Patient Selection and Treatment Monitoring for Autoimmune Disorders

Abstract

The present invention relates to V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or mRNA as a diagnostic marker for selecting patients suffering from autoimmune disorders for treatment and as a means for monitoring the success of treatment of autoimmune disorders. The present invention relates to methods for patient selection and treatment monitoring, combined diagnostic and therapeutic applications, which involve the determination of VISTA protein and / or mRNA in patient samples, and diagnostic kits suitable for use in the methods of the invention.

Description

[Technical Field]

[0001] The present invention relates to V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) protein or mRNA as a diagnostic marker for selecting patients suffering from autoimmune disorders for treatment and as a means for monitoring the success of treatment of autoimmune disorders. The present invention relates to methods for patient selection and treatment monitoring, combined diagnostic and therapeutic applications, which involve the determination of VISTA protein and / or mRNA in patient samples, and diagnostic kits suitable for use in the methods of the invention. [Background technology]

[0002] In autoimmune diseases, a patient's immune system loses the ability to distinguish between the patient's own body's ("self") proteins and foreign proteins. This results in antibody and / or T-cell responses that recognize "self" determinants, such as proteins, carbohydrates, and nucleic acids, resulting in the formation of immune complexes and the continuous activation of the immune system as "self" structures are persistently produced and recognized as foreign. This chronic condition can persist for years, ultimately resulting in severe tissue and organ damage and, in some cases, even death of the patient. There are many different autoimmune disorders that affect the body in different ways. For example, the brain is affected in individuals with multiple sclerosis, the digestive tract in individuals with Crohn's disease, and the synovial membranes, bone, and cartilage of various joints in individuals with rheumatoid arthritis. Autoimmune disorders lead to the progressive destruction of one or more body tissues, resulting in abnormal organ growth or altered organ function. Autoimmune disorders can affect a single organ or tissue type or multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include red blood cells, blood vessels, connective tissue, endocrine glands (eg, the thyroid or pancreas), muscles, joints, and skin.

[0003] Examples of inflammatory and / or autoimmune disorders include, but are not limited to, primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune hemolytic anemia (AIHA), type 1 diabetes, pemphigus vulgaris, Hashimoto's thyroiditis, autoimmune inner ear disease, myasthenia gravis, pernicious anemia, Addison's disease, dermatomyositis, Sjogren's syndrome, dermatomyositis, multiple sclerosis, Reiter's syndrome, Graves' disease, autoimmune hepatitis, familial adenomatous polyposis, and ulcerative colitis.

[0004] The V-set immunoregulatory receptor (VSIR), first described by [Non-Patent Document 1] and named "V-domain Ig suppressor of T cell activation" (VISTA), is an immunoglobulin (Ig) superfamily ligand that negatively regulates T cell responses. VISTA is primarily expressed in hematopoietic cells, and VISTA expression is highly regulated in antigen-presenting cells (APCs) and T cells in the bone marrow. Wang et al. demonstrated that soluble VISTA-Ig fusion protein or VISTA expression in APCs inhibited T cell proliferation and cytokine production in vitro, and that a VISTA-specific monoclonal antibody interfered with VISTA-induced suppression of T cell responses by VISTA-expressing APCs in vitro. These findings indicate that VISTA has functional activities that are not redundant with those of other Ig superfamily members. Wang et al. further hypothesized that VISTA may play a role in the development of autoimmunity and immune surveillance in cancer. VISTA has since been recognized as a broad-spectrum negative checkpoint regulator for cancer immunotherapy (Non-Patent Document 2).

[0005] VISTA has been associated with acquired resistance to anti-PD-1 therapy in patients with metastatic melanoma (Non-Patent Document 3; published online August 4, 2017), and has also been associated with compensatory inhibitory pathways in prostate tumors after treatment with ipilimumab (Non-Patent Document 4). Furthermore, the immune checkpoint protein VISTA has also been described to critically regulate the IL-23 / IL-17 inflammatory axis (Non-Patent Document 5).

[0006] New approaches are needed to address the very early stages after clinical onset, with the ultimate goal being disease prevention. To improve treatment, we must address antigen-specific autoimmunity, which regulates the development of joint inflammation. Autoimmune responses include citrullinated proteins in genetically predisposed individuals, from preclinical to early stages after clinical onset. Furthermore, immune responses emerge against joint proteins, including the major joint protein type II collagen (COL2), and against citrullinated COL2. COL2 is particularly noteworthy because it is not only a major structural protein in cartilage but also an autoantigenic component expressed in the central lymphoid organs, the thymus and bone marrow. Indeed, autoimmune arthritis can be induced in rodents by immunization with COL2 using a potent adjuvant (collagen-induced arthritis [CIA] model). Experimental arthritis depends on major histocompatibility complex class II (MHCII) interactions. CIA in mice is associated with the MHCII allele Aq and strictly depends on T cell recognition of the galactosylated 259-273 COL2 epitope. 259~273 Peptide-specific T cells express DRB1 * COL2-reactive T cells are detectable in both healthy individuals and RA patients, and the immunodominant COL2 259~273They primarily recognize peptides glycosylated at positions 264 and / or 270. The function of these frequently occurring COL2-reactive T cells is unclear, as they may have regulatory or pathogenic functions. However, human DRB1 * In humanized mice expressing 0401 MHCII, COL2 259~273 CIA mediated by specific T cells occurs.

[0007] Patent Document 1 discloses an HLA-DR / COL2 peptide complex containing a chondroitin-binding peptide sequence at the C-terminus of a polypeptide containing an HLA-DR alpha chain and / or an HLA-DR beta chain, where the COL2 peptide is fused to the N-terminus of the HLA-DR alpha chain or HLA-DR beta chain via a linker peptide. This complex is used to treat chronic inflammatory diseases, such as arthritis, in human patients.

[0008] Patent document 2 discloses in situ glycosylated MHC II / COL2 peptide complexes, i.e., complexes that are naturally glycosylated during recombinant protein expression in host cells. Patent document 2 also relates to a method for producing glycosylated MHC II / COL2 peptide complexes in mammalian cells, as well as the use of such post-translationally modified, preferably glycosylated, MHC / COL2 complexes in the treatment of rheumatoid arthritis.

[0009] Various co-inhibitory immune receptors have been characterized as being upregulated in natural and induced regulatory T cells. These include CTLA-4, PD-1, Lag-3, Tim-3, and TIGIT. The immunoregulatory functions of these molecules have been well characterized, and their expression in CD4+ T cells is commonly used as a marker of the regulatory T cell phenotype. These markers and additional molecules, such as the transcription factors FOXP3, FR4 (folate receptor 4), and CD49b (α2 integrin chain), CD73 (ecto-5'-nucleotidase), and secretion of interleukin-10 (reviewed in 2), have been tested as indicators of pharmacodynamic effects on CD4+ T cell populations in the development of vaccine approaches. The above markers proved suitable for characterizing the lymphocyte phenotype induced by therapeutic vaccination against collagen-induced arthritis (CIA) using a mouse homolog of the human MHCII / galCOL2 peptide complex, suggesting that the lymphocyte phenotype resembles type 1 regulatory (TR1) cells. However, consistent upregulation of the entire set of TR1 markers by in vitro stimulation of patient-derived PBMCs with each human vaccine was not observed. Therefore, as suggested by the prior art summarized above, the use of an established set of prior art markers for induced regulatory cells alone does not enable immune monitoring of innovative treatments for autoimmune disorders. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2021 / 028347 [Patent Document 2] International Publication No. 2021 / 028350 [Non-patent literature]

[0011] [Non-Patent Document 1] Wang et al (2011; J Exp Med 208:777) [Non-patent document 2] Lines et al, 2014; Cancer Immunol Res 2: 510 [Non-patent document 3] Kakavand et al, 2017; Modern Pathol 89, doi: 10.1038 / modpathol.2017.89 [Non-patent document 4] Gao et al, 2017; Nat Med 23: 551 [Non-Patent Document 5] Li et al, 2017; Sci Rep 7: 1485 Summary of the Invention [Problem to be solved by the invention]

[0012] It is therefore an object of the present invention to provide novel means that allow for the effective selection of patients suffering from autoimmune disorders such as rheumatoid arthritis and monitoring of treatment success, thereby enabling clinicians to effectively control patient therapy. [Means for solving the problem]

[0013] The problems associated with the prior art described above are solved in a general aspect of the present invention by VISTA protein or RNA as a detectable biomarker indicative of treatment susceptibility and success. In broad terms and as a brief description, the main aspects of the present invention can be described as follows:

[0014] In a first aspect, the present invention provides a method of evaluating a treatment for an autoimmune disorder in a subject undergoing or having undergone the treatment, comprising: (a) providing a biological test sample containing T cells of a subject; (b) (i) determining the expression level of V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or VISTA RNA in T cells of the biological test sample, or (ii) determining the number or percentage of VISTA protein and / or VISTA RNA positive T cells in the biological test sample; (c) comparing the value determined in step (b)(i) and / or step (b)(ii) with a reference; Preferably, the reference for (i) is a predetermined level of VISTA protein or VISTA RNA, or the reference for (i) is a level of VISTA protein or VISTA RNA determined in T cells of a control sample, for example a biological sample comprising T cells of the subject at a time point before treatment or during treatment but before obtaining the biological test sample, and / or Preferably, the reference to (ii) is a predetermined number / percentage of VISTA protein and / or VISTA RNA positive T cells, or the reference to (ii) is the number / percentage of VISTA protein and / or VISTA RNA positive T cells in a control sample, e.g., a biological control sample comprising the subject's T cells, obtained at a time point before treatment or during treatment but before obtaining the biological test sample. The process and Including, An increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective references indicates successful treatment. Regarding the method.

[0015] In a second aspect, the present invention provides a method for assessing whether a subject is susceptible to a treatment for an autoimmune disorder in a subject suffering from an autoimmune disorder and not yet receiving treatment, comprising: (a) providing a biological test sample containing T cells of a subject; (b) (i) determining the expression level of V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or VISTA RNA in T cells of the biological test sample, or (ii) determining the number or percentage of VISTA protein and / or VISTA RNA positive T cells in the biological test sample; (c) comparing the value determined in step (b)(i) and / or step (b)(ii) with a reference; Preferably, the reference for (i) is a predetermined level of VISTA protein or VISTA RNA, or the reference for (i) is a level of VISTA protein or VISTA RNA determined in T cells of a control sample, for example a biological sample comprising T cells of the subject at a time point before treatment or during treatment but before obtaining the biological test sample, and / or Preferably, the reference to (ii) is a predetermined number / percentage of VISTA protein and / or VISTA RNA positive T cells, or the reference to (ii) is the number / percentage of VISTA protein and / or VISTA RNA positive T cells in a control sample, e.g., a biological control sample comprising the subject's T cells, obtained at a time point before treatment or during treatment but before obtaining the biological test sample. The process and Including, An increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective references indicates that the subject is susceptible to treatment. Regarding the method.

[0016] In a third aspect, the present invention relates to compounds or compositions for use in the treatment of an autoimmune disorder in a subject, wherein the compounds and compositions are immunomodulatory and upon administration negatively affect a T cell mediated immune response in the subject, and the treatment further comprises obtaining a biological test sample from the subject at least once before, during, and / or after administration of the compound or composition, and the subject is stratified as being successfully treated or susceptible to treatment by the method of any one of claims 1 to 30 using the biological test sample from the subject.

[0017] The elements of the present invention are described below. While these elements are listed with specific embodiments, it is understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be construed as supporting and encompassing embodiments that combine two or more explicitly described embodiments, or combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, all permutations and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0018] In a first aspect, the present invention provides a method of evaluating a treatment for an autoimmune disorder in a subject undergoing or having undergone the treatment, comprising: (a) providing a biological test sample containing T cells of a subject; (b) (i) determining the expression level of V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or VISTA RNA in T cells of the biological test sample, or (ii) determining the number or percentage of VISTA protein and / or VISTA RNA positive T cells in the biological test sample; (c) comparing the value determined in step (b)(i) and / or step (b)(ii) with a reference; Preferably, the reference for (i) is a predetermined level of VISTA protein or VISTA RNA, or the reference for (i) is a level of VISTA protein or VISTA RNA determined in T cells of a control sample, for example a biological sample comprising T cells of the subject at a time point before treatment or during treatment but before obtaining the biological test sample, and / or Preferably, the reference to (ii) is a predetermined number / percentage of VISTA protein and / or VISTA RNA positive T cells, or the reference to (ii) is the number / percentage of VISTA protein and / or VISTA RNA positive T cells in a control sample, e.g., a biological control sample comprising the subject's T cells, obtained at a time point before treatment or during treatment but before obtaining the biological test sample. The process and Including, An increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective references indicates successful treatment. Regarding the method.

[0019] Thus, the present invention provides the use of VISTA expression (protein or RNA) in T lymphocytes as a quantitative biomarker for treatment-induced regulatory phenotype CD4+ T cells in patients with an (auto)immune disease, namely rheumatoid arthritis (RA). The present invention is based on the identification of VISTA as a biomarker whose upregulation indicates a regulatory phenotype of CD4+ T cells and which can be used to measure the impact of therapeutic intervention with T cell-directed immunomodulatory agents, i.e., protein complexes consisting of MHC (major histocompatibility complex) II proteins and peptides bound in their binding groove for selective T cell receptor (TCR)-mediated activation of subsets of T lymphocytes. This biomarker is useful for predicting responsiveness to treatment and is also a parameter of efficacy in immunomonitoring of initiated therapy.

[0020] Accordingly, in a second aspect, the present invention provides a method for assessing whether a subject is susceptible to a treatment for an autoimmune disorder in a subject suffering from an autoimmune disorder and not yet receiving treatment, comprising: (a) providing a biological test sample containing T cells of a subject; (b) (i) determining the expression level of V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or VISTA RNA in T cells of the biological test sample, or (ii) determining the number or percentage of VISTA protein and / or VISTA RNA positive T cells in the biological test sample; (c) comparing the value determined in step (b)(i) and / or step (b)(ii) with a reference; Preferably, the reference for (i) is a predetermined level of VISTA protein or VISTA RNA, or the reference for (i) is a level of VISTA protein or VISTA RNA determined in T cells of a control sample, for example a biological sample comprising T cells of the subject at a time point before treatment or during treatment but before obtaining the biological test sample, and / or Preferably, the reference to (ii) is a predetermined number / percentage of VISTA protein and / or VISTA RNA positive T cells, or the reference to (ii) is the number / percentage of VISTA protein and / or VISTA RNA positive T cells in a control sample, e.g., a biological control sample comprising the subject's T cells, obtained at a time point before treatment or during treatment but before obtaining the biological test sample. The process and Including, An increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective references indicates that the subject is susceptible to treatment. Regarding the method.

[0021] Information about "V-domain Ig inhibitor of T cell activation" (VISTA) from Non-Patent Document 1 is described above, and "V-set immunoregulatory receptor," or "VSIR" (or "VISTA"), as a protein, is a member of the immunoglobulin superfamily in the context of the present invention. Relevant information about the human VISTA gene can be found in Entrez Gene ID: 64115; HGNC ID: 30085; Genome Coordinates for assembly GRCh38:CM000672.2: Chromosome 10: 71,747,559-71,773,498 reverse strand, and information about the human VISTA protein is available in UniProt: Q9H7M9 (e.g., Entry version of Nov. 2022). In the context of the present invention, the VISTA protein is generally approximately 50 kDa, a type I transmembrane protein, and has one IgV domain. Preferably (e.g., as the human VISTA protein), it comprises the amino acid sequence set forth in SEQ ID NO: 1. With reference to SEQ ID NO: 1, amino acids 1-32 represent the N-terminal signal peptide, amino acids 33-194 form the extracellular domain (SEQ ID NO: 2), amino acids 195-215 form the helical transmembrane (TM) region, and amino acids 216-311 form the cytoplasmic domain. The extracellular domain (ECD) of (human) VISTA forms an Ig-like V-type domain (SEQ ID NO: 3) between amino acids 33 and 168.

[0022] The human VISTA gene is located at chromosome 10q22.1 and has orthologs (e.g., is conserved) in many species, including chimpanzees and other great apes, rhesus monkeys, cynomolgus and green monkeys, marmosets, dogs, pigs, cattle, and mice. In particular, the amino acid sequence of the mouse VISTA protein can be derived under UniProt identifier Q9D659, entry version Nov 2023 (77.2% identical to the human VISTA protein). The closest human paralog to human VISTA is programmed cell death 1 ligand 1, CD274, or PD-L1 (24.8% identical to human VSIR). In some embodiments of the present invention, the term "VISTA" also relates to variants of the human VISTA protein that have an amino acid sequence that is substantially identical to, or has at least 70%, 75%, or 80%, preferably 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., at least 90% or 95% sequence identity) to, the amino acid sequence set forth in SEQ ID NO: 1, as determined, for example, using the algorithms described elsewhere herein, and that (preferably) retain the same or substantially the same biological activity as the respective reference VISTA. Preferred variants of the VISTA protein include sequence variants resulting from sequence polymorphisms and mutations between and within populations of the respective species, relative to the wild-type sequence of VISTA (e.g., SEQ ID NO: 1). A preferred variant of the VISTA protein is the VISTA variant D187E (e.g., relative to SEQ ID NO: 1) (corresponding to variant dbSNP: rs3747869). Depending on the context (unless more specifically indicated), the term VISTA may refer to a VISTA protein (e.g., a VISTA protein as described above) or to an mRNA molecule encoding such a VSIR protein (VISTA RNA or VISTA mRNA).

[0023] In certain embodiments of the invention, VISTA is human VISTA, preferably a protein comprising the amino acid sequence of SEQ ID NO: 1, or a protein having no more than two, no more than four, no more than six or no more than eight amino acid substitutions, insertions or deletions compared to this sequence, for example no more than one, no more than two or no more than three, for example no more than one.

[0024] In the context of VISTA variants, the present invention includes embodiments in which the VISTA variant is a protein comprising an amino acid sequence having at least 80%, 85%, 90%, 92%, 95% or 97% sequence identity (particularly at least 92% or 95% sequence identity) to the sequence of SEQ ID NO: 1.

[0025] In the context of other VISTA variants, the present invention also encompasses embodiments in which the VISTA variant is selected from the group consisting of orthologs (or paralogs) of VISTA. In certain such embodiments, the VISTA variant comprises the extracellular domain (ECD) of a VISTA protein, e.g., the ECD of a human VISTA protein.

[0026] In certain embodiments of the present invention, the extracellular domain (ECD) of VISTA is the ECD of the human VISTA protein, for example, the VISTA of the human VSIR protein (the ECD of the human VSIR (VISTA) protein) is an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3 (preferably SEQ ID NO: 3), or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95% or 97% sequence identity (preferably at least 92% or 95% sequence identity) to these sequences, and / or having no more than two, no more than four, no more than six or no more than eight amino acid substitutions, insertions or deletions compared to these sequences, for example no more than one, no more than two or no more than three, for example no more than one.

[0027] Thus, in a preferred embodiment, the term "VISTA" refers to human VISTA or a VISTA variant as described above. In another embodiment, VISTA may alternatively or additionally be understood to refer to human VISTA.

[0028] In certain embodiments of the invention, detection of VISTA protein or mRNA is performed by analyzing a sample containing an immune cell or multiple immune cells. The term "immune cell" is art-recognized to describe any cell involved in the immune system of an organism, particularly a mammal, such as a human. Leukocytes (white blood cells) are immune cells involved in the innate immune system, while cells of the adaptive immune system are a special type of white blood cell known as lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, while T cells are involved in cell-mediated immune responses. In a preferred embodiment of the invention, the immune cell is a myeloid cell, such as a T cell (e.g., a CD4+ T cell), and in particular (where an increased cell-mediated immune response mediated by T cells is associated with an autoimmune disorder, such as in the context of RA), the T cell may be a cytotoxic T cell (TC, also known as cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD4+ T cell or killer T cell). CTLs are T cells that are involved in killing target cells, infected cells (especially virally infected cells), or otherwise damaged cells, or in the context of pathological inflammatory responses and autoimmune disorders, healthy "self" cells.

[0029] As used herein, the term "biomarker" is intended to encompass substances used as indicators of a biological state, including genes (and the nucleotide sequences of such genes), mRNAs (and the nucleotide sequences of such mRNAs), and proteins (and the amino acid sequences of such proteins). A preferred biomarker of the present invention is VISTA, preferably human VISTA or a variant or fragment thereof. VISTA biomarkers can be detected based on their gene, protein, or RNA.

[0030] In a further preferred embodiment, the method of the present invention may comprise detecting or quantifying the expression level of one or more additional biological markers determined in the biological test sample, for example in step (b). "Additional" biomarkers, in the context of the present invention, are biomarkers that are not human VISTA. The one or more biological markers are selected from CTLA-4, PD-1, Lag-3, Tim-3, TIGIT, FOXP3, FR4 (folate receptor 4), and CD49b (a2 integrin chain), CD73 (ecto-5'-nucleotidase), and interleukin-10.

[0031] Treatment for autoimmune disorders in the context of the invention described herein preferably involves administration of an immunomodulatory agent to a subject suffering from an autoimmune disorder. The terms "treatment" or "treating," as used herein, refer to obtaining beneficial or desired results, including clinical outcomes, in a subject suffering from an autoimmune disorder. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) disease, prevention of disease spread, delay or slowing of disease progression, improvement or alleviation of the condition, reduction in disease recurrence, and remission (whether partial or complete). "Treating" and "treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. "Treating" and "treatment," as used herein, also encompass prophylactic treatment.

[0032] More importantly, the agent administered to the subject is selected from agents that negatively affect T cell-mediated immune responses in the subject. Such agents or treatments can be selected from immunosuppressive agents and / or treatments aimed at inducing regulatory T cells. Treg-enhancing treatments are known in the art and include the drugs rapamycin (type 1 diabetes), metformin (systemic lupus erythematosus), and N-acetylcysteine ​​(systemic lupus erythematosus and multiple sclerosis).

[0033] Other Treg agonist approaches in the art are based on antigen-specific Treg cell therapy and interleukin 2 (IL2). Treg cell enhancement therapy in the context of the present invention is reviewed in Sharabi, A., Tsokos, M., Ding, Y. et al. Nat Rev Drug Discov 17, 823-844 (2018). https: / / doi.org / 10.1038 / nrd.2018.148, which is incorporated herein in its entirety.

[0034] A preferred Treg-enhancing agent of the present invention is a protein or protein complex that binds to a T cell receptor suspected of being expressed on the subject's T cells. For example, the agent is preferably a protein complex comprising a complex of an MHC class II protein or an antigenic peptide-binding derivative or fragment thereof and an antigenic peptide containing an epitope that specifically mediates binding to a T cell receptor (or a paratope on a T cell receptor).

[0035] Thus, the autoimmune disease of choice in the context of the present invention is one that is mediated by T cells in the subject. In such cases, treatment involves the administration of a peptide-MHC protein complex that specifically binds to a T cell receptor expressed on naive T cells or on T cells that regulate, drive, or mediate the autoimmune disease.

[0036] A preferred treatment is the treatment of rheumatoid arthritis, as described in the prior art document WO 02 / 047900. In particular, a composition comprising a recombinant HLA-DR / COL2 peptide complex comprising (a) the extracellular region of the HLA-DR alpha chain comprising at least the alpha 1 domain, (b) the extracellular region of the HLA-DR beta chain comprising at least the beta 1 domain, and (c) a type II collagen peptide (COL2 peptide) optionally fused to the N-terminus of the HLA-DR alpha chain or the HLA-DR beta chain via a linker peptide, preferably fused to the HLA-DR beta chain, wherein the COL2 peptide is selected from the group consisting of AGFKGEQGPKG (SEQ ID NO: 4), AGFKGEQ The present invention also encompasses compositions comprising an amino acid sequence selected from the group consisting of AGFKGEXGPKG (SEQ ID NO: 6), AGFKGXQGPKG (SEQ ID NO: 7), AGFKXEQGPKG (SEQ ID NO: 8), AGFKGEXGPXG (SEQ ID NO: 9), AGFKGXQGPXG (SEQ ID NO: 10), and AGFKXEQGPXG (SEQ ID NO: 11), wherein the HLA-DR / COL2 peptide complex comprises a chondroitin-binding peptide at the C-terminus of a polypeptide comprising an HLA-DR alpha chain and / or an HLA-DR beta chain. In this context, the autoimmune disorder treated with such a complex is preferably selected from chronic inflammatory diseases in human patients, in particular chronic inflammatory joint diseases and / or arthritis. Preferably, the composition used to treat a chronic inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, non-radiographic axial spondyloarthritis, ankylosing spondylitis, juvenile idiopathic arthritis, relapsing polychondritis, systemic lupus erythematosus, Lyme disease, Meniere's disease, autoimmune inner ear disease (AIED), or Still's disease.

[0037] The term "biological test sample" is selected from whole blood, plasma, serum, synovial fluid, saliva, urine, stool, tears, hair, skin, any other bodily fluid, tissue sample (e.g., biopsy material), and cell extracts thereof. Therefore, a "biological test sample" can be selected from any tissue or bodily fluid sample. Examples of tissue samples include (but are not limited to) oral cells, brain samples, skin samples, or organ samples (e.g., liver). The term "bodily fluid" refers to any fluid present in the body, including, but not limited to, blood, plasma, serum, synovial fluid, lymphatic fluid, urine, saliva, or cerebrospinal fluid. If necessary, the biological sample may be obtained by pretreatment, for example, by homogenization or extraction. Such pretreatment can be appropriately selected by those skilled in the art depending on the biological sample to be subjected to the treatment. A preferred biological test sample is a synovial fluid sample.

[0038] The diagnostic / monitoring methods of the present invention, in all aspects or embodiments, may preferably be ex-vivo or in-vitro methods.

[0039] In some embodiments, the biological test sample provided in step (a) is a sample from a subject prior to obtaining the biological test sample, a sample from a subject that has undergone treatment for an autoimmune disorder, or a sample from a subject that is still undergoing treatment for an autoimmune disorder.

[0040] The detection of the presence or absence of the biomarkers of the present invention, or the determination of any amount or level, can be achieved using any known prior art method. Such detection and quantification methods depend on the nature of the biomarker, and therefore vary depending on whether the biomarker (preferably VISTA) is detected based on VISTA protein, VISTA RNA, or any specific genetic change in the VISTA gene region. For example, the level or expression of VISTA protein is determined immunologically using a VISTA-specific antibody, which may be fluorescently labeled or detectable by a fluorescently labeled secondary antibody, or the expression level of VISTA RNA is detected using a polymerase chain reaction (PCR)-based method, such as reverse transcription (RT) PCR, more preferably quantitative RT-PCR (qRT-PCR), or RNA sequencing.

[0041] Generally, in preferred embodiments, immunological detection of VISTA protein can be performed using immunostaining of cell or tissue samples, Western blotting, ELISA, or fluorescence activated cell scanning (FACS). Such immunological methods involve the use of VISTA-specific antibodies.

[0042] As used herein, the term "antibody" can be understood in the broadest sense as any immunoglobulin (Ig) capable of binding to its epitope. Full-length "antibodies" or "immunoglobulins" are generally heterotetrameric glycoproteins of approximately 150 kDa composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an amino-terminal variable domain (VH) followed by three carboxy-terminal constant domains (CH). Each light chain has an N-terminal variable domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) alternating with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to cells or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system. Other forms of antibodies include heavy-chain antibodies, which are composed of only two heavy chains and lack the two light chains typically found in antibodies. Heavy-chain antibodies include hcIgG (IgG-like) antibodies from camelids, such as dromedaries, camels, llamas, and alpacas, and IgNAR antibodies from cartilaginous fish (e.g., sharks). Yet another form of antibody is the single domain antibody (sdAb, also called Nanobody by its developer, Ablynx), which is an antibody fragment consisting of a single monomeric variable antibody domain. Single domain antibodies are generally made from heavy chain antibodies, but may also be derived from conventional antibodies.

[0043] Preferably, antibodies in the context of the present invention are antibodies or antigen-binding fragments thereof that specifically bind to a VISTA protein or VISTA variant protein as described elsewhere herein. Preferably, antibodies of the present invention are capable of specifically binding to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 3, and therefore are capable of facilitating the detection of said protein.

[0044] In some embodiments, the determination and / or diagnosis methods of the present invention may comprise, for example, in a further step, comparing the detected amount (e.g., protein or mRNA) (or its activity) of the applicable biomarker (i.e., VISTA or a variant thereof) with a standard value or cut-off value, wherein a detected amount greater than the standard value or cut-off value indicates a phenotype (or risk of developing a phenotype) associated with the undesirable presence of VISTA-positive T cells (or cells positive for a variant of VISTA), and / or associated with cellular resistance to a cell-mediated immune response in the subject, and / or associated with (e.g., abnormal) expression or activity of VISTA (or a variant) in the subject. Such a standard value or cut-off value may be determined using a control assay, or may be previously determined from one or more values ​​obtained from a test, such as, for example, the assays performed in the Experimental Section herein below, or from a plurality of samples with known phenotypes. For example, a cutoff value for a diagnostic test can be determined by analyzing samples obtained from patients in the context of a controlled clinical trial and determining the cutoff according to the desired (or obtained) sensitivity and / or specificity of the test.

[0045] Examples of methods useful for detecting the applicable biomarker (i.e., VISTA or a variant thereof) (e.g., its presence, absence, or amount) include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), flow cytometry-based assays such as fluorescence-activated cell sorting (FACS), and radioimmunoassays (RIAs), using antibodies (e.g., VISTA-specific antibodies), such as antibodies or antigen-binding fragments thereof, that specifically bind to such applicable biomarker (preferably a VISTA protein or a variant thereof).

[0046] For such methods, monoclonal or polyclonal antibodies can be used. Examples of VISTA monoclonal antibodies are known in the art. The term "polyclonal antibody," as used herein, refers to a mixture of antibodies that are genetically distinct because they have been produced by plasma cells derived from multiple somatic recombination and clonal selection events, and that generally recognize different epitopes of the same antigen.

[0047] Alternatively, the presence of an applicable biomarker (i.e., VISTA or a variant thereof) may be detected by detecting the presence of mRNA encoding such applicable biomarker, or a fragment of such mRNA. Methods for detecting the presence of such mRNA (or fragment) can include PCR (e.g., quantitative RT-PCR), hybridization (e.g., hybridization to an Illumina chip), nucleic acid sequencing, etc. Such methods may involve or include using one or more nucleic acids described herein, such as PCR primers or probes, or hybridization probes, that bind (e.g., specifically) to such mRNA.

[0048] For such detection, determination, or diagnostic applications, antibodies or nucleic acids are typically labeled with a detectable labeling group. Generally, labeling groups fall into various classes depending on the assay in which they are to be detected: a) isotopic labels, which may be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags, etc.). Suitable labeling groups include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 Examples of suitable labeling groups include fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined peptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antibody or nucleic acid via a spacer arm of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used. For example, an antibody or nucleic acid can be labeled with a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags, etc.).

[0049] Thus, in certain embodiments of the detection / diagnostic methods (or kits therefor), the means (e.g., detector) (e.g., antibody or nucleic acid) for detecting the protein or mRNA of the applicable biomarker (e.g., VISTA) is labeled, e.g., coupled to a detectable label. The term "label" or "labeling group" refers to any detectable label, including those described herein.

[0050] In certain embodiments, the detection / diagnosis methods of the present invention involve immunohistochemical (IHC) or immunocytochemical (ICC) assays. The terms "IHC" and "ICC" are art-recognized and encompass techniques used to identify the location of antigen expression dependent on specific epitope-antibody interactions. IHC generally refers to the use of tissue sections, while ICC generally describes the use of cultured cells or cell suspensions and is therefore preferred. In both methods, positive staining is generally visualized using a molecular label (e.g., a label that may be fluorescent or chromogenic). Briefly, the sample is generally fixed to preserve cellular integrity and then incubated with a blocking reagent to prevent nonspecific binding of the antibody. The sample is then generally incubated with a primary antibody (and sometimes a secondary antibody) to visualize the signal for analysis at the microscopic level.

[0051] Accordingly, such embodiments of the detection / diagnostic methods of the invention may comprise preparing a subject IHC or ICC prepared tissue or cells (e.g., present in a biological sample obtained from a subject), wherein detection of antibody binding to an applicable biomarker (i.e., VISTA or a variant thereof) expressed by the tissue or cells of said IHC or ICC preparation preferably indicates (i) susceptibility to (or likelihood of successful treatment for) an autoimmune disease, disorder, or condition associated with enhanced regulatory T cell activity.

[0052] Preferably, antibodies used in such methods (antibodies that specifically bind to VISTA or its variants) are validated. For example, the antibody is validated to (detectably) bind to the applicable biomarker (i.e., VISTA or its variants) expressed by the cells and / or tissues of the validation IHC or ICC preparation, but not to (detectably) bind to a control IHC or ICC preparation of control cells and / or tissues that do not express such applicable biomarker. Preferably, the control cells are cells and / or tissues that have undergone gene knockdown or gene knockout for the applicable biomarker (i.e., VISTA or its variants), and more preferably, the gene knockdown or gene knockout cells and / or tissues have undergone gene knockdown or gene knockout for such applicable biomarker by siRNA or shRNA.

[0053] In a third aspect, the present invention relates to compounds or compositions for use in the treatment of an autoimmune disorder in a subject, wherein the compounds and compositions are immunomodulatory and upon administration negatively affect a T cell mediated immune response in the subject, and the treatment further comprises obtaining a biological test sample from the subject at least once before, during and / or after administration of the compound or composition, and wherein the subject is stratified as being successfully treated or susceptible to treatment by a method according to the first and second aspects described elsewhere herein using the subject's biological test sample.

[0054] In certain embodiments of the third aspect, an elevated level of (i) and / or an increased number / percentage of (ii) compared to the respective references is preferably an indication that the subject should receive further administration of the compound or composition.

[0055] In another embodiment of the third aspect, a decrease in the level of (i) and / or a decrease in the number / percentage of (ii) compared to the respective references is preferably an indication for the subject to receive further administration of the compound or composition.

[0056] Treatments are as described herein above, including the proposed agents and therapies that enhance regulatory T cell function and activity.

[0057] As used herein, the terms "of the invention," "in accordance with the invention," "according to the invention," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0058] As used herein, the term "comprising" is to be interpreted as encompassing both "including" and "consisting of," with both meanings specifically intended, and thus referring to individually disclosed embodiments according to the present invention. As used herein, "and / or" shall be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. For example, "A and / or B" shall be taken as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if individually defined herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision understood by a person skilled in the art to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the specified numerical value of ±20%, ±15%, ±10%, and, for example, ±5%. As will be understood by a person skilled in the art, the specific deviation regarding the numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than man-made or engineered technical effects. As will be understood by one skilled in the art, the specific such deviations regarding the numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than man-made or engineered technical effects. When an indefinite or definite article is used when referring to a singular noun, such as "a," "an," or "the," this includes the plural of that noun unless specifically stated otherwise.

[0059] It will be understood that adapting the teachings of the present invention to a particular problem or environment, and incorporating variations of the present invention or additional features thereto (such as further aspects and embodiments), is within the skill of one of ordinary skill in the art in light of the teachings contained herein.

[0060] Unless otherwise dictated by context, the feature descriptions and definitions presented above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0061] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0062] The drawings show: [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1 shows that a tolerogenic collagen peptide-based vaccine attenuates autoimmune pathologies (CIA, DTH, and CAIA) in a T cell-dependent manner. CIA in vaccinated mice was assessed by the mean clinical score of arthritis severity (left panel) and the incidence of arthritis (percentage of affected mice [right panel]) at different days post-immunization (DPIM). QB mice were immunized with 100 μg of COL2 on day 0 and boosted with 50 μg of COL2 on day 35. On day 7, mice were vaccinated subcutaneously with Aq-peptide complexes using an osmotic pump (n = 10 for Aq-galCOL2, n = 13 for Aq-CLIP). [Figure 2] 10 shows photographs depicting histological examination of the ankle joint of a QB mouse treated with Aq-galCOL2. [Figure 3] Photographs showing histological examination of ankle joints of mice treated with Aq-CLIP, demonstrating cellular infiltration and cartilage / bone destruction. [Figure 4] 1 is a graph showing anti-COL2 IgG serum levels assessed by ELISA on day 35 during CIA. [Figure 5] 1 is a graph showing DTH response (ear swelling) after treatment with Aq-galCOL2 complexes. [Figure 6]Graphs showing the mean clinical score (left panel) and incidence (right panel) of arthritis severity after transfer of T cells from Aq-galCOL2-treated and Aq-CLIP-treated QB mice (n=5 per group). [Figure 7] Figure 1 shows CAIA after transfer of CD4+ T cells from Aq-galCOL2-treated and Aq-CLIP-treated BC mice (n=5 per group). Mice were immunized with COL2 on day 5 and vaccinated with 100 μg of either Aq-galCOL2 or Aq-CLIP using an osmotic pump. On day 15, purified CD4+ T cells were transferred into BQ.Cia9i recipients, who were injected with an arthritogenic CAIA cocktail (M2139 and ACC1 anti-COL2 antibodies) on the same day as T cell transfer. Results are expressed as mean ± SEM. DPIN, days post-injection. [Figure 8] This graph shows that treatment with Aq-galCOL2 complexes reduces the frequency of galCOL2-specific T cells. HCQ3tg mice were immunized with COL2 / CFA and implanted with osmotic pumps loaded with Aq-peptide complexes (2001D, 100 μg) 5 days after immunization. On day 12 after immunization, T cells in the dLN were analyzed by flow cytometry using galCOL2 peptide / Aq tetramer staining. [Figure 9] Figure 1 shows that vaccination with Aq-galCOL2 complexes induces a tolerogenic phenotype in galCOL2-specific CD4+ T cells. Upregulation of Foxp3, CD73, FR4, and PD1 was assessed by either galCOL2 peptide / Aq tetramer staining and immunophenotyping or the congenic marker CD45.1. [Figure 10]Figure 1 shows that BQ.HCQ3tg.Rag1- / - T cells from vaccinated mice suppress anti-CD3 / CD28-induced polyclonal T cell proliferation. GalCOL2-specific CD4+ T cells (suppressor cells) from vaccinated BQ.HCQ3tg.Rag1- / - mice were cocultured in vitro with 1 x 105 purified CD45.2-expressing CFSE-labeled CD4+ T cells (responder cells) from BQ mice at a 1:3 ratio. FACS analysis was performed 3 days after stimulation with anti-CD3 / CD28 antibodies. Quantification (left panel) and representative gating (right panel) are shown. [Figure 11] Figure 1 shows a graph showing the induction of a unique T regulatory phenotype by the Aq-galCOL2 complex in BQ.HCQ3tg.MMCtg mice. BQ.HCQ3tg.MMCtg mice were immunized with COL2 / CFA and implanted with osmotic pumps loaded with Aq-peptide complexes (2001D, 100 μg) 5 days post-immunization. At 12 days post-immunization, splenic gal-COL2 / Aq tetramer CD4 T cells were analyzed for expression of CD49b and LAG3 (markers of the Tr1 phenotype) by flow cytometry (gating on live CD3CD4galCOL2 / Aq tetramer cells). [Figure 12]Volcano plots comparing the proteomic profiles of galCOL2-specific T cells treated with Aq-CLIP and Aq-galCOL2 conjugates. BQ.HCQ.3 tg mice were immunized with COL2 / CFA. Five days after immunization, osmotic pumps (Model 2001D) loaded with 100 μg of either Aq-galCOL2 conjugates (n=3) or control Aq-CLIP conjugates (n=4) were implanted. Ten days after immunization, iLN cells were harvested, and Ag-specific CD4+ T cells were labeled with galCOL2 / Aq tetramers and sorted using a BD FACS Aria flow FACS sorter. Sorted cells were subjected to LC-MS / MS. Proteins involved in T cell tolerance and T cell effector function that were significantly up- or down-regulated and substantially up- or down-regulated are highlighted in blue. Rrgac, Ras-related GTP-binding protein C; Dgka, diacylglycerol kinase alpha; Vista, V-domain immunoglobulin inhibitor of T-cell activation. [Figure 13] Figure 1 shows that vaccination with Aq-galCOL2 resulted in increased expression of VISTA, CD73, FR4, and PD1 in gal-COL2-specific T cells from wt mice. BQ wt mice were immunized with COL2 / CFA and implanted with osmotic pumps loaded with Aq-peptide complexes (2001D, 100 μg, n = 3 per group) 5 days after immunization. Ten days after immunization, splenocytes and dLN cells were harvested, stained with PE-labeled galCOL2 / Aq tetramer, and enriched using anti-PE magnetic beads. Phenotypic analysis of enriched galCOL2-specific CD4+ T cells was performed by flow cytometry (gating on live CD4+galCOL2 / Aq tetramer+ cells). [Figure 14]Figure 1 shows that VISTA is required for the suppressive effect of tolerogenic galCOL2-specific cells. Osmotic pumps (2001D, 100 μg, n=3 per group) loaded with Aq-peptide complexes were implanted subcutaneously into BQ.HCQ3tg mice. Six days after implantation, splenocytes were harvested, CD4+ T cells were purified, and cocultured with purified CFSE-labeled CD4+ T cells from BQ mice in the presence or absence of blocking antibodies targeting VISTA, CD73, or PD1. FACS analysis was performed 3 days after stimulation with anti-CD3 / CD28 antibodies. Quantification (left panel) and representative gating (right panel) are shown. Results are expressed as mean ± SD. [Figure 15]Peripheral blood mononuclear cells (PBMCs) from HLA DRB1*0401 RA patients were seeded in 24-well plates (1 × 10 cells, 1 ml / well) and stimulated with 1% penicillin / streptomycin (Gibco) in TexMACS medium (Milteny Biotec) for 24 hours under the following conditions: For stimulation with the soluble DR4-galCol2 construct, wells were preincubated with 15% FCS / medium for 6 hours and then washed with PBS (Gibco). As a control, PBMCs were stimulated by coating with anti-human CD3 (αCD3, Ultra-LEAF purified anti-human CD3, clone OKT3, BioLegend) and soluble anti-human CD28 (αCD28, 1 μg / ml, LEAF purified anti-human CD28, clone CD28.2, BioLegend). Pre-coating of wells with anti-CD3 antibody was performed by incubating with 1 μg / ml αCD3 (1 ml / well) for 6 hours, followed by washing with PBS (twice). Under control conditions, cells were left untreated. To test for active substances, cells were stimulated with recombinant DRA / DRB1COL2259-274 peptide complex at a concentration of 5 μg / ml in medium. After 24 hours, cells were detached from the plate, transferred to FACS tubes, and rinsed with 1 ml of PBS. The samples were then centrifuged at 1900 rpm for 5 minutes. The cell pellet was incubated with 2 μl of human FcR blocker (Milteny Biotec) for 10 minutes at 4°C. The cells were then washed with 2 ml of PBS. The resuspended cell pellet was stained with 2 μl of anti-human VISTA, FITC (Invitrogen, clone: ​​B7H5DS8), 1 μl of anti-human CD4, BV650 (BioLegend, clone: ​​OKT4), and 0.2 μl of zombie NIR (BioLegend) for 20 min at 4°C in the dark, after which the cells were washed with 2 ml of FACS wash solution (PBS + 0.5% BSA).Finally, CD4+ T cells were then analyzed for VISTA staining by flow cytometry (FACS Fortessa [BD; Franklin Lakes, NJ] in single-cell suspensions using FlowJo Software [Treestar; Ashland, OR]) according to a live / dead marker (Zombie NIR, Biolegend #77184) and gating on CD4+ cells in the live population, corresponding to the Fluorescence Minus One (FMO) control. The top two rows show the specific effect of DR4-galCol2 on the expansion of the VISTA-positive subset of CD4+ T cells for two samples (P203 and P502), as shown in the highlighted area in the upper right quadrant of the panel (including double-positive cells), compared with unstimulated PBMCs or PBMCs activated with anti-CD3 / anti-CD28 antibodies. The bottom row shows the results of stimulation of PMNCs from sample P141. PMNCs from sample P141 did not show upregulation of VISTA in T cells in response to in vitro challenge with the DR4-galCOL2 vaccine. Thus, the data indicate that there is specific upregulation of VISTA in a subset of CD4+ cells in DR4-galCOL2-stimulated cells, although the extent may vary between individual patients. DETAILED DESCRIPTION OF THE INVENTION

[0064] SEQ ID NO: 1 shows the amino acid sequence of human type V immunoglobulin domain-containing T cell activation inhibitor: MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHH SEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVGILCLPLILLLVYKQRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI

[0065] SEQ ID NO: 2 shows the ECD of the human VISTA protein (UniProt identifier Q9H7M9): FKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAA

[0066] SEQ ID NO: 3 shows the Ig-like V-type domain of the human VISTA protein (UniProt identifier Q9H7M9): FKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAMELQV [Example]

[0067] Certain aspects and embodiments of the present invention will now be described, by way of example, with reference to the description, figures, and tables set forth herein. Such examples of methods, uses, and other aspects of the present invention are merely representative and should not be construed as limiting the scope of the present invention to only such representative examples.

[0068] The examples show:

[0069] Example 1:A q -galCOL2 complex reverses autoimmune arthritis In mice, galCOL2 259~273 Peptide-loaded A q Molecule (A q It has previously been shown that two intravenous (iv) injections of A-galCOL2 mediate protection from arthritis. Based on in vitro and in vivo stability experiments (not shown), we conclude that A-galCOL2 mediates protection from arthritis. q To prolong the availability of -galCOL2, we used an osmotic ALZET pump instead of an IV injection. Consistent with previous data, A q Subcutaneous (sc) administration of the -galCOL2 complex not only significantly reduced the severity and incidence of CIA, but also significantly delayed the onset of arthritis (Fig. 1). In this and further in vivo experiments (unless otherwise noted), A released by the osmotic pump was q -CLIP complex served as a negative control.

[0070] At the end of the experiment, arthritis scoring data were confirmed by histological analysis of the paws (Figures 2 and 3). Furthermore, anti-COL2 IgG antibody responses were observed in A. q -galCol2 treatment group q -reduced compared with the CLIP-treated control group (Figure 4).

[0071] Example 2:A q -galCOL2 complex targets antigen-specific T cells Since RA and animal models of RA involve Th1-mediated immunity, q We investigated the potential of -galCOL2 to down-regulate COL2-specific Th1 cell-mediated immune responses. We used a model of delayed-type hypersensitivity (DTH) reaction induced by sensitization and challenge with COL2, in which antigen-specific Th1 cells are required to elicit a hypersensitivity response. Challenge with COL2 significantly increased ear thickness in COL2-sensitized mice, and ear swelling was observed in A. q This was attenuated by administration of -galCOL2 (Fig. 5), indicating that COL2-specific Th1 cells were functionally inactivated.

[0072] Next, adoptive T cell transfer experiments were performed to confirm the A q We tested whether suppressive effects could be transferred from T cells derived from mice treated with -galCOL2. Three groups of QB mice (donor, 5 mice per group) were treated with A in phosphate-buffered saline (PBS). q -galCOL2, A in PBS q -CLIP, or PBS alone was received via osmotic pump on day 0. At the same time (day 0), another group of QB mice (recipients) was immunized with COL2 in complete Freund's adjuvant (CFA). q Seven days after -galCOL2 injection, purified CD4+ T cells from the donor's spleen and draining lymph nodes (dLN) were transferred intravenously into the immunized recipient. q T cells from mice that received the -galCOL2 complex were effective in inducing and preventing the development of arthritis (FIG. 6).

[0073] Finally, A qWe explored whether T cells from mice vaccinated with -galCOL2 could suppress arthritis independently of other T or B cells. Therefore, collagen antibody-induced arthritis (CAIA) was induced by intraperitoneal injection of an arthritis-inducing cocktail consisting of the M2139 monoclonal antibody directed against COL2 and citrullinated COL2, and the ACC1 monoclonal antibody, and the disease was boosted with lipopolysaccharide 6 days after injection. BQ donors were immunized with COL2 and boosted with A 5 days after immunization. q -galCOL2 or A q On day 15 after immunization, CD4+ T cells were purified and transferred intravenously into naive recipients, who were then injected with an arthritogenic antibody cocktail on the same day after T cell transfer. As shown in Figure 7, A q T cells from donors treated with -galCOL2 attenuated the severity of CAIA and delayed the onset of the disease. q -galCOL2 treatment generates regulatory cells from the natural T cell pool that can actively suppress joint inflammation mediated by pathogenic autoantibodies.

[0074] Example 3:A q Treatment with -galCOL2 expands antigen-specific CD4+ regulatory T cells Since RA and animal models of RA involve Th1-mediated immunity, q We investigated the potential of -galCOL2 to down-regulate COL2-specific Th1 cell-mediated immune responses. We used a model of delayed-type hypersensitivity (DTH) reaction induced by sensitization and challenge with COL2, in which antigen-specific Th1 cells are required to elicit a hypersensitivity response. Challenge with COL2 significantly increased ear thickness in COL2-sensitized mice, and ear swelling was observed in A. q This was alleviated by administration of -galCOL2, indicating that COL2-specific Th1 cells were functionally inactivated.

[0075] We then characterized the tolerogenic T cells that mediate the vaccination effect. To assess the phenotype of COL2-specific T cells, we administered glycosylated forms of COL2 in the context of Aq and peptide / MHCII tetramers. 259~273 We used the BQ mouse strain, which expresses a transgenic (tg) αβ TCR (HCQ3tg) that specifically recognizes the peptide. q We found that administration of galCOL2 to BQ.HCQ3tg mice resulted in a decrease in the number of galCOL2-specific T cells (Figure 8). q Expression of cell surface markers associated with a regulatory phenotype (CD73, folate receptor 4 [FR4], programmed death-1 [PD-1], and forkhead box P3 [FoxP3]) was increased in galCOL2-specific T cells from BQ.HCQ3tg mice treated with -galCOL2 (Figure 9). Indeed, the phenotype of these cells was similar to that observed in BQ.HCQ3tg.MMCtg mice. MMCtg mice express the immunodominant T cell epitope of rat / human COL2 (i.e., possess 266E). As a result, MMCtg mice, when immunized with rat COL2, exhibit T cell tolerance and are less susceptible to arthritis compared with non-tolerized wild-type (wt) mice.

[0076] Functional assays were performed to confirm the q We investigated whether COL2-specific T cells treated with -galCOL2 exert a dominant suppressive effect on activated polyclonal T cells. HCQ3tg recombination activating gene 1 (RAG1)-deficient mice, which contain only HCQ3-specific T cells, were cultured in A. q Four days after vaccination, the mice were treated with BQ.HCQ3tg.RAG1 - / -GalCOL2-specific T cells were enriched from mouse spleens and co-cultured with purified carboxyfluorescein N-succinimidyl ester (CFSE)-labeled CD4+ T cells from wild-type BQ mice activated with anti-CD3 and anti-CD28 antibodies. After 3 days of cell culture, polyclonal T cell proliferation was assessed by fluorescence-activated cell sorting (FACS). As shown in Figure 10, A q Tolerogenic galCOL2-specific T cells generated in vivo by administration of -galCOL2 efficiently suppressed the activation and proliferation of non-cognate T cells.

[0077] Notably, A q Administration of the -galCOL2 complex to BQ.HCQ3tg.MMCtg mice induced a Tr1-regulatory phenotype (LAG3 and CD49b double positive) in galCOL2 T cells (Figure 11). q To provide a more systematic characterization of the regulatory phenotype of galCOL2-specific CD4+ T cells elicited by treatment with -galCOL2, we compared the proteomic changes in these cells (Figure 12). q GalCOL2-specific CD4+ T cells from BQ.HCQ.3tg mice treated with galCOL2 / A were analyzed. q The tetramers were selected by FACS and analyzed by liquid chromatography and tandem mass spectrometry (LCMS / MS). qThe anergic phenotype of galCOL2-specific CD4+ T cells from mice vaccinated with -galCOL2 was confirmed (reflected by CD73 and FR4 overexpression and reduced responsiveness to TCR stimulation), revealing overexpression of diacylglycerol kinase α (DGKα), an enzyme crucial for anergy induction by diacylglycerol depletion, and reduced expression of Ras-related GTP-binding protein C (Rragc), a small G protein that mediates a critical step in the activation of the mechanistic target of rapamycin (mTOR) signaling cascade, which has been reported to drive T cell activation and differentiation into Th1, Th2, and Th17 effector T cell lineages. Conversely, inactivation of mTOR in T cells affects their regulatory function, which is also evident in galCOL2-specific T cells. q In response to galCOL2 treatment, q We found enhanced expression of the surface molecule V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), which may critically contribute to the tolerogenic potency of antigen-specific CD4+ T cells treated with -galCOL2.

[0078] VISTA is a potent negative regulator of T cell responses, actively quiescent naive T cells and myeloid cells, inhibiting T cell activation and cytokine production. Furthermore, genetic deficiency of VISTA in T cells reduces inducible Treg differentiation, suggesting that VISTA is required for the generation and stability of inducible Tregs. Immunization with COL2 / CFA and A q Immunophenotyping of antigen-specific CD4+ T cells enriched with galCOL2 peptide-MHCII-tetramers recovered from BQ mice treated with galCOL2 confirmed overexpression of VISTA (Figure 10). Importantly, immunization with COL2 and A q Phenotypic analysis of peptide / MHCII tetramer-enriched galCOL2-specific CD4+ T cells from wt BQ mice vaccinated with -galCOL2 revealed a regulatory T cell phenotype identical to that found in BQ.HCQ.3tg mice (Figure 13).

[0079] Furthermore, treatment with anti-VISTA neutralizing antibodies reduced the suppressive potency of tolerized galCOL2-specific HCQ3tg T cells in an ex vivo suppression assay, suggesting that A q The data show a clear impact of VISTA on the therapeutic efficacy of -galCOL2. In contrast, blocking antibodies against CD73 or PD1 had minimal or no effect, respectively (Figure 14). q This demonstrates that treatment with -galCOL2 generates antigen-specific regulatory T cells. This was observed in both wild-type (D266) and MMC (266E) models, with T cells in the latter model being expressed after immunization of mice with COL2 or A. q -galCOL2 treatment, the cells had a tolerized phenotype.

[0080] Example 4:A q Treatment with -galCOL2 expands antigen-specific CD4+ regulatory T cells Human DR4-galCOL2 construct, DRB1 * We investigated the potential of DR4-galCol2 to induce upregulation of the immunoinhibitory receptor VISTA in CD4+ T cell subsets in peripheral blood mononuclear cells (PBMCs) from RA patients carrying the 0401 allele. The aim of these studies was to address the question of whether VISTA could faithfully mirror the pharmacodynamic effects of DR4-galCol2 on the differentiation of responding T cell subsets in PBMCs toward a tolerogenic phenotype. Blood samples for this study were obtained from RA patients who fulfilled the corresponding American College of Rheumatology 1987 revised RA classification criteria and provided prior written consent to be analyzed as part of a study ethically approved by the Institutional Review Board of the University Hospital Frankfurt. Study patients were genotyped for DRB1 * The patient tested positive for 0401 carriage status.

[0081] As shown in Figure 15, measurement of lymphocyte fractions staining double positive for FITC-labeled anti-CD4 mAbs and VISTA-specific mAbs conjugated with BV650 by fluorescence-activated flow cytometry (FACS) demonstrated upregulation of VISTA in CD4+ lymphocyte subsets by 24 hours of stimulation with the DR4-glCOL2 construct at a concentration of 5 μg / ml. In a pilot study of two samples, a detectable increase in the percentage of VISTA-positive CD4+ T cell subsets was detected and was specifically induced by stimulation with the DR4-galCOL2 construct, but not by the anti-CD3 / anti-CD28 antibody combination, a standard stimulus for T cell activation, or under 24 hours of culture in unstimulated medium, which served as a control. However, in an initial study limited to four samples, we also detected a lack of response of PBMCs to challenge with DR4-galCOL2 with respect to VISTA upregulation in CD4+ T lymphocytes, as exemplified by the results of analysis of sample P141 shown in Figure 15.

[0082] In summary, this study demonstrates that VISTA is a marker that is upregulated on the cell surface of CD4+ T cell subsets in response to challenge with the MHCII-galCOL2 construct in mice (in vivo) and humans (in vitro). Thus, therapeutic vaccine A in mice q We were able to demonstrate the functional role of VISTA in the tolerogenic phenotype induced by each of the mouse variants of -galCOL2. q Transfer of regulatory T cell subsets from donor mice vaccinated with -galCOL2 to naive recipients protected the latter from arthritis induction, and the data suggest that VISTA contributes significantly to the vaccination effect. Parallel studies on human PBMCs demonstrated that A qThis study demonstrates the ability of human DR4-galCOL2, a homologous variant of DR4-galCOL2, to specifically upregulate VISTA in CD4+ T cell subsets in RA patients, a target population for future therapeutic applications. Results indicate that the response is stimulus-specific but variable between individual patients. Thus, VISTA expression in CD4+ T lymphocytes is a marker of the tolerogenic T cell phenotype. This marker can therefore be used in pretreatment in vitro assays to test the individual sensitivity of a patient's PBMCs to various immunomodulatory drugs aimed at inducing regulatory T cells, and can also improve personalized treatment response prediction and treatment stratification. Furthermore, VISTA can also be used as an ex vivo marker to quantify the size of "tolerogenic" CD4+ subsets in immune monitoring during long-term follow-up before and after the initiation of patient treatment to support therapeutic decisions, such as determining the timing of retreatment or the need for treatment modifications. [Explanation of symbols]

[0083] Drawing translation Figure 1 Arthritis score (All animals) Arthritis insidence Incidence of arthritis Figure 4 anti-rCOL2 IgG anti-rCOL2 IgG control Figure 5 ear swelling time (h) time (hour) control Figure 6 Arthritis score (All animals) Arthritis insidence Incidence of arthritis PBS without T cells T cells control T cells A q -galCOL2 T cell A q -galCOL2 Figure 7 Arthritis score (All animals) Incidence of arthritis T cells T cells Figure 8 galCOL2 T cells galCOL2 T cells % of live CD3 / CD4 cells Control Figure 9 FoxP3+ cells FoxP3+ cells % of galCOL2 T cells % of galCOL2 T cells Control Figure 10 % of dividing cells Count Figure 11 % of galCOL2 T cells % of galCOL2 T cells Control Figure 12 log2 fold change -log 10(p.value) -log 10(p.value) Figure 13 % of galCOL2 T cells % of galCOL2 T cells Control Figure 14 % of proliferating cells anti Vista anti-VISTA anti 73 anti-73 anti PD1 anti-PD1 Count Count Figure 15 Unstimulated Unstimulated antiCD3 and antiCD28 anti-CD3 & anti-CD28

Claims

1. 1. A method of evaluating a treatment for an autoimmune disorder in a subject undergoing or having undergone said treatment, comprising: (a) providing a biological test sample comprising T cells from said subject; (b) (i) determining the expression level of V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or VISTA RNA in T cells of the biological test sample, or (ii) determining the number or percentage of VISTA protein and / or VISTA RNA positive T cells in the biological test sample; (c) comparing the value determined in step (b)(i) and / or step (b)(ii) with a reference; Preferably, the reference for (i) is a predetermined level of VISTA protein or VISTA RNA, or the reference for (i) is a level of VISTA protein or VISTA RNA determined in T cells of a control sample, for example a biological sample comprising T cells of said subject at a time point before treatment or during treatment but before said biological test sample is obtained, and / or Preferably, the reference to (ii) is a predetermined number / percentage of VISTA protein and / or VISTA RNA positive T cells, or the reference to (ii) is the number / percentage of VISTA protein and / or VISTA RNA positive T cells in a control sample, for example a biological control sample comprising T cells from the subject, obtained at a time point before treatment or during treatment but before obtaining the biological test sample. The process and Including, An increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective reference indicates successful treatment. method.

2. 1. A method of assessing whether a subject is susceptible to a treatment for an autoimmune disorder in a subject who has an autoimmune disorder and has not yet received said treatment, comprising: (a) providing a biological test sample comprising T cells from said subject; (b) (i) determining the expression level of V-type immunoglobulin domain-containing inhibitor of T-cell activation (VISTA) protein or VISTA RNA in T cells of the biological test sample, or (ii) determining the number or percentage of VISTA protein and / or VISTA RNA positive T cells in the biological test sample; (c) comparing the value determined in step (b)(i) and / or step (b)(ii) with a reference; Preferably, the reference for (i) is a predetermined level of VISTA protein or VISTA RNA, or the reference for (i) is a level of VISTA protein or VISTA RNA determined in T cells of a control sample, for example a biological sample comprising T cells of said subject at a time point before treatment or during treatment but before said biological test sample is obtained, and / or Preferably, the reference to (ii) is a predetermined number / percentage of VISTA protein and / or VISTA RNA positive T cells, or the reference to (ii) is the number / percentage of VISTA protein and / or VISTA RNA positive T cells in a control sample, for example a biological control sample comprising T cells from the subject, obtained at a time point before treatment or during treatment but before obtaining the biological test sample. The process and Including, an increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective references indicates that the subject is susceptible to the treatment; method.

3. 3. The method of claim 1 or 2, wherein the treatment is the administration of an immunomodulatory agent to the subject, preferably wherein the modulating agent negatively affects a T cell-mediated immune response in the subject.

4. 4. The method of claim 3, wherein the modulator is a protein or protein complex that binds to a T cell receptor suspected to be expressed in the subject's T cells, for example, the modulator is a protein complex comprising a complex of an MHC class II protein or an antigenic peptide-binding derivative or fragment thereof and an antigenic peptide comprising an epitope that specifically mediates binding to a T cell receptor (or a paratope on a T cell receptor).

5. The method of any one of claims 1 to 4, wherein the autoimmune disease is mediated by T cells in the subject.

6. 6. The method of any one of claims 1 to 5, wherein the treatment comprises enhancing regulatory T cell function or activity, e.g., by administering a peptide-MHC protein complex that specifically binds to a T cell receptor expressed on naive T cells or T cells that regulate, drive, or mediate an autoimmune disease.

7. 7. The method of any one of claims 1 to 6, wherein the biological test sample is selected from whole blood, plasma, serum, synovial fluid, saliva, urine, stool, tears, hair, skin, any other bodily fluid, tissue sample (e.g., biopsy), and cellular extracts thereof.

8. The method of claim 7, wherein the biological test sample is a synovial fluid sample.

9. A method according to any one of claims 1 to 8, wherein VISTA is a protein having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or is a homologue, ortholog, or paralog of the VISTA protein set forth in SEQ ID NO:

1.

10. A method according to any one of claims 1 to 9, wherein the level or expression of the VISTA protein is determined immunologically, for example using a VISTA-specific antibody which may be fluorescently labeled or which is detectable by a fluorescently labeled secondary antibody, or wherein the expression level of the VISTA RNA is detected using a polymerase chain reaction (PCR)-based method, for example reverse transcription (RT) PCR, more preferably quantitative RT-PCR (qRT-PCR), or RNA sequencing.

11. 11. The method of claim 10, wherein the VISTA protein is determined immunologically using immunostaining, Western blotting, ELISA or fluorescence activated cell scanning (FACS) of a cell or tissue sample.

12. 12. The method of any one of claims 1 to 11, further comprising testing one more biomarker, preferably said one or more biological markers being selected from CTLA-4, PD-1, Lag-3, Tim-3, TIGIT, FOXP3, FR4 (folate receptor 4) and CD49b (a2 integrin chain), CD73 (ecto-5'-nucleotidase) and interleukin-10.

13. 12. Compounds or compositions for use in the treatment of an autoimmune disorder in a subject, wherein the compounds and compositions are immunomodulatory and upon administration negatively affect a T cell mediated immune response in the subject, the treatment further comprising obtaining a biological test sample from the subject at least once before, during, and / or after administration of the compound or composition, wherein the subject is stratified as being successfully treated or susceptible to treatment by the method of any one of claims 1 to 11 using the subject's biological test sample.

14. 14. The compound or composition of claim 13, wherein an increase in the level of (i) and / or an increase in the number / percentage of (ii) compared to the respective references indicates that the subject should receive further administration of the compound or composition.

15. 14. The compound or composition of claim 13, wherein a decrease in the level of (i) and / or a decrease in the number / percentage of (ii) compared to the respective references indicates that the subject should receive further administration of the compound or composition.

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