Method for treating kidney diseases or disorders

Abstract

Methods and kits for treating kidney-related diseases or disorders, and complications, such as Berger's disease and kidney transplant rejection, are provided herein. Also provided are methods for predicting or measuring a response to treatment by measuring the level of a biomarker in a sample, and methods for modulating biomarker levels.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 365,448, filed May 27, 2022, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application is filed electronically with a sequence listing. The sequence listing is provided as a file entitled "ELDN.011WO.xml", created on May 1, 2023, with a size of 27,088 bytes. The electronic form information of the sequence listing is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] Background Field The present disclosure relates to methods of treating autoimmune disorders in a subject, including, for example, kidney - related diseases, disorders, or complications, such as kidney - related autoimmune disorders including Berger's disease or kidney transplantation. Methods of predicting and / or measuring a response to treatment, including measuring the levels of one or more biomarkers, are also provided. Embodiments also relate to methods of modulating the levels of one or more biomarkers.

[0004] Description of Related Art The kidneys are essential organs for filtering blood and regulating mineral content in a subject. Many diseases and disorders are associated with the kidneys, such as chronic kidney disease (CKD), diabetic kidney disease, acute kidney injury, kidney failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cysts, kidney infections, kidney stones, and IgA nephropathy, among others.

[0005] IgA nephropathy, also known as Berger's disease or IgAN, is a type of chronic kidney disease. Over approximately 10 - 20 years, IgA accumulates in the glomeruli of the subject, as a result of which the kidney's filtration system is blocked. Next, this activates the immune system, causing general inflammation around the glomeruli of the kidney. Aggressive IgAN can also affect other major organs such as the liver, skin, and cardiovascular system.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Severe kidney disease in an individual can result in the need for a kidney organ transplant. However, an individual usually has to be transplanted with a kidney provided by a donor whose ABO blood type and cross-match (human leukocyte antigen - HLA) are compatible. Even if a proper kidney transplant is successful, the individual has to continue taking immunosuppressive drugs for the rest of their life. Otherwise, the risk of rejecting the new organ due to an immune reaction becomes high. Another possible complication after kidney transplantation is graft-versus-host disease, where the donor's white blood cells still remaining inside the newly transplanted kidney recognize the new host as a foreign body and attack the individual's somatic cells. The high risk of complications after kidney transplantation still remains a significant barrier in the medical field.

Means for Solving the Problems

[0007] Overview A method for predicting the response of a subject having a kidney-related disease or disorder to treatment with a compound against CD40L or CD40 is disclosed in the present application. In some embodiments, the method comprises collecting a sample from the subject; and measuring the concentration of at least one biomarker present in the sample, wherein the at least one biomarker is CXCL9, CXCL10, IgM, C3, IgA, IgE, CD40L, and CD40; and predicting from the concentration of the at least one biomarker that the subject may be responsive to treatment of the kidney-related disease or disorder with a compound against CD40L or CD40. In some embodiments, the kidney-related disease or disorder includes inflammation. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the compound blocks the interaction between CD40 and CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is AT-1501. In some embodiments, the compound is MR1 or 5c8. In some embodiments, the kidney-related disease or disorder is selected from the group consisting of colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, transplant immune response, kidney transplantation, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephrosis, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, and IgA nephropathy. In some embodiments, the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease. In some embodiments, the sample is blood, urine, saliva, or mucus. In some embodiments, the sample is urine.

[0008] Also disclosed herein are methods of treating a subject having a kidney-related disease or disorder. In some embodiments, the method comprises administering a compound to CD40L or CD40 in a therapeutically effective dose. In some embodiments, the kidney-related disease or disorder includes inflammation. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the compound blocks the interaction of CD40 and CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is AT-1501. In some embodiments, the compound is MR1 or 5c8. In some embodiments, the compound is administered at least once every three weeks. In some embodiments, the compound is administered at a dose of 200-100 mg / kg; preferably at a dose of 1-20 mg / kg. In some embodiments, the kidney-related disease or disorder is colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, transplant immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephrosis, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy. In some embodiments, the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease. In some embodiments, the method further comprises administering a second pharmaceutically effective compound. In some embodiments, the second compound blocks the interaction of CD28 and CD86 or CD28 and CD80. In some embodiments, the second compound targets at least one of a biomarker selected from the group consisting of interleukins, cytokines, and inflammation-inducing markers. 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 by intravenous, intramuscular, or subcutaneous injection. In some embodiments, a compound that targets CD40L or CD40 comprises: (1) a CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; (2) a CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; and a CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; a heavy chain variable region (V H ). And (4) a CDRL1 domain set forth in SEQ ID NO: 16 or 17; (5) a CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; and (6) a CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20; a light chain variable region (V L ). It is also an antibody that includes. In some embodiments, a compound that targets CD40L or CD40 comprises a heavy chain variable region (V H ) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4; and a light chain variable region (V L ) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8; an antibody comprising.

[0009] Methods for modulating the concentration of at least one biomarker in a subject are also disclosed herein. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound against CD40L or CD40, and the biomarker is selected from the group consisting of IgA, IgE, CD40L, CD40, C3, CXCL9, CXCL10, and IgM. In some embodiments, the concentration of at least one biomarker increases after administration of the compound. In some embodiments, the concentration of at least one biomarker decreases after administration of the compound. In some embodiments, after administration of the compound, the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases. In some embodiments, the subject has a kidney-related disease or disorder. In some embodiments, the kidney-related disease or disorder includes inflammation. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the compound blocks the interaction between CD40 and CD40L. In some embodiments, the compound is an anti-CD40L antibody or an anti-CD40 antibody. In some embodiments, the compound is AT-1501. In some embodiments, the compound is MR1 or 5c8. In some embodiments, the kidney-related disease or disorder is colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, transplant immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy. In some embodiments, the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease. In some embodiments, in some embodiments, the compound is administered once every at least three weeks. In some embodiments, the compound is administered at a dose of 200-100 mg / kg; preferably at a dose of 1-20 mg / kg.In some embodiments, the compound is administered orally, parenterally, or topically. In some embodiments, the compound is administered parenterally. In some embodiments, the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection. In some embodiments, a compound that targets CD40L or CD40 comprises: (1) a CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; (2) a CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; and (3) a CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; a heavy chain variable region (V H ). And (4) a CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; (5) a CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; and (6) a CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20; a light chain variable region (V L ). An antibody. In some embodiments, a compound that targets CD40L or CD40 comprises a heavy chain variable region (V having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4 H ); and a light chain variable region (V having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8 L ); an antibody comprising. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0011] Detailed Description In the following detailed description, reference is made to the accompanying drawings which form a part hereof. In the drawings, like reference numerals generally identify like components unless specified otherwise in the context. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be 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 the aspects of the present disclosure, generally described and illustrated in the drawings herein, can be arranged, substituted, combined, separated, and designed in a wide variety of configurations, all of which are explicitly contemplated herein. All references cited herein are hereby incorporated by reference in their entirety into this specification and for the specific disclosure herein to which they are cited.

[0012] The present disclosure describes a method of treating a patient having an autoimmune disorder, such as a kidney-related autoimmune disorder, e.g., a kidney disease, disorder, or complication, by administering a therapeutically effective amount of a compound that blocks the interaction of CD40 and CD40 ligand (CD40L). The present disclosure also describes a method of treating a patient by co-administering a compound that blocks the interaction of CD40 and CD40L with a compound that blocks the interaction of CD28 and CD86 or CD28 and CD80.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, patent applications, published applications, and other publications referenced herein are hereby incorporated by reference in their entirety unless otherwise specified. In the event of multiple definitions of terms herein, the terms in this section shall control unless otherwise specified.

[0014] It will be understood by those skilled in the art that starting materials, biological and chemical materials, biological and chemical reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be used in the practice of the present disclosure without undue experimentation. All functional equivalents known in the art of any such materials and methods are intended to be encompassed by the present disclosure.

[0015] As used herein, "a" or "an" can mean one or more.

[0016] As used herein, the term "about" or "approximately" has its ordinary meaning as understood by those skilled in the art, and thus means that a value includes the inherent variability of the error of the method used to determine the value, or the variability present among multiple determinations.

[0017] The use of the conjunction "or" is used without distinction from "at least one of". For example, if a composition contains A or B, the method must include at least one of A and B, but may also include both A and B. Similarly, a composition containing "A, B, C, or D" must include at least one of the group of A, B, C, or D, but may also include all of A, B, C, or D, or any combination thereof.

[0018] Amino acid substitutions are indicated by rules by which the original amino acid, the amino acid of the designated sequence, and the substituting amino acid are identified. For example, C11S indicates that the cysteine at position 11 of the polypeptide sequence is substituted with serine.

[0019] A humanized antibody is an antibody produced from a non-human species whose protein sequence has been modified to enhance its similarity to antibody variants that are naturally produced in humans. The process of "humanization" is applied to monoclonal antibodies developed for administration to humans (e.g., antibodies developed as anti-cancer drugs).

[0020] Currently, it is common to humanize non-human antibodies by inserting the relevant CDRs from antibodies made in non-human animals into a human antibody "scaffold". The "direct" generation of a humanized antibody can be achieved by inserting appropriate CDR-encoding segments (which confer the desired binding properties) into a human antibody scaffold. This is accomplished by recombinant DNA methods that are expressed in mammalian cells using appropriate vectors. That is, after an antibody has been developed to have the desired properties in a mouse (or other non-human), the DNA encoding that antibody can be isolated, cloned into a vector, and sequenced. Thereafter, the DNA sequences corresponding to the CDRs of the antibody can be determined. Once the exact sequences of the desired CDRs are known, strategies can be devised to appropriately insert these sequences into constructs containing DNA for human antibody variants. Prior to insertion into the scaffold, the CDRs may be modified, for example, to enhance specificity.

[0021] The term "human antibody" means an antibody that possesses an amino acid sequence corresponding to the sequence of an antibody produced by a human, and / or an antibody that has been produced using any technique for making human antibodies. By this definition of a human antibody, humanized antibodies that contain non-human antigen-binding residues are expressly excluded.

[0022] As used herein, the term "variant" is defined as a modified or altered form of a wild-type sequence, e.g., in which one or more amino acids can be replaced with other amino acids or non-amino acids that do not substantially affect the function. In some embodiments, a variant can contain a modified side chain with respect to at least one amino acid residue.

[0023] As used herein, the term "antigen" is defined as an entity that can stimulate the production of antibodies and can specifically bind to them, and / or an entity that can induce an immune system response. For example, a cell surface protein or a specific linear or non-linear portion thereof. In this specification, the term may be abbreviated as "Ag".

[0024] The term "antigen-binding antibody fragment" means a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fd fragment, dAb fragment, Fab’-SH, F(ab’)2; bispecific antibodies; triabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments, and minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides). The antigen-binding fragments disclosed in this application bind to antigen CD40L.

[0025] Antigen-binding fragments of antibodies generally contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR that is adjacent to or within a framework that has one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH domain and the VL domain can be positioned relative to each other in an appropriate arrangement. For example, the variable region can be a dimer and can contain dimers such as VH-VH, VH-VL or VL-VL. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH domain or VL domain.

[0026] Examples of isolated antibodies include bispecific antibodies in which each arm or antigen-binding fragment of the antibody binds to a different target or epitope.

[0027] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and mean an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that contains an Fc region.

[0028] "5c8" means a murine anti-human antibody produced by a hybridoma that binds to CD40L and is available from ATCC under accession number HB10916 and is described in U.S. Patent No. 5,474,771. "hu5c8" means a humanized version of 5c8, the sequence of which is disclosed in Karpusas, et al., Structure vol.9, pp 321-329, (2001).

[0029] The term "specifically binds" and the like means that an antibody or an antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (KD) of about 3000 nM or less (i.e., the smaller the KD, the stronger the binding), about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 1 nM or less, or about 0.5 nM.

[0030] Specific binding to a particular antigen or epitope is, for example, at least about 1×10 -4 M, at least about 1×10 -5 M, at least about 1×10 -6 M, at least about 1×10 -7 M, at least about 1×10 -8 M, at least about 1×10 -9 M, or alternatively at least about 1×10 -10 M, at least about 1×10 -11 M, at least about 1×10 -12Can be represented by an antibody having a KD for an antigen or epitope, where KD represents the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more, relative to its antigen or epitope, for a control molecule. Also, specific binding to a particular antigen or epitope can be shown, for example, by an antibody having a Ka for the epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more, relative to a control, where Ka refers to the association rate of a particular antibody-antigen interaction.

[0031] The term "neutralizing antibody" includes an antibody that can inhibit and / or neutralize the biological activity of CD40L, for example, an anti-CD40L antibody or an antigenic fragment thereof that inhibits, prevents, or reduces the binding of CD40L to CD40, and thus inhibits or reduces the signal transduction pathway induced by CD40L, and / or inhibits or reduces the binding of CD40L to CD40.

[0032] The terms "antagonistic antibody" or "antagonist antibody" are used interchangeably herein and include an antibody that can inhibit and / or neutralize the biological signal transduction activity of CD40L, as described for the neutralizing antibodies above.

[0033] The terms and expressions used herein are used as terms for explanation rather than for limitation, and there is no intention in the use of such terms and expressions to exclude equivalents or portions thereof of the features shown and described. However, it is recognized that various modifications are possible within the scope of the methods and compositions disclosed herein. Accordingly, although aspects of the present disclosure are specifically disclosed by preferred embodiments, exemplary embodiments, and various embodiments that may include any features, it should be understood that modifications and changes to the concepts disclosed herein may be made by those skilled in the art. Such modifications and changes are considered to be within the scope of the embodiments of the present disclosure as set forth in the appended claims and as defined by the appended claims.

[0034] As used herein, reference is made to the percent identity between polypeptide or amino acid sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for the optimal alignment of the two sequences. Identity can be measured as "local identity" or "global identity". Local identity refers to the degree of sequence relatedness between polypeptides and is determined by the matches between strings of such sequences. Global identity refers to the degree of sequence relatedness of a polypeptide compared to the full length of a reference polypeptide. Unless otherwise specified, as used herein, identity means global identity. For the purposes of the present disclosure, the percent global identity is calculated using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. There are numerous publicly available software programs incorporating the Needleman and Wunsch algorithm, such as the GAP program of the GCG software package.

[0035] CD40L is also known as CD154, gp39, T-BAM, 5c8 antigen, or TNF-related activation protein (TRAP).

[0036] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are used to mean including the stated step or element or group of steps or elements but not to mean excluding any other step or element or group of steps or elements. "Consisting of" means including anything following the phrase "consisting of" and being limited to what follows the phrase. Thus, the phrase "consisting of" means that the recited elements are required or essential and that no other elements can be present. "Consisting essentially of" means including the elements recited after the phrase and being limited to other elements that do not interfere with or contribute to the specified activity or action as disclosed for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential but that other elements are optional and may or may not be present depending on whether or not they substantially affect the activity or action of the recited elements.

[0037] As used herein, the terms "function" and "functional" have their plain and ordinary meaning as understood in the context of this specification and mean biological, enzymatic, or therapeutic function.

[0038] As used herein, the term "yield" of any given substance, compound, or material has its plain and ordinary meaning as understood in view of this specification, and means the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material can be 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, at least 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, at least about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or less of the expected total amount, about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or less of the expected total amount, including all decimals between the numbers. The yield can be affected by the efficiency of the reaction or process, unwanted side reactions, decomposition, the quality of the starting materials, compounds, or materials, or losses of the desired substance, compound, or material during the manufacturing process.

[0039] As used herein, the term "isolated" has its plain and ordinary meaning as understood in view of this specification, and means a substance and / or entity that is (1) separated from at least a portion of the components with which it was associated when first produced (whether naturally and / or produced in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity can be separated from other components with which it was initially associated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% (ranges including and / or spanning the foregoing values).In some embodiments, the agent to be isolated is at 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% purity, at about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% purity, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% purity, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% purity, at 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less purity, or at about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less purity, at substantially 100% purity, or at 100% purity (ranges including and / or spanning the foregoing values). As used herein, an "isolated" substance can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can mean a cell that is not contained within a multicellular organism or tissue.

[0040] The term "isolated protein" or "isolated polypeptide" (e.g., an isolated antibody or an isolated antigen-binding fragment) refers to a protein or polypeptide that, by virtue of its origin or derivation, is not associated with the naturally associated components that accompany it in its native state; a protein or polypeptide that is substantially free of other proteins from the same species; a protein or polypeptide expressed by cells from a different species; or a protein or polypeptide that does not occur naturally. Thus, a polypeptide that is chemically synthesized or synthesized in a cell line different from the cell in which it naturally occurs is "isolated" from its naturally associated components. A protein can also be made substantially free of its naturally associated components by isolation using protein purification techniques well known in the art.

[0041] As used herein, "in vivo" is considered to have its plain and ordinary meaning as understood in view of this specification, and means the performance of a method within a living organism, typically an animal, a mammal such as a human, and a plant, or within living cells that make up these living organisms, as contrasted with tissue extracts or dead organisms.

[0042] As used herein, "ex vivo" is considered to have its plain and ordinary meaning as understood in view of this specification, and means the performance of a method outside of a living organism with little modification of natural conditions.

[0043] As used herein, "in vitro" is considered to have its plain and ordinary meaning as understood in view of this specification, and means the performance of a method outside of biological conditions, for example, in a Petri dish or a test tube.

[0044] As used herein, "nucleic acid", "nucleic acid molecule" or "nucleotide" refers to polynucleotides or oligonucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, exonuclease action, and synthetic production. Nucleic acid molecules can be composed of monomers that are natural nucleotides (such as DNA and RNA) or analogs of natural nucleotides (e.g., enantiomeric forms of natural nucleotides), or a combination of both. Modified nucleotides can have alterations at the sugar moiety and / or at the pyrimidine or purine base moiety. Examples of sugar modifications include substitution of one or more hydroxyl groups with, for example, halogen, alkyl groups, amines, and azide groups, or they can be functionalized as ethers or esters. Further, the entire sugar moiety can be replaced with stereochemically and electronically similar structures such as azasugars and carbocyclic sugar analogs. Examples of modifications at the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other known heterocyclic substitutions. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoranilothioate, phosphororanilidate, phosphoramidate, etc. The term "nucleic acid molecule" also encompasses so-called "peptide nucleic acids" containing natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded.

[0045] As used herein, the terms "peptide", "polypeptide", and "protein" have their plain and ordinary meaning as understood in view of this specification, and mean macromolecules composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structures, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, biologically produced by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating nucleic acid templates, mutations in peptides, polypeptides, and proteins, such as substitutions, deletions, truncations, additions, duplications, or fusions of multiple peptides, polypeptides, or proteins, can be effected.These fusions of multiple peptides, polypeptides, or proteins can be adjacent in the same molecule, or, for example, between linkers, repeat sequences, epitopes, or tags, or between any other sequences of any length of from 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 in length, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases or less in length, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases or less in length, or within a range defined by any two of the foregoing lengths, and can be linked with additional amino acids. As used herein, the term "downstream" of a polypeptide has its plain and ordinary meaning as understood in the context of this specification and means the sequence that is after the C-terminus of the preceding sequence.As used herein, the term "upstream" of a polypeptide has its plain and ordinary meaning as understood in view of this specification, and means the sequence that is before the N-terminus of the subsequent sequence.

[0046] As used herein, the term "gene" has its plain and ordinary meaning as understood in view of this specification, and generally means a portion of a nucleic acid that encodes a protein or functional RNA; however, the term optionally includes regulatory sequences. It will be understood by those skilled in the art that the term "gene" can 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 references to nucleic acids that do not encode proteins but encode functional RNA molecules such as tRNA and miRNA. In some cases, a gene includes regulatory sequences involved in transcription, or message production or composition. In other embodiments, a gene includes a transcriptional sequence that encodes a protein, polypeptide, or peptide. In light of the terminology described herein, an "isolated gene" can include one or more transcribed nucleic acids, regulatory sequences, coding sequences, etc., that are isolated substantially away from other such sequences such as other native genes, regulatory sequences, polypeptides, or peptide coding sequences. In this regard, the term "gene" is used herein for brevity to mean a nucleic acid that includes a transcribed nucleotide sequence and its complementary sequence. As will be understood by those skilled in the art, this functional term "gene" includes both genomic sequences, RNA or DNA sequences, or smaller engineered nucleic acid segments that include nucleic acid segments of the non-transcribed portion of the gene, including, but not limited to, non-transcribed promoter or enhancer regions of the gene. The smaller engineered gene nucleic acid segments can be expressed or adapted to express proteins, polypeptides, domains, peptides, fusion proteins, variants, and / or the like using nucleic acid manipulation techniques.

[0047] As used herein, the terms "individual", "subject", "host", or "patient" have their ordinary meaning as understood by one of ordinary skill in the art and thus include humans or non-human mammals. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, primates such as Simians (chimpanzees, apes, monkeys), humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0048] The dimensions and values disclosed herein are not to 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 around that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm".

[0049] The following abbreviations are used herein: superoxide dismutase 1 (SOD1); T cell receptor (TCR); major histocompatibility complex antigen presenting cell (APC); phosphate buffered saline (PBS), complementarity determining region (CDR). "IP" means intraperitoneal and "IV" means intravenous.

[0050] R1 is a hamster monoclonal antibody that binds to murine CD40 ligand. As used herein, "wild-type" means a non-transgenic mouse. As used herein, "small molecule" means a compound having a molecular weight of less than 2000 daltons. As used herein, "treatment" or "treating" includes prophylactic and therapeutic treatment. The phrase "therapeutically effective amount" means an amount of a compound or a pharmaceutically acceptable salt thereof that is sufficient, when administered alone or in combination with another pharmaceutical or treatment, to suppress or ameliorate a disorder or symptom being treated in a particular subject or population of subjects.

[0051] An "effective amount" of an agent, e.g., a pharmaceutical formulation, means an amount effective to achieve a desired therapeutic or prophylactic result at the required dosage and for the required period of time.

[0052] "Affinity" means the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" means the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally represented by the equilibrium dissociation constant (KD), k off / k on ratio. KD and affinity are inversely proportional. The K value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), and the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Affinity can be measured by common methods known in the art, such as the methods described herein. Specific, illustrative, and exemplary embodiments for measuring binding affinity can be measured by radioimmunoassay (RIA), surface plasmon resonance (SPR) on a BIAcore® device (GE Healthcare Europe GmbH, Glattbrugg, Switzerland), using human CD40 ligand polypeptide as a sample, and capturing the antibody on a Protein A-binding CM5 research grade sensor chip (GE Healthcare Europe GmbH, Glattbrugg, Switzerland; BR-1000-14). Other methods include radioimmunoassay and binding equilibrium exclusion methods. The binding equilibrium exclusion method is a general immunoassay platform that can measure the equilibrium dissociation constant for antigen / antibody interactions, as well as the association and dissociation rate constants.

[0053] TMB is an abbreviation for 3,3',5,5'-tetramethylbenzidine.

[0054] As used herein, "CDR domain" means an antibody complementarity determining region, with or without flanking sequences.

[0055] "Effector function" means a biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocyte activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. One effector function is, but not limited to, the ability of the Fc or constant region of an antibody to bind to a protein, such as an Fc receptor (FcR) (e.g., the high-affinity Fc region (CD64) of the IgG receptor Ia (FCγRIa), the low-affinity immunoglobulin γ Fc region receptor IIa (FCγRIIa) (CD32), the low-affinity immunoglobulin γ Fc region receptor IIIa (FCγRIIIA) (CD16a), the low-affinity immunoglobulin γ Fc region receptor IIIb (FCγRIIIb) (CD16b), etc.). In some embodiments where the antibody and its antigen-binding fragment have an Fc domain, the Fc domain has been engineered to reduce or eliminate one or more Fc effector functions. In preferred embodiments, the Fc domain has been engineered to reduce or eliminate platelet activation and / or platelet aggregation, and the associated risk of thromboembolism.

[0056] Identification of candidate compounds The co-stimulatory pathway includes the binding of CD40 on B cells to CD40L (also known as CD154, gp39, T-BAM, 5c8 antigen, CD40CR and TRAP) on T cells, along with other interactions. Human CD40 is expressed on mature B cells, as well as on macrophages, dendritic cells, fibroblasts and activated endothelial cells. Blockade of CD40:CD40L binding is thought to promote the development of type I helper T cell responses.

[0057] Using compounds that block these interactions and inhibit co-stimulatory signals, the immunomodulatory effects of blocking one or more of CD40L, CD80, or CD86 in preclinical models of transplantation and autoimmunity have been demonstrated by significant substances of the study. Blocking of CD40L function using blocking antibodies or adenoviral expression of CD40L-Ig improves allograft survival for 30 to 90 days. Similar studies that transiently block CD80 / CD86 on APCs with CTLA4-Ig or adenoviral expression of CTLA4-Ig also improve allograft survival. Graft rejection in these models is transient, and graft rejection occurs over time. Prevention of longer-term graft rejection can be achieved by both blocking the co-stimulatory pathway with CTLA4-Ig and blocking CD40L activation of APCs with anti-CD40L antibodies.

[0058] Blocking antibodies against CD40L or genetic deletion of CD40L in mice have demonstrated that CD40L improves disease progression, survival, and surrogate markers in preclinical models of experimental allergic encephalomyelitis (EAE), multiple sclerosis, collagen-induced arthritis, and systemic lupus erythematosus. Blockade of CD40:CD40L binding appears to reduce the ability of nitric oxide-producing macrophages, which mediate much of the inflammatory-inducing activity of macrophages.

[0059] From such studies, it is thought that blocking of CD40:CD40L interactions and / or blocking of CD28:CD80 or CD28:CD86 interactions may regulate the immune response.

[0060] Immunohistochemical data have been shown to correlate well with gene expression data, and from these data, macrophages are identified as antigen-presenting cell-infiltrating skeletal muscle during disease progression in G93A mice.

[0061] To determine the presence and location of potential antigen-presenting cells in the gastrocnemius muscle from G93A and wild-type animals, immunohistochemistry was performed on gastrocnemius muscle tissues collected on day 110 from G93A and wild-type mice. Immediately after collection, the tissues were embedded in OCT. Frozen sections were stained with H&E and hybridized with antibodies against myelin (anti-S100b antibody), or antibodies against T hematopoietic cell lineages, such as T cells (anti-CD3 antibody), B cells (CD45R pan B cell antibody), and macrophages (anti-CD11b antibody). On day 110, there was infiltration of CD11b-positive macrophages, and the macrophages were found to be localized to the axons of the nerves innervating the skeletal muscle. The localization of macrophages was not dispersed throughout the muscle, suggesting that the inflammation was not caused by muscle atrophy or muscle fiber remodeling.

[0062] To confirm the identity of monocytic cells in the skeletal muscle on day 110 and to clarify the relationship of these cells compared to non-monocytic cells, additional immunohistochemistry was performed with a panel of antibodies specific for the macrophage lineage. The anti-S100b antibody was used to label myelin-related axons innervating the skeletal muscle. All of the macrophage-specific antibodies (anti-CD11b antibody, anti-CD86 antibody, and anti-MAC1 antibody) localized macrophages to the axons of the nerves innervating the skeletal muscle of G93A mice, and no macrophages were present on the axons of wild-type animals. The localization of macrophages was specific to the nerves innervating the muscle, and no macrophages were present in the muscle fibers of the muscle.

[0063] Gene expression data suggest that genes associated with the costimulatory pathway are transiently increased during disease progression in both the spinal cord and skeletal muscle. To characterize the timing of macrophage infiltration into skeletal muscle, immunohistochemistry was performed on gastrocnemius muscle sections from G93A mice at 60, 80, and 100 days. There was no evidence of macrophage infiltration or localization to axons at day 60. Macrophages became apparent at day 80 and localized to axons innervating the muscle described above for skeletal muscle at day 110. The number of macrophages increased between days 80 and 100, and macrophage accumulation was specific to axons innervating skeletal muscle.

[0064] To quantify the increase in macrophage infiltration, representative sections from 5 G93A animals and 5 wild-type animals were hybridized with an anti-CD86 antibody and the number of macrophages per 10,000 square microns was counted. The number of macrophages in wild-type animals was the same at 60, 80, and 110 days. Macrophages transiently accumulated in skeletal muscle between 80 and 100 days, and there were few macrophages in wild-type skeletal muscle.

[0065] In summary, immunohistochemistry data correlated very well with gene expression data, and macrophages were identified as antigen-presenting cell-infiltrated skeletal muscle during disease progression in the G93A mouse model. The unexpected finding is that clophage infiltration appears to be specifically targeted to axons innervating skeletal muscle such that it is localized by labeling with antibodies against myelin and macrophages.

[0066] The tissue level of MR1 directed against mouse CD40L was determined using a sandwich format matrix match, non-competitive enzyme immunoassay (ELISA). A 7-point standard curve was included on each plate. Standards were prepared using purified MR1 spiked into a PBS dilution solution. The PBS dilution solution was a matrix matched to normal mouse tissue at an equal dilution of the unknown sample to correct for non-specific effects arising from tissue lysates.

[0067] For pharmacokinetic analysis, 84 plasma samples were collected over a two-week period after administration (10 mg / kg, IP) in both female and male G93A mice.

[0068] The elimination half-life was similar in females (23 days) and males (22 days) and was similar to the half-life for typical mouse IgG2-based antibodies in mice. No signs of anti-hamster antibody response were seen.

[0069] Females showed a slightly smaller volume of distribution of MR1 than males and thus higher plasma levels when administered the same dose of 10 mg / kg. Males showed faster clearance and a higher volume of distribution. Thus, males would require a higher dose to achieve similar plasma levels. Figure 1A shows MR1 concentration over time using a linear concentration (Y) axis. Figure 1B shows MR1 concentration over time using a log concentration (Y) axis.

[0070] Thirty-six female G93A mice were litter matched and randomized into study groups. Eighteen G93A mice were placed in the MRI treatment group and 18 other G93A mice were placed in the control group. The study days were based on days from birth.

[0071] A single injection of 56 μg of MR1 was administered intraperitoneally (IP) on day 50. Following the bolus injection, weekly maintenance injections of 18 μg of MR1 were administered by IP injection. The doses were prepared in a total volume of 200 μl in vehicle (phosphate buffered saline (PBS, pH 7.3)). 200 μl of PBS was administered to control animals. Starting on day 54, the animals were monitored daily throughout the course of the study and daily body weight measurements were taken.

[0072] Animals transgenic for mutant SOD1 gene exhibit normal body weight (BW) characteristics as neonates and gain weight normally compared to non-transgenic animals into adulthood. Depending on the nature of the genetic mutation in the transgene and the copy number of the mutant transgene, weight loss becomes apparent in adult animals and persists until death. Analysis of weight loss in the treatment and control groups can provide insight into the putative therapeutic effect on the onset and progression rate of the disease. To evaluate the effect of MR1 treatment on body weight, two summary parameters are examined: (1) the change in BW from the start of the study until the peak weight is reached, which may reflect the effect on disease onset; and (2) the change in BW from the peak weight until death, which may reflect the effect on disease progression.

[0073] Figure 2 shows the survival time (time-to-event) curves for the MR1 treatment group and the control group for comparison from day 40 until the peak weight is reached. The median time to peak weight for the control group was 50 days compared to 51 days for the MR1 treatment group. This difference was not significant when analyzed by the Kaplan Meier method using the log rank and Wilcoxon statistical models, Cox proportional hazards, or parametric statistical tests.

[0074] The survival curves of the comparison MR1 treatment group and the control group for the time from peak body weight to death are shown in Figure 3. The time from peak body weight to death was statistically significantly 15 days later in the MR1-treated animals. Control animals had a 2.4 - 4.7-fold higher risk of dying immediately after reaching peak body weight compared to MR1-treated animals. The median time from peak body weight to death in the control group was 26 days, while in the MR1 treatment group it was 41 days. In this example, the significance of each analysis was calculated in several ways to better evaluate the significance. When analyzed using several approaches, the delay was statistically significant (Kaplan - Meier, log-rank p = 0.0110 and Wilcoxon, p = 0.0069; Cox proportional hazards model p = 0.05151; parametric statistical model, p = 0.0122). Based on the body weight data, MR1 seems to have little effect on the onset of the disease in the G93A mouse model, but has a dramatic effect on slowing the rate of weight loss from peak body weight to death.

[0075] The survival period of the treated animals was 13 days later than that of the control animals. The survival curves of the comparison MR1 treatment and the control group for the time from peak body weight to death are shown in Figure 4. The median survival period of the control group was 128 days, while the median survival period of the MR1 treatment group was 141 days. Control animals had a 2.8 - 3.2-fold higher risk of dying earlier than MR1. When analyzed using several approaches, the delay was statistically significant (Kaplan - Meier, log-rank (p = 0.0040) and Wilcoxon test (p = 0.0109); Cox proportional hazards model (p = 0.0060); parametric analysis (p = 0.0049).

[0076] Sixty female G93A mice and thirty-six male G93A mice were littermates and were randomly assigned to the treatment group or the control group. Thirty female mice and eighteen male mice were treated with MR1 starting on day 50. The study days were based on the days from birth.

[0077] In each of the female and male, a single bolus injection of 5.22 mg / kg or 6.75 mg / kg of MR1 was administered intraperitoneally on the 50th day. Following the bolus injection, female was injected with 1 mg / kg of MR1 weekly, and male was injected with 1.34 mg / kg of MR1 weekly by IP injection. The dose was prepared in a total volume of 200 μl in excipient (phosphate buffered saline (PBS, pH 7.3)). 200 μl of PBS was administered to the control animals. The animals were monitored for body weight and the above-mentioned euthanasia criteria.

[0078] Figure 5A shows the survival time curves of the comparative MR1 treatment and control groups for the time from the 40th day to reaching the peak body weight. The median time to the peak body weight of the control group was 49 days compared to 53 days for the MR1 treatment group. This difference was not significant when analyzed by the Kaplan-Meier method using the log-rank and Wilcoxon statistical models, Cox proportional hazards, or parametric statistical tests.

[0079] Figure 5B shows the survival time curves of the comparative MR1 treatment group and control group for the time from the peak body weight to death. The time from the peak body weight to death was statistically significantly 6 days later in the MR1-treated animals. The median time from the peak body weight to death in the control group was 29 days, while it was 35 days in the MR1 treatment group. In this example, the significance of each analysis was calculated in several ways to better evaluate 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 seems to have little effect on the onset of the disease in the G93A mouse model, but has a dramatic effect on slowing down the rate of body weight loss from the peak body weight to death.

[0080] The age at which the MR1 group and control group animals progressed to neurological score 2, and the number of days at neurological score 2 are shown in Figure 5C. The time to reach score 2 in the control group was 113 days, and in the MR1 treatment group it was 121 days. Based on the neurological score data, MR1 delayed disease onset by approximately 8 days in the G93A mouse model, and this delay was statistically significant when analyzed using several statistical models: Kaplan–Meier, log-rank (p = 0.0038) and Wilcoxon (p = 0.0017); and the Cox proportional hazards model (p = 0.0010).

[0081] The survival period of the treated animals was 9 days later than that of the control animals. The median survival period of the control group was 124 days, and the median survival period of the MR1 treatment group was 133 days as shown in Figure 5D. This delay was statistically significant when analyzed using several statistical models: Kaplan–Meier, log-rank (p = 0.0043) and Wilcoxon test (p = 0.0040); and the Cox proportional hazards model (p = 0.0030).

[0082] The examples and embodiments described herein are for illustrative purposes only, and in light of them, various modifications or changes may be proposed to those skilled in the art and are understood to be within the spirit and scope of this application and the scope of the appended claims.

[0083] Nucleic acid sequences encoding the heavy and light chains of the antibody of interest were designed to be suitable for expression in mammalian cells such as Chinese hamster ovary (CHO) cells. The nucleic acids were then artificially synthesized and ligated into the antibody expression vector BPJPuro using standard molecular biology techniques. BPJPuro is a dual-gene mammalian expression vector optimized for selectable and stable expression of immunoglobulins in Chinese hamster ovary (CHO) cells. The vector was then transfected into CHO cells and appropriate transfectants were selected.

[0084] Similarly, a nucleic acid encoding a heavy chain having the amino acid sequence of SEQ ID NO: 9 (SEQ ID NO: 10) and a nucleic acid encoding a light chain having the amino acid sequence of SEQ ID NO: 7 (SEQ ID NO: 8) were synthesized and ligated into the antibody expression vector BPJPuro.

[0085] The resulting expression vector encoding the heavy and light chains was transfected into a CHO system (CHO SA, Cellectis SA, Paris, France) using liposome-mediated transfection. Stable transfectants were isolated by puromycin selection and subcloned to provide a clonal cell line. Candidate cell lines were adapted to serum-free suspension culture medium and screened for IgG production and robust growth. One of the cell lines was selected and named JB5. The cell line was cultured in a pilot-scale bioreactor, and antibody JB5 was purified from the conditioned medium by sequential concentration, protein A / G affinity chromatography, and size exclusion chromatography.

[0086] Using a three-component sandwich ELISA assay, the binding kinetics of the JB5 antibody to the parental antibody hu5c8 were determined. All washes were performed using three washes with 250 μl of PBS. A 96-well polystyrene plate was coated with 100 μl / well of JB5 or hu5c8 antibody (2 μg / ml) at 4 °C for 16 hours. The plate was washed and then blocked with 2% bovine serum albumin / PBS for 1 hour at room temperature. The plate was washed and recombinant human CD40L protein (Santa Cruz Biotechnology, Santa Cruz, California, USA) was added to the plate titrated by two-fold dilutions starting at 2000 ng / ml. After binding and washing, 100 μl of biotinylated goat anti-human CD40L polyclonal antibody (200 ng / ml) and 100 μl of streptavidin-horseradish peroxidase conjugate were used at 100 ng / ml to detect the bound D40L protein. Colorimetric quantitative detection was performed with chromagen TMB (3,3’,5,5’-tetramethylbenzidine), and spectrophotometric analysis of the absorption at 450 nm was performed. From the resulting binding curve (Figure 6), it can be seen that JB5 (●) has very similar CD40L binding to the parental antibody hu5c8 (■). A control protein CTLA4-IgG1 (▼) having the same Fc domain as JB5 showed no significant binding. The calculated EC50 values for hu5c8 and JB5 were 114 nM and 137 nM, respectively. JB5-R28K and JB5-K74R showed binding similar to JB5.

[0087] To compare the CD40L binding of antibodies from all 16 clones against 5c8 or AT-1501 (a humanized monoclonal antibody that inhibits CD40 ligand, also known as Teprotumumab), a binding assay was performed using 2 clones, and 5c8 and AT-1501 were run on the same 96-well assay plate. A three-component sandwich ELISA assay was used to determine the binding levels of the antibodies of the present disclosure in comparison to the reference antibodies 5c8-19 and AT-1501. A 96-well polystyrene plate was coated with recombinant human CD40L (BioLegend catalog number 591706) at 2 μg / ml in PBS, and 50 μl / well was added to a Costar 96-well half-area high-binding assay plate (Corning 3690) and incubated overnight at 4°C. The plate was blocked with (1×) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. Binding curves of 5C8 or AT1501 (starting from 2 μg / ml, serially diluted 2-fold) were added (50 μl / well) and incubated for 1 hour at room temperature. The plate was washed and incubated with HRP-(Fab2) donkey anti-human IgG (Fc specific) (Jackson Immuno. 709-036-098) at a dilution of 1:10,000 (50 μl / well) for 1 hour at room temperature. The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) was added (50 μl / well). 2 SO 4 Using 25 μl, the color development was stopped after 5 minutes at room temperature. The plate was read on a Molecular Devices SpectraMax M5 plate reader using the SoftMax Pro 6.2.2 program to determine the absorbance at 450 nm.

[0088] The relative CD40L binding ability was calculated as follows: [IC50 (clone) / IC50 (5c8 or AT-1501) × 100%]. The ranked potencies of 16 clones against 5c8 are shown in Figure 13A, and the ranked potencies against AT-1501 are shown in Figure 13B. The resulting binding curves are shown in Figures 14A - 14Q (comparison of 16 clones against 5c8, Figure 14H shows the comparison between AT-1501 and 5c8) and Figures 15A - 15Q (comparison of 16 clones against AT-1501, Figure 15H shows the comparison between AT-1501 and 5c8). The IC50, LCL, UCL, and relative potencies of each clone are shown in Table 1 (comparison with 5c8) and Table 2 (comparison with AT-1501).

[0089]

Table 1

[0090]

Table 2

[0091]

Table 3

[0092]

Table 4

[0093] The 16 VH / VL antibody clones were generated with IgG1Fc having two mutations, P238S and N297G (SEQ ID NO: 21). These antibody clones were assayed for Fc effector functions with respect to binding to human FcγRI, FcγRIIa, and FcγRIIIa.

[0094] The anti-CD40L antibody (abatacept included as a negative control) was diluted to 2 μg / ml in (1×) PBS, and 50 μl / well was added to a Costar 96-well half-area high-binding assay plate (Corning 3690) and incubated overnight at 4°C. The plate was blocked with (1×) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. Binding curves of recombinant human FcγRI, IIa, IIIa, and IIIb (starting from 5 μg / ml, serially diluted 2-fold) were added (50 μl / well) and incubated for 1 hour at room temperature. The plate was washed and incubated with mouse anti-human CD16 (anti-FcRIII); CD32 (anti-FcRIIa) or CD64 (anti-FcRI) (eBioSciences / Invitrogen 14-0168-82; 16-0329-81; 14-0649-82) at 2 μg / ml (50 μl / well) for 1 hour at room temperature. The plate was washed and incubated with HRP-(Fab2) goat anti-mouse IgG (Fc-specific) (Jackson Immuno. 116-036-071) at a dilution of 1:10,000 for 1 hour at room temperature. The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) was added (50 μl / well). 2N H 2 SO 4 (25 μl / well) was used to stop the color development after 5 minutes at room temperature. The plate was read using a Molecular Devices SpectraMax M5 plate reader with the SoftMax Pro 6.2.2 program, and the absorbance was determined at 450 nm.

[0095] As can be seen in the drawings, the clones disclosed in the present application are negative for binding to three Fc receptors, 5c8 binds to FcγRI and FcγRIIa, but does not bind to FcγRIIIa or FcγRIIIb. Figures 16A and 16B each show the binding to FcγRIa for eight antibody clones, which are compared to the binding curves of 5c8, AT-1501 and abatacept. Figures 17A and 17B each show the binding to FcγRIIa for eight antibody clones, which are compared to the binding curves of 5c8, AT-1501 and abatacept. Figures 18A and 18B each show the binding to FcγRIIIa for eight antibody clones, which are compared to the binding curves of 5c8, AT-1501 and abatacept. Figures 18C and 18D each show the binding to FcγRIIIb for eight antibody clones, which are compared to the binding curves of 5c8, AT-1501 and abatacept (Figures 16A, 17A, 18A and 18B show the results from antibody clones 4-4, 5-3, 6-6, 7-5, 8-3, 8-4, 10-1 and 10-4; Figures 16B, 17B, 18C and 18D show the results from antibody clones 11-5, 12-4, 13-2, 15-1, 15-4, 16-3, 17-1 and 18-2).

[0096] The anti-CD40L antibody (abatacept included as a negative control) was diluted to 2 μg / ml in (1×) PBS and 50 μl / well was added to a Costar 96 well half area high binding assay plate (Corning 3690) and incubated overnight at 4°C. The plate was blocked with (1×) PBS / 1.0% BSA (140 μl / well) for 1 hour at room temperature to prevent background binding. The binding curve of native human C1q protein (Abcam ab96363) serially diluted 2-fold from 10 μg / ml was added for 1 hour at room temperature (50 μl / well). The plate was washed and HRP-goat anti-human C1q (Abcam ab46191) was added at a dilution of 1:400 (50 μl / well) and incubated for 1 hour at room temperature. The plate was washed and TMB substrate (Surmodics BioFX TMBW-1000-01) was added (50 μl / well). 2N H 2 SO4 (25 μl / well) was used and the color development was stopped after 5 minutes at room temperature. The plate was read on a Molecular Devices SpectraMax M5 plate reader using the SoftMaxPro 6.2.2 program, and the absorbance was determined at 450 nm.

[0097] As can be seen in the drawings, the antibodies from all 16 antibody clones of the present disclosure were negative for binding to C1q, whereas 5c8 showed significant binding. Figures 19A and 19B show the binding to C1q for eight antibody clones, compared to the binding curves of 5c8, AT-1501, and abatacept, respectively. Figure 19A shows the results from antibody clones 4-4, 5-3, 6-6, 7-5, 8-3, 8-4, 10-1, and 10-4; Figure 19B shows the results from antibody clones 11-5, 12-4, 13-2, 15-1, 15-4, 16-3, 17-1, and 18-2.

[0098] Since gene expression data indicated the involvement of the costimulatory factor pathway, the efficacy of MRI was evaluated in the G93A model. MR1 binds to CD40L, and thus its interaction with CD40, which is involved in the costimulatory pathway related to the immune response, is blocked. It has been reported in the literature that MR1 is effective in the treatment of rheumatoid arthritis and graft-versus-host disease, both of which have strong immunologic components. Rheumatoid arthritis is an autoimmune disease, and graft-versus-host disease occurs when the host body initiates an active immune response against the transplanted tissue.

[0099] The method according to the present disclosure includes treating a patient having an autoimmune disorder, such as a kidney-related autoimmune disorder such as a kidney-related disease, disorder or complication, by administering to the patient a compound that blocks the interaction of CD40L and CD40 and / or blocks the interaction of CD28 and CD80 and / or blocks the interaction of CD28 and CD86. In some embodiments, the method includes treating a patient having colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, kidney transplantation, transplant immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy by administering to the patient a compound that blocks the interaction of CD40L and CD40. In some embodiments, the method includes treating the patient by administering an anti-CD40L antibody. In some embodiments, the method includes treating a patient having an autoimmune disease. Non-limiting examples of autoimmune diseases include 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.

[0100] The therapeutic compounds provided herein include compounds that block the interaction of CD40 and CD40L. For example, many animal studies have described agents that can interfere with CD40:CD40L binding (see, e.g., U.S. Patent Application Publication No. 2005 / 158314 and U.S. Patent No. 7,173,046, which are incorporated herein by reference). Many anti-CD40L antibodies have been made and characterized (see, e.g., U.S. Patent No. 5,876,950 to Bristol-Myers Squibb, which is incorporated herein by reference). Anti-CD40L antibodies useful in the methods of the present disclosure include, but are not limited to, hamster monoclonal antibodies available from MR1, Taconic (Hudson, NY), and BD Biosciences (San Jose, CA); the humanized antibody 5c8 described in U.S. Patent No. 5,474,771 (incorporated herein by reference); the hamster-human chimeric antibody, IDEC131 / E6040, a humanized monoclonal antibody that includes the CDRs of the mouse monoclonal antibody clone 24-31 and human gamma-1 heavy chain and human kappa light chain, available from Ancell (Catalog X353-020, Bayport, Minn.); ABI793; Sgn-40; ImxM90 (Immunex); ImxM91 (Immunex); ImxM92 (Immunex); and anti-CD40L mAbs commercially available from Genzyme (Cambridge, Mass., Catalog No. 80-3703-01); Also, anti-CD40L mAbs are commercially available from PharMingen (San Diego, Catalog No. 33580D). Embodiments according to the present disclosure include methods of treating patients having a kidney-related autoimmune disease, disorder, or complication, including administering a therapeutically effective amount of any compound capable of binding to CD40L, such as an anti-CD40L antibody. One embodiment is that the method includes treating a patient having a kidney-related autoimmune disease, disorder, or complication and administering a therapeutically effective amount of an anti-CD40L antibody selected from MR1, 5c8, IDEC131 / E6040, clone 24-31, ABI793, ImxM90, ImxM91, ImxM92, or Sgn-40.In one embodiment, the antibody is 5c8. In another embodiment, the antibody is MR1.

[0101] In some embodiments, the kidney-related disease, disorder, or complication is colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, kidney transplantation, transplant immune response, transplant rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy by administering to the patient a compound that blocks the interaction of CD40L and CD40. In some embodiments, the method of treating a patient is a method by administering an anti-CD40L antibody. In some embodiments, the method includes treating a patient having an autoimmune disease. Non-limiting examples of autoimmune diseases include 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 anti-CD40L antibody is MR1. In some embodiments, the anti-CD40L antibody is 5c8.

[0102] In some embodiments, the method of treatment includes 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’), single-chain antibody, polypeptide, fusion construct of polypeptides, etc. In some embodiments, the compound is a small molecule compound capable of blocking the CD40:CD40L interaction. In some embodiments, these compounds include either BIO3417 or a compound disclosed in U.S. Patent No. 7,173,046 having the ability to block the CD40:CD40L interaction.

[0103] Compounds that block the CD40:CD40L interaction can be administered in combination with other compounds. Thus, in some embodiments, the methods of treating a patient described herein include administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD80:CD28 interaction. In some embodiments, the method includes administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD86:CD28 interaction. In some embodiments, the compound that blocks the CD80:CD28 interaction is galiximab, or H1f1&h3d1, or 16C10, or 7C10. In some embodiments, the compound that blocks the CD86:CD28 interaction is a CTLA4-Ig protein conjugate such as abatacept or belatacept. Embodiments according to the present disclosure include administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD80:CD28 interaction, or administering a compound that blocks the CD40L:CD40 interaction in combination with a compound that blocks the CD86:CD28 interaction in a therapeutically effective amount, a method of treating a patient having an autoimmune disorder such as a kidney-related autoimmune disorder including a kidney-related disease, disorder, or complication, wherein the kidney-related disease, disorder or complication is colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, kidney transplantation, transplant immune response, transplant rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy by administering to the patient a compound that blocks the CD40L:CD40 interaction, the method also includes. In some embodiments, the method includes administering an anti-CD40L antibody. In some embodiments, the method includes treating a patient having an autoimmune disease.Non-limiting examples of autoimmune diseases include systemic lupus erythematosus, type 1 diabetes, myasthenia gravis, inflammatory bowel disease, immune thrombocytopenic purpura, rheumatoid arthritis, psoriasis, Addison's disease, Crohn's disease, uveitis, multiple sclerosis, hemolytic anemia, and Graves' disease. In some embodiments, the compound that blocks the interaction of CD40L and CD40 is MR1, and the compounds that block the interaction of CD28 and CD86, or CD28 and CD80 are abatacept, galiximab or belatacept. In some embodiments, the compound that blocks the interaction of CD40L and CD40 is 5c8. In some embodiments, the method comprises administering MR1 in a therapeutically effective amount in combination with abatacept or belatacept. In some embodiments, the method comprises administering 5c8 in a therapeutically effective amount in combination with abatacept or belatacept.

[0104] Kidney-related autoimmune diseases, disorders, and conditions The present disclosure relates to the treatment of a subject having one or more autoimmune diseases, including kidney-related autoimmune diseases, disorders, and / or conditions. In some embodiments, the autoimmune disease is a kidney-related disease, disorder, or condition. In some embodiments, the kidney-related disease, disorder, or condition is a complication after kidney transplantation. In some embodiments, the kidney-related disease, disorder, or condition is kidney transplant rejection. In some embodiments, the kidney-related disease, disorder, or condition is graft-versus-host disease. In some embodiments, the kidney-related disease, disorder, or condition is chronic kidney disease. In some embodiments, the kidney-related disease, disorder, or condition is nephrosis. In some embodiments, the kidney-related disease, disorder, or condition is IgA nephropathy. In some embodiments, the kidney-related disease, disorder, or condition is a kidney autoimmune disease. In some embodiments, the kidney-related disease, disorder, or condition is inflammation or is accompanied by inflammation.

[0105] The compounds disclosed herein that target CD40 or CD40L would be understood to be useful for any disease, disorder, or medical condition in which the kidney is impaired. Non-limiting examples of kidney-related diseases, disorders, and medical conditions include: colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, kidney transplantation, transplant immune response, transplant rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephrosis, interstitial nephritis, membranoproliferative glomerulonephritis, simple kidney cysts, kidney infections, kidney stones, IgA nephropathy, 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, Graves' disease, Abdelhalden-Kaufmann-Lignac syndrome (nephropathic cystinosis), abdominal compartment syndrome, acetaminophen-induced nephrotoxicity, acute renal failure / acute kidney injury, acute renal failure after exercise (ALPE), renal hypouricemia, acute lobar nephronia, acute phosphate nephropathy, acute tubular necrosis, adenine phosphoribosyltransferase deficiency, adenovirus nephritis, Alagille syndrome, alkaptonuria, Alport syndrome, Alström syndrome, amyloidosis, ANCA vasculitis associated with endocarditis and other infections, angiomyolipoma, anabolic steroid abuse, analgesic nephropathy, anorexia nervosa and kidney disease, angiotensin antibodies and focal segmental glomerulosclerosis, anticoagulant-related nephropathy, antiphospholipid syndrome, anti-LRP2 nephropathy, anti-brush border nephropathy, anti-TNF-α therapy-related glomerulonephritis, APOL1 mutations, apparent mineralocorticoid excess syndrome, aristolochic acid nephropathy, Chinese herb nephropathy, Balkan endemic nephropathy, arteriovenous malformations and fistulas of the ureter, atypical hemolytic uremic syndrome, autosomal dominant hypocalcemia, Bardet-Biedl syndrome, Bartter syndrome, Bassolt and acute kidney injury, beer potomania, beeturia, β-thalassemia kidney disease, bile cast nephropathy, Birt-Hogg-Dubé syndrome, BK polyomavirus nephropathy in native kidneys, bladder rupture, detrusor muscle dyssynergia, bladder tamponade, bodybuilder's glomerulopathy, Border-Crossers nephropathy,Bourbon virus and acute kidney injury, BRASH syndrome, acute renal failure, Byetta and renal failure, C1q nephropathy, C3 glomerulopathy, C4 glomerulopathy, CAKUT (congenital anomalies of the kidney and urinary tract), renal calyceal diverticulum, calcineurin inhibitor nephrotoxicity, Callilepsis Laureola poisoning, cannabinoid hyperemesis acute renal failure, capillary leak syndrome, Carconoid syndrome and renal failure, cardiorenal syndrome, carfilzomib-induced kidney injury, CFHR5 nephropathy, Charcot-Marie-Tooth disease with glomerulopathy, cholesterol embolism, Churg-Strauss syndrome, chyluria, ciliopathy, cisplatin nephrotoxicity, cold diuresis, colistin nephrotoxicity, collagen fibrillary glomerulopathy, collapsing glomerulopathy, CMV-associated collapsing glomerulopathy, combination antiretroviral (cART)-associated nephropathy, congenital adrenal hyperplasia, 17-α-hydroxylase / 17,20-lyase deficiency, congenital anomalies of the kidney and urinary tract (CAKUT), congenital nephrotic syndrome, congestive renal failure, Conn's syndrome (primary aldosteronism or Seldin-Mainzer disease), contrast-induced nephropathy, copper sulfate poisoning, coronavirus (COVID-19)-associated renal failure and kidney diseases, cortical necrosis, crizotinib-related acute kidney injury, Crying Kidneys, cryocryoglobulinemia, cryoglobulinemia, crystal globulin-induced nephropathy, crystal deposition histiocytosis, cystic kidney disease, acquired cystic kidney disease, focal renal failure, cystinuria, cystine accumulation disease, dasatinib-induced nephrotic-range proteinuria, deferasirox (Exjade) nephrotoxicity, membranoproliferative glomerulonephritis (MPGN type 2), Dent disease (X-linked recessive nephrolithiasis), DHA crystalline nephropathy, disequilibrium syndrome, diabetic kidney disease, diffuse mesangial sclerosis, diuresis, Djenkolism, duplicated renal pelvis and ureter, EAST syndrome, ectopic kidney, edema, swelling, end-stage kidney disease, Erdheim-Chester disease, Fabry disease, familial hypocalciuric hypercalcemia, Fanconi syndrome, Fraser syndrome, fibronectin glomerulopathy, fibrillary glomerulonephritis and immunotactoid glomerulopathy, fibrillary glomerulonephritis, Congo, Frey syndrome, Fluid Overload, hypervolemia, focal segmental glomerulosclerosis, focal sclerosis, focal glomerulosclerosis,Nodular segmental glomerulosclerosis with complete heart block (familial), Galloway-Mowat syndrome, giant cell (temporal) arteritis with renal involvement, pregnancy hypertension, Gitelman syndrome, glomerular disease, glomerulotubular reflux, glomerulonephritis, glycosuria, Goodpasture syndrome, Green Smoothie Cleanse nephropathy, HANAC syndrome, Harvoni (ledipasvir-sofosbuvir)-induced kidney injury, hair dye ingestion and acute kidney injury, hantavirus infection podocytopathy, heat stress nephropathy, hematuria (blood in urine), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), hemophagocytic syndrome, hemorrhagic cystitis, hemorrhagic fever with renal syndrome (HFRS, hantavirus renal disease, Korean hemorrhagic fever, epidemic hemorrhagic fever, epidemic nephropathy), hemosiderinuria, paroxysmal nocturnal hemoglobinuria and hemosiderosis associated with hemolytic anemia, hepatic glomerulosclerosis, hepatic central vein occlusion, sinusoidal obstruction syndrome, hepatitis C virus-related kidney disease, hepatocyte nuclear factor 1β-related kidney disease, hepatorenal syndrome, herbal supplement and kidney disease, high altitude kidney disorder syndrome, hypertension and kidney disease, HIV-related immune complex kidney disease (HIVICK), HIV-related nephropathy (HIVAN), Holocaust kidney disease, HNF1B-related autosomal dominant tubulointerstitial kidney disease, horseshoe kidney (fusion of kidneys), Hunter ulcer, hydrophilic polymer embolism, hydroxychloroquine-induced renal phospholipidosis, hyperaldosteronism, hypercalcemia, hyperkalemia, hypermagnesemia, hypernatremia, hyperoxaluria, hyperphosphatemia, hypocalcemia, hypocomplementemic urticarial vasculitis syndrome, hypokalemia, hypokalemia-induced renal dysfunction, hypokalemic periodic paralysis, hypomagnesemia, hyponatremia, hypophosphatemia, hypertension, hypertension, monogenic, ice tea nephropathy, ifosfamide nephrotoxicity, IgA nephropathy, IgG4 nephropathy, immersion diuresis, immune checkpoint therapy-related interstitial nephritis, infliximab-related kidney disease, interstitial cystitis, painful bladder syndrome, interstitial nephritis, interstitial nephritis, karyomegalic, IBMARK syndrome, JC virus nephropathy, Joubert syndrome, ketamine-related bladder dysfunction, kidney-based immune disease, kidney stones, nephrolithiasis, kidney stones caused by light chainsKimura disease, mushroom toxicity of black tea, renal lead poisoning and lead-related nephrotoxicity, lecithin cholesterol acyltransferase deficiency (LCAT deficiency), leptospirosis renal disease, light chain deposition disease, monoclonal immunoglobulin deposition disease, light chain proximal tubulopathy, Riedel syndrome, Lightwood-Albright syndrome, lipoprotein glomerulopathy, lithium nephrotoxicity, LMX1B mutation-related hereditary FSGS, loin pain hematuria, lupus, systemic lupus erythematosus, lupus nephritis, lupus nephritis, lupus nephritis with positive anti-neutrophil cytoplasmic antibody serum, lupus podocytopathy, lupus-like nephritis, lupus nephritis, Lyme disease-related glomerulonephritis, lysinuric protein intolerance, lysozyme nephrosis, malaria nephropathy, malignant disease-related renal disease, malignant hypertension, malacoplakia, Marfan syndrome and renal disease, McKusick-Kaufman syndrome, urethral meatus stricture, medullary cystic kidney disease, uroguanylin-related nephropathy, juvenile hyperuricemic nephropathy type 1, medullary sponge kidney, megaureter, MELAS syndrome, membranoproliferative glomerulonephritis, membranous nephropathy, membranous nephropathy with globules, membranous glomerulopathy with Masked IgG Kappa Deposit, Mesoamerican nephropathy, metabolic acidosis, metabolic alkalosis, methotrexate-related renal failure, microscopic polyangiitis, milk-alkali syndrome, minimal change disease, monoclonal immunoglobulin abnormality important for the kidney, protein abnormality in blood, mouthwash toxicity, MUC1 nephropathy, polycystic dysplastic kidney, multiple myeloma, myeloproliferative tumor and glomerulopathy, nail-patella syndrome, NARP syndrome, nephrocalcinosis, Nephrocystin-1 gene deletion and ESRD, nephrogenic systemic fibrosis, nephronophthisis caused by Nephrocystin-1 gene deletion, nephroptosis (floating kidney, kidney ptosis), nephrotic syndrome, neurogenic bladder, nodular glomerulosclerosis, non-gonococcal urethritis, nutcracker syndrome, oligomeganephronia, orofaciodigital syndrome, orotic aciduria, orthostatic hypotension, orthostatic proteinuria, osmotic diuresis, osmotic nephrosis, ovarian hyperstimulation syndrome, oxalate nephropathy, page kidney, papillary necrosis, papillary nephropathy syndrome (renal coloboma syndrome, isolated hypoplastic kidney), peritoneal-renal syndrome, POEMS syndrome, posterior urethral valve, podocyte entrapment glomerulopathy, post-infectious glomerulonephritis, post-streptococcal glomerulonephritis, post-infectious glomerulonephritis, atypical post-infectious glomerulonephritis (IgA dominant), mimicking IgA nephropathy,Polyarteritis nodosa, polycystic kidney disease, posterior urethral valve, obstructive posterior urethral stricture, preeclampsia, propofol infusion syndrome, proliferative glomerulonephritis with monoclonal IgG deposition (Nasr disease), propolis (bee wax)-related renal failure, proteinuria (protein in urine), pseudohyperaldosteronism, pseudohypobicarbonatemia, pseudohypoparathyroidism, pseudoporphyria, psoriasis and kidney disease, pulmonary-renal syndrome, purple urine bag syndrome, pyelonephritis (kidney infection), pyonephrosis, radiation nephropathy, refeeding syndrome, reflux nephropathy, rapidly progressive glomerulonephritis, autosomal dominant tubulointerstitial kidney disease caused by REN mutation, renal abscess, perinephric abscess, renal agenesis, acute kidney injury associated with renal arcuate vein microthrombosis, renal artery aneurysm, renal artery dissection, idiopathic renal artery stenosis, renal cell carcinoma, renal cyst, renal hypouricemia with exercise-induced acute kidney injury, renal infarction, renal osteodystrophy, renal tubular acidosis, renin mutation and autosomal dominant tubulointerstitial kidney disease, renin-secreting tumor (juxtaglomerular cell tumor), reset osmostat, retroperitoneal fibrosis, rhabdomyolysis, rhabdomyolysis associated with bariatric surgery, rheumatoid arthritis-related kidney disease, rucaparib-related creatinine elevation, sarcoidosis kidney disease, Saturday night acute kidney injury, schistosomiasis and glomerular disease, Shprintzen-Goldberg craniosynostosis syndrome, scleroderma renal, ophisthorchiasis polycystic kidney syndrome, Exner syndrome, Sézary syndrome, sickle cell nephropathy, Sjögren's syndrome and kidney disease, Sri Lankan farmer kidney disease, star fruit nephrotoxicity, kidney transplantation, kidney disease after hematopoietic cell transplantation, kidney disease associated with stem cell transplantation, TAFRO syndrome, decreased oral intake (Tea and Toast) hyponatremia, tenofovir-induced nephrotoxicity, thin basement membrane disease, benign familial hematuria, thrombotic microangiopathy with monoclonal immunoglobulin abnormality, trench nephritis, autosomal dominant thrombotic microangiopathy and CKD, trigonitis, tuberous sclerosis, tuberous sclerosis (TSC) and autosomal dominant polycystic kidney disease (ADPKD) (TSC2 / PKD1 contiguous gene syndrome), tubular dysgenesis, immune complex tubulointerstitial nephritis, tumor lysis syndrome, uremia, uremic optic neuropathy, ureteral cyst, ureteral tumor, urethral caruncle, urethral stricture, urinary incontinence, urinary tract infection, urinary tract obstruction, urogenital fistula, vasomotor nephropathy, vesicointestinal fistula, vesicoureteral reflux, von Hippel-Lindau disease, Waldenström macroglobulinemia glomerulonephritis, Wegener granulomatosis,Granulomatosis with polyangiitis, Wunderlich syndrome, Zellweger syndrome, and cerebrohepatorenal syndrome are included.

[0106] Pharmaceutical compositions and administration methods For treating any of the above diseases, the pharmaceutical compositions used according to the methods of the present disclosure can be formulated by conventional techniques using one or more physiologically acceptable carriers. The pharmaceutically acceptable carrier is partly determined by the particular composition being administered and by the particular method used for administration of the composition. Accordingly, there are a wide variety of suitable formulations for the compounds useful in the methods of the present disclosure (see, e.g., Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro et al. Eds., Lippincott Williams and Wilkins, 2000).

[0107] Formulations suitable for oral administration include, for example, solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multiparticulates or nanoparticles, liquids, or powders; troches (such as liquid-filled); chewables; gels; rapid-dissolution dosage forms; films; ovules; sprays; and buccal / mucoadhesive patches.

[0108] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0109] According to the present disclosure, the compound can be administered by any suitable means, which may vary depending on the type of disease being treated and the nature of the compound itself. For example, the compound can be administered orally, parenterally, or topically. For proteins such as antibodies, the route of administration preferably includes parenteral administration, such as intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Preferably, parenteral administration is performed by injection, most preferably by intravenous, intramuscular, or subcutaneous injection. The amount administered varies depending on various factors, such as clinical symptoms, the weight of the individual, and whether other drugs are being administered. Determination of the appropriate dosage form, dosage, and route of administration is within the level of those skilled in the pharmaceutical and medical arts, and it should be understood that this is described below.

[0110] In an exemplary pharmaceutical composition containing an anti-CD40L antibody or an antigen-binding fragment thereof, the composition is formulated as a sterile, preservative-free solution of the anti-CD40L antibody or an antigen-binding fragment thereof for intravenous or subcutaneous administration. The formulation can be supplied as a disposable, prefilled pen, such as a disposable prefilled glass syringe containing about 1 mL, or as a disposable vial for institutional use. Preferably, the pharmaceutical composition containing the anti-CD40L antibody or an antigen-binding fragment thereof is transparent or colorless and has a pH in the range of about 5.0 to about 6.9, preferably about 5.0 to about 6.5, and more preferably about 5.0 to about 6.0. In various embodiments, the formulation containing the pharmaceutical composition, when reconstituted and administered to a subject, can contain about 500 mg to about 1 mg, or about 400 mg to about 10 mg, or about 300 mg to about 30 mg, or about 200 mg to about 50 mg of the anti-CD40L antibody or an antigen-binding fragment thereof per mL of the solution.

[0111] The selection of a dosing regimen for treatment varies depending on several factors such as the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix, the patient's overall health status, the patient's medical history, etc. Preferably, the dosing regimen minimizes the amount of therapeutic agent delivered to the patient that is consistent with an acceptable level of side effects. Thus, the amount of biologic delivered is partly dependent on the particular entity and the severity of the medical condition being treated. It should be understood that the determination of the appropriate dosage form, dosage, and route of administration is within the level of those skilled in the fields of pharmacy and medicine.

[0112] Dosage and Exposure Levels The pharmaceutical formulations of the present disclosure may contain from about 0.001 to about 200 mg / kg of an anti-CD40L antibody or an antigen-binding fragment thereof, for example, from about 0.001 to about 100 mg / kg, or from about 0.001 to about 50 mg / kg, or from about 0.001 to about 10 mg / kg of an anti-CD40L antibody or an antigen-binding fragment thereof by intravenous injection, and may be administered as a bolus, and the remainder of the antibody dosage may be administered by intravenous injection. A predetermined dosage of the anti-CD40L antibody or an antigen-binding fragment thereof may be administered over a period of time, for example, 1 hour to 2 hours to 5 hours.

[0113] In a further embodiment, a portion of the dosage is administered by subcutaneous injection and / or infusion in the form of a bolus, and the remainder is administered by infusion of the antibody formulation. In some exemplary dosages, the antibody formulation may be subcutaneously administered at dosages in the range of from about 0.001 to about 200 mg / kg, for example, from about 0.001 to about 100 mg / kg, or from about 0.001 to about 50 mg / kg, or from about 0.001 to about 10 mg / kg of an anti-CD40L antibody or an antigen-binding fragment thereof by intravenous injection. In some embodiments, the dosage may be administered as a bolus, and the remainder of the antibody dosage may be administered by subcutaneous or intravenous injection. A predetermined dosage of the anti-CD40L antibody or an antigen-binding fragment thereof may be administered over a period of, for example, 1 hour, or 2 hours, or 3 hours, or 4 hours or 5 hours, or more.

[0114] Combination Therapy The antibodies or antibody fragments described herein can be administered alone (monotherapy) or in combination, i.e., in combination with other agents. For example, in one embodiment, the combination therapy can include one or more additional therapeutic agents. In another embodiment, the combination therapy includes a standard therapy that may or may not include an additional therapeutic agent (substantially consisting of an antibody or an antibody fragment thereof).

[0115] Additional or combined administration (co - administration) includes simultaneous administration of any of the antibodies or antigen - binding fragments thereof described herein and one or more agents in the same or different dosage forms, or separate administration (e.g., sequential administration) of a polypeptide and one or more agents. As a result of such simultaneous or sequential administration, preferably, the polypeptide and one or more agents are present simultaneously in the treated patient.

[0116] Kits and Manufactured Articles There is further provided a kit comprising an antibody or an antibody - binding fragment thereof described herein and instructions for use. The kit typically includes a reagent in a predetermined amount and a packaged combination with a label description and label indicating the purpose of use of the contents of the kit. The terms label and instructions include a document or record on the kit or supplied with the kit, or associated with the kit at any point during its manufacture, transportation, sale, or use. It can take the form defined by a government agency that regulates the manufacture, use, or sale of a pharmaceutical or biological product, and the notice reflects approval by the agency for manufacture, use, or sale for human administration or veterinary use. The label or instructions can also include advertising brochures and pamphlets, package materials, and audio or video instructions.

[0117] Anti - CD40L Antigen - Binding Molecules Some embodiments provided herein relate to modified forms of the anti - CD40L antibody hu5c8 that include the variable light - chain and variable heavy - chain CDR sequences of hu5c8 with an Fc domain modified to prevent platelet activation, within a human IgG1 consensus framework.

[0118] Table 3 provides the content of the sequence numbers referred to in this application (and shown in FIGS. 7-12).

[0119] [Table 5]

[0120] One embodiment (Embodiment A) is an isolated antibody that binds to CD40L and comprises a light chain and a heavy chain, the light chain of which comprises a light chain variable region having an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, and the heavy chain of which comprises a heavy chain variable region and an Fc region, the heavy chain variable region having an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with SEQ ID NO: 2, and the Fc region having an amino acid sequence having at least at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with SEQ ID NO: 3, the Fc region of which comprises one substitution or combination of substitutions selected from the group consisting of C11S, C14S, and P23S.

[0121] Another embodiment (Embodiment B) is the isolated antibody according to Embodiment A, wherein the Fc region further comprises the amino acid substitution C5S.

[0122] In variant forms of Embodiments A and B, the antibody comprises a light chain variable region that does not include any of the substitutions T33W, S26D, and Q27E.

[0123] In other variants of Embodiments A and B, the light chain variable region comprises the substitution R28K.

[0124] In some variants of Embodiments A and B, the CDRs of the heavy and light chains have the sequences shown in Table 4.

[0125] [Table 6]

[0126] In still other variants of Embodiments A and B, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15). In other embodiments, the light chain variable region comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO: 15), and one or both of the amino acid sequences YASNLES (SEQ ID NO: 16) and QHSWEIPPT (SEQ ID NO: 17).

[0127] In some variants of Embodiments A and B, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 1. In still other embodiments, the light chain variable region consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 7. In other embodiments, the light chain consists of the amino acid sequence of SEQ ID NO: 7. In still other embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 11. In still other embodiments, the light chain consists essentially of the amino acid sequence of SEQ ID NO: 11. In still other embodiments, the light chain consists of the amino acid sequence of SEQ ID NO: 11.

[0128] In other embodiments 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 embodiments of Embodiments A and B, the light chain variable region does not contain any of the substitutions T33W, S26D, and Q27E, and the heavy chain variable region does not contain any of the substitutions T30H, Y33W, or S54N.

[0129] In other variant forms of Embodiments A and B, the heavy chain variable region contains the substitution K74R. In one embodiment, the heavy chain variable region contains 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).

[0130] In another embodiment, the heavy chain variable region contains 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 contains the amino acid sequence of SEQ ID NO: 6. In other embodiments, the heavy chain variable region consists essentially of the amino acid sequence of SEQ ID NO: 6. In still other embodiments, the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 6.

[0131] Some embodiments provided herein relate to an isolated antibody in which the light chain contains the amino acid sequence of SEQ ID NO: 1 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 9.

[0132] Some embodiments provided herein relate to an isolated antibody in which the light chain consists of the amino acid sequence of SEQ ID NO: 7 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 9.

[0133] Some embodiments provided herein relate to an isolated antibody in which the light chain variable region contains the amino acid sequence of SEQ ID NO: 5 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 9.

[0134] Yet another embodiment is an isolated antibody in which the light chain consists of the amino acid sequence of SEQ ID NO: 11 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 9.

[0135] Another embodiment is an isolated antibody in which the light chain consists of the amino acid sequence of SEQ ID NO: 7 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 13.

[0136] Another embodiment is an isolated antibody in which the light chain consists of the amino acid sequence of SEQ ID NO: 11 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 13.

[0137] Some embodiments of the present application relate to the modification of biomarkers in a subject using the treatment of a compound that targets CD40 / CD40L. In some embodiments, the biomarker is a protein involved in CD40 / CD40 activity. In some embodiments, the biomarker is CD40. In some embodiments, the biomarker is CD40L. In some embodiments, the biomarker is NFL. In some embodiments, the biomarker is an inflammation-inducing marker or is related to inflammation. In some embodiments, the biomarker is a cytokine and / or an interleukin. In some embodiments, the biomarker is one or more of IgA, IgE, CD40L, C3, CXCL9, CXCL10, and / or IgM.

[0138] In some embodiments, the concentration of one or more biomarkers is increased by treatment with a compound that targets CD40 / CD40L. In some embodiments, the concentration of one or more biomarkers is decreased by treatment with a compound that targets CD40 / CD40L. In some embodiments, the concentration of at least one type of biomarker is increased and the concentration of at least one other type of biomarker is decreased by treatment with a compound that targets CD40 / CD40L. In some embodiments, the baseline level of at least one type of biomarker serves as a predictor of the response of a subject having a kidney-related disease, disorder, or condition to treatment with a compound that targets CD40 / CD40L.

[0139] Some embodiments provided herein are further described in the alternatives listed below.

[0140] 1. A method for predicting the response of a subject having a kidney-related disease or disorder to treatment with a compound against CD40L or CD40, comprising Collecting a sample from a subject; Measuring the concentration of at least one biomarker present in the sample, wherein the at least one biomarker is CXCL9, CXCL10, IgM, C3, IgA, IgE, CD40L, and CD40; and predicting from the concentration of the at least one biomarker that the subject may be responsive to treatment of a kidney-related disease or disorder with a compound against CD40L or CD40; A method comprising:

[0141] 2. The method of alternative 1, wherein the kidney-related disease or disorder includes inflammation.

[0142] 3. The method according to alternative 1 or 2, wherein the subject is a mammal and / or a human.

[0143] 4. The method according to any one of alternatives 1 to 3, wherein the compound blocks the interaction between CD40 and CD40L.

[0144] 5. The method according to any one of alternatives 1 to 4, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0145] 6. The method according to any one of alternatives 1 to 5, wherein the compound is AT-1501 or 5c8.

[0146] 7. The method according to any one of alternatives 1 to 6, wherein the compound is MR1.

[0147] 8. The method according to any one of alternatives 1 to 7, wherein the kidney-related disease or disorder is selected from the group consisting of colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, transplant immune response, kidney transplantation, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stones, and IgA nephropathy.

[0148] 9. A method according to any one of alternative 1 to 8, wherein the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease.

[0149] 10. A method according to any one of alternative 1 to 9, wherein the sample is blood urine, saliva, or mucus.

[0150] 11. A method according to alternative 10, wherein the sample is urine.

[0151] 12. A method for treating a subject having a kidney-related disease or disorder, comprising administering a compound against CD40L or CD40 in a therapeutically effective dose.

[0152] 13. A method according to alternative 12, wherein the kidney-related disease or disorder includes inflammation.

[0153] 14. A method according to alternative 12 or 13, wherein the subject is a mammal and / or a human.

[0154] 15. A method according to any one of alternative 12 to 14, wherein the compound blocks the interaction between CD40 and CD40L.

[0155] 16. A method according to any one of alternative 12 to 15, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0156] 17. A method according to any one of alternative 12 to 16, wherein the compound is AT-1501.

[0157] 18. A method according to any one of alternative 12 to 17, wherein the compound is MR1 or 5c8.

[0158] 19. A method according to any one of alternative 12 to 18, wherein the compound is administered at least once every three weeks.

[0159] 20. The method according to any one of alternatives 12 to 19, wherein the compound is administered at a dose of 200 to 100 mg / kg; preferably at a dose of 1 to 20 mg / kg.

[0160] 21. The method according to any one of alternatives 12 to 20, wherein the kidney-related disease or disorder is colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, transplant immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephrosis, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy.

[0161] 22. The method according to any one of alternatives 12 to 21, wherein the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease.

[0162] 23. The method according to any one of alternatives 12 to 22, further comprising administering a second pharmaceutically effective compound.

[0163] 24. The method of alternative 23, wherein the second compound blocks the interaction of CD28 with CD86 or CD28 with CD80.

[0164] 25. The method of alternative 23, wherein the second compound targets at least one of the biomarkers selected from the group consisting of interleukins, cytokines, and inflammation-inducing markers.

[0165] 26. The method of alternative 23, wherein the second compound is CTLA4-Ig fusion protein, abatacept, belatacept, or galiximab.

[0166] 27. The method according to any one of alternatives 12 to 26, wherein the compound is administered orally, parenterally, or topically.

[0167] 28. The method of alternative 27, wherein the compound is administered parenterally.

[0168] 29. The method of alternative 28, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

[0169] 30. A compound that targets CD40L or CD40 is: (a) i) A CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; ii) A CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; iii) A CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; and a heavy chain variable region (VH) comprising: (b) i) A CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; ii) A CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; iii) A CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20; and a light chain variable region (VL) comprising: and is an antibody comprising: The method according to any one of alternatives 1 to 29.

[0170] 31. A compound that targets CD40L or CD40 is: (a) A heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4; (b) A light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8; and is an antibody comprising: The method according to any one of alternatives 1 to 30.

[0171] 32. A method for modulating the concentration of at least one biomarker in a subject, comprising: administering to the subject a therapeutically effective amount of a compound that binds to CD40L or CD40, A method wherein the biomarker is selected from the group consisting of IgA, IgE, CD40L, CD40, C3, CXCL9, CXCL10, and IgM.

[0172] 33. The method of alternative 32, wherein the concentration of at least one biomarker increases after administration of the compound.

[0173] 34. The method of alternative 32, wherein the concentration of at least one biomarker decreases after administration of the compound.

[0174] 35. The method of alternative 32, wherein the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases after administration of the compound.

[0175] 36. The method according to any one of alternatives 32 to 35, wherein the subject has a kidney-related disease or disorder.

[0176] 37. The method of alternative 36, wherein the kidney-related disease or disorder includes inflammation.

[0177] 38. The method according to any one of alternatives 32 to 37, wherein the subject is a mammal and / or a human.

[0178] 39. The method according to any one of alternatives 32 to 38, wherein the compound blocks the interaction between CD40 and CD40L.

[0179] 40. The method according to any one of alternatives 32 to 39, wherein the compound is an anti-CD40L antibody or an anti-CD40 antibody.

[0180] 41. The method according to any one of alternatives 32 to 40, wherein the compound is AT-1501.

[0181] 42. The method according to any one of alternatives 32 to 41, wherein the compound is MR1 or 5c8.

[0182] 43. The method according to any one of alternatives 36 to 42, wherein the kidney-related disease or disorder is colitis, drug-induced lupus nephritis, lupus nephritis, nephrosis, chronic kidney disease, graft-versus-host disease, transplantation immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal insufficiency, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephrosis, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stone, or IgA nephropathy.

[0183] 44. The method according to any one of alternatives 36 to 43, wherein the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease.

[0184] 45. The method according to any one of alternatives 32 to 44, wherein the compound is administered once every at least three weeks.

[0185] 46. The method according to any one of alternatives 32 to 45, wherein the compound is administered at a dose of 200 to 100 mg / kg; preferably at a dose of 1 to 20 mg / kg.

[0186] 47. The method according to any one of alternatives 32 to 46, wherein the compound is administered orally, parenterally, or topically.

[0187] 48. The method according to alternative 47, wherein the compound is administered parenterally.

[0188] 49. The method according to alternative 47, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

[0189] 50. The compound that targets CD40L or CD40 is: (a) i) A CDRH1 domain comprising the sequence set forth in SEQ ID NO: 9; ii) A CDRH2 domain comprising the sequence set forth in SEQ ID NO: 10, 11, 12, 13, or 14; iii) A CDRH3 domain comprising the sequence set forth in SEQ ID NO: 15; A heavy chain variable region (VH) comprising: (b) i) A CDRL1 domain comprising the sequence set forth in SEQ ID NO: 16 or 17; ii) A CDRL2 domain comprising the sequence set forth in SEQ ID NO: 18 or 19; iii) A CDRL3 domain comprising the sequence set forth in SEQ ID NO: 20; A light chain variable region (VL) comprising: An antibody comprising: according to any one of alternatives 33 to 49.

[0190] 51. A compound that targets CD40L or CD40 is: (a) A heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4; (b) A light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 8; An antibody comprising: according to any one of alternatives 32 to 50.

Examples

[0191] Embodiments are further defined in the following examples. It should be understood that these examples are merely illustrative. From the above discussion and these examples, one of ordinary skill in the art can identify the essential features and, without departing from the spirit and scope, make various changes and modifications to the embodiments and adapt them to various applications and conditions. Therefore, in addition to those shown and described herein, modifications to the embodiments will be apparent to one of ordinary skill in the art from the foregoing description. Such modifications are also intended to be within the scope of the appended claims. The disclosure of each reference described herein is incorporated herein by reference in its entirety and with respect to the disclosure referenced herein.

[0192] Example 1 Clinical trial in kidney transplantation Four human subjects with IgAN (Berger's disease) were kidney transplanted and monitored for 12 weeks. At the end of the 12 weeks, urine samples were collected from each subject. These samples were then screened for changes in inflammatory markers by comparing their levels in the urine of each subject before kidney transplantation.

[0193] The IgAN-related inflammatory markers IgA, IgE, CD40L, and C3 were significantly decreased in the patients after 12 weeks (Figures 20A - 20D). Furthermore, the systemic transplantation inflammatory markers CXCL9, CXCL10, IgM, and C3 were decreased after 12 weeks (Figures 21A - 21D). Assuming that CD40L and CD40L-related proteins are significantly decreased in the subjects when a healthy kidney is transplanted, subsequent treatment with a CD40L-targeting compound such as the compounds disclosed herein may be useful for promoting recovery and reducing inflammation in the subjects.

[0194] Example 2 Phase 1B Human Clinical Trial The clinical trial was expanded to a single-dose level study for 52 weeks with up to 12 participants. The details of the study design are as shown in Figure 22. Immunosuppressive therapy was administered to the participants using an ATG induction therapy + CNI-free maintenance therapy that included mycophenolic acid, corticosteroids, and tegoprubart instead of tacrolimus. The drugs were administered as follows:

[0195] rATG: Administered at 1.5 mg / kg / IV for 4 doses (total 6 mg / kg). The first dose was initiated before revascularization, and the remaining 3 doses (total 4.5 mg / kg) were within 5 - 10 days after transplantation in accordance with the labeled instructions.

[0196] Corticosteroids: Tapered to 5 mg of prednisone (or equivalent) daily until 28 days after transplantation according to standard treatment.

[0197] MMF: BID or MPS 1000 mg: BID 720 mg

[0198] Tegopurivat: On days 1, 3, 7, 14, 21, and 28, and then every 21 days thereafter, 20 mg / kg by IV injection.

[0199] The first 4 participants selected for the study were 2 females and 2 males, aged 60 - 77 years (Figure 23). These participants each received a kidney transplant and were subsequently dosed as described above. Renal function was monitored for the next 120 days using estimated glomerular filtration rate (eGFR) (Figures 24 - 25). As shown in Figure 25, Tegopurivat was administered at 4 different time points. So far, renal function has been significantly improved and the hazard ratio is low.

[0200] The section headings used in this specification are for purposes of organization and should in no way be construed as limiting the subject matter described. All documents and similar materials described in this application, including but not limited to patents, patent applications, articles, books, academic papers, and Internet web pages, are hereby expressly incorporated by reference in their entirety for any purpose, such as the disclosure specifically referenced herein. If the definitions of terms in the incorporated references are thought to be different from those provided in the teachings of the present invention, the definitions provided in the teachings of the present invention shall control. It should be understood that the presence of "about" before temperatures, concentrations, times, etc. considered in the teachings of the present invention is such that minor and insignificant deviations are within the scope of the teachings of the present invention herein.

[0201] The embodiments described in this specification are disclosed in the context of specific embodiments and examples. However, the present disclosure extends beyond the specifically disclosed embodiments to cover other alternative embodiments and / or the use of the disclosed content and its obvious modifications and equivalents, which will be understood by those skilled in the art. Further, although some variations of the embodiments are shown and described in detail, other modifications within the scope of the present disclosure will be readily understood by those skilled in the art based on the present disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments can be made and may still fall within the scope of the present disclosure. It should be understood that the various features and aspects of the disclosed embodiments can be combined with each other or replaced with each other to form various modes or embodiments. Therefore, it is intended that the scope of the present disclosure should not be limited by the specific embodiments disclosed above.

[0202] However, since various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art, it should be understood that this detailed description showing preferred embodiments is presented by way of example only.

[0203] The technical terms used in the descriptions shown in this specification are not intended to be construed in a limiting or restrictive manner. Rather, the technical terms are simply used in combination with the detailed description of the embodiments of the system, method, and related components. Further, an embodiment may include several new features, none of which are, by themselves, the cause of its desirable characteristics or considered essential for the implementation of the embodiments described in this specification.

Claims

1. A pharmaceutical composition for treating a subject having a kidney-related disease or disorder, A pharmaceutical composition comprising a compound for CD40L or CD40 in a therapeutically effective dose.

2. The pharmaceutical composition according to claim 1, wherein the kidney-related disease or disorder includes inflammation.

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

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

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

6. The pharmaceutical composition according to claim 1, wherein the compound is AT-1501.

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

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

9. The pharmaceutical composition according to claim 1, wherein the compound is administered in a dose of 200 to 100 mg / kg; preferably in a dose of 1 to 20 mg / kg.

10. The pharmaceutical composition according to claim 1, wherein the kidney-related disease or disorder is colitis, drug-induced lupus nephritis, lupus nephritis, nephropathy, chronic kidney disease, graft-versus-host disease, transplant immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stones, or IgA nephropathy.

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

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

13. The pharmaceutical composition according to claim 12, wherein the second compound blocks the interaction between CD28 and CD86 or between CD28 and CD80.

14. The pharmaceutical composition according to claim 12, wherein the second compound targets at least one of the biomarkers selected from the group consisting of interleukins, cytokines, and pro-inflammatory markers.

15. The pharmaceutical composition according to claim 12, wherein the second compound is a CTLA4-Ig fusion protein, abatacept, beratacept, or galiximab.

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

17. The pharmaceutical composition according to claim 16, wherein the compound is administered parenterally.

18. The pharmaceutical composition according to claim 17, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

19. The compound that targets CD40L or CD40 is: (a) i) CDRH1 domain containing the sequence described in Sequence ID No. 18; ii) CDRH2 domain containing the sequence described in Sequence ID No. 19; iii) CDRH3 domain containing the sequence described in Sequence ID No. 20; The heavy chain variable region (VH) includes, (b) i) CDRL1 domain containing the sequence described in Sequence ID No. 15; ii) CDRL2 domain containing the sequence described in Sequence ID No. 16; iii) CDRL3 domain containing the sequence described in Sequence ID No. 17; The light chain variable region (VL) includes, The pharmaceutical composition according to claim 1, comprising an antibody or an antigen-binding fragment thereof.

20. The compound that targets CD40L or CD40 is: (a) A heavy chain variable region (VH) having the amino acid sequence described in SEQ ID NO: 2 or 6; (b) A light chain variable region (VL) having the amino acid sequence described in SEQ ID NO: 1 or 5; The pharmaceutical composition according to claim 1, comprising an antibody or an antigen-binding fragment thereof.

21. A pharmaceutical composition for adjusting the concentration of at least one biomarker in a subject: It contains a therapeutically effective amount of a compound for CD40L or CD40, A pharmaceutical composition in which the biomarker is selected from the group consisting of IgA, IgE, CD40L, CD40, C3, CXCL9, CXCL10, and IgM.

22. The pharmaceutical composition according to claim 21, wherein the concentration of at least one biomarker increases after administration of the compound.

23. The pharmaceutical composition according to claim 21, wherein the concentration of at least one biomarker decreases after administration of the compound.

24. The pharmaceutical composition according to claim 21, wherein, after administration of the compound, the concentration of at least one biomarker increases and the concentration of at least one other biomarker decreases.

25. The pharmaceutical composition according to claim 21, wherein the subject has a kidney-related disease or disorder.

26. The pharmaceutical composition according to claim 25, wherein the kidney-related disease or disorder includes inflammation.

27. The pharmaceutical composition according to claim 21, wherein the target is a mammal and / or a human.

28. The pharmaceutical composition according to claim 21, wherein the compound blocks the interaction between CD40 and CD40L.

29. The pharmaceutical composition according to claim 21, wherein the compound is an anti-CD40L antibody or an antigen-binding fragment thereof, or an anti-CD40 antibody or an antigen-binding fragment thereof.

30. The pharmaceutical composition according to claim 21, wherein the compound is AT-1501.

31. The pharmaceutical composition according to claim 21, wherein the compound is MR1 ​​or 5c8.

32. The pharmaceutical composition according to claim 25, wherein the kidney-related disease or disorder is colitis, drug-induced lupus nephritis, lupus nephritis, nephropathy, chronic kidney disease, graft-versus-host disease, transplant immune response, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stones, or IgA nephropathy.

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

34. The pharmaceutical composition according to claim 21, wherein the compound is administered at least once every three weeks.

35. The pharmaceutical composition according to claim 21, wherein the compound is administered in a dose of 200 to 100 mg / kg; preferably in a dose of 1 to 20 mg / kg.

36. The pharmaceutical composition according to claim 21, wherein the compound is administered orally, parenterally, or topically.

37. The pharmaceutical composition according to claim 36, wherein the compound is administered parenterally.

38. The pharmaceutical composition according to claim 36, wherein the compound is administered by injection, most preferably by intravenous, intramuscular, or subcutaneous injection.

39. The compound that targets CD40L or CD40 is (a) i) CDRH1 domain containing the sequence described in Sequence ID No. 18; ii) CDRH2 domain containing the sequence described in Sequence ID No. 19; iii) CDRH3 domain containing the sequence described in Sequence ID No. 20; The heavy chain variable region (VH) includes, (b) i) CDRL1 domain containing the sequence described in Sequence ID No. 15; ii) CDRL2 domain containing the sequence described in Sequence ID No. 16; iii) CDRL3 domain containing the sequence described in Sequence ID No. 17; The light chain variable region (VL) includes, The pharmaceutical composition according to claim 21, comprising an antibody or an antigen-binding fragment thereof.

40. The compound that targets CD40L or CD40 is: (a) A heavy chain variable region (VH) having the amino acid sequence described in SEQ ID NO: 2 or 6; (b) A light chain variable region (VL) having the amino acid sequence described in SEQ ID NO: 1 or 5; The pharmaceutical composition according to claim 21, comprising an antibody or an antigen-binding fragment thereof.

41. A method for assisting in predicting the response of a subject with kidney-related disease or impairment to treatment with a compound against CD40L or CD40, The method involves measuring the concentration of at least one biomarker present in a sample from the subject, wherein the at least one biomarker is CXCL9, CXCL10, IgM, C3, IgA, IgE, CD40L, and CD40; Based on the concentration of at least one of the aforementioned biomarkers, it can be predicted that the subject may be responsive to treatment of kidney-related diseases or disorders with a compound targeting CD40L or CD40; A method that includes this.

42. The method according to claim 41, wherein the kidney-related disease or disorder includes inflammation.

43. The method according to claim 41, wherein the subject is a mammal and / or a human.

44. The method according to claim 41, wherein the compound blocks the interaction between CD40 and CD40L.

45. The method according to claim 41, wherein the compound is an anti-CD40L antibody or an antigen-binding fragment thereof, or an anti-CD40 antibody or an antigen-binding fragment thereof.

46. The method according to claim 41, wherein the compound is AT-1501 or 5c8.

47. The method according to claim 41, wherein the compound is MR1.

48. The method according to claim 41, wherein the kidney-related disease or disorder is selected from the group consisting of colitis, drug-induced lupus nephritis, lupus nephritis, nephropathy, chronic kidney disease, graft-versus-host disease, transplant immune response, kidney transplant, graft rejection, atherosclerosis, diabetic kidney disease, acute kidney injury, renal failure, polycystic kidney disease, hematuria, Fabry disease, lupus nephritis, Alport syndrome, IgM nephropathy, interstitial nephritis, membranoproliferative glomerulonephritis, simple renal cyst, kidney infection, kidney stones, and IgA nephropathy.

49. The method according to claim 41, wherein the kidney-related disease or disorder is an autoimmune disease, and the autoimmune disease is 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, or Graves' disease.

50. The method according to claim 41, wherein the sample is blood, urine, saliva, or mucus.

51. The method according to claim 50, wherein the sample is urine.

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