some embodiments, after administration of the compound, the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is an inflammation, a neurodegenerative disease, or a neuromuscular disease. In some embodiments, the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia. In some embodiments, the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the subject is a mammal and / or a human. In some embodiments, the compound blocks the interaction between CD40 and CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is tegolizumab. In some embodiments, the compound is MR1 or 5c8. In some embodiments, the method comprises administering at a therapeutically effective dose such that the compound in the plasma of the subject is maintained at a concentration of 5 to 100 μg / mL; preferably, at a dose of 30 to 60 μg / mL.In some embodiments, the compound is administered at least once every two weeks. In some embodiments, the compound is administered for a period of at least 12 weeks. In some embodiments, the compound is administered at a dose of 200 - 100 mg / kg; preferably at a dose of 1 - 8 mg / kg; most preferably at a dose of 2 - 4 mg / kg. In some embodiments, the compound is administered orally, parenterally, or topically. In some embodiments, the compound is administered parenterally. In some embodiments, the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection. In some embodiments, 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 immune response, graft rejection, and atherosclerosis. In some embodiments, 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, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease. In some embodiments, the compound that targets CD40L or CD40 comprises a heavy chain variable region (V H ) comprising: (1) a CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; (2) a CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; and (3) a CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; and further comprises a light chain variable region (V L ) comprising: (4) a CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; (5) a CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; and (6) a CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20. In some embodiments, the compound that targets CD40L or CD40 is an antibody comprising a heavy chain variable region (V H ) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4; and a light chain variable region (V L ) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8.

[0011] Methods for maintaining or improving the ALS-FRS score of a subject having ALS are also disclosed herein. In some embodiments, the method comprises administering a compound against CD40L or CD40 in a therapeutically effective dose, and by administering the therapeutically effective dose, the compound in the plasma of the subject is maintained at a concentration of 5 μg / mL and 100 μg / mL; preferably, maintained at a dose of 30 μg / mL to 60 μg / mL.

[0012] Methods for pre-screening and treating a subject having a disease or disorder with a compound against CD40L or CD40 are also disclosed herein. In some embodiments, the method comprises collecting a sample from the subject; detecting the concentration of at least one biomarker present in the body fluid, wherein the at least one biomarker is NFL, MMP9, MMP3, IL6, IL18, IgA, TNFR2, TNFa, IL2ra, factor VIII, CRP, SAP, MCP1, En-Raged, MIG, vWF, IgE, IL8, IP10, C3, or MMP2; screening the concentration of the at least one biomarker against the concentration of the at least one biomarker in a subject without the disease or disorder; and administering a compound against CD40L or CD40 in a therapeutically effective dose so as to maintain the concentration of the compound at a dose of 5 to 100 μg / mL; preferably, at a dose of 30 to 60 μg / mL. In some embodiments, the compound is administered to the subject when the concentration of the at least one biomarker in the subject is significantly different from the concentration of the at least one biomarker in a subject without the disease or disorder.

Brief Description of the Drawings

[0013] Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0014] Detailed Description In the following detailed description, reference is made to the accompanying drawings which form a part hereof. In the drawings, like reference numerals generally identify like components unless specified otherwise in the context. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter shown herein. Generally, the aspects of the present disclosure described herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in a variety of configurations, all of which are readily understood to be expressly contemplated herein. All references cited herein are hereby expressly incorporated by reference in their entirety into this specification and for the specific disclosure referenced herein.

[0015] The present disclosure describes a method of treating a patient having a neurodegenerative disease or a neuromuscular disease by administering a therapeutically effective amount of a compound that blocks the interaction of CD40 and CD40L. The present disclosure also describes a method of treating a patient by co-administering a compound that blocks the interaction of CD40 and CD40L with a compound that blocks the interaction of CD28 and CD86, or CD28 and CD80. Some embodiments relate to methods of predicting and / or measuring a response to treatment, such as measuring the level of one or more biomarkers. Embodiments also relate to methods of modulating the level of one or more biomarkers.

[0016] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, patent applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless otherwise specified. In the event that there are multiple definitions of a term herein, the terms in this section shall control unless otherwise specified.

[0017] It will be understood by those skilled in the art that starting materials, biological and chemical materials, biological and chemical reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be used in the practice of the embodiments without undue experimentation. All functional equivalents known in the art of any such materials and methods are intended to be included in the present disclosure.

[0018] As used herein, "a" or "an" can mean one or more.

[0019] As used herein, the term "about" or "approximately" has its ordinary meaning as understood by one of ordinary skill in the art, and thus a value includes the inherent variability of the error of the method used to determine the value, or the variability that exists among a plurality of determinations.

[0020] The use of the conjunction "or" is used interchangeably with "at least one of". For example, if a composition contains A or B, the method must contain at least one of A and B, but may also contain both A and B. Similarly, a composition containing "A, B, C or D" must contain at least one of the group of A, B, C or D, but may also contain all of A, B, C or D, or any combination thereof.

[0021] Amino acid substitutions are indicated by the rules by which the original amino acid, the amino acid of the designated sequence, and the substituted amino acid are identified. For example, C11S indicates that the cysteine at position 11 of the polypeptide sequence is substituted with serine.

[0022] A humanized antibody is an antibody produced from a non-human species whose protein sequence has been modified to enhance its similarity to antibody variants that are naturally produced in humans. The process of "humanization" is applied to monoclonal antibodies developed for administration to humans (e.g., antibodies developed as anti-cancer drugs).

[0023] Currently, it is common to humanize non-human antibodies by inserting the relevant CDRs from antibodies made in non-human animals into a human antibody "scaffold". The "direct" generation of humanized antibodies can be achieved by inserting appropriate CDR-encoding segments (which confer the desired binding properties) into a human antibody "scaffold". This is accomplished by recombinant DNA methods that are expressed in mammalian cells using appropriate vectors. That is, after the antibody has been developed to have the desired properties in a mouse (or other non-human), the DNA encoding the antibody can be isolated, cloned into a vector, and sequenced. Subsequently, the DNA sequences corresponding to the CDRs of the antibody can be determined. Once the exact sequences of the desired CDRs are known, strategies can be devised to appropriately insert these sequences into constructs containing DNA for human antibody variants. Before insertion into the scaffold, the CDRs may be modified, for example, to enhance specificity.

[0024] The term "human" antibody means an antibody having an amino acid sequence corresponding to the sequence of an antibody produced by a human, and / or an antibody produced using any technique for making a human antibody. By this definition of a human antibody, humanized antibodies containing non-human antigen-binding residues are expressly excluded.

[0025] As used herein, the term "variant" is defined as a modified or altered form of a wild-type sequence, e.g., in which one or more amino acids may be substituted with other amino acids or non-amino acids that do not substantially affect the function. In some embodiments, a variant may contain a modified side chain with respect to at least one amino acid residue.

[0026] As used herein, the term "antigen" is defined as an entity that can stimulate the production of antibodies and can specifically bind to them, and / or an entity that can induce an immune system response. For example, a cell surface protein or a specific linear or non-linear portion thereof. In this specification, the term may be abbreviated as "Ag".

[0027] "Antigen-binding antibody fragment" means a molecule other than an intact antibody that contains a part of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fd fragments, dAb fragments, Fab’-SH, F(ab’)2; bispecific antibodies; triabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments, and minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides). The antigen-binding fragments disclosed in this application bind to the antigen CD40L.

[0028] Antigen-binding fragments of antibodies generally contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR that is adjacent to or within a framework that has one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH domain and the VL domain can be positioned relative to each other in an appropriate arrangement. For example, the variable region can be a dimer and can contain a dimer such as VH-VH, VH-VL, or VL-VL. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH domain or VL domain.

[0029] Examples of isolated antibodies include bispecific antibodies in which each arm or antigen-binding fragment of the antibody binds to a different target or epitope.

[0030] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and mean an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that contains an Fc region.

[0031] "5c8" means a murine anti-human antibody produced by a hybridoma that binds to CD40L and is available from the ATCC under accession number HB10916 and is described in U.S. Patent No. 5,474,771. "hu5c8" means a humanized version of 5c8, the sequence of which is disclosed in Karpusas, et al., Structure vol.9, pp 321-329, (2001).

[0032] The term "specifically binds" and the like mean that an antibody or its antigen-binding fragment forms a relatively stable complex with an antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (KD) of about 3000 nM or less (i.e., the smaller the KD, the stronger the binding), about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 1 nM or less, or about 0.5 nM.

[0033] Specific binding to a particular antigen or epitope can be shown, for example, by an antibody having a KD for the antigen or epitope of at least about 1×10 -4 M, at least about 1×10 -5 M, at least about 1×10 -6 M, at least about 1×10 -7 M, at least about 1×10 -8 M, at least about 1×10 -9 M, or instead at least about 1×10 -10 M, at least about 1×10 -11 M, at least about 1×10 -12 M, or more, where KD represents the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD for the control molecule that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more compared to that antigen or epitope. Also, specific binding to a particular antigen or epitope can be shown, for example, by an antibody having a Ka for the epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more compared to the control, where Ka refers to the association rate of a particular antibody-antigen interaction.

[0034] The term "neutralizing antibody" includes an antibody that can inhibit and / or neutralize the biological activity of CD40L, for example, an anti-CD40L antibody or an antigenic fragment thereof that inhibits or prevents or reduces the binding of CD40L to CD40, and thus inhibits or reduces the signal transduction pathway induced by CD40L, and / or inhibits or reduces the binding of CD40L to CD40.

[0035] The terms "antagonistic antibody" or "antagonist antibody" are used interchangeably herein and include an antibody that can inhibit and / or neutralize the biological signal transduction activity of CD40L as described for the neutralizing antibodies above.

[0036] The terms and expressions used in this specification are used as explanatory terms rather than limiting terms, and in using such terms and expressions, it is not intended to exclude any equivalents or portions thereof of the features shown and described, but it is recognized that various modifications are possible within the scope of the present invention as claimed. Accordingly, although aspects of the present disclosure are specifically disclosed by preferred embodiments, exemplary embodiments, and various embodiments that may include any features, it should be understood that modifications and changes to the concepts disclosed herein may be made by those skilled in the art. Such modifications and changes are considered to be within the scope of the embodiments of the present disclosure as set forth in the appended claims and as defined by the appended claims.

[0037] This specification refers to the percent identity between polypeptides or amino acid sequences. The percent 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, which need to be introduced for the optimal alignment of the two sequences. Identity can be measured as "local identity" or "global identity". Local identity refers to the degree of sequence relatedness between polypeptides and is determined by the matches between the strings of such sequences. Global identity refers to the degree of sequence relatedness of a polypeptide compared to the full length of a reference polypeptide. Unless otherwise specified, as used herein, identity means global identity. For the purposes of the present disclosure, the percentage of global identity is calculated using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. There are numerous publicly available software programs incorporating the Needleman and Wunsch algorithm, such as the GAP program of the GCG software package.

[0038] CD40L is also known as CD154, gp39, T-BAM, 5c8 antigen, or TNF-related activation protein (TRAP).

[0039] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are used to mean including the stated step or element or group of steps or elements but not to mean excluding any other step or element or group of steps or elements. "Consisting of" means including anything following the phrase "consisting of" and being limited to what follows the phrase. Thus, the phrase "consisting of" means that the recited elements are required or essential and that no other elements may be present. "Consisting essentially of" means including the elements recited after the phrase and being limited to other elements that do not interfere with or contribute to the specified activity or action of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential but that other elements are optional and may or may not be present depending on whether or not they substantially affect the activity or action of the recited elements.

[0040] As used herein, the terms "function" and "functional" have their plain and ordinary meaning as understood in the context of this specification and mean a biological, enzymatic, or therapeutic function.

[0041] As used herein, the term "yield" of any given substance, compound, or material has its plain and ordinary meaning as understood in view of the present specification, and means the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material can be 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, at least 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, at least about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or less of the expected total amount, about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or less of the expected total amount, including all decimals between the numbers. The yield can be affected by the efficiency of the reaction or process, unwanted side reactions, decomposition, the quality of the starting materials, compounds, or materials, or losses of the desired substance, compound, or material during the manufacturing process.

[0042] As used herein, the term "isolated" has its plain and ordinary meaning as understood in view of this specification, and means a substance and / or entity that is (1) separated from at least a portion of the components with which it was associated when first produced (whether naturally and / or produced in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from other components with which they were initially associated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% (ranges including and / or spanning the foregoing values).In some embodiments, the agent to be isolated has a purity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, a purity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less, or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less, a purity of substantially 100%, or 100% (ranges including and / or spanning the foregoing values). As used herein, an "isolated" substance can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can mean a cell that is not contained within a multicellular organism or tissue.

[0043] The terms "isolated protein" or "isolated polypeptide" (e.g., an isolated antibody or an isolated antigen-binding fragment) refer to a protein or polypeptide that, by virtue of its origin or derivation, is not associated with the naturally associated components that accompany it in its native state; a protein or polypeptide that is substantially free of other proteins from the same species; a protein or polypeptide expressed by cells from a different species; or a protein or polypeptide that does not occur naturally. Thus, a polypeptide that is chemically synthesized or synthesized in a cell line different from the cell in which it naturally occurs is "isolated" from its naturally associated components. Proteins can also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0044] As used herein, "in vivo" is considered to have its plain and ordinary meaning as understood in view of this specification, and means the performance of a method within a living organism, typically an animal, a mammal such as a human, and a plant, or within a living cell that constitutes such a living organism, as contrasted with a tissue extract or a dead organism.

[0045] As used herein, "ex vivo" is considered to have its plain and ordinary meaning as understood in view of this specification, and means the performance of a method outside of a living organism with little modification of natural conditions.

[0046] As used herein, "in vitro" is considered to have its plain and ordinary meaning as understood in view of this specification, and means the performance of a method outside of biological conditions, for example, in a Petri dish or a test tube.

[0047] As used herein, "nucleic acid", "nucleic acid molecule" or "nucleotide" refers to polynucleotides or oligonucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, exonuclease action, and synthetic production. Nucleic acid molecules can be composed of monomers that are natural nucleotides (such as DNA and RNA) or analogs of natural nucleotides (e.g., enantiomeric forms of natural nucleotides), or combinations of both. Modified nucleotides can have alterations at the sugar moiety and / or at the pyrimidine or purine base moiety. Examples of sugar modifications include substitution of one or more hydroxyl groups with, for example, halogen, alkyl groups, amines, and azide groups, or they can be functionalized as ethers or esters. Further, the entire sugar moiety can be replaced with sterically and electronically similar structures such as azasugars and carbocyclic sugar analogs. Examples of modifications at the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other known heterocyclic substitutions. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphorosenoates, phosphorodiselenoates, phosphoranoanilothioates, phosphororanilidates, phosphoramidates, and the like. The term "nucleic acid molecule" also encompasses so-called "peptide nucleic acids" containing natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded.

[0048] As used herein, the terms "peptide", "polypeptide", and "protein" have their plain and ordinary meaning as understood in view of this specification, and mean macromolecules composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, biologically produced by ribosomal complexes using a nucleic acid template, although chemical synthesis is also available. By manipulating the nucleic acid template, mutations in peptides, polypeptides, and proteins, such as substitutions, deletions, truncations, additions, duplications, or fusions of multiple peptides, polypeptides, or proteins, can be performed.These fusions of multiple peptides, polypeptides, or proteins can be adjacent in the same molecule, or, for example, between linkers, repeat sequences, epitopes, or tags, or between any other sequences of any length defined by any length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases or less, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases or less or any length within the range defined by any two of the foregoing lengths and can be linked with extra amino acids. As used herein, the term "downstream" of a polypeptide has its plain and ordinary meaning as understood in the context of this specification and means the sequence that is after the C-terminus of the preceding sequence.As used herein, the term "upstream" of a polypeptide has its plain and ordinary meaning as understood in view of the present specification, and means the sequence that is before the N-terminus of a subsequent sequence.

[0049] As used herein, the term "gene" has its plain and ordinary meaning as understood in view of the present specification, and generally means a portion of a nucleic acid that encodes a protein or functional RNA; however, the term optionally includes regulatory sequences. It will be understood by those skilled in the art that the term "gene" may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will further be understood that the definition of a gene includes references to nucleic acids that do not encode proteins but encode functional RNA molecules such as tRNA and miRNA. In some cases, a gene includes regulatory sequences involved in transcription, or message production or composition. In other embodiments, a gene includes a transcriptional sequence that encodes a protein, polypeptide, or peptide. In light of the terminology described herein, an "isolated gene" may include one or more transcriptional nucleic acids, regulatory sequences, coding sequences, etc., that are isolated substantially away from other such sequences, such as other native genes, regulatory sequences, polypeptides, or peptide coding sequences. In this regard, the term "gene" is used herein for brevity to mean a nucleic acid that includes a transcribed nucleotide sequence and its complementary sequence. As will be understood by those skilled in the art, this functional term "gene" includes both genomic sequences, RNA or DNA sequences, or smaller engineered nucleic acid segments that include nucleic acid segments of the non-transcribed portion of a gene, including, but not limited to, non-transcribed promoter or enhancer regions of a gene. The smaller engineered gene nucleic acid segments can be expressed or adapted to express, using nucleic acid manipulation techniques, proteins, polypeptides, domains, peptides, fusion proteins, variants, and / or the like.

[0050] As used herein, the terms "individual", "subject", "host", or "patient" have their ordinary meaning as understood by one of ordinary skill in the art and thus include humans or non-human mammals. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, primates such as Simians (chimpanzees, apes, monkeys), humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0051] The dimensions and values disclosed herein should not be understood to be strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range around that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm".

[0052] The following abbreviations are used herein: amyotrophic lateral sclerosis (ALS); superoxide dismutase 1 (SOD1); T cell receptor (TCR); major histocompatibility complex (MHC) antigen-presenting cell (APC); phosphate-buffered saline (PBS), complementarity-determining region (CDR). "IP" means intraperitoneal and "IV" means intravenous.

[0053] MR1 is a hamster monoclonal antibody that binds to mouse CD40 ligand. As used herein, "wild-type" means non-transgenic mice. As used herein, "small molecule" means a compound having a molecular weight of less than 2000 daltons. As used herein, "treatment" or "treating" includes prophylactic and therapeutic treatment. The phrase "therapeutically effective amount" means an amount of a compound or a pharmaceutically acceptable salt thereof that is sufficient, when administered alone or in combination with another pharmaceutical or treatment, to suppress or ameliorate a disorder or condition being treated in a particular subject or population of subjects.

[0054] An "effective amount" of an agent, e.g., a pharmaceutical formulation, means an amount effective to achieve a desired therapeutic or prophylactic result at the required dosage and for the required period of time.

[0055] "Affinity" means the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" means the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally represented by the equilibrium dissociation constant (KD), k off / k on ratio. KD and affinity are inversely proportional. The K value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), and the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Affinity can be measured by common methods known in the art, such as the methods described herein. Specific, illustrative, and exemplary embodiments for measuring binding affinity can be measured by radioimmunoassay (RIA), surface plasmon resonance (SPR) on a BIAcore® instrument (GE Healthcare Europe GmbH, Glattbrugg, Switzerland), using human CD40 ligand polypeptide as a sample, by capturing the antibody on a Protein A-binding CM5 research grade sensor chip (GE Healthcare Europe GmbH, Glattbrugg, Switzerland; BR-1000-14). Other methods include radioimmunoassay and binding equilibrium exclusion methods. The binding equilibrium exclusion method is a general immunoassay platform that can measure the equilibrium dissociation constant for antigen / antibody interactions, as well as the association and dissociation rate constants.

[0056] "ALS-TDI" is the abbreviation of the ALS Therapy Development Institute. "hSOD1G93A preclinical mouse model", "hSOD1G93A mouse model", "G93A preclinical mouse model" and "G93A mouse model" have the same meaning in this specification. "hSOD1G93A mouse" and "G93A mouse" have the same meaning in this specification.

[0057] TMB is the abbreviation of 3,3’,5,5’-tetramethylbenzidine.

[0058] As used herein, "CDR domain" means an antibody complementarity determining region, with or without flanking sequences.

[0059] "Effector function" means a biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocyte activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. One effector function is, but not limited to, the ability of the Fc or constant region of an antibody to bind to a protein, such as an Fc receptor (FcR) (e.g., the high-affinity Fc region of IgG receptor Ia (FCγRIa) (CD64) (SEQ ID NO: 34), the low-affinity immunoglobulin γ Fc region receptor IIa (FCγRIIa) (CD32) (SEQ ID NO: 35), the low-affinity immunoglobulin γ Fc region receptor IIIa (FCγRIIIA) (CD16a) (SEQ ID NO: 36), the low-affinity immunoglobulin γ Fc region receptor IIIb (FCγRIIIb) (CD16b) (SEQ ID NO: 37), etc.). In some embodiments where the antibody and its antigen-binding fragment have an Fc domain, the Fc domain has been engineered to reduce or eliminate one or more Fc effector functions. In preferred embodiments, the Fc domain has been engineered to reduce or eliminate platelet activation and / or platelet aggregation and the attendant risk of thromboembolism.

[0060] In vivo Evaluation of Compounds The compounds used in the methods of the present disclosure can have their efficacy evaluated using the G93A mouse model [see Tu PH et al. Proc Natl Acad Sci USA 1996; 93:3155-60 and Gurney ME. et al. J Neurol Sci 1997;152(Suppl 1):S67-73]. This model was created by inserting 23 copies of the human SOD1 gene containing a mutation in which the 93rd glycine was replaced with alanine into the mouse genome. These mice accurately utilize the most prominent points of the pathology of the human sporadic and familial forms of the disease, and thus this model is the best available option for testing disease altering interventions.

[0061] At birth, there is no distinguishable phenotypic expression of abnormality in G93A mice. Until approximately 90 days of age, no visible disease symptoms appear, after which they experience progressive hindlimb function loss, resulting in complete paralysis around 134 days of age and death. Muscle atrophy is caused by the death or dysfunction of motor neurons, and these cell deaths are associated with and partly caused by interactions with surrounding cells including microglia and astrocytes. A significant increase in astrocytes first appears around 80 days of age, and neuroinflammation mainly mediated by microglia appears around 100 days of age and spreads until death.

[0062] While human disease can begin in any motor area, mouse disease invariably first affects the cervical and lumbar regions. In G93A mice, the number of motor neurons has significantly decreased by approximately the time of visible symptom onset at 85 days, reaching more than a 50% loss at the time of death. Biochemical abnormalities in the cytoskeleton, neurofilaments, axonal transport, Golgi apparatus, endoplasmic reticulum, mitochondria, apoptosis machinery, proteasome, and cytosolic protein processing are observed in neurons during the course of the disease.

[0063] Whereas human diseases can begin in any motor region, mouse diseases invariably first affect the lumbar and sacral regions. In G93A mice, the number of motor neurons is significantly reduced by the time of visible symptom onset at approximately 85 days, reaching more than 50% loss at the time of death. Biochemical abnormalities in the cytoskeleton, neurofilaments, axonal transport, Golgi apparatus, endoplasmic reticulum, mitochondria, apoptosis machinery, proteasome, and cytosolic protein processing are observed in neurons during the course of the disease.

[0064] To date, there have been at least 50 publications describing therapeutic agents to extend the lifespan of this mouse. However, therapeutic agents other than riluzole have not shown corresponding clinical efficacy. The ALS Therapy Development Institute describes the screening of optimized therapeutic agents in the G93A mouse model that controls for noise variables [Scott S. et al. Amyotrophic Lateral Sclerosis 2008; 9: 4-15, incorporated herein by reference]. Scott et al. describe the minimal test design of the G93A mouse model that addresses and manages the noise generated by intrinsic biological confounding variables. In the validation of this test design, 9 compounds that were reported to be effective in preclinical models but subsequently failed in human clinical trials were evaluated at various doses. Some of these molecules are anti-inflammatory molecules that inhibit tumor necrosis factor signaling (TNF) and microglial activation, such as Celebrex®, minocycline, thalidomide, and creatine. Celebrex® was reported to improve lifespan by 19% (24 days) in the G93A model, but in a high-powered study, no change in survival time could be detected (1.8 days, 0.52%) (Scott et al.). Similar results were obtained for minocycline (previously reported; improvement 15.8%; high-powered study -0.60%), creatine (previously reported as 17.8%; high-powered study 0.67%), and thalidomide (previously reported as a 16% improvement in survival time; high-powered study -1.9%).

[0065] Identification of Candidate Compounds Genome-wide expression profile analysis was performed on wild-type and G93A mice at various time points during disease progression. The identified genes that were differentially expressed between the two groups were analyzed, and the resulting data were used to focus on the selection of drugs to screen. Among the differentially expressed genes, genes involved in immune response and cell adhesion such as CD86, CD44, ICAM, ITGAM, ITGA, ITGAX, ITGB2, H2-K1 (MHC II), H2-AB1 (MHC II), H2-D1 (MHCII), and H2-Eb1 (MHC II) were included. From these data, it can be seen that the inflammatory signature increases during disease progression, which is consistent with the involvement of the co-stimulatory pathway. The co-stimulatory pathway includes, in part, cell-type interactions mediated by the CD28 / CD80 or CD28 / CD86 or CD40 / CD40L interactions identified in gene expression analysis.

[0066] To identify molecular pathways applicable to therapeutic development, changes in gene expression patterns were characterized during disease progression in the G93A mouse model. Whole-genome transcriptional profiling was studied using the Affymetrix GeneChip® Mouse Expression Set 430vII MOE430vII gene chip. Using a longitudinal study design, G93A skeletal muscle and spinal cord were compared with non-transgenic littermates. In the G93A mouse model, symptom onset was first seen as tail paralysis starting at approximately day 75, with progressive paralysis of the hindlimbs, then the forelimbs, and finally the diaphragm. The average survival period of the G93A animal colony was 134 days. In the longitudinal study design, gastrocnemius muscle (gastrocnemius) and spinal cord were collected from G93A animals and wild-type littermates at days 30, 50, 60, 80, 90, 100, 110, and 120 (day 0 is the birth date). For each time point, tissues were collected from 5 wild-type animals and 5 G93A animals and independently processed for a total of 160 tissues.

[0067] In accordance with the IACUC protocol, the animals were euthanized at the appropriate time points described above. Tissues were immediately harvested and snap-frozen in liquid nitrogen. The frozen tissues were stored at -80 °C. Total RNA was simultaneously isolated from the tissues using the Qiagen RNaEasy kit as described by the manufacturer. The isolated total RNA was amplified using standard T7 linear amplification, and biotin-labeled nucleotides were incorporated using the Ambion MessageAMP II T7 in vitro transcription kit. The labeled probes were fragmented and hybridized to the Affymetrix GeneChip® Mouse Expression Set 430vII according to the manufacturer's protocol. The Genechip was washed in the Affymetrix GeneChip® Fluids Station 450 to remove unhybridized probes. The Genechip was scanned using the Affymetrix GeneChip® 3000 7G scanner.

[0068] All computer processing and modeling were performed using the commercially available R development language version 2.6 from Bioconductor. The datasets for the spinal cord and gastrocnemius muscle were analyzed independently. The Affymetrix CEL files were used for all data preprocessing. All CEL files in each dataset were quality-controlled using the Bioconductor vignettes SIMPLE AFFY, AFFY, and AFFY PLM. The statistical changes in gene expression between G93A tissues and wild-type tissues at a given time point were evaluated using the LIMMA package. A Bayesian model was used to determine the significance of the expression changes between groups.

[0069] From these data, it can be seen that the inflammatory signature increases during disease progression. These changes in gene expression reflect the activation of antigen-presenting cells such as dendritic cells, macrophages, and B cells. The immune response that worsens the disease progression of ALS can be improved by blocking the interaction. By blocking the CD-28 / CD80 or CD-28 / CD86 or CD40 / CD40L interaction, the co-stimulatory pathway can be inhibited.

[0070] The co-stimulatory pathway includes, among other interactions, the binding of CD40 on B cells to CD40L (also known as CD154, gp39, T-BAM, 5c8 antigen, CD40CR, and TRAP) on T cells. Human CD40 is expressed on mature B cells, as well as on macrophages, dendritic cells, fibroblasts, and activated endothelial cells. Blocking of CD40:CD40L binding is thought to promote the generation of type I helper T cell responses.

[0071] Using compounds that block these interactions and inhibit co-stimulatory signals, an immunomodulatory effect of blocking one or more of CD40L, CD80, or CD86 in preclinical models of transplantation and autoimmunity has been demonstrated by significant substances in the study. Blocking of CD40L function by using blocking antibodies or adenoviral expression of CD40L-Ig improves the allograft survival period for 30 to 90 days. Similar studies that transiently block CD80 / CD86 on APCs with CTLA4-Ig or adenoviral expression of CTLA4-Ig also improve the allograft survival period. Graft rejection in these models is transient, and graft rejection occurs over time. Prevention of graft rejection for a longer period can be achieved by both blocking the co-stimulatory pathway with CTLA4-Ig and blocking the CD40L activation of APCs with anti-CD40L antibodies.

[0072] Blocking antibodies against CD40L or gene deletion of CD40L in mice have been demonstrated to improve disease progression, survival, and surrogate markers in preclinical models of experimental allergic encephalomyelitis (EAE), multiple sclerosis, collagen-induced arthritis, and systemic lupus erythematosus models. Blockade of CD40:CD40L binding appears to reduce the ability of nitric oxide-producing macrophages, which mediate much of the inflammatory-inducing activity of macrophages.

[0073] From such studies, it is thought that blocking CD40:CD40L interactions and / or blocking CD28:CD80 or CD28:CD86 interactions may regulate the immune response.

[0074] Immunohistochemical data have been shown to correlate well with gene expression data, and from these data, macrophages are identified as antigen-presenting cell infiltrating skeletal muscle during disease progression in G93A mice.

[0075] To determine the presence and location of potential antigen-presenting cells in gastrocnemius muscles from G93A and wild-type animals, immunohistochemistry was performed on gastrocnemius muscle tissue collected on day 110 from G93A and wild-type mice. Immediately after collection, the tissue was embedded in OCT. Frozen sections were stained with H&E and hybridized with antibodies against myelin (anti-S100b antibody), or antibodies against T hematopoietic cell lineages, such as T cells (anti-CD3 antibody), B cells (CD45R pan B cell antibody), and macrophages (anti-CD11b antibody). On day 110, there was infiltration of CD11b-positive macrophages, and the macrophages appear to localize to the axons of the nerves innervating the skeletal muscle. The localization of macrophages is not dispersed throughout the muscle, suggesting that the inflammation is not due to muscle atrophy or muscle fiber remodeling.

[0076] On day 110, additional immunohistochemistry was performed with a panel of antibodies specific for the macrophage lineage to confirm the identity of monocyte-derived cells in skeletal muscle and to clarify the relationship of these cells compared to non-monocyte-derived cells. The anti-S100b antibody was used to label myelin-associated axons innervating skeletal muscle. All macrophage-specific antibodies (anti-CD11b antibody, anti-CD86 antibody, and anti-MAC1 antibody) localized macrophages to the axons of the nerves innervating the skeletal muscle of G93A mice, and no macrophages were present on the axons of wild-type animals. The localization of macrophages was specific to the nerves innervating the muscle, and no macrophages were present in the muscle fibers of the muscle.

[0077] Gene expression data suggest that genes associated with the co-stimulatory pathway are transiently increased during disease progression in both the spinal cord and skeletal muscle (Figures 1A - 1B). To characterize the timing of macrophage infiltration into skeletal muscle, immunohistochemistry was performed on gastrocnemius muscle section specimens from G93A mice at days 60, 80, and 100. There was no evidence of macrophage infiltration and localization to axons at day 60. Macrophages became apparent at day 80 and localized to the axons innervating the skeletal muscle as described above for skeletal muscle at day 110. The number of macrophages increased between days 80 and 100, and the accumulation of macrophages was specific to the axons innervating the skeletal muscle.

[0078] To quantify the increase in macrophage infiltration, representative sections from 5 G93A and 5 wild-type animals were hybridized with an anti-CD86 antibody, and the number of macrophages per 10,000 square microns was counted. The number of macrophages in wild-type animals was the same at the 60-day, 80-day, and 110-day time points. As can be seen in Figure 2, macrophages transiently accumulated in skeletal muscle between days 80 and 100, and few macrophages were present in wild-type skeletal muscle.

[0079] In summary, immunohistochemical data highly correlates with gene expression data, and macrophages are identified as antigen-presenting cell-infiltrated skeletal muscle during disease progression in the G93A mouse model. The unexpected finding is that macrophage infiltration seems to be specifically targeted to the axons innervating skeletal muscle such that it is localized by labeling with antibodies against myelin and macrophages.

[0080] Pharmacokinetic analysis of MR1 in G93A tissues Using a sandwich format matrix-matched, non-competitive enzyme immunoassay (ELISA), the tissue levels of MR1 directed against mouse CD40L were determined. A 7-point standard curve was included on each plate. Standards were prepared using purified MR1 spiked into PBS dilution solution. The PBS dilution solution was a matrix matched to normal mouse tissue at an equal dilution of the unknown samples to correct for non-specific effects arising from tissue lysates.

[0081] For pharmacokinetic analysis, 84 plasma samples were collected over a 2-week period after administration (10 mg / kg, IP) in both male and female G93A mice.

[0082] The elimination half-lives were similar in females (23 days) and males (22 days) and were similar to the half-life for a typical mouse IgG2-based antibody in mice. No signs of an anti-hamster antibody response were seen.

[0083] Females showed a slightly smaller volume of distribution of MR1 than males and thus higher plasma levels when administered the same dose of 10 mg / kg. Males showed a faster clearance and a higher volume of distribution. Thus, males would require a higher dose to achieve similar plasma levels. Figure 1A shows the MR1 concentration over time using a linear concentration (Y)-axis. Figure 1B shows the MR1 concentration over time using a log concentration (Y)-axis.

[0084] Thirty-six female G93A mice were litter matched and randomly assigned to the study groups. Eighteen G93A mice were placed in the MRI treatment group and the other eighteen G93A mice were placed in the control group. The study days were based on the days from birth.

[0085] A single injection of 56 μg of MR1 was administered intraperitoneally (IP) on day 50. Following the bolus injection, a weekly maintenance injection of 18 μg of MR1 was administered by IP injection. The doses were prepared in a total volume of 200 μl in excipient (phosphate buffered saline (PBS, pH 7.3)). 200 μl of PBS was administered to the control animals. Starting on day 54, the animals were monitored daily throughout the course of the study, and daily body weight measurements and neurological scores were taken.

[0086] For each mouse, neurological scores of both hindlimbs were evaluated daily from 50 days of age. A scale of 0 - 4 (Scott et al., ALS Journal Jan 2008) was used for the neurological scores. Briefly, animals designated a score of 0 had complete extension of the hindlimbs away from the midline when the mouse was suspended by its tail, and the mouse could be suspended 2 - 3 times and hold this for 2 seconds. If the mouse showed collapse or partial collapse of limb extension relative to the midline during tail suspension, or showed tremors in the hindlimbs, the animal was scored 1. If the toes curled at least twice during a 12-inch walk, or if part of the foot was dragged along the cage bottom / table, the animal was scored 2. If there was strong paralysis or minimal joint movement, or if the feet were not used for forward movement, the animal was scored 3. If the animal could not return to its original state from either side within 30 seconds, the animal was scored 4. When one hindlimb was scored 2, diet pellets were placed on the bedding. When both hindlimbs were scored 2, Nutra-Gel® (Bio-Serve number S4798) was provided as food in addition to the diet pellets on the bedding, and a long absorbent tube was placed in the water bottle.

[0087] The date and cause of death were recorded for each mouse. For humane reasons, animals were monitored closely and, using the criteria for severe moribundity, were euthanized as moribund just prior to actual death. To determine survival time reliably and humanely, a moribund state (neurological score 4), defined as the mouse being unable to right itself 30 seconds after being placed on its side, was used. Moribund mice were scored as "dead" and euthanized using carbon dioxide.

[0088] The standard procedure is to exclude non-ALS-related deaths in both the treatment and control groups prior to statistical analysis. In this case, animal deaths in either the control or treatment group were due to ALS. Thus, there were no animals terminated due to non-ALS-related death.

[0089] Transgenic mutant SOD1 animals exhibit normal body weight (BW) characteristics as neonates and gain weight normally compared to non-transgenic animals into adulthood. Depending on the nature of the genetic mutation in the transgene and the copy number of the mutant transgene, weight loss becomes apparent in adult animals and continues until death. Analysis of weight loss in the treatment and control groups can provide insight into the putative therapeutic effect on disease onset and progression rate. To evaluate the effect of MR1 treatment on body weight, two summary parameters were examined: (1) the change in BW from the start of the study until the attainment of peak weight, which may reflect the effect on disease onset; and (2) the change in BW from peak weight until death, which may reflect the effect on disease progression.

[0090] The survival time (time-to-event) curves of the comparative MR1 treatment group and the control group from the 40th day until peak body weight is reached are shown in Figure 2. The median time to peak body weight for the control group was 50 days compared to 51 days for the MR1 treatment group. This difference was not significant when analyzed by the Kaplan Meier method using the log rank and Wilcoxon statistical models, the Cox proportional hazards, or parametric statistical tests.

[0091] The survival time curves of the comparative MR1 treatment group and the control group from peak body weight until death are shown in Figure 3. The time from peak body weight until death was statistically significantly 15 days later in the MR1-treated animals. Control animals had a 2.4 - to 4.7-fold higher risk of dying immediately after reaching peak body weight compared to MR1-treated animals. The median time from peak body weight until death in the control group was 26 days, whereas it was 41 days in the MR1 treatment group. The significance of each analysis in this example was calculated in several ways to better evaluate the significance. When analyzed using several approaches, the delay was statistically significant (Kaplan Meier, log rank p = 0.0110 and Wilcoxon, p = 0.0069; Cox proportional hazards model p = 0.05151; parametric statistical model, p = 0.0122). Based on the body weight data, MR1 appears to have little effect on the onset of the disease in the G93A mouse model, but has a dramatic effect on slowing the rate of weight loss from peak body weight until death.

[0092] The timing of disease onset was also characterized by analyzing the daily neurological scores of the saline-treated and MR1-treated groups. At the start of the study (day 50), all animals had a neurological score of 0, without observable symptoms or paralysis. Disease onset can be characterized by examining the progression of the neurological score from a score of 0 to a score of 2, where the animal is clearly dragging its hind limbs. The time-to-event plot for the age at which MR1 and control group animals progressed to a neurological score of 2, and the survival time for the number of days at a neurological score of 2, is shown in Figure 4. The median time at a neurological score of 2 for each group is plotted in Figure 5. The time to reach a score of 2 in the control group was 115 days, and in the MR1-treated group it was 122 days. Based on the neurological score data, MR1 delayed disease onset by approximately 7 days in the G93A mouse model, and when analyzed using several approaches, the delay was statistically significant (Kaplan–Meier, log-rank (p = 0.0378) and Wilcoxon (p = 0.0591); Cox proportional hazards model (p = 0.0521); parametric analysis (p = 0.0582)). The number of days spent at each neurological score level is plotted in Figure 5 against the median age at that score.

[0093] The survival period of the treated animals was 13 days later than that of the control animals. The survival curves of the MR1-treated and control groups for the time from peak body weight to death are shown in Figure 6. The median survival period of the control group was 128 days, and that of the MR1-treated group was 141 days. Control animals had a 2.8 - to 3.2-fold higher risk of dying earlier than MR1. When analyzed using several approaches, the delay was statistically significant (Kaplan–Meier, log-rank (p = 0.0040) and Wilcoxon test (p = 0.0109); Cox proportional hazards model (p = 0.0060); parametric analysis (p = 0.0049).

[0094] Sixty female G93A mice and thirty-six male G93A mice were littermates and were randomly assigned to the treatment or control groups. Thirty female mice and eighteen male mice were treated with MR1 starting on day 50. The study days were based on the days from birth.

[0095] In each of the females or males, a single bolus injection of 5.22 mg / kg or 6.75 mg / kg of MR1 was administered intraperitoneally on day 50. Following the bolus injection, females were injected with 1 mg / kg of MR1 weekly and males were injected with 1.34 mg / kg of MR1 weekly by IP injection. The doses were prepared in a total volume of 200 μl in vehicle (phosphate buffered saline (PBS, pH 7.3)). 200 μl of PBS was administered to the control animals. The animals were monitored for body weight, neurological score, non-related ALS death, and the above euthanasia criteria.

[0096] The survival time curves of the MR1-treated and control groups for the time from day 40 to reach peak body weight are shown in FIG. 7A. The median time to peak body weight in the control group was 49 days compared to 53 days in the MR1-treated group. This difference was not significant when analyzed by the Kaplan-Meier method using the log-rank and Wilcoxon statistical models, Cox proportional hazards, or parametric statistical tests.

[0097] The survival curves of the comparison MR1 treatment group and the control group for the time from peak body weight to death are shown in Figure 7B. The time from peak body weight to death was statistically significantly 6 days later in the MR1-treated animals. The median time from peak body weight to death in the control group was 29 days, while in the MR1 treatment group it was 35 days. The significance of each analysis in this example was calculated in several ways to better evaluate the significance. When analyzed using several statistical models: Kaplan–Meier, log-rank (p = 0.0413) and Wilcoxon (p = 0.0732); and the Cox proportional hazards model (p = 0.0460), the delay was statistically significant. Based on the body weight data, MR1 seems to have little effect on the onset of the disease in the G93A mouse model, but has a dramatic effect on slowing the rate of weight loss from peak body weight to death.

[0098] Figure 7C shows the age at which the MR1 group and control group animals progressed to neurological score 2, and the number of days at neurological score 2. The time to reach score 2 in the control group was 113 days, while in the MR1 treatment group it was 121 days. Based on the neurological score data, MR1 delays disease onset by approximately 8 days in the G93A mouse model, and when analyzed using several statistical models: Kaplan–Meier, log-rank (p = 0.0038) and Wilcoxon (p = 0.0017); and the Cox proportional hazards model (p = 0.0010), the delay is statistically significant.

[0099] The survival period of the treated animals was 9 days later than that of the control animals. The median survival period of the control group was 124 days, while the median survival period of the MR1 treatment group was 133 days as shown in Figure 7D. When analyzed using several statistical models: Kaplan–Meier, log-rank (p = 0.0043) and Wilcoxon test (p = 0.0040); and the Cox proportional hazards model (p = 0.0030), the delay is statistically significant.

[0100] The examples and embodiments described herein are for illustrative purposes only, and in light of which various modifications or changes may be proposed to those skilled in the art, and it is understood that they are included within the spirit and scope of the present application and the scope of the appended claims.

[0101] Nucleic acid sequences encoding the heavy and light chains of the antibody of interest 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 expression of immunoglobulins in Chinese hamster ovary (CHO) cells. The vector was then transfected into CHO cells and appropriate transfectants were selected.

[0102] Similarly, a nucleic acid encoding a heavy chain having the amino acid sequence of SEQ ID NO: 9 (SEQ ID NO: 10) and a nucleic acid encoding a light chain having the amino acid sequence of SEQ ID NO: 7 (SEQ ID NO: 8) were synthesized and ligated into the antibody expression vector BPJPuro.

[0103] 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. 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 named JB5. The cell line was cultured in a pilot-scale bioreactor and the antibody JB5 was purified from the conditioned medium by sequential concentration, protein A / G affinity chromatography, and size exclusion chromatography.

[0104] The binding kinetics of the JB5 antibody to the parental antibody hu5c8 was determined using a three-component sandwich ELISA assay. All washes were performed using three washes with 250 μl of PBS. A 96-well polystyrene plate was coated with 100 μl / well of JB5 or hu5c8 antibody (2 μg / ml) at 4 °C for 16 hours. The plate was washed and then blocked with 2% bovine serum albumin / PBS for 1 hour at room temperature. The plate was washed and recombinant human CD40L protein (Santa Cruz Biotechnology, Santa Cruz, California, USA) was added to the plate titrated by two-fold dilutions starting at 2000 ng / ml. After binding and washing, 100 μl of biotinylated goat anti-human CD40L polyclonal antibody (200 ng / ml) and 100 μl of streptavidin-horseradish peroxidase conjugate were used at 100 ng / ml to detect the bound D40L protein. Colorimetric quantitative detection was performed with chromagen TMB (3,3’,5,5’-tetramethylbenzidine) and spectrophotometric analysis of the absorbance at 450 nm was performed. From the resulting binding curve (Figure 8), it can be seen that JB5 (●) has very similar CD40L binding to the parental antibody hu5c8 (■). The control protein CTLA4-IgG1 (▼) having the same Fc domain as JB5 showed no significant binding. The calculated EC50 values for hu5c8 and JB5 were 114 nM and 137 nM, respectively. JB5-R28K and JB5-K74R showed binding similar to JB5.

[0105] To compare the CD40L binding of antibodies from all 16 clones against 5c8 or AT-1501, a binding assay was performed using two clones together with 5c8 and AT-1501, and was carried out on the same 96-well assay plate. A three-component sandwich ELISA assay was used to determine the binding levels of the antibodies of the present disclosure compared to the reference antibodies 5c8-19 and AT1501. A 96-well polystyrene plate was coated with recombinant human CD40L (BioLegend catalog number 591706) at 2 μg / ml in PBS, 50 μl / well was added to a Costar 96-well half-area high-binding assay plate (Corning 3690), and incubated overnight at 4°C. The plate was blocked with (1×) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. The binding curves of 5C8 or AT1501 (starting from 2 μg / ml, serially diluted 2-fold) were added (50 μl / well) and incubated for 1 hour at room temperature. The plate was washed and incubated with HRP-(Fab2) donkey anti-human IgG (Fc specific) (Jackson Immuno. 709-036-098) at a dilution of 1:10,000 (50 μl / well) for 1 hour at room temperature. The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) was added (50 μl / well). 2 SO 4 Using 25 μl, the color development was stopped after 5 minutes at room temperature. The plate was read on a Molecular Devices SpectraMax M5 plate reader using the SoftMax Pro 6.2.2 program to determine the absorbance at 450 nm.

[0106] The relative CD40L binding ability was calculated as follows: [IC50 (clone) / IC50 (5c8 or AT-1501) × 100%]. The ranked potencies of 16 clones against 5c8 are shown in Fig. 15A, and the ranked potencies against AT-1501 are shown in Fig. 15B. The obtained binding curves are shown in Figs. 16A-16Q (comparison of 16 clones against 5c8, Fig. 16H shows the comparison between AT-1501 and 5c8) and Figs. 17A-17Q (comparison of 16 clones against AT-1501, Fig. 17H shows the comparison between AT-1501 and 5c8). The IC50, LCL, UCL, and relative potencies of each clone are shown in Table 1 (comparison with 5c8) and Table 2 (comparison with AT-1501).

[0107]

Table 1

[0108]

Table 2

[0109]

Table 3

[0110]

Table 4

[0111] The 16 VH / VL antibody clones were generated with IgG1 Fc having two mutations, P238S and N297G (SEQ ID NO: 21). These antibody clones were assayed for Fc effector functions against binding to human FcγRI, FcγRIIa, and FcγRIIIa.

[0112] The anti-CD40L antibody (abatacept included as a negative control) was diluted to 2 μg / ml in (1×) PBS, and 50 μl / well was added to a Costar 96-well half-area high-binding assay plate (Corning 3690) and incubated overnight at 4°C. The plate was blocked with (1×) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. Binding curves of recombinant human FcγRI, IIa, IIIa, and IIIb (starting from 5 μg / ml, serially diluted 2-fold) were added (50 μl / well) and incubated for 1 hour at room temperature. The plate was washed and incubated with mouse anti-human CD16 (anti-FcRIII); CD32 (anti-FcRIIa) or CD64 (anti-FcRI) (eBioSciences / Invitrogen 14-0168-82; 16-0329-81; 14-0649-82) at 2 μg / ml (50 μl / well) for 1 hour at room temperature. The plate was washed and incubated with HRP-(Fab2) goat anti-mouse IgG (Fc-specific) (Jackson Immuno. 116-036-071) at a dilution of 1:10,000 for 1 hour at room temperature. The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) was added (50 μl / well). 2 SO 4 (25 μl / well) was used to stop the color development after 5 minutes at room temperature. The plate was read on a Molecular Devices SpectraMax M5 plate reader using the SoftMax Pro 6.2.2 program, and the absorbance was determined at 450 nm.

[0113] As seen in the figures, the clones disclosed in this application are negative for binding to three Fc receptors. 5c8 bound to FcγRI and FcγRIIa, but did not bind to FcγRIIIa or FcγRIIIb. Figures 18A and 18B each show the binding to FcγRIa for eight antibody clones, compared to the binding curves of 5c8, AT-1501, and abatacept. Figures 19A and 19B each show the binding to FcγRIIa for eight antibody clones, compared to the binding curves of 5c8, AT-1501, and abatacept. Figures 20A and 20B each show the binding to FcγRIIIa for eight antibody clones, compared to the binding curves of 5c8, AT-1501, and abatacept. Figures 20C and 20D each show the binding to FcγRIIIb for eight antibody clones, compared to the binding curves of 5c8, AT-1501, and abatacept (Figures 18A, 19A, 20A, and 20B show the results from antibody clones 4-4, 5-3, 6-6, 7-5, 8-3, 8-4, 10-1, and 10-4; Figures 18B, 19B, 20C, and 20D show the results from antibody clones 11-5, 12-4, 13-2, 15-1, 15-4, 16-3, 17-1, and 18-2).

[0114] The anti-CD40L antibody (abatacept, included as a negative control) was diluted to 2 μg / ml in (1×) PBS, and 50 μl / well was added to a Costar 96-well half-area high-binding assay plate (Corning 3690) and incubated overnight at 4°C. The plate was blocked with (1×) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. A binding curve of native human C1q protein (Abcam ab96363) serially diluted 2-fold from 10 μg / ml was added at room temperature for 1 hour (50 μl / well). The plate was washed, and HRP-goat anti-human C1q (Abcam ab46191) was added at a dilution of 1:400 (50 μl / well) and incubated at room temperature for 1 hour. The plate was washed, and the TMB substrate (Surmodics BioFX TMBW-1000-01) was added (50 μl / well). 2N H 2 SO4 (25 μl / well) was used and the color development was stopped after 5 minutes at room temperature. The plate was read using the SoftMaxPro 6.2.2 program on a Molecular Devices SpectraMax M5 plate reader, and the absorbance was determined at 450 nm.

[0115] As seen in the drawings, antibodies from all 16 antibody clones of the present disclosure were negative for binding to C1q, whereas 5c8 showed significant binding. Figures 21A and 21B show the binding to C1q for eight antibody clones, compared to the binding curves of 5c8, AT-1501, and abatacept, respectively. Figure 21A shows the results from antibody clones 4-4, 5-3, 6-6, 7-5, 8-3, 8-4, 10-1, and 10-4; Figure 21B shows the results from antibody clones 11-5, 12-4, 13-2, 15-1, 15-4, 16-3, 17-1, and 18-2.

[0116] Since gene expression data indicated the involvement of the costimulatory pathway, the efficacy of MRI was evaluated in the G93A model. MR1 binds to CD40L, and thus blocks that interaction of CD40, which is involved in the costimulatory pathway related to the immune response. It has been reported in the literature that MR1 is effective in the treatment of rheumatoid arthritis and graft-versus-host disease, both of which have strong immunologic components. Rheumatoid arthritis is an autoimmune disease, and graft-versus-host disease occurs when the host body initiates an active immune response against the transplanted tissue.

[0117] ALS has an immune component mediated by the activation of microglia and astrocytes, but is not considered an autoimmune disease. Some anti-inflammatory drugs such as TNFα-inhibitors, Celebrex®, minocycline, and thalidomide have not been able to show efficacy in preclinical or clinical trials. Therefore, it was unexpected to discover that MR1 showed efficacy in the G93A ALS model.

[0118] The method according to the present disclosure includes treating a patient having a neurodegenerative and / or neuromuscular disease by administering to the patient a compound that blocks the interaction of CD40L and CD40 and / or blocks the interaction of CD28 and CD80 and / or blocks the interaction of CD28 and CD86. One embodiment is a method of treating a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, and spinocerebellar ataxia by administering to the patient a compound that blocks the interaction of CD40L and CD40. In another embodiment, the method of treating a patient is by administering an anti-CD40L antibody.

[0119] In some embodiments, the therapeutic compound can be any compound that blocks the interaction between CD40 and CD40L. For example, many animal studies have described agents that can interfere with CD40:CD40L binding (see, e.g., U.S. Patent Application Publication No. 2005 / 158314 and U.S. Patent No. 7,173,046, which are incorporated herein by reference). Also, for example, many anti-CD40L antibodies have been generated and characterized (see, e.g., U.S. Patent No. 5,876,950 to Bristol-Myers Squibb, which is incorporated herein by reference). Anti-CD40L antibodies useful in the methods of the present disclosure include, but are not limited to, hamster monoclonal antibodies available from MR1, Taconic (Hudson, NY), and BD Biosciences (San Jose, CA); the humanized antibody 5c8 described in U.S. Patent No. 5,474,771 (incorporated herein by reference); the hamster-human chimeric antibody, IDEC131 / E6040, a humanized monoclonal antibody that includes the CDRs of the mouse monoclonal antibody clone 24-31 and human γ-1 heavy chain and human kappa light chain, available from Ancell (Catalog No. X353-020, Bayport, Minn.); ABI 793; Sgn-40; ImxM90 (Immunex); ImxM91 (Immunex); ImxM92 (Immunex); and anti-CD40L mAbs commercially available from Genzyme (Cambridge, Mass., Catalog No. 80-3703-01); Also, anti-CD40L mAbs are commercially available from PharMingen (San Diego, Catalog No. 33580D). Embodiments according to the present disclosure include methods of treating a patient having a neurodegenerative disease or a neuromuscular disease, including administering an anti-CD40L antibody in a therapeutically effective amount. One embodiment is a method of treating a patient having a neurodegenerative disease or a neuromuscular disease, the method comprising administering in a therapeutically effective amount an anti-CD40L antibody selected from MR1, 5c8, IDEC131 / E6040, clone 24-31, ABI793, ImxM90, ImxM91, ImxM92, or Sgn-40. In one embodiment, the antibody is 5c8.In another embodiment, the antibody is MR1.

[0120] In some embodiments, the neurodegenerative or neuromuscular disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia. One embodiment is a method of treating a patient having amyotrophic lateral sclerosis, comprising administering an anti-CD40L antibody in a therapeutically effective amount. In certain embodiments, the anti-CD40L antibody is MR1. In another specific embodiment, the anti-CD40L antibody is 5c8.

[0121] In another embodiment, the method of treatment comprises administering an anti-CD40 antibody in a therapeutically effective amount. In some embodiments, the anti-CD40L compound is a Fab fragment, F(ab’) 2 , F(ab’), single-chain antibody, polypeptide, fusion construct of polypeptides, and the like. In some embodiments, the compound is a small molecule compound capable of blocking the CD40:CD40L interaction. In other embodiments, these compounds include either BIO3417 or a compound disclosed in U.S. Patent No. 7,173,046 having the ability to block the CD40:CD40L interaction.

[0122] Compounds that block the CD40:CD40L interaction can be administered in combination with other compounds. Thus, another embodiment is a method of treating a patient by administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD80:CD28 interaction. In one embodiment, the compound that blocks the CD80:CD28 interaction is galiximab, or H1f1 & h3d1, or 16C10, or 7C10. In one embodiment, the compound that blocks the CD86:CD28 interaction is a CTLA4-Ig protein conjugate such as abatacept or belatacept. Embodiments according to the present disclosure include administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD80:CD28 interaction, or administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD86:CD28 interaction in a therapeutically effective amount, a method of treating a patient having a neurodegenerative disease or a neuromuscular disease, wherein the neurodegenerative disease or neuromuscular disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia. In certain embodiments, the compound that blocks the CD40L:CD40 interaction is MR1, and the compound that blocks the CD28:CD86 interaction or the CD28:CD80 interaction is abatacept, galiximab or belatacept. In other embodiments, the compound that blocks the CD40L:CD40 interaction is 5c8. Another embodiment is a method of treating a patient having amyotrophic lateral sclerosis, comprising administering a therapeutically effective amount of MR1 in combination with abatacept or belatacept. Another embodiment is a method of treating a patient having amyotrophic lateral sclerosis, comprising administering a therapeutically effective amount of 5c8 in combination with abatacept or belatacept.

[0123] Pharmaceutical Compositions and Methods of Administration To treat any of the above-mentioned diseases, the pharmaceutical compositions used according to the methods of the present disclosure can be formulated by conventional techniques using one or more physiologically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and by the particular method used to administer the composition. Thus, there are a variety of suitable formulations for the compounds useful in the methods of the present disclosure (see, e.g., Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro et al. Eds., Lippincott Williams and Wilkins, 2000).

[0124] Formulations suitable for oral administration include, for example, solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multiparticulates or nanoparticles, liquids, or powders; lozenges (such as liquid-filled); chewables; gels; rapid-dissolving dosage forms; films; ovules; sprays; and buccal / mucoadhesive patches.

[0125] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may contain suspending, solubilizing, thickening, stabilizing, and preserving agents.

[0126] According to the present disclosure, the compound can be administered by any suitable means, which may vary depending on the type of disease being treated and the nature of the compound itself. For example, the compound can be administered orally, parenterally, or topically. For proteins such as antibodies, the route of administration preferably includes parenteral administration, such as intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Preferably, parenteral administration is performed by injection, most preferably by intravenous, intramuscular, or subcutaneous injection. The amount administered varies depending on various factors, such as clinical symptoms, the weight of the individual, and whether other drugs are being administered. Determination of the appropriate dosage form, dosage, and route of administration is within the level of those skilled in the fields of pharmacy and medicine, and it should be understood that this is described below.

[0127] In an exemplary pharmaceutical composition containing an anti-CD40L antibody or an antigen-binding fragment thereof, the composition is formulated as a sterile, preservative-free solution of the anti-CD40L antibody or an antigen-binding fragment thereof for intravenous or subcutaneous administration. The formulation can be supplied as a disposable, prefilled pen, such as a disposable prefilled glass syringe containing about 1 mL, or as a disposable vial for institutional use. Preferably, the pharmaceutical composition containing the anti-CD40L antibody or an antigen-binding fragment thereof is transparent or colorless and has a pH in the range of about 5.0 to about 6.9, preferably about 5.0 to about 6.5, and more preferably about 5.0 to about 6.0. In various embodiments, the formulation containing the pharmaceutical composition, when reconstituted and administered to a subject, may contain about 500 mg to about 1 mg, or about 400 mg to about 10 mg, or about 300 mg to about 30 mg, or about 200 mg to about 50 mg of the anti-CD40L antibody or an antigen-binding fragment thereof per mL of the solution.

[0128] The selection of a dosing regimen for treatment varies depending on several factors, such as the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix, the overall health of the patient, and the patient's medical history. Preferably, the dosing regimen minimizes the amount of therapeutic agent delivered to the patient that is consistent with an acceptable level of side effects. Thus, the amount of biologic delivered depends in part on the particular entity and the severity of the medical condition being treated. It is to be understood that the determination of the appropriate dosage form, dosage, and route of administration is within the level of those of ordinary skill in the pharmaceutical and medical arts.

[0129] Dosage and Exposure The pharmaceutical formulations of the present disclosure can contain from about 0.001 to about 200 mg / kg of an anti-CD40L antibody or an antigen-binding fragment thereof, for example, from about 0.001 to about 100 mg / kg, or from about 0.001 to about 50 mg / kg, or from about 0.001 to about 10 mg / kg of an anti-CD40L antibody or an antigen-binding fragment thereof by intravenous injection, and can be administered as a bolus, and the remainder of the antibody dosage can be administered by intravenous injection. A given dosage of an anti-CD40L antibody or an antigen-binding fragment thereof can be administered over a period of time, for example, 1 hour to 2 hours to 5 hours.

[0130] In a further embodiment, a portion of the dosage is administered by subcutaneous injection and / or infusion in the form of a bolus, and the remainder is administered by infusion of the antibody formulation. In some exemplary dosages, the antibody formulation can be subcutaneously administered at dosages in the range of from about 0.001 to about 200 mg / kg of an anti-CD40L antibody or an antigen-binding fragment thereof by intravenous injection, for example, from about 0.001 to about 100 mg / kg, or from about 0.001 to about 50 mg / kg, or from about 0.001 to about 10 mg / kg. In some embodiments, the dosage can be administered as a bolus, and the remainder of the antibody dosage can be administered by subcutaneous or intravenous injection. A given dosage of an anti-CD40L antibody or an antigen-binding fragment thereof can be administered over a period of time, for example, 1 hour, or 2 hours, or 3 hours, or 4 hours or 5 hours, or more.

[0131] In some embodiments, the dosage is administered at a constant rate so as to maintain a steady state in the plasma. In some embodiments, the dosage is administered every two weeks. In some embodiments, the compound is administered such that the plasma of the subject maintains the concentration of the compound at a dosage of 1 μg / mL to 1 mg / mL. In some embodiments, the compound is administered such that the plasma of the subject maintains the concentration of the compound at a dosage of 1 μg / mL to 500 μg / mL. In some embodiments, the compound is administered such that the plasma of the subject maintains the concentration of the compound at a dosage of 1 μg / mL to 250 μg / mL. In some embodiments, the compound is administered such that the plasma of the subject maintains the concentration of the compound at a dosage of 5 μg / mL to 100 μg / mL. In some embodiments, the compound is administered such that the plasma of the subject maintains the concentration of the compound at a dosage of 30 μg / mL to 60 μg / mL.

[0132] Biomarker Some embodiments of the present application relate to modifying biomarkers in a subject with a compound that targets CD40 / CD40L. In some embodiments, the biomarker is a protein involved in CD40 / CD40L activity. In some embodiments, the biomarker is CD40. In some embodiments, the biomarker is CD40L. In some embodiments, the biomarker is NFL. In some embodiments, the biomarker is an inflammation-inducing marker or a marker associated with inflammation. In some embodiments, the biomarker is associated with ALS. In some embodiments, the biomarker is associated with neurodegenerative or neuromuscular diseases or disorders. In some embodiments, the biomarker is a cytokine and / or an interleukin. In some embodiments, the biomarker is one or more of NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrinogen, IgE, ICAM, factor VIII, Ena-78, Pal1, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10, and MMP2.

[0133] In some embodiments, the compound that targets CD40 / CD40L increases the concentration of one or more biomarkers. In some embodiments, treatment with a compound that targets CD40 / CD40L reduces the concentration of one or more biomarkers. In some embodiments, treatment with a compound that targets CD40 / CD40L increases the concentration of at least one biomarker and reduces the concentration of at least one other biomarker. In some embodiments, the baseline level of at least one biomarker serves as a predictor of the response of a subject having a disease, disorder, or condition to treatment with a compound that targets D40 / CD40L.

[0134] Combination therapy The antibodies or antibody fragments described herein can be administered alone (monotherapy) or in combination, i.e., in combination with other agents. For example, in one embodiment, the combination therapy can include one or more additional therapeutic agents. In another embodiment, the combination therapy includes standard therapy that may or may not include an additional therapeutic agent (substantially consisting of an antibody or an antibody fragment thereof).

[0135] Additional or combined administration (co - administration) includes simultaneous administration of any of the antibodies or antigen - binding fragments thereof described herein and one or more agents in the same or different dosage forms, or separate administration (e.g., sequential administration) of the polypeptide and one or more agents. As a result of such simultaneous or sequential administration, preferably, the polypeptide and one or more agents are present simultaneously in the treated patient.

[0136] Kits and Manufactured Articles There is further provided a kit comprising an antibody or an antibody - binding fragment thereof described herein and instructions for use. The kit typically includes a reagent in a predetermined amount and a packaged combination of a label description and a label indicating the purpose of use of the contents of the kit. The terms label and instructions include a document or record on the kit or supplied with the kit, or associated with the kit at any point during its manufacture, transportation, sale, or use. It can take a form defined by a government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, and the notice reflects approval by the agency for manufacture, use, or sale for human administration or veterinary use. The label or instructions can also include advertising brochures and pamphlets, package materials, and audio or video instructions.

[0137] Anti - CD40L Antigen - Binding Molecules Some embodiments provided herein relate to an antibody that is a modified form of the anti - CD40L antibody hu5c8, comprising the variable light - chain and variable heavy - chain CDR sequences of hu5c8 with an Fc domain modified to prevent platelet activation and a human IgG1 consensus framework.

[0138] Table 3 provides the content of the sequence numbers referenced in this application.

[0139]

Table 5

[0140] One embodiment (Embodiment 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 having an amino acid sequence with 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 heavy chain variable region and an Fc region, the heavy chain variable region having an amino acid sequence with 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: 2, and the Fc region having an amino acid sequence with at least 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, and the Fc region comprises one substitution or combination of substitutions selected from the group consisting of C11S, C14S, and P23S.

[0141] Another embodiment (Embodiment B) is the isolated antibody according to Embodiment A, wherein the Fc region further comprises the amino acid substitution C5S.

[0142] In variant forms of Embodiments A and B, the antibody comprises a light chain variable region that does not include any of the substitutions T33W, S26D, and Q27E.

[0143] In other variants of Embodiments A and B, the light chain variable region contains the substitution R28K.

[0144] In some variants of Embodiments A and B, the CDRs of the heavy and light chains have the sequences shown in Table 4.

[0145] [Table 6]

[0146] In still other variants of Embodiments A and B, the light chain variable region contains the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15). In other embodiments, the light chain variable region contains one or both of the amino acid sequences ICRRASQRVSSSTYSYMH (SEQ ID NO: 15), and YASNLES (SEQ ID NO: 16) and QHSWEIPPT (SEQ ID NO: 17).

[0147] In some variants of Embodiments A and B, the light chain variable region contains the amino acid sequence of SEQ ID NO: 1. In still other embodiments, the light chain variable region consists of the amino acid sequence 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 of the amino acid sequence of SEQ ID NO: 7. In still other embodiments, the light chain contains the amino acid sequence of SEQ ID NO: 11. In still other embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 11. In still other embodiments, the light chain consists of the amino acid sequence of SEQ ID NO: 11.

[0148] In other embodiments of Embodiments A and B, the antibody contains a heavy chain variable region that does not contain 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 contain any of the substitutions T33W, S26D, and Q27E. In other embodiments of Embodiments A and B, the light chain variable region does not contain any of the substitutions T33W, S26D, and Q27E, and the heavy chain variable region does not contain any of the substitutions T30H, Y33W, or S54N.

[0149] In other variant forms 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).

[0150] 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 still 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 other embodiments, the heavy chain variable region consists essentially of the amino acid sequence of SEQ ID NO: 6. In still other embodiments, the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 6.

[0151] Some embodiments provided herein relate to an isolated antibody in which 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.

[0152] Some embodiments provided herein relate to an isolated antibody in which 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.

[0153] Some embodiments provided herein relate to an isolated antibody in which 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.

[0154] Still another embodiment is an isolated antibody in which 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.

[0155] Yet another embodiment is an isolated antibody in which 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.

[0156] Another embodiment is an isolated antibody in which 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.

[0157] Some embodiments provided herein relate to the alternatives listed below.

[0158] A method of predicting the response of a subject having a disease or disorder to treatment with a compound against CD40L or CD40, comprising: collecting a sample from the subject; measuring the concentration of at least one biomarker present in the sample, wherein the at least one biomarker is NFL, MMP9, MMP3, IL6, IL18, IgA, TNFR2, TNFa, IL2ra, Factor VIII, CRP, SAP, MCP1, En-Raged, MIG, vWF, IgE, IL8, IP10, C3, and MMP2; predicting from the concentration of the at least one biomarker that the subject may be responsive to treatment of the disease or disorder with a compound against CD40L or CD40.

[0159] The method of alternative 1, wherein the disease or disorder is an inflammatory disease, a neurodegenerative disease, or a neuromuscular disease.

[0160] The method of alternative 2, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia.

[0161] The method of alternative 3, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

[0162] The method according to any one of alternatives 1 to 4, wherein the subject is a mammal and / or a human.

[0163] The method according to any one of alternatives 1 to 5, wherein the compound blocks the interaction between CD40 and CD40L.

[0164] The method according to any one of alternatives 1 to 6, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0165] The method according to any one of alternatives 1 to 7, wherein the compound is tegapertide.

[0166] The method according to any one of alternatives 1 to 8, wherein the compound is MR1 or 5c8.

[0167] A method of treating a subject having a disease or disorder, comprising administering a compound against CD40L or CD40 in a therapeutically effective dose, wherein upon administration of the therapeutically effective dose, the compound in the plasma of the subject is maintained at a concentration of 5 to 100 μg / mL; preferably at a dose of 30 to 60 μg / mL.

[0168] The method of alternative 10, wherein the disease or disorder is an inflammatory disease, a neurodegenerative disease, or a neuromuscular disease.

[0169] The method of alternative 11, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia.

[0170] The method of alternative 12, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

[0171] The method according to any one of alternatives 10 to 13, wherein the subject is a mammal and / or a human.

[0172] The method according to any one of alternatives 10 to 14, wherein the compound blocks the interaction between CD40 and CD40L.

[0173] The method according to any one of alternatives 10 to 15, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0174] The method according to any one of alternatives 10 to 16, wherein the compound is tegapertide.

[0175] The method according to any one of alternatives 10 to 17, wherein the compound is MR1 or 5c8.

[0176] The method according to any one of alternatives 10 to 18, wherein the compound is administered once every at least two weeks.

[0177] The method according to any one of alternatives 10 to 19, wherein the compound is administered for a period of at least 12 weeks.

[0178] The method according to any one of alternatives 10 to 20, wherein the compound is administered at a dose of 200 to 100 mg / kg; preferably at a dose of 1 to 8 mg / kg; most preferably at a dose of 2 to 4 mg / kg.

[0179] The method according to any one of alternatives 10 to 21, further comprising administering a second pharmaceutically effective compound.

[0180] The method according to alternative 22, wherein the second compound blocks the interaction of CD28 and CD86 or the interaction of CD28 and CD80.

[0181] The method according to alternative 22, wherein the second compound targets at least one of the biomarkers selected from the group consisting of interleukin, cytokine, and inflammation-inducing markers.

[0182] The method according to alternative 24, wherein the second compound targets at least one of the biomarkers selected from the group consisting of NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrinogen, IgE, ICAM, factor VIII, Ena-78, Pal1, lanthes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, and MMP2.

[0183] The method of alternative 22, wherein the second compound is CTLA4-Ig fusion protein, abatacept, belatacept, or galiximab.

[0184] The method according to any one of alternatives 10 to 26, wherein the compound is administered orally, parenterally, or topically.

[0185] The method of alternative 27, wherein the compound is administered parenterally.

[0186] The method of alternative 28, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

[0187] The method according to any one of alternatives 1 to 29, 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 immune response, graft rejection, and atherosclerosis.

[0188] The method according to any one of alternatives 1 to 30, 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, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease.

[0189] The compound targeting CD40L or CD40 is an antibody comprising (a) a heavy chain variable region (V H ) comprising i) a CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; ii) a CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; and iii) a CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; and (b) a light chain variable region (V L ) comprising i) a CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; ii) a CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; and iii) a CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20, according to any one of alternatives 1 to 31.

[0190] A method according to any one of alternatives 1 to 32, wherein the compound targeting CD40L or CD40 is an antibody comprising (a) a heavy chain variable region (V H ) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4, and (b) a light chain variable region (V L ) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8.

[0191] A method of modulating the concentration of at least one biomarker in a subject, comprising administering to the subject a therapeutically effective amount of a compound against CD40L or CD40, wherein the at least one biomarker is NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrinogen, IgE, ICAM, factor VIII, Ena-78, Pal1, lactoferrin, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10, or MMP2.

[0192] The method of alternative 34, wherein the concentration of at least one biomarker increases after administration of the compound.

[0193] The method of alternative 34, wherein the concentration of at least one biomarker decreases after administration of the compound.

[0194] The method of alternative 34, wherein the concentration of at least one biomarker increases after administration of the compound and at least one other biomarker decreases after administration of the compound.

[0195] The method according to any one of alternatives 34 to 37, wherein the subject has a disease or disorder.

[0196] The method of alternative 38, wherein the disease or disorder is an inflammatory disease, a neurodegenerative disease, or a neuromuscular disease.

[0197] The method of alternative 39, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia.

[0198] The method of alternative 40, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

[0199] The method according to any one of alternatives 34 to 41, wherein the subject is a mammal and / or a human.

[0200] The method according to any one of alternatives 34 to 42, wherein the compound blocks the interaction between CD40 and CD40L.

[0201] The method according to any one of alternatives 34 to 43, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0202] The method according to any one of alternatives 34 to 44, wherein the compound is tegoprubart.

[0203] The method according to any one of alternatives 34 to 45, wherein the compound is MR1 or 5c8.

[0204] The method according to any one of alternatives 34 to 46, wherein the compound in the plasma of the subject is maintained at a concentration of 5 to 100 μg / mL; preferably at a dose of 30 to 60 μg / mL, by administration of a therapeutically effective dose.

[0205] The method according to any one of alternatives 34 to 47, wherein the compound is administered at least once every two weeks.

[0206] The method according to any one of alternatives 34 to 48, wherein the compound is administered for a period of at least 12 weeks.

[0207] The method according to any one of alternatives 34 to 49, wherein the compound is administered at a dose of 200 to 100 mg / kg; preferably at a dose of 1 to 8 mg / kg; most preferably at a dose of 2 to 4 mg / kg.

[0208] The method according to any one of alternatives 34 to 50, wherein the compound is administered orally, parenterally, or topically.

[0209] The method according to alternative 51, wherein the compound is administered parenterally.

[0210] The method according to alternative 52, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

[0211] The method according to any one of alternatives 34 to 53, 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 immune response, graft rejection, and atherosclerosis.

[0212] The method according to any one of alternatives 34 to 54, 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, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease.

[0213] The compound targeting CD40L or CD40 is an antibody comprising (a) a heavy chain variable region (V H ) comprising i) a CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; ii) a CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; and iii) a CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; and (b) a light chain variable region (V L ) comprising i) a CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; ii) a CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; and iii) a CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20, according to any one of alternatives 34 to 55.

[0214] A compound that targets CD40L or CD40, wherein the method according to any one of alternatives 34 to 56 is an antibody comprising (a) a heavy chain variable region (V H ) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4, and (b) a light chain variable region (V L ) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8.

[0215] A method of maintaining or improving the ALS-FRS score in a subject having ALS, comprising administering a compound against CD40L or CD40 in a therapeutically effective dose, wherein by administering the therapeutically effective dose, the compound in the plasma of the subject is maintained at a concentration of 5 to 100 μg / mL; preferably at a dose of 30 to 60 μg / mL.

[0216] A method of pre-screening and treating a subject having a disease or disorder with a compound against CD40L or CD40, comprising collecting a sample from the subject; measuring the concentration of at least one biomarker present in the body fluid, wherein the at least one biomarker is NFL, MMP9, MMP3, IL6, IL18, IgA, TNFR2, TNFa, IL2ra, factor VIII, CRP, SAP, MCP1, En-Raged, MIG, vWF, IgE, IL8, IP10, C3, or MMP2; screening at least one biomarker concentration against the concentration of at least one biomarker in a subject without the disease or disorder; administering the compound in a therapeutically effective dose such that the plasma of the subject maintains the concentration of the compound at a dose of 5 to 100 μg / mL, preferably at a dose of 30 to 60 μg / mL.

[0217] The method of alternative 59, wherein the compound is administered when the concentration of at least one biomarker in the subject is significantly different from the concentration of at least one biomarker in a subject without the disease or disorder.

Examples

[0218] Examples The embodiments provided herein are further defined in the following examples. It should be understood that these examples are merely illustrative. From the above discussion and these examples, those skilled in the art can identify the essential features of the present disclosure and, without departing from its spirit and scope, make various changes and modifications to the embodiments of the present disclosure and adapt it to various uses and conditions. Therefore, in addition to what is shown and described herein, modifications to the embodiments will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be within the scope of the appended claims. The disclosure of each reference described herein is hereby incorporated by reference in its entirety and for the disclosure referred to herein in this specification.

[0219] Example 1 Clinical Trial Design The efficacy of compounds targeting CD40L or CD40 was evaluated in a clinical trial. A total of 54 human subjects were involved; these subjects were selected based on a positive ALS diagnosis or a diagnosis that was possible, probable, or definite according to the laboratory-supported revised El Escorial criteria. Each subject also had a total ALSFRS-R score of 35 or more and was less than 24 months from initial diagnosis. Further, each subject selected for the study had undergone tracheotomy or used a permanent assisted ventilation system. The demographics of the selected subjects are shown in Table 5.

[0220] [Table 7]

[0221] There were four dose cohorts: 1 mg / kg (n = 9), 2 mg / kg (n = 9), 4 mg / kg (n = 18), and 8 mg / kg (n = 18). As shown in Figure 22, for each subject, two biological samples of blood were collected as baseline data either 28 days or 4 days before the first administration of the compound. On day 0, another two biological samples were collected. These biological samples consisted of one plasma sample for pK / ADA analysis and one plasma sample for biomarker level analysis.

[0222] Following the collection of the biological samples, each subject immediately received an IV infusion of compound AT-1501 (also known as tegoprubart) at either 1 mg / kg, 2 mg / kg, 4 mg / kg, or 8 mg / kg. This pattern of administration of AT-1501 at set doses was repeated every 14 days for a total of 101 days. Infusions were performed on days 0, 15, 29, 43, 57, and 71. Two additional biological samples were collected 2 hours after the infusion.

[0223] After the dose study was completed, the endpoints to be analyzed included: safety and tolerability, pharmacokinetics, and anti-drug antibody levels, CD40L target binding (target engagement), changes in inflammatory markers, and changes in ALSFRS-R, respiratory function, and neurofilament light chain (NFL) levels.

[0224] Safety Over 90% of the subjects completed the clinical trial as summarized in Table 6. One subject marked "other" in the table was eligible at screening but did not receive the administration and was replaced by another participant instead.

[0225]

Table 8

[0226] The safety of AT-1501 was evaluated by counting the number of treatment-emergent adverse events (TEAEs) that occurred in subjects over the course of the clinical trial. The results are summarized in Table 7. Overall, tegoprubart was well tolerated, with no serious treatment-related adverse events or significant antidrug antibody responses.

[0227]

Table 9

[0228] The detailed symptoms of adverse effects and the percentage of subjects who self-reported those symptoms are summarized in Table 8.

[0229]

Table 10

[0230] Example 2 Pharmacokinetics and Antidrug Antibody Concentrations As discussed in Example 1, plasma biosamples were collected from subjects before dosing and 2 hours after infusion dosing. Infusions were performed on Days 0, 15, 29, 43, 57, and 71 of the clinical trial. These biosamples were used in part to evaluate the concentration of the AT-1501 drug level present in the blood. An overlay of the AT-1501 levels for each subject over time is shown in Figure 23. From this analysis, the half-life of AT-1501 was determined to exceed 14 days. As a result, the exposure level increased in subjects over time and thus did not reach a steady state.

[0231] Furthermore, there was no evidence of ADA neutralization. Among 215 samples from 7 subjects, 9 ADA were present (Figures 24A - 24I). When the subject was away from the test substance, 4 out of 9 ADA were detected at week 14. One subject (102201) had an existing ADA titer before the test. Table 9 shows the ADA - positive subjects and the time points of appearance. All 9 ADA had very low titers and were at the detection cut - off point in the assay. These ADA were non - neutralizing and did not affect the level of the drug tegoprubart (AT - 1501) administered to those subjects.

[0232]

Table 11

[0233] Example 3 General biomarker analysis Each biological sample collected from the subjects during the clinical trial was also evaluated for changes in molecular biomarkers. These markers included markers used as measurements of CD40L target engagement (CXCL13, IgA, and CD40L) and as measurements of inflammation (TNF - α, IL - 6, En - Raged MCP - 1, IL - 1, IL1 - β, and CRP). Two methods of analysis were used to examine the biomarkers: (1) calculating a regression model for all subjects, plotting all specimens, and (2) normalizing the data and then calculating the fold - change of the biomarkers over time and correcting for the false discovery rate.

[0234] Before analysis, data pre - processing was performed to exclude outliers such as subjects with scores less than 4 in the bulbar sub - domain and ALSFRS less than 35 on day 0. The subjects excluded as outliers were summarized in Table 10.

[0235]

Table 12

[0236] Furthermore, proteins with weak positive signals were filtered. These proteins included: AFP, CA-125, CA-19-9, eotaxin-1, EPO, G-CSF, GH, GM-CSF, hCG, INFg, IL1-a, IL1-b, IL10, IL12-p40, IL12-p70, IL-13, IL-17, IL1-ra, IL-2, IL-3, IL-4, IL-5, IL-6 (Luminex), IL-7, MCP-2, MICA, MIP1-A, PSA-F, SCF, and TNFa (Luminex).

[0237] Analysis revealed that a significant number of proteins were downregulated after tegopuribart treatment (Figure 25). The degree of protein increase or downregulation was dose-dependent.

[0238] Biomarkers of target engagement Engagement of CD40L and CD40 by AT-1501 was evaluated by monitoring the levels of CXCL13, CD40L, and CD40 in the subjects. AT-1501 exposure reduced all three protein levels in a dose-dependent manner (Figures 26A, 26B, 27A, 27B, 28A, and 28B). The decrease was statistically significant at doses of 4 mg / kg and 8 mg / kg. The maximum decrease was achieved by day 2 after administering AT-1501 to the subjects. This demonstrated that the CD40L / CD40 target was significantly bound by the treatment.

[0239] For the change in concentration after treatment of the subject with AT-1501, the levels of inflammatory cytokine biomarkers and other related stress proteins were also evaluated. Among those evaluated, CD40L, CXCL13, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, IL2r, CXCL10, CD40, and B2M had the greatest reduction in protein concentration after AT-1501 treatment (Figures 29A - 29L). A significant reduction in concentration was also detected for the proteins VCAM, IL-18, IL-16, SAP, MIP1-β, MDC, C3, CRP, fibrinogen, IgE, ICAM, and MCP1 (Figures 30A - 30L). Exposure of the subject to AT-1501 increased the concentration of several proteins; these included IL-6, ENA-78, Rantes, MMP3, and MMP9 (Figures 31A - 31E). Overall, treatment of the subject with AT-1501 reduced the expression of more than 50% of the proteins examined that are associated with inflammation and co-stimulatory signaling.

[0240] The concentration of the neurofilament light chain (NFL) level was also evaluated at week 12 (Figure 32). There was no statistically significant change in NFL concentration at any dose. However, the NFL level at the time of enrollment was not correlated with the rate of progression.

[0241] Example 4 ALS Functional Rating Scale During the clinical trial, every two weeks, the subject was evaluated by a clinician for changes in its ALS Functional Rating Scale (ALS-FRS). The change in ALS-FRS score was plotted for all subjects over time; overall, each cohort deteriorated at a rate equivalent to that of the general population of untreated patients supplied by the database Pro-ACT (Figure 33A).

[0242] Next, the data were corrected to exclude patients who no longer met the initial enrollment criteria at week 12 (Figure 33B). This decreased the overall error for each group, and the overall trend of the ALS-FRS scores strongly correlated with the proportion of untreated patients.

[0243] The data were further corrected to exclude patients with treatment-related effects (TE) (Figure 33C). This mostly reduced the decline in FRS for subjects administered low levels of AT-1501, but had no significant effect on the higher dose cohort.

[0244] Finally, the data were corrected only to include patients who appeared to respond best to treatment with AT-1501 (hereinafter referred to as "responders") (Figure 33D). This most significantly shifted the data, and subjects in the treatment group, particularly in the 4 mg / kg dose cohort, had a reduced decline in ALS-FRS scores compared to untreated.

[0245] Table 11 shows the overall slopes of monthly changes in ALS-FRS for human subjects by dosing group. The columns are: "All" (patients are not excluded), "EC" (patients not meeting enrollment criteria are excluded), "TE" (patients with treatment-related adverse effects are excluded), and "Res" (only patients who responded positively to drug treatment are included); The "Pro-Act" column shows ALS-FRS data for untreated patients obtained from the Pro-ACT database.

[0246]

Table 13

[0247] Responder vs Non-responder Why did some patients respond well to AT-1501 (referred to as "responders"), while others did not (referred to as "non-responders")? To answer this question, for each individual, the ALS-FRS score was plotted over time. The ALS-FRS score values of 16 of these individuals (8 responders and 8 non-responders) are as shown in Table 12 and are plotted in graphical form in Figure 34.

[0248]

Table 14

[0249] As can be seen from Figure 34 and the above table, the subgroup of "responders" had no significant change in ALS-FRS from baseline to 12 weeks. On the other hand, the "non-responders" declined at a rate consistent with untreated patients. This indicates that while the "non-responder" group did not obtain a detectable benefit from AT-1501, the "responders" acquired significant resistance to the decline in ALS-FRS score when treated with AT-1501.

[0250] Next, the protein concentrations in each subject before treatment with AT-1501 (baseline values) were compared by multiple linear regression analysis. As shown in Figure 35 and Tables 13 - 14, there were several proteins that were present at significantly higher concentrations in the plasma of subjects who would later have a positive response to treatment with AT-1501. Table 13 shows the F statistic and p value of the difference in protein mean concentration for each protein at baseline plasma levels, normalized to intensity. The difference in each protein at baseline was also analyzed using linear regression as a predictor of the slope of the ALS-FRS change; these values were as shown in Table 14. Among these, the initial concentration of NFL (not shown in Figure 35 due to scale differences) with an F statistic of nearly 50 points was the most significant. Also, the most significant initial protein concentrations were MMP9, MMP3, Il2ra, TNF-α, and IL-18.

[0251]

Table 15

[0252]

Table 16

[0253] As shown in FIGS. 35 and Tables 12 - 13, there were statistically significant differences in protein marker expression between non - responders and responders prior to any treatment. None of these protein levels were significantly correlated with covariates such as race, gender, age, or treatment time.

[0254] This finding is highly significant because early assessment of the concentrations of these proteins in the plasma of ALS patients could serve as a predictor of patient responsiveness to AT - 1501 treatment. That is, individuals with biomarkers at concentrations consistent with the responder cohort may potentially benefit significantly from treatment with compounds that target CD40L / CD40.

[0255] Summary Overall, tegoprubart has demonstrated a good safety profile with linear pK and dose - proportionality with a low incidence of ADA. Furthermore, tegoprubart has demonstrated target engagement with dose - dependent decreases in the T - cell marker CD40L and the B - cell markers CXCL13, IgA, and IgE, as well as an increase in patient response with dose escalation. Tegoprubart also decreased three of the four predicted and selected pro - inflammatory markers TNFa, MCP1, and En - Raged in a dose - dependent manner while increasing patient response. IL1b was below the detection limit of multiplex ELISA and IL6 was not down - regulated by tegoprubart.

[0256] Tegoprubart reduced the expression of 23 out of 32 assayed inflammatory proteins and clearly demonstrated downstream regulation of inflammatory signaling, which has been described for CD40L inhibition in multiple preclinical models of autoimmunity and transplantation. The decrease in inflammatory signaling trended towards an improvement in disease progression, as measured by the ALSFRS. Several proteins were identified by linear regression that correlated with changes in ALSFRS over time.

[0257] Again, from the findings reported in this application, it has been shown that initial quantification of select proteins in the plasma of ALS patients can serve as a predictor of patient responsiveness to treatment with compounds that target CD40L / CD40.

[0258] The section headings used herein are for purposes of organization and should in no way be construed as limiting the subject matter described. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, academic papers, and Internet web pages, are hereby expressly incorporated by reference in their entirety for any purpose, such as the disclosure specifically referenced herein. Where definitions of terms in incorporated references appear to differ from those provided in the teachings of the present invention, the definitions provided in the teachings of the present invention shall control. It should be understood that the term "about" may precede temperatures, concentrations, times, etc. contemplated in the teachings of the present invention so that minor and insignificant deviations are within the scope of the teachings of the present invention herein.

[0259] Although this disclosure is presented in the context of specific embodiments and examples, it will be understood by those skilled in the art that the disclosure extends beyond the specifically disclosed embodiments to cover other alternative embodiments, and / or the use of embodiments and their obvious modifications and equivalents. Further, while some variations of the disclosure are shown and described in detail, other modifications within the scope of the disclosure will be readily understood by those skilled in the art based on this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments can be made and remain within the scope of the embodiments. It should be understood that the various features and aspects of the disclosed embodiments can be combined with or replaced by one another to form various modes or embodiments. Accordingly, it is intended that the scope of the disclosure not be limited by the specific embodiments disclosed above.

[0260] However, since various changes and modifications within the spirit and scope of the disclosure will be apparent to those skilled in the art, it should be understood that this detailed description showing preferred embodiments is presented by way of example only.

[0261] The terminology used in the description presented herein is not intended to be construed in a limiting or restrictive sense. Rather, the terminology is simply used in combination with a detailed description of embodiments of systems, methods, and related components. Further, an embodiment can include several new features, none of which, by itself, is the cause of the desired characteristics or is considered essential to the implementation of the embodiment.

Claims

1. A pharmaceutical composition for treating a subject with a disease or disorder: The pharmaceutical composition comprises a compound relative to CD40L or CD40 in a therapeutically effective dose, wherein administration of the therapeutically effective dose maintains the compound in the target plasma at a concentration of 5 to 100 μg / mL; preferably at a dose of 30 to 60 μg / mL.

2. The pharmaceutical composition according to claim 1, wherein the disease or disorder is inflammation, neurodegenerative disease, or neuromuscular disease.

3. The pharmaceutical composition according to claim 2, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia.

4. The pharmaceutical composition according to claim 3, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

5. The pharmaceutical composition according to claim 1, wherein the subject is a mammal and / or a human.

6. The pharmaceutical composition according to claim 1, wherein the compound blocks the interaction between CD40 and CD40L.

7. The pharmaceutical composition according to claim 1, wherein the compound is an anti-CD40L antibody or an antigen-binding fragment thereof, or an anti-CD40 antibody or an antigen-binding fragment thereof.

8. The pharmaceutical composition according to claim 1, wherein the compound is tegopulvat.

9. The pharmaceutical composition according to claim 1, wherein the compound is MR1 ​​or 5c8.

10. The pharmaceutical composition according to claim 1, wherein the compound is administered at least once every two weeks.

11. The pharmaceutical composition according to claim 1, wherein the compound is administered for a period of at least 12 weeks.

12. The pharmaceutical composition according to claim 1, wherein the compound is administered in a dose of 200 to 100 mg / kg; preferably 1 to 8 mg / kg; most preferably 2 to 4 mg / kg.

13. The pharmaceutical composition according to claim 1, wherein the treatment further comprises administering a second pharmaceutically effective compound.

14. The pharmaceutical composition according to claim 13, wherein the second compound blocks the interaction between CD28 and CD86 or the interaction between CD28 and CD80.

15. The pharmaceutical composition according to claim 13, wherein the second compound targets at least one biomarker selected from the group consisting of interleukins, cytokines, and pro-inflammatory markers.

16. The pharmaceutical composition according to claim 15, wherein the second compound targets at least one of the biomarkers selected from the group consisting of NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrogen, IgE, ICAM, factor VIII, Ena-78, Pal1, Lantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, and MMP2.

17. The pharmaceutical composition according to claim 13, wherein the second compound is a CTLA4-Ig fusion protein, abatacept, beratacept, or galiximab.

18. The pharmaceutical composition according to claim 1, wherein the compound is administered orally, parenterally, or topically.

19. The pharmaceutical composition according to claim 18, wherein the compound is administered parenterally.

20. The pharmaceutical composition according to claim 19, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

21. The pharmaceutical composition according to claim 1, 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 immune response, graft rejection, and atherosclerosis.

22. The pharmaceutical composition according to claim 1, 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, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease.

23. The compound that targets CD40L or CD40 is (a) i) CDRH1 domain containing the sequence described in Sequence ID No. 18; ii) CDRH2 domains containing the sequence described in Sequence ID No. 19; and iii) CDRH3 domain containing the sequence described in Sequence ID No. 20; Heavy chain variable region including (V H )and, (b) i) CDRL1 domain containing the sequence described in Sequence ID No. 15; ii) CDRL2 domains containing the sequence described in Sequence ID No. 16; and iii) CDRL3 domain containing the sequence described in Sequence ID No. 17; Light chain variable region including (V L )and, The pharmaceutical composition according to claim 1, comprising an antibody or an antigen-binding fragment thereof.

24. The compound that targets CD40L or CD40 is (a) Heavy chain variable region having the amino acid sequence described in SEQ ID NO: 2 or 6 (V H )and, (b) Light chain variable region having the amino acid sequence described in SEQ ID NO: 1 or 5 (V L )and, A pharmaceutical composition according to any one of claims 1 to 23, comprising an antibody or an antigen-binding fragment thereof.

25. A pharmaceutical composition for adjusting the concentration of at least one biomarker in a subject: The pharmaceutical composition comprises a therapeutically effective amount of CD40L or a compound for CD40, wherein the at least one biomarker is NFL, CXCL13, CD40L, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, Il2r, CXCL10, CD40, B2M, VCAM, Il-18, Il-16, SAP, MIP1β, MDC, C3, CRP, fibrogen, IgE, ICAM, factor VIII, Ena-78, Pal1, Lantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10, or MMP2.

26. The pharmaceutical composition according to claim 25, wherein the concentration of at least one of the biomarkers increases after administration of the compound.

27. The pharmaceutical composition according to claim 25, wherein the concentration of at least one of the biomarkers decreases after administration of the compound.

28. The pharmaceutical composition according to claim 25, wherein, after administration of the compound, the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases.

29. The pharmaceutical composition according to claim 25, wherein the subject has a disease or disorder.

30. The pharmaceutical composition according to claim 29, wherein the disease or disorder is inflammation, neurodegenerative disease, or neuromuscular disease.

31. The pharmaceutical composition according to claim 30, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia.

32. The pharmaceutical composition according to claim 31, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

33. The pharmaceutical composition according to claim 25, wherein the subject is a mammal and / or a human.

34. The pharmaceutical composition according to claim 25, wherein the compound blocks the interaction between CD40 and CD40L.

35. The pharmaceutical composition according to claim 25, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

36. The pharmaceutical composition according to claim 25, wherein the compound is tegopulvat.

37. The pharmaceutical composition according to claim 25, wherein the compound is MR1 ​​or 5c8.

38. The pharmaceutical composition according to claim 25, wherein, upon administration of the therapeutically effective dose, the compound in the target plasma is maintained at a concentration of 5 to 100 μg / mL; preferably at a dose of 30 to 60 μg / mL.

39. The pharmaceutical composition according to claim 25, wherein the compound is administered at least once every two weeks.

40. The pharmaceutical composition according to claim 25, wherein the compound is administered for a period of at least 12 weeks.

41. The pharmaceutical composition according to claim 25, wherein the compound is administered in a dose of 200 to 100 mg / kg; preferably 1 to 8 mg / kg; most preferably 2 to 4 mg / kg.

42. The pharmaceutical composition according to claim 25, wherein the compound is administered orally, parenterally, or topically.

43. The pharmaceutical composition according to claim 42, wherein the compound is administered parenterally.

44. The pharmaceutical composition according to claim 43, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

45. The pharmaceutical composition according to claim 25, 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 immune response, graft rejection, and atherosclerosis.

46. The pharmaceutical composition according to claim 25, wherein the disease or disorder is an autoimmune disease selected from the group consisting of erythema of systemic lupus erythematosus, type 1 diabetes mellitus, myasthenia gravis, inflammatory bowel disease, immune thrombocytopenic purpura, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease.

47. The compound that targets CD40L or CD40 is (a) i) CDRH1 domain containing the sequence described in Sequence ID No. 18; ii) CDRH2 domains containing the sequence described in Sequence ID No. 19; and iii) CDRH3 domain containing the sequence described in Sequence ID No. 20; Heavy chain variable region including (V H )and, (b) i) CDRL1 domain containing the sequence described in Sequence ID No. 15; ii) CDRL2 domains containing the sequence described in Sequence ID No. 16; and iii) CDRL3 domain containing the sequence described in Sequence ID No. 17; Light chain variable region including (V L )and, The pharmaceutical composition according to claim 25, comprising an antibody or an antigen-binding fragment thereof.

48. The compound that targets CD40L or CD40 is (a) Heavy chain variable region having the amino acid sequence described in SEQ ID NO: 2 or 6 (V H )and, (b) Light chain variable region having the amino acid sequence described in SEQ ID NO: 1 or 5 (V L )and, The pharmaceutical composition according to claim 25, comprising an antibody or an antigen-binding fragment thereof.

49. A pharmaceutical composition for maintaining or improving the ALS-FRS score of a person with ALS: The pharmaceutical composition comprises a compound relative to CD40L or CD40 in a therapeutically effective dose, wherein administration of the therapeutically effective dose maintains the concentration of the compound in the target plasma at a concentration of 5 to 100 μg / mL; preferably at a dose of 30 to 60 μg / mL.

50. A method for assisting in pre-screening subjects having a disease or disability, The method involves detecting the concentration of at least one biomarker present in a sample from the subject, wherein the at least one biomarker is NFL, MMP9, MMP3, IL6, IL18, IgA, TNFR2, TNFa, IL2ra, factor VIII, CRP, SAP, MCP1, En-Raged, MIG, vWF, IgE, IL8, IP10, C3, or MMP2; Screening the concentration of at least one biomarker against the concentration of at least one biomarker in subjects without the aforementioned disease or disorder; If the concentration of at least one biomarker in the subject is significantly different from the concentration of at least one biomarker in a subject without disease or impairment, it is determined that it is necessary to administer the compound to CD40L or CD40 in a therapeutically effective dose such that the plasma of the subject maintains a concentration of the compound at a dose of 5 to 100 μg / mL; preferably 30 to 60 μg / mL; Methods that include...

51. A method for assisting in predicting the response of a subject with a disease or disorder to treatment with a compound against CD40L or CD40, The method involves measuring the concentration of at least one biomarker present in a sample from the subject, wherein the at least one biomarker is NFL, MMP9, MMP3, IL6, IL18, IgA, TNFR2, TNFa, IL2ra, factor VIII, CRP, SAP, MCP1, En-Raged, MIG, vWF, IgE, IL8, IP10, C3, and MMP2; Based on the concentration of at least one of the aforementioned biomarkers, it can be predicted that the subject may be responsive to treatment of the disease or disorder with a compound for CD40L or CD40; A method that includes this.

52. The method according to claim 51, wherein the disease or disorder is inflammation, neurodegenerative disease, or neuromuscular disease.

53. The method according to claim 52, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, or spinocerebellar ataxia.

54. The method according to claim 53, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

55. The method according to claim 51, wherein the subject is a mammal and / or a human.

56. The method according to claim 51, wherein the compound blocks the interaction between CD40 and CD40L.

57. The method according to claim 51, wherein the compound is an anti-CD40L antibody or an antigen-binding fragment thereof, or an anti-CD40 antibody or an antigen-binding fragment thereof.

58. The method according to claim 51, wherein the compound is tegopulvat.

59. The method according to claim 51, wherein the compound is MR1 ​​or 5c8.

60. The method according to claim 51, 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 immune response, graft rejection, and atherosclerosis.

61. The method according to claim 51, 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, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease.

62. The compound that targets CD40L or CD40 is (a) i) CDRH1 domain containing the sequence described in Sequence ID No. 18; ii) CDRH2 domains containing the sequence described in Sequence ID No. 19; and iii) CDRH3 domain containing the sequence described in Sequence ID No. 20; The heavy chain variable region (VH) includes, (b) i) CDRL1 domain containing the sequence described in Sequence ID No. 15; ii) CDRL2 domains containing the sequence described in Sequence ID No. 16; and iii) CDRL3 domain containing the sequence described in Sequence ID No. 17; The light chain variable region (VL) includes, The method according to claim 51, wherein the antibody contains or an antigen-binding fragment thereof.

63. The compound that targets CD40L or CD40 is (a) A heavy chain variable region (VH) having the amino acid sequence described in SEQ ID NO: 2 or 6, (b) A light chain variable region (VL) having the amino acid sequence described in SEQ ID NO: 1 or 5, The method according to any one of claims 51 to 62, wherein the antibody contains or an antigen-binding fragment thereof.

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