Methods for treating amyotrophic lateral sclerosis

Abstract

Provided herein are methods and kits for the treatment of nervous system degenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal amyotrophy, Kenedia disease, and spinal cerebellar ataxia. Also provided are methods of predicting or measuring response to treatment by measuring the level of a biomarker in a sample, as well as methods of modulating the level of a biomarker.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 365,458, filed on May 27, 2022, which is incorporated by reference in its entirety.

[0003] References to sequence listings

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file titled "ELDN.010WO.xml" created on April 20, 2023, which is 27,094 bytes in size. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety.

[0005] background

[0006] field

[0007] The present disclosure relates to methods for treating neurodegenerative diseases such as amyotrophic lateral sclerosis. Methods of predicting and / or measuring responses to treatment are also provided, comprising measuring the level of one or more biomarkers. Embodiments also relate to methods of regulating the level of one or more biomarkers.

[0008] Related technical notes

[0009] Amyotrophic lateral sclerosis (ALS) (sometimes called Lou Gehrig's disease) is a progressive, fatal neurological disorder characterized by muscle fiber atrophy caused by degeneration of motor neurons in the spine and brain. ALS affects approximately 30,000 U.S. citizens, of which only about 10% of cases are classified as familial forms of ALS. In a subgroup of familial patients with mutations in the metabolic enzyme superoxide dismutase 1 (SOD1), pathological progression may be attributed to unknown functional gain associated with the mutant form of the enzyme (SOD1 dependency) (Rosen, 1993). However, in most 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). Therefore, the remaining 90% of ALS cases are classified as sporadic cases without a well-characterized genetic component or causative factor.

[0010] Because the cause of the sporadic form of the disease is unknown, researchers have turned to transgenic strategies to create laboratory models of the disease. The identification of the SOD1 gene has led to the generation of transgenic rodent models of ALS. Humans carrying 23 copies of SOD1 G93AThe transgenic transgenic mouse strain ("G93A mouse") is the most widely used ALS mouse model and is accepted as the standard model for ALS treatment research ("G93A mouse") (see Tu PH et al. (1996) Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 93:3155-3160 and Gurney ME (1997) J Neuro Sci [Journal of Neuroscience] 152 Suppl 1:S67-S73).

[0011] Although ALS is characterized by the loss of motor neurons in the spinal cord (causing muscle atrophy), the disease itself also manifests itself as changes in axonal transport, protein aggregation, excitotoxicity, astrocytosis, mitochondrial dysfunction, microglial activation and synaptic remodeling. The presence of microglial activation, astrocytosis and infiltrating inflammatory cells from the periphery has been well described. There is an 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 of ALS. Although little is known about the role of infiltrating immune cells, recent work will show that infiltrating T cell populations are neuroprotective rather than cytotoxic. Although ALS has an immune component mediated by the activation of microglia and astrocytes, it is not considered an autoimmune disorder. Unlike diseases such as rheumatoid arthritis or systemic lupus erythematosus that have been described to involve specific immunomodulatory pathways (e.g., costimulatory pathways), ALS has not yet been described to involve such pathways.

[0012] Currently, doctors have limited options for treating ALS. At this time, riluzole is the only drug that the FDA has approved for the treatment of ALS. In clinical trials, riluzole showed only a slight benefit in modestly increasing survival time. Therefore, there is a pressing need for effective therapies for ALS.

[0013] Overview

[0014] Disclosed herein is a method for predicting the response of a subject with a disease or disorder to treatment with a compound directed against CD40L or CD40. In some embodiments, the method comprises 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; wherein the concentration of the at least one biomarker predicts that the subject is likely to respond to treatment of the disease or disorder with a compound directed against CD40L or CD40. In some embodiments, the disease or disorder is 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's 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 with 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.

[0015] Also disclosed herein is a method for treating a subject with a disease or disorder. In some embodiments, the method includes administering a therapeutically effective dose of a compound for CD40L or CD40, wherein the administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL. In some embodiments, the disease or disorder is 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 with CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is teiglut. 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 between 200 mg / kg and 100 mg / kg, preferably at a dose between 1 mg / kg and 8 mg / kg, most preferably at a dose between 2 mg / kg and 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 between CD28 and CD86 or between 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 pro-inflammatory 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 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 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, immune graft response, transplant 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 is an antibody comprising a heavy chain variable region (V H ), the heavy chain variable region comprises: (1) a CDRH1 domain comprising the sequence shown in SEQ ID NO:9; (2) a CDRH2 domain comprising the sequence shown in SEQ ID NO:10, 11, 12, 13 or 14; and (3) a CDRH3 domain comprising the sequence shown in SEQ ID NO:15; and further comprises a light chain variable region (V L ), the light chain variable region comprises: (4) a CDRL1 domain comprising the sequence shown in SEQ ID NO: 16 or 17; (5) a CDRL2 domain comprising the sequence shown in SEQ ID NO: 18 or 19; and (6) a CDRL3 domain comprising the sequence shown in SEQ ID NO: 20. In some embodiments, the compound targeting CD40L or CD40 is an antibody comprising a heavy chain variable region (V H ), the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; and the light chain variable region (V L ), the light chain variable region having an amino acid sequence as shown in SEQ ID NO:5, 6, 7 or 8.

[0016] Also disclosed herein is a method for regulating the concentration of at least one biomarker in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a compound for 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, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10 or MMP2. In some embodiments, the concentration of the at least one biomarker increases after the compound is administered. In some embodiments, the concentration of the at least one biomarker decreases after the compound is administered. 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 suffers from a disease or disorder. In some embodiments, the disease or disorder is 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's 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 with CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is teiglut. In some embodiments, the compound is MR1 ​​or 5c8. In some embodiments, the method includes administering the therapeutically effective dose to maintain the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably at a dose between 30 μg / mL and 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 between 200 mg / kg and 100 mg / kg, preferably at a dose between 1 mg / kg and 8 mg / kg, and most preferably at a dose between 2 mg / kg and 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 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, immune graft response, transplant 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 is an antibody comprising a heavy chain variable region (V H ), the heavy chain variable region comprises: (1) a CDRH1 domain comprising the sequence shown in SEQ ID NO:9; (2) a CDRH2 domain comprising the sequence shown in SEQ ID NO:10, 11, 12, 13 or 14; and (3) a CDRH3 domain comprising the sequence shown in SEQ ID NO:15; and further comprises a light chain variable region (V L ), the light chain variable region comprises: (4) a CDRL1 domain comprising the sequence shown in SEQ ID NO: 16 or 17; (5) a CDRL2 domain comprising the sequence shown in SEQ ID NO: 18 or 19; and (6) a CDRL3 domain comprising the sequence shown in SEQ ID NO: 20. In some embodiments, the compound targeting CD40L or CD40 is an antibody comprising a heavy chain variable region (V H ), the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; and the light chain variable region (V L ), the light chain variable region having an amino acid sequence as shown in SEQ ID NO:5, 6, 7 or 8.

[0017] Also disclosed herein is a method for maintaining or improving the ALS-FRS score of a subject with ALS. In some embodiments, the method comprises administering a therapeutically effective dose of a compound directed against CD40L or CD40, wherein administering the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

[0018] Also disclosed herein is a method for pre-screening a subject for a disease or disorder and treating the subject with a compound directed to CD40L or CD40. In some embodiments, the method comprises collecting a sample from the subject; detecting the concentration of at least one biomarker present in the 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 for the concentration of the at least one biomarker in subjects not suffering from the disease or disorder; and administering a therapeutically effective dose of a compound directed to CD40L or CD40 such that the concentration of the compound in the subject's plasma is maintained at a dose between 5 μg / mL and 100 μg / mL; preferably at a dose between 30 μg / mL and 60 μg / mL. In some embodiments, the compound is administered to the subject if the concentration of the at least one biomarker in the subject is significantly different than the concentration of the at least one biomarker in subjects not suffering from the disease or disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A MR1 concentration over time is shown using a linear concentration (Y) axis.

[0020] Figure 1B MR1 concentration over time is shown using the log concentration (Y) axis.

[0021] Figure 2 is a graph of the average daily body weight measurements for the control and treatment groups starting on day 40 until peak body weight was reached.

[0022] Figure 3 Mean daily body weight measurements from peak body weight until death are depicted for the control and treatment groups.

[0023] Figure 4 Time to disease onset based on time to progression to a neurologic function score of 2 from study entry is depicted.

[0024] Figure 5 is a graph showing the median age of each group at a given neurological function score of 0 to 4 (X-axis) and the number of days each group was stable at a given score (Y-axis).

[0025] Figure 6 Kaplan-Meier survival plots for the control and treatment groups as calculated by the Cox proportional hazards model are shown.

[0026] Fig. 7Ais a graph of average daily body weight measurements for the control and MR1 treated groups starting on day 40 until peak body weight was reached.

[0027] Figure 7B is a graph depicting the mean daily body weight measurements from peak body weight until death for the control and MR1 treated groups.

[0028] Figure 7C Shown is the time to disease onset based on the time to progression to a neurological function score of 2 in the control and MR1-treated groups.

[0029] Fig.7D Mean daily body weight measurements from peak body weight until death are shown for the control and MR1 treated groups.

[0030] Figure 8 is a graph showing the relative binding of JB5 antibody (circles, dashed line), hu5c8 antibody (squares-solid line) and control CTLA4-IgG1 (triangles) to human CD40L.

[0031] Fig. 9 Provided are the amino acid sequences of hu5c8 and JB5 VL region (SEQ ID NO: 1), hu5c8 and JB5 VH region (SEQ ID NO: 2), Fc region (SEQ ID NO: 3), JB5 Fc region (SEQ ID NO: 4), JB5-R28K VL region (SEQ ID NO: 5), JB5-K74R VH region (SEQ ID NO: 6), and the JB5 light chain amino acid sequence (SEQ ID NO: 7).

[0032] Fig.10 Provided is a synthetic nucleotide sequence encoding the light chain of the anti-CD40L antibody JB5 (SEQ ID NO: 8), with capital letters representing exons of the synthetic gene and lowercase letters representing intron sequences of the synthetic gene; and also provided is a heavy chain amino acid sequence of the anti-CD40L antibody JB5 (SEQ ID NO: 9).

[0033] Fig.11 A synthetic nucleic acid sequence encoding the heavy chain of the anti-CD40L antibody JB5 is provided (SEQ ID NO: 10), with capital letters representing exons of the synthetic gene and lowercase letters representing intronic sequences of the synthetic gene.

[0034] Fig.12Provided are the amino acid sequence of the anti-CD40L antibody JB5-R28K (SEQ ID NO: 11); a synthetic nucleic acid sequence encoding the light chain of the anti-CD40L antibody JB5-R28K (SEQ ID NO: 12), with capital letters representing exons of the synthetic gene and lowercase letters representing intron sequences of the synthetic gene; and also provided is the heavy chain amino acid sequence of the anti-CD40L antibody JB5-K74R (SEQ ID NO: 13).

[0035] Fig.13 A synthetic nucleic acid sequence encoding the heavy chain of the anti-CD40L antibody JB5-K74R is provided (SEQ ID NO: 14), with capital letters representing exons of the synthetic gene and lowercase letters representing intronic sequences of the synthetic gene.

[0036] Fig.14 Provided are the amino acid sequences of the CDRs of the heavy and light chains of the anti-CD40L antibody JB5 (SEQ ID NOs: 15-20, respectively) and the amino acid sequence of the heavy chain of hu5C8 (SEQ ID NO: 21).

[0037] Fig.15A is a bar graph showing the ranked potency of 16 antibody clones compared to the anti-CD40L antibody 5c8. The ranked potency is IC50 clone / IC50 x 100 of 5c8.

[0038] Fig. 15B is a bar graph showing the ranked potency of 16 antibody clones compared to the anti-CD40L antibody AT- 1501. Rank potency is IC50 clone / IC50 x 100 of AT-1501.

[0039] Figures 16A-16G and Figure 16I-16Q The binding curves of antibodies from each clone are shown along with the binding curve of 5c8. Fig.16H Binding curves of AT-1501 and 5c8 are shown.

[0040] Figures 17A-17G and Figure 17I-17Q The binding curves of antibodies from each clone are shown along with the binding curve of AT-1501. Fig.17H Binding curves of AT-1501 and 5c8 are shown.

[0041] Fig.18A and Fig.18B ] are graphs each showing binding of eight antibody clones, 5c8, AT-1501 and Abatacept to FcγRI. The only antibody with significant binding was the 5c8 antibody.

[0042] Fig.19A and Fig.19B ] are graphs each showing binding of eight antibody clones, 5c8, AT-1501 and Abatacept to FcγRIIa. The only antibody with significant binding was the 5c8 antibody.

[0043] Fig. 20A and Fig. 20B ] are graphs each showing binding of eight antibody clones, 5c8, AT-1501 and Abatacept to FcγRIIIa. No antibody showed significant binding to FcγRIIIa.

[0044] Fig. 20C and Fig.20D ] are graphs each showing binding of eight antibody clones, 5c8, AT-1501 and abatacept to FcγRIIIb. No antibody showed significant binding to FcγRIIIb.

[0045] Fig.21A and Fig. 21B are graphs, each showing the binding of eight antibody clones, 5c8, AT-1501 and Abatacept to C1q. The only antibody with significant binding to C1q was the 5c8 antibody.

[0046] Fig. 22 A schematic diagram showing the time points at which AT-1501 was administered to human subjects as part of the clinical trial outlined in Example 1. Blood (biological samples) were collected from subjects on days -28 to -4 for a "pre-screen" baseline assessment, and then subjects were administered dosing every two weeks for a total of 101 days. On each dosing day, blood was collected for pre-screening before drug administration, and again 2 hours after infusion.

[0047] Fig.23 Concentrations of AT-1501 over time in subjects dosed at 1 mg / kg, 2 mg / kg, 4 mg / kg or 8 mg / kg are shown. The X-axis is the number of days since initial dosing, and the Y-axis is the concentration of AT-1501 present in plasma (in μg / mL).

[0048] Figure 24A-Figure 24I Nine ADA positive samples, the concentration of AT-1501 in the blood and the time of detection of ADA (circles) are shown. These figures show that patient 103202 ( Fig.24A )、104202( Fig. 24B )、105202( Fig.24C )、105205( Fig.24D )、105206( Fig.24E )、108202( Fig.24F )、108203( Figure 24G )、108204( Fig.24H ) and 108205( Fig.24I ) positive detection of ADA titer in plasma.

[0049] Fig.25 Shown is a heat map of protein changes in subjects following exposure to 1, 2, 4, or 8 mg / kg of AT-1501.

[0050] Figure 26A-26B Shown are the percent changes from baseline in the biomarker CXCL13 in human subjects following 4, 6, 8, 10 and 12 weeks of exposure to 1, 2, 4 or 8 mg / kg of AT-1501. Fig.26B Here is a closer look at the Week 12 data.

[0051] Figure 27A-27B Shown are the percent changes from baseline in the biomarker CD40L in human subjects following 4, 6, 8, 10 and 12 weeks of exposure to 1, 2, 4 or 8 mg / kg of AT-1501. Fig.27B is a close-up view of the data for week 12. "*" indicates a p-value between 0.05 and 0.01; "**" indicates a p-value between 0.01 and 0.001; and "***" indicates a p-value less than 0.001.

[0052] Figure 28A-28B Shown are the percent changes from baseline in the biomarker CD40 in human subjects following 4, 6, 8, 10 and 12 weeks of exposure to 1, 2, 4 or 8 mg / kg of AT-1501. Fig.28B is a close-up view of the data for week 12. "*" indicates a p-value between 0.05 and 0.01; "**" indicates a p-value between 0.01 and 0.001; and "***" indicates a p-value less than 0.001.

[0053] Figure 29A-29L The percentage change in protein concentration in the plasma of human subjects after 12 weeks of treatment with compounds targeting CD40L / CD40 is shown. Each graph shows the mean change in protein in patients dosed with 1, 2, 4, or 8 mg / kg of AT-1501. The corresponding graphs each show the highest decrease in total protein concentration among the proteins evaluated, and these graphs are for CD40L ( Fig.29A )、CXCL13( Fig.29B )、CXCL9( Fig.29C )、TNF-α( Fig.29D )、En-Raged( Fig.29E )、TNFR2( Fig.29F )、IgM( Figure 29G )、IgA( Fig.29H )、Il2r( Fig.29I )、CXCL10( Fig.29J )、CD40( Figure 29K ) and B2M( Figure 29L ). “*” indicates a p-value between 0.05 and 0.01; “**” indicates a p-value between 0.01 and 0.001; “***” indicates a p-value less than 0.001.

[0054] Figure 30A-Figure 30L The percentage change in protein concentration in the plasma of human subjects after 12 weeks of treatment with a compound targeting CD40L / CD40 is shown. Each graph shows the mean change in protein in patients dosed with 1, 2, 4, or 8 mg / kg of AT-1501. The corresponding graphs each show the reduction in total protein concentration for: VCAM ( Fig. 30A )、IL-18( Fig. 30B )、IL-16( Fig. 30C )、SAP( Fig.30D )、MIP1-β( Fig.30E )、MDC( Fig.30F )、C3( Figure 30G )、CRP( Fig. 30H ), fibrinogen ( Fig. 30I )、IgE( Fig. 30J )、ICAM( Figure 30K ) and MCP1( Figure 30L ). “*” indicates a p-value between 0.05 and 0.01; “**” indicates a p-value between 0.01 and 0.001; “***” indicates a p-value less than 0.001.

[0055] Figure 31A-Figure 31E The percentage change in protein concentration in the plasma of human subjects after 12 weeks of treatment with a compound targeting CD40L / CD40 is shown. Each graph shows the mean change in protein in patients dosed with 1, 2, 4, or 8 mg / kg of AT-1501. The corresponding graphs each show the increase in total protein concentration for: IL-6 ( Fig.31A )、ENA-78( Fig.31B )、RANTES( Fig.31C )、MMP3( Fig.31D ) and MMP9( Fig.31E ). “*” indicates a p-value between 0.05 and 0.01; “**” indicates a p-value between 0.01 and 0.001; “***” indicates a p-value less than 0.001.

[0056] Fig.32Shown are the percent changes in plasma NFL concentrations in human subjects following 12 weeks of treatment with AT-1501 at doses of 1, 2, 4, or 8 mg / kg.

[0057] Figure 33A-Figure 33D The total change in ALS-FRS scores over 11 weeks for the human subject cohort is shown. ALS-FRS scores were assessed by a clinician. The data were separated according to those human subject cohorts that received 1 mg / kg of AT-1501 treatment, 2 mg / kg of AT-1501 treatment, 4 mg / kg of AT-1501 treatment, 8 mg / kg of AT-1501 treatment, or no treatment "Proact". Fig.33A It is a composite score of all patients involved in a clinical trial. Fig.33B Scores for patients who no longer met recruitment criteria were removed. Fig.33C Scores for patients reporting treatment-related adverse events were removed. Fig.33D All patient scores except those with the best response to treatment were removed.

[0058] Fig.34 Shown is the change in ALS-FRS scores over 7 weeks in human subjects. In this case, subjects were not pooled and the data were grouped according to responders (grey) and non-responders (dark grey) to treatment with AT-1501.

[0059] Fig.35 Nested dot plots of protein concentrations (in log2) of subjects before treatment are shown. Values ​​are separated by type of protein (X-axis) and by non-responders (left, light grey) and responders (right, dark grey) to subsequent treatment with AT-1501. The horizontal line in each cluster represents the mean of that cluster. DETAILED DESCRIPTION

[0060] Reference is made to the accompanying drawings forming a part thereof in the following detailed description. Unless the context otherwise dictates, in the accompanying drawings, similar symbols typically identify similar components. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be restrictive. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that, as generally described herein and shown in the accompanying drawings, aspects of the present disclosure may be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein. All references cited herein are expressly incorporated herein by reference in their entirety, and are used for the specific disclosures cited herein.

[0061] The present disclosure describes methods of treating patients with neurodegenerative or neuromuscular disorders by administering a therapeutically effective amount of a compound that blocks the interaction of CD40 with CD40L. The present disclosure also describes methods of treating patients by co-administering a compound that blocks the interaction of CD40 with CD40L with a compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80. Some embodiments relate to methods of predicting and / or measuring a response to treatment, the methods comprising measuring the level of one or more biomarkers. Embodiments also relate to methods of modulating the level of one or more biomarkers.

[0062] Abbreviations and definitions

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Unless otherwise indicated, all patents, applications, published applications and other publications cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, in the case where there are multiple definitions for a term herein, those in this section shall prevail.

[0064] Those of ordinary skill in the art will appreciate 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 to practice the embodiments without resorting to undue experimentation. All functional equivalents known in the art for any such materials and methods are intended to be included in this disclosure.

[0065] As used herein, "a" or "an" may mean one or more than one or more than one.

[0066] As used herein, the terms "about" or "approximately" have their ordinary meaning as understood by those skilled in the art, thereby indicating that a value includes the inherent variation of error in the method being employed to determine the value or the variation that exists among multiple determinations.

[0067] The use of the conjunction "or" is used interchangeably with "at least one of which". For example: in the case where a composition comprises A or B, the composition must comprise at least one of A and B, but may also comprise both A and B. Similarly, a composition comprising "A, B, C or D" must comprise at least one of the group of A, B, C and D, but may also comprise all or any combination of A, B, C and D.

[0068] Amino acid substitutions are indicated by convention wherein the original amino acid, the position of the amino acid in the designated sequence, and the replacement amino acid are identified, for example C11S would indicate that the cysteine ​​at position 11 of the polypeptide sequence is substituted with serine.

[0069] Humanized antibodies are antibodies produced from non-human species whose protein sequences have been modified to increase their similarity to naturally occurring antibody variants in humans. The "humanization" process is often applied to the development of monoclonal antibodies for administration to humans (e.g., antibodies developed as anti-cancer drugs).

[0070] At present, non-human antibodies are usually humanized by inserting the relevant CDR from the antibody produced in non-human animals into the human antibody "scaffold". The "direct" generation of humanized antibodies can be completed by inserting the appropriate CDR coding segment (responsible for the required binding properties) into the human antibody "scaffold". This can be achieved by using the recombinant DNA method of the appropriate carrier and expressing in mammalian cells. That is to say, after the antibody is developed to have the required properties in mice (or other non-humans), the DNA encoding the antibody can be separated, cloned into a vector and sequenced. The DNA sequence corresponding to the antibody CDR can then be determined. Once the precise sequence of the required CDR is known, it is possible to design a strategy for appropriately inserting these sequences into the construct containing the DNA of the human antibody variant. CDR can also be changed before being inserted into the scaffold, for example to increase specificity.

[0071] The term "human" antibody refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or has been prepared using any technique for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0072] The term "variant" as used herein is defined as a modified or altered form of a wild-type sequence, for example, wherein one or more amino acids can be substituted by other one or more amino acids or one or more non-amino acids that do not substantially significantly affect function. In some embodiments, a variant may contain an altered side chain of at least one amino acid residue.

[0073] The term "antigen" as used herein is defined as an entity that can stimulate the production of antibodies and specifically bind to them and / or an entity that triggers an immune system response. For example, a cell surface protein or a specific linear or nonlinear portion thereof. The term herein can be abbreviated as "Ag".

[0074] "Antigen-binding antibody fragment" refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody that binds to the 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; diabodies; triabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments, minimal recognition units composed of amino acid residues that mimic the hypervariable region of antibodies (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides). Antigen-binding fragments as disclosed in the present application bind to the antigen CD40L.

[0075] The antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or any amino acid composition, and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains can be in any suitable arrangement relative to each other. For example, the variable region can be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0076] Isolated antibodies include bispecific antibodies, in which each arm of the antibody or antigen-binding fragment binds to a different target or epitope.

[0077] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region.

[0078] "5c8" refers to a mouse anti-human antibody that binds CD40L and is produced by a hybridoma that is available from the ATCC under accession number HB 10916 and described in U.S. Pat. No. 5,474,771. "hu5c8" refers to a humanized form of 5c8, the sequence of which is disclosed in Karpusas et al., Structure, Vol. 9, pp. 321-329, (2001).

[0079] The term "specific binding" and the like means that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (KD) of about 3000 nM or less (i.e., a smaller KD indicates tighter 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.

[0080] Specific binding for a particular antigen or epitope can be exhibited, for example, by an antibody having a KD for the antigen or epitope of at least about 1 x 10 -4 M, at least about 1x 10 -5 M, at least about 1x 10 -6 M, at least about 1x 10 -7 M, at least about 1x 10 -8 M, at least about 1x 10 -9 M, alternatively at least about 1 x 10 -10 M, at least about 1x 10 -11 M, at least about 1x 10 -12 5,000, 10,000, or more, wherein KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD that is 20 times, 50 times, 100 times, 500 times, 1000 times, 5,000 times, 10,000 times, or more, greater than an antigen or epitope. In addition, specific binding to a specific antigen or epitope can be exhibited, for example, by an antibody having a Ka to an antigen or epitope that is at least 20 times, 50 times, 100 times, 500 times, 1000 times, 5,000 times, 10,000 times, or more, greater than an epitope relative to a control, wherein Ka refers to the association rate of a specific antibody-antigen interaction.

[0081] The term "neutralizing antibody" includes antibodies that are capable of inhibiting and / or neutralizing the biological activity of CD40L, such as anti-CD40L antibodies or antigenic fragments thereof that inhibit or prevent or attenuate the binding of CD40L to CD40 and thereby inhibit or reduce the signaling pathway triggered by CD40L and / or inhibit or reduce the binding of CD40L to CD40.

[0082] The terms "antagonist antibody" or "antagonist antibody" are used equivalently herein and include antibodies that are capable of inhibiting and / or neutralizing the biological signaling activity of CD40L, as described above for neutralizing antibodies.

[0083] The terms and expressions that have been employed herein are used as illustrative and not limiting terms, and it is not intended that any equivalents of the features shown and described, or portions thereof, be excluded by the use of such terms and expressions, but it will be appreciated that various modifications are possible within the scope of the claimed embodiments. It should therefore be understood that, although aspects of the present disclosure have been specifically disclosed by various embodiments that may include preferred embodiments, exemplary embodiments, and optional features, modifications and variations of the concepts disclosed herein may be resorted to by those skilled in the art. Such modifications and variations are considered to be within the scope of the embodiments of the present disclosure as described and as may be defined by the appended claims.

[0084] In this specification, the identity percentage between polypeptide or amino acid sequence is mentioned.The identity percentage 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, and it is necessary to introduce them for the optimal comparison of two sequences.Identity can be measured as "local identity" or "overall identity".Local identity refers to the degree of sequence correlation between polypeptides, as determined by the matching between such sequence strings.Overall identity refers to the degree of sequence correlation of polypeptides compared with the full length of the reference polypeptide.Unless otherwise indicated, as used herein, identity means overall identity.For the purpose of this disclosure, the percentage of overall identity is calculated using the Blossum 62 scoring matrix (gap penalty is 12, gap extension penalty is 4, and frameshift gap penalty is 5) using Needleman and Wunsch ((1970) J.Mol.Biol. [Molecular Biology] 48:444-453) algorithm. There are a number of publicly available software programs that incorporate the Needleman and Wunsch algorithm, such as the GAP program in the GCG software package.

[0085] CD40L is also known as CD154, gp39, T-BAM, 5c8 antigen, or TNF-related activator protein (TRAP).

[0086] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean including a stated step or element or group of steps or elements, but not excluding any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present. "Consisting essentially of" means including any element listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the present disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

[0087] The terms "function" and "functionality" as used herein have their plain and ordinary meaning as understood in light of the present specification, and refer to a biological function, an enzyme function, or a therapeutic function.

[0088] As used herein, the term "yield" of any given substance, compound, or material has its simple and ordinary meaning as understood in accordance with this specification, and refers to 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 is, is about, is at least, is at least about, is not more than, or is not more than about 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including all decimals therebetween, of the expected total amount. The yield may be affected by the efficiency of the reaction or process, unwanted side reactions, degradation, the amount of input substance, compound, or material, or the loss of the desired substance, compound, or material during any step of the production.

[0089] As used herein, the term "isolated" has its plain and ordinary meaning as understood in light of this specification, and refers to substances and / or entities that have been (1) separated from at least some of the components with which they were originally associated (whether in nature and / or 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 originally associated by equal to, about, at least, at least about, not greater than, or not greater 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% (or a range including and / or spanning the foregoing values). In some embodiments, the isolated agent is, is about, is at least, is at least about, is not more than, or is not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% (or a range including and / or spanning the above values) pure. As used herein, an "isolated" substance can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0090] The term "isolated protein" or "isolated polypeptide" (e.g., an isolated antibody or isolated antigen-binding fragment) is a protein or polypeptide that, by its origin or source of derivation, is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by cells from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell from which it is naturally derived will be "isolated" from its naturally associated components. A protein can be rendered substantially free of naturally associated components by separation using protein purification techniques well known in the art.

[0091] As used herein, "in vivo" is given its plain and ordinary meaning as understood in light of this specification, and refers to methods performed within living organisms (typically animals, mammals (including humans) and plants) or living cells that make up these living organisms (as opposed to tissue extracts or dead organisms).

[0092] As used herein, "ex vivo" is given its plain and ordinary meaning as understood in light of this specification, and refers to a method performed outside a living organism with little change to natural conditions.

[0093] As used herein, "in vitro" is given its plain and ordinary meaning as understood in light of this specification, and refers to methods performed outside of biological conditions (eg, in a culture dish or test tube).

[0094] As used herein, "nucleic acid", "nucleic acid molecule" or "nucleotide" refers to a polynucleotide or oligonucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), an oligonucleotide, a fragment produced by the polymerase chain reaction (PCR), and a fragment produced by any of ligation, cleavage, endonuclease action, exonuclease action, and synthesis. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA) or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides) or a combination of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyls, amines, and azido groups, or sugars can be functionalized as ethers or esters. In addition, the entire sugar moiety can be replaced by a spatially and electronically similar structure (such as azasugars and carbocyclic sugar analogs). Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be connected by phosphodiester bonds or analogs of such connections. Phosphodiester-connected analogs include phosphorothioates, phosphorodithioates, selenophosphates, diselenphosphates, aniline thiophosphates, phosphoranilidates, phosphoramidates, etc. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which include naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded.

[0095] The terms "peptide", "polypeptide" and "protein" as used herein have their simple and ordinary meanings as understood in light of this specification, and refer to 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, structures, transport, defense, hormones or signal transduction. Peptides, polypeptides and proteins are usually, but not always, biologically produced by ribosome complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide and protein mutations (such as substitutions, deletions, truncations, additions, duplications or fusions of more than one peptide, polypeptide or protein) can be performed. These fusions of more than one peptide, polypeptide or protein can be contiguously linked in the same molecule, or with additional amino acids (e.g., linkers, repeat sequences, epitopes or tags) therebetween, or any other sequence that is, is about, is at least, is at least about, is no more than, or is no more than 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 long, or any length within the range defined by any two of the above lengths. The term "downstream" as used herein with respect to a polypeptide has its simple and common meaning as understood in accordance with this specification, and refers to the sequence after the C-terminus of the preceding sequence. The term "upstream" as used herein with respect to a polypeptide has its plain and ordinary meaning as understood in light of the present specification, and refers to a sequence preceding the N-terminus of a subsequent sequence.

[0096] The term "gene" as used herein has its simple and common meaning as understood according to this specification, and generally refers to a part of a nucleic acid encoding a protein or functional RNA; however, the term may optionally encompass regulatory sequences. It will be understood by those of ordinary skill in the art that the term "gene" may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will be further understood that the definition of a gene includes reference 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 that are involved in transcription or information production or composition. In other embodiments, a gene comprises a transcribed sequence encoding a protein, polypeptide, or peptide. Consistent with the terms described herein, an "isolated gene" may include one or more transcribed nucleic acids, regulatory sequences, coding sequences, etc. that are substantially separated from other such sequences (such as other naturally occurring genes, regulatory sequences, polypeptide or peptide coding sequences, etc.). In this regard, the term "gene" is used to simply refer to a nucleic acid comprising a transcribed nucleotide sequence and its complementary sequence. As will be understood by those skilled in the art, this functional term "gene" includes genomic sequences, RNA or cDNA sequences, or smaller engineered nucleic acid segments, including nucleic acid segments of non-transcribed portions of genes, including but not limited to non-transcribed promoter or enhancer regions of genes. Smaller engineered gene nucleic acid segments can express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, mutants and / or the like using nucleic acid manipulation techniques.

[0097] As used herein, the terms "individual", "subject", "host" or "patient" have their usual meaning as understood by those skilled in the art, and thus include human or non-human mammals. The term "mammal" is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates (including simians (chimpanzees, apes, monkeys), humans), cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice or guinea pigs.

[0098] The dimensions and values ​​disclosed herein should not be understood as being 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 surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm".

[0099] 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.

[0100] MR1 is a hamster monoclonal antibody that binds to the mouse CD40 ligand. "Wild type" as used herein means a non-transgenic mouse. "Small molecule" as used herein means a compound with a molecular weight of less than 2000 Daltons. "Treatment" or "treating" as used herein includes preventive treatment and therapeutic treatment. The phrase "therapeutically effective amount" means an amount of a compound or a pharmaceutically acceptable salt thereof sufficient to inhibit or allow improvement in a particular subject or subject population for a disorder or condition to be treated by administration alone or in combination with another agent or treatment.

[0101] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0102] "Affinity" refers to 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 indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (KD) (k off / k on KD and affinity are inversely related. The KD value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), so the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Affinity can be measured by conventional methods known in the art (including those described herein). Specific, illustrative and exemplary embodiments for measuring binding affinity can be by radioimmunoassay (RIA); Surface plasmon resonance (SPR) on a GE Healthcare Europe GmbH, Zurich, Switzerland instrument (GE Healthcare Europe GmbH, Zurich, Switzerland) was measured using human CD40 ligand polypeptide as the analyte by capturing the antibody on a protein A-coupled CM5 research grade sensor chip (GE Healthcare Europe GmbH, Zurich, Switzerland; BR-1000-14). Other methods may include radioimmunoassay and kinetic exclusion assay. Kinetic exclusion assay is a general immunoassay platform that is capable of measuring the equilibrium dissociation constant as well as the association rate constant and dissociation rate constant of antigen / antibody interactions.

[0103] "ALS-TDI" is an abbreviation for ALS Therapy Development Institute. As used herein, "hSOD1G93A preclinical mouse model", "hSOD1G93A mouse model", "G93A preclinical mouse model" and "G93A mouse model" have the same meaning. As used herein, "hSOD1G93A mouse" and "G93A mouse" have the same meaning.

[0104] TMB is the abbreviation of 3,3′,5,5′-tetramethylbenzidine.

[0105] "CDR domain" as used herein means the complementarity determining region of an antibody with or without flanking sequences.

[0106] "Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. An effector function is the ability of the Fc or constant region of an antibody to bind to proteins including, but not limited to, Fc receptors (FcR) (e.g., high affinity IgG Fc region receptor Ia (FCγRIa) (CD64) (SEQ ID NO: 34), low affinity immunoglobulin γ Fc region receptor IIa (FCγRIIa) (CD32) (SEQ ID NO: 35), low affinity immunoglobulin γ Fc region receptor IIIa (FCγRIIIA) (CD16a) (SEQ ID NO: 36), low affinity immunoglobulin γ Fc region receptor IIIb (FCγRIIIb) (CD16b) (SEQ ID NO: 37)). In some embodiments, where the antibody and antigen-binding fragment thereof have an Fc domain, the Fc domain has been engineered to reduce or eliminate one or more Fc effector functions. In a preferred embodiment, the Fc domain has been engineered to reduce or eliminate platelet activation and / or platelet aggregation and the attendant risk of thromboembolism.

[0107] In vivo evaluation of compounds

[0108] The G93A mouse model can be used to evaluate the efficacy of compounds used in the methods of the present disclosure [see TuPH 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 is constructed by inserting 23 copies of the human SOD1 gene (which contains a glycine to alanine mutation at position 93) into the mouse genome. These mice accurately reproduce most of the major points of the pathology of both the human sporadic and familial forms of the disease, making this model the best currently available option for testing disease-modifying interventions.

[0109] There is no discernible abnormal phenotypic expression in G93A mice at birth. Visible signs of disease are not expressed until about 90 days of age, so it experiences progressive loss of hind limb function, causing complete paralysis and death at about 134 days of age. Although muscle wasting is caused by the death or dysfunction of motor neurons, the death of these cells is related to peripheral cells (including microglia and astrocytes) and is partially caused by the interaction with peripheral cells. Significant astrocytosis first occurs at about 80 days of age, and neuroinflammation mainly mediated by microglia occurs at about 100 days of age, and expands until death.

[0110] Although human disease may start in any motor area, mouse disease reliably affects the cervical and lumbar regions first. In G93A mice, the number of motor neurons has already dropped significantly at the onset of visible symptoms (approximately on day 85), and more than 50% loss is reached at the time of death. Abnormalities in the cytoskeleton, neurofilaments, axonal transport, Golgi bodies, endoplasmic reticulum, mitochondria, apoptotic machinery, proteasomes, and cytoplasmic protein processing are observed in neurons during the course of the disease.

[0111] Although human disease may start in any motor area, mouse disease first reliably affects the lumbar and sacral regions. In G93A mice, the number of motor neurons has already dropped significantly at the onset of visible symptoms (approximately at day 85), and more than 50% loss is reached at the time of death. Abnormalities in cytoskeleton, neurofilaments, axonal transport, Golgi bodies, endoplasmic reticulum, mitochondria, apoptotic machinery, proteasomes, and cytoplasmic protein processing are observed in neurons during the course of the disease.

[0112] To date, there have been at least 50 publications describing therapeutic agents that extend the lifespan of this mouse. However, with the exception of riluzole, no therapeutic agent has shown corresponding clinical efficacy. The ALS Therapy Development Institute has described optimized therapeutic drug screening in the G93A mouse model to control noise variables [Scott S. et al. Amyotrophic Lateral Sclerosis 2008; 9: 4-15, which is hereby incorporated by reference]. Scott et al. described a minimal study design for the G93A mouse model that addresses and manages noise caused by inherent confounding biological variables. In validating this study design, nine compounds that had previously been reported to be effective in preclinical models and subsequently failed in human clinical trials were evaluated at different doses to the greatest extent possible. Several of these molecules are anti-inflammatory molecules that inhibit tumor necrosis factor (TNF) signaling and microglia activation, including Minocycline, thalidomide, and creatine. In the G93A model, lifespan was increased by 19% (24 days), however, a high-powered study failed to detect any change in survival (1.8 days, 0.52%) (Scott et al.). Similar results were obtained with minocycline (previous report, 15.8% increase; high-powered study, -0.60%), creatine (previous report, 17.8%; high-powered study, 0.67%), and thalidomide (previous report, 16% increase in survival; high-powered study, -1.9%).

[0113] Identification of candidate compounds

[0114] Whole genome expression profile analysis was performed on wild-type mice and G93A mice at different time points during disease progression.Genes identified as differentially expressed between the two groups were analyzed, and the resulting data were used to focus on the selection of drugs to be screened.Differentially expressed genes include genes related to immune response and cell adhesion, including CD86, CD44, ICAM, ITGAM, ITGA ITGAX, ITGB2, H2-K1 (MHC II), H2-AB1 (MHC II), H2-D1 (MHCII) and H2-Eb1 (MHCII).These data show that inflammatory features increase during disease progression, and are consistent with the involvement of costimulatory pathways.Costimulatory pathways are related to interactions between cell types via CD28 / CD80 or CD28 / CD86 or CD40 / CD40L interactions (some of which are identified in gene expression analysis).

[0115] To identify molecular pathways suitable for therapeutic development, changes in gene expression patterns during disease progression were characterized in the G93A mouse model. Mouse expression group 430vII MOE430vII gene chip studies whole genome transcriptional profile analysis.Adopt the longitudinal study design that G93A skeletal muscle and spinal cord are compared with non-transgenic littermates.In the G93A mouse model, symptom onset is first observed as the tail paralysis that begins about the 75th day, wherein hind limbs, then forelimbs, finally diaphragm appear progressive paralysis.The average survival period of the G93A animal group is 134 days.Longitudinal study design collects calf muscle (gastrocnemius) and spinal cord from G93A animals and wild-type littermates at the 30th day, the 50th day, the 60th day, the 80th day, the 90th day, the 100th day, the 110th day and the 120th day (the 0th day is the date of birth).For each time point, tissues were collected from 5 wild-type and 5 G93A animals, and 160 tissues were processed independently in total.

[0116] Animals were euthanized at the appropriate time points described above according to IACUC protocols. Tissues were immediately harvested and snap frozen in liquid nitrogen. Frozen tissues were stored at -80°C. Total RNA was simultaneously isolated from tissues using the Qiagen RNaEasy kit as described by the manufacturer. The isolated total RNA was amplified using the Ambion Message AMPII T7 in vitro transcription kit using standard T7 linear amplification incorporating biotinylated nucleotides. The labeled probes were fragmented and amplified with Affymetrix according to the manufacturer's protocol. Mouse expression group 430vII hybridization. In Affymetrix The gene chip was washed in Fluids Station 450 to remove unhybridized probe. Scanning gene chips in 3000 7G scanner.

[0117] All computational processing and modeling were performed using the R development language 2.6 version from Bioconductor. Spinal cord and gastrocnemius muscle data sets were analyzed independently. All data preprocessing was performed using Affymetrix CEL files. All CEL files in each data set were quality controlled using Bioconductor vignette SIMPLE AFFY, AFFY and AFFY PLM. The LIMMA package was used to assess the statistical changes in gene expression between G93A and wild-type tissues at a given time point. The Bayesian model was used to determine the significance of expression changes between each group.

[0118] These data show that inflammatory signatures increase during disease progression. These gene expression changes reflect activation of antigen presenting cells such as dendritic cells, macrophages, and B cells. Blocking the interaction may improve the immune response that exacerbates disease progression in ALS. Co-stimulatory pathways may be inhibited by blocking CD-28 / CD80 or CD-28 / CD86 or CD40 / CD40L interactions.

[0119] The co-stimulatory pathway, along with other interactions, involves 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 macrophages, dendritic cells, fibroblasts and activated endothelial cells. Blockade of CD40:CD40L binding is thought to promote the formation of type I T helper cell responses.

[0120] Using compounds that block these interactions and inhibit costimulatory signals, a large amount of work has demonstrated the immunomodulatory effects of blocking one or more of CD40L, CD80 or CD86 in preclinical models of transplantation and autoimmunity. Blocking CD40L function with blocking antibodies or expressing CD40L-Ig with adenovirus increases the allograft transplantation period by 30 to 90 days. Similar studies that block CD80 / CD86 on APCs with CTLA4-Ig or express CTLA4-Ig with adenovirus transiently increase allograft transplant survival. Transplant rejection in these models is transient, and graft rejection ensues over time. Long-term suppression of transplant rejection can be achieved by blocking costimulatory pathways with CTLA4-Ig and blocking CD40L activation of APCs with anti-CD40L antibodies.

[0121] Blocking antibodies against CD40L or genetic deletion of CD40L in mice have demonstrated that CD40L improves disease progression, survival, and surrogate markers of disease in preclinical models of experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis, collagen-induced arthritis, and systemic lupus. Blockade of CD40:CD40L binding appears to reduce the ability of macrophages to produce nitric oxide, which mediates many of the pro-inflammatory activities of macrophages.

[0122] From such studies it appears that blocking the CD40:CD40L interaction and / or blocking the CD28:CD80 or CD28:CD86 interaction can modulate the immune response.

[0123] The immunohistochemical data showed good correlation with the gene expression data and identified macrophages in G93A mice as antigen-presenting cells that infiltrate skeletal muscle during disease progression.

[0124] In order to determine the presence and location of potential antigen presenting cells in the gastrocnemius from G93A and wild-type animals, immunohistochemistry was performed on the gastrocnemius tissue harvested from G93A and wild-type mice on the 110th day. The tissue was embedded in OCT immediately after harvesting. The frozen sections were stained with H&E and hybridized with antibodies against myelin (anti-S100b antibody) or antibodies against hematopoietic cell lineages (including T cells (anti-CD3 antibody), B cells (CD45R pan-B cell antibody) and macrophages (anti-CD11b antibody). At the 110th day, there was CD11b positive macrophage infiltration, and the macrophages appeared to be located in the axons of the nerves that innervate skeletal muscle. The location of macrophages is not scattered throughout the muscle, which indicates that inflammation is not due to muscle atrophy or muscle fiber remodeling.

[0125] In order to confirm the identity of mononuclear cell lineage cells in skeletal muscle at the 110th day and to illustrate the relevance of these cells compared with non-transgenic animals, additional immunohistochemistry was performed with a group of antibodies specific to the macrophage lineage. Anti-S100b antibody was used to mark the myelin-related axons that dominate skeletal muscle. All macrophage-specific antibodies (anti-CD11b antibody, anti-CD86 antibody and anti-MAC1 antibody) localized macrophages to the axons of the nerves that dominate the skeletal muscle of G93A mice, while there were no macrophages on the axons of wild-type animals. The location of macrophages is unique to the nerves that dominate muscle, and there are no macrophages on the muscle fibers of muscle.

[0126] Gene expression data showed that genes associated with co-stimulatory pathways were transiently increased during disease progression in both the spinal cord and skeletal muscle ( Figure 1A-1B ). To characterize the time of macrophage infiltration into skeletal muscle, immunohistochemistry was performed on gastrocnemius sections from the 60th, 80th and 100th days in G93A mice. There was no evidence of macrophage infiltration and localization to axons at the 60th day. Macrophages were evident at the 80th day, and as previously described for skeletal muscle at the 110th day, they were localized to axons that innervate muscle. The number of macrophages increased between the 80th and 100th days, and the accumulation of macrophages was unique to axons that innervate skeletal muscle.

[0127] To quantify the increase in macrophage infiltration, representative sections from 5 G93A and 5 wild-type animals were hybridized with anti-CD86 antibody and the number of macrophages per 10,000 square microns was counted. The number of macrophages was the same in wild-type animals at the 60, 80, and 110 day time points. As can be seen in Figure 2 As observed in Figure 2, macrophages transiently accumulated in skeletal muscle between days 80 and 100, and very few macrophages were present in wild-type skeletal muscle.

[0128] In summary, the immunohistochemical data correlated very well with the gene expression data and identified macrophages as antigen presenting cells that infiltrate skeletal muscle during disease progression in the G93A mouse model. An unexpected finding was that the macrophage infiltration appeared to specifically target axons innervating skeletal muscle, as localized by labeling with antibodies against myelin and macrophages.

[0129] Pharmacokinetic analysis of MR1 in G93A tissues

[0130] A matrix-matched non-competitive enzyme-linked immunosorbent assay (ELISA) in a sandwich format was used to determine tissue levels of MR1 against murine CD40L. A seven-point standard curve was included on each plate. Standards were prepared using purified MR1 spiked into a PBS diluent solution. The PBS diluent solution was matrix-matched to normal mouse tissue at equal dilutions of unknown samples to correct for any nonspecific effects caused by tissue lysates.

[0131] In both female and male G93A mice, 84 plasma samples were collected over a two-week period following dosing (10 mg / kg, IP) for pharmacokinetic analysis.

[0132] The elimination half-life was similar in females (23 days) and males (22 days) and is similar to the half-life of a typical mouse IgG2-based antibody in mice. No evidence of anti-hamster antibody responses was observed.

[0133] Females showed a slightly smaller volume of distribution for MR1 compared to males, and therefore showed higher plasma levels when given the same 10 mg / kg dose. Males showed faster clearance and a higher volume of distribution. Therefore, males would require a higher dose to achieve similar plasma levels. Figure 1A MR1 concentration over time is shown using a linear concentration (Y) axis. Figure 1B MR1 concentration over time is shown using the log concentration (Y) axis.

[0134] Thirty-six female G93A mice were litter-matched and randomly divided into two study groups. 18 G93A mice were placed in the MRI treatment group, and the other 18 G93A mice were placed in the control group. The study days were based on days from birth.

[0135] A single injection of 56 μg MR1 was administered intraperitoneally (IP) on day 50. After the bolus, weekly maintenance injections of 18 μg MR1 were administered by intraperitoneal injection. Doses were prepared in vehicle (phosphate buffered saline (PBS, pH 7.3)) to a total volume of 200 μl. Control animals were administered 200 μl PBS. Starting from day 54, animals were monitored daily throughout the study, and daily body weight measurements and neurological function scores were measured.

[0136] Starting from 50 days of age, the neurological function scores of the two hind legs of each mouse were assessed every day. Neurological function scores were scored on a scale of 0-4. (Scott et al., ALS Journal [ALS Magazine] January 2008). In short, when the mouse was suspended by its tail, and the mouse could hold it for 2 seconds, when suspended 2-3 times, the hind legs of the animal with a score of 0 were fully extended away from the lateral midline. When the animal showed collapse or partial collapse (weakness) of the legs to the lateral midline during tail suspension or tremor of the hind legs, the animal was scored as 1. When the toes curled at least twice during 12-inch walking, or any part of the foot dragged along the bottom of the cage / table, the animal was scored as 2. When the animal had rigid paralysis or minimal joint movement, or did not use the foot to move forward, the animal was scored as 3. When the animal could not self-correct within 30 seconds from either side, the animal was scored as 4. If a hind leg was scored as 2, food pellets were left on the litter. If both hind legs are scored 2, provide Nutra- (Bio-Serve #S4798) was used as food, and a long straw was placed on the water bottle.

[0137] The date and cause of death of each mouse were recorded. For humanitarian reasons, animals were closely monitored before actual death using the criteria for severe moribundity, and were put to death when moribund. In order to reliably and humanely determine the duration of survival, moribundity was used, defined as mice being unable to self-correct after being placed on one side for 30 seconds (neurological function score of 4). Moribund mice were scored as "death" and euthanized using carbon dioxide.

[0138] The standard procedure is to remove non-ALS-related deaths in both the treatment and control groups before statistical analysis. In this case, all animal deaths in the control or treatment groups are attributed to ALS. Therefore, no animals are censored due to non-ALS-related deaths.

[0139] Mutant SOD1 transgenic animals exhibit normal body weight (BW) characteristics as neonates and gain weight normally into adulthood compared to non-transgenic animals. Depending on the nature of the genetic mutation in the transgene and the number of copies of the mutant transgene, weight loss becomes evident in adult animals and persists until death. Analysis of weight loss in treatment and control groups can provide insights into the effects of putative treatments on the rate of disease onset and progression. To assess the effects of MR1 treatment on body weight, two summary parameters were examined: (1) the change in BW from the start of the study to peak body weight, which can reflect the effect on disease onset; and (2) the change in BW from peak body weight to death, which can reflect the effect on disease progression.

[0140] The time-to-event curves from day 40 to the time to peak body weight for the comparative MR1-treated and control groups are shown in Figure 2 The median time to peak body weight was 50 days in the control group compared with 51 days in the MR1-treated group. This difference was not significant when analyzed by Kaplan-Meier using log-rank and Wilcoxon statistical models, Cox proportional hazards models, or parametric statistical tests.

[0141] The time-to-event curves from peak body weight to death for the comparative MR1-treated and control groups are shown in Figure 3 In animals treated with MRI, the time from peak body weight to death was statistically significantly later by 15 days. The risk of control animals dying sooner after reaching peak body weight was 2.4 to 4.7 times higher than that of animals treated with MR1. The median time from peak body weight to death in the control group was 26 days, while it was 41 days in the MR1-treated group. The significance of each analysis in this example was calculated in several ways to better evaluate the significance. The delay was statistically significant when analyzed using several methods. (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 a small effect on the onset of disease in the G93A mouse model, but has a significant effect in slowing the rate of weight loss from peak body weight until death.

[0142] The time to disease onset was also characterized by analyzing daily neurological scores for the saline and MR1 treated groups. At the beginning of the study (day 50), all animals had a neurological score of 0 with no observable symptoms or paralysis. Disease onset can be characterized by examining the progression of neurological scores from a neurological score of 0 to a neurological score of 2 (when the animal visibly drags its hind limbs). The time-to-event graphs of the age at which the MR1 and control animals progressed to a neurological score of 2 and the number of days to a neurological score of 2 are shown in Figure 4The median time to a neurological score of 2 for each group is plotted in Figure 5 The time to reach a score of 2 was 115 days in the control group and 122 days for the MR1 treated group. Based on the neurological function score data, MR1 delayed disease onset in the G93A mouse model by approximately 7 days, and the delay was statistically significant when analyzed using several methods. (Kaplan-Meier, log-rank (p=0.0378) and Wilcoxon (p=0.0591); Cox proportional hazards model (p=0.0521); parametric analysis (p=0.0582)). Figure 5 The number of days spent at each neurological function score level is plotted against the median age at that score level.

[0143] The survival time of treated animals was 13 days later than that of control animals. The time-event curves from peak body weight to death for the comparative MR1 treated and control groups are shown in Figure 6 The median survival time was 128 days for the control group and 141 days for the MR1-treated group. The risk of more rapid death was 2.8 to 3.2 times higher for control animals than for MR1-treated animals. The delay was statistically significant when analyzed using several methods. (Kaplan-Meier, log-rank (p=0.0040) and Wilcoxon tests (p=0.0109); Cox proportional hazards model (p=0.0060); parametric analysis (p=0.0049)).

[0144] Sixty female and 36 male G93A mice were litter-matched and randomly assigned to treatment or control groups. Thirty female mice and 18 male mice were treated with MR1 starting on day 50. Study days were based on birth.

[0145] On day 50, a single bolus of 5.22 mg / kg or 6.75 mg / kg MR1 was administered intraperitoneally in females or males, respectively. After the bolus, females received weekly injections of 1 mg / kg MR1 and males received weekly injections of 1.34 mg / kg MR1 via intraperitoneal injection. Doses were prepared in vehicle (phosphate buffered saline (PBS, pH 7.3)) to a total volume of 200 μl. Control animals were administered 200 μl PBS. Animals were monitored for body weight, neurological scores, non-related ALS deaths, and euthanasia criteria as previously described.

[0146] The time-to-event curves from day 40 to the time to peak body weight for the comparative MR1-treated and control groups are shown in Fig. 7AThe median time to peak body weight was 49 days in the control group compared with 53 days in the MR1-treated group. This difference was not significant when analyzed by Kaplan-Meier using log-rank and Wilcoxon statistical models, Cox proportional hazards models, or parametric statistical tests.

[0147] The time-to-event curves from peak body weight to death for the comparative MR1-treated and control groups are shown in Figure 7B In the MRI-treated animals, the time from peak body weight to death was statistically significantly delayed by 6 days. The median time from peak body weight to death in the control group was 29 days, while it was 35 days in the MR1-treated group. The significance of each analysis in this example was calculated in several ways to better assess the significance. The delay was statistically significant when analyzed using several statistical models: Kaplan-Meier, log-rank (p=0.0413) and Wilcoxon (p=0.0732); and Cox proportional hazards model (p=0.0460). Based on the body weight data, MR1 appears to have a small effect on the onset of disease in the G93A mouse model, but has a significant effect in slowing the rate of weight loss from peak body weight until death.

[0148] The time-to-event graphs of the age at which MR1 and control animals progressed to a neurologic score of 2 and the number of days to a neurologic score of 2 are shown in Figure 7C The time to score 2 was 113 days in the control group and 121 days for the MR1 treated group. Based on the neurological function score data, MR1 delayed the onset of disease in the G93A mouse model by approximately 8 days, and the delay was statistically significant when analyzed using the following analytical models: Kaplan-Meier, log-rank (p=0.0038) and Wilcoxon (p=0.0017); and Cox proportional hazards model (p=0.0010).

[0149] The survival of treated animals was 9 days later than that of control animals. The median survival of the control group was 124 days, and the median survival of the MR1 treated group was 133 days. Fig.7D The delay was statistically significant when analyzed using several statistical models: Kaplan-Meier, log-rank (p=0.0043), and Wilcoxon tests (p=0.0040); and the Cox proportional hazards model (p=0.0030).

[0150] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

[0151] The nucleic acid sequences encoding the heavy and light chains of the desired antibodies are designed to be suitable for expression in mammalian cells, such as Chinese hamster ovary (CHO) cells. The nucleic acids are then artificially synthesized and connected to the antibody expression vector BPJPuro using standard molecular biology techniques. BPJPuro is a dual-gene mammalian expression vector optimized for selective and stable expression of immunoglobulins in Chinese hamster ovary (CHO) cells. The vector is then transfected into CHO cells, and stable transfectants are selected.

[0152] Similarly, nucleic acid encoding the heavy chain having the amino acid sequence of SEQ ID NO: 9 (SEQ ID NO: 10) and nucleic acid encoding the 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.

[0153] The obtained expression vector encoding heavy chain and light chain is transfected into CHO system (CHO SA, Cellectis SA, Paris, France) using liposome-mediated transfection. Stable transfectants are separated by puromycin selection and subcloned to provide clonal cell lines. Candidate cell lines are adapted to serum-free suspension culture, and are screened for IgG production and robust growth. A cell line is selected and named JB5, which is cultivated in a pilot scale bioreactor, and antibody JB5 is purified from conditioned medium by sequential concentration, protein A / G affinity chromatography and size exclusion chromatography.

[0154] The binding kinetics of the JB5 antibody relative to the parent antibody hu5c8 were determined using a three-part sandwich ELISA assay. All washes were performed using 3 washes of 250 μl PBS. 96-well polystyrene plates were coated with 100 μl / well JB5 or hu5c8 antibodies (2 μg / ml) for 16 hours at 4°C. The plates were washed and then blocked with 2% bovine serum albumin / PBS for 1 hour at room temperature. The plates were washed and recombinant human CD40L protein (Santa Cruz Biotechnology, Santa Cruz, California, USA) was added to the plates and titrated by 2-fold dilution 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 (100 ng / ml) were used to detect the bound CD40L protein. Colorimetric detection was performed using the colorimetric reagent TMB (3,3′,5,5′-tetramethylbenzidine), and the absorbance at 450 nm was analyzed spectrophotometrically. The resulting binding curve ( Figure 8) shows that JB5 (circles) has highly similar CD40L binding relative to the parent antibody hu5c8 (squares). The control protein CTLA4-IgG1 (triangles) with the same Fc domain as JB5 did not show significant binding. The calculated EC50s for hu5c8 and JB5 were 114 and 137 nM, respectively. JB5-R28K and JB5-K74R showed similar binding to that of JB5.

[0155] To compare the CD40L binding of antibodies from all 16 clones compared to 5c8 or AT-1501, a binding assay was run using 2 clones (5c8 and AT-1501 run on the same 96-well assay plate). A three-part sandwich ELISA assay was used to determine the binding level of the antibodies of the present disclosure compared to reference antibodies 5c8-19 and AT1501. A 96-well polystyrene plate was coated with recombinant human CD40L (BioLegend catalog number 591706) (using 2 μg / ml in PBS) and 50 μl / well was added to a Costar 96-well 1 / 2 area high binding assay plate (Corning 3690) and incubated overnight at 4°C. The plate was blocked with (1X) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. The binding curve of 5C8 or AT1501 (serial 2-fold dilution from 2 μg / ml) (50 μl / well) was added and incubated at room temperature for 1 hour. The plate was washed and incubated at room temperature for 1 hour with 1:10,000 dilution of HRP-(Fab2) donkey anti-human IgG (Fc specific) (Jackson Immuno. 709-036-098) (50 μl / well). The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) (50 μl / well) was added. After 5 minutes at room temperature, 25 μl 2NH2SO4 was used to stop the color development. The plate was read on a Molecular Devices SpectraMaxM5 plate reader using the SoftMax Pro 6.2.2 program to determine the absorbance at 450 nm.

[0156] The relative CD40L binding potency was calculated as follows: [IC50 (clone) / IC50 (5c8 or AT-1501) x 100%]. The ranked potency of the 16 clones compared to 5c8 is shown in Fig.15A and the ranking efficacy compared to AT-1501 is shown in Fig. 15B The resulting binding curve is shown in Figure 16A-16Q (Comparison of 16 clones with 5c8, Fig.16Hshows a comparison between AT-1501 and 5c8) and Figure 17A-17Q (Comparison of 16 clones with AT-1501, Fig.17H The IC50, LCL, UCL and relative potency of each clone are shown in Table 1 (comparison with 5c8) and Table 2 (comparison with AT-1501).

[0157] Table 1:

[0158]

[0159]

[0160]

[0161] Table 2:

[0162]

[0163]

[0164] 16 VH / VL antibody clones were constructed using IgG1 Fc (SEQ ID NO: 21) with two mutations P238S and N297G. These antibody clones were assayed for Fc effector function binding to human FcγRI, FcγRIIa and FcγRIIIa.

[0165] Anti-CD40L antibodies (including abatacept as a negative control) were diluted to 2 μg / ml in (1X) PBS and 50 μl / well were added to Costar 96-well 1 / 2 area high binding assay plates (Corning 3690) for overnight incubation at 4°C. The plates were blocked with (1X) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. A binding curve of recombinant human FcγRI, IIa, IIIa and IIIb (serial 2-fold dilutions starting from 5 μg / ml) (50 μl / well) was added and incubated for 1 hour at room temperature. The plate was washed and incubated at room temperature for 1 hour with 2 μg / ml of mouse anti-human CD16 (anti-FcRIII); CD32 (anti-FcRIIa) or CD64 (anti-FcRI) (eBioSciences / Invitrogen 14-0168-82; 16-0329-81; 14-0649-82) (50 μl / well). The plate was washed and incubated at room temperature for 1 hour with 1:10,000 dilution of HRP-(Fab2) goat anti-mouse IgG (Fc specific) (Jackson ImmunoResearch 116-036-071) (50 μl / well). The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) (50 μl / well) was added. After 5 min at room temperature, the color development was stopped with (25 μl / well) 2NH2SO4. Plates were read on a Molecular Devices SpectraMax M5 plate reader using the SoftMax Pro 6.2.2 program to determine absorbance at 450 nm.

[0166] As can be observed in the figures, the clones disclosed in the present application were negative for binding to the three Fc receptors, whereas 5c8 bound to FcγRI, FcγRIIa, but not to FcγRIIIa or FcγRIIIb. Fig.18A and Fig.18B Binding of eight antibody clones to FcγRIa is shown individually in comparison with the binding curves of 5c8, AT-1501 and abatacept. Fig.19A and Fig.19B Binding of eight antibody clones to FcγRIIa is shown individually in comparison to the binding curves of 5c8, AT-1501 and abatacept. Fig. 20A and Fig. 20B Binding of eight antibody clones to FcγRIIIa is shown individually in comparison to the binding curves of 5c8, AT-1501 and abatacept. Fig. 20C and Fig.20DEach shows the binding of eight antibody clones to FcγRIIIb compared with the binding curves of 5c8, AT-1501 and abatacept. Fig.18A , Fig.19A , Fig. 20A and Fig. 20B Results from antibody clones 4-4, 5-3, 6-6, 7-5, 8-3, 8-4, 10-1, and 10-4 are shown; Fig.18B , Fig.19B , Fig. 20C and Fig.20D Results from antibody clones 11-5, 12-4, 13-2, 15-1, 15-4, 16-3, 17-1, and 18-2 are shown).

[0167] Anti-CD40L antibodies (including abatacept as a negative control) were diluted to 2 μg / ml in (1X) PBS and 50 μl / well were added to Costar 96-well 1 / 2 area high binding assay plates (Corning 3690) for overnight incubation at 4°C. The plates were blocked with (1X) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. Native human C1q protein (Abcam ab96363) (serial 2-fold dilutions starting from 10 μg / ml) (50 μl / well) was added and the binding curve was continued for 1 hour at room temperature. The plates were washed and HRP-sheep anti-human C1q (Abcam ab46191) at a dilution of 1:400 was added (50 μl / well) and incubated for 1 hour at room temperature. The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) (50 μl / well) was added. After 5 minutes at room temperature, the color development was stopped with (25 μl / well) 2NH2SO4. 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.

[0168] As can be observed in the figure, antibodies from all sixteen antibody clones disclosed in the present application were negative for binding to C1q, whereas 5c8 showed significant binding. Fig.21A and Fig. 21B Binding of eight antibody clones to C1q is shown in comparison with the binding curves of 5c8, AT-1501 and abatacept respectively. Fig.21A Results from antibody clones 4-4, 5-3, 6-6, 7-5, 8-3, 8-4, 10-1, and 10-4 are shown; Fig. 21B Results from antibody clones 11-5, 12-4, 13-2, 15-1, 15-4, 16-3, 17-1, and 18-2 are shown.

[0169] Since genetic expression data indicated that costimulatory pathways were involved, the efficacy of MRI was evaluated in the G93A model. MR1 binds to CD40L, thereby blocking its interaction with CD40, which participates in costimulatory pathways involved in immune responses. It has been reported in the literature that MR1 is effective in treating rheumatoid arthritis and graft-versus-host disease, both of which have strong immune components. Rheumatoid arthritis is an autoimmune disease, and graft-versus-host disease occurs when the host's body mounts a strong immune response to the transplanted tissue.

[0170] Although ALS has an immune component mediated by activation of microglia and astrocytes, it is not considered an autoimmune disorder. Several anti-inflammatory drugs have failed to show efficacy in preclinical or clinical testing, including TNFα inhibitors, Minocycline and thalidomide. Therefore, it was unexpected to find that MR1 showed efficacy in the G93A ALS model.

[0171] Methods according to the present disclosure include methods of treating patients with neurodegenerative disorders and / or neuromuscular disorders by administering to the patient a compound that blocks the interaction of CD40L with CD40 and / or blocks the interaction of CD28 with CD80 and / or blocks the interaction of CD28 with CD86. One embodiment is a method of treating patients with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, and spinocerebellar ataxia by administering to the patient a compound that blocks the interaction of CD40L with CD40. In another embodiment, the method of treating the patient is by administering an anti-CD40L antibody.

[0172] In some embodiments, therapeutic compounds include any compound that blocks the interaction of CD40 with CD40L. For example, many animal studies describe agents that can block CD40:CD40L binding (see, e.g., US2005158314 and US7173046, which are hereby incorporated by reference). Also, for example, many anti-CD40L antibodies have been generated and characterized. (See, e.g., U.S. Pat. No. 5,876,950 to Bristol-Myers Squibb, which is hereby incorporated by reference). Anti-CD40L antibodies that can be used in the methods of the present disclosure include, but are not limited to, MR1, a hamster monoclonal antibody available from Taconic (Hudson, NY) and BD Biosciences (San Jose, CA); 5c8, a humanized antibody described in U.S. Pat. No. 5,474,771 (which is hereby incorporated by reference); hamster-human chimeric antibody IDEC 131 / E6040, a humanized monoclonal antibody comprising a human gamma-1 heavy chain and a human kappa light chain having the CDRs of murine monoclonal antibody clone 24-31, commercially available from Ancell (Catalog No. X353-020, Bayport, MN); ABI 793; Sgn-40; ImxM90 (Immunex); ImxM91 (Immunex); ImxM92 (Immunex); and anti-CD40L mAb commercially available from Genzyme (Cambridge, Mass., catalog number 80-3703-01). Also commercially available is anti-CD40L mAb from PharMingen (San Diego, catalog number 33580D). Embodiments according to the present disclosure include methods of treating a patient suffering from a neurodegenerative disorder or a neuromuscular disorder comprising administering a therapeutically effective amount of an anti-CD40L antibody. One embodiment is a method of treating a patient with a neurodegenerative disorder or a neuromuscular disorder, the method comprising administering a therapeutically effective amount of an anti-CD40L antibody selected from the group consisting of MR1, 5c8, IDEC131 / E6040, clone 24-31, ABI 793, ImxM90, ImxM91, ImxM92, or Sgn-40. In one embodiment, the antibody is 5c8. In another embodiment, the antibody is MR1.

[0173] In some embodiments, the neurodegenerative disorder or neuromuscular disorder is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, or spinocerebellar ataxia. One embodiment is a method of treating a patient with amyotrophic lateral sclerosis, the method comprising administering a therapeutically effective amount of an anti-CD40L antibody. In a specific embodiment, the anti-CD40L antibody is MR1. In another specific embodiment, the anti-CD40L antibody is 5c8.

[0174] In another embodiment, the method of treatment comprises administering a therapeutically effective amount of an anti-CD40 antibody. In some embodiments, the anti-CD40L compound is a Fab fragment, F(ab')2, F(ab'), a single-chain antibody, a polypeptide, a fusion construct of a polypeptide, etc. In some embodiments, these compounds are small molecule compounds capable of blocking CD40:CD40L interactions. In other embodiments, these compounds include BIO3417 or any compound disclosed in U.S. Patent No. 7,173,046 that has the ability to block CD40:CD40L interactions.

[0175] 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 suffering from a neurodegenerative disorder or a neuromuscular disorder, the method comprising administering a combination of a compound that blocks the interaction of CD40L with CD40 and a compound that blocks the interaction between CD80 and CD28. Another embodiment is a method of treating a patient by administering a combination of a compound that blocks the interaction of CD40L with CD40 and a compound that blocks the interaction between CD86 and CD28. In one embodiment, the compound that blocks the interaction of CD80 with CD28 is galiximab, or H1f1 and h3d1, or 16C10, or 7C10. In one embodiment, the compound that blocks the interaction between CD86 and CD28 is a CTLA4-Ig protein conjugate, such as abatacept or belatacept. Embodiments according to the present disclosure also include methods for treating patients with neurodegenerative disorders or neuromuscular disorders, the method comprising administering a combination of a compound that blocks the interaction of CD40L with CD40 and a compound that blocks the interaction between CD80 and CD28, or administering a therapeutically effective amount of a compound that blocks the interaction of CD40L with CD40 and a compound that blocks the interaction between CD86 and CD28, wherein the neurodegenerative disorder or neuromuscular disorder is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multifocal motor neuropathy, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, or spinocerebellar ataxia. In certain embodiments, the compound that blocks the interaction of CD40L with CD40 is MR1, and the compound that blocks the interaction between CD28 and CD86 or between CD28 and CD80 is abatacept, galiximab, or belatacept. In other embodiments, the compound that blocks the interaction of CD40L with CD40 is 5c8. Another embodiment is a method of treating a patient with amyotrophic lateral sclerosis, the method comprising administering a therapeutically effective amount of MR1 in combination with abatacept or belatacept. Another embodiment is a method of treating a patient with amyotrophic lateral sclerosis, the method comprising administering a therapeutically effective amount of 5c8 in combination with abatacept or belatacept.

[0176] Pharmaceutical compositions and methods of administration

[0177] For treating any of the foregoing disorders, one or more physiologically acceptable carriers can be used to formulate a pharmaceutical composition for use in accordance with the methods of the present disclosure in a conventional manner. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and by the particular method used to administer the composition. Accordingly, 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 Edition, edited by Gennaro et al., Lippincott Williams and Wilkins, 2000).

[0178] Formulations suitable for oral administration include, for example, solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particles, liquids or powders; lozenges (including liquid-filled); chewable tablets; gels; rapid-dissolving dosage forms; films; ovules; sprays; and oral / mucoadhesive patches.

[0179] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostatic agents and solutes that render the formulation isotonic with the blood of the intended recipient), and aqueous and non-aqueous sterile suspensions (which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives).

[0180] In accordance with the present disclosure, the compounds can be administered by any suitable means, which can vary depending on the type of disorder being treated and the nature of the compound itself. For example, the compounds can be administered orally, parenterally or topically. For proteins (such as antibodies), the route of administration preferably includes parenteral, such as intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous. Preferably, parenteral administration is by injection, most preferably intravenous, intramuscular or subcutaneous injection. The amount to be administered will depend on a variety of factors such as clinical symptoms, the weight of the individual and whether other medications are being administered. It should be understood that the determination of the appropriate dosage form, dose and route of administration is within the level of ordinary skill in the pharmaceutical and medical arts and is described below.

[0181] 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 its antigen-binding fragment for intravenous or subcutaneous administration. The formulation can be provided as a disposable prefilled pen, as a disposable (e.g., containing about 1 mL) prefilled glass syringe, or as a disposable mechanism use vial. Preferably, the pharmaceutical composition containing the anti-CD40L antibody or its antigen-binding fragment is transparent and colorless, with a pH of about 5.0 to about 6.9, preferably a pH of about 5.0 to about 6.5, and even more preferably a pH range of about 5.0 to about 6.0. In various embodiments, when reconstituted and administered to a subject, the formulation containing the pharmaceutical composition can contain from about 500 mg to about 1 mg, or from about 400 mg to about 10 mg, or from about 300 mg to about 30 mg, or from about 200 mg to about 50 mg of anti-CD40L antibody or its antigen-binding fragment per mL of solution.

[0182] The administration scheme of selecting therapeutic agent depends on several factors, including the serum or tissue turnover rate of entity, symptom level, immunogenicity of entity and the accessibility of target cells in biological matrix, the general health status of patient, the previous medical history of patient, etc. Preferably, the administration scheme maximizes the amount of therapeutic agent delivered to the patient, consistent with the acceptable side effect level. Therefore, the amount of biological preparation delivered depends in part on the severity of the specific entity and the disease treated. It should be understood that the determination of appropriate dosage form, dosage and administration route is within the level of ordinary technicians in the pharmaceutical field and medical field.

[0183] Dose and exposure

[0184] The pharmaceutical formulations of the present disclosure may contain from about 0.001 to about 200 mg / kg of an anti-CD40L antibody or antigen-binding fragment thereof, for example, from about 0.001 mg / kg to about 100 mg / kg, or from about 0.001 mg / kg to about 50 mg / kg, or from about 0.001 mg / kg to about 10 mg / kg of an anti-CD40L antibody or antigen-binding fragment thereof may be given as a bolus, and the remainder of the antibody dose may be administered by intravenous injection. A predetermined dose of an anti-CD40L antibody or antigen-binding fragment thereof may be administered, for example, over a period of one to two to five hours.

[0185] In other embodiments, a portion of the dose is administered by subcutaneous injection and / or infusion as a bolus, and the remainder of the dose is administered by infusion of the antibody formulation. In some exemplary doses, the antibody formulation may be administered subcutaneously at a dose ranging from about 0.001 to about 200 mg / kg (e.g., from about 0.001 mg / kg to about 100 mg / kg, or from about 0.001 mg / kg to about 50 mg / kg, or from about 0.001 mg / kg to about 10 mg / kg) of an intravenous injection of an anti-CD40L antibody or antigen-binding fragment thereof. In some embodiments, the dose may be given as a bolus, and the remainder of the antibody dose may be administered by subcutaneous or intravenous injection. A predetermined dose of an anti-CD40L antibody or antigen-binding fragment thereof may be administered, for example, over a period of one hour, or a period of two hours, or a period of three hours, or a period of four hours, or a period of five hours or more.

[0186] In some embodiments, the dose is administered at a constant rate, such as to maintain a steady state in the plasma. In some embodiments, the dose is administered every two weeks. In some embodiments, the compound is administered so that the concentration of the compound in the subject's plasma is maintained at a dose between 1 μg / mL and 1 mg / mL. In some, the compound is administered so that the concentration of the compound in the subject's plasma is maintained at a dose between 1 μg / mL and 500 μg / mL. In some embodiments, the compound is administered so that the concentration of the compound in the subject's plasma is maintained at a dose between 1 μg / mL and 250 μg / mL. In some embodiments, the compound is administered so that the concentration of the compound in the subject's plasma is maintained at a dose between 5 μg / mL and 100 μg / mL. In some embodiments, the compound is administered so that the concentration of the compound in the subject's plasma is maintained at a dose between 30 μg / mL and 60 μg / mL.

[0187] Biomarkers

[0188] Some embodiments of the present application relate to modifying biomarkers in a subject by treatment with a compound targeting 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 a pro-inflammatory marker or is associated with inflammation. In some embodiments, the biomarker is associated with ALS. In some embodiments, the biomarker is associated with neurodegeneration or neuromuscular disease or disorder. 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.

[0189] In some embodiments, treatment with a compound targeting CD40 / CD40L increases the concentration of one or more biomarkers. In some embodiments, treatment with a compound targeting CD40 / CD40L decreases the concentration of one or more biomarkers. In some embodiments, treatment with a compound targeting CD40 / CD40L increases the concentration of at least one biomarker and decreases 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 with a disease, disorder or condition to treatment with a compound targeting CD40 / CD40L.

[0190] Combination therapy

[0191] The antibodies or antibody fragments thereof 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 may include one or more additional therapeutic agents. In another embodiment, the combination therapy includes standard of care treatment, which may or may not include additional therapeutic agents (consisting essentially of the antibody or antibody fragment thereof).

[0192] Adjunctive or combined administration (co-administration) includes simultaneous administration of any antibody or antigen-binding fragment thereof described herein and one or more agents in the same or different dosage forms, or separate administration of the polypeptide and one or more agents (e.g., sequential administration). Such simultaneous or sequential administration preferably results in both the polypeptide and one or more agents being present in the treated patient at the same time.

[0193] Kits and Products

[0194] A test kit containing an antibody or its antigen-binding fragment as described herein and instructions for use is further provided. Test kits typically include a packaged combination of a predetermined amount of reagents and instructions and labels indicating the intended use of the contents of the test kit. The term label or instructions includes any written or recorded material provided on or with the test kit or otherwise accompanying the test kit at any time during the manufacture, transportation, sale or use of the test kit. It can be in a form prescribed by a government agency that manages the manufacture, use or sale of a drug or biological product, and the announcement reflects the agency's approval of manufacture, use or sale for use in humans or veterinary use. Labels or instructions can also cover advertising leaflets and brochures, packaging materials, and audio or video instructions.

[0195] Anti-CD40L antigen binding molecules

[0196] Some embodiments provided herein relate to antibodies that are modified forms of the anti-CD40L antibody hu5c8, comprising a human IgG1 consensus framework having variable light and variable heavy chain CDR sequences of hu5c8, wherein the Fc domain is modified to prevent platelet activation.

[0197] Table 3 provides a description of the SEQ ID NOs cited in this application.

[0198] Table 3:

[0199]

[0200]

[0201] 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 comprising an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, and the heavy chain comprises a variable heavy chain region and an Fc region, wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 2, and the Fc region comprises an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. ID NO: 3 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, wherein the Fc region comprises a substitution or a combination of substitutions selected from the group consisting of: C11S, C14S and P23S.

[0202] Another embodiment (embodiment B) is the isolated antibody according to embodiment A, wherein the Fc region further comprises the amino acid substitution C5S.

[0203] In a variation of Examples A and B, the antibody comprises a light chain variable region that does not contain any of the substitutions T33W, S26D and Q27E.

[0204] In other variations of Examples A and B, the light chain variable region comprises the substitution R28K.

[0205] In some variations of Examples A and B, the CDRs of the heavy and light chains have the sequences listed in Table 4.

[0206] Table 4:

[0207]

[0208]

[0209] In yet other variations of embodiments A and B, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15). In still other embodiments, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15) and one or two of the amino acid sequences YASNLES (SEQ ID NO: 16) and QHSWEIPPT (SEQ ID NO: 17).

[0210] In some variations of embodiments A and B, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 1. In yet other embodiments, the light chain variable region consists of the amino acids of SEQ ID NO: 1. In some embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 7. In other embodiments, the light chain consists of the amino acid sequence of SEQ ID NO: 7. In still other embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 11. In yet other embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 11. In still other embodiments, the light chain consists of the amino acid sequence of SEQ ID NO: 11.

[0211] In other variations of embodiments A and B, the antibody comprises 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 variations 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.

[0212] In yet other variations 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).

[0213] 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 yet 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.

[0214] Some embodiments provided herein relate to an isolated antibody, wherein the light chain comprises the amino acid sequence of SEQ ID NO:1 and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0215] Some embodiments provided herein relate to an isolated antibody wherein the light chain consists of the amino acid sequence of SEQ ID NO:7 and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0216] Some embodiments provided herein relate to an isolated antibody, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:5, and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0217] Still another embodiment is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:11, and the heavy chain consists of the amino acid sequence of SEQ ID NO:9.

[0218] Yet another embodiment is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:7, and the heavy chain consists of the amino acid sequence of SEQ ID NO:13.

[0219] Another embodiment is an isolated antibody, wherein the light chain consists of the amino acid sequence of SEQ ID NO:11, and the heavy chain consists of the amino acid sequence of SEQ ID NO:13.

[0220] Some embodiments provided herein involve the alternatives enumerated below.

[0221] A method for predicting the response of a subject having a disease or disorder to treatment with a compound directed against CD40L or CD40, the method 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; wherein the concentration of the at least one biomarker predicts that the subject is likely to respond to treatment of the disease or disorder with a compound directed against CD40L or CD40.

[0222] The method of Alternative 1 wherein the disease or disorder is inflammation, a neurodegenerative disease, or a neuromuscular disease.

[0223] The method of Alternative Embodiment 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's disease or spinocerebellar ataxia.

[0224] The method of Alternative Embodiment 3, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

[0225] The method of any one of Alternatives 1-4, wherein the subject is a mammal and / or a human.

[0226] The method of any of Alternatives 1-5, wherein the compound blocks the interaction of CD40 and CD40L.

[0227] The method of any of Alternatives 1-6, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0228] The method of any one of Alternatives 1-7, wherein the compound is tigrut.

[0229] The method of any one of Alternatives 1-8, wherein the compound is MR1 ​​or 5c8.

[0230] A method of treating a subject suffering from a disease or disorder, the method comprising: administering a therapeutically effective dose of a compound directed against CD40L or CD40, wherein administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

[0231] The method of Alternative 10 wherein the disease or disorder is inflammation, a neurodegenerative disease, or a neuromuscular disease.

[0232] The method of Alternative Embodiment 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's disease, or spinocerebellar ataxia.

[0233] The method of Alternative Embodiment 12 wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

[0234] The method of any one of Alternatives 10-13, wherein the subject is a mammal and / or a human.

[0235] The method of any of Alternatives 10-14, wherein the compound blocks the interaction of CD40 and CD40L.

[0236] The method of any of Alternatives 10-15, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0237] The method of any of Alternatives 10-16, wherein the compound is tigrut.

[0238] The method of any of Alternatives 10-17, wherein the compound is MR1 ​​or 5c8.

[0239] The method of any of Alternatives 10-18, wherein the compound is administered at least once every two weeks.

[0240] The method of any of Alternatives 10-19, wherein the compound is administered for a period of at least 12 weeks.

[0241] The method of any of Alternatives 10-20, wherein the compound is administered at a dose between 200 mg / kg and 100 mg / kg, preferably at a dose between 1 mg / kg and 8 mg / kg, most preferably at a dose between 2 mg / kg and 4 mg / kg.

[0242] The method of any of Alternatives 10-21, further comprising administering a second pharmaceutically effective compound.

[0243] The method of Alternative Embodiment 22, wherein the second compound blocks the interaction between CD28 and CD86 or between CD28 and CD80.

[0244] The method of Alternative Embodiment 22 wherein the second compound targets at least one of the biomarkers selected from the group consisting of an interleukin, a cytokine, and a pro-inflammatory marker.

[0245] A method as described in Alternative Embodiment 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, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9 and MMP2.

[0246] The method of Alternative Embodiment 22, wherein the second compound is CTLA4-Ig fusion protein, abatacept, belatacept or galiximab.

[0247] The method of any of Alternatives 10-26, wherein the compound is administered orally, parenterally or topically.

[0248] The method of Alternative Embodiment 27 wherein the compound is administered parenterally.

[0249] A method as described in Alternative 28 wherein the compound is administered by injection, most preferably intravenous, intramuscular or subcutaneous injection.

[0250] The method of any of Alternatives 1-29, 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, immune graft response, transplant rejection, and atherosclerosis.

[0251] The method of any of Alternatives 1-30, 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.

[0252] The method of any one of Alternatives 1-31, wherein the compound targeting CD40L or CD40 is an antibody comprising: (a) a heavy chain variable region (V H ), the heavy chain variable region comprising: i) a CDRH1 domain comprising the sequence shown in SEQ ID NO: 9; ii) a CDRH2 domain comprising the sequence shown in SEQ ID NO: 10, 11, 12, 13 or 14; and iii) a CDRH3 domain comprising the sequence shown in SEQ ID NO: 15; and (b) a light chain variable region (V L ), the light chain variable region comprises: i) a CDRL1 domain comprising the sequence shown in SEQ ID NO: 16 or 17; ii) a CDRL2 domain comprising the sequence shown in SEQ ID NO: 18 or 19; and iii) a CDRL3 domain comprising the sequence shown in SEQ ID NO: 20.

[0253] The method of any one of Alternatives 1-32, wherein the compound targeting CD40L or CD40 is an antibody comprising: (a) a heavy chain variable region (V H ), the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; and (b) a light chain variable region (V L ), the light chain variable region having an amino acid sequence as shown in SEQ ID NO:5, 6, 7 or 8.

[0254] A method for modulating the concentration of at least one biomarker in a subject, the method comprising: administering to the subject a therapeutically effective amount of a compound directed 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, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10 or MMP2.

[0255] The method of Alternative Embodiment 34 wherein the concentration of the at least one biomarker increases following administration of the compound.

[0256] The method of Alternative Embodiment 34 wherein the concentration of the at least one biomarker decreases following administration of the compound.

[0257] The method of Alternative Embodiment 34 wherein following administration of the compound, the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases.

[0258] The method of any of Alternatives 34-37, wherein the subject has a disease or disorder.

[0259] The method of Alternative Embodiment 38 wherein the disease or disorder is inflammation, a neurodegenerative disease, or a neuromuscular disease.

[0260] The method of Alternative Embodiment 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's disease, or spinocerebellar ataxia.

[0261] The method of Alternative Embodiment 40 wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

[0262] The method of any one of Alternatives 34-41, wherein the subject is a mammal and / or a human.

[0263] The method of any of Alternatives 34-42, wherein the compound blocks the interaction of CD40 and CD40L.

[0264] The method of any of Alternatives 34-43, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0265] The method of any one of Alternatives 34-44, wherein the compound is tigrut.

[0266] The method of any of Alternatives 34-45, wherein the compound is MR1 ​​or 5c8.

[0267] The method of any of Alternatives 34-46, wherein administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

[0268] The method of any of Alternatives 34-47, wherein the compound is administered at least once every two weeks.

[0269] The method of any one of Alternatives 34-48, wherein the compound is administered for a period of at least 12 weeks.

[0270] The method of any of Alternatives 34-49, wherein the compound is administered at a dose between 200 mg / kg and 100 mg / kg, preferably at a dose between 1 mg / kg and 8 mg / kg, most preferably at a dose between 2 mg / kg and 4 mg / kg.

[0271] The method of any of Alternatives 34-50, wherein the compound is administered orally, parenterally or topically.

[0272] The method of Alternative Embodiment 51 wherein the compound is administered parenterally.

[0273] A method as described in Alternative Embodiment 52, wherein the compound is administered by injection, most preferably intravenous, intramuscular or subcutaneous injection.

[0274] The method of any of Alternatives 34-53, 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, immune graft response, transplant rejection, and atherosclerosis.

[0275] The method of any of Alternatives 34-54, 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.

[0276] The method of any of Alternatives 34-55, wherein the compound targeting CD40L or CD40 is an antibody comprising: (a) a heavy chain variable region (V H ), the heavy chain variable region comprising: i) a CDRH1 domain comprising the sequence shown in SEQ ID NO: 9; ii) a CDRH2 domain comprising the sequence shown in SEQ ID NO: 10, 11, 12, 13 or 14; and iii) a CDRH3 domain comprising the sequence shown in SEQ ID NO: 15; and (b) a light chain variable region (V L ), the light chain variable region comprises: i) a CDRL1 domain comprising the sequence shown in SEQ ID NO: 16 or 17; ii) a CDRL2 domain comprising the sequence shown in SEQ ID NO: 18 or 19; and iii) a CDRL3 domain comprising the sequence shown in SEQ ID NO: 20.

[0277] The method of any of Alternatives 34-56, wherein the compound targeting CD40L or CD40 is an antibody comprising: (a) a heavy chain variable region (V H ), the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; and (b) a light chain variable region (V L ), the light chain variable region having an amino acid sequence as shown in SEQ ID NO:5, 6, 7 or 8.

[0278] A method for maintaining or improving the ALS-FRS score of a subject with ALS, the method comprising: administering a therapeutically effective dose of a compound directed against CD40L or CD40, wherein administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

[0279] A method for pre-screening a subject for a disease or disorder and treating the subject with a compound directed against CD40L or CD40, the method comprising: collecting a sample from the subject; detecting the concentration of at least one biomarker present in the 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 subjects not suffering from the disease or disorder; and administering a therapeutically effective dose of a compound directed against CD40L or CD40 such that the concentration of the compound in the subject's plasma is maintained at a dose between 5 μg / mL and 100 μg / mL; preferably between 30 μg / mL and 60 μg / mL.

[0280] The method of Alternative Embodiment 59, wherein the compound is administered to the subject if the concentration of the at least one biomarker in the subject is significantly different than the concentration of the at least one biomarker in subjects not suffering from the disease or disorder.

[0281] Examples

[0282] The embodiments provided herein are further defined in the following examples. It should be understood that these examples are given by way of illustration only. According to the above discussion and these examples, those skilled in the art can determine the essential features of the present disclosure, and without violating its spirit and scope, various changes and modifications of the embodiments of the present disclosure can be made to adapt them to various uses and conditions. Therefore, in addition to those modifications shown and described herein, the various modifications of the embodiments will become clear to those skilled in the art according to the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference shown herein is incorporated herein by reference in its entirety, and is used for the disclosures cited herein.

[0283] Example 1

[0284] Clinical study design

[0285] The effectiveness of compounds targeting CD40L or CD40 was evaluated in clinical trials. A total of 54 human subjects were involved; these subjects were selected based on a positive ALS diagnosis, or a possible, probable, or definite diagnosis derived from the laboratory-supported revised El Escorial criteria. Each subject also had an ALSFRS-R total score of 35 or higher and no more than 24 months had passed since their initial diagnosis. In addition, each subject selected for the trial had a tracheostomy or used a permanent assisted ventilation system. The demographics of the selected subjects are shown in Table 5.

[0286] Table 5:

[0287]

[0288]

[0289] There are four dosing cohorts: 1 mg / kg (n=9), 2 mg / kg (n=9), 4 mg / kg (n=18), 8 mg / kg (n=18). Fig. 22 Schematically summarized, two blood biosamples were collected from each subject as baseline data at -28 days or -4 days before the first administration of the compound. Two subsequent biosamples were collected on day 0. These biosamples consisted of 1 plasma sample for analysis of pK / ADA and 1 plasma sample for analysis of biomarker levels.

[0290] Immediately following biosample collection, each subject received an intravenous infusion of 1 mg / kg, 2 mg / kg, 4 mg / kg, or 8 mg / kg of the compound AT-1501 (also known as tigrut). This pattern of collecting plasma biosamples followed by administration of AT-1501 at a set dose was repeated every 14 days for a total of 101 days. Infusions were given on Days 0, 15, 29, 43, 57, and 71. Two hours after the infusion, two more biosamples were collected.

[0291] After dosing was completed, endpoints analyzed included: safety and tolerability, pharmacokinetics, and anti-drug antibody levels, CD40L target engagement, changes in pro-inflammatory markers, and changes in ALSFRS-R, respiratory function, and neurofilament light chain (NFL) levels.

[0292] Security

[0293] More than 90% of the subjects completed the clinical trial, as summarized in Table 6. One subject (noted under "Other" in the table) was eligible at screening, but did not receive a dose and was replaced by another participant instead.

[0294] Table 6:

[0295]

[0296] The safety of AT-1501 was evaluated by counting the number of treatment-emergent adverse events (TEAEs) in subjects during the course of the clinical trial. The results are summarized in Table 7. Overall, tigrut was well tolerated, with no treatment-related serious adverse events and no significant anti-drug antibody responses.

[0297] Table 7:

[0298]

[0299]

[0300] Specific symptoms of adverse reactions and the percentages of subjects who self-reported these symptoms are summarized in Table 8.

[0301] Table 8:

[0302]

[0303]

[0304] Example 2

[0305] Pharmacokinetics and anti-drug antibody concentrations

[0306] As discussed in Example 1, plasma biosamples were collected from subjects before and two hours after dosing by infusion. Infusions were performed on Days 0, 15, 29, 43, 57, and 71 of the clinical trial. These biosamples were used, in part, to assess the concentration of AT-1501 drug levels present in the blood. Fig.23 Shown is the superposition of the levels of AT-1501 in the plasma of each subject over time. Based on this analysis, the half-life of AT-1501 was determined to be greater than 14 days. Therefore, the exposure levels of the subjects increased over time and therefore steady state was not reached.

[0307] In addition, there was no evidence of neutralization of ADA. Nine ADA were present in 215 samples from seven subjects ( Figure 24A-Figure 24I ). Four of these nine ADA were detected at week 14 when the subject was no longer using the test article. One subject (102201) had a pre-existing ADA titer before the trial. The subjects and time of appearance of ADA positives are shown in Table 9. All nine ADAs had very low titers and were at the detection cutoff in the assay. These ADAs are non-neutralizing and do not affect the levels of the drug ticagrelor (AT-1501) given to these subjects.

[0308] Table 9:

[0309]

[0310]

[0311] Example 3

[0312] Universal Biomarker Analysis

[0313] Changes in molecular biomarkers were also assessed for each biological sample collected from subjects during the clinical trial. These markers included those used as measures of CD40L target engagement (CXCL13, IgA, and CD40L) and those used as measures of inflammation (TNF-α, IL-6, En-Raged, MCP-1, IL-1, IL1-β, and CRP). The following two analytical methods were used to examine biomarker changes: (1) regression models were calculated and all analytes for all subjects were plotted, and (2) data were normalized and then the fold changes of biomarkers over time were calculated and corrected for the false discovery rate.

[0314] Prior to analysis, data preprocessing was performed to remove outliers, such as subjects with scores less than 4 in the bulbar subdomain and subjects with an ALSFRS less than 35 on Day 0. The subjects removed as outliers are summarized in Table 10.

[0315] Table 10:

[0316]

[0317] In addition, proteins with rare positive signals were filtered. These proteins include: 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).

[0318] After analysis, a significant number of proteins were found to be downregulated after ticagrelor treatment ( Fig.25 ). The extent of protein increase or downregulation is dose-dependent.

[0319] Biomarkers of target engagement

[0320] The engagement of CD40L and CD40 by AT-1501 was assessed by monitoring the levels of CXCL13, CD40L, and CD40 in the subjects. AT-1501 exposure reduced the levels of all three proteins in a dose-dependent manner ( Fig.26A , Fig.26B , Fig.27A , Fig.27B , Fig.28A and Fig.28B ). The reduction was statistically significant at the 4 mg / kg and 8 mg / kg doses. The maximum reduction was achieved on the second day of AT-1501 administration to the patient. This demonstrates that the CD40L / CD40 target was significantly engaged after treatment.

[0321] Pro-inflammatory cytokine biomarkers

[0322] Changes in the concentrations of pro-inflammatory markers and other related stress proteins following treatment of subjects with AT-1501 were also assessed. Of those assessed, CD40L, CXCL13, CXCL9, TNF-α, En-Raged, TNFR2, IgM, IgA, IL2r, CXCL10, CD40, and B2M had the highest decreases in protein concentrations following AT-1501 treatment ( Figure 29A-29L A significant decrease in the concentrations of proteins VCAM, IL-18, IL-16, SAP, MIP1-β, MDC, C3, CRP, fibrinogen, IgE, ICAM, and MCP1 was also detected ( Figure 30A-Figure 30L ). After subjects were exposed to AT-1501, concentrations of several proteins increased; these included IL-6, ENA-78, RANTES, MMP3, and MMP9 ( Figure 31A-Figure 31E ). Overall, treatment of subjects with AT-1501 reduced the expression of more than 50% of the examined proteins associated with inflammatory and co-stimulatory signaling.

[0323] Neurofilament light chain (NFL) levels were also assessed at week 12 ( Fig.32 There were no statistically significant changes in NFL concentrations at any dose. However, NFL levels at recruitment did correlate with progression rate.

[0324] Example 4

[0325] ALS Functional Rating Scale

[0326] Every two weeks during the clinical trial, the subjects were assessed by clinicians for changes in the ALS Functional Rating Scale (ALS-FRS). Changes in ALS-FRS scores over time were plotted for all subjects; overall, each cohort deteriorated at a rate comparable to the general rate of untreated patients, as provided by the database Pro-ACT ( Fig.33A ).

[0327] Next, the data were amended to exclude patients who no longer met the initial recruitment criteria at week 12 ( Fig.33B ). Although this reduced the overall error in each group, the overall trend in ALS-FRS scores was strongly correlated with the rate in untreated patients.

[0328] The data were further amended to exclude patients with treatment-related responses (TE) ( Fig.33C This primarily reduced the decline in FRS in subjects given low levels of AT-1501, but had no significant effect in the higher dose cohort.

[0329] Finally, the data were amended to include only those patients who showed the best response to treatment with AT-1501 (hereinafter referred to as "responders") ( Fig.33D ). This most significantly altered the data, with a reduced decrease in ALS-FRS scores in treated subjects (particularly those in the 4 mg / kg dose cohort) compared to untreated subjects.

[0330] The overall slope of the monthly change in ALS-FRS for human subjects by dose group is shown in Table 11. The columns are: "All" (no patients are excluded), "EC" (those patients who do not meet the recruitment criteria are excluded), "TE" (those patients with treatment-related adverse reactions are excluded), and "Res" (only those patients who have a positive response to drug treatment are included). The "Pro-Act" row represents the ALS-FRS data for untreated patients taken from the Pro-ACT database.

[0331] Table 11:

[0332] Group all EC EC / TE Res 1mg / kg -0.83 -0.83 -063 -0.63 2mg / kg -0.94 -0.94 -0.75 -0.75 4mg / kg -0.5 -0.48 -0.48 -0.36 8mg / kg -1.79 -0.93 -0.93 -0.88 ProAct -0.83 -0.83 -0.83 -0.83

[0333] Responders and non-responders

[0334] Why do some patients appear to respond well to AT-1501 (referred to as "responders"), while others do not (referred to as "non-responders")? To answer this question, the ALS-FRS score for each individual was plotted over time. The ALS-FRS score values ​​for 16 of these individuals (8 responders and 8 non-responders) are shown in Table 12 and are plotted in graphical form at Fig.34 middle.

[0335] Table 12:

[0336]

[0337]

[0338] If you can Fig.34 As observed in the table above, the ALS-FRS of the "responder" subgroup did not change significantly from baseline to week 12. On the other hand, the "non-responders" declined at a rate consistent with untreated patients. This suggests that the "non-responder" group did not gain a detectable benefit from AT-1501, while the "responder" group gained significant resistance to the decline in ALS-FRS scores after treatment with AT-1501.

[0339] Next, multiple linear regression analysis was performed to compare the protein concentrations of each subject before treatment with AT-1501 (baseline values). Fig.35 As shown in Tables 13-14, several proteins were present at significantly higher concentrations in the plasma of subjects who subsequently had a positive response to treatment with AT-1501. Table 13 shows the F statistic and p-value for the difference in mean protein concentration of each protein at baseline plasma levels, as normalized for intensity. Linear regression was also used to analyze the difference in each protein at baseline as a predictor of the slope of the ALS-FRS change; these values ​​are shown in Table 14. Of these, the most significant was the initial concentration of NFL (not shown in Table 14 due to scaling differences). Fig.35 ), which has an F statistic of almost 50 points. In addition, the most significant initial protein concentrations include MMP9, MMP3, Il2ra, TNF-α, and IL-18.

[0340] Table 13:

[0341]

[0342]

[0343] Table 14:

[0344] protein F-statistic p-value NFL 44.08 1.41E-10 MMP9 10.77 0.00113 MMP3 10.76 0.001138 IL6 9.978 0.001716 IL18 9.746 0.00194 IgA 9.537 0.002167 TNFR2 7.211 0.007575 TNFa 7.077 0.01629 IL 5.651 0.01796 Factor VIII 5.302 0.02186 CRP 4.108 0.0434

[0345] like Fig.35 As shown in Tables 12-13, there were statistically significant differences in protein marker expression between non-responders and responders before any treatment. None of these protein levels were significantly associated with covariates such as race, sex, age, or treatment duration.

[0346] This finding is extremely meaningful because early assessment of the concentration of these proteins in the plasma of ALS patients can serve as a predictor of responsiveness of these patients to treatment with AT-1501. That is, if an individual has a biomarker at a concentration that corresponds to the responder cohort, they may benefit significantly from treatment with a compound targeting CD40L / CD40.

[0347] Summarize

[0348] Overall, ticagliptin demonstrated a good safety profile with linear pK and dose proportionality with low ADA incidence. In addition, ticagliptin demonstrated dose-dependent reductions in target engagement and T cell markers CD40L and B cell markers CXCL13, IgA and IgE, as well as increased patient responses with increasing doses. Tigliptin also reduced 3 of 4 prospectively selected proinflammatory markers, TNFa, MCP1 and En-Raged, in a dose-dependent manner, with increased patient responses. IL1b was below the limit of detection in multiplex ELISA, and IL6 was not downregulated by ticagliptin.

[0349] Tigret reduced the expression of 23 of the 32 proinflammatory proteins assayed, clearly demonstrating downstream regulation of proinflammatory signaling that has been described in multiple preclinical models of autoimmunity and transplantation by inhibition of CD40L. Reduction in proinflammatory signaling trended toward improvement in disease progression, as measured by the ALS FRS. Linear regression identified several proteins that correlated with changes in the ALS FRS over time.

[0350] Likewise, the findings reported in this application suggest that early quantification of selected proteins in the plasma of ALS patients can serve as predictors of the responsiveness of these patients to treatment with compounds targeting CD40L / CD40.

[0351] As used herein, section headings are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All documents and similar materials cited in this application (including but not limited to patents, patent applications, articles, books, papers and internet web pages) are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically cited herein. When the definition of a term in the incorporated reference appears to be different from the definition provided in this teaching, the definition provided in this teaching should be used as the standard. It will be understood that there is an implicit "about" before the temperature, concentration, time, etc. discussed in this teaching, so that slight and non-substantial deviations are within the scope of the teaching herein.

[0352] Although the present disclosure has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of embodiments and obvious modifications and equivalents thereof. In addition, although several variations of the present disclosure have been shown and described in detail, other modifications based on the present disclosure (which are within the scope of the present disclosure) will be easily clear to those skilled in the art. It is also contemplated that various combinations or sub-combinations may be made to the specific features and aspects of the embodiments, and these combinations or sub-combinations still fall within the scope of the embodiments. It should be understood that the various features and aspects of the disclosed embodiments may be combined or replaced with each other to form different modes or embodiments. Therefore, it is intended that the scope of the present disclosure should not be limited by the above-mentioned specifically disclosed embodiments.

[0353] It should be understood, however, that this detailed description, while indicating preferred embodiments, is given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

[0354] The terms used in the specification presented herein are not intended to be interpreted in any limited or restrictive manner. Instead, the terms are used only in conjunction with the detailed description of the embodiments of the systems, methods, and related components. In addition, the embodiments may include several novel features, no single feature of which is solely responsible for its desired attributes or is considered necessary to practice the embodiments.

Claims

1. A method of predicting the response of a subject suffering from a disease or disorder to treatment with a compound directed against CD40L or CD40, the method 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; wherein the concentration of the at least one biomarker predicts that the subject is likely to respond to treatment of the disease or disorder with a compound directed against CD40L or CD40.

2. The method of claim 1, wherein the disease or disorder is inflammation, a neurodegenerative disease, or a neuromuscular disease.

3. The method of 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's disease, or spinocerebellar ataxia.

4. The method of claim 3, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

5. The method of claim 1, wherein the subject is a mammal and / or a human.

6. The method of claim 1, wherein the compound blocks the interaction of CD40 and CD40L.

7. The method of claim 1, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

8. The method of claim 1, wherein the compound is ticagglutide.

9. The method of claim 1, wherein the compound is MR1 ​​or 5c8.

10. A method of treating a subject suffering from a disease or disorder, the method comprising: A therapeutically effective dose of a compound directed against CD40L or CD40 is administered, wherein administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

11. The method of claim 10, wherein the disease or disorder is inflammation, a neurodegenerative disease, or a neuromuscular disease.

12. The method of claim 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's disease, or spinocerebellar ataxia.

13. The method of claim 12, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

14. The method of claim 10, wherein the subject is a mammal and / or a human.

15. The method of claim 10, wherein the compound blocks the interaction of CD40 and CD40L.

16. The method of claim 10, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

17. The method of claim 10, wherein the compound is ticagglutide.

18. The method of claim 10, wherein the compound is MR1 ​​or 5c8.

19. The method of claim 10, wherein the compound is administered at least once every two weeks.

20. The method of claim 10, wherein the compound is administered for a period of at least 12 weeks.

21. The method of claim 10, wherein the compound is administered at a dose between 200 mg / kg and 100 mg / kg, preferably at a dose between 1 mg / kg and 8 mg / kg, most preferably at a dose between 2 mg / kg and 4 mg / kg.

22. The method of claim 10, further comprising administering a second pharmaceutically effective compound.

23. The method of claim 22, wherein the second compound blocks the interaction between CD28 and CD86 or between CD28 and CD80.

24. The method of claim 22, wherein the second compound targets at least one of the biomarkers selected from the group consisting of: an interleukin, a cytokine, and a pro-inflammatory marker.

25. The method of claim 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, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, and MMP2.

26. The method of claim 22, wherein the second compound is CTLA4-Ig fusion protein, abatacept, belatacept, or galiximab.

27. The method of claim 10, wherein the compound is administered orally, parenterally, or topically.

28. The method of claim 27, wherein the compound is administered parenterally.

29. The method of claim 28, wherein the compound is administered by injection, most preferably intravenous, intramuscular or subcutaneous injection.

30. The method of 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, immune graft response, transplant rejection, and atherosclerosis.

31. The method of 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.

32. The method of claim 1, wherein the compound that targets CD40L or CD40 is an antibody comprising: (a) Heavy chain variable region (V H ), the heavy chain variable region comprises: i) a CDRH1 domain comprising the sequence shown in SEQ ID NO:9; ii) a CDRH2 domain comprising the sequence shown in SEQ ID NO: 10, 11, 12, 13 or 14; and iii) a CDRH3 domain comprising the sequence shown in SEQ ID NO: 15; and (b) Light chain variable region (V L ), the light chain variable region comprises: i) a CDRL1 domain comprising the sequence shown in SEQ ID NO: 16 or 17; ii) a CDRL2 domain comprising the sequence shown in SEQ ID NO: 18 or 19; and iii) a CDRL3 domain comprising the sequence shown in SEQ ID NO:

20.

33. The method of any one of claims 1-32, wherein the compound that targets CD40L or CD40 is an antibody comprising: (a) Heavy chain variable region (V H ), wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; and (b) Light chain variable region (V L ), wherein the light chain variable region has an amino acid sequence as shown in SEQ ID NO:5, 6, 7 or 8.

34. A method of modulating the concentration of at least one biomarker in a subject, the method comprising: The subject is administered a therapeutically effective amount of a compound directed 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, Rantes, TIMP1, vWF, Il-6, MCP1, MMP3, MMP9, MCP1, MIG, vWF, Il-8, IP10 or MMP2.

35. The method of claim 34, wherein the concentration of the at least one biomarker increases after administration of the compound.

36. The method of claim 34, wherein the concentration of the at least one biomarker decreases after administration of the compound.

37. The method of claim 34, wherein after administration of the compound, the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases.

38. The method of claim 34, wherein the subject suffers from a disease or disorder.

39. The method of claim 38, wherein the disease or disorder is inflammation, a neurodegenerative disease, or a neuromuscular disease.

40. The method of claim 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's disease, or spinocerebellar ataxia.

41. The method of claim 40, wherein the neurodegenerative disease or neuromuscular disease is amyotrophic lateral sclerosis (ALS).

42. The method of claim 34, wherein the subject is a mammal and / or a human.

43. The method of claim 34, wherein the compound blocks the interaction of CD40 and CD40L.

44. The method of claim 34, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

45. The method of claim 34, wherein the compound is ticagglutide.

46. ​​The method of claim 34, wherein the compound is MR1 ​​or 5c8.

47. The method of claim 34, wherein administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

48. The method of claim 34, wherein the compound is administered at least once every two weeks.

49. The method of claim 34, wherein the compound is administered for a period of at least 12 weeks.

50. The method of claim 34, wherein the compound is administered at a dose between 200 mg / kg and 100 mg / kg, preferably at a dose between 1 mg / kg and 8 mg / kg, most preferably at a dose between 2 mg / kg and 4 mg / kg.

51. The method of claim 34, wherein the compound is administered orally, parenterally, or topically.

52. The method of claim 51, wherein the compound is administered parenterally.

53. The method of claim 52, wherein the compound is administered by injection, most preferably intravenously, intramuscularly or subcutaneously.

54. The method of claim 34, 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, immune graft response, transplant rejection, and atherosclerosis.

55. The method of claim 34, 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.

56. The method of claim 34, wherein the compound that targets CD40L or CD40 is an antibody comprising: (a) Heavy chain variable region (V H ), the heavy chain variable region comprises: i) a CDRH1 domain comprising the sequence shown in SEQ ID NO:9; ii) a CDRH2 domain comprising the sequence shown in SEQ ID NO: 10, 11, 12, 13 or 14; and iii) a CDRH3 domain comprising the sequence shown in SEQ ID NO: 15; and (b) Light chain variable region (V L ), the light chain variable region comprises: i) a CDRL1 domain comprising the sequence shown in SEQ ID NO: 16 or 17; ii) a CDRL2 domain comprising the sequence shown in SEQ ID NO: 18 or 19; and iii) a CDRL3 domain comprising the sequence shown in SEQ ID NO:

20.

57. The method of claim 34, wherein the compound that targets CD40L or CD40 is an antibody comprising: (a) Heavy chain variable region (V H ), wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; and (b) Light chain variable region (V L ), wherein the light chain variable region has an amino acid sequence as shown in SEQ ID NO:5, 6, 7 or 8.

58. A method of maintaining or improving the ALS-FRS score of a subject suffering from ALS, the method comprising: A therapeutically effective dose of a compound directed against CD40L or CD40 is administered, wherein administration of the therapeutically effective dose maintains the compound in the subject's plasma at a concentration between 5 μg / mL and 100 μg / mL; preferably a dose between 30 μg / mL and 60 μg / mL.

59. A method of prescreening a subject for a disease or disorder and treating the subject with a compound directed against CD40L or CD40, the method comprising: collecting a sample from the subject; detecting the concentration of at least one biomarker present in the 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 not suffering from the disease or disorder; as well as A therapeutically effective dose of a compound directed against CD40L or CD40 is administered such that the concentration of the compound in the subject's plasma is maintained at a dose between 5 μg / mL and 100 μg / mL; preferably between 30 μg / mL and 60 μg / mL.

60. The method of claim 59, wherein the compound is administered to the subject if the concentration of the at least one biomarker in the subject is significantly different than the concentration of the at least one biomarker in subjects not suffering from the disease or disorder.

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