Treatment of myocarditis and myositis after immune checkpoint therapy

Abstract

Provided herein are method for identifying a subject as having or at risk of having myocarditis and / or myositis based on the detection of the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, and / or on the detection of the level of expression of a TNF signaling via NFκB, an interferon-α signaling, an interferon-γ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling in CD45+ immune cells. The identification of such patients is used in methods of treating a subject having or at risk of having myocarditis and / or myositis, methods of treating cancer, and methods of treating immune checkpoint therapy (ICT)-related myocarditis and / or ICT-related myositis. The methods of treatment include the administration of a therapeutic compound to inhibit IL-1β and TNF.

Description

PATENT ATTORNEY DOCKET NO.: MDA1240-1WO TREATMENT OF MYOCARDITIS AND MYOSITIS AFTER IMMUNE CHECKPOINT THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 533,839, filed August 21, 2023. The disclosure of the prior application is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety. BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0002] The present invention relates generally to immune checkpoint therapy-induced myocarditis and myositis, and more specifically to identification and treatment of a subject having or at risk of having immune checkpoint therapy-induced myocarditis and myositis. BACKGROUND INFORMATION

[0003] Immune checkpoint therapies (ICTs) induce anti-tumor T cell responses and long-term survival in a subset of patients. However, ICTs can also trigger life-threatening autoimmunity, termed immune-related adverse events (irAEs), that often require ICT discontinuation. Immune- related myocarditis, inflammation of the heart, is a rare irAE affecting approximately 1% of patients and carries up to 40% mortality with standard first-line treatment with corticosteroids. This often co-occurs with immune-related myositis (inflammation of skeletal muscle) and myasthenia gravis (MG, antibody-mediated disruption of neuromuscular transmission). The molecular pathways and immune subsets underlying these irAEs are incompletely understood and remain a critical unmet need for developing rational immunosuppressive treatment strategies to ameliorate these toxicities. The first report of fatal immune-related myocarditis and myositis reported intracardiac infiltration of CD4+and CD8+T cells and CD68+macrophages in patient autopsy specimens, with shared T cell receptor (TCR) clones among heart, skeletal muscle, and tumor, suggesting that antigen cross-reactivity may contribute to pathogenesis. Consistent with these findings, Į-myosin-expanded TCRs were identified in cardiac and skeletal muscle of patients with myocarditis, further supporting the role of antigen-specific CD8+T cells 1 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO in disease pathogenesis. Nonetheless, T cells do not function in isolation, requiring interaction of TCRs with MHC-bound peptide antigens expressed on antigen-presenting cells (APCs) and costimulatory signals for optimal activation, differentiation, and proliferation. Myeloid cells, which encompass diverse cell types including macrophages and dendritic cells (DCs) that can fulfill these functions, have previously been shown to contribute to irAEs such as colitis and arthritis. However, their role in myocarditis and myositis remains undefined, despite their significant presence in inflamed muscle tissues.

[0004] Provided herein, is the characterization of the cardiac and skeletal muscle tissue microenvironments obtained utilizing single-cell transcriptomics in living patients with clinically-proven severe immune-related myocarditis and myositis, as well as the comparative analyses performed on similar tissues from patients who were clinically ruled out for immune- related myocarditis and myositis to dissect the effects of ICT exposure, complemented by anatomic histopathologic studies and evaluation of serum immunoglobulins. Optimization of biopsy techniques allowed the safe and rapid collection of fresh heart and skeletal muscle tissues in living, severely ill patients early in their disease course. Such analyses lead to the discovery of candidate molecular pathways and immune subpopulations in immune-related myocarditis and myositis as targets for developing biologically rational immunosuppressive therapeutic strategies. SUMMARY OF THE INVENTION

[0005] The present invention is based on the seminal discovery that during immune checkpoint therapy-induced myocarditis and / or myositis, muscles are infiltrated with increased levels of CD45+ immune cells characterized by a TNF and IL-1ȕ signaling signature, two pathways that can be targeted to identify patients having or at risk of having ICT-induced myocarditis and / or myositis, as well as to treat patients having ICT-induced myocarditis and / or myositis and cancer. In illustrative examples herein, the TNF is TNF-alpha.

[0006] In one embodiment, the present invention provides a method for identifying a subject as having or at risk of having myocarditis and / or myositis including detecting in a sample from the subject a level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of the cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of the subject 2 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO having or at risk of having myocarditis and / or myositis, thereby identifying a subject as having or at risk of having myocarditis and / or myositis.

[0007] In one aspect, the subject has or is at risk of having myocarditis and myositis. In another aspect. the subject has previously received immunotherapy. In some aspects, the subject has previously received immune checkpoint therapy (ICT). In another aspect, the myocarditis and / or myositis is ICT-induced myocarditis and / or ICT-induced myositis. In one aspect, the sample is selected from a muscle biopsy, a blood sample, or a serum sample. In various aspects, the muscle biopsy is an endomyocardial biopsy or a quadricep biopsy. In many aspects, the muscle biopsy is a needle biopsy. In some aspects, the muscle is a cardiac or a skeletal muscle. In other aspects, the cytotoxic CD8+ T cells are clonally expanded activated cytotoxic CD8+ T cells. In other aspects, the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myocarditis and / or myositis express higher levels of LAG3, TIGIT, PDCD1, HAVCR2, TOX, GZMB, GNLY, NKG7, CST7, CCL5, CCL4 and / or CCL4L2 than the cytotoxic CD8+ T-cells in a sample from a subject not having or not at risk of having myocarditis and / or myositis. In one aspect, the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myocarditis are characterized by an increased TNF signaling via NF^B signature, IL2 / STAT5 signature and / or hypoxia signature. In another aspect, the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myositis are characterized by an increased interferon-Į signature. In one aspect, the inflammatory myeloid cells in the sample from a subject having or at risk of having myocarditis and / or myositis express higher levels of TGFB1, HIF1A, CXCL2, CXCL3 and / or CXCL8 than the inflammatory myeloid cells in a sample from a subject not having or not at risk of having myocarditis and / or myositis. In another aspect, the inflammatory myeloid cells in the sample from a subject having or at risk of having myocarditis and / or myositis are characterized by an increased TNF signaling via NF^B signature, interferon-Į signature, interferon-Ȗ signature and / or a complement signature. In one aspect, the inflammatory myeloid cells express IL-1ȕ and / or TNF in higher levels than myeloid cells in a subject not having or at risk of having myocarditis and / or myositis. In another aspect, the inflammatory myeloid cells express C1QA, C1QB, C1QC and / or C2 at higher levels than myeloid cells in a subject not having or at risk of having myocarditis and / or myositis. In some aspects, the inflammatory myeloid cells are CD68+ cells. In various aspects, the inflammatory myeloid cells are IL-1ȕ+TNF+ myeloid cells. In other aspects, an 3 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO increased level of inflammatory myeloid cells is associated with increased serum troponin T level in the subject. In one aspect, frequency of IL-1ȕ+TNF+ myeloid cells in the sample in indicative of the subject’s overall survival and / or clinical outcome. In some aspects, an increased frequency of IL-1ȕ+TNF+ myeloid cells is indicative of a shortened overall survival of the subject, and / or with an increased risk to develop major adverse cardiovascular event. In another aspect, serum titers of autoantibodies against muscle antigens are increased in subject having or at risk of having myocarditis and / or myositis as compared to serum titers of autoantibodies against muscle antigens in subject not having or not being at risk of having myocarditis and / or myositis. In one aspect, identifying includes performing single-cell transcriptomics analysis, anatomic histopathology analysis and / or evaluation of serum immunoglobulins. In another aspect, the method further includes administering to the subject identified as having or at risk of having myocarditis and / or myositis, a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT. In some aspects, the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug.

[0008] In another embodiment, the invention provides a method for identifying a subject as having or at risk of having myocarditis and / or myositis including detecting in CD45+ immune cells in a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon- Į signaling, an interferon-Ȗ signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling in CD45+ immune cells from a sample obtained from a control subject that is not having or at risk of having myocarditis and / or myositis, wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, IL2 / STAT5 signaling, hypoxia signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, an IL2 / STAT5 signaling, hypoxia signaling and / or complement signaling in CD45+ immune cells in a sample obtained from a control subject is indicative of the subject having or at risk of having myocarditis and / or myositis, thereby identifying a subject as having or at risk of having myocarditis and / or myositis.

[0009] In one aspect, CD45+ immune cells include cytotoxic CD8+ T-cells and / or inflammatory myeloid cells. In another aspect, the method further includes administering to the subject identified as having or at risk of having myocarditis and / or myositis, a therapeutic 4 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO compound to inhibit IL-1ȕ, TNF and / or JAK / STAT. In some aspects, the therapeutic compound that inhibits IL-1ȕ, TNF or JAK / STAT is an antibody or small molecule drug.

[0010] In an additional embodiment, the invention provides a method of treating a subject having or at risk of having myocarditis and / or myositis including administering to the subject a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT, wherein the subject has previously received immunotherapy or immune checkpoint therapy, thereby treating a subject having or at risk of having myocarditis and / or myositis.

[0011] In one aspect, the subject is identified as having or at risk of having myocarditis and / or myositis by a method including: detecting in a sample from the subject the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of the cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of the subject having or at risk of having myocarditis and / or myositis.

[0012] In yet another embodiment, the invention provides a method of treating cancer in a subject including a) administering to the subject an immune checkpoint therapy (ICT) and optionally an anti-cancer treatment, b) identifying a risk for the subject to develop ICT-related myocarditis and / or ICT-related myositis, and c) administering to the subject identified as having or at risk of developing ICT-related myocarditis and / or immune-related myositis in b) a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT in combination with the ICT and optionally the anti-cancer treatment, thereby treating cancer in the subject.

[0013] In one aspect, identifying a risk for the subject to develop ICT-related myocarditis and / or ICT-related myositis includes detecting in a sample from the subject the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of the cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of a subject having or at risk of having myocarditis and / or myositis; and / or detecting in CD45+ immune cells from a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling and / or a complement signaling in CD45+ immune cells in a sample obtained from a control subject that is not having or 5 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO at risk of having myocarditis and / or myositis, wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon- Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling in CD45+ immune cells in a sample obtained from a control subject is indicative of the subject having or at risk of having myocarditis and / or myositis.

[0014] In one embodiment, the invention provides a method of treating immune checkpoint therapy (ICT)-related myocarditis and / or ICT-related myositis in a subject including a) identifying the myocarditis and / or myositis as ICT-related myocarditis and / or ICT-related myositis, and b) administering to the subject identified in a) as having ICT-related myocarditis and / or ICT-related myositis a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT in combination with the ICT, thereby treating ICT-related myocarditis and / or ICT-related myositis in a subject.

[0015] In some aspects, the subject has cancer. In various aspects, the cancer is selected from, but not limited to, metastatic melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, malignant pleural mesothelioma, esophageal squamous carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, small cell lung cancer, esophageal carcinoma, gastric cancer (or gastroesophageal junction cancer), bladder cancer, breast cancer, lung, brain, stomach, colon or rectal cancer, liver, pancreatic cancer, prostate cancer, leukemia, melanoma, ovarian cancer, renal or urothelial carcinoma. In some aspects, the subject is a human. In various aspects, the subject has previously received an immune checkpoint therapy (ICT). In some aspects, the ICT is selected from the group consisting of anti-CTLA-4, anti-PD-L1, anti-PD-1, anti-LAG3 monotherapy and a combination thereof. In some aspects, the ICT is selected from but not limited to ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, spartalizumab and a combination thereof. In some aspects, the IL-1ȕ inhibitor is rilonacept, canakinumab or glyburide. In other aspects. the TNF inhibitor is infliximab, adalimumab, etanercept, golimumab, certolizumab, thalidomide or a combination thereof. In other aspects, the JAK / STAT inhibitor is ruxolitinib. 6 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGs 1A-1C illustrate the clinical characteristics of patients with confirmed toxicities. FIG.1A is a graph representing the distribution of irAE cases. FIG.1B is a bar graph representing serum troponin T measurements in confirmed irAE cases compared to controls. FIG.1C is a graph bar representing serum creatine kinase (CK) measurements in confirmed irAE cases compared to controls.

[0017] FIG. 2 is a schematic representation of the overall cellular landscape of immune checkpoint therapy (ICT)-induced myocarditis and myositis. It shows an overview of enrolled patients and collected specimens.

[0018] FIGs 3A-3P illustrate the analysis of CD45- subpopulations in cardiac muscle. FIG.3A is a bar graph illustrating the frequency of CD45- fibroblasts in immune-related myocarditis versus control cardiac muscle tissues. FIG. 3B is a bar graph illustrating the frequency of CD45- endothelial cells in immune-related myocarditis versus control cardiac muscle tissues. FIG.3C is a bar graph illustrating the frequency of CD45- cardiomyocytes in immune-related myocarditis versus control cardiac muscle tissues. FIG.3D is a bar graph illustrating the frequency of CD45- pericyte / smooth muscle cells in immune-related myocarditis versus control cardiac muscle tissues. FIG. 3E is a bar graph illustrating the frequency of CD45- undefined cells in immune-related myocarditis versus control cardiac muscle tissues. FIG.3F is a bar graph illustrating the frequency of fibroblast C1 in cardiac muscle. FIG.3G is a bar graph illustrating the frequency of fibroblast C2 in cardiac muscle. FIG.3H is a bar graph illustrating the frequency of fibroblast C3 in cardiac muscle. FIG.3I is a bar graph illustrating the frequency of fibroblast C4 in cardiac muscle. FIG. 3J is a bar graph illustrating the frequency of endothelial C1 in cardiac muscle. FIG.3K is a bar graph illustrating the frequency of endothelial C2 in cardiac muscle. FIG. 3L is a bar graph illustrating the frequency of endothelial C3 in cardiac muscle. FIG.3M is a bar graph illustrating the frequency of endothelial C4 in cardiac muscle. FIG.3N is a bar graph illustrating the frequency of endothelial C5 in cardiac muscle. FIG.3O is a bar graph illustrating the frequency of endothelial C6 in cardiac muscle. FIG.3P is a bar graph illustrating the frequency of endothelial C7 in cardiac muscle.

[0019] FIGs 4A-4L illustrate the analysis of CD45- subpopulations in cardiac muscle. FIG. 4A is a bar graph illustrating the frequency of CD45- skeletal muscle cells in immune-related 7 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO myositis versus control skeletal muscle tissues. FIG.4B is a bar graph illustrating the frequency of CD45- FAP cells in immune-related myositis versus control skeletal muscle tissues. FIG. 4C is a bar graph illustrating the frequency of CD45- endothelial cells in immune-related myositis versus control skeletal muscle tissues. FIG.4D is a bar graph illustrating the frequency of CD45- pericyte / smooth muscle cells in immune-related myositis versus control skeletal muscle tissues. FIG. 4E is a bar graph illustrating the frequency of CD45- satellite cells in immune-related myositis versus control skeletal muscle tissues. FIG.4F is a bar graph illustrating the frequency of CD45- fibro adipogenic precursor (FAP) cell subset C1 in immune-related myositis versus control skeletal muscle tissues. FIG.4G is a bar graph illustrating the frequency of CD45- fibro adipogenic precursor (FAP) cell subset C2 in immune-related myositis versus control skeletal muscle tissues. FIG. 4H is a bar graph illustrating the frequency of CD45- fibro adipogenic precursor (FAP) cell subset C3 in immune-related myositis versus control skeletal muscle tissues. FIG.4I is a bar graph illustrating the frequency of CD45- fibro adipogenic precursor (FAP) cell subset C4 in immune-related myositis versus control skeletal muscle tissues. FIG.4J is a bar graph illustrating the frequency of CD45- endothelial cells subset C1 in immune-related myositis versus control skeletal muscle tissues. FIG. 4K is a bar graph illustrating the frequency of CD45- endothelial cells subset C2 in immune-related myositis versus control skeletal muscle tissues. FIG. 4L is a bar graph illustrating the frequency of CD45- endothelial cells subset C3 in immune-related myositis versus control skeletal muscle tissues.

[0020] FIGs 5A-5J illustrate that analysis of total CD45+ immune cell populations. FIG.5A is a bar graph illustrating the frequency of CD45+ cells in cardiac muscle from patients with myocarditis compared with controls. FIG.5B is a bar graph illustrating the frequency of CD45+ total T cells in cardiac muscle from patients with myocarditis compared with controls. FIG.5C is a bar graph illustrating the frequency of CD45+ total NK cells in cardiac muscle from patients with myocarditis compared with controls. FIG. 5D is a bar graph illustrating the frequency of CD45+ total B cells in cardiac muscle from patients with myocarditis compared with controls. FIG.5E is a bar graph illustrating the frequency of CD45+ total myeloid cells in cardiac muscle from patients with myocarditis compared with controls. FIG. 5F is a bar graph illustrating the frequency of CD45+ cells in skeletal muscle from patients with myositis compared with controls. FIG.5G is a bar graph illustrating the frequency of CD45+ total T cells in skeletal muscle from 8 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO patients with myositis compared with controls. FIG.5H is a bar graph illustrating the frequency of CD45+ total NK cells in skeletal muscle from patients with myositis compared with controls. FIG. 5I is a bar graph illustrating the frequency of CD45+ total B cells in skeletal muscle from patients with myositis compared with controls. FIG.5J is a bar graph illustrating the frequency of CD45+ total myeloid cells in skeletal muscle from patients with myositis compared with controls.

[0021] FIG. 6 shows the top upregulated (left) and downregulated (right) differentially expressed genes (DEG) in CD45+ cells in myocarditis versus myositis samples.

[0022] FIGs 7A-7B illustrate the top DEGs in CD45+ cells. FIG. 7A is a volcano plot illustrating the differentially expressed genes in CD45+ immune cells in cardiac muscle. FIG.7B is a volcano plot illustrating the differentially expressed genes in CD45+ immune cells in skeletal muscle.

[0023] FIGs 8A-8B illustrate immune cell subsets in ICT-induced myocarditis and myositis. FIG. 8A is a bar graph illustrating comparison of individual cluster frequency in myocarditis versus control samples. FIG. 8B is a bar graph illustrating comparison of individual cluster frequency in myositis versus control samples ranked by Log2FC.

[0024] FIGs 9A-9B illustrate that cytotoxic CD8+ T cells expressing activation markers are enriched in myocarditis and myositis. FIG. 9A is a bar graph illustrating individual T cell subclusters present in differential frequencies in myocarditis versus control cardiac muscle specimens. FIG.9B is a bar graph illustrating individual T cell subclusters present in differential frequencies in myositis versus control skeletal muscle specimens.

[0025] FIG. 10 is a bar graph illustrating a comparison of cytotoxic CD8+ T cell subset in patient-matched myocarditis and myositis tissues by showing the frequency of cytotoxic CD8+ T cell subpopulations in patient-matched myocarditis versus myositis samples.

[0026] FIGs 11A-11B illustrate that IL-1B+TNF+ myeloid cells are specifically enriched in ICT-induced myocarditis. FIG. 11A shows bar graphs illustrating the frequency of individual myeloid cell subclusters in myocarditis versus control cardiac muscle specimens. FIG. 11B is a bar graph illustrating the frequency of individual myeloid cell subclusters in myositis versus control skeletal muscle specimens. 9 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0027] FIG. 12 illustrates that differentially expressed genes (DEGs) in IL1B+ Myeloidcell subsets and association of frequency of cytotoxic CD8+ T cells with clinical outcomes. The graph shows the association of frequency of cytotoxic CD8+ T cells with clinical outcomes.

[0028] FIGs 13A-13D illustrate that IL-1B+TNF+ myeloid cells are associated with poorer clinical outcomes. FIG.13A is a bar graph illustrating the frequency of IL1B+TNF+ myeloid cell subpopulations in patient-matched myocarditis versus myositis samples. FIG. 13B is a graph illustrating the association of frequency of IL1B+TNF+ myeloid cells with serum troponin T. FIG. 13C is a graph illustrating the association of frequency of IL1B+TNF+ myeloid cells with clinical outcomes. FIG. 13D is a graph illustrating the overall survival stratified by frequency of IL1B+TNF+ myeloid cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is based on the seminal discovery that during immune checkpoint therapy-induced myocarditis and / or myositis, muscles are infiltrated with increased levels of CD45+ immune cells characterized by a TNF and IL-1ȕ signaling signature, two pathways that can be targeted to identify patients having or at risk of having ICT-induced myocarditis and / or myositis, as well as to treat patients having ICT-induced myocarditis and / or myositis and cancer.

[0030] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0031] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0032] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. 10 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0033] As used herein, the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated. For example, and based on the context, the value can vary up or down by 5-10%. For example, for a value of about 100, means 90 to 110 (or any value between 90 and 110).

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0036] In one embodiment, the present invention provides a method for identifying a subject as having or at risk of having myocarditis and / or myositis including detecting in a sample from the subject a level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of the cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of the subject having or at risk of having myocarditis and / or myositis, thereby identifying a subject as having or at risk of having myocarditis and / or myositis.

[0037] Myocarditis, or inflammatory cardiomyopathy, is an acquired cardiomyopathy due to inflammation of the heart muscle. Symptoms can include, by way of example, shortness of breath, chest pain, decreased ability to exercise, advanced atrioventricular heart block, ventricular tachycardia, ventricular fibrillation, sudden cardiac death and an irregular heartbeat. The duration of problems can vary from hours to months. Complications may include heart failure due to dilated cardiomyopathy or cardiac arrest. Myocarditis is often due to a viral infection, but other causes include bacterial infections, certain medications, toxins, and autoimmune disorders. Treatment depends on both the severity and the cause, medications such as ACE inhibitors, beta blockers, and diuretics are often used. Corticosteroids or intravenous immunoglobulin (IVIG) may also be 11 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO useful in certain cases. In severe cases an implantable cardiac defibrillator or heart transplant may be recommended.

[0038] Myositis is a rare disease that involves inflammation of the muscles. It can present with a variety of symptoms such as skin involvement (i.e., rashes), muscle weakness, and other organ involvement. Systemic symptoms such as weight loss, fatigue, and low fever can also present. There are various causes for myositis including injury (a mild form of myositis can occur with hard exercise. A more severe form of muscle injury, called rhabdomyolysis, is also associated with myositis, this is a condition where injury to the patient's muscles causes them to quickly break down), medicines (a variety of different medicines can cause myositis, including statins), infection (the most common infectious cause of myositis is viral infections, such as the common cold), or an autoimmune disorder (in case of abnormal immune response to a functioning body part, in this case the muscles).

[0039] In one aspect, the subject has or is at risk of having myocarditis and myositis.

[0040] The present invention is based on the discovery that myocarditis and myositis are characterized by an infiltration of a specific subset of immune cell in the inflamed muscles. The invention therefore provides a method of identifying subject having or at risk of having myocarditis and / or myositis including detecting in a sample from the subject the level of one or more of these immune cells. By “level of immune cells” it is meant that the number, or percent of immune cells in the sample is compared to a number, percent or level in a sample obtained from a control sample. A control sample can be a sample obtained from any subject that is known not to have and not to be at risk of having myocarditis and / or myositis.

[0041] The terms “immunoreactive cell” or “immune cells” in the context of the present invention relate to a cell which has been identified as being present at increased levels in subject having or at risk of having myocarditis and / or myositis. An “immune cell” preferably is a CD45+ cell, including cytotoxic CD8+ T-cells, inflammatory myeloid cells and / or plasma B cells.

[0042] A cytotoxic T cell, also referred to as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell is a T lymphocyte that kills cancer cells, cells that are infected by intracellular pathogens (such as viruses or bacteria), or cells that are damaged in other ways. Most cytotoxic T cells express T-cell receptors (TCRs) that can recognize a specific antigen (a molecule capable of stimulating an immune response that is often produced by cancer cells, 12 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO viruses, bacteria, or intracellular signals). In the context of the present invention, CD8+ T-cells that are present at higher levels in subject having or at risk of having myocarditis and / or myositis have been characterized and found to display a specific signature of genes expression.

[0043] In various aspect, the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myocarditis and / or myositis express higher levels of LAG3, TIGIT, GZMB, PDCD1, HAVCR2, TOX, GNLY, NKG7, CST7, CCL5, CCL4 and / or CCL4L2 than cytotoxic CD8+ T-cells in a sample from a subject not having or not at risk of having myocarditis and / or myositis. In other aspects, the cytotoxic CD8+ T cells are clonally expanded activated cytotoxic CD8+ T cells.

[0044] In other aspects, the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myocarditis are characterized by an increased TNF signaling via NF^B signature, an interferon-Į, IL2 / STAT5 signature and / or hypoxia signature.

[0045] The tumor necrosis factor (TNF) superfamily refers to a superfamily of cytokines that can cause cell death. All TNF superfamily members form homotrimeric (or heterotrimeric in the case of LT-alpha / beta) complexes that are recognized by their specific receptors. Examples of TNF super family members include TNF-Į, TNF-ȕ, lymphotoxin-alpha, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L and TRAIL.

[0046] Interleukin-2 (IL-2) is a cytokine signaling molecule that regulates the activities of white blood cells (leukocytes, often lymphocytes). IL-2 is part of the natural response to microbial infection, and in discriminating between foreign ("non-self") and "self". IL-2 mediates its effects by binding to IL-2 receptors, expressed by lymphocytes. The major sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells.

[0047] IL-2 transduction can take place via 3 different signaling pathway: (1) the JAK-STAT pathway, (2) the PI3K / Akt / mTOR pathway and (3) the MAPK / ERK pathway. IL-2 binding to its receptor leads to the activation of Janus kinases JAK1 and JAK3 which subsequently phosphorylate T338 on CD122. This phosphorylation recruits STAT transcription factors, predominantly STAT5, which dimerize and migrate to the cell nucleus where they bind to DNA.

[0048] IL2 is a critical immunomodulatory cytokine that both positively and negatively affects the differentiation of individual Th cell subsets. IL-2 signals are propagated, in part, via activation of STAT5, which functions as a key regulator of CD4+ T cell gene programs. 13 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0049] Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Examples of IFNs include IFN-Į, IFN-ȕ, IFN-İ, IFN-^ and IFN-Ȗ.

[0050] A myeloid cell is a type of blood cell that originates in the bone marrow. As a myeloid cell matures into an adult blood cell, it will take on a specific role as a basophil, eosinophil, erythrocyte, macrophage, monocyte, neutrophil, or platelet. In the context of the present invention, inflammatory myeloid cells that are present at higher levels in subject having or at risk of having myocarditis and / or myositis have been characterized and found to display a specific signature of gene expression.

[0051] In one aspect, the inflammatory myeloid cells in the sample from a subject having or at risk of having myocarditis and / or myositis express higher levels of TGFB1, HIF1A, CXCL2, CXCL3 and / or CXCL8 than the inflammatory myeloid cells in a sample from a subject not having or not at risk of having myocarditis and / or myositis.

[0052] In another aspect, the inflammatory myeloid cells in the sample from a subject having or at risk of having myocarditis and / or myositis are characterized by an increased TNF signaling via NF^B signature, interferon-Į signature, interferon-Ȗ signature and / or complement signature.

[0053] The complement system, also known as complement cascade, is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen's cell membrane. It is part of the innate immune system, which is not adaptable and does not change during an individual's lifetime. The complement system can, however, be recruited and brought into action by antibodies generated by the adaptive immune system.

[0054] In one aspect, the inflammatory myeloid cells express IL-1ȕ and / or TNF in higher levels than myeloid cells in a subject not having or at risk of having myocarditis and / or myositis. 14 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0055] In another aspect, the inflammatory myeloid cells express C1QA, C1QB, C1QC and / or C2 at higher levels than myeloid cells in a subject not having or at risk of having myocarditis and / or myositis.

[0056] In some aspects, the inflammatory myeloid cells are CD68+ cells.

[0057] In various aspects, the inflammatory myeloid cells are IL-1ȕ+TNF+ myeloid cells.

[0058] In other aspects, an increased level of inflammatory myeloid cells is associated with increased serum troponin T level in the subject. In one aspect, frequency of IL-1ȕ+TNF+ myeloid cells in the sample in indicative of the subject’s overall survival and / or clinical outcome. In some aspects, an increased frequency of IL-1ȕ+TNF+ myeloid cells is indicative of a shortened overall survival of the subject, and / or with an increased risk to develop major adverse cardiovascular event.

[0059] The present invention is based on the discovery that levels of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that are higher than the levels in a subject not having or at risk of having myocarditis and / or myositis is indicative of a subject having or at risk of having myocarditis and / or myositis, and subject with such higher levels of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells are identified as having or at risk of having myocarditis and / or myositis. Consequently, subjects that present levels of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that are equivalent, or lower than the levels in a subject not having or at risk of having myocarditis and / or myositis are identified as not having and at risk of having myocarditis and / or myositis.

[0060] In one aspect, the subject has previously received immunotherapy.

[0061] The term “immunotherapy” refers to any type of therapy that includes modulating the immune system or the immune response. Modulating the immune system includes inducing, stimulating, or enhancing the immune system as well as reducing, suppressing, or inhibiting the immune system. Immunotherapy can be active or passive. Passive immunotherapy relies on the administration of monoclonal antibodies directed against the target to eliminate. For example, tumor-targeted monoclonal antibodies have demonstrated clinical efficacy to treat cancer. Active immunotherapy aims to induce a cellular immunity and establish immunological memory against the target agent. Active immunotherapy includes but is not limited to vaccination, immune modulators. 15 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0062] In some aspects, the subject has previously received immune checkpoint therapy.

[0063] “Immune checkpoint therapy (ICT),” or “checkpoint inhibitor therapy” is a form of cancer treatment that uses immune checkpoints which affect immune system functioning. Immune checkpoints can be stimulatory or inhibitory. Tumors can use these checkpoints to protect themselves from immune system attacks. Checkpoint therapy can block inhibitory checkpoints, thereby restoring immune system function. Checkpoint proteins include, but are not limited to, programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), A2AR (Adenosine A2A receptor), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (B and T Lymphocyte Attenuator, or CD272), IDO (Indoleamine 2,3-dioxygenase), KIR (Killer-cell Immunoglobulin- like Receptor), LAG3 (Lymphocyte Activation Gene-3), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), and VISTA (V-domain Ig suppressor of T cell activation).

[0064] In some aspects, the ICT is selected from the group consisting of anti-CTLA-4, anti-PD- L1, anti-PD-1, anti-LAG3 monotherapy and a combination thereof.

[0065] CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that, functioning as an immune checkpoint, downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation - a phenomenon which is particularly notable in cancers. CTLA4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits an inhibitory signal to T cells. CTLA4 is homologous to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA-4 binds CD80 and CD86 with greater affinity and avidity than CD28 thus enabling it to outcompete CD28 for its ligands. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. CTLA4 is also found in regulatory T cells and contributes to its inhibitory function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4.

[0066] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a cell surface receptor that plays an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune checkpoint and guards against autoimmunity through a dual mechanism of 16 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO promoting apoptosis (programmed cell death) in antigen-specific T-cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).

[0067] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DC) in response to LPS and GM-CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling, whereas in resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD-L1 is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma upon treatment with IFN-Ȗ. PD-L2 expression is more restricted and is expressed mainly by DCs and a few tumor lines.

[0068] Lymphocyte-activation gene 3, also known as LAG-3 was designated CD223 (cluster of differentiation 223); it is a cell surface molecule with diverse biologic effects on T cell function. It is an immune checkpoint receptor and as such is the target of various drug development programs by pharmaceutical companies seeking to develop new treatments for cancer and autoimmune disorders. In soluble form it is also being developed as a cancer drug in its own right. LAG-3 may be a better checkpoint inhibitor target than CTLA-4 or PD-1 since antibodies to these two checkpoints only activate effector T cells, and do not inhibit Treg activity, whereas an antagonist LAG-3 antibody can both activate T effector cells (by downregulating the LAG-3 inhibiting signal into pre-activated LAG-3+ cells) and inhibit induced (i.e., antigen-specific) Treg suppressive activity. Combination therapies are also ongoing involving LAG-3 antibodies and CTLA-4 or PD- 1 antibodies.

[0069] In many aspects, the ICT is selected from, by way of example, relatlimab, ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, spartalizumab and a combination thereof.

[0070] Depending on the subject and the cancer type affecting the subject, the cancer treatment may involve the use of ICT, which can be used alone (e.g., one ICT) or in combination with one another (e.g., 2, 3, or more ICT combined).

[0071] Myocarditis and / or myositis have multiple causes and can results from a multitude of etiologies. One of the causes for myocarditis and / or myositis include treatment with ICT. In such cases, the myocarditis and / or myositis are referred to as ICT-induced myocarditis and / or ICT- 17 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO induced myositis. In various aspect, the myocarditis and / or myositis is ICT-induced myocarditis and / or ICT-induced myositis.

[0072] Levels of cytotoxic CD8+ T-cells and inflammatory myeloid cells can be detected in a variety of samples obtained from the subject.

[0073] As used herein, a “sample” or “biological sample” is meant to refer to any “biological specimen” collected from a subject, and that is representative of the content or composition of the source of the sample, considered in its entirety. A sample can be collected and processed directly for analysis or be stored under proper storage conditions to maintain sample quality until analyses are completed. Ideally, a stored sample remains equivalent to a freshly collected specimen. The source of the sample can be an internal organ, vein, artery, or even a fluid. Non-limiting examples of sample include blood, plasma, saliva, organ biopsy, cerebrospinal fluid (CSF), muscle biopsy and skin biopsy.

[0074] In one aspect, the sample is selected from a muscle biopsy, a blood sample, or a serum sample. In various aspects, the muscle biopsy is an endomyocardial biopsy or a quadricep biopsy. In many aspects, the muscle biopsy is a needle biopsy. In some aspects, the muscle is a cardiac or a skeletal muscle.

[0075] An autoantibody is an antibody produced by the immune system that is directed against one or more of the individual's own proteins. Many autoimmune diseases are associated with the production of such antibodies. Antibodies are produced by B cells in two ways: (i) randomly, and (ii) in response to a foreign protein or substance within the body. Initially, one B cell produces one specific kind of antibody. In either case, the B cell is allowed to proliferate or is killed off through the process of clonal deletion. Normally, the immune system is able to recognize and ignore the body's own healthy proteins, cells, and tissues, and to not overreact to non-threatening substances in the environment, such as foods. Sometimes, the immune system ceases to recognize one or more of the body's normal constituents as "self," leading to production of pathological autoantibodies. Autoantibodies may also play a nonpathological role; for instance, they may help the body to destroy cancers and to eliminate waste products. In the context of the instant invention, with the increased levels of plasma B cells in subject having or at risk of having myocarditis and / or myositis. It has been discovered that those patients present higher serum titers of autoantibodies. 18 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0076] In another aspect, serum titers of autoantibodies against muscle antigens are increased in subject having or at risk of having myocarditis and / or myositis as compared to serum titers of autoantibodies against muscle antigens in subject not having or not being at risk of having myocarditis and / or myositis.

[0077] There are many methods known in the art that can be used to identify a subject as having or at risk of having myocarditis and / or myositis by detecting the level of cytotoxic CD8+ T-cells, inflammatory myeloid cells and / or plasma B cells in a sample from the subject. One of skill in the art would easily decipher among the techniques available in the art the one that are the most appropriate, notably depending on the nature of the sample used.

[0078] In one aspect, identifying includes performing single-cell transcriptomics analysis, anatomic histopathology analysis and / or evaluation of serum immunoglobulins.

[0079] In another aspect, the method further includes administering to the subject identified as having or at risk of having myocarditis and / or myositis, a therapeutic compound to inhibit IL-1ȕ and / or TNF.

[0080] In another embodiment, the invention provides a method for identifying a subject as having or at risk of having myocarditis and / or myositis including detecting in CD45+ immune cells in a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon- Į signaling, an interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling and / or a complement signaling in CD45+ immune cells from a sample obtained from a control subject that is not having or at risk of having myocarditis and / or myositis, wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling in CD45+ immune cells in a sample obtained from a control subject is indicative of the subject having or at risk of having myocarditis and / or myositis, thereby identifying a subject as having or at risk of having myocarditis and / or myositis.

[0081] In one aspect, CD45+ immune cells include cytotoxic CD8+ T-cells and / or inflammatory myeloid cells. 19 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0082] In another aspect, the method further includes administering to the subject identified as having or at risk of having myocarditis and / or myositis, a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT.

[0083] In an additional embodiment, the invention provides a method of treating a subject having or at risk of having myocarditis and / or myositis including administering to the subject a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT, wherein the subject has previously received immunotherapy or immune checkpoint therapy, thereby treating a subject having or at risk of having myocarditis and / or myositis.

[0084] The term “subject” as used herein refers to any individual or patient to which the subject methods are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be a non-human animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject. In some aspects, the subject is a human.

[0085] The term "treatment" is used interchangeably herein with the term "therapeutic method" or “therapy” and refers to 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic conditions or disorder, and / or 2) prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those at risk, and needing preventive measures).

[0086] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor, or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., treatment of myocarditis and / or myositis, or treatment of cancer). The effective amount can be determined as described herein.

[0087] The terms “administration of” and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in 20 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO need of treatment. Administration routes can be enteral, topical, or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration.

[0088] The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained-release formulations, lipid complexes, etc.

[0089] In some aspects administration can be in combination with one or more additional therapeutic agents. The phrases “combination therapy”, “combined with” and the like refer to the use of more than one medication or treatment simultaneously to increase the response. The composition of the present invention (e.g., the therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT) might for example be used in combination with other drugs or treatment in use to treat myocarditis and / or myositis, as well as in combination with drugs or treatment in use to treat cancer. Such therapies can be administered prior to, simultaneously with, or following administration of the therapeutic compound to inhibit IL-1ȕ. TNF and / or JAK / STAT. That is, each therapeutic compound (e.g., the therapeutic compound that inhibits IL-1ȕ, and the therapeutic compound that inhibits TNF), can each be administered individually, subsequently or concurrently, with one another, with another drug or treatment in use to treat myocarditis and / or myositis, and / or with another drug or treatment in use to treat cancer.

[0090] Interleukin-1 beta (IL-1ȕ) also known as leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, lymphocyte activating factor and other names, is a cytokine protein that in humans is encoded by the IL1B gene. There are two genes for interleukin-1 (IL-1): IL-1 alpha and IL-1 beta. IL-1ȕ precursor is cleaved by cytosolic caspase 1 (interleukin 1 beta convertase) to form mature IL-1ȕ. IL-1ȕ is a member of the interleukin 1 family of cytokines. This cytokine is produced by activated macrophages, monocytes, and a subset of dendritic cells known 21 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO as slanDC, as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1 / ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. The induction of cyclooxygenase-2 (PTGS2 / COX2) by this cytokine in the central nervous system (CNS) is found to contribute to inflammatory pain hypersensitivity. This gene and eight other interleukin 1 family genes form a cytokine gene cluster on chromosome 2. IL-1ȕ, in combination with IL-23, induced expression of IL-17, IL-21 and IL-22 by Ȗį T cells. This induction of expression is in the absence of additional signals. That suggests IL-1ȕ is involved in modulation of autoimmune inflammation.

[0091] Tumor necrosis factor (TNF, cachexin, or cachectin) is an adipokine and a cytokine. TNF is a member of the TNF superfamily, which consists of various transmembrane proteins with a homologous TNF domain. As an adipokine, TNF promotes insulin resistance, and is associated with obesity-induced type 2 diabetes. As a cytokine, TNF is used by the immune system for cell signaling. If macrophages detect an infection, they release TNF to alert other immune system cells as part of an inflammatory response. TNF signaling occurs through two receptors: TNFR1 and TNFR2. TNFR1 is constitutively expressed on most cell types, whereas TNFR2 is restricted primarily to endothelial, epithelial, and subsets of immune cells. TNFR1 signaling tends to be pro- inflammatory and apoptotic, whereas TNFR2 signaling is anti-inflammatory and promotes cell proliferation. Suppression of TNFR1 signaling has been important for treatment of autoimmune disease, whereas TNFR2 signaling promotes wound healing.

[0092] TNF-Į exists as a transmembrane form (mTNF-Į) and as a soluble form (sTNF-Į). sTNF-Į results from enzymatic cleavage of mTNF-Į, by a process called substrate presentation. mTNF-Į is mainly found on monocytes / macrophages where it interacts with tissue receptors by cell-to-cell contact. sTNF-Į selectively binds to TNFR1, whereas mTNF-Į binds to both TNFR1 and TNFR2. TNF-Į binding to TNFR1 is irreversible, whereas binding to TNFR2 is reversible. The primary role of TNF is in the regulation of immune cells. TNF, as an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, and inflammation, inhibit tumorigenesis and viral replication, and respond to sepsis via IL-1 and IL-6-producing cells.

[0093] In some aspects, the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug. 22 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0094] As used herein the terms “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. “Antibody,” as used herein, encompasses any polypeptide comprising an antigen-binding site regardless of the source, species of origin, method of production, and characteristics. Antibodies include natural or artificial, mono- or polyvalent antibodies including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, and antibody fragments. “Antibody fragments” include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’ and F(ab’)2, Fc fragments or Fc-fusion products, single-chain Fvs (scFv), disulfide-linked Fvs (sdfv) and fragments including either a VL or VH domain; diabodies, tribodies and the like (Zapata et al. Protein Eng.8(10):1057- 1062

[1995] ).

[0095] The term “molecule” includes, but is not limited to, small molecules (including small molecules that do not have optimal cell-permeability), and larger molecules including lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines.

[0096] A small molecule or micro molecule is a low molecular weight (^ 1000 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Many drugs are small molecules; the terms are equivalent and used interchangeably. Pharmacology usually restricts the term "small molecule" to molecules that bind specific biological macromolecules and act as an effector, altering the activity or function of the target. Small molecules can have a variety of biological functions or applications, serving as cell signaling molecules, drugs in medicine, pesticides in farming, and in many other roles. These compounds can be natural (such as secondary metabolites) or artificial (such as antiviral drugs); they may have a beneficial effect against a disease (such as drugs) or may be detrimental (such as teratogens and carcinogens). One advantage that small molecule drugs (SMDs) have over "large molecule" biologics is that many small molecules can be taken orally whereas biologics generally require injection or another parenteral administration. 23 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0097] In some aspects, the IL-1ȕ inhibitor is rilonacept, canakinumab or glyburide. In other aspects. the TNF inhibitor is infliximab, adalimumab, etanercept, golimumab, certolizumab, thalidomide, or a combination thereof. In other aspects, the JAK / STAT inhibitor is ruxolitinib.

[0098] In one aspect, the subject is identified as having or at risk of having myocarditis and / or myositis by a method described herein.

[0099] In some aspect, identifying the subject as having or at risk of having myocarditis and / or myositis includes detecting in a sample from the subject the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of the = subject having or at risk of having myocarditis and / or myositis.

[0100] Alternatively, or in addition, identifying the subject as having or at risk of having myocarditis and / or myositis includes detecting in CD45+ immune cells from a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon-Į signaling, an interferon- Ȗ signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, a IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling in CD45+ immune cells in a sample obtained from a control subject that is not having or at risk of having myocarditis and / or myositis, wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, a IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, a IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling in CD45+ immune cells in a sample obtained from a control subject is indicative of the subject having or at risk of having myocarditis and / or myositis.

[0101] In yet another embodiment, the invention provides a method of treating cancer in a subject including a) administering to the subject an immune checkpoint therapy (ICT) and optionally an anticancer treatment, b) identifying a risk for the subject to develop ICT-related myocarditis and / or ICT-related myositis, and c) administering to the subject identified as having or at risk of developing ICT-related myocarditis and / or immune-related myositis in b) a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT in combination with the ICT and optionally the anticancer treatment, thereby treating cancer in the subject. 24 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0102] The term “chemotherapeutic agent,” or “anti-cancer treatment” as used herein refers to any therapeutic agent used to treat cancer. The skilled artisan will understand that often use more than one agent. In certain aspects, a chemotherapeutic agent or radiation is administered with the antibodies and small molecules described in the present invention. Chemotherapeutic and antineoplastic agents are well known cytotoxic agents, and include: (i) anti-microtubules agents comprising vinca alkaloids (vinblastine, vincristine, vinflunine, vindesine, and vinorelbine), taxanes (cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel), epothilones (ixabepilone), and podophyllotoxin (etoposide and teniposide); (ii) antimetabolite agents comprising anti-folates (aminopterin, methotrexate, pemetrexed, pralatrexate, and raltitrexed), and deoxynucleoside analogues (azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, doxifluridine, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, mercaptopurine, nelarabine, pentostatin, tegafur, and thioguanine); (iii) topoisomerase inhibitors comprising Topoisomerase I inhibitors (belotecan, camptothecin, cositecan, gimatecan, exatecan, irinotecan, lurtotecan, silatecan, topotecan, and rubitecan) and Topoisomerase II inhibitors (aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicinm, merbarone, mitoxantrone, novobiocin, pirarubicin, teniposide, valrubicin, and zorubicin); (iv) alkylating agents comprising nitrogen mustards (bendamustine, busulfan, chlorambucil, cyclophosphamide, estramustine phosphate, ifosamide, mechlorethamine, melphalan, prednimustine, trofosfamide, and uramustine), nitrosoureas (carmustine (BCNU), fotemustine, lomustine (CCNU), N-Nitroso-N-methylurea (MNU), nimustine, ranimustine semustine (MeCCNU), and streptozotocin), platinum-based (cisplatin, carboplatin, dicycloplatin, nedaplatin, oxaliplatin and satraplatin), aziridines (carboquone, thiotepa, mytomycin, diaziquone (AZQ), triaziquone and triethylenemelamine), alkyl sulfonates (busulfan , mannosulfan, and treosulfan), non-classical alkylating agents (hydrazines, procarbazine, triazenes, hexamethylmelamine, altretamine, mitobronitol, and pipobroman), tetrazines (dacarbazine, mitozolomide and temozolomide); (v) anthracyclines agents comprising doxorubicin and daunorubicin. Derivatives of these compounds include epirubicin and idarubicin; pirarubicin, aclarubicin, and mitoxantrone, bleomycins, mitomycin C, mitoxantrone, and actinomycin; (vi) enzyme inhibitors agents comprising FI inhibitor (Tipifarnib), CDK inhibitors (Abemaciclib, Alvocidib, Palbociclib, Ribociclib, and Seliciclib), PrI inhibitor (Bortezomib, Carfilzomib, and 25 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO Ixazomib), PhI inhibitor (Anagrelide), IMPDI inhibitor (Tiazofurin), LI inhibitor (Masoprocol), PARP inhibitor (Niraparib, Olaparib, Rucaparib), HDAC inhibitor (Belinostat, Panobinostat, Romidepsin, Vorinostat), and PIKI inhibitor (Idelalisib); (vii) receptor antagonist agent comprising ERA receptor antagonist (Atrasentan), Retinoid X receptor antagonist (Bexarotene), Sex steroid receptor antagonist (Testolactone); (viii) ungrouped agent comprising Amsacrine, Trabectedin, Retinoids (Alitretinoin Tretinoin) Arsenic trioxide, Asparagine depleters (Asparaginase / Pegaspargase), Celecoxib, Demecolcine Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane, Oblimersen, Omacetaxine mepesuccinate, and Eribulin; and anti-vascular endothelial growth factor inhibitors such as Avastin, Sutent, and the like.

[0103] In one aspect, identifying a risk for the subject to develop ICT-related myocarditis and / or ICT-related myositis includes detecting in a sample from the subject the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of the subject having or at risk of having myocarditis and / or myositis; and / or detecting in CD45+ immune cells in a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, a IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, a IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling in CD45+ immune cells in a sample obtained from a control subject that is not having or at risk of having myocarditis and / or myositis, wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon- Ȗ signaling, a IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling in CD45+ immune cells in a sample obtained from a control subject is indicative of the subject having or at risk of having myocarditis and / or myositis.

[0104] In one embodiment, the invention provides a method of treating immune checkpoint therapy (ICT)-related myocarditis and / or ICT-related myositis in a subject including a) identifying the myocarditis and / or myositis as ICT-related myocarditis and / or ICT-related myositis, and b) administering to the subject identified in a) as having ICT-related myocarditis and / or ICT-related 26 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO myositis a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT in combination with the ICT, thereby treating ICT-related myocarditis and / or ICT-related myositis in a subject.

[0105] In some aspects, the subject has cancer.

[0106] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. In 2015, about 90.5 million people had cancer, about 14.1 million new cases occur a year and it caused about 8.8 million deaths (15.7% of deaths). The most common types of cancer in males are lung cancer, prostate cancer, colorectal cancer, and stomach cancer. In females, the most common types are breast cancer, colorectal cancer, lung cancer and cervical cancer.

[0107] The term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 100 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure environmental pollutant, tobacco and or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof.

[0108] As used herein, “neoplasm” or “tumor” including grammatical variations thereof, means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, pancreatic, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0109] Exemplary cancers described by the national cancer institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant 27 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS— Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, 28 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non- Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, 29 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

[0110] In various aspects, the cancer is selected from metastatic melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, malignant pleural mesothelioma, esophageal squamous carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, small cell lung cancer, esophageal carcinoma, gastric cancer (or gastroesophageal junction cancer), bladder cancer, breast cancer, or urothelial carcinoma.

[0111] Presented below are examples discussing the impact of increased levels of CD45+ immunes cells in subject having or at risk of having myocarditis and / or myositis, contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES EXAMPLE 1 RATIONAL AND METHODS

[0112] The mechanisms underlying these irAEs that affect normal muscle tissues remain poorly understood. To address this, cardiac and skeletal muscle tissues of 58,523 cells collected from 20 ICT-treated patients within 96 hours of clinical presentation for suspected immune-related myocarditis and myositis were evaluated by single-cell RNA sequencing (scRNA-seq).

[0113] scRNA-seq of the affected cardiac tissues revealed enrichment of CD8+T cells with a cytotoxic phenotype and a population of inflammatory IL-1B+TNF+myeloid cells. tissue-resident myeloid cells that expressed FcȖRIIIa, which recognizes IgG antibodies and regulates complement activation were identified. Immunohistochemistry (IHC) analyses of the affected cardiac tissues identified CD8+T cells and CD68+myeloid cells, and the presence of pan-IgG and 30 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO the complement product C4d, implicating plasma B cells in disease pathogenesis. Consistent with this, half of the patients with confirmed myocarditis had high serum titers of autoantibodies against muscle antigens. scRNA-seq of affected skeletal muscles revealed identical populations of CD8+T cells and inflammatory myeloid cells as were found in the affected cardiac muscles. These results nominate immune subpopulations potentially contributing to disease pathogenesis that may inform rational treatments for immune-related myocarditis and myositis.

[0114] Serum autoantibodies have been identified in irAEs, suggestive of B cell contribution to pathogenesis. Furthermore, although prior studies have shown T cells and myeloid cells in inflamed muscle tissue, their specific subtype and role in disease pathogenesis have yet to be defined. This understanding may contribute to the development of more effective immunosuppressive strategies. This work aimed at assessing whether or not in addition to T cells, plasma B and myeloid cells may contribute to the pathogenesis of ICT-induced myocarditis, myositis, and MG.

[0115] Patients with suspected ICT-induced myocarditis, myositis, and MG were enrolled on an IRB-approved laboratory protocol. Diagnosis required: 1) symptoms requiring hospitalization; 2) elevated serum markers (creatine kinase ^5x normal, troponin .99th percentile or detection of autoantibodies for MG); and 3) positive biopsy, electromyography, or cardiac MRI. Patients underwent standard diagnostic testing (including endomyocardial and / or skeletal muscle biopsy) and treatment as clinically indicated. As detailed below, 23 patients were enrolled (n=13, confirmed irAE; n=10, negative control). Most patients received combination ICT (10 / 13, 77%). Anti-striated muscle and / or acetylcholine receptor binding antibodies were detected in 7 / 13 patients (54%), including 3 without MG (known to have both T and B cell etiologies). Single cell RNA sequencing of patient tissue (confirmed irAEs: cardiac n=10, skeletal n=9; control: cardiac n=4, skeletal n=5) identified specific populations of myeloid and CD8+ T cells conserved between inflamed cardiac and skeletal muscle. Specific populations of T cells, myeloid cells, and plasma B cells were found to be potential targets for rational immunosuppressive strategies in ICT-induced myocarditis and myositis and will be tested in murine models to inform future clinical trial design.

[0116] Study Design and Patients

[0117] . Patients hospitalized at The University of Texas MD Anderson Cancer Center from November 2020 to February 2022 with suspected immune-related myocarditis, myositis, and 31 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO myasthenia gravis provided written informed consent to an institutional review board (IRB)- approved laboratory protocol (MD Anderson PA13-0291) for immune monitoring studies in accordance with the Declaration of Helsinki. All patients underwent standard diagnostic testing and treatment as clinically indicated. Patients with suspected immune-related myocarditis underwent endomyocardial biopsy by an interventional cardiologist with at least four cores sampled. Patients with suspected immune-related myositis underwent skeletal muscle (quadriceps) biopsy by an interventional radiologist. Cardiac and skeletal muscle pathology specimens were reviewed by a clinical pathologist to assign a clinicopathologic diagnosis. All biopsies were obtained within 96 hours of clinical presentation. Due to the life-threatening nature of these toxicities, corticosteroids were not withheld prior to biopsy. In most cases, biopsies were obtained within 24 hours of corticosteroid initiation. Confirmed irAE diagnosis required all three of the following criteria: 1. symptoms requiring hospitalization; 2. Elevated serum markers (creatine kinase [CK, muscle enzyme] ^5x upper limit of normal, troponin-I or troponin-T [cardiac enzyme] >99th percentile or presence of autoantibodies known to be associated with MG); 3. positive muscle tissue biopsy, electromyography (EMG), or cardiac magnetic resonance imaging (MRI).

[0118] Single-Cell RNA Sequencing

[0119] scRNA-seq was performed using the 10x Genomics Chromium Single Cell Controller. Briefly, single-cell suspensions were prepared from cardiac and skeletal muscle biopsies as described. Cells were resuspended in freezing media containing 90% AB serum (derived from donors with AB blood type) and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until analysis. For scRNA-seq analysis, cells were thawed, washed, and droplet-separated using the Chromium Single Cell 3ƍ v.3 Reagent Kit (10x Genomics) or 5ƍ v.2 Reagent Kit (10X Genomics) with the 10x Genomics microfluidic system creating cDNA library with individual barcodes for individual cells. Barcoded cDNA transcripts from patients were pooled and sequenced using the NovaSeq 6000 Sequencing System (Illumina). For single-cell TCR sequencing, an aliquot of cDNA generated by 5ƍ v.2 Reagent Kit was used to enrich for V(D)J regions using the Chromium Single Cell V(D)J Enrichment Kit Human T Cell (10X Genomics) according to the manufacturer’s protocol. V(D)J region-enriched libraries were pooled and sequenced using the NovaSeq 6000 Sequencing System (Illumina). 32 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0120] Overview of 3’ Single-Cell RNA sequencing analysis

[0121] Raw scRNA-seq reads generated by Illumina sequencer were demultiplexed into FASTQ and aligned to GRCh38 reference genome to generate count matrices using Cell Ranger v6.0.0 analysis pipelines (10x Genomics). The preliminarily processed data were used for downstream analysis by Seurat v4.3.0. Low-quality and dead cells which had <200 or >2,500 unique transcripts or >5% of mitochondrial transcripts were filtered out. Multiple datasets (control, myocarditis, and myositis) were combined, normalized, identified variable features, and applied principal component analysis (PCA) using Seurat. Batch effect correction of combined data was performed using Harmony algorithm v0.1.1. Following batch correction, we applied FindClusters with a resolution of 0.5 to identify cell clusters and further non-linear dimension reduction was performed with uniform manifold approximation and projection (UMAP).

[0122] Comparison of transcriptional programs in immune cell subsets

[0123] Differentially expressed genes (DEGs) of certain immune cell subsets under different clinical conditions (control samples, myocarditis, myositis, patients with MG, etc.) were identified using the function FindMarkers within R package Seurat Then, GSEA analysis was performed against the Molecular Signature Database (MSigDB) with the R package fgsea (version 1.28.0). The results were visualized with the R package ggplot2 (version 3.4.0) Specifically, for the comparison of MG+ vs. MG- samples in the myositis datasets, due to the imbalanced cell count distribution among samples involved in the comparison, and to avoid potential bias, random down- sampling was performed on the samples with significantly higher cell counts for 3-times independently, and the downstream analysis results based on the 3 down-sampled datasets were further assessed to guarantee the consistency among different down-sampling attempts. The representative GSEA results were shown in Supplementary Figure 18. Comparison of myeloid cell subsets with previously reported subsets in human heart. The signatures of different myeloid cell subsets reported in the previous study by LitviĖuková et al. were generated by extracting the top 30 DEGs in each cluster. Then, the signature scores were calculated using the function AddModuleScore in the R package Seurat (v4.3.0). The results were then visualized with the package pheatmap (v1.0.12) in R. Comparison of transcriptomic profiles among cell types in myocarditis and myositis samples. To assess the transcriptome similarity and phenotypic 33 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO relationships among the cell types and states identified in the myocarditis and myositis datasets, the CD45+ and CD45- cells from the two datasets were merged, followed by library size normalization, dimension reduction, batch correction and unsupervised clustering analysis. The distribution of the cells from the two datasets in the UMAP space was visualized using the R package ggplot2 (version 3.4.0) and the DimPlot function in Seurat. In addition, we performed a hierarchical clustering analysis. Briefly, the average gene expression values for each cell cluster were calculated using the AverageExpression function in the R package Seurat (version 4.3.0), and a dendrogram was drawn based on hierarchical clustering to show the relationships of cell types defined in the two datasets. Furthermore, overlapping DEGs from the two datasets (myocarditis and myositis) were identified within the same cell type. Overrepresentation analysis was then performed on these overlapping DEGs against the hallmark gene sets in the MSigDB using the R package clusterProfiler (version 4.4.4). Briefly, the analysis of overrepresented pathways was performed with the function enricher. Then, the results were visualized with the function dotplot in clusterProfiler.

[0124] 5’ scRNA-seq data analysis

[0125] Raw data preprocessing and quality control

[0126] The raw sequencing data were preprocessed (demultiplexing cell barcodes, read alignment, and generation of gene count matrix) using the Cell Ranger Single Cell Software Suite (version 7.1.0) provided by 10× Genomics. The human reference genome GRCh38 was used for sequence alignment. Subsequent analysis was performed using the Seurat software (version 4.3.0).

[0010] For quality filtering, genes detected in fewer than 10 cells and cells with transcripts aligned to less than 200 genes were removed. Cells with 15% or more of their transcripts aligned to mitochondrial genes were also filtered out. Furthermore, cells with transcripts aligned to 6,000 or more genes were removed to exclude potential doublets or multiplets. Following these steps, a total of 10,861 cells were retained for downstream analysis. Following this initial clustering, we removed likely doublets and multiplets, as we previously described.

[0127] Library size normalization, dimension reduction, batch correction and unsupervised clustering

[0128] Log normalization on the library size of each cell was applied to the gene count matrix. 2,000 highly variable genes were identified with the function FindVariableFeatures in Seurat. The 34 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO data were then scaled, and a linear dimension reduction based on these highly variable genes was conducted using the RunPCA function in Seurat.

[0010] An elbow plot was drawn, and the number of principle components used for subsequent analysis was determined accordingly. Batch effects were evaluated, and batch correction was conducted with Harmony (version 0.1.1) software in R.

[0011] A nearest-neighbor graph was constructed using the FindNeighbors function in Seurat.

[0010] Following this, unsupervised clustering was performed with the Louvain algorithm at a resolution of 0.1, resulting in the identification of 12 cell clusters. Non-linear dimension reduction was performed using the Uniform Manifold Approximation and Projection (UMAP) method. Differential gene expression analysis was then conducted with the FindAllMarkers function in Seurat, using the Wilcoxon Rank Sum test, with the min.pct parameter set to 0.25. Major cell types were further determined based on a manual review of DEGs and the expression of classical lineage markers in each cluster. By checking multiple lineage markers, we observed no additional doublets or multiplets at the major cell type level.

[0129] Sub-clustering analysis of T & NK cells

[0130] We further performed sub-clustering on T & NK cells, following the same procedures as described above. Cell clusters that simultaneously expressing discrepant lineage makers (e.g., cells in the T-cell cluster showed expression of B cell markers) were identified and removed. We performed several rounds of filtering as we previously reported until no obvious doublets or multiplets were observedn DEGs were identified with the same steps as above. Top 30 most significant DEGs were carefully reviewed. Besides, the expressions of canonical markers for T cell types and states, as reported previously, were manually checked, and bubble plots were generated accordingly. T cell types and states were then inferred by integrating the above information, and annotations were added to each cluster with the AddMetaData function in Seurat.

[0131] scTCR-seq data analysis

[0132] In total, 9 samples from 8 patients were included for TCR repertoire analysis, including 4 patients with myocarditis (4388, 4400, 4582, 4658), 1 patient with both myocarditis and myositis (4420), and 3 control patients (4398, 4554, 4659). The preprocessing of raw sequencing data was conducted using the Cell Ranger software (version 7.1.0, 10× Genomics) with the cellranger vdj function. The pipeline encompassed several steps, including cell barcode 35 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO demultiplexing, aligning reads to the human reference genome GRCh38, assembling TCRs, and identifying the V(D)J gene usage and sequences of the framework regions and complementary determining regions (CDRs). For downstream analysis, the R package scRepertoire (version 1.7.0) was used.

[0019] As part of the quality control, only productive TCRĮ and TCRȕ chains were kept, and cells with multiple TCRĮ or TCRȕ chains were removed. TCRs with exactly the same VJ gene usage and CDR3 sequences on both TCRĮ and TCRȕ chains were assigned as the same clonotype. The clonal size of TCRs was classified as hyperexpanded (clonal proportion > 0.10), large (0.05 < clonal proportion ^ 0.10), medium (0.01 < clonal proportion ^ 0.05), small (clonal proportion ^ 0.01) and singleton (with only 1 cell expressing the clonotype). The inverse Simpson index was used to assess clonal diversity. It is calculated as follows:

[0133] where ^ represents the Simpson index, ^irepresents the proportion of clonotype i in the repertoire, and R represents the total number of clonotypes.

[0134] To assess the level of clonal overlap between samples, the Jaccard index was employed, which is calculated as follows:

[0135] where A and B represents the two samples being compared; J(A, B) denotes the Jaccard index between A and B. A Ŋ B represents the size of the intersection between A and B, and A ^ B represents the size of the union of A and B. Heatmaps were used to visualize the Jaccard scores between samples. In addition, alluvial plots were employed to illustrate the overlap of the top TCR clones across different samples. The TCR clonotype information was further integrated with the 5’ scRNA-seq data based on the unique cell barcodes, using the AddMetaData function in Seurat.

[0010] The clonal size distribution in each T cell cluster was visualized using the R package ggplot2 (version 3.4.0).

[0136] Cell-cell communication analysis 36 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO

[0137] Cell-cell communication analysis was conducted using the NicheNet method, which utilizes the information of active target signaling pathways to infer cellular interactions.

[0020] The analysis was carried out using the nichenetr package (version 1.1.1) in R. Cellular interactions in different conditions (myocarditis vs. normal myocardium, myositis vs. normal skeletal muscle) were compared using the nichenet_seuratobj_aggregate function. Additionally, the comparison of cellular interactions across different target cell types was performed with the nichenet_seuratobj_cluster_de function, also part of the nichenetr package. For the identification of active ligands, only upregulated target genes in the condition or cell type of interest were taken into consideration.

[0138] Serum Autoantibodies

[0139] IgGs were evaluated in patient serum at the time of hospitalization using Clinical Laboratory Improvement Amendments (CLIA)-certified striated muscle and myasthenia gravis / Lambert-Eaton myasthenia syndrome antibody panels, including autoantibodies for acetylcholine receptor (muscle) binding antibody with reflex testing to acetylcholine receptor modulating antibody.

[0140] Immunohistochemistry

[0141] At least three samples from the right ventricular endomyocardial biopsy and quadricep biopsy were fixed in phosphate-buffered formalin and embedded in a single paraffin block. Sections were then made for evaluation with light microscopy including staining for hematoxylin and eosin (H&E), trichrome and immunohistochemistry (IHC). IHC staining was performed for CD4 (Agilent IR649, Clone 4B12), CD8 (Agilent GA623, Clone C8 / 144B), CD20 (Agilent GA604, Clone L26), CD68 (Agilent GA609, Clone KP1), pan-IgG (Agilent IR512, polyclonal), and C4d (Biorad 0300-0230, polyclonal). One sample from right ventricular endomyocardial biopsy was fixed in glutaraldehyde and electron microscopy was performed (results not presented).

[0142] Statistical analysis

[0143] Normality assumption of data was assessed using Shapiro-Wilk test. For datasets with normal distributions (p > 0.05), a two-tailed Welch's t-test was employed. For datasets with non- normal distributions (p < 0.05), a two-tailed Mann-Whitney U-test was used. For the comparison of cell proportions in the patient-matched dataset, Wilcoxon matched-pairs signed rank tests 37 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO were performed. Statistical tests and data visualizations were performed using Prism 9.4.1(GraphPad Software) and R statistical software (version 4.2.0)."

[0144] Data Availability

[0145] Data are available upon reasonable request. The data generated in this study are publicly available in the European Genome-phenome Archive (EGA) under accession no. EGAS00001007511. All data relevant to the study are included in the article or uploaded as supplementary material. EXAMPLE 2 PATIENT CHARACTERISTICS AND TISSUE SPECIMENS

[0146] Clinical data and tissue samples from 20 living patients with cancers treated with ICTs and admitted to the hospital with suspected immune-related myocarditis and / or myositis on the basis of clinical symptoms (e.g. dyspnea, fatigue) and / or laboratory values (elevated troponin-T or troponin-I [cardiac enzymes] or creatine kinase [CK, muscle enzyme]), and who also had evaluable tissue for further analysis were collected (Table 1). A confirmed irAE diagnosis of myocarditis and / or myositis required all three of the following criteria: 1. symptoms requiring hospitalization; 2. elevated serum markers (CK ^5x upper limit of normal, troponin-I or troponin-T >99th percentile or presence of autoantibodies known to be associated with MG); and 3. positive muscle tissue biopsy, electromyography (EMG), or cardiac magnetic resonance imaging (MRI).

[0147] A total of 11 patients were confirmed to have an irAE diagnosis (myocarditis plus myositis, n=10; myocarditis-only, n=1 [patient 4388]) (Table 1) (FIG.1A). The 9 patients that were treated with ICT and clinically ruled out for immune-related myocarditis and myositis were utilized as controls (cardiac muscle, n=4; skeletal muscle, n=5). Serum markers of tissue injury were consistent with the adjudicated diagnoses. The median serum levels of troponin-T, a component of the troponin complex of proteins integral to contraction of skeletal and cardiac muscles and commonly utilized as a measurement of cardiac tissue damage, were 1,056 ng / mL in the confirmed irAE group (95% CI: 485-4,613) and 60 ng / mL in the control group (95% CI: 19-358) (reference range: <18 ng / mL, p<0.0001) (FIG. 1B). Similarly, the median levels of creatine kinase (CK), a protein that catalyzes conversion of phosphocreatine that is used as an energy reservoir in tissues that rapidly consume adenosine triphosphate (ATP) such as skeletal muscle and is a measure of 38 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO muscle damage, were 1,373 U / L in the confirmed irAE group (95% CI: 182-6,012) and 235 U / L in the control group (95% CI: 59-717) (reference range: <180 U / L, p=0.0097) (FIG.1C). The most common cancers in this patient cohort were renal cell carcinoma (RCC) and melanoma (Supplementary Table 1).

[0148]

[0149] Although all patients received anti-PD-(L)1 based-therapy, the most common ICT received in patients with confirmed irAEs was the combination of anti-CTLA-4 plus anti-PD-(L)1 (n=4, 36%) (Table 1). The median number of cycles of ICT in the confirmed irAEs group was 1 (interquartile range [IQR]: 1-2) and in the control group was 4 (IQR: 3-8) (Table 1).

[0150] Table 1: Baseline Clinical Characteristics. IQR=interquartile range. Patients with Control Confirmed irAEs Patients Age at irAE Onset (IQR) 74 (64-77) 72 (66-76) Sex, n (%) Male 9 (82) 4 (44) Female 2 (18) 5 (56) Race, n (%) White 9 (82) 7 (78) Hispanic / Latino 2 (18) 2 (22) Body Mass Index (BMI, kg / m2[%]) Normal (BMI 18-25) 0 (0) 2 (22) Overweight (BMI 25-30) 6 (55) 4 (44) Obese (BMI ^30) 5 (45) 3 (33) Tobacco History, n (%) 5 (45) 3 (33) Comorbidities, n (%) Coronary artery disease 3 (27) 3 (30) Hypertension 9 (82) 5 (56) Type 2 diabetes mellitus 2 (1) 4 (44) Cancer, n (%) Renal cell carcinoma 3 (27) 2 (22) Melanoma 3 (27) 2 (22) Urothelial carcinoma 1 (9) 4 (44) Prostate carcinoma 1 (9) 0 (0) Pancreatic adenocarcinoma 1 (9) 0 (0) Hepatocellular carcinoma 1 (9) 0 (0) Head and neck squamous cell carcinoma 1 (9) 0 (0) 39 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO Small cell lung carcinoma 0 (0) 1 (11) Immune Checkpoint Therapy (ICT) - n, (%) Anti-CTLA-4 + Anti-PD-(L)1 4 (36) 2 (22) Anti-PD-(L)1 + Other 2 (18) 3 (33) Anti-PD-(L)1 Monotherapy 3 (23) 2 (22) Anti-CTLA-4 + Anti-PD-(L)1 + Other 1 (8) 2 (20) Anti-PD-(L)1 + Anti-LAG3 1 (8) 0 (0) Median Cycles of ICT (IQR) 1 (1-2) 4 (3-8) EXAMPLE 3 OVERALL CELLULAR LANDSCAPE OF IMMUNE-RELATED MYOCARDITIS AND MYOSITIS

[0151] Given that immune-related myocarditis and myositis are rare, often concurrently occurring irAEs both involving muscle tissues, it was surmised that they may also share molecular pathways and immune cell subsets in contributing to disease pathogenesis. Thus, to comprehensively define the phenotype and putative function of immune cells within the inflamed tissue microenvironment, single-cell RNA sequencing (scRNA-seq) was performed on cardiac (n=10 myocarditis; n=4 control) and skeletal muscle tissues (n=6 myositis; n=5 control) (FIG. 2A). Of the 11 total patients with confirmed irAEs, 10 patients had overlapping myocarditis and myositis, and 1 patient (4388) had confirmed myocarditis only (FIG.1A). Paired patient-matched cardiac and skeletal muscle tissues were obtained in five patients (FIG.2A). A total of 27,594 live cells were evaluable for further analyses, with 4,591 live cells from normal cardiac tissue and 23,003 live cells from myocarditis tissue. From skeletal muscle (quadriceps), a total of 30,929 live cells were obtained, with 14,561 live cells from normal skeletal muscle and 16,368 live cells from myositis tissue. To ensure the evaluation of cell subsets was not skewed by differing cell numbers in toxicity versus control tissues, the cells in the toxicity groups were downsampled to match the number of cells in each control group, and they were visualized under uniform manifold approximation and projection (UMAP) embeddings.

[0152] To ensure that the biopsy methods obtained representative cardiac and skeletal muscle samples in both the toxicity and control groups, the CD45- non-immune cell compartment was first evaluated. Within cardiac muscle, 22,411 cells consisting of cardiomyocytes, endothelial 40 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO cells, fibroblasts, and pericytes / smooth muscle were obtained. Within skeletal muscle, 12,762 CD45- cells that were comprised of skeletal muscle cells, endothelial cells, satellite cells (muscle stem cells), fibro-adipogenic precursors (FAPs), and pericytes / smooth muscle, which have previously been described in single-cell atlases of human skeletal muscle were obtained. Within these subpopulations, there were no difference in frequencies between control and immune-related myocarditis tissues (FIGs 3A-3P) nor between control and immune-related myositis samples (FIGs 4A-4L). To formally compare the CD45- cell types in the cardiac and skeletal muscle samples, hierarchical clustering based on Euclidean distance of expression was performed, which demonstrated as expected greater similarities for smooth muscle and endothelial cell clusters than for fibroblast and FAP subsets between cardiac and skeletal muscles.

[0153] Next, the CD45+ immune cell compartment (n=5,183 CD45+ cells in cardiac muscle samples; n=18,167 CD45+ cells in skeletal muscle samples) was analyzed. There was a trend towards a higher frequency of CD45+ cells in both immune-related myocarditis and myositis specimens compared to control samples (FIGs 5A-5B). It should be noted that most patients received at least one dose of intravenous corticosteroids prior to biopsy, which may have attenuated the frequency of live immune cell populations within the inflamed muscles (Table 1). To evaluate the transcriptional profiles of CD45+ immune cells in inflamed muscle tissues, the top upregulated and downregulated differentially expressed genes between myocarditis and myositis samples were quantified, and it was observed that a minority of genes were shared (FIG. 6). To determine the identity of these transcriptional differences, gene set enrichment analysis (GSEA) on all pooled CD45+ immune cells was performed. Within both cardiac and skeletal muscle, the TNF-Į signaling via NF-KB and IFN-Ȗ response pathways were observed to be highly upregulated, in addition to the complement pathway in skeletal muscle. Consistent with these findings, genes associated with cytotoxic T cells (CD8A), T cell activation (LAG3), type I interferon (ISG15), and type II interferon (IFNG) were significantly differentially expressed in CD45+ cells in ICT- induced myocarditis and myositis compared to controls (FIGs 7A-7B). To account for individual patient variation and isolate transcriptional pathways within CD45+ immune cells that were differentially expressed in myocarditis versus myositis, patient-matched myocarditis and myositis samples were directly compared. The top pathways differentially expressed in myocarditis meeting a padj<0.05 threshold included hallmark hypoxia and IL2 / STAT5 signaling pathways. 41 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO EXAMPLE 4 IMMUNE CELL SUBSETS IN ICT-INDUCED MYOCARDITIS AND MYOSITIS

[0154] To evaluate the overall immune cell populations in myocarditis and myositis, the frequency of total lymphoid (T cell, NK cell, or B cell) and myeloid cell frequency within CD45+ cells in myocarditis versus control cardiac muscle samples and myositis and control skeletal muscle samples were compared (FIG. 5A and 5B, respectively), and there were no observed differences in frequency. Therefore, individual immune cell clusters were subclassified and distinct clusters of CD8+ T cells (cytotoxic, GZMK-expressing, and proliferating), CD4+ T cells (naïve / central memory, Treg, cytotoxic), NK cells, naïve B cells, and myeloid cells (including SELL+ [L-selectin] macrophages, IL1B+ TNF+ myeloid cells, and conventional type 2 dendritic cells [cDC2s], professional APCs that prime CD4+ T cells) were identified in cardiac muscle (FIG. 8A). Similarly, within skeletal muscle, clusters of CD8+ T cells (cytotoxic, proliferating, and GZMK-expressing), CD4+ T cells (naïve / central memory), NK cells, and myeloid cells (including SELL+ myeloid, IL1B+TNF+ myeloid, and cDC2 populations were identified (FIG. 8B). To formally compare the CD45+ cell types in the cardiac and skeletal muscle samples, hierarchical clustering based on Euclidean distance of expression was performed and shared clustering of the majority of specific cell subtypes was observed in cardiac and skeletal muscle (FIGs 5A and 5B). Consistent with this observation, pooling of patient-matched myocarditis and myositis samples revealed overall similarity of the immune cell subclusters in both compartments. EXAMPLE 4 CLONALLY EXPANDED CYTOTOXIC CD8+ T CELLS EXPRESSING ACTIVATION MARKERS ARE ENRICHED IN MYOCARDITIS AND MYOSITIS.

[0155] Based on prior evidence strongly implicating CD8+ T cells in immune-related myocarditis and myositis, the first focus was on T cell populations. In both immune-related myocarditis (FIG.9A) and myositis (FIG.9B), a significantly higher frequency of a population of CD8+ T cells expressing genes associated with T cell activation (LAG3, TIGIT, PDCD1, HAVCR2, TOX) and cytotoxicity (GZMB, GNLY, NKG7, CST7) was observed. Furthermore, this population of activated, cytotoxic CD8+ T cells expressed genes for chemokines, including 42 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO CCL5, CCL4, and CCL4L2). This expression profile is in line with a recent study identifying a circulating population of cytotoxic CD8+ T effector cells re-expressing CD45RA (TEMRA) cells in the peripheral blood of patients with immune-related myocarditis; these have not previously been directly shown in cardiac tissue from patients. As similar marker genes and frequencies were observed within this population in immune-related myocarditis and myositis, whether specific transcriptional pathways distinguished these populations in inflamed cardiac and skeletal muscle was investigated. The most highly upregulated hallmark transcriptional pathway within the cytotoxic CD8+ T cell population in myocarditis compared to control cardiac muscle was TNF-Į signaling via NF-kB, whereas in myositis compared to control skeletal muscle, the most highly upregulated pathway was interferon-Į response. To further delineate similarities and differences within this cytotoxic CD8+ T cell population across organ sites, patient-matched myocarditis and myositis samples were compared and similar clustering of this subset was observed. Consistent with this, this subset was found to be present in similar frequency in patient-matched myocarditis and myositis tissue (FIG. 10). Within the patient-matched specimens, the hallmark hypoxia, IL2 / STAT5, and TNF-Į signaling via NF-^B signaling pathways were observed to be more highly expressed in myocarditis than in myositis. In line with this, a subset of differentially expressed genes were shared between the myocarditis and myositis samples, although the transcriptional profiles were not identical, suggestive of context-dependent gene expression within the cardiac and skeletal muscle compartments.

[0156] Previous studies demonstrated the presence of shared TCR clones between involved heart and skeletal muscles in patients with ICT-induced myocarditis and myositis; however, the specific phenotype of these T cells in inflamed tissue has not previously been described. Single- cell TCR sequencing (scTCR-seq) data from 9 samples (n=5 myocarditis, n=1 myositis, n=2 control cardiac muscle, and n=1 control skeletal muscle) were therefore analyzed, including one patient with matched myocarditis and myositis samples. For these samples, paired 5’ scRNA-seq data were also generated. A total of 1,477 cells with productive TCR Į / ȕ chains after removing cells with multiple TCRs were identified. Clonotypes were defined based on VJ gene usage and CDR3 nucleotide sequence, and evidence of TCR clonal expansion was observed in toxicity cases. Consistent with prior reports, TCR clonal overlap was observed in cardiac and skeletal muscles in the only evaluable patient with matched samples from myocarditis and myositis. The specific 43 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO phenotype associated with these TCR clonotypes was evaluated, and expanded TCR clones were mapped primarily to a population of cytotoxic CD8+ T cells expressing CCL4 and CCL5, consistent with the population an increased in frequency in myocarditis and myositis tissues was observed. Taken together, these findings demonstrate that clonally expanded, cytotoxic CD8+ T cells are enriched in cardiac and skeletal muscles of patients with ICT-induced myocarditis and myositis. EXAMPLE 5 INFLAMMATORY IL1B+TNF+ MYELOID CELLS SPECIFICALLY ENRICHED IN ICT-INDUCED MYOCARDITIS ARE ASSOCIATED WITH POORER CLINICAL OUTCOMES

[0157] The myeloid cell compartment was evaluated next, as infiltration of myeloid cells within the inflamed muscle tissue has been reported but their phenotype and putative function have not yet been fully described (FIGs 11A and 11B). There were no observed differences in the frequencies of C1Q+ myeloid populations expressing tissue-resident signatures between the myocarditis and myositis samples compared to control samples (FIGs 11A and 11B).

[0158] It was further observed a significantly higher frequency (p=0.03) of HLA- DRAloCD86+ myeloid cells expressing the pro-inflammatory cytokines IL1B and TNF, the pleiotropic cytokine TGFB1, and the hypoxia-response factor HIF1A within the immune-related myocarditis samples (FIG. 11A). This population expressed CXCL2 (also called macrophage inflammatory protein [MIP]-2Į, chemotactic for granulocytes and hematopoietic stem cells), CXCL3 (MIP-2ȕ, which is chemotactic for monocytes), and CXCL8 (IL-8, mediates neutrophil chemotaxis). This population was also identified within skeletal muscle tissues but was not significantly increased (p=0.93) in myositis compared to control samples (FIG.11B). Quantitative comparison of the transcriptomes of these populations between myocarditis and myositis samples demonstrated that a plurality of differentially expressed genes were shared (FIG.12). Furthermore, within the patient-matched myocarditis and myositis samples, the IL1B+TNF+ myeloid cell subsets were clustered similarly and were present in comparable frequencies in myocarditis and myositis tissues (FIG. 13A). We sought to determine if specific transcriptional pathways distinguished these myeloid cell populations in inflamed cardiac and skeletal muscle. Within 44 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO cardiac muscle, the most highly expressed transcriptional pathways in this population of myeloid cells were IFN-Į response, TNF-Į signaling via NF-^B, and IFN-Ȗ response. In skeletal muscle, TNF-Į signaling via NF-^B was the most highly expressed transcriptional pathway, however the IFN-Į response and IFN-Ȗ response pathways were not significantly upregulated in myositis. Consistent with this, direct comparison of matched myocarditis and myositis samples identified significantly increased interferon-Į response pathway in myocarditis and significantly increased TNF-Į signaling via NF-^B in myositis. As this population of IL1B+TNF+ myeloid cells has not previously been identified in these toxicities and was found to be significantly increased in myocarditis, the association with severity of myocarditis and clinical outcomes was evaluated. A trend toward higher median serum troponin-T levels associating with increased frequency of the IL1B+TNF+ myeloid cell subset was observed(p=0.09) (FIG. 13B). Furthermore, a higher frequency of the IL1B+TNF+ myeloid cell subset was associated with poorer clinical outcomes, defined as an occurrence of a major adverse cardiovascular event (a composite of heart failure, arterial thrombus, life threatening arrhythmia, pulmonary embolism or unexplained sudden cardiac death) or death before next anti-cancer therapy, whereas a higher frequency of the cytotoxic CD8+ T cell population was not associated with poorer clinical outcomes (FIG. 13C). Moreover, an increased frequency of IL1B+TNF+ myeloid cells (above the median) was founds associated with a trend toward shorter overall survival (p=0.062) (FIG.13D). The duration of survival is consistent with reports from our group and others that non-fatal irAEs are associated with improved overall outcomes following ICTs. To evaluate potential interactions between these myeloid cells, T cells, and stromal cells, computational cell-cell communication analyses were performed and predicted interactions of cytotoxic CD8+ T cell-derived IFN-Ȗ and IL1B+TNF+ myeloid cell-derived IL1ȕ on endothelial cells were identified, consistent with endothelial stress responses.

[0159] Finally, as myocarditis and myositis have been reported to occur in conjunction with myasthenia gravis (MG) as part of an overlap syndrome, the transcriptional programs within CD45+ cells of patients with all three toxicities (myocarditis plus myositis plus MG) versus those without MG (myocarditis plus myositis) were evaluated. Increased expression of the TNFĮ signaling pathway was observed in myocarditis samples from patients with MG, potentially consistent with greater severity of disease, however no significant differences were observed in 45 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO skeletal muscle samples. Given the small sample size of these subgroups, the comparisons are necessarily exploratory. EXAMPLE 6 PUTATIVE INTERACTIONS OF CYTOTOXIC CD8+ T CELLS, INFLAMMATORY IL1B+TNF+ MYELOID CELLS AND ENDOTHELIAL CELLS WITHIN INFLAMED MUSCLE TISSUES

[0160] ICTs offer the promise of long-term survival benefit to subsets of patients with cancer but can induce life-threatening irAEs. The focus of the present study was on immune-related myocarditis and myositis, rare irAEs affecting muscle tissues that carry high mortality. There is an unmet need to understand the molecular pathways and immune subsets underlying these irAEs to inform rational, front-line immunosuppressive treatments. Single-cell analyses of cardiac and skeletal muscle tissues were performed at the time of hospitalization since performing baseline endomyocardial and quadriceps muscle biopsies in otherwise healthy patients represented an unacceptable risk. The strength of the approach is that these technically challenging biopsies were obtained early in the course of toxicity in living patients, before progression to mortality, which may lead to cellular breakdown and confound evaluation of the inflamed microenvironment. Furthermore, all patients except for one had overlapping myocarditis and myositis, and a skew in our dataset was not observe from the one patient with myocarditis- only. It should be noted that clinically necessary treatments could not be withheld prior to biopsy. Therefore, most patients received at least one dose of intravenous corticosteroids, the first-line treatment for these irAEs, potentially limiting yield of CD45+ cells in these needle biopsies. Despite these limitations, molecular pathways and immune subsets in myocarditis and myositis that may inform investigation into rational treatments for these irAEs were identified. The data identified the enrichment of clonally expanded cytotoxic CD8+ T cells in both myocarditis and myositis, acknowledging that this finding of shared T cell clones was derived from the one patient who had paired cardiac and skeletal muscle tissue evaluable for TCR sequencing. This finding is consistent with prior reports identifying clonal expansion of CD8+ T cell subsets in peripheral blood from patients with myocarditis, and identification of Į-myosin-specific TCR clonotypes within inflamed heart and skeletal muscle of patients. increased frequency of inflammatory myeloid cells 46 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO expressing genes for the TNF-Į and IL-1ȕ cytokines specifically in immune-related myocarditis were further identified. It should be noted that this population of myeloid cells was also identified in myositis but was not significantly different in frequency between myositis and control samples, possibly due to the higher cellular frequency observed in both groups. TNF-Į promotes further release of inflammatory cytokines, upregulation of endothelial adhesion molecules, and regulation of leukocyte migration.

[0161] In fact, TNF inhibitors have successfully been utilized as second-line treatment of immune- related myocarditis; however, their clinical applicability is limited by the increased risk of infection and congestive heart failure (CHF), particularly at higher doses. Another second-line approach is blockade of the T cell CD28-CD80 / 86 costimulatory pathway with the CTLA-4 Ig fusion protein, abatacept, which has been shown to provide clinical benefit in a case series of patients with immune-related myocarditis alone and in combination with Janus kinase 1 (JAK1) and JAK2 inhibitor, ruxolitinib. The identification of upregulated IL6 / JAK / STAT3 signaling in the data supports this role of ruxolitinib. Furthermore, the data suggest that abatacept may function through disruption of costimulatory signaling via CD86 provided by the identified population of inflammatory IL-1B+TNF+ myeloid cells. Myeloid cells have been implicated in the development of irAEs, such as colitis and arthritis, and have heterogeneous effects on anti-tumor immunity. Therefore, it is important to understand the relative contributions of myeloid subpopulations to toxicities and anti-tumor immune responses. A recently published study identified a population of CD68+CD16a+ macrophages in human myocarditis samples co-localizing with CXCL9 and CXCL10 mRNA, consistent with the instant identification of a population of myeloid cells expressing tissue-resident signatures and FcȖRIIIa (CD16a). However, the present study is the first to identify IL-1B-expressing myeloid cells associated with poorer clinical outcomes in immune- related myocarditis, a strength of single cell RNA sequencing analysis of patient tissue specimens. Indeed, single-cell RNA sequencing analyses of other organ sites affected by irAEs have yielded unique insights into potential mechanisms of these toxicities—for example, evaluation of colon tissues from patients with ICT-induced colitis determined that a high proportion of colitis- associated CD8+ T cells with cytotoxic and proliferative phenotypes originated from tissue- resident populations, consistent with the early-onset of this irAE. Furthermore, single-cell analysis of synovial fluid samples in patients with ICT-induced arthritis implicated clonally expanded 47 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO CXCR3hiCD8+ T cells in synovial fluid that may be recruited by CXCL9 / 10 / 11 / 16 secreted by myeloid cells, underscoring the heterogeneity of potential mechanisms contributing to irAEs.

[0162] The findings described herein suggest that the IL-1 pathway is an attractive target for selective immunosuppression in myocarditis. IL-1 encompasses two cytokines: IL-1Į, which primarily functions in local inflammatory responses, and IL-1ȕ, which is produced chiefly by myeloid cells (including macrophages, neutrophils and mast cells) and exerts systemic effects. Engagement of the IL-1 receptor initiates multiple downstream events, including production of inflammatory cytokines such as IL-6, induction of fever, promotion of leukocyte migration, and supporting expansion and effector functions of CD4+ and CD8+ T cells. Indeed, in a murine model of ICT-induced myocarditis, IL-1ȕ blockade was found to prevent myocarditis and reduce cardiac fibrosis. However, it should also be noted that IL-1 signaling has not been found to be consistently upregulated in all murine models of myocarditis. Furthermore, the role of inflammatory IL-1 signaling in non-ICT-related heart disease, including viral myocarditis and coronary artery disease, has also previously been recognized. In fact, IL-1 pathway inhibitors are currently in clinical use for the treatment of pericarditis, inflammation of the lining of the heart, and an anti-IL-1ȕ antibody (canakinumab) was shown to reduce cardiovascular events in patients with prior myocardial infarction. The present data further implicate B cell responses, with secretion of IgGs that recognize muscle antigens potentially engaging with FcȖRIIIa (CD16a) on C1qhiMRC1+ myeloid cells leading to downstream activation of the complement pathway, as observed both transcriptionally and at the protein level, with deposition of the complement split product C4d. These findings provide a rationale for B cell-directed therapies such as plasma exchange (to remove autoantibodies) and anti-CD20 antibodies (to deplete B cells) in myocarditis, myositis, and other associated irAEs such as myasthenia gravis, Guillain-Barre syndrome (GBS), vasculitis, and glomerulonephritis and may suggest a role for direct complement-targeting therapies in these toxicities. This potential role for B cells in irAEs is aligned with recent reports of B cells organized into tertiary lymphoid structures (TLS) contributing to anti-tumor immune responses with ICTs. In summary, populations of clonally expanded cytotoxic CD8+ T cells expressing activation markers shared in cardiac and skeletal muscle and specific myeloid cell subsets that may be involved in immune-related toxicities via the IL-1 and classical complement pathways were identified. These results nominate candidate clinically actionable targets for investigation in these 48 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO severe irAEs, to ultimately advance the complementary goals of minimizing toxicity and maximizing efficacy of ICTs.

[0163] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims. 49 ACTIVE\1612701606.1

Claims

PATENT ATTORNEY DOCKET NO.: MDA1240-1WO What is claimed is:

1. A method for identifying a subject as having or at risk of having myocarditis and / or myositis comprising: detecting in a sample from the subject a level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level the cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of the subject having or at risk of having myocarditis and / or myositis, thereby identifying a subject as having or at risk of having myocarditis and / or myositis.

2. The method of claim 1, wherein the subject has or is at risk of having myocarditis and myositis.

3. The method of claim 1, wherein the subject has previously received immunotherapy.

4. The method of claim 3, wherein the subject has previously received immune checkpoint therapy (ICT).

5. The method of claim 4, wherein the myocarditis and / or myositis is ICT-induced myocarditis and / or ICT-induced myositis.

6. The method of claim 1, wherein the sample is selected from a muscle biopsy, a blood sample, or a serum sample.

7. The method of claim 6, wherein the muscle biopsy is an endomyocardial biopsy or a quadricep biopsy.

8. The method of claim 6, wherein the muscle biopsy is a needle biopsy.

9. The method of claim 6, wherein the muscle is a cardiac or a skeletal muscle.

10. The method of claim 1, wherein the cytotoxic CD8+ T cells are clonally expanded activated cytotoxic CD8+ T cells.

11. The method of claim 1, wherein the cytotoxic CD8+ T cells in the sample from the subject having or at risk of having myocarditis and / or myositis express higher levels of LAG3, TIGIT, 50 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO PDCD1, HAVCR2, TOX, GZMB, GNLY, NKG7, CST7, CCL5, CCL4 and / or CCL4L2 than the cytotoxic CD8+ T-cells in a sample from a subject not having or not at risk of having myocarditis and / or myositis.

12. The method of claim 1, wherein the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myocarditis are characterized by an increased TNF signaling via NF^B signature, IL2 / STAT5 signature and / or hypoxia signature.

13. The method of claim 1, wherein the cytotoxic CD8+ T-cells in the sample from a subject having or at risk of having myositis are characterized by an increased interferon-Į signature.

14. The method of claim 1, wherein the inflammatory myeloid cells in the sample from a subject having or at risk of having myocarditis and / or myositis express higher levels of TGFB1, HIF1A, CXCL2, CXCL3 and / or CXCL8 than the inflammatory myeloid cells in a sample from a subject not having or not at risk of having myocarditis and / or myositis.

15. The method of claim 1, wherein the inflammatory myeloid cells in the sample from a subject having or at risk of having myocarditis and / or myositis are characterized by an increased TNF signaling via NF^B signature, interferon-Į signature, interferon-Ȗ signature and / or a complement signature.

16. The method of claim 1, wherein the inflammatory myeloid cells express IL-1ȕ and / or TNF at higher levels than myeloid cells in a subject not having or at risk of having myocarditis and / or myositis.

17. The method of claim 1, wherein the inflammatory myeloid cells express C1QA, C1QB, C1QC and / or C2 at higher levels than myeloid cells in a subject not having or at risk of having myocarditis and / or myositis.

18. The method of claim 1, wherein the inflammatory myeloid cells are CD68+ cells.

19. The method of claim 1, wherein the inflammatory myeloid cells are IL-1ȕ+TNF+ myeloid cells. 51 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO 20. The method of claim 1, wherein an increased level of inflammatory myeloid cells is associated with increased serum troponin T level in the subject.

21. The method of claim 1, wherein frequency of IL-1ȕ+TNF+ myeloid cells in the sample in indicative of the subject’s overall survival and / or clinical outcome.

22. The method of claim 21, wherein an increased frequency of IL-1ȕ+TNF+ myeloid cells is indicative of a shortened overall survival of the subject, and / or with an increased risk to develop major adverse cardiovascular event.

23. The method of claim 1, wherein serum titers of autoantibodies against muscle antigens are increased in subject having or at risk of having myocarditis and / or myositis as compared to serum titers of autoantibodies against muscle antigens in subject not having or not being at risk of having myocarditis and / or myositis.

24. The method of claim 1, wherein identifying comprises performing single-cell transcriptomics analysis, anatomic histopathology analysis and / or evaluation of serum immunoglobulins.

25. The method of claim 1, further comprising administering to the subject identified as having or at risk of having myocarditis and / or myositis, a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT.

26. The method of claim 25, wherein the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug.

27. A method for identifying a subject as having or at risk of having myocarditis and / or myositis comprising: detecting in CD45+ immune cells in a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling in CD45+ immune cells from a sample obtained from a control subject that is not having or at risk of having myocarditis and / or myositis, 52 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, IL2 / STAT5 signaling, hypoxia signaling and / or complement signaling in CD45+ immune cells in a sample obtained from a control subject is indicative of the subject having or at risk of having myocarditis and / or myositis, thereby identifying a subject as having or at risk of having myocarditis and / or myositis.

28. The method of claim 27, wherein CD45+ immune cells comprise cytotoxic CD8+ T-cells and / or inflammatory myeloid cells.

29. The method of claim 27, further comprising administering to the subject identified as having or at risk of having myocarditis and / or myositis, a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT.

30. The method of claim 29, wherein the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug.

31. A method of treating a subject having or at risk of having myocarditis and / or myositis comprising administering to the subject a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT, wherein the subject has previously received immunotherapy or immune checkpoint therapy, thereby treating a subject having or at risk of having myocarditis and / or myositis.

32. The method of claim 31, wherein the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug.

33. The method of claim 31, wherein the subject is identified as having or at risk of having myocarditis and / or myositis by a method comprising: detecting in a sample from the subject the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, 53 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO wherein a level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of a subject having or at risk of having myocarditis and / or myositis.

34. A method of treating cancer in a subject comprising: a) administering to the subject an immune checkpoint therapy (ICT) and optionally an anti- cancer treatment, b) identifying a risk for the subject to develop ICT-related myocarditis and / or ICT-related myositis, and c) administering to the subject identified as having or at risk of developing ICT-related myocarditis and / or immune-related myositis in b) a therapeutic compound to inhibit IL-1ȕ and TNF in combination with the ICT and optionally the anti-cancer treatment, thereby treating cancer in the subject.

35. The method of claim 34, wherein identifying a risk for the subject to develop ICT-related myocarditis and / or ICT-related myositis comprises: detecting in a sample from the subject the level of cytotoxic CD8+ T-cells and / or inflammatory myeloid cells, wherein a level of the cytotoxic CD8+ T-cells and / or inflammatory myeloid cells that is higher than the level in a subject not having or at risk of having myocarditis and / or myositis is indicative of a subject having or at risk of having myocarditis and / or myositis; and / or detecting in CD45+ immune cells from a sample from the subject a level of expression of a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling and / or a complement signaling, as compared to a TNF signaling via NF^B, an interferon-Į signaling, an interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or a complement signaling in CD45+ immune cells from a sample obtained from a control subject that is not having or at risk of having myocarditis and / or myositis, wherein an increase in TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling, as compared to TNF signaling via NF^B, interferon-Į signaling, interferon-Ȗ signaling, an IL2 / STAT5 signaling, a hypoxia signaling and / or complement signaling in CD45+ immune cells from a sample obtained 54 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO from a control subject is indicative of a subject having or at risk of having myocarditis and / or myositis.

36. The method of claim 26, wherein the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug.

37. A method of treating immune checkpoint therapy (ICT)-related myocarditis and / or ICT- related myositis in a subject comprising: a) identifying the myocarditis and / or myositis as ICT-related myocarditis and / or ICT-related myositis, and b) administering to the subject identified in a) as having ICT-related myocarditis and / or ICT-related myositis a therapeutic compound to inhibit IL-1ȕ, TNF and / or JAK / STAT in combination with the ICT, thereby treating ICT-related myocarditis and / or ICT-related myositis in a subject.

38. The method of claim 37, wherein the therapeutic compound that inhibits IL-1ȕ, TNF and / or JAK / STAT is an antibody or small molecule drug.

39. The method of claim 1, 27, 31 or 37, wherein the subject has cancer.

40. The method of claim 39, wherein the cancer is selected from metastatic melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, malignant pleural mesothelioma, esophageal squamous carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, small cell lung cancer, esophageal carcinoma, gastric cancer (or gastroesophageal junction cancer), bladder cancer, breast cancer, lung cancer, brain cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, melanoma, ovarian cancer, renal cancer or urothelial carcinoma.

41. The method of claim 1, 27, 31, 34 or 37, wherein the subject is a human.

42. The method of claim 27, 31, 34 or 37, wherein the subject has previously received an immune checkpoint therapy (ICT).

43. The method of claim 4 or 42, wherein the ICT is selected from the group consisting of anti- CTLA-4, anti-PD-L1, anti-PD-1, anti-LAG3 monotherapy and a combination thereof. 55 ACTIVE\1612701606.1PATENT ATTORNEY DOCKET NO.: MDA1240-1WO 44. The method of claim 4 or 42, wherein the ICT is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, spartalizumab and a combination thereof.

45. The method of claim 26, 29, 32, 36 or 38, wherein the IL-1ȕ inhibitor is rilonacept, canakinumab or glyburide.

46. The method of claim 26, 29, 32, 36 or 38, wherein the TNF inhibitor is infliximab, adalimumab, etanercept, golimumab, certolizumab, thalidomide or a combination thereof.

47. The method of any of claims 12-46 wherein the TNF is TNF-alpha.

48. The method of claim 26, 29, 32, 36 or 38, wherein the JAK / STAT inhibitor is ruxolitinib. 56 ACTIVE\1612701606.1

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