Small molecule inhibitors of OCA-b and OCA-t

Abstract

The present disclosure is concerned with bisamidine compounds that inhibit OCA-B and / or OCA-T. The disclosed bisamidine compounds and compositions can be useful in the treatment of a variety of diseases and disorders such as an autoimmune disease and cancer. The compounds can also be useful for the treatment or prevention of an immune-related adverse event (irAE) in a cancer patient undergoing immunotherapy such as, for example, a rash, diarrhea, colitis, hypothyroidism, and pneumonitis. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

Attorney Docket No. 21101.0501 PlSMALL MOLECULE INHIBITORS OF OCA-B AND OCA-TSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under R01 AI162929 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This Application claims the benefit of U.S. Application No. 63 / 753,322, filed on February 3, 2025, the contents of which are incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing submited on February’ 2, 2026 as a xml file named“21101.0501P1.xml,” created on January 30, 2026, and having a size of 4,562 bytes is hereby incorporated by reference pursuant to 37 C. F. R. § 1.52(e)(5).BACKGROUND

[0004] Fully 1 in 15 US individuals suffer from some form of autoimmunity, frequently with associated chronic morbidity, and there remain few curative or preventative autoimmune therapies. Multiple sclerosis is a debilitating autoimmune disease in which the host immune system is directed towards antigens associated with glial cells and myelinated axons of the central nervous system (CNS). It affects >2.5 million individuals worldwide, with over a million diagnosed in tire United States. Disturbingly, these numbers are increasing.Pathologically, MS is characterized by leukocyte CNS infiltration, inflammation, neuronal damage, and demyelination. Recently deployed treatments, such as injectable B cell depleting and integrin-blocking monoclonal antibodies, have made a significant impact, but are expensive, do not perform well in all patients and are associated with side effects stemming from nonspecific immunosuppression. Most critically, enough time has elapsed that progression independent of relapse activity (PIRA) has been detected, suggesting that these expensive top-line injectables don't treat this most dangerous facet of the disease. Therapeutic options for patients with primary-progressive and secondary-progressive MS, the most aggressive forms of the disease (Dobson R, Giovannoni G. Multiple sclerosis - a review, EurAttorney Docket No. 21101.0501 PlJ Neurol. 2019 Jan;26(1):27-40; Granqvist M, et al., JAMA Neurol. 2018 Mar 1;75(3):320-327), are even more limited.

[0005] Unlike these treatments, an ideal therapy would block MS while preserving normal immune function. Attaining such a treatment is the long-term goal in the field. The proposal addresses this therapeutic gap, as the findings indicate that targeting the transcriptional coregulator OCA-B (Bob.l / OBF-1, gene symbol Pou2af could provide an avenue to specifically block autoreactive CD4+ T cells while sparing normal immunity. OCA-B is a B-and T cell-restricted transcription coactivator associated with Th17 cells (Yosef N, et al., Nature. 2013 Apr 25;496(7446):461-8). Th17 cells, in particular IL-17 / IFNy dual-producers, are major mediators of MS (Axtell RC, et al., Nat Med. 2010 Apr; 16(4): 406- 12; Schnell A, et al., Cell. 2021 Dec 22;184(26):6281-6298.e23).

[0006] The MHC class II locus has the strongest genetic influence on MS, implicating CD4+ T cells as primary drivers of disease pathogenesis, (Patsopoulos NA. Cold Spring Harb PerspectMed. 2018 Jul 2;8(7):a028951). CD4+T cells are often found at the periphery of active MS lesions and can be thought of as master regulators, whereas cytotoxic T cells and other cell types migrate deeper into tissues to mediate white matter damage (Rumble JM, et al., J Exp Med. 2015 Jan 12;212(1):23-35). In CD4 + T cells, OCA-B docks with the transcription factor Octi (gene symbol Pou2fl ) to regulate targets including 112, Ifng, Icos, Csf2 (Gmcsf), Tbx21 (Tbet) and 1117a (Shakya A, et al., J Biol Chem. 2011 Jan 7;286(1):450-9; Shakya A, et al., J Exp Med. 2015 Nov 16;212(12):2115-31). Upon T cell activation, these genes are activated by pathways that converge on tire broadly expressed transcription factors NF-AT, AP-1 and NF-KB. These well-characterized factors are the primary "on" switches. Drugs that block these activities, e.g., cyclosporin, effectively eliminate target gene expression. Such drugs represent clinical equivalents "sledgehammers" and have utility in many contexts, but also have drawbacks including global immunosuppression and toxicity due to the broad expression of their targets, e.g., NF-ATc3 in the heart. By contrast, OCA-B expression is confined to lymphocytes, and the mechanism identified controls gene expression only in cases of repeated antigen exposure, such as that observed in autoimmunity. Agents targeting this pathway would therefore be more analogous to precision “scalpels” that selectively inhibit overactive, pathogenic T cell responses. Without wishing to be bound by theory, it is hypothesized that OCA-B is a druggable factor that coordinately regulates target genes encoding cytokines and other immunomodulatory proteins to control MS pathogenesis. A corollary of the hypothesis is that OCA-B pharmaceutical inhibition will selectively inhibit autoimmunity while sparing baseline immune function.Attorney Docket No. 21101.0501 Pl

[0007] Both OCA-B and Octi, the transcription factor with which OCA-B docks, are highly conserved between mice and humans. Human polymorphisms affecting Octi (and therefore OCA-B) binding to the TNFA and CTLA4 loci are associated with IBD and SLE, respectively (van Heel DA, et al., Hum Mol Genet. 2002 May 15; 11( 11): 1281-9; Cunninghame Graham DS, et al., Hum Mol Genet. 2006 Nov 1; 15(21):3195-205). More recent work identified additional polymorphisms in Octi binding sites within the human IL 13, IL 18 and HLA-C loci (Leon Rodriguez DA, et al., PLoS Negl Trop Dis. 2016 Mar 30; 10(3):e0004580; Kiesler P, et al., Hum Mol Genet. 2009 Dec 1; 18(23):4513-20; Vince N, et al., Am J Hum Genet. 2016 Dec 1;99(6): 1353-1358). These findings were collated with other studies into two meta-analyses showing a strong association between Octi / OCA-B binding site polymorphisms and autoimmunity including type-1 diabetes (T1D) and MS. It has been shown that OCA-B loss in T cells protects mice against T1D, supporting a broader role for OCA-B in autoimmunity. Two additional studies directly link polymorphisms in the human OCAB (POU2AF1) locus itself with. It 'as also found that OCA-B expression is elevated in pathogenic T cells from patients with secondary progressive MS, an aggressive MS subtype for which few treatments are available. Collectively, these studies associate human OCA-B with autoimmunity generally and MS in particular,

[0008] Recent evidence shows that pathogenic memory / stem-like T cells promote and sustain autoimmunity. Targeting these cells offers a potential therapeutic window in which disease can be inhibited while preserving baseline immunity. Recent findings show that OCA-B selectively promotes pathogenic maturation of stem-like T cells during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Using a reporter mouse, it was also shown that viable OCA-B -expressing CD4+T cells are selectively encephalitogenic in an EAE model, express high levels of Tcf1, as well as genes associated with pathogenic T cells e.g., Rorc, c-Maf, Il23r. In a relapsing-remitting EAE model, OCA-B T cell deficiency specifically protects mice from relapse. Using single-cell RNA-seq and flow cytometry, it was shown that the protection observed in T cell knockouts was associated with reduced numbers of stem-like T cells and reduced proinflammatory cytokine production.

[0009] It is relatively easy to “cure” autoimmunity in laboratory mice, e.g., by disrupting aspects of T cell development, antigen recognition, signaling or response. These manipulations result in global immunosuppression. In contrast to the dramatic protection observed with OCA-B T cell loss in EAE models, responses to CNS infection w'ere preserved. Intracranial infection was used with JHMV, a glial-tropic β-coronavirus which induces a clinical disease similar to EAE but without self antigen, to test the effect of OCA-BAttorney Docket No. 21101.0501 PlT cell loss on clinical severity, baseline immune responses and viral clearance. None were affected. This therapeutic window is not observed with, e.g., NF-AT or NF-KB loss, which cause global immunosuppression, and identifies OCA-B as a legitimate therapeutic target. A likely side effect of therapies targeting this pathway would be an inability to generate new memory CD4+ T cells, however this would be preferable to global immunosuppression. Further, immune repertoires are largely formed by the age at which most individuals are diagnosed with MS. A similar therapeutic window is observed with Octi, the transcription factor with which OCA-B26. While supporting the biology of the pathway, Octi itself is a poor drug target as it is widely expressed and important for embryonic development and adult somatic stem cell function. OCA-B's T and B cell-restricted expression, and the lack of a phenotype outside lymphoid cells in OCA-B germline-deficient mice, makes it the superior drug target. Because OCA-B is much more highly expressed in B compared to T cells, a calibrated dose of competitive inhibitor could selectively affect T cells while sparing known B cell-intrinsic OCA-B functions such as germinal center formation. Together, these findings suggest OCA-B as a promising therapeutic target for autoimmune disease.

[0010] OCA-B may also be a valid therapeutic target in B cell lymphoma. Beyond this, OCA-B is a member of a group of tissue-restricted transcription coregulators, known as OCA proteins: OCA-B, OCA-T1 / 2. OCA-T is a master regulator of tuft cells and a robust therapeutic target in small cell lung cancers originating from this cell type. For all three proteins, whole-body null mice are healthy, viable and fertile. Inhibitors could therefore be developed for autoimmune, B cell lymphoma and small cell lung cancer indications, however no small molecules targeting OCA proteins in either a clinical or laboratory setting are available.

[0011] Accordingly, there remains a need for compounds and compositions that inhibit OCA-B and / or OCA-T, which can be used for the treatment of autoimmune diseases and cancers, as well as for the treatment or prevention of immune-related adverse events (irAEs).SUMMARY

[0012] In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to bisamidine compounds that inhibit OCA-B and / or OCA-T. The disclosed bisamidine compounds can be useful in the treatment of a variety of diseases and disorders such as, for example, autoimmune diseases (e.g, primary biliary' cirrhosis, psoriasis, Addison disease. Celiac disease, dermatomyositis, GravesAttorney Docket No. 21101.0501 Pldisease, Hashimoto thyroiditis, inflammatory bowel disease, multiple sclerosis (MS), myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, systemic lupus erythematosus, and type I diabetes) and cancer (e.g., a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, kidney cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, lung cancer, a glioma, and plasma cell neoplasm (myeloma). The disclosed bisamidine compounds can also be useful in tire treatment or prevention of irAEs in a cancer patient undergoing immunotherapy. Examples of irAEs include, but are not limited to, a rash, diarrhea, colitis, hypothyroidism, and pneumonitis.

[0013] Thus, disclosed are methods of treating an autoimmune di sease in a subject in need thereof, the method comprising administering to the subject a compound having a structure selected from:or a pharmaceutically acceptable salt thereof.

[0014] Also disclosed are methods of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a compound having a structure represented by a formula:Attorney Docket No. 21101.0501 Plwherein X1is selected from O and S; wherein R1is selected from hydrogen and halogen; and wherein each of R2and R3is independently selected from -C(=NH)NHR10and Cyl; wherein each occurrence of R10is independently selected from hydrogen and C1-C4 alkyl; and wherein each occurrence of Cy1is independently a structure:wherein n is selected from 0 and 1; wherein each of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that when each of R2and R3is -C(=NH)NHR10, then either R1is halogen, R10is hydrogen, or R1is halogen and R10is hydrogen.

[0015] Also disclosed are compounds having a structure represented by a formula:wherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen.

[0016] Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of a disclosed compound or a pharmaceutically salt thereof, and a pharmaceutically acceptable carrier.

[0017] Also disclosed are methods of inhibiting OCA-B and / or OCA-T in a cell, the method comprising contacting the cell with an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof.Attorney Docket No. 21101.0501 Pl

[0018] Also disclosed are methods of inhibiting OCA-B and / or OCA-T in a subject, the method comprising administering to the subject an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof.

[0019] Also disclosed are methods of treating cancer in a subject in need thereof the method comprising administering to the subject an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof.

[0020] Also disclosed are kits comprising an effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof, and one or more of: (a) an agent known to treat an autoimmune disease; (b) a chemotherapeutic agent; (c) instructions for administering the compound in connection with treating an autoimmune disease, cancer, and / or an immune- related adverse event (irAE); and (d) instructions for treating an autoimmune disease, cancer, and / or an immune-related adverse event (irAE).

[0021] Also disclosed are methods of treating or preventing an immune-related adverse event (irAE) in a cancer patient undergoing immunotherapy, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:wherein X3is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each of R2and R3is independently selected from –C(=NH)NHR10or Cy1; wherein each occurrence of R10is independently selected from hydrogen or C1-C4 alkyl; and wherein each occurrence of Cy1is independently selected from a structure:wherein each occurrence of n is independently selected from 0 or 1; and wherein each occurrence of R20ais independently selected from hydrogen or halogen and each occurrence of R20bis independently selected from hydrogen or halogen, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 21101.0501P1

[0022] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.BRIEF DESCRIPTION OF THE FIGURES

[0023] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0024] FIG. 1A-J show representative data illustrating that OCA-B loss protects animals from chronic EAE.

[0025] FIG. 2A and FIG. 2B show representative data illustrating reclustered CD4 f nuclei gene expression and an analysis of peripheral blood CD4+ T effector cell bulk RNAseq.

[0026] FIG. 3A-H show representative data illustrating minimal differences in cytokine expressing CD4+ T cell, CD8+ T cell, B cell, macrophage, or microglial cell counts at peak chronic EAE in OCA-B knockouts.

[0027] FIG. 4A-K show representative data illustrating that loss of OCA-B does not strongly alter initial Thl or Th 17 polarization in vitro.

[0028] FIG. 5A-F show representative data illustrating that OCA-B promotes Thl and Thl7 adoptive -transfer EAE through recall response.

[0029] FIG. 6A-G show representative data illustrating that OCA-B is dispensable for T cell response to CNS infection with a neurotropic virus.

[0030] FIG. 7A-F show representative data illustrating minimal changes in immune responses to intracranial JHMV infection in the absence of OCA-B.

[0031] FIG. 8A-H show representative data illustrating that OCA-B expression marks encephalitogenic stem-like CD4+T cells.Attorney Docket No. 21101.0501 Pl

[0032] FIG. 9A-D show representative data for sorting OCA-B-mCherry reporter T cells and additional analyses of the effects of transferring OCA-Bhi versus OCA-Blo CD4+ cells.

[0033] FIG. 10A-I show representative data illustrating that OCA-B promotes relapsing¬ remitting EAE through stem -like CD4’ T cells.

[0034] FIG. 11A and FIG. 11B show representative data from individual NOD mouse EAE clinical scores.

[0035] FIG. 12A-C show representative data illustrating remission scRNA-seq cluster annotation, heat map of gene enrichments by cluster, and additional feature plots.

[0036] FIG. 13A and FIG. 13B show representative data illustrating EAE remission flow cytometry evaluating PD-1 expression in CNS infiltrating CD4+ T cells.

[0037] FIG. 14A-F show' representative data illustrating that OCA-B promotes disease relapse through control of pathogenic stem-like Thl7 differentiation.

[0038] FIG. 15A-C show representative data illustrating scRNA-seq cluster annotation genes, heat map of NOD. EAE gene enrichments by cluster, and TCR clonotype overlap between conditions at the relapse timepoint.

[0039] FIG. 16A and FIG. 16B show representative data of example relapse feature plots and a pseudotime analysis of relapse single cell RNA seq,

[0040] FIG. 17A-H show' representative data illustrating that OCA-B deletion in T cells confers protection against PD-1 blockade-induced diabetes in NOD mice.

[0041] FIG. 18A-F show representative data illustrating that OCA-B deletion in T cells protects NOD mice from spontaneous and PD-1 blockade-induced diabetes.

[0042] FIG. 19 show s representative data illustrating that OCA-B deletion in T cells leaves B16F10 tumor growth and immune response unaltered.

[0043] FIG. 20A-H show representative data illustrating that OCA-B in T cells is dispensable for anti-tumor immunity in C57BL / 6 mice.

[0044] FIG. 21A and FIG. 21B show representative data illustrating that OCA-B deletion in T cells decreases the TCM- and naive-like CD4+and CD8+T cell populations in MC38.

[0045] FIG. 22A-I show representative data illustrating that OCA-B in T cells is dispensable for anti -tumor immunity in NOD mice.

[0046] FIG. 23A-C show additional representative data illustrating that OCA-B deletion in T cells confers protection against PD-1 blockade-induced diabetes in NOD mice.

[0047] FIG. 24A-I show' representative data illustrating cellular inhibition of OCA-B by¬ small molecule DNA minor groove binders.Attorney Docket No. 21101.0501P1

[0048] FIG. 25 shows representative data illustrating the effect of staurosporine on luciferase-based transcription activity in SupTl cells.

[0049] FIG. 26A-H show representative data illustrating cellular inhibition of OCA-B by additional small molecule DNA minor groove binders.

[0050] FIG. 27A-H show representative data showing that DB1033 inhibits OCA-B-Octl- DNA complex formation,

[0051] FIG. 28A-F show representative data illustrating the effect of DB 1033 and DB2232 on PD-1 blockade -induced diabetes in NOD mice.

[0052] FIG. 29A-E show representative data illustrating that DB1033 inhibits PD-1 blockade-induced diabetes in NOD mice.

[0053] FIG. 30 shows a representative schematic illustrating a primary OCA-B targeting strategy.

[0054] FIG. 31 shows a representative schematic for three OCA-B inhibitor strategies.

[0055] FIG. 32 shows representative data illustrating that SupTl cells express OCA-B at similar levels to activated primary mouse CD4 cells.

[0056] FIG. 33A-C show a representative schematic of the two compound screening assay systems.

[0057] FIG. 34A-E show representative data of hits from a computational chemical screen of 139,735 compounds.

[0058] FIG. 35A and FIG. 35B show data illustrating OCA-B expression in CD4+ T cells in human MS lesions.

[0059] FIG. 36 shows a representative schematic of the hit advancement strategy.

[0060] FIG. 37A-C show a representative schematic and data showing Octl's DNA binding domain and OCA-B form a ternary complex on DNA that can be detected by EMSA.

[0061] FIG. 38A-F show a representative schematic and data showing that an Octl-OCA-B chimera forms a stable complex on DNA.

[0062] FIG. 39 shows representative data illustrating that Chimeric Octl-OCA-B in complex with DNA is disrupted by DB1033.

[0063] FIG. 40A-C show a representative schematic and data for a crosslinked chimeric protein and mutant to be used in DEL screening.

[0064] FIG. 41 shows a representative schematic illustrating the Protein-Metabolite interaction core workflow.

[0065] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practiceAttorney Docket No. 21101.0501 Plof the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0066] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0067] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic me thods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0068] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for in terpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0069] Throughout this application, various publications are referenced, The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is notAttorney Docket No. 21101.0501 Plentitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.A. DEFINITIONS

[0070] As used in the specification and the appended claims, the singular forms “a,” “an"’ and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

[0071] As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0072] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0073] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to beAttorney Docket No. 21101.0501 Plsuch. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0074] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0075] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0076] As used herein, “ICso” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. in one aspect, an IC50can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein. In a further aspect, IC50refers to the half-maximal (50%) inhibitory concentration (IC) of a substance.

[0077] As used herein, “ECso” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% agonism of a biological process, or component of a process, including a protein, subuni t, organelle, ribonucleoprotein, etc. In one aspect, an EC50can refer to the concentration of a substance that is required for 50% agonism in vivo, as further defined elsewhere herein. In a further aspect, EC50 refers to the concentration of agonist that provokes a response halfway between the baseline and maximum response,

[0078] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0079] As used herein, the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.Attorney Docket No. 21101.0501 Pl

[0080] As used herein, the term "‘treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i. e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc,),

[0081] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0082] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

[0083] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenousAttorney Docket No. 21101.0501 Pladministration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0084] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve tire desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; tire rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0085] As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. A dosage forms can comprise inventive a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharmaceutically acceptable excipient, such as a preservative, buffer, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts ofAttorney Docket No. 21101.0501 Plphosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxy ethylene9- 10 nonyl phenol, sodium desoxycholate), solution and / or cryo / lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethyl silozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). A dosage form formulated for injectable use can have a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, suspended in sterile saline solution for injection together with a preservative.

[0086] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0087] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed pro tocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form, which may be supplied on computer readable memory' device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

[0088] As used herein, the term “therapeutic agent” includes any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and / or physiologic effect by local and / or systemic action. The term thereforeAttorney Docket No. 21101.0501 Plencompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14thedition), the Physicians' Desk Reference (64thedition), and The Pharmacological Basis of Therapeutics (12thedition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; anti-ALS agents such as entry inhibitors, fusion inhibitors, non¬ nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors, NCP7 inhibitors, protease inhibitors, and integrase inhibitors; analgesics and analgesic combinations, anorexics, antiinflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, ei ther ribonucleotides (RNA) or deoxyribonucleo tides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. Tire term "therapeutic agent" also includes without limitation, medicaments; vitamins; mineral supplements; substances usedAttorney Docket No. 21101.0501 Plfor the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or prodrugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0089] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i. e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0090] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary' derivatives include salts, esters, amides, sal ts of esters or amides, and N-oxides of a paren t compound.

[0091] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of sui table aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by tire inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly (anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions whichAttorney Docket No. 21101.0501 Plare compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0092] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary', individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0093] In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0094] The term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.Attorney Docket No. 21101.0501 Pl

[0095] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, -butyl, / -butyl, w-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a Cl alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, Cl -CIO alkyl, and the like up to and including a C1-C24 alkyl.

[0096] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below, Tire term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. Tire term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxyalkyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0097] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particularAttorney Docket No. 21101.0501P1substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0098] Hie term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0099] The term “polyalkylene group” as used herein is a group having two or more CH2groups linked to one another. The polyalkylene group can be represented by the formula —(CH2)a—, where “a” is an integer of from 2 to 500.

[0100] Tire terms “alkoxy” and “alkoxy!” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as — OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as — OA1— OA2or — OA1— (OA2)a— OA3, where “a” is an in teger of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0101] Hie term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C, Tire alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cy cloalky nyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0102] Tire term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to,Attorney Docket No. 21101.0501 Plcyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbomenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0103] Hie term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, and, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0104] Tire term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0105] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized π electrons above and below the plane of the molecule, where the π clouds contain (4n+2) π electrons. A further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.Attorney Docket No. 21101.0501 Pl

[0106] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. Tire aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, — NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon¬ carbon bond. For example, biaryl can be two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0107] The term “aldehyde” as used herein is represented by the formula — C(O)H.Throughout this specification “C(O)” is a shorthand notation for a carbonyl group, i.e., C=O.

[0108] The terms “amine” or “amino” as used herein are represented by the formula ——NA1A2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is —NH2.

[0109] The term “alkylamino” as used herein is represented by the formula — NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like,

[0110] Tire term “dialkylamino” as used herein is represented by tire formula — N(-alkyl)2 where alkyl is a described herein. Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N -ethyl -N -methylamino group, N-methyl-N -propylamino group, N-ethyl-N-propylamino group and the like.

[0111] Tire term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0112] Hie term “ester” as used herein is represented by the formula ——OC(O)A1or ——C(O)OA1, where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,Attorney Docket No. 21101.0501 Plaryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula —(A1O(O)C-A2-C(O)O)a— or —(A1O(O)C-A2-OC(O))a—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

[0113] The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. Tire term “polyether” as used herein is represented by the formula — (A1O-A2O)a —, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0114] The terms “halo,” “halogen,” and “halide” as used herein can be used interchangeably and refer to F, Cl, Br, or I.

[0115] Tire terms “pseudohahde,” “pseudohalogen,” and “pseudohalo” as used herein can be used interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanate, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0116] The term “heteroalkyl” as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0117] Hie term “heteroaryl” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, A’-methylpyrrolyl, quinolinyl,Attorney Docket No. 21101.0501P1isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[l,2-b]pyridazinyl, imidazo[l,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0118] The terms “heterocycle"’ and “heterocyclyl” as used herein can be used interchangeably and refer to single and multi -cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle"’ and “polycyclic heterocycle.” Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3- oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2, 3 -thiadiazole, 1,2,5-thiadiazole, and 1, 3, 4-thiadiazole, triazole, including, 1,2, 3 -triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazme, pyrazine, triazine, including 1,2,4-triazine and 1,3,5-triazine, tetrazine, including 1,2, 4, 5 -tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0119] Tire term “bicyclic heterocycle” or “bicyclic heterocyclyl” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring. BicyclicAttorney Docket No. 21101.0501 Plheterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[l,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-l,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, lH-pyrazolo[4,3-c]pyridin-3-yl; lH-pyrrolo[3,2-b]pyridin-3-yi; and 1H-pyrazolo[3,2-b]pyridin-3-yl.

[0120] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized, and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0121] Tire terms "‘hydroxyl” and “hydroxyl” as used herein are represented by the formula —OH.

[0122] The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0123] The term “azide” or “azido” as used herein is represented by the formula —N3.

[0124] The term “nitro” as used herein is represented by the formula — NO2.

[0125] Tire term “nitrile” or “cyano” as used herein is represented by the formula — CN.

[0126] Tire term “silyl” as used herein is represented by the formula — SiA1A2A3, where A1, A2, and A3can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0127] The term “sulfo-oxo” as used herein is represented by the formulas —S(O)A1, — S(O)? A1, - -OS(O)2. A1, or —OS(O)2OA1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a shorthand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, lire term “sulfone” as used herein is represented by theAttorney Docket No. 21101.0501P1formula A1S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, lire term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0128] The term “thiol” as used herein is represented by the formula — SH.

[0129] “R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

[0130] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” -whether preceded by the term “optionally” or not, means that one or more hydrogen of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, tire substituent may be either tire same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0131] The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0132] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CFb o- R0; (CH?)<v40Ro; -0(CH2)o-4R°, - O (CH2)O-4C(0)OR°; -(CH2)O-4CH(OR°)?.; -(CH2.)O-4SR°; -(CH2)O- d’h. which may beAttorney Docket No. 21101.0501P1substituted with R°; –(CH2)0–4O(CH2)0–1Ph which may be substituted with R°; –CH=CHPh, which may be substituted with R°; -(CH2)o 40(CH2)o i-pyridyl which may be substituted with R°; –NO2; –CN; –N3; –(CH2)0–4N(R°)2; –(CH2)0–4N(R°)C(O)R°; -N(R°)C(S)R°; - (CH2)o-4N(R°)C(0)NR°2; -N(R°)C(S)NR°?.; -(CII2)W4N(RO)C(0)0R°; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; –(CH2)0–4C(O)R°; –C(S)R°; –(CH2)0–4C(O)OR°; –(CH2)0–4C(O)SR°; –(CH2)0–4C(O)OSiR°3; –(CH2)0–4OC(O)R°; –OC(O)(CH2)0–4SR–, SC(S)SR°; –(CH2)0–4SC(O)R°; –(CH2)0–4C(O)NR°2; –C(S)NR°2; - C(S)SR°; -(CH2)O^OC(0)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(0)CH2C(0)Ro; - C(NOR°)R°; –(CH2)0–4SSR°; –(CH2)0–4S(O)2R°; –(CH2)0–4S(O)2OR°; –(CH2)0–4OS(O)2R°; - S(0)2NRO2; -(CH2)(W4S(0)RO; -N(RO)S(0)2NR°2; -N(R°)S(0)2RO; -N(OR°)R°; - C(NH)NR°2; -P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(ORC)2; SIR°3; -(CI-4 straight or branched alkylene)O-N(R°)2; or -(C1-4 straight or branched aikylene)C(O)O-~N(R°)2, wherein each R° may be substituted as defined below' and is independently hydrogen, Ci-6 aliphatic, -CH Ph, -0(CH2)o 1PI1, -CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently- selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or ary l mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below'.

[0133] Suitable monovalent substituents on R° (or the ring formed by taking tw o independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)O2R*, -(haloR*), -(CH2)o 2OH, -(CH2)o 2OR8, -(CH2)o2CH(OR*)2; -O(haloR’), -CN, — 3, -(CH2)O2C(O)R®, -(CH2)O2C(O)OH, -(CH2)O2C(O)OR*, -(CH2)O SR* -(CH2)o-2SH, -(CH2)o-2NH2, -(CH2)O 2NHR*, -(CH2)O-2NR*2, -NO2, -SiR*3, -OSiR*3, -C(O)SR* -(Ci-4 straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci 4 aliphatic, -CFbPb, -0(CH2)o 1PI1, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from ni trogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and S.

[0134] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNRV2, =NNHC(0)R*, =NNHC(0)0RAttorney Docket No. 21101.0501P1=NNHS(O)2R*, =NR*, =NOR*, –O(C(R*2))2–3O–, or –S(C(R*2))2–3S–, wherein each independent occurrence of R* is selected from hydrogen, Ci 6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2 3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5 -6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0135] Suitable substituents on the aliphatic group of R’ include halogen, - R”, -(haloR*), -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)OR’, -NH2, -NHR“, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CIRPh, -0(CH2)o-iPh, or a 5 -6 -membered saturated, partially unsaturated, or aryl ring having 0 -4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0136] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -RT, -NR12, -C(O)Rt, -C(O)ORr, -C(O)C(O)RT, -C(O)CH2C(O)Rt, - S(O)2RT, -S(O)2NRT2, --C(S)NR'2, -C(NH)NRT2, or -N(Rt)S(O)2Rt; wherein each R:is independently hydrogen, C1 6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of RT, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0137] Suitable substituents on the aliphatic group of RTare independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or w'here preceded by “halo” is substituted only with one or more halogens, and is independently Ci 4 aliphatic, -CH2PI1, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or ary l ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0138] Hie term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bondingAttorney Docket No. 21101.0501 Plelectrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate.

[0139] Tire terms “hydrolysable group” and “hydrolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999).

[0140] The term “organic residue” defines a carbon-containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

[0141] A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4- thiazolidinedione radical in a particular compound has the structure:Oregardless of whether thiazolidinedione is used to prepare the compound. In some embodiments the radical (for example an alkyl) can be further modified (i. e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” lire number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary’ elsewhere herein.

[0142] “Organic radicals,” as the term is defined and used herein, contain one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-Attorney Docket No. 21101.0501P112 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bound to at least some of the carbon atoms of the organic radical. One example of an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2 -naphthyl radical. In some embodiments, an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, wherein the terms are defined elsewhere herein. A few non-limiting examples of organic radicals that include heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals and the like.

[0143] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

[0144] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture.Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0145] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light, in describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about itsAttorney Docket No. 21101.0501 Plchiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane), The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0146] When the disclosed compounds contain one chiral center, the compounds exist in two enantiomeric forms. Unless specifically stated to the contrary, a disclosed compound includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixture. The enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step can liberate the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.Attorney Docket No. 21101.0501P1

[0147] Designation of a specific absolute configuration at a chiral carbon in a disclosed compound is understood to mean that the designated enantiomeric form of the compounds can be provided in enantiomeric excess (e.e.). Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%, for example, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. In one aspect, the designated enantiomer is substantially free from the other enantiomer. For example, the “R” forms of the compounds can be substantially free from the “S” forms of the compounds and are, thus, in enantiomeric excess of the “S” forms. Conversely, " S'" forms of the compounds can be substantially free of “R” forms of the compounds and are, thus, in enantiomeric excess of the “R” forms.

[0148] When a disclosed compound has two or more chiral carbons, it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to four optical isomers and two pairs of enantiomers ((S, S) / (R, R) and (R, S) / (S, R)). The pairs of enantiomers (e.g., (S, S) / (R, R)) are mirror image stereoisomers of one another. The stereoisomers that are not mirror-images (e.g., (S, S) and (R, S)) are diastereomers. The diastereoisomeric pairs can be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. Unless otherwise specifically excluded, a disclosed compound includes each diastereoisomer of such compounds and mixtures thereof.

[0149] The compounds according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug forming moieties. For instance, the hydroxymethyl position may form mono-, di- or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander etal., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al,, PCT WO 2000 / 041531, p, 30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.

[0150] “Derivatives” of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof. Tire “combinations” mentioned in this context are refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, and solvates. Examples of radio¬Attorney Docket No. 21101.0501P1actively labeled forms include compounds labeled with tritium, phosphorous-32, iodine-129, carbon- 11, fluorine- 18, and the like.

[0151] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically labeled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,35S,18F and36C1, respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0152] The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary' number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.

[0153] Hie term “co-crystal"’ means a phy sical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework in the crystalline lattice. In certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. Do PharmaceuticalAttorney Docket No. 21101.0501P1Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et. al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p- toluenesulfonic acid and benzene sulfonic acid.

[0154] It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.keto form enol form amide form imidic acid form Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. As another example, pyrazoles can exist in two tautomeric forms, N1-unsubstituted, 3-A3and N1-unsubstituted, 5-A3as shown below.A4A4N-N N-NH HUnless stated to the contrary, the invention includes all such possible tautomers,

[0155] It is known that chemical substances form solids that are present in different states of order, which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.

[0156] In some aspects, a structure of a compound can be represented by a formula:which is understood to be equivalent to a formula:Rn(a)Rn(d)wherein n is typically an integer. That is, R” is understood to represent five independent substituents, R”(a), R"(b), R"(c), R”(d), R”(e). By “independent substituents,” it is meant that eachAttorney Docket No. 21101.0501 PlR substituent can be independently defined. For example, if in one instance R”(a)is halogen, then R"(b)is not necessarily halogen in that instance.

[0157] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N. J.), Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry’, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0158] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and tire number or type of embodiments described in the specification.

[0159] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and di scussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and eveiy combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary’. Thus, if aAttorney Docket No. 21101.0501 Plclass of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0160] It is understood that the compositions disclosed herein have certain functions.Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve tire same result.B. COMPOUNDS

[0161] In one aspect, the invention relates to bisamidine compounds for use in the treatment of autoimmune diseases such as, for example, primary biliary cirrhosis, psoriasis, Addison disease, Celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory bowel disease, multiple sclerosis (MS), myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, systemic lupus erythematosus, and type I diabetes.

[0162] In one aspect, the invention relates to bisamidine compounds for use in the treatment of cancer such as, for example, a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, non-small cell lung carcinoma, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, plasma cell neoplasm (myeloma).

[0163] In one aspect, the invention relates to bisamidine compounds for use in the treating or preventing irAEs such as, for example, a rash, diarrhea, colitis, hypothyroidism, and pneumonitis, in a cancer patient undergoing immunotherapy.Attorney Docket No. 21101.0501 Pl

[0164] It is contemplated that each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed compound can be provided by the disclosed methods. It is also understood that the disclosed compounds can be employed in the disclosed methods of using.1. STRUCTURE

[0165] In one aspect, disclosed are compounds having a structure selected from:andor a pharmaceutically acceptable salt thereof,

[0166] In one aspect, disclosed are compounds having a structure represented by a formula:wherein X1is selected from O and S; wherein R1is selected from hydrogen and halogen; and wherein each of R2and R3is independently selected from -C(=NH)NHR10and Cy1; wherein each occurrence of R10is independently selected from hydrogen and C1-C4 alkyl; and wherein each occurrence of Cy1is independently a structure:Attorney Docket No. 21101.0501P1wherein n is selected from 0 and 1; wherein each of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that when each of R2and R3is ~C(=NH)NHR10, then either R1is halogen, R10is hydrogen, or R1is halogen and R10is hydrogen.

[0167] In one aspect, disclosed are compounds having a structure represented by a formula:wherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen.

[0168] In one aspect, disclosed are compounds having a structure represented by a formula:wherein X3is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each of R2and R3is independently selected from ~C(=NH)NHRi0or Cy1; wherein each occurrence of R10is independently selected from hydrogen or C1-C4 alkyl; and wherein each occurrence of Cy1is independently selected from a structure:wherein each occurrence of n is independently selected from 0 or 1; and wherein each occurrence of R20ais independently selected from hydrogen or halogen and each occurrenceAttorney Docket No. 21101.0501P1of R20bis independently selected from hydrogen or halogen, or a pharmaceutically acceptable salt thereof.

[0169] In various aspects, the compound is:or a pharmaceutically acceptable salt thereof.

[0170] In various aspects, the compound is:or a pharmaceutically acceptable salt thereof.

[0171] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0172] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0173] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501P1or a pharmaceutically acceptable salt thereof.

[0174] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0175] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0176] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0177] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

[0178] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0179] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0180] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501P1provided that either Rlis halogen, each occurrence of R20aand R20bis independently selected from halogen, or R1is halogen and each occurrence of R'103and R20bis independently selected from halogen.

[0181] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof,

[0182] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0183] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof,

[0184] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

[0185] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0186] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0187] In various aspects, tire compound is selected from:Attorney Docket No. 21101.0501P1or a pharmaceutically acceptable salt thereof.

[0188] In various aspects, the compound is selected from:Attorney Docket No. 21101.0501 PlAttorney Docket No. 21101.0501P1or a pharmaceutically acceptable salt thereof.

[0189] In one aspect, n is independently selected from 0 or 1. In a further aspect, n is 0. In a still further aspect, n is 1,a. X1GROUPS

[0190] In one aspect, X1is selected from O and S. In a further aspect, X1is O. In a still further aspect, X1is S.b. X2GROUPSAttorney Docket No. 21101.0501P1

[0191] In one aspect, X2is selected from O, S, and Se. in a further aspect, X2is selected from O and S. In a still further aspect, X2is selected from S and Se. In yet a further aspect, X2is selected from O and Se. In an even further aspect, X2is O. In an even still further aspect, X2is S. In yet an even further aspect, X2is Se.c. X3GROUPS

[0192] In one aspect, X3is selected from O, S, and Se. In a further aspect, X3is selected from O and S. In a still further aspect, X3is selected from S and Se. In yet a further aspect, X3is selected from O and Se. In an even further aspect, X3is O. In an even still further aspect, X3is S, In yet an even further aspect, X3is Se,d. R1GROUPS

[0193] In one aspect, R1is selected from hydrogen and halogen. In a further aspect, R' is selected from hydrogen, -F, -Cl, and -Br. In a still further aspect, Rlis selected from hydrogen, -F, and -Cl. In yet a further aspect, R1is selected from hydrogen and -F.

[0194] In various aspects, R1is hydrogen.

[0195] In various aspects, R1is halogen. In a further aspect, R' is selected from -F, -Cl, and -Br. In a still further aspect, Rlis selected from -F, and -Cl. In yet a further aspect, R1is -F.e. R2AND R3G OUPS

[0196] In one aspect, each of R2and R3is independently selected from -C(=NH)NHR10and Cy1. In a further aspect, each of R2and R3is -C(=NH)NHR10. In a still further aspect, each of R2and R3is Cy1.

[0197] In various aspects, each of R2and R3is -C(=NH)NHR10. In a further aspect, each of R2and R3is -C(=NH)NII2

[0198] In various aspects, each of R2and R3is -C(:::NH)NH(C1-C4 alkyl). In a further aspect, each of R2and R3is independently selected from -C(=NH)NHCHs, - C(=NH)NHCH2CH3, -C(=NH)NHCH2CH2CH3, and -C(=NH)NHCH(CH3)2. In a still further aspect, each of R2and R3is independently selected from -C(=NH)NHCH3 and - C(:::NH)NHCH2CH3. In yet a further aspect, each of R2and R3is C(::NH)NHCH3. In an even further aspect, each of R2and R3is -C(=NH)NHCH2CH3. In an even still furtherAttorney Docket No. 21101.0501 Plaspect, each of R2and R’ is C(::NH)NHCH2CH2CH3. In yet an even further aspect, each of R2and R3is -C(=NH)NHCH(CH3)2.f. R10GROUPS

[0199] In one aspect, each occurrence of R10is independently selected from hydrogen and C1-C4 alkyl. In a further aspect, each occurrence of R!0is independently selected from hydrogen, methyl, ethyl, propyl, and isopropyl. In a still further aspect, each occurrence of R10is independently selected from hydrogen, methyl, and ethyl. In yet a further aspect, each occurrence of R10is independently selected from hydrogen and methyl.

[0200] In various aspects, each occurrence of R10is independently C1-C4 alkyl. In a further aspect, each occurrence of R10is independently selected from methyl, ethyl, propyl, and isopropyl. In a still further aspect, each occurrence of Ri0is independently selected from methyl, and ethyl. In yet a further aspect, each occurrence of R10is methyl.

[0201] In various aspects, each occurrence of R!0is hydrogen.g. R?" ' AND R20BGROUPS

[0202] In one aspect, each of R20aand R20bis independently selected from hydrogen and halogen. In a further aspect, each of R2uaand R20bis independently selected from hydrogen, -F, -CI, and -Br. In a still further aspect, each of R20aand R20bis independently selected from hydrogen, F, and -Cl. In yet a further aspect, each of R20aand R20bis independently selected from hydrogen and -F.

[0203] In various aspects, each of R20aand R20bis hydrogen.

[0204] In various aspects, each of R20aand R20bis halogen, in a further aspect, each of R20aand R20bis independently selected from -F, -Cl, and -Br. In a still further aspect, each of R20aand R2ubis independently selected from -F, and -Cl, In yet a further aspect, each of R20aand R20bis -F.h. CY1GROUPS

[0205] In one aspect, each occurrence of Cy1is independently a structure:R20a4>^NHR20bnAttorney Docket No. 21101.0501 Plwherein n is selected from 0 and 1.

[0206] In various aspects, Cy1is a structure:

[0207] In various aspects, Cy' is a structure:

[0208] In various aspects, Cy' is a structure:

[0209] In various aspects, Cy1is a structure:F2. EXAMPLE COMPOUNDS

[0210] In one aspect, a compound can be present as one or more of the following structures:Attorney Docket No. 21101.0501P1or a pharmaceutically acceptable salt thereof.3. PROPHETIC COMPOUND EXAMPLES

[0211] The following compound examples are prophetic, and can be prepared using the synthesis methods described herein above and other general methods as needed as would be known to one skilled in the art. It is anticipated that the prophetic compounds would be active as inhibitors of OCA-B and / or OCA-T, and such activity can be determined using the assay methods described herein below.

[0212] In one aspect, a compound can be selected from:Attorney Docket No. 21101.0501 PlAttorney Docket No. 21101.0501P1or a pharmaceutically acceptable salt thereof.

[0213] It is contemplated that one or more compounds can optionally be omitted from the disclosed invention.

[0214] It is understood that the disclosed compounds can be used in connection with the disclosed methods, compositions, kits, and uses.

[0215] It is understood that pharmaceutical acceptable derivatives of the disclosed compounds can be used also in connection with the disclosed methods, compositions, kits, and uses. The pharmaceutical acceptable derivatives of the compounds can include any suitable derivative, such as pharmaceutically acceptable salts as discussed below, isomers, radiolabeled analogs, tautomers, and the like.C. METHODS OF MAKING A COMPOUND

[0216] The compounds of this invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein.

[0217] Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the following Reaction Schemes, as described and exemplified below. In certain specific examples, the disclosed compounds can be prepared by Route I as described and exemplified below. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting.1. ROUTE I

[0218] In one aspect, substituted pyrimidinyl analogs can be prepared as shown below.Attorney Docket No. 21101.0501P1SCHEME 1A.

[0219] Compounds are represented in generic form with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.SCHEME IB.

[0220] In one aspect, compounds of type 1.8, and similar compounds, can be prepared according to reaction Scheme IB above. Thus, compounds of type 1.8 can be prepared by a condensation reaction of two aryl 1,2 diamines e.g., 1.5 and 1.6 as shown above, and a dialdehyde, e.g., 1.7 as shown above. Appropriate aryl 1,2-diamines and appropriate dialdehydes are commercially available or prepared by methods known to one skilled in the art. The condensation reaction is carried out in an appropriate solvent, e.g., 1,4 benzoquinone and ethanol, at an appropriate temperature, e.g., reflux.Attorney Docket No. 21101.0501 PlD. PHARMACEUTICAL COMPOSITIONS

[0221] In one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0222] Thus, in one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a compound having a structure represented by a formula:wherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen.

[0223] In various aspects, tire compounds and compositions of tire invention can be administered in pharmaceutical compositions, which are formulated according to the intended method of administration. Tire compounds and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, a pharmaceutical composition can be formulated for local or systemic administration, e.g., administration by intravenous, topical, or oral administration.

[0224] The nature of the pharmaceutical compositions for administration is dependent on the mode of administration and can readily be determined by one of ordinary skill in the art. In various aspects, the pharmaceutical composition is sterile or sterilizable. The therapeutic compositions featured in the invention can contain carriers or excipients, many of which are known to skilled artisans. Excipients that can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. The nucleic acids, polypeptides,Attorney Docket No. 21101.0501 Plsmall molecules, and other modulatory compounds featured in the invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. A modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, for injection, or for ingestion; gels or powders can be made for ingestion or topical application. Methods for making such formulations are well known and can be found in, for example. Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA 1990.

[0225] In one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a compound

[0226] In various aspects, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. Tire pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0227] In various aspects, the pharmaceuti cal compositions of this invention can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds,

[0228] Tire pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0229] In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the likeAttorney Docket No. 21101.0501 Plcan be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0230] A tablet containing the composition of this invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0231] The pharmaceutical compositions of the present invention comprise a compound of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy,

[0232] Pharmaceutical compositions of the present invention suitable for paren teral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to preven t the detrimental grow th of microorganisms.

[0233] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol,Attorney Docket No. 21101.0501 Plpropylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0234] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouthwashes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and w ater, together w ith about 5 wl% to about 10 wl% of the compound, to produce a cream or ointment having a desired consistency.

[0235] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein tire carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Tire suppositories can be conveniently formed by first admixing tire composition with the softened or melted carrier) s) followed by chilling and shaping in molds.

[0236] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including antioxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient.Compositions containing a compound of the invention, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0237] In a further aspect, an effective amount is a therapeutically effective amount. In a still further aspect, an effective amount is a prophylactically effective amount.

[0238] In a further aspect, the pharmaceutical composition is administered to a mammal. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient.

[0239] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.Attorney Docket No. 21101.0501 PlMETHODS OF TREATING AN AUTOIMMUNE DISEASE

[0240] In one aspect, disclosed are methods of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a compound having a structure selected from:or a pharmaceutically acceptable salt thereof.

[0241] In one aspect, disclosed are methods of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a compound having a structure represented by a formula:wherein X1is selected from O and S; wherein R' is selected from hydrogen and halogen; and wherein each of R2and R3is independently selected from -C(=NH)NHR10and Cy1; wherein each occurrence of R10is independently selected from hydrogen and C1-C4 alkyl; and wherein each occurrence of Cy' is independently a structure:Attorney Docket No. 21101.0501 Plwherein n is selected from 0 and 1; wherein each of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that when each of R2and R3is ~C(=NH)NHR10, then either R1is halogen, R10is hydrogen, or R1is halogen and R10is hydrogen.

[0242] In various aspects, the compound is:or a pharmaceutically acceptable salt thereof.

[0243] In various aspects, the compound is:or a pharmaceutically acceptable salt thereof.

[0244] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0245] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0246] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

[0247] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0248] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0249] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0250] In various aspects, the compound is selected from:Attorney Docket No. 21101.0501 PlAttorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

[0251] In various aspects, the subject is a mammal. In a further aspect, the subject is a human.

[0252] In various aspects, the subject has been diagnosed with a need for treatment of the autoimmune disease prior to the administering step.

[0253] In various aspects, the method further comprising the step of identifying a subject in need of treatment of the autoimmune disease.

[0254] In various aspects, the effective amount is a therapeutically effective amount. In a further aspect, the effective amount is a prophylactically effective amount.Attorney Docket No. 21101.0501 Pl

[0255] In various aspects, the autoimmune disease is selected from primary biliary cirrhosis, psoriasis, Addison disease, Celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory bowel disease, multiple sclerosis (MS), myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, systemic lupus erythematosus, and type I diabetes. In a further aspect, the autoimmune disease is MA. In a still further aspect, the autoimmune disease is type I diabetes.F. METHODS OF INHIBITING OCA-B AND / OR OCA-T IN A CELL

[0256] In one aspect, disclosed are methods of inhibiting OCA-B and / or OCA-T in a cell, the method comprising contacting the cell with an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.

[0257] Thus, in one aspect, disclosed are methods of inhibiting OCA-B and / or OCA-T in a cell, the method comprising contacting the cell with an effective amount of a compound having a structure represented by a formula:wherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen, or a pharmaceutically acceptable salt thereof.

[0258] In various aspects, the method inhibits OCA-B and OCA-T. In a further aspect, the method inhibits OCA-B. In a still further aspect, the method inhibits OCA-T.

[0259] In various aspects, the cell is mammalian. In a further aspect, the cell is human.

[0260] In various aspects, the cell has been isolated from a human prior to the administering step.

[0261] In various aspects, contacting is via administration to a subject.Attorney Docket No. 21101.0501 Pl

[0262] In various aspects, the subject has been diagnosed with a need for inhibition of OCA-13 and / or OCA-T activity prior to the administering step.

[0263] In various aspects, the subject has been diagnosed with a need for treatment of disease or disorder associated with OCA-B and / or OCA-T activity.G. METHODS OF INHIBITING OCA-B AND / OR OCA-T IN A SUBJECT

[0264] In one aspect, disclosed are methods of inhibiting OCA-B and / or OCA-T in a subject, the method comprising administering to the subject an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.

[0265] Thus, in one aspect, disclosed are methods of inhibiting OCA-B and / or OCA-T in a subject, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:wherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen, or a pharmaceutically acceptable salt thereof.

[0266] In various aspects, the method inhibits OCA-B and OCA-T In a further aspect, the method inhibits OCA-B, In a still further aspect, the method inhibits OCA-T.

[0267] In various aspects, the subject has been diagnosed with a need for inhibition of OCA-B and / or OCA-T activity prior to the administering step.

[0268] In various aspects, the subject has been diagnosed with a need for treatment of a disease or disorder associated w ith OCA-B and / or OCA-T activity prior to the administering step.

[0269] In various aspects, the method further comprising the step of identifying a subject in need of treatment of a disease or disorder associated with OCA-B and / or OCA-T activity.Attorney Docket No. 21101.0501 PlH. METHODS OF TREATING CANCER IN A SUBJECT

[0270] In one aspect, disclosed are methods of treating cancer in a subject in a subject, the method comprising administering to tire subject an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof.

[0271] Thus, in one aspect, disclosed are methods of treating cancer in a subject in a subject, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:wherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen, or a pharmaceutically acceptable salt thereof.

[0272] In various aspects, the subject is a mammal. In a further aspect, the subject is a human.

[0273] In various aspects, the subject has been diagnosed with a need for treatment of cancer prior to tire administering step.

[0274] In various aspects, the method further comprising the step of identifying a subject in need of treatment of cancer.

[0275] In various aspects, tire effective amount is a therapeutically effective amount. In a further aspect, the effective amount is a prophylactically effective amount,

[0276] In various aspects, the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, kidney cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, chronicAttorney Docket No. 21101.0501 Plmyeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, lung cancer, a glioma, and plasma cell neoplasm (myeloma). In a further aspect, the cancer is lung cancer. In a further aspect, the cancer is non-small cell lung cancer (NSCLC).

[0277] In various aspects, the cancer is a lymphoma. In a further aspect, the lymphoma is diffuse large B cell lymphoma.I. METHODS OF TREATING OR PREVENTING AN IMMUNE-RELATED ADVERSE EVENT IN CANCER PATIENT UNDERGOING IMMUNOTHER PY

[0278] In one aspect, disclosed are methods of treating or preventing an immune-related adverse event (irAE) in a cancer patient undergoing immunotherapy, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:wherein X3is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each of R2and R3is independently selected from –C(=NH)NHR10or Cy1; wherein each occurrence of R10is independently selected from hydrogen or C1-C4 alkyl; and wherein each occurrence of Cy1is independently selected from a structure:wherein each occurrence of n is independently selected from 0 or 1; and wherein each occurrence of R20ais independently selected from hydrogen or halogen and each occurrence of R20bis independently selected from hydrogen or halogen, or a pharmaceutically acceptable salt thereof.

[0279] In various aspects, X3is selected from O and S and wherein when each of R2and R3is independently selected from –C(=NH)NHR10, then either R1is halogen, R10is hydrogen, or R1is halogen and R10is hydrogen.

[0280] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plprovided that either R1is halogen, each occurrence of R20aand R20bis independently selected from halogen, or R1is halogen and each occurrence of R20aand R20bis independently selected from halogen.

[0281] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0282] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof,

[0283] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0284] In various aspects, the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof,

[0285] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0286] In various aspects, the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0287] In various aspects, the compound is selected from:Attorney Docket No. 21101.0501 PlAttorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

[0288] In various aspects, the compound is selected from:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

[0289] In various aspects, the subject is a mammal. In a further aspect, the subject is a human.

[0290] In various aspects, the subject has been diagnosed with a need for treatment of the autoimmune disease prior to the administering step.

[0291] In various aspects, the method further comprising the step of identifying a subject in need of treatment of the autoimmune disease.

[0292] In various aspects, the effective amount is a therapeutically effective amount. In a further aspect, the effective amount is a prophylactically effective amount.

[0293] In various aspects, the irAE is selected from a rash, diarrhea, colitis, hypothyroidism, and pneumonitis.

[0294] In various aspects, the cancer patient is undergoing immunotherapy with an immune checkpoint inhibitor. In a further aspect, the immune checkpoint inhibitor is selected from pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, relatlimab. cemiplimab, dostarlimab, tremelimumab, retifanlimab, and toripalimab.J. Kus

[0295] In one aspect, disclosed are kits comprising a disclosed compound or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) an agent known to treat an autoimmune disease; (b) a chemotherapeutic agent; (c) instructions for administering the compound in connection with treating an autoimmune disease, cancer, and / or an immune-related adverse event (irAE); and (d) instructions for treating an autoimmune disease, cancer, and / or an immune-related adverse event (irAE).

[0296] Thus, in one aspect, disclosed are kits an effective amount of a compound having a structure represented by a formula:Attorney Docket No. 21101.0501 Plwherein X2is selected from O, S, and Se; wherein R1is selected from hydrogen and halogen; and wherein each occurrence of n is independently selected from 0 and 1; wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen, or a pharmaceutically acceptable salt thereof, provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen, or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) an agent known to treat an autoimmune disease; (b) a chemotherapeutic agent; (c) instructions for administering the compound in connection with treating an autoimmune disease, cancer, and / or an immune-related adverse event (irAE); and (d) instructions for treating an autoimmune disease, cancer, and / or an immune-related adverse event (irAE),

[0297] In various aspects, the agent known to treat an autoimmune disease is selected from a corticosteroid (e.g., prednisone, methylprednisolone)), a disease-modifying antirheumatic drug (DMARD) (e.g, methotrexate, sulfasalazine, leflunomide), a biologic (e.g., adalimumab, infliximab, secukinumab), an immunosuppressant (azathioprine, cyclosporine, mycophenolate mofetil), a janus kinase (JAK) inhibitor (e.g., tofacitinib), and a nonsteroidal anti-inflammatory drug (NSAID) (ibuprofen, naproxen).

[0298] In various aspects, the chemotherapeutic agent is selected from an alkylating or crosslinking agent, an antimetabolite agent, an antineoplastic antibiotic agent, a mitotic inhibitor agent, an mTor inhibitor agent, a kinase inhibitor, and an immune checkpoint inhibitor.

[0299] In various aspects, the antineoplastic antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt thereof.

[0300] In various aspects, the antimetabolite agent is selected from gemcitabine, 5- fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 21101.0501 Pl

[0301] In various aspects, the alkylating or cross-linking agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharmaceutically acceptable salt thereof.

[0302] In various aspects, the mitotic inhibitor agent is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etoposide, vincristine, ixabepilone, vinorelbine, vinblastine, mitoxantrone, and teniposide, or a pharmaceutically acceptable salt thereof.

[0303] In various aspects, the mTor inhibitor agent is selected from everolimus, siroliumus, and temsirolimus, or a pharmaceutically acceptable salt thereof.

[0304] In various aspects, the kinase inhibitor is selected from BCR-ABL inhibitors (e.g., imatinib, dasatinib, nilotinib, bosutinib, ponatinib), EGFR inhibitors (e.g., erlotinib, gefitinib, afatinib, osimertinib, dacomitinib), ALK Inhibitors (e.g., crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, VEGFR inhibitors (e.g., sunitinib, sorafenib, pazopanib, axitimb, regorafenib, lenvatinib, cabozantinib), BRAF inhibitors (e.g., vemurafenib, dabrafenib, encorafenib), MEK inhibitors (e.g., trametinib, cobimetinib, binimetinib), HER2 inhibitors (e.g., lapatinib, neratinib, tucatinib), CDK4 / 6 inhibitors (e.g., palbociclib, ribociclib, abemaciclib), BTK inhibitors (e.g., ibrutinib, acalabrutinib, zanubrutinib, pirtobrutinib), PI3K inhibitors (e.g., idelalisib, copanlisib, duvelisib, alpelisib), FLT3 inhibitors (e.g., midostaurin, gilteritinib, quizartinib), and JAK inhibitors (e.g., ruxolitinib, fedratinib, pacritinib, momelolitinib), or a pharmaceutically acceptable salt thereof,

[0305] In various aspects, the immune checkpoint inhibitor is selected from pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, relatlimab. cemiplimab, dostarlimab, tremelimumab, retifanlimab, and toripalimab.

[0306] In various aspects, the compound and the agent known to treat an autoimmune disease are co-packaged. In a further aspect, the compound and the agent known to treat an autoimmune disease are co-formulated.

[0307] In various aspects, the compound and the chemotherapeutic agent are co-packaged. In a further aspect, the compound and the chemotherapeutic agent are co-formulated.

[0308] The foregoing description illustrates and describes the disclosure. Additionally, the disclosure shows and describes only the preferred embodiments but, as mentioned above, it is to be understood that it is capable to use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the invention concepts as expressed herein, commensurate with the above teachings and / or the skill or knowledge of the relevant art. The embodiments described herein above are further intendedAttorney Docket No. 21101.0501 Plto explain best modes known by applicant and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the invention to the form disclosed herein. Also, it is intended to the appended claims be construed to include alternative embodiments.

[0309] All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application w ere specifically and individually indicated to be incorporated by¬ reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls.K. EXAMPLES

[0310] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0311] The Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way.1. OCA-B PROMOTES PATHOGENIC MATURATION OF STEM-LIKE CD4+T CELLS AND AUTOIMMUNE DEMYELINATION

[0312] There are 2.8 million individuals worldwide with multiple sclerosis (MS), with almost 1 million cases occurring in the United States (Walton C, et al. Mult Scler.2020:26(14): 1816-1821), Despite recent advances, the development of new therapies that block MS w'hile sparing mechanisms that prevent infection and viral recrudescence remains a critical goal. GWAS have identified the HLA loci as the most important determinant of MS pathogenesis, emphasizing the impact of T cells (Wtccc CC, et al. Nature.2011;476(7359):214-219; IMSGC. Am J Hum Genet. 2005;77(3):454-467). The associationAttorney Docket No. 21101.0501 Plwith the MHC class II locus in particular indicates that myelin-specific CD4+ T cells are key regulators of disease.

[0313] The predominant preclinical model of MS is experimental autoimmune encephalomyelitis (EAE), in which laboratory animals are immunized with myelin proteins or peptides to activate antigen-specific CD4* T cells that infiltrate the CNS and mediate disease, EAE within the C57BL / 6 mouse background establishes chronic disease in which Thl and Thl7 effector CD4+ T cell subsets are pathogenetic mediators (Adorini L, et al. Autoimmunity. 1996;23(1):53 -68; Langrish CL, et al. J Exp Med. 2005;201(2): 233 -240). After accessing the CNS, these cells secrete IFN-y, IL- 17, GM-CSF, and other inflammatory mediators and chemoattractants γ that recruit the cell types responsible for driving demyelination (Brucklacher-Waldert V, et al. Brain. 2009; 132(pt 12):3329— 3341; Montes M, et al. Clin Immunol. 2009:130(2): 133-144; Hartmann FJ, et al. Nat Commun.2014;5(l):5056).

[0314] Myelin-reactive T cells from the blood of patients with MS show stem / memory-like properties, with reduced need for costimulation (Scholz C, et al. J Immunol.1998;160(3): 1532-1538; Bums JB, et al. Cell Immunol. 2001;212(l):44-50; Pelfrey CM, et al. J Immunol. 2000; 165(3): 1641- 1651; Lovett-Racke AE, et al. J Clin Invest.1998; 101 (4):725— 730). Consistent with these findings, autoreactive and bystander memory¬ phenotype CD4+ T cells preferentially transfer demyelinating disease in mouse models (Lee HG, et al. Nat Commun. 2019; 10(1): 709; Williams JL, et al. J Neuroimmunol. 2011:234(1-2):84-9; Elyaman W, et al. Am J Pathol. 2008; 173(2):411-422). Although these studies have identified vital CD4+ T cell populations in EAE, further research on the mechanisms sustaining these populations and driving their pathogenic activity is required to develop more precise therapies.

[0315] Octi, a POU domain transcription factor encoded by Pou2fl, has been linked to autoimmune disease. GWAS studies have identified Octi binding site polymorphisms associated with a predisposition for autoimmune diseases, including MS (Maurano MT, et al. Science. 2012;337(6099):l 190-1195; Graham DSC, et al. Hum Mol Genet.2006;15(21):3195-3205; van Heel DA. Hum Mol Genet. 2002; 11(11): 1281-1289; Farh KK, et al. Nature. 2015;518(7539):337- 343). In CD4* T cells, Octi promotes the development of both CD4+ memory (Shakya A, et al. J Exp Med. 2015;212(12): 2115—2131) and EAE pathogenesis and demyelination (Kim H, et al. J Neuroinflammation. 2019; 16( 1): 133). In contrast, Octi loss preserves immune responses to the neurotrophic viral infection (Kim H, et al. J Neuroinflammation. 2019; 16(1): 133). These observations highlight a promising pathwayAttorney Docket No. 21101.0501 Plthat, when targeted, may limit autoimmune CNS demyelination without the broad immune suppression elicited by many current therapeutics. However, Octi is widely expressed and regulates both embryonic development and somatic stem cell function (Vazquez-Arreguin K, et al. PLoS Genet. 2019;15(5):el007687 Perovanovic J, et al. Sci Signal.2023:16(781):eadd5750; Sebastiano V, et al. Development. 2010; 137(21):3551—3560), making it a poor therapeutic target.

[0316] The lymphoid-restricted transcription cofactor OCA-B regulates transcription by docking with Octi and, in B cells, an additional related factor, Oct2 (Hwang SS, et al.Biochim Biophys Acta. 2016; 1859(6):825— 831). In B cells, OCA-B is essential for B cell maturation and germinal center formation (Teitell MA. Trends Immunol. 2003;24(10):546-553; Shi W, et al. Nat Immunol. 2015; 16(6):663— 673; Kim U, et al. Nature.1996;383(6600):542-547). In contrast to Octi, OCA-B whole-body knockouts are viable and fertile (Kim U, et al. Nature. 1996;383(6600):542-547), Polymorphisms inthe OCAB (POU2AF1) locus are associated with autoimmune diseases, including MS (The GAMES Collaborative Group. J Neuroimmunol. 2006; 179(1-2): 108-116; Nakamura M, et al. Am J Hum Genet. 2012;91(4):721-72.8). Although its expression is 50- to 100-fold lower in T cells compared with B cells (Sun W, et al. Proc Natl Acad Sci U S A.2024;121 (9):e2309153121), targeting either OCA-B by genetic deletion in T cells or its downstream functions with a membrane-permeable peptide mimic suppresses type-1 diabetes (Kim H, et al, J Exp Med. 2021;218(3): 133). OCA-B interacts with target genes such as 112, If g, 1121, Csf2 (Gmcsf), Tnfrsf4 (0x40), Icos, and Ctla4, but is dispensable for their expression after primary activation of T cells. Instead, OCA-B regulates these targets under a narrow range of conditions, such as after secondary stimulation in culture (Shakya A, et al. J Exp Med. 2015;212(12):2115— 2131). Many of these targets are directly implicated in MS pathogenesis (Brucklacher-Waldert V, et al. Brain. 2009;132(pt 12):3329— 3341; Montes M, et al. Clin Immunol. 2009;130(2): 133-144; Hartmann FJ, et al. Nat Commun.2014;5( 1):5056; Tzartos JS, et al. Am J Pathol. 2011; 178(2): 794-802; Ndhlovu LC, et al. J Immunol. 2001;167(5):299I-2999; Hurwitz AA, et al. Proc Natl Acad Sci U S A.2002;99(5):3013-3017; Arellano G, et al. Front Immunol. 2015;6:492; Rottman JB, et al. Nat Immunol. 2001;2(7):605-611; Rouse M, et al. Immunobiology. 2013;218(4):674-682.Duncker PC, et al. J Immunol. 2018;200(3):966-973), OCA-B controls expression of Tbx21 (Tbet), which encodes a key regulator of Thl effector responses but also promotes Th 17 differentiation and may interact with RORyt, a master regulator of Th 17 cells (Shakya A, et al. J Exp Med. 2,015;2,12(12):2115-2,131; Yosef N, et al. Nature. 2013;496(7446):461-Attorney Docket No. 21101.0501 Pl468; Ikegami I, et al. Biochem Biophys Res Commun. 2019:514(4): 1167- 1171). In the context of MS, the only prior OCA-B work used whole-body knockouts and showed partial protection in chronic C57BL / 6 EAE models (Ikegami I, et al. Biochem Biophys Res Commun.2019:514(4): 1167—1171). Cumulatively, these findings indicate OCA-B plays poorly defined but possibly important roles in MS pathogenesis.

[0317] Here, it has been shown that OCA-B within the T cell compartment drives EAE by promoting pathogenic maturation of stem-like CD4+ T cells. In C57BL / 6 mice, T cell OCA-B loss substantially protects mice from chronic EAE while preserving responses to the John H. Muller strain of murine hepatitis virus (JHMV), a neurotrophic coronavirus. Using passive transfer assays in which reactive T cells are primed with antigen in donor mice, polarized in culture, and then re-encounter antigen in naive recipients, it has been shown that OCA-B is critical for the infiltration of pathogenic cells into the CNS and the manifestation of clinical disease. Using an OCA-B reporter mouse, it has been found that OCA-B expressing CD4+ T cells taken from mice primed with myelin oligodendrocyte glycoprotein (MOG) peptide preferentially display pathogenic stem-like gene expression patterns and preferentially transfer demyelinating disease, highlighting OCA-B as both a marker and promoter of encephalitogenic autoimmune CD4+T cell activity. Intriguingly, T cell conditional OCA-B knockout in EAE models on the autoimmune-prone NOD background, which develop relapsing-remitting MS (RRMS) (Baker D, et al. Ann Clin Transl Neurol. 2019;6(8): 1362— 1372), specifically protects against relapse. Single cell transcriptomic analysis of CD3&, CNS-infiltrating T cells at remission and relapse indicates that OCA-B promotes disease relapse through control of stem -like T cell populations, driving them to pathogenic Th17 differentiation. Cumulatively, these findings mark OCA-B as a critical mediator of the pathogenic maturation of autoreactive T cells and a promising potential therapeutic target, a. METHODSi. SEX AS A BIOLOGICAL VARIABLE

[0318] The objective of this study was to determine the role of the transcriptional regulator OCA-B in demyelinating disease. OCA-B T cell conditional knockout mice and OCA-B reporter mice were used in multiple mouse-demyelinating-disease models to evaluate the impact of T cell OCA-B expression on clinical disease manifestation and the phenotypes of neuroinflammatoiy cells, particularly T cells. Male and female animals were used forAttorney Docket No. 21101.0501 Plexperiments except in active EAE experiments, for which the better model of spinal cord demyelination and inflammation is female mice.ii. ANALYSIS OF A PUBLICLY AVAILABLE HUMAN CONTROL AND MS SINGLE-NUCLEI RNA-SEQ DATASET

[0319] Initial evaluation of POU2AF1 expression in T cells was conducted in the interactive web browser created by the authors of the dataset availableat https: / / malhotralab.shinyapps.io / MS broad / . Briefly, P0U2AF1 differential gene expression was filtered to show T cell gene expression by diagnosis with a minimal threshold cutoff of 10 cells / sample. Cleaned, aligned matrix files were then downloadedfrom https: / / zenodo.org / records / 8338963 and subsequently processed by using the 10X Genomics CellRanger pipeline and further analyzed using the 10X Genomics Loupe Browser 7 software. A total of 19,906 CD4+cells were then subclustered and evaluatedfor POU2AF1 and IL1R1 gene expression by diagnosis.iii. CHRONIC C57BL / 6 EAE

[0320] Female C57BL / 6 littermate animals (11-12 weeks old) were used for all experiments. For adoptive-transfer experiments using the OCA-B-mCherry reporter allele (31). 11 -week-old female mice were used for chronic EAE and 9-week-old male mice were used as MOG35-55-primed adoptive -transfer donors. EAE was induced on day 0 by two 100 pL s.c. injections into both hind flanks of 0.5 pmol / mL MOG35 55 peptide (University of Utah Peptide Core) emulsified in CFA, which was composed of incomplete Freund’s adjuvant (77145, Thermo Fisher) together with 4 mg / mL heat-killed Mycobacterium tuberculosis (DF3114338, Fisher Scientific). Animals were given two 400-ng i.p. injections of B. pertussis toxin (181, List Biological laboratories) on days 0 and 2.iv. CLINICAL SCORING

[0321] EAE clinical scores were determined using a 0-5 scale with 0.5 scoring intervals: 0.5, tip of tail is limp or tail muscle strain observed; 1, completely limp tail; 1.5, completely limp tail and at least 1 hind limb consistently falls through wire rack when animal is dropped; 2, completely limp tail and 1 hind leg drags without movement beyond hip; 2.5, completely limp tail and both hind limbs drag without movement beyond hip; 3, complete hind limb paralysis; 3,5, complete hind limb paralysis with flat-laying hind quarters and hump in upperAttorney Docket No. 21101.0501 Pltorso; 4, complete hind limb and partial forelimb paralysis, minimal movement around cage; 4.5, complete hind limb and partial forelimb paralysis, no movement around cage; 5, seizure or death. Animals scoring a 4 or higher for longer than 48 hours were euthanized and scored 5 for the remainder of the experiment. JHMV clinical disease was evaluated using a previously described 0-4 point scale (Lane IE, et al. J Virol. 2000;74(3): 1415-1424).v. IMMUNOFLUORESCENCE AND HISTOLOGY

[0322] Spinal columns were isolated from C57BL / 6 Ocabfl / fl and Ocabfl / fl; CD4-Cre animals after EAE or JHMV-induced demyelination and fixed overnight in 4% paraformaldehyde at 4°C. Spinal cords were subsequently isolated and cryoprotected in 30% sucrose for 3-5 days. Thoracic and lumbar sections were embedded in OCT compound (Thermo Fisher Scientific) and frozen and stored at -80°C. Spinal sections (8 pm) were cut by cryostat and stained with Luxol fast blue (212170250, Thermo Fisher) along with hematoxylin (8495, ENG Scientific) and eosin (E511-25, Fisher Chemical). Demyelination was assessed by dividing the area of demyelinated white matter by total white matter, as previously described (Blanc CA, et al. Am J Pathol. 2015; 185( 10):2819- -2832; Plaisted WC, et al. PLoS One.2016;! l(6):e0157620), For immunofluorescence, spinal cord sections were blocked in 5% normal goat serum (005-000-121, Jackson ImmunoResearch) and 0.1% Tween-20 for 1 hour at room temperature. Sections were stained overnight at 4°C with primary antibody rabbit anti-MBP (1: 1000; PA 1050, Boster), followed by secondary antibody goat anti-rabbit conjugated with Alexa Fluor 488 (1:750; Al 1008, Invitrogen) for 2 hours at room temperature. Primary and secondary' antibodies were diluted in 1% BSA and 0.1% Tween-20. Coverslips were placed on slides using Fluoromount-G with DAPI (00495952, Invitrogen). Sections were imaged with a Zeiss Axioscan 7 Microscope Slide Scanner using a *20 objective.vi. FLOW CYTOMETRY AND SPECTRAL CYTOMETRY

[0323] Spinal cords and brains were harvested at peak disease or a specified time point and mechanically dissociated by passing through a 100 pm cell strainer, A 30% / 70% Percoll gradient (catalog 17544501, Cytiva) was used to enrich mononuclear cells. Cells from collected spleens and lymph nodes (cervical, brachial, axillary, and inguinal) were passed through a 70 pm strainer, and erythrocytes were lysed by ammonium -chlori de-potassium lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). For intracellular staining,Attorney Docket No. 21101.0501 Plcells were cultured in complete medium (RPMI 1640, 10% FBS, 1% penicillin / streptomycin, Glutamax) supplemented with 1 pL / mL brefeldin A (555029, Golgiplug, BD), 50 ng / mL phorbol myristate acetate (P1585, Sigma-Aldrich), and 1 pg / mL ionomycin (10634, Sigma-Aldrich) for 4 hours. Mouse I-A(b) MOGss-49 tetramers (GWYRSPFSRVVH (SEQ ID NOT: 1)) (Keeler GD, et al. Gene therapy-induced antigen-specific tregs inhibit neuro-inflammation and reverse disease in a mouse model of multiple sclerosis. Mol Ther. 2018;26(l): 173-183; Tu E, et al. T cell receptor-regulated TGF-P type i receptor expression determines T cell quiescence and activation. Immunity. 2018;48(4): 745— 759) and control I-A(b) human class II-associated invariant chain peptide (CLIP) tetramers (PV SKMRMATPLLMQA (SEQ ID NO: 2)) conjugated to allophycocyanin (APC) or phycoerythrin (PE) -were synthesized by the NIH tetramer core facility. Cells were stained with control CLIP or MOG38-49 tetramers for 1 hour at 37°C prior to live / dead, surface, and intracellular staining. Cultured cells were fixed using Cytofix / Cytopemi (554714, BD Biosciences) according to manufacturer’s protocol, and stained for intracellular cytokines in Perm / Wash buffer (BD Biosciences).

[0324] Antibodies used for flow cytometry included CD45-Percp (30F11; 103129, Biolegend); CDllb-APC / Cy7 (MI / 70; 101225, Biolegend); CD19-BUV661 (ID3; 612971, BD); CD19-FITC (103 / CD19: 152404, Biolegend); NK1 1-PE-Cy5 (PK136; 108715, Biolegend); CD3e-BV605 (17A2; 100237, Biolegend); CD3e-FITC (145-2C11; 100305, Biolegend); TCRb-BV570 (H57-597; 109231, Biolegend); CD4-BV711 (RM4-5; 100557, Biolegend): CD4-BUV395 (GK1.5: 565974, BD): CD8a-APC (53-6.7; 100712, Biolegend); CD8a-BUV737 (53-6.7; 612759, BD); I-A / I-E-BUV805 (M5 / 144.15.2; 748844, BD);CD62L-PE-Cy7 (MEL-14; 25-0621-82, eBioscience); CD44-Percp-Cy5.5 (IM7; 45-0441-80, eBioscience); Ly-108-BUV563 (13G3; 7741436, BD); FR4-BUV496 (12A5; 750415, BD); CD73-V450 (Ty / 23; 561544, BD); IL-23R-BV421 (12B2B64; 150907, Biolegend); CXCR6-BV711 (SA051D1; 151111, Biolegend); PD-l-APC / Fire810 (29F.1A12: 135251, Biolegend); IFN-γ–PE–Cy7 (XMG1.2; 25-7311-82, eBioscience); IFN-y APC (XMG1.2; 17-7311-81, eBioscience): IL-17a-BV605 (TCI 1 -18H10.1; 506927, Biolegend); IL-17A-BV650 (TC11-18H10.1; 506929, Biolegend); TNF-a-FITC (MP6-XT22; 506304, Biolegend); and GM-CSF-PE / Dazzle594 (MP1-22E9; 505421, BioLegend). Flow cytometry samples were profiled using a BD Fortessa (BD Biosciences) or an Aurora spectral flow cytometer (Cytek). FiowJo software (BD Biosciences) was used for data analysis.vii. PREPOLARIZATION PROFILING AND MIXED ADOPTIVE TRANSFERAttorney Docket No. 21101.0501 Pl

[0325] 10-week-old C57BL / 6 Ocaly1'1and Ocabfl / fl; CD4-Cre littermates were immunized with MOG55 55 in CFA, as described earlier in Methods. After 14 days, spleen and lymph node (cervical, axillary', brachial, and inguinal) cells were isolated and cultured in complete medium (RPMI 1640, 10% FBS, 1% penicillin / streptomycin, Glutamax) supplemented with 1 µL / mL brefeldin A (Golgiplug; 555029, BD), 50 ng / mL phorbol myristate acetate (P1585, Sigma-Aldrich), and 1 pg / mL ionomycin (10634, Sigma-Aldrich) for 4 hours. Cells were stained for surface markers, fixed using the Foxp3 / Transcription Factor Staining Buffer Kit (00-5523-00, Thermo Fisher), and stained for intracellular markers (IFN-y and IL-17).viii. TH1 AND TH17 POLARIZATION AND ADOPTIVE-TRANSFER C57BL / 6 EAE

[0326] 10 -12 week-old female C57BL / 6 littermate animals were used for all experiments. Donor animals were immunized with MOG35 -55 in CFA, as described earlier in Methods. At 10-14 days after immunization, spleen and lymph node (cervical, axillary', brachial, and inguinal) cells were collected and placed into mixed culture with MOG peptide and cytokines at 5.0 x 106cells / mL. For Th1 conditions, complete medium was supplemented with 50 pg / mL MOG35 55, 6 ng / mL miIL-6 (216-16, Peprotech), and 2 ng / mL recombinant mouse (rm) IFN-y (315-05, Peprotech). For Th17 conditions, complete medium was supplemented with 50 pg / mL MOG35 55, 8 ng / mL rmIL-23 (1887-ML-010, R& D Systems), 10 ng / mL rmIL-1α (211-11A, Peprotech), and 10 pg / mL anti-IFN-y (BE0055, BioXcell XMG1.2). After 4 days of respective culture, CD4+T cells were purified using negative magnetic selection and resuspended in PBS at 1.5 × 106cells / mL. A 100 pL cell suspension was injected i.p. into age-matched male C57BL / 6J recipient mice (The Jackson Laboratory, 000664) (~13 weeks old at time of disease induction). Clinical disease was scored for 15 days.ix. JHMV

[0327] Ocabfl / fland Ocabfl / fl;CD4-Cre C57BL / 6 mice were anesthetized using isoflurane. Mice were intracranially injected with 1500 PFU of JHMV (strain V34) suspended in 30 pL of Hank’s balanced salt solution (Thermo Fisher). Clinical disease -was assessed for 21 days using a previously described scale (Lane IE, et al. J Virol. 2000;74(3): 1415-1424). Mice were sacrificed at 7, 12, and 21 dpi to assess viral titers within brain homogenates using a plaque assay previously described (Dickey LL, et al. J Neuroinflammation. 2016; 13( l):240).Attorney Docket No. 21101.0501 PlSpinal cords were taken on 12 and 21 dpi to assess demyelination by DAPI / MBP and by Luxol fast blue and hematoxylin and eosin histology, respectively.x. OCA-B-MCHERRY ADOPTIVE-TRANSFER EAE

[0328] OCA-B -mCherry reporter allele animals (Sun W, et al. Proc Natl Acad Sci U S A.2024;121(9):e2309153121) were primed for 14 days with MOG35 55 in CFA, as described previously in Methods. After priming, spleen and lymph nodes were collected and CD4+T cells were purified by magnetic negative selection (Miltenyi Bio kit and LS columns). Non–CD4+T cells were removed from the LS column and placed on ice for subsequent in vitro culture. Purified CD4+T cells were separated by OCA -B-m Cherry7expression (PE-Texas Red) using FACS on a BD Aria cell sorter. OCA-B-mCherry-positive and -negative CD4+T cells were placed into mixed culture in complete medium with the previously purified non- CD4+T cells at a 1: 10 ratio (CD4 ‘ to non-CD4 ) at 1.0 × 106cells / mL. The culture was supplemented with 20 pg / mL MOG35 55 and 0.5 ng / mL IL-12p70 (210-12, Peprotech). After 2 days of culture, CD4+T cells were again purified through negative magnetic selection. Purified CD4 T cells from OCA-B -mCherry positive and negative cultures were resuspended in PBS at 8.8 * 106cells / mL. A 100 pL cell suspension was injected i.p. into age-matched male C57BL / 6J recipients. Clinical disease was assessed for 24 days.xi. BULK RNA-SEQ

[0329] OCA-B-mCherry reporter mice (n = 9) were primed for 14 days with MOG35 55 in CFA. After priming, lymph nodes (cervical, axillary, brachial, and inguinal) were isolated and mCherry -positive and -negative CD4+T cells were collected by FACS. Total RNA, 3 groups (pooled, n = 3 mice each), was isolated using the Quick RNA micro kit (R1050, Zymo Research) with DNase treatment (79254, Qiagen). RNA concentration was measured with a Qubit RNA IIS Assay Kit (Q32855, Fisher Scientific). RNA quality was evaluated with an RNA ScreenTape Assay (5067-5579 and 5067-5580, Agilent Technologies). Total RNA samples (5-500 ng) were hybridized with NEBNext rRNA Depletion Kit v2 (E7400, New England Biolabs) to diminish rRNA from the samples. Stranded RNA-Seq libraries were prepared as described using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L, New England Biolabs). Purified libraries were qualified on a 4150 TapeStation (Agilent Technologies) using a D1000 ScreenTape assay (5567-5582 and 5067-5583, Agilent Technologies). The molarity of adapter-modified molecules was defined byAttorney Docket No. 21101.0501 Plquantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (KK4824, Roche). Individual libraries were normalized to 5 nM. NovaSeq 150 x 150 bp sequencing libraries were chemically denatured and applied to an NovaSeq flow cell using the XP workflow (20043131, Illumina). After transfer to an Illumina NovaSeq 6000 instrument, 150 x 150 cycle paired-end sequencing was performed using a S4 reagent Kit vl.5 (20028312, Illumina). Between 8 million and 11 million reads were generated per sample and were aligned to the GRCm38 mouse reference genome. Differential gene expression and pathway analysis were conducted using the DESeq2 and Enrichr R packages.xii. NOD-EAE

[0330] Female littermate OcaV^ and Ocabfl / fl;CD4-Cre animals (15—18 weeks old) backcrossed to a NOD strain background (Kim H, et al. J Exp Med. 2021;218(3): 133) were used for all experiments. Diabetic mice (blood glucose > 150 mg / dL) were excluded from tire analysis. EAE was induced by two 100 pL s.c, injections, into both hind flanks, of 0.5 pmol / mL MOG35-55 peptide emulsified in incomplete Freuds adjuvant (77145, Thermo Fisher) supplemented with 4 mg / mL heat-killed M. tuberculosis (DF3114338, Fisher Scientific) to form CFA. Animals were given two 400-ng i.p. injections of B. pertussis toxin (81, List Biological Laboratories) on days 0 and 2. Clinical disease was assessed over 47 days using the same 5 -point scale described previously under Clinical Scoring. Remission time points were determined to be between days 22 and 26; first relapse was determined to be between days 26 and 39,xiii. SCRNA-SEQ

[0331] NOD.Ocabfl / fland NOD.Ocabfl / fl;CD4-Cre animals were induced with EAE and monitored for disease progression. Brains and spinal cords were isolated on day 24 (remission) and day 33 (relapse), and processed for FACS sorting. Viable cells were sorted by CD3e using a FACS Aria (BD Bioscience). Cells from each condition were isolated from 3-4 mice and combined. Cells were processed using the 5' 10X Genomics Chromium platform according to the manufacturer’s instructions. Paired-end high-throughput sequencing (n = 125 cycles) was performed using a NovaSeq instrument (Illumina).Sequencing reads were processed with the 10X Genomics CellRanger pipeline and further analyzed using the Seurat R package. Analysis of cells used a standard filtering protocol removing cells with unique feature counts of more than 5000 or less than 100, as well as cellsAttorney Docket No. 21101.0501 Plwith greater than 10% mitochondrial counts (indicative of dead cells). No more than 10% of total cells were removed by this process. Cells were subjected to unsupervised hierarchical clustering followed by UMAP to visualize clusters with similar gene expression and their relative positions. RNA velocity analysis was performed using velocyto. R, version 0.6, to model cell-state transitions using the guidelines provided in La Manno et al. (La Manno G, et al. Nature. 2018;560(7719):494— 498) and the associated repositoryat https: / / github.com / velocyto-team / velocyto.R, Branch: HEAD, Commit ID:83e6ed92c2d9c9640122dcebf8ebbb5788165a21. High-dimensional velocity vectors were graphed on the predefined Seurat UMAP projection using localized average grid projections. Pseudotime analysis was conducted using the Seurat and Monocle 3 packages. Integrated data were divided into subsets based on genotype, and Monocle 3 w'as used to calculate trajectory, order cells, and plot pseudotime for each condition.xiv. STATISTICS

[0332] Multiple 2-tailed student’s / -tests were used to determine statistical differences in EAE clinical scores, and all error bars denote ± SEM unless otherwise noted. Two-way ANOVA was used to determine differences in weight loss throughout disease progression. GraphPad Prism software was used for all statistics and graphing. Two-tailed Student’s / tests 'ere used to determine statistical differences in cell populations observed by flow cytometry, and all error bars represent ± SD unless otherwise noted.xv. STUDY APPROVAL

[0333] All animal experiments were performed in strict accordance with the NIH guide for the Care and Use of Laboratory animals and institutional guidelines for animal care at tire University of Utah under approved protocol 00001553.xvi. DATA AVAILABILITY

[0334] All RNA-Seq, single-cell sequencing, and single-cell T cell V(D)J sequencing data associated with the findings of this study have been deposited to National Center of Biotechnology Information’s GEO database under accession code GSE243727. Data values for all graphs and values behind any reported means are reported in the Supporting Data Values file.Attorney Docket No. 21101.0501 Plb. RESULTS

[0335] Elevated expression of OCAB in CD4’ T cells from secondary-progressive human MS lesions. To determine if the expression of the gene encoding OCA-B is elevated in human MS samples, high-throughput, single-nucleus RNA-Seq data of glial and immune cells were mined from a recent study that used single-nucleus RNA-Seq to evaluate gene expression in brain tissue from 54 patients with MS and 26 control participants without MS (Macnair W, et al. Neuron. 2025;113(3):396— 410). The 1 RRMS sample had farhigher POU2AF1 expression compared with the control (FIG. 1A–J). There were more primary-progressive MS samples; interestingly, only 1 sample had detectable expression of POU2AF1, perhaps reflecting imperfections in diagnoses. Most dramatically, a subset of secondary-progressive (SPMS) samples showed significantly elevated OCA-B expression (FIG. 1 A). CD4+cell nuclei from this dataset were then reclustered. Uniform manifold approximation and projection (UMAP) feature plots revealed that theelevated POU2AF1 expression found in T cell nuclei of patients with SPMS was confined to 1 specific CD4+cluster that also expresses the pathogenic marker IL1R1 (FIG. IB), This cluster was also marked by the expression of the T cell activation marker CD44 and the transcription factors LEF, ETS1, BCL6, and MAF (FIG. 2A). To assess ifelevated POU2 AF1 expression was restricted to CNS -infiltrating T cells, a recent bulk RNA-Seq dataset was analyzed (Sumida TS, et al. Sci Transl Med. 2024;16(762):eadp1720) comparing peripheral-blood memory T effector cells (i.e., CD4+CD25low / ne8CD127hiCD45RO‘) from 20 patients with RRMS and 20 healthy control participants. Similar to CNS T cells, POU2AF1 expression in peripheral CD4+T cells was elevated in patients with MS compared with control participants without MS (FIG. 2B). These findings identify elevated OCAB mRNA expression in T cells from patients with MS across multiple datasets,

[0336] Reclustered CD4+ nuclei gene expression and analysis of peripheral blood CD4+ T effector cell bulk RNAseq is shown in FIG. 2A and FIG. 2B. FIG. 2A shows additional feature plots of reclustered CD4+ T cell nuclei from single -nucleus RNAseq data of secondary-progressive MS (SPMS) patient lesions and controls (McNair et al. 2025 Neuron 113:396) showing cluster gene expression of CD4, CD44, LEF1, ETS1, BCL6, and MAF. FIG. 2B shows quantification of POU2AF1 counts from bulk RNAseq data of CD4+ T effector cells isolated from the peripheral blood of control (HC Teff) and MS (MS Teff) patients (Sumida et al. 2024 Sci. Trans. Med. 16:eapd1720). All data represent mean ±SDAttorney Docket No. 21101.0501P1and Welch’s T-test was used to determine statistical significance for data with unequal variance.i. OCA-B PROMOTES CHRONIC EAE IN C57BL / 6 MICE

[0337] To understand the impact of OCA-B in T cell mediated autoimmunity,an Ocab conditional allele crossed to the CD4-Cre driver was used, which efficiently deletes OCA-B in T cells (Kim H, et al. J Exp Med. 2021:218(3): 133). EAE in C57BL / 6 female Occ? A'yCD4-Cre mice and littermate Ocab^ controls were induced through s.c. injection of MOG peptide in CFA followed by i.p. injections of Bordetellapertussis toxin, Ocabfl / fl;CD4-Cre mice had reduced chronic EAE clinical scores and weight loss compared with Ocabfl / fl(FIG. 1C and FIG. ID). Spinal cords were taken at peak of disease (day 15) for immunofluorescence and histological analyses. Myelin basic protein (MBP) immunofluorescence showed a significant decrease in demyelinated white matter in spinal sections from Ocab“; CD4-Cre mice compared with littermate Ocab!l,!!controls, and DAPI staining showed simultaneously decreased immune cell infiltration (FIG. IE and FIG.IF). Ocabfl / fl;CD4-Cre mice also had a significant decrease in demyelination as measured by luxol fast blue (LFB) in combination with H& E staining (FIG. 3A and FIG. 3B). Flow cytometric profiling at peak disease showed decreased CD4+ T cell CNS infiltration in Ocabfl / fl;CD4-Cre mice; however, no differences were observed in other cell populations, anergic T cells (CD4+FR4+CD73+), or in the relative frequencies of the proinflammatory cytokines IFN-y, IL- 17, GM-CSF, or CD44 (FIG. 1G and FIG. 1H and FIG. 3C-H)

[0338] Minimal differences in cytokine expressing CD4+ T cell, CD8+ T cell, B cell, macrophage or microglial cell counts at peak chronic EAE in OCA-B knockouts is shown in FIG. 3A-H. FIG. 3A shows representative spinal cord LFB / H& E staining of thoracic sections taken from mice 15 days after EAE induction. Areas of demyelination are outlined in black and marked by increased loss of LFB (blue) staining and visibility of H& E (pink) within the white matter. FIG. 3B shows quantification of the % demyelination from LFB / H& E histology. Significance was ascribed by two-tailed student’s t-test, FIG. 3C shows quantification of the frequency of CNS infiltrating CD4+ T cells between Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two-tailed student’s t-test. FIG. 3D shows quantification of frequency and count of CD8a positive T cells between Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two-tailed student’s t-test. FIG. 3E show's quantification of the number of B cells, macrophages and microglia. Significance wasAttorney Docket No. 21101.0501 Plascribed by two-tailed student’s t-test. FIG. 3F shows representative flow cytometry plots comparing the expression of FR4 and CD 73 within CNS infiltrating CD4+ T cells from Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. FIG. 3G shows quantification of the frequency of CMS infiltrating FR4 f CD73- and FR4 + CD73+ CD4+ T cells within Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two-tailed student's t-test. FIG. 3H shows quantification of frequency and count of IFNg, IL- 17, GM-CSF, and CD44 expressing CD4+ T cells from Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two- tailed student’s t-test. All data represent mean ±SD.

[0339] MOG-specific CD4+T cells was studied using MHC class II (I-Ab) MOG38-49tetramers. At peak disease, OcaA’i; CD4-Cre mice had a trending reduction in MOG tetramer-specific cell frequency (P = 0.055) and a significant reduction in numbers (P = 0.003) compared with controls (FIG. II and FIG. 1 J). These results indicate the protection conferred by OCA-B loss in T cells is marked by modest decreases in MOG-specific CD4+ T cells within the CNS, resulting in fewer pathogenic T cells driving demyelination.

[0340] OCA-B loss protects animals from chronic EAE is shown in FIG. 1A-J. FIG. 1A shows CD4 T cell nuclei from single-nucleus RNA-Seq data of patients with primaryprogressive (PPMS), RRMS, or SPMS lesions and from control participants (Macnair W, et al. Neuron. 2025; 113(3): 396— 410) were identified and tested in silicofor POU2AF1 expression, which is displayed as counts per million. Significance was ascribed by Welch’s t test. FIG. IB shows the CD4+T cell nuclei were reclustered and displayed as UMAP feature plots showing POU2AF1 (left) and IL1R1 (right) expression in control (CTR) and SPMS brain tissue. Red arrow highlights a likely pathogenicCD4+population expressing high levels of IL1R1. FIG. 1C Ocabfl / fl(n = 16)and Ocabfl / fl;CD4-Cre (n = 22) mice were injected with MOG35 55 peptide in CFA and pertussis toxin to induce EAE. Clinical scores were evaluated after EAE induction to determine disease progression. Significance was ascribed by multiple 2-tailedStudent’s t tests, FIG. ID shows animal weights were recorded to evaluate weight loss as a measurement of disease progression. Significance was ascribed by 2-way ANOVA. FIG. IE representative DAPI / MBP immunostaining of thoracic spinal cord sections taken from mice 15 days after EAE induction. Areas of demyelination are marked by decreased MBP staining (green) and often coincide with increased cellular infiltration (blue). FIG. IF Quantification of demyelination in Ocabfl / fl(n = 5) and Ocabfl / fl;CD4-Cre mice (n = 6). Significance as ascribed by 2-tailed Student’s t test. FIG. 1G shows brain and spinal cords were isolated on day 15 of EAE and analyzed by spectral cytometry. Representative flow cytometry plots ofAttorney Docket No. 21101.0501 PlCD45 TCRP+CNS infiltrating T cells showing frequency of CD4+and CD8+T cells. FIG.1H shows quantification of the number of CNS infiltrating CD4+T cells. Ocab,1 / ’1(n = 9) and Oca&^; CD4-Cre (n = 13). Significance was ascribed bv 2-tailed Student’s t test. FIG.301 shows representative plots showing frequency of MOG38-49 tetramer-positive CD4+T cells. FIG. 1J shows quantification of the frequency and count of MOG38-49 tetramerpositive ( 1)4 T cells. Ocabfl / fl(n = 9) and Ocabfl / fl;CD4-Cre (n = 13). Significance was ascribed by 2-tailed Student’s t test. For clinical scores and normalized weights, values represent mean ± SEM. All other values represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.ii. OCA-B IS ESSENTIAL FOR EAE MEDIATED BY ADOPTIVE TRANSFER OF Till - AND TH17-POLARIZED T CELLS

[0341] MS is driven by autoreactive CD4+Thl and Thl7 cells within the CNS that recruit CD8+ T cells, macrophages, microglia, and neutrophils, which together mediate white-matter damage (Rumble JM, et al. J Exp Med. 2015;212(1):23— 35). To investigate the impact of OCA-B on autoimmune Thl and Thl7 populations, adoptive-transfer EAE models were used with female Oc<ty‘ra; CD4-Cre and Ocab littermate controls. Mice were primed with MOG35–55in CFA for 14 days. After MOG priming, CD4+T cells were purified from the lymph nodes of primed experimental or control donors (FIG. 4A). No differences between genotypes in CD4+T cell viability, count, or effector cytokine (IFN-y and IL- 17) expression were observed (FIG. 4B-E)

[0342] To investigate if OCA-B expression affects Thl- or Thl7-mediated adoptive transfer EAE, CD4+T cells purified from donors were polarized in vitro into Thl and Till 7 cells, as previously described (Kroenke MA, et al. J Exp Med. 2008:205(7): 1535-1541; Grifka-Walk HM, et al. Eur J Immunol. 2013;43( 11):2824— 2831). Via i.p. injection, 3 × 106Th1- or Th17-polarized CD4+ cells were transferred into naive, age-matched, wild-type, male C57BL / 6 recipients. EAE onset in mice receiving either Thl- or Thl7-polarized control cells was rapid and severe (FIG. 5 A and FIG. 5B, and FIG. 4F and FIG. 4G), In contrast to control cells, and in contrast to the mild protection observed with OCA-B deficiency in standard C57BL / 6 EAE, mice adoptively transferred with T cells lacking OCA-B were almost completely protected from clinical disease regardless of their polarization state (FIG. 5A and FIG. 4F and FIG. 4G). Interestingly, the ability to initially polarize CD4+T cells into Th1 and Th17 effectors in vitro was only minimally affected by OCA-B loss (FIG. 4H-J), whereas cellsAttorney Docket No. 21101.0501 Pltaken from the CNS of engrafted recipient mice at day 15 not only showed decreased CNS infiltration but also reduced IL- 17 expression (FIG. 5C-F). This effect appeared to be specific to IL-17, because no significant difference in IFN-y was observed (FIG. 4K). These results indicate a central role for OCA-B in promoting robust neuroinflammatory T cell responses upon antigen re-encounter in adoptive -transfer EAE models.

[0343] Loss of OCA-B does not strongly alter initial Thl or Th 17 polarization in vitro is shown in FIG. 4A-K. FIG. 4A shows experimental schematic for assessing CD4+ T cell differences between Ocabfl / fl and Ocabfl / fl; CD4-Cre prior to in vitro Thl or Th 17 polarization. Lymph nodes (cervical, brachial, axillary', and inguinal) were harvested from Ocabfl / fl (n=4) and Ocabfl / fl; CD4-Cre (n=4) mice following 14 days of MOG35-55 / CFA priming and analyzed by flow cytometry. FIG. 4B shows frequency of viable cells isolated from Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two-tailed student’s t-test. FIG. 4C shows total CD4+ T cell numbers isolated from Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two-tailed student’s t-test. FIG. 4D show's quantification of the frequency oflFNg expressing CD4+ T cells isolated from Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance was ascribed by two tailed student’s t- test, FIG. 4E shows quantification of the frequency of IL- 17 expressing CD4+ T cells derived from Ocabfl / fl and Ocabfl / fl; CD4-Cre mice. Significance w'as ascribed by two-tailed student’s t-test. FIG. 4F shows normalized weights of recipient mice following adoptive transfer of Ocabfl / fl or Ocabfl / fl; CD4-Cre Thl -polarized MOG-reactive CD4+ T cells. Significance 'as ascribed by Two-way ANOVA. FIG. 4G show s normalized weights of recipient mice following adoptive transfer of Ocabfl / fl or Ocabfl / fl; CD4-Cre Thl7 cells. Significance was ascribed by Two-way AM OVA FIG. 4H shows representative flow cytometry plots showing IFNg and IL-17 expressing CD4+ T cells from Ocabfl / fl (n=5) and Ocabfl / fl; CD4-Cre (n=5) mice after Till and Th 17 in vitro polarization. FIG. 41 shows quantification oflFNg expressing CD4+ T cells after Thl polarization. Significance was ascribed by two-tailed student’s t-test. FIG. 4A shows quantification of IL-17 expressing CD4+ T cells after Thl7 polarization. Significance 'as ascribed by two-tailed student’s t- test. FIG. 4K shows quantification of the frequency oflFNg expressing CD4+ T cells isolated from the spinal cords of Th 17 adoptive transfer EAE recipient mice. Significance was ascribed by two-tailed student’s t-test. Normalized weight variance is displayed as mean ±SEM. All other data represent mean ±SD.

[0344] OCA-B promotes Till and Till 7 adoptive-transfer EAE through recall response is shown in FIG. 5A-F. FIG. 5A and FIG. 5B show Ocabfl / fland Ocabfl / fl;CD4-Cre mice wereAttorney Docket No. 21101.0501P1primed with MOG35-55 peptide in CFA for 10—14 days. Cells from the spleens and lymph nodes of primed mice were isolated and cultured for 4 days in Th1 or Th17 polarizing conditions. Thl- or Thl7-polarized cells (n = 3.0 x 106) were injected i.p. into C57BL / 6 wild-type recipient mice (Th1: n = 5 Ocabfl / fl, n = 5 Ocabfl / fl;CD4-Cre) (Th17: n = 3 Ocabfl / fl, n = 4 Ocabfl / fl;CD4-Cre). Clinical scores were assessed for 14-15 days. Significance was ascribed by multiple 2-tailed Student’s t tests. FIG. 5C shows At Thl 7 adoptive-transfer EAE endpoint, brains and spinal cords were analyzed by flow cytometry'. Quantification of brain- and spinal cord -infiltrating CD4+T cells after Thl 7 adoptive transfer. Significance was ascribed by 2-tailed Student’s t test. FIG. 5D shows representative flow cytometry plots showing the frequency of IFN-y- and IL-17-expressing CD4+T cells within the spines of Thl7-recipient mice. FIG. 5E shows quantification of IL- 17 frequency in spinal cord-infiltrating CD4+T cells. Significance was ascribed by 2-tailed Student’s t test. FIG.5F shows quantification of the total count of IL-17+CD4+T cells within the spinal cord of recipient mice. Significance was ascribed by 2-tailed Student’s t test. For clinical scores, values represent mean ± SEM. All other values represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.iii. OCA-B IS DISPENSABLE FOR NEUROTROPIC VIRAL CLEARANCE.

[0345] To investigate if T cell OCA-B deletion affects the ability to mount immune responses and control CNS viral infection, male Ocabfl / fl;CD4-Cre and Ocabfl / fllittermate controls were intracranially inoculated with 1500 PFU of JHMV, a β-coronavirus adapted to infect glial cells (namely, astrocytes, microglia, and oligodendrocytes) in the mouse brain and spinal cord. Inoculation with JHMV results in acute encephalomyelitis and chronic demyelination similar to autoantigen-driven EAE. Disease severity was determined by evaluating clinical scores over 21 days postinfection (dpi). Ocabfl / fl;CD4-Cre animals showed negligible differences in clinical scores compared with Ocabfl / fllittermates (FIG. 6A).

[0346] Spinal cords were taken on 12 and 21 dpi for immunofluorescence and histological analysis to investigate differences in demyelination. MBP immunofluorescence at 12 dpi showed similar demyelination and immune infiltration between Ocabfl / fland Ocabfl / fl;CD4-Cre spinal sections (FIG. 6B and FIG. 6C). Similarly, histology of Luxol fast blue -stained brain tissue at 21 dpi showed no significant differences in demyelination between OCA-B- deficient and control infected animals (FIG. 7A and FIG. 7B). Flow cytometric analysis of brain tissue showed no significant differences in infiltrating CD4+T cell numbers or IFN-yAttorney Docket No. 21101.0501P1expression at 7, 12, or 21 dpi (FIG. 6D-F, and FIG. 7C). Similarly, no differences in either total CD8+T cell numbers or the percentage or numbers of IFN-y-expressing CD8+T cells were observed (FIG. 7D-F). Brain hemisphere homogenates were collected on 7, 12, and 21 dpi to measure viral titers and clearance. Ocabfl / fl;CD4-Cre and Ocabfl / fllittermate control animals had similar titers at dpi 7 and 12, and virus was undetectable at day 21 regardless of genotype (FIG. 6G). This result suggests OCA-B is dispensable for baseline immune responses to and clearance of neurotropic viruses.

[0347] Data illustrating that OCA-B is dispensable for T cell response to CNS infection with a neurotropic virus is shown in FIG. 6A-G. FIG. 6A shows Ocabfl / fl(n = 19)and Ocabfl / fl;CD4-Cre (n = 17) mice were injected intracranially with 1500 PFU JHMV, Clinical scores were recorded for 21 dpi. Significance was ascribed by Multiple 2-tailed student’s t tests. FIG. 6B shows representative DAPI / MBP immunofluorescence staining of spinal cord sections at 12 dpi. Demyelinated areas are marked by decreased MBP staining (green) and often coincide with increased cellular infiltration (blue). FIG. 6C shows quantification of percent demyelination in spinal cord sections (n = 7 per group). Significance was ascribed by 2-tailed Student’s t test. Half-brains were taken at 7, 12, and 21 dpi for flow cytometric and viral titer analysis. FIG. 6D shows quantification of CD4+T cell counts within the half-brains at 7. 12, and 21 dpi. Ocabfl / fl(n = 7-9 / time point) and Ocabfl / fl;CD4-Cre (n = 6-7 / time point). Significance was ascribed by 2-tailed Student’s t test. FIG. 6E shows representative plots showing the frequency of IFN-y-expressing CD4+T cells at 7, 12, and 21 dpi. FIG. 6F shows quantification of frequency of IFN-y-expressing CD4+Tcells. Ocabfl / fl(n = 7-9 / time point) and Ocabfl / fl;CD4-Cre (n = 6-7 / time point). Significance was ascribed by 2-tailed Student’s t test. FIG. 6G shows viral titers of half-brains at dpi 7, 12, and 21 (not detected), Ocabfl / fl(n = 7-9 / time point) and Ocabfl / fl;CD4-Cre (n = 6-7 / time point). Significance was ascribed by 2-tailed Student’s t test. These data represent the combined results of 2 independent experiments. For clinical scores, values represent mean ± SEM. All other values represent mean ± SD. ns = P > 0.05,

[0348] Minimal changes in immune responses to intracranial JHMV infection in the absence of OCA-B is shown in FIG. 7A-F. FIG. 7A shows representative LFB / H& E histology of spinal cord sections at dpi 21. FIG. 7B shows demyelination quantification of LFB / H& E histology at dpi 21, Significance was ascribed by two-tailed student’s t-test. FIG. 7C shows quantification of the number of IFNg expressing CD4+ T cells at dpi 7, 12 and 21.Significance was ascribed by two-tailed student’s t-test. FIG. 7D shows quantification of the number of CD8+ T cells at dpi 7, 12 and 21. Significance was ascribed by two-tailedAttorney Docket No. 21101.0501 Plstudent’s t-test. FIG. 7E shows quantification of the frequency of IFNg expressing CD8+ T cells at dpi 7, 12 and 21. Significance was ascribed by two-tailed student’s t-test. FIG. 7F shows quantification of the number of IFNg expressing CD8+ T cells at dpi 7, 12 and 21. Significance was ascribed by two-tailed student’s t-test. All data represent mean ±SD.iv. OCA-B EXPRESSING CD4+T CELLS DISPLAY PATHOGENIC STEMLIKE PROPERTIES AND PREFERENTIALLY TRANSFER DEMYELINATING DISEASE

[0349] A recently described OCA-B-mCherry reporter mouse (Sun W, et al. Proc Natl Acad Set U SA. 2024; I21(9):e2309153121) was used to evaluate OCA-B expression within CNS infiltrating T cells. EAE was induced in female C57BL / 6 homozygous OCA-B-mCherry reporter mice with MOG35-55 and CNS-infiltrating (4)4 T cells assessed by flow cytometry at peak clinical disease, m Cherry -positive T cells showed increased CD44 and CD62L coexpression, indicative of increased memory-like cells in the OCA-B-expressing population (FIG. 8A). The association of OCA-B expression with a memory-like profile was observed in the brain, spine, and draining cervical lymph nodes (FIG. 8B). Interestingly, the frequency of CD44+CD62L ( *1)4 T cells was elevated within the OCA-B-expressing population specifically within the cervical lymph nodes (FIG. 9A).

[0350] Memory-like CD4+ T cells have been implicated as major orchestrators of autoimmune demyelination in mice and humans (Scholz C, et al. J Immunol.1998;160(3): 1532-1538; Burns JB, et al. Cell Immunol. 2001;212(l):44-50; Pelfrey CM, et al. J Immunol. 2000; 165(3): 1641— 1651; Lovett-Racke AE, et al. J Clin Invest.1998; 101 (4):725-730; Williams JL, et al. J Neuroimmunol. 2011;234( 1-2): 84-92; Williams JL, et al. J Neuroimmunol. 2011;234(l-2): 84-92; Schnell A, et al. Cell. 2021; 184(26):6281-6298). To see if this memory -like profile of OCA-B-expressing CD4 T cells is specific to CNS infiltrating cells, flow cytometry was performed using draining lymph node and splenic T cells from MOG-primed OCA-B-m Cherrys reporter mice. Similar to OCA-B* CNS-infiltrating cells, OCA-B-expressing CD4* T cells taken from the cervical lymph node and spleen had increased CD44 and CD62L co-expression (FIG. 8C and FIG. 8D).

[0351] OCA-B-mCherry positive and negative populations were next evaluated by RNA-Seq of CD4* T cells isolated from the lymph nodes of 14-day MOG-primed mice. CD4* T cells were sorted based on m Cherry expression, as w'ell as lack of CD8, lack of CD 19, expression of CD4, and viability (FIG. 9B). Approximately 1500 genes w'ere upregulated andAttorney Docket No. 21101.0501 Plapproximately 350 were downregulated in OCA-B -expressing cells compared with OCA-B negative cells. Upregulated genes in OCA-B-expressing cellsincluded Cxcr6, Slamf6, Il23r, Rorc, Ifng, Bcl6, and Il17a, and downregulated genes included Foxp3, Il7r, and Ccr9 (FIG. 8E and FIG. 8F). Expression of Tcf7 (which encodes TCF1) was high within both OCA-B-positive and -negative populations. Gene ontology and pathway analysis of upregulated genes show enrichment for terms associated with autoimmune diseases, including lupus, autoimmune thyroid disease, and type 1 diabetes (FIG. 8G).

[0352] To further assess how OCA-B may transcriptionally regulate these differentially regulated genes, previously published Oct1 / OCA-B ChIP-Seq dataset was analyzed (Shakya A, et al. Octi and OCA-B are selectively required for CD4 memory T cell function. J Exp Med. 2015;212( 12):2115-2131). After realignment to the mm39 reference genome, Oct1 and OCA-B co-localized peaks were observed both near the transcription start site and within gene body of Tcf7, Bcl6, and Bach2 (FIG. 9C). These findings indicate OCA-B likely directly regulates at least a subset of the differentially expressed genes identified in gene knockout experiments.

[0353] To determine if OCA-B expression can be used to prospectively identify viable pathogenic CD4+ T cell populations, OCA-B-mCherry reporter mice were used as donors in passive-transfer EAE. Male and female littermates (11 weeks old), homozygous for the OCA- B-mCherry reporter, were primed for 14 days with MOG,, in CFA, as previously described (Grifka-Walk HM, et al. Eur J Immunol. 2013;43(l 1):2824— 2831 ). 9 × 106mCherry-negative and -positive cells were cultured in Till polarizing conditions and subsequently transferred them into age-matched male C57BL / 6 recipients, as previously described (Williams JL, et al. J Neuroimmunol. 2011;234(l-2):84-92). Disease progression was measured by clinical score and normalized weight. Recipients of OCA-B-expressing mCherry-positive cells had increased clinical disease severity and weight loss compared with mCherry-negative control recipients (FIG. 8H and FIG. 9D). These findings indicate OCA-B marks CD4+T cells with pathogenic stem-like gene expression and can be used to prospectively identify peripheral CD4+ T cells with enhanced encephalitogenic properties.

[0354] Data illustrating that OCA-B expression marks encephalitogenic stem-like CD4+T cells is shown in FIG. 8A-H. FIG. 8A shows OCA-B-mCherry mice were injected with MOG35 55 peptide in CFA and pertussis toxin to induce EAE. Cervical lymph nodes (CLNs), brains, and spines were isolated at peak disease (day 15) and analyzed by flow cytometry for mCherry, CD44, and CD62L expression. FIG. 8B shows CD44+- and CD62L+-expressingAttorney Docket No. 21101.0501 Pl( 1)4 T cells were quantified based on mCherry expression. Mean values are shown from 5 biological replicates. Significance was ascribed by 2-tailed Student’s ttest. FIG. 8C shows OCA-B-m Cherry mice were injected with MOG35-55 peptide in CFA, and after 14 days, CD4 T cells within CLNs and spleens were profiled by flow cytometry for mCherry, CD44, and CD62L expression. FIG. 8D shows quantification of CD44 and CD62L expression of OCA-B- negative and -positive CD4+T cells from MOG-primed OCA-B-m Cherry’ mice, n = 4-6 biological replicates from 2 combined replicate experiments are shown.Significance was ascribed by 2-tailed Student’s t test. FIG. 8E shows Heatmap showing relative expression of the top 40 differentially expressed genes from OCA-B-positive and - negative CD4+T cells from bulk RNA-Seq of OCA-B-positive and -negative CD4+T cells from MOG35-55 primed OCA-B-mCherry reporter mice. FIG. 8F shows Volcano plot of differentially expressed genes between OCA-B-positive and -negative I' cells (P < 0.05 and log2-fold change >1 data points are colored). FIG. 8G shows Gene Ontology terms associated with OCA-B-positive and -negative groups. FIG. 8H shows EAE clinical scores of C57BL / 6 w ild-type mice after passive transfer of OCA-B-positive or -negative Thl polarized CD4+T cells. Significance was ascribed by multiple 2-tailed Student’s t tests. Clinical score values represent mean ± SEM. All other values represent mean ± SD. ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. KEGG, Kyoto Encyclopedia of Genes and Genomes.

[0355] Sorting OCA-B-m Cherry’ reporter T cells and additional analysis of the effects of transferring OCA-Bhi versus OCA-Blo CD4+ cells is shown in FIG. 9A-D. FIG. 9A show s quantification of the frequency of CD44+ CD62- CD4+ T cells within the cervical lymph nodes of OCA-B-mCherry reporter mice at peak EAE by OCA-B-mCherry expression. Significance was ascribed by two-tailed student’s t-test. FIG. 9B shows gating strategy for sorting of OCA-B+ and OCA-B- CD4+ T cells. FIG. 9C shows mm39 ChIP-seq tracks showing peaks for Octi (blue), OCA-B (red), along with vertebrate conservation (black) at Tcf7, Bcl6, and Bach2. FIG. 9D show’s normalized weights of recipient mice following adoptive transfer of OCA-B-mCherry positive or negative Thl cells. Significance was ascribed by Two-way AN OVA. Normalized weight is represented by mean ±SEM. All other data represent mean ±SD.V. OCA-B PROMOTES NEUROINFLAMMATION IN RELAPSING¬ REMITTING EAE MODELS VIA CONTROL OF STEM / MEMORY-LIKE T CELL POPULATIONSAttorney Docket No. 21101.0501P1

[0356] In contrast to the chronic form of EAE that develops in C57BL / 6 mouse models. EAE on the NOD background exhibits a relapsing-remitting pattern that more closely mimics human MS Baker D, et al. Ann Clin Transl Neurol. 2019;6(8): 1362-1372). EAE was induced in female NOD.Ocabfl / fl;CD4-Cre and littermate control NOD.Ocabfl / flmice (15—18 weeks old) (Kim H, et al. J Exp Med. 2021;218(3): 133) with MOG35-55. Clinical scores and weights were recorded for 47 days to observe initial disease onset, relapse, and relapse recover}-. No significant differences in clinical scores w ere observed between groups at disease onset (days 10—19). However, after the primary remission, NOD.Ocabfl / fl;CD4-Cre animals were almost completely protected against disease relapse (FIG. 10A). Individual NOD mouse clinical scores are shown in FIG. 11. Normalized weights of animals also reflected the protective effect of OCA-B loss in T cells at relapse (FIG. 10B).

[0357] Individual NOD mouse EAE clinical scores is shown in FIG. 11A and FIG. 11B. FIG. 11A shows clinical scores of individual NOD. Ocabfl / fl mice following EAE induction. FIG. 11B shows clinical scores of individual NOD. Ocabfl / fl; CD4-Cre mice follow ing EAE induction.

[0358] To understand differences in CNS -infiltrating T cells underlying relapse protection at population and transcriptional levels, single-cell RNA-Seq (scRNA-Seq) of CD3ε+T cell isolates from CNS (brain and spinal cord) at remission (day 24) and peak relapse (day 33) were performed. Cells from 3–6 NOD.Ocabfl / fl;CD4-Cre and NOD.Ocabfl / fllittermate controls were collected and combined for analysis. A total of 7799 (remission) and 9224 (relapse) Ocab knockout and 6009 (remission) and 7525 (relapse) control cells passed filtering and were used for analysis, with an average per cell read depth ranging from 90,000 to 190,000. For remission, UMAP clustering using both genotypes revealed 16 clusters corresponding to a broad T cell repertoire consisting of NKT cells (the largest cluster), γδ T cells with a Th17-like gene signature (gd17), and different CD4 and CD8- subsets (FIG. 10C and FIG. 12A). Noteworthy clusters based on mRNA expression included proliferating (Ki67, Pcna, Mcm2), Th1-like (Tbx21, Ifng, Tnf, Il12rb2), and Treg (Foxp3, Ctla4) (FIG. 12A and FIG. 12B). Feature plots for Foxp3 andMki67 are shown in FIG. 10D. Additional feature plots for Cxcr6, 1123 r, and Bach2 are shown in FIG. 12C. Bach2 was notably poorly expressed in all clusters. Notably, there was a large decrease in NKT cell numbers in knockout mice (36% control vs. 14% experimental). Within this cluster, changes in gene expression between control and conditional knockout groups were minimal. Most NKT cells from both groups had a limited inflammatory cytokine response, consistent with the lack of clinical disease at remission. The decrease in the large NKT population in the experimentalAttorney Docket No. 21101.0501 Plgroup proportionally increases the percentage contribution of the other clusters; therefore, similar or decreased percentages of these cells identify reduced populations, which correlate with protection from relapse (e.g., proliferating T cells). Other clusters, such as Tregs, increased disproportionately. Differential gene expression analysis of key clusters (Treg, Thl7-like, proliferating, and Hi 1 -like) revealed increased expression of Slamf6 in Tregs lacking OCA-B, whereas OCA-B-deficient proliferating and Th1-like cells had decreased expression of genes associated with quiescence (Btg1 and Samhd1), memory / sternness (Slamf6, Tcf7, and Sell), and pathogenicity Tcf7 and Tox co-expression) (FIG. 10E).Spectral cytometry showed no significant difference in the frequency of CNS-infiltrating CD4+ T cells or their expression of PD-1 between groups at remission (FIG. 10F and FIG.13A and FIG. 13B). However, OCA-B-deficient CD4+T cells did show a significant reduction in CXCR6 and IFN-y / GM-CSF co-expression, indicative of reduced Th17 pathogenicity (FIG. 10G-I). These results indicate loss of OCA-B in T cells selectively reduces proliferation and differentiation of pathogenic stem / memory-like CD4+ T cells during disease remission.

[0359] Data illustrating that OCA-B promotes relapsing-remitting EAE through stem-like CD4+T cells is shown in FIG. 10A-I. FIG. 10A shows NOD.Ocabfl / fl(n = 19) and NOD.Ocabfl / fl;CD4-Cre (n = 15) mice were injected with MOG35-55 peptide in CFA and pertussis toxin to induce EAE. Clinical scores of animals representing relapsing-remitting disease progression are shown as a function of time. Significance was ascribed by multiple 2-tailed Student’s / tests. FIG. 10A shows animal weights were recorded to determine weight loss throughout initial disease and relapse. Significance was ascribed by 2-way ANOVA. FIG. 10C shows scRNA-Seq was performed on pooled CD3ε+cells isolated from the brain and spine of NOD.Ocabfl / fl(n = 3) and NOD.Ocabfl / fl;CD4-Cre (n = 4) 24 days after EAE induction (remission time point). Cell populations were plotted in a UMAP using the Seurat R package, and percentages are shown for each cluster. Clusters were identified through differential gene expression analysis. FIG. 10D shows feature plots comparing the expression of Foxp3 and Mki67 among clusters. FIG. 10E shows violin plots showing the expression of Btgl, Samhdl, Slamf6, Tcf7, Sell, and Tox within the Treg, Thl7-like, proliferating, and Th 1 -like clusters. FIG. 10F shows CNS cells isolated at remission time point (day 24) were analyzed by spectral cytometry. Representative flow cytometry plots and quantification showing the frequency of C D4 T cells between control and experimental groups. Significance was ascribed by 2-tailed Student’s / test. FIG. 10G shows representative flow cytometry plots and quantification showing the frequency of CXCR6 expressingAttorney Docket No. 21101.0501P1CD4+T cells. Significance was ascribed by 2-tailed Student’s t test. FIG. 10H shows representative flow cytometry plots showing the frequency of CD4+T cells expressing IFN-y and GM-CSF. FIG. 101 shows quantification of ( 1)4 T cells expressing IFN-y and GM-CSF. Significance was ascribed by 2-tailed Student’s ttest. For clinical scores, data represent mean ± SEM. All other data represent mean ± SD. ns = P > 0.05, *P < 0.05.

[0360] Additional remission scRNA-seq cluster annotation, heat map of gene enrichments by cluster, and additional feature plots is shown in FIG. 12A-C. FIG. 12A shows violin plots showing cluster gene expression of CD3e, Cd4, Cd8a, Ncrl(NKp46), Cxcr6, Tcf7, and Tcrg-V6 at EAE remission. FIG. 12B shows heatmap showing the top 10 genes expressed by cluster at remission EAE timepoint. FIG. 12C shows UMAP feature plots showing expression of Cxcr6, 1123 r, and Bach2 amongst clusters at remission.

[0361] Data illustrating EAE remission flow cytometry evaluating PD-1 expression in CNS infiltrating CD4+ T cells is shown in FIG. 13A and FIG. 13B. FIG. 13 shows a representative flow cytometry plots showing the frequency of PD-1 expressing CD4+ T cells. FIG. 13B shows a quantification of the frequency of PD-1 + CD4+ T cells within the central nervous system of control and experimental mice at EAE remission. Significance was ascribed by two-tailed student’s t-test. All data represent mean ±SD.

[0362] At relapse, multiple CNS-infiltrating stem-like and effector CD4+ and CD8 control T cell populations were present (FIG. 14A, FIG. 14A and FIG. 15A). T cell receptor (TCR) sequencing revealed an expansion of a subset of TCR clones in the OCA-B-deficient CD8 clusters, whereas most other clusters were highly polyclonal FIG. 14B. Comparing the 10 most expanded TCR clonotypes between knockouts and controls, several clonotypes were shared but differentially expanded (FIG. 15C). A Ccr9 stem-like cluster expressing the gut-homing chemokine receptor Ccr9, as well as Ccr7, Bach2 ', and Tcf7, notably appeared at relapse (FIG. 14C). TCR clonotype analysis indicated that this population was highly polyclonal (FIG. 14B). Additionally, a stem -like CD4-2 cluster expressing Tcf7, Ccr7, Bach2, Cd44, and low-level Cxcr6, and a prominent Th 17 -like clusterexpressing Tcf7, Cd44, Il1r1, Lgals3, and higher levels of Cxcr6 were identified (FIG. 14C). The latter cluster likely contains pathogenic effector cells.

[0363] Data illustrating scRNA-seq cluster annotation genes, heat map of NOD. EAE gene enrichments by cluster, and TCR clonotype overlap between conditions at the relapse timepoint is shown in FIG. 15A-C. FIG. 15A shows violin plots showing cluster gene expression of CD3e, Cd4, Cd8a, Ncrl(NKp46), Cxcr6, Tcf7, and Tcrg-V6 at EAE relapse. FIG. 15B shows heatmap showing the top 10 genes expressed by cluster at relapse EAEAttorney Docket No. 21101.0501 Pltimepoint. FIG. 15C shows alluvial plot showing top 10 TCR clonotype gene overlap between Ocabfl / fl and Ocabfl / fl; CD4-Cre groups. FIG. 15C also shows UMAP feature showing the expression of ( 1)4. CD8a, Tox, Self Ccr7, and Ccr4 amongst clusters at relapse timepoint.

[0364] In the absence of OCA-B, NKT cell populations were normalized compared with the remission time point, and several clusters showed major changes in the percentage of cells, including most prominently within the Ccr9 stem-like cluster, which was much more prevalent (FIG. 14D). Feature plots comparing OCA-B T cell - deficient mice and littermate controls for Ccr9 and the transcription factors Tcf7 and Bach2 are shown in FIG. 14D. The cells were also marked by the expression of Cd4, Tox, Ccr7, and Ccr4, and limited expression of Sell (FIG. 16A).

[0365] RNA velocity analysis of spliced and unspliced transcripts (La Manno G, et al.Nature. 2018;560(7719):494— 498) identified clusters of T cells, most of which had static gene expression profiles consistent with terminal differentiation but some with differentiating or transitional gene expression (FIG. 14E). Notably, OCA-B knockout cells had increased transcriptional stasis within the Ccr9 stem-like and proliferating clusters, and a decreased transcriptional directionality of Th 17 cells tow'ard gd!7 cells, the latter of which express high levels of pathogenic markers, including LgalsS, Cxcr6, and Illrl (FIG. 15A and FIG. 15B). Pseudotime analysis identified a transcriptional capacity in the control Ccr9-expressing population to differentiate into pathogenic Th l7-like cells, whereas cells lacking OCA-B were limited in this capacity, possibly resulting in the accumulation of these cells (FIG. 16B). These changes culminated in the Thl7-like effector knockout cells expressing reduced pathogenicity -associated genes such as Pdcdl, Tigit, and Illrl (FIG. 14F). Together, these results suggest OCA-B mediates the transition of nonpathogenic stem-like cells to pathogenic Thl7 cells, which ultimately drive disease relapse.

[0366] Data illustrating that OCA-B promotes disease relapse through control of pathogenic stem-like Till 7 differentiation is shown in FIG. 14A-F. FIG. 14A shows scRNA and TCR sequencing were performed on pooled CD3s+cells isolated from the brain and spine ofNOD.Ocabfl / fl(n = 5) and NOD.Ocabfl / fl;CD4-Cre (n = 6) mice 33 days after EAE induction (relapse time point). Cell populations were plotted in a UMAP using the Seurat R package and percentages are shown for each cluster. Clusters were identified through differential gene expression analysis. FIG. 14B show s UMAP TCR clonotype expansionbetween Ocabfl / fland Ocabfl / fl;CD4-Cre among clusters. FIG. 14C show's violin plots showing expression of Ccr9, Bach2, Ccr7, Tcf7, Cd44, Cxcr6, Illrl, and Lgals3 in the Ccr9 stem-likeAttorney Docket No. 21101.0501 PlCD4, stem-like CD4-2, and Thl7-like clusters. FIG. 14D shows feature plots comparing the expression of Ccr9, Tcf7, and Bach2 among clusters. FIG. 14E shows UMAPs showing RNA velocity of spliced and unspliced transcripts by experimental condition. FIG. 14F shows violin plots showing expression of Pdcdl, Tigit, and Illrl in the Thl7-like cluster between control and experimental groups.

[0367] Example relapse feature plots and pseudotime analysis of relapse single cell RNA seq is shown in FIG. 16A and FIG. 16B. FIG. 16A shows UMAP feature showing the expression of CD4, CD8a, Tox, Sell, Ccr7, and Ccr4 amongst clusters at relapse timepoint. FIG. 16B shows pseudotime analysis of relapse single cell from root starting node at or near the Ccr9- expressing stem-like cluster.c. DISCUSSION

[0368] The development of therapies that inhibit autoimmunity while minimizing impacts on normal immune function remains a critical unmet goal in the field. In this study, it was shown that expression of mRNA encoding the transcriptional coregulator OCA-B is elevated in CD4+T cells from SPMS brain tissue and from effector memory-like, peripheral-blood CD4+ T cells from patients with RRMS. It was shown that OCA-B promotes the pathogenic maturation of T cells and establishment of CNS autoimmunity, particularly episodes of relapse. Loss of OCA-B attenuates these populations in chronic EAE, in adoptive -transfer EAE, and in NOD. EAE during remission. During relapse, OCA-B T cell knockout mice are almost completely protected and accumulate a population of CD4 T cellsexpressing Ccr9, Bach2, and Tcf7. OCA-B expression also marks viable pathogenic stem / memory-like CD4+ T cells that preferentially transfer EAE. Furthermore, the effects of modulating OCA-B are selective, because responses to infection with a neurotropic coronavirus that generates similar clinical symptoms but lacks an autoantigen were largely unaffected.

[0369] OCA-B is a member of a small family of transcription coregulators that also includes OCA-T1, OCA-T2, and IκBζ (Alpsoy A, et al. Mol Cell. 2024;84(6): 1149-1157; Wu XS, et al. Nature. 2022;607(7917): 169-175). It becomes induced upon CD4* T cell activation and docks with Octi to directly regulate immunomodulatory genes suchas Il2, Ifng, Il17a, Tbx21 (T-bet), and Csf2 (Gmcsf). Rather than functioning as a primary activator of these genes, OCA-B is required for their robust expression upon antigen reencounter (Shakya A, et al. J Exp Med. 2015;212(12):2115-2131; Kim II, et al. J Exp Med.Attorney Docket No. 21101.0501 Pl2021;218(3): 133). Notably, unlike transcription factors such as NF-AT, AP-1 and NF-KB, Octi and OCA-B are dispensable for normal primary T cell responses (Shakya A, et al. J Exp Med. 2015;212(12):2115-2131). These findings predict important roles for OCA-B in CDA T cells, specifically in situations of antigen re-encounter, which is a necessary feature of autoimmunity. Conditional T cell knockout of Octi reduces EAE severity, with reduced proinfl ammatory cytokine expression and increased anergy (Kim H, et al. J Neuroinflammation. 2019; 16(1): 133). Deletion of Octi in T cells was dispensable for neurotropic viral clearance (Kim H, et al. J Neuroinflammation. 2019; 16(1): 133): however, the ubiquitous expression of Octi in adult tissue limits its translational potential. In contrast, OCA-B expression is largely confined to B and T cells, and whole-body knockout mice are viable and fertile (Schubart DB, et al. Nature. 1996;383(6600):538-542). OCA-B is highly expressed in B cells and required for germinal center formation (Schubart DB, et al. Nature.1996;383(6600):538-542; Qin XF, et al. EMBO J. 1998; 17(17):5066-5075). OCA-B is expressed in CD4 T cells at levels 50- to 100-fold less than in B cells (Kim H, et al. J Exp Med. 2021;218(3): 133; Heng TSP, et al. Nat Immunol. 2008;9( 10): 1091-1094; Zwilling S, et al. Science. 1997;277(5323):221–225), which suggests that with proper therapeutic dosing, OCA-B could be inhibited selectively in T cells with minimal impact to the B cell compartment. Short-term administration of an OCA-B peptide mimic that inhibits OCA-B’ s downstream effector functions reverses spontaneous type-1 diabetes in NOD mice (Kim H, et al. J Exp Med. 2021;218(3): 133). However, toxicity would limit the peptide mimic’s efficacy, including in EAE models. Further research is required for the development of safe and effective OCA-B inhibitors.

[0370] OCA-B whole-body knockout animals are partially protected from EAE (Ikegami I, et al. Biochem Biophys Res Commun. 2019;514(4):1167–1171); however, the known activity of OCA-B in B cells made i t unclear if the protection is T cell derived. It is shown that OCA-B promotes EAE pathogenesis through T cells. Opportunistic infections and reactivation of latent CNS-tropic viruses are noteworthy issues for MS therapeutics that focus on global immune suppression as a therapeutic mechanism. When OCA-B T cell conditional knockout mice are challenged with the neurotropic coronavirus JHMV, there is little difference in their ability to mount immune responses and control virus replication. These data are consistent with previous findings that OCA-B loss in T cells preserves lymphocytic choriomeningitis virus acute viral infection response, instead selectively affecting memory recall responses (Shakya A, et al. J Exp Med. 2015;212( 12):2115-2131; Sun W, et al. Proc Natl Acad Sci U S A. 2024;121(9):e2309153121). This finding provides a potential therapeutic window inAttorney Docket No. 21101.0501 Plwhich targeting the low levels of OCA-B in T cells could be used to blunt autoimmunity while sparing baseline immune responses.

[0371] Unlike tire significant but modest protection observed in chronic C57BL / 6 EAE, OCA-B loss in adoptive transfer EAE almost completely protects recipient animals from disease. This may indicate that adoptive-transfer EAE models involve more autoantigen reexposures, because transferred T cells must re-encounter antigen in a naive host before promoting disease. Protection was observed regardless of whether donor cells were Thl or Th 17 polarized, indicating that OCA-B functions through a mechanism that parallels rather than enforces the Thl / Thl7 paradigm. Therefore, other mechanisms were sought that might explain why OCA-B T cell knockouts are so strongly protected.

[0372] Autoimmune pathogenesis and maintenance have been increasingly associated with stem- or progenitor-like T cell subpopulations (Hughes EP, et al. Trends Immunol.2024:45(3): 158-166; Schnell A. Immunol Rev. 2024;325(l):9-22). Recent findings indicate an emerging role for stem / memory-like T cells in promoting autoimmunity (Schnell A, et al. Cell. 2021;184(26):6281-6298; Gearty SV. Nature. 2022;602(7895): 156-161; Li Y, et al. Nat Immunol. 2024;25(7): 1231-1244; Sato Y, et al. Sci Transl Med. 2023; 15(712): eadh0380). For example, myelin-specific CD4+ T cells in patients with MS show signs of a previously activated, memory-like phenotype compared with healthy controls (Scholz C, et al. J Immunol. 1998; 160(3): 1532— 1538; Burns JB, et al. Cell Immunol. 2001;212(1):44–50; Lovett-Racke AE, et al. J Clin Invest. 1998; 101 (4):725-730). Furthermore, memory-like Thl and Till 7 cells are increased in the peripheral blood of patients with MS and display- increased pathogenicity' compared with conventional Th 17 cells (Paroni M, et al. J Allergy Clin Immunol. 2017;140(3):797-808). Similarly, in EAE models, peripheral central memorylike CD4 CD44 CD62L. T cells confer increased disease severity' upon transfer (Williams JL., et al. J Neuroimmunol. 201 l;234(l-2):84-92). Both Octi and OCA-B promote the functionality of long-lived CD4+memory T cells following antigenic rechallenge (Sun W, et al. Proc Natl Acad Sci USA. 2024;121(9):e2309153121; Johns TG, et al. J Immunol.1995;154(10):5536- 5541). An OCA-B-mCherry reporter mouse allele, which effectively labels memory progenitor CD4 T cells during acute infection (Sun W, et al. Proc Natl Acad Sci U S A. 2024;121(9):e2309153121), also labels CD4 T cells that preferentially confer EAE. OCA-Bhicells taken from the CNS at peak EAE selectively express stem / memory markers. Further profiling OCA-BhiCD4 T cells suggests that OCA-B promotes the transition of nonpathogenic stem -like cells to a pathogenic state associated with markers such as Cxcr6, Slamf6, Iftig, and III 7a. Elevated Poulafl (Ocab) transcripts are also found inAttorney Docket No. 21101.0501 Plpathogenic Thl7 cell populations (Gao Y, et al. Nat Immunol. 2023;24(8): 1331-1344). The connection between OCA-B expression and pathogenic memory / stem-like T cells likely has not been appreciated before because OCA-B is expressed at levels too low to be reproducibly captured with many current scRNA-Seq technologies.

[0373] Relapsing-remitting EAE models have been developed using the Swiss Jim Lambert and autoimmune-prone NOD backgrounds (Baker D, et al. Ann Clin Transl Neurol.2019;6(8): 1362-1372; McRae BL, et al. J Neuroimmunol. 1992;38(3):229-240). In NOD mice, MOG35–55EAE has been described as relapsing-remitting with minimal initial disease that increases in severity upon relapse (Bernard CCA, et al. J Mol Med (Berl).1997;75(2):77-88; Anderson AC, et al. J Immunol. 2012;188(5):2084-2092). Using this model with the conditional Pou2afl allele backcrossed to the NOD background (Kim H, et al. J Exp Med. 2021;218(3): 133), it was found that OCA-B T cell loss minimally affects initial clinical presentation; instead, it protects animals specifically at relapse. The finding that OCA-B loss in conventional C57BL / 6 EAE results in partial protection intermediate between the initial and relapse phases of NOD. EAE suggests that chronic C57BL / 6 EAE may not model either initial presentation or relapse accurately but instead represents a superposition of the 2.

[0374] Single-cell RNA expression profiling at remission identifies a substantial loss of Slamf6, Tcf7 (encoding TCF1), and Sell from proliferative stem-like CD4 T cells in the OCA-B-deficient condition. Although most research on TCF1 -expressing stem-like T cells focuses on CD8+ populations with the capacity for self-renewal and differentiation into effector cells, analogous descriptions have been made for CD4+populations (Schnell A, et al. Cell. 2021;184(26):6281-6298; Nish SA, et al. J Exp Med. 2017;214(l):39-47; Mammadli M, et al. Int J Mol Sci. 2023;24(5):4326). Indeed, autoimmune CD4 T cells expressing these memory / stem-like markers have been reported as an encephalitogenic reservoir that plays a key role in EAE pathogenicity (Schnell A, et al. Cell. 2021;184(26):6281–6298). In the same study, bulk RNA-Seq associated OCA-B expression within this stem-like reservoir (Schnell A, et al. Cell. 2021; 184(26):6281-6298).

[0375] scRNA-Seq at relapse identified multiple emergent populations of effector T cells compared with at remission. Notably, there was also an accumulation of a unique population of CD4- cells in OCA-B T cell-deficient mice. These cells are conspicuously marked by expression not only of Ccr4 but also of Ccr9, which encodes a gut-homing chemokine receptor. CCR9 memory-like CD4+ T cells have been linked to MS and EAE protection (Kadowaki A, et al. Brain. 2019; 142(4):916— 931). The cells also express Bach2, aAttorney Docket No. 21101.0501 Pltranscription regulator that recently was associated with non-pathogenic, immunomodulatory Thl7-polarized cells in the context of C57BL / 6 EAE (Thakore PI, et al. Nat Immunol.2024;25(8): 1395-1410). Bach2 expression was largely concentrated in the CCR9 stem-like cluster, which is more numerous in the knockout condition. Consistently, in bulk RNA-Seq, Bach2 mRNA was upregulated in OCA-B cells (P < 10 ). although with a small (+1.74) fold change. Similarly, recent work has associated Pou2afl (Ocab) with pathogenic Thl7 / Thl cells compared with the nonpathogenic Bac7?2-expressing cells (Thakore PI, et al. Nat Immunol. 2024;25(8):1395–1410). In B cells, OCA-B regulates Bach2 (Song S, et al. Blood. 2021;137(21):2920–2934): however, the interplay between OCA-B and Bach2 and how it influences the pathogenic maturation of Till 7 cells merit additional research. Velocity and pseudotime analysis indicates that these cells are normally transcriptionally inclined to progress toward an highly pathogenic 11117 population but that progression stalls in mice whose T cells lack OCA-B. Consistently, the Th 17 effector population in OCA-B T cell knockout mice lacks expression of key genes such as III rl and Pdcdl, both of which are associated with pathogenicity in CNS -infiltrating T1117 cells (Schnell A, et al. Cell.2021;184(26):6281-6298; Sha Y, Markovic-Plese S. Front Immunol. 2016;7:543). The cumulative alterations in these populations in OCA-B knockouts likely explains why these mice are strongly protected against disease relapse. In conclusion, this study indicates a prominent role for OCA-B in driving stem-like CD4+ T cell populations toward a pathogenic state while playing a minimal role in acute antiviral responses to a CNS infection.2. OCA-B / POU2AF1 EXPRESSION IN MOUSE T CELLS 1 PROMOTES PD-1BLOCKADE-INDUCED AUTOIMMUNITY BUT IS DISPENSABLE FOR ANTI-TUMOR IMMUNITY

[0376] Type 1 diabetes (T1D) affects 1.8 million people in the United States and is increasing worldwide (National Diabetes Statistics Report. (2024) Centers for Disease Control and Prevention; Ogrotis, I., et al., (2023) Medicina (Kaunas) 59; Gregory, G. A., et al., (2022) Lancet Diabetes Endocrinol 10, 741-760). It is characterized by autoimmune destruction of pancreatic P cells and consequent inability to produce insulin (Cooke, D. W., and Plotnick, L. (2008). Pediatr Rev 29, 374-384; quiz 385). While T1D symptoms can be managed with insulin injections, they must be taken for life. Transplantation of islets of Langerhans can restore insulin-independence to T1D patients, but results in reactivation of islet-specific memory cells T cells, P cell destruction and transplant rejection(Sutherland,Attorney Docket No. 21101.0501 PlD. E., et al., (1984) Trans Assoc Am Physicians 97, 80-87; Vendrame, F, et al., (2010). Diabetes 59, 947-957). Similarly, engraftment of islets from non-obese diabetic (NOD) mice with severe combined immunodeficiency (NOD. SCID) into diabetic NODs briefly restores normoglycemia, but results in robust memory autoimmune responses and complete loss of grafted islet function in 10-14 days(Wang, Y., et al., (1991). Proc Natl Acad Sci U S A 88, 527-531). Human therapies aim to minimize graft rejection or block the initial emergence of T1D in at-risk patients altogether while sparing normal immune function (Lemmark, A., and Larsson, H. E. (2013) Nat Rev Endocrinol 9, 92-103).

[0377] In humans, a-PD-1 therapy as well as therapies directed against a number of other inhibitory T cell receptors (immune checkpoint inhibitors or ICIs) are now used for treatment of a variety of cancer indications (Tawbi, H. A., et al., (2022) J Med 386, 24-34; Hamid, O, et al., (2013) N Engl J Med 369, 134-144; Cortes, J., et al., (2022). N Engl J Med 387, 217- 226), The development and wide adoption of ICIs has resulted in a concomitant increase in associated unwanted immune-related adverse events, or irAEs, affecting a variety of tissues (e.g., skin, rheumatoid, pulmonary, gut, cardiac), some of which can be permanent (Tawbi, H. A., et al, (2022) J Med 386, 24-34; Postow, M. A, et al, (2015) J Clin Oncol 33, 1974- 1982; Berti, A, et al, (2021) Crit Rev Oncol Hematol 162, 103351; Chen, T. W, et al, (2015) Ann Oncol 26, 1824-1829; Postow, M. A et al, (2018) N Engl J Med 378, 158-168; Young, A, et al, (2018) Cancer Immunol Res 6, 1445-1452; Molina, G. E, et al, (2021) Oncologist 26, 514-522; Stamatouli, A. M, et al, (2018) Diabetes 67, 1471-1480; Quandt, Z. et al, (2021) Annu Rev Med 72, 313-330). IrAEs include endocrinopathies such as thyroiditis (Osorio, JC, et al., (2017) Ann Oncol 28, 583-589), and less frequently hypophysitis, autoimmune diabetes and other diseases (Postow, M. A et al, (2018) N Engl J Med 378, 158-168; Molina, G. E, et al, (2021) Oncologist 26, 514-522). A critical gap in the field is tire identification of treatment strategies that block the emergence of irAEs while preserving anti-tumor immunity.

[0378] Polymorphisms in the HLA-DR and HLA-DQ genes encoding MHC class II have the strongest genetic influence on T1D, accounting for 40-50% of familial risk (Noble, J. A, and Valdes, A. M. (2011) Curr Diab Rep 11, 533-542). These findings implicate CD4’ T cells as primary drivers of pathogenesis. CD4+T cells direct other cell types such as CD8+T cells and macrophages to infiltrate pancreatic islets and cause [3-cell destruction (Hu, H, et al, (2020) Proc Natl Acad Sci U S A 117, 31319-31330). Consistent with these findings, transient T cell depletion using a-CD3e antibodies (Teplizumab-mzwv) significantly delays T1D onset in at- risk individuals in human clinical trials (Sims, E. K, et al, (2021) Sci Transl Med 73; Ramos,Attorney Docket No. 21101.0501 PlE. L, et al., (2023) N Engl J Med 389, 2151-2161). Though promising, such treatments are limited by tire resulting global immunosuppression. An ideal alternative would selectively deplete pathogenic T cell function while preserving beneficial immune function, including immunity against tumors.

[0379] OCA-B is a B cell- and T cell-restricted transcription coactivator (Zwilling, S, et al., (1997) Science 277, 221-225; Sun, W., et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121). In CD4+T cells, OCA-B docks with the transcription factor Octi (gene symbol Pou2fl) to regulate targets including 112, Ifng, Icos, Csf2 (Gmcsf) and Tbx21 (Tbet) (Shakya, A., et al., (2011) J Biol Chem 286, 450-459; Shakya, A et al., (2015) J Exp Med 212, 2115-2131). Unlike transcription factors such as NFAT, AP-l and NF-KB, OCA-B controls the expression of these genes specifically during repeated antigen exposures. Agents targeting this pathway would selectively inhibit over-active T cell responses to antigen reexposure but preserve acute immune response. Consistent with this prediction, in the absence of either OCA-B or its cognate transcription factor Octl / Pou2fl, primary T cell responses to systemic and glial-tropic viruses (LCMV and JHMV, respectively) and viral clearance are normal (Sun, W, et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121; Hughes, E. P., et al,, (2025) J Clin Invest), however fewer central memory’ (TCM) ( D I T cells form, and those that do respond poorly to antigenic re-challenge (Sun, W, et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121; Sun, W, et al, (2024) Proc Natl Acad Sci U S A 121, e2309153121). Three studies directly link polymorphisms in the human OCAB (POU2AF1) locus with autoimmune disease (Nakamura, M, et al, (2012) Am J Hum Genet 91, 721-728; Goris, A, et al, (2003) J Neuroimmunol 143, 65-69; Ban, M, et al, (2006) J Neuroimmunol 179, 108-116). Human polymorphisms affecting Octi (and therefore OCA-B) binding to the TNFA and CTLA4 loci are associated with inflammatory bow’el disease and systemic lupus erythematosus, respectively (van Heel, D. A, et al, (2002) Hum Mol Genet 11, 1281-1289; Cunninghame Graham, D. S, et al, (2006). Hum Mol Genet 15, 3195-3205). More recent work identified additional polymorphisms in Octi binding sites within the human IL13, IL18 and HLA-C loci (Leon Rodriguez, D. A, et al, (2016) PLoS Negl Trop Dis 10, e0004583; Kiesler, P, et al, (2009) Hum Molec Genet 18, 4513-4520; Vince, N, et al, (2016) Am J Hum Genet 99, 1353-1358). Additionally, two meta-analyses show a strong association betw een Octl / OCA-B binding site polymorphisms and autoimmunity including T1D (Farh, K. K, et al, (2015) Nature 518, 337-343; Maurano, M. T, et al, (2012) Science 337, 1190- 1195). Collectively, these studies link OCA-B with human autoimmunity.Attorney Docket No. 21101.0501P1

[0380] OCA-B loss in T cells protects mice against spontaneous T1D onset (Kim, H., et al., (2021). J Exp Med 218, e20200533). Single-cell RNA-sequencing (scRNA-seq) reveals that protection is associated with loss of autoreactive clones but maintenance of the overall pancreatic islet immune architecture. These findings suggest that targeting OCA-B could be used to treat T1D early in the disease course while sparing baseline immunity. OCA-B’s lymphoid-restricted expression pattern (Zwilling, S. et al., (1997) Science 277, 221-225; Sun, W., et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121 ), its dispensability for primary antiviral responses (Sun, W., et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121; Shakya, A et al., (2015) J Exp Med 212, 2115-2131; Hughes, E. P., et al., (2025) J Clin Invest), and its specificity for Octl / Pou2fl and Oct2 / Pou2f2 complexes with DNA (Strubin, M., et al., (1995) Cell 80, 497-506; Gstaiger, M., et al., (1995) Nature 373, 360-362; Chasman, D, et al., (1999) Genes Dev. 13, 2650-2657) make it a promising therapeutic target that could be used to specifically block autoreactive CD4+T cells while sparing normal immunity. OCA-B peptide mimics linked to membrane -penetrating peptides can reduce inflammation and hyperglycemia in recent-onset T1D NOD mice, but also show significant toxicity (Kim, H., et al., (2021). J Exp Med 218, e20200533). An outstanding question is whether OCA-B can be successfully targeted by more drug-like small molecules. It also remains to be determined if OCA-B promotes diabetes driven by TCI therapy, and / or if OCA-B promotes anti-tumor immunity, including that promoted by 1CI.a. MATERIALS AND METHODSi. LABORATORY MICE

[0381] All mice used in this study were non-obese diabetic (NOD) background, wi th the exception of B16-F10 and MC38 tumor injections which used C57BL / 6 mice. Pou2afl (Ocab) conditional (floxed) mice on the C57BL / 6 and NOD backgrounds were generated as described previously (Sun, W., et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121; Kim, H., et al., (2021). J Exp Med 218, e20200533). All animal experiments were approved by the University of Utah Institutional Animal Care and Use Committee (IACUC approval 00001553).ii. A-PD-1-INDUCED DIABETES

[0382] 250 pg a-PD-1 (clone RMP1-14, Cat# BE0146, Bio X Cell) or rat IgG2a isotype control (Cat# BE0089, Bio X Cell) was interperitoneally (Ip.) administered intoAttorney Docket No. 21101.0501 PlNOD.fl / florfl / fl;CD4-Cre on day 0, 4. 8, and 12 (or on day 0, 4, 8, 12, and 16 as indicated). Antibody was diluted in PBS for injection of 200 pL per mouse.

[0383] Histology. Salivary glands and lacrimal glands were isolated and fixed in 10% formalin. After 24-72 hr, tissues were transferred to 70% ethanol and embedded in paraffin, sectioned at 5~7 m, and H& E staining was performed. Slides were examined and imaged using an Olympus BX53 microscope with UPlanFL objectives, Olympus DP73 camera, and Olympus cellSens imaging software,iii. SECONDARY LYMPHOID ORGAN AND TUMOR HARVEST

[0384] Spleens and lymph nodes were ground and filtered through a 70 pm nylon cell strainer (VWR #76327-100). Red blood cells were removed by Ammonium-Chloride-Potassium (ACK) lysis buffer (10 mM KHCO3, 150 mM NH4Cl, and 0.1 mM Na2EDTA) MC38 tumors were manually disrupted by grinding between the frosted end of slide glass in a 6-well plate with 3 mL of PBS plus 4% FBS, and filtered through a 40 pm nylon cell strainer (VWR #76327-098), 9495 tumors were mechanically disrupted in 6-well-plate and digest in RPMI media containing 16mg / mL collagenase D (Sigma-Aldrich #11088858001) and lOmg / mL Dnase (Sigma-Aldrich #10104159001) at 37°C in CO2 incubator for 45 minutes and filtered through a 40 pm nylon cell strainer, spined in 37.5% percoll gradient, and red blood cells (RBCs) were lysed to produce single-cell suspensions.iv. PANCREATIC ISLET HARVEST

[0385] Lymphocytes were isolated based on a protocol from Jing et al (Jing, Y., et al., (2023) bioRxiv. 10.1101 / 2023.01.17.523999). Pancreata were harvested and perfused with collagenase 4 (Worthington #LS004188) at 37°C, and islets were picked using a 10 pL pipette. Islet single-cell suspensions were obtained by vortexing in cell dissociation buffer (ThermoFisher Scientific #13151014).v. FLOW CYTOMETRY

[0386] Single-cell samples were stained with antibodies (1: 100 diluted in FACS buffer) for flow cytometric analysis. For intracellular staining, cells were cultured in RPMI1640 (ThermoFisher Scientific #11875093) supplemented with 10% FBS, 1% penicillin / streptomycin (ThermoFisher Scientific #15140122) and 1% GlutaMAX (ThermoFisher Scientific #35050061), 1 pL / mL Brefeldin A (BD Biosciences #555029), 50Attorney Docket No. 21101.0501 Plng / mL phorbol 12-myristate 13-acetate (Sigma-Aldrich #P 1585) and 1 ug / mL ionomycin (Sigma-Aldrich #10634) for 4 hr. Cells were stained for surface markers and fixed using Cytofix / Cytoperm (BD Biosciences #554714) following the manufacture’s protocol, and resuspended in Perm / Wash buffer for intracellular cytokine staining. For intranuclear staining, cells were stained for surface markers, fixed using the Foxp3 / Transcription Factor Staining Buffer Kit (ThermoFisher Scientific #00-5523-00) and stained for intranuclear markers. Flow cytometry- was performed using a BD Fortessa (BD Biosciences). Data analysis was performed using Flow Jo software (BD Biosciences).vi. CELL LINES

[0387] Mouse MC38 cell line was a gift from Ryan O'Connell (University of Utah). B 16- F10 cells were a gift from the lab of Matthew A. Williams (University of Utah). MC38 cells were grown in DMEM (ThermoFisher Scientific #11965092) supplemented with 10% FBS.1% penicillin / streptomycin, 1% MEM-NEAA (ThermoFisher Scientific #11140050) and 1% HEPES-C1 pH 7.3 (ThermoFisher Scientific #15630130). B16-F10 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. NOD 9495 cells were grown similar to a prior report (Young, A,, et al,, (2025). in prep) in DMEM (no glutamine, no sodium pyruvate, ThermoFisher Scientific #11960044) supplemented with 10% FBS (ThermoFisher Scientific #A5256701) 1% penicillin / streptomycin, 1% sodium pyruvate, 1% 100× MEM-NEAA, 1% GlutaMAX (ThermoFisher Scientific #35050061). SupTl human T lymphoblast cells were grown in RPMI1640 supplemented with 5% FBS, 5% calf serum (Sigma), 1% penicillin / streptomycin and 1% GlutaMAX at 37°C with 5% CO2.vii. TUMOR CELL LINE ENGRAFTMENT

[0388] All tumor experiments used 6- to 10-week-old C57BL / 6 or 6~8-week-old NOD background mice. For injections of B16-F10 and MC38, after trypsinization, cultured tumor cells were resuspended in PBS and 1×106and 5×105tumor cells were s.c. injected into the hind flank of mice, respectively. For the injection of NOD 9495 tumor line, after trypsinization, cultured tumor cells were resuspended in PBS and 1.5×106tumor cells were s.c. injected into the hind flank of mice as described (Young, A., et al., (2025). in prep).viii. SMALL MOLECULESAttorney Docket No. 21101.0501 Pl

[0389] Synthesis of DB 1033, DB2232, DB2658, DB2379, DB2559, and DB2385 has been previously reported (Liu, Y., et al., (2012) J Am Chem Soc 134, 5290-5299). All compounds were dissolved in 100% DMSO and stored at 5mM at -80°C for later use. For cell-based assays, stock solutions or DMSO vehicle controls were diluted in culture media. For EMSA, compounds were diluted in 0.6X buffer D (Tantin, D., et al., (2008) Genome Res 18, 631-639; Wang, V. E., et al., (2004) Mol Cell Biol 24, 1022-1032). For mouse injections, sterile PBS was used to dissolve the compounds.ix. GENERATION OF SUPT1 LUCIFERASE REPORTER CELL LINES

[0390] Reporter constructs were generated using a modified secreted nano-luciferase vector, pNL2.3 (Promega #N108A), with the hygromycin resistance cassette replaced with puromycin resistance cassette (to generate pNL2.3-Puro). Gene blocks (Integrated DNA Technologies) were synthesized containing 5 canonical Octi binding motifs (ATGCAAAT (SEQ ID NO: 3), 5 xOct ) or mutant motifs (CAAACTAC (SEQ ID NO: 4), 5xMut) upstream of a minimal CMV promoter. Gene blocks were subcloned into the multiple cloning site of pNL2.3-Puro using EcoRV and Hindlll restriction sites. To generate stable transfectants, human SupTl cells were electroporated using Neon (ThermoFisher Scientific) with 1 pg of either the pNL2.3-Puro empty-vector (EV), pNL2.3-Puro xOct (5xOct), or pNL2.3-Puro 5xMut (5xMut) construct. Electroporation parameters were: 1700 V, 10 milliseconds, x3 pulses. Electroporated cells were allowed to recover for 2 days, then selected for stable DNA integration and expanded in complete media containing 1 pg / mL puromycin (ThermoFisher Scientific #A1113803) for 2 weeks. Cells were then single cell-cloned by limited dilution in complete media containing 1 pg / mL puromycin.x. SUPT1 OCA-B ACTIVITY ASSAYS

[0391] 1.0×1055xOct and 5xMut reporter cells were seeded into wells of 96-well plates were cultured in 100 pl, complete RPMI in complete medium with serially diluted compounds for 18-20 hr. Supernatant was collected and mixed with 1:50 buffer-diluted Nano-Gio luciferase substrate (Cat# 1110, Promega). Luminescence was measured using a Modulus Microplate Multimode Reader (Promega).xi. IN VIVO TREATMENT OF PD-1 BLOCKADE-INDUCED DIABETESAttorney Docket No. 21101.0501 Pl

[0392] NOD. Oca ^and NODZ^? - / 7; CD4-Cre mice were i.p. administered with a-PD-1 and 15 mg / kg of DB1033 was administered i.p. to the mice at day 0, 4, 8, 11, and 12. Mice were sacrificed on day 24.xii. STATISTICAL ANALYSES

[0393] Two-tailed student's t-tests were used to determine all statistical differences. All error bars represent ^standard deviations unless otherwise noted. All error bars tumor size represents ±SEM unless otherwise denoted. For all figures, ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, > < 0.001; ****,? < 0.0001.xiii. CONSTRUCTION AND PURIFICATION OF RECOMBINANT OCT1 DNA BINDING DOMAIN OCT1-FUSION PROTEINS

[0394] A cDNA for the Octi DNA binding domain (comprising POUs and POUH subdomains) fused to OCA-BI-44 was ordered from IDT and included an N-terminal 6-His, consecutive C-terminal Twinstrep and FLAG tags, the cDNAs were cloned into the pET28a vector (invitrogen) between the Ncol and Hindlll sites. The plasmids were transformed into Rosetta 2 (DE3) pLysS competent cells (Sigma) using 50 pg / ml kanamycin as a selectable marker. Protein was expressed using 1 mM IPTG (Sigma) at 37°C for three hr. The cells were lysed in buffer containing 20 mM HEPES, pH 7.4 (Sigma), 300 mM NaCl (Sigma), 5% glycerol (Sigma) and 1 mM DTT (ThermoFisher Scientific) containing EDTA-free protease inhibitor cocktail (Sigma) at 2× concentration. Protein was bound to a Histrap column (Cytiva) using the same buffer and eluted with buffer containing 400 mM imidazole (Sigma). The protein was further purified by size exclusion chromatography on a Superdex increase 75 10 / 300 GL column (Cytiva.inc). The column was loaded using PBS containing 1 mM DTT. The protein was analyzed on 12% SDS-PAGE gel visualized by Coomassie staining.xiv. NANOBRET

[0395] The NanoBRET donor plasmid was made by cloning nLuc in-frame with the N- terminus of full-length human Octi between the Ncol and Xbal restriction sites of pACE-MAM2 (Geneva Biotech). The acceptor plasmid was made by cloning full-length human OCA-B containing a C-terminal HaloTag into pHTC vector (Promega) between the EcoRI and Xhol sites. nanoBRET was performed using the NanoBRET protein: protein interaction kit (Promega) following the manufacturer protocol. Briefly, 2×106293T cells were plated inAttorney Docket No. 21101.0501 Pl6 -w ell plates were transfected with a total of 2 pg of donor plus acceptor plasmid at a ratio of 1: 100. After 24 hr, cells were harvested and counted. 2×105resuspended cells were seeded into the wells of 96-well plates using DMEM lacking phenol red (ThermoFisher Scientific) containing 10% FBS. After 1 hr, DB1033 or DMSO vehicle control were also added at the indicated concentrations in biological triplicate. After 24 hr, the Nano-Glo substrate and Haloligand were added before measuring BRET ratio using plate reader (Spectromax iD5, Molecular Devices Inc.). The miliBRET ratio was plotted with respect to the DB1033 concentration.xv. EMSA

[0396] Published protocols (Tantin, D., et al., (2008) Genome Res 18, 631-639; Wang, V. E., et al., (2004) Mol Cell Biol 24, 1022-1032) -were used for EMSA. The octamer sequence probe was 5’ labeled w ith Cy-5 and used at a final concentration of 40 nM. The probe was incubated with 10 nM Octl-OCA B chimeric protein ±equimolar OCA B (1-44) peptide over ice for 15 min prior to the addition of 100 nM-400 nM DB1033, then further incubated over ice for another 15 min. Reaction mixtures were electrophoresed through 6% native polyacry lamide gels. Images were collected using a Molecular Dynamics Typhoon scanner using the Cy-5 fluorescence filter.xvi. NANODSF

[0397] Tryptophan fluorescence at 330 nm and 350 nm were simultaneously monitored over a temperature range from 25°C to 95°C using a NanoTemper Technologies nanoDSF instrument with an excitation wavelength of 280 nm based on published procedures (Magnusson, A. O., et al., (2019). FEES J 286, 184-204. 10.1111 / febs.14696). The Octl-OCA B chimeric protein complexed with double-stranded DNA containing an Octl-OCA-B binding site was incubated on ice for 15 min. Protein lacking DNA was used as a control, The effect of DB1033 compound or DMSO control on the protein DNA complex were measured by comparing the melting point of the protein / DNA complex ±DB1033 compound. 10 µl from each sample was loaded into each capillary avoiding any air bubbles. The fluorescence intensity ratio and its first derivative were calculated with the manufacturer’s software (PR ThermControl, version 2.1.2). The smoothened curves and the first derivative were plotted in using Microsoft Excel software. The peak from the first-order derivative indicates the melting temperature of the sample protein sample.Attorney Docket No. 21101.0501 Plb. RESULTSi. OCA-B T CELL KNOCKOUT PROTECTS NOD MICE AGAINST DIABETES INDUCED BY A-PD-1 ANTIBODY TREATMENT

[0398] T cell conditional OCA-B loss protects NOD mice against spontaneous T1D (Kim, H., et al., (2021). J Exp Med 218, e20200533). Modulating the PD-1 pathway induces diabetes and other endocrinopathies in NOD mice (Ansari, M J., et al., (2003) J Exp Med 198, 63-69; Fife, B. T., et al., (2006) J Exp Med 203, 2737-2747; Zhou, J., et al., (2016) Sci Rep 6, 39105; Ippolito, S., et al., (2021) Thyroid 31, 1839-1849). To test the effect of OCA-B T cell loss in diabetes induced by PD-1 blockade, 8-week-old male NOD mice were treated with four injections of a-PD-1 antibody. OCA-B loss in T cells completely blocked a-PD-1- induced diabetes development (FIG. 17A and FIG. 17B). Treatment of 12-week-old female NOD mice with a-PD-1 antibodies resulted in less complete protection, with half the mice failing to develop diabetes and the other half showing delayed onset (FIG. 18 A and FIG.18B). Flow cy tometry revealed a significant reduction in CD45+leukocytes in a-PD-1 antibody treated knockout mice (FIG. 17C and FIG. 17D). Also observed was a skewing from CD8+to CD4+T cells in the islets of 8-week-old knockouts (FIG. 17E and FIG. 17F), consistent with changes in immune profiles reflecting diabetes progression. At 12 weeks, islet PD-1 TIM-3+CD8+T cells were decreased in the OCA-B deficient condition ((FIG. 18C and FIG. 18D). PD-1 blockade is known to normally induce exhausted CD8’ T cell proliferation and differentiation in the pancreas, promoting the expansion of both TPEX and TEX cells but also skewing the islet total population in favor of TEX. The finding of decreased TIM-3 expression could therefore be explained if OCA-B loss blocks this pathogenic maturation. To test this, SEAMF6 and CD39 were used to distinguish TPEX and TEX CD8+T cells at both 8 and 12 weeks. OCA-B loss resulted in a marked retardation in the ability of PD-1+CD8+TPEX cells within the islets of 8-week-old mice to differentiate into TEX (FIG. 17G and FIG. 17H). Similar findings were made at 12 weeks ((FIG. 18E and FIG. 18F) Cumulatively, these results indicate that OCA-B loss in T cells blocks the induction of diabetes by a-PD-1 treatment, and blocks the pathogenic maturation of a TpEx-like T cell population in the pancreatic islets.

[0399] OCA-B deletion in T cells confers protection against PD-1 blockade-induced diabetes in NOD mice is shown in FIG. 17A-H. FIG. 17A shows Kaplan -Mei er plot of diabetes-free mice in 8-week-old male Ocab^'b (n=7) and Ocabfl / fl;CD4-Cre (n=7) NOD mice. 250 pg a-Attorney Docket No. 21101.0501P1PD-1 was administered by intraperitoneal (i.p.) injection at day 0, 4, 8 and 12. Blood glucose levels were measured during different stages of diabetes development. FIG. 17B shows blood glucose levels of individual mice from (A). FIG. 17C shows islets were prepared from 8-week-old treated female knockout and littermate control mice 5 days after one dose of a-PD-1 antibody and used in flow cytometry. Representative flow cytometry plots depicting total infiltrating CD45+leukocytes for control and knockout mice, FIG. 17D shows quantification of total infiltrating CD45+islet leukocyte flow cytometry data from NOD.Ocabfl / fl(n=4) and NOD.Ocabfl / fl;CD4-Cre mice (n=3). FIG. 17E shows representative flow cytometry plots depicting CD4 and CD8 islet T cells. Cells were pre-gated using a-CD3 staining. FIG. 17F shows quantification of flow cytometry data showing the percentages of CD4+and CD8+T cells in islets from NOD. OcaA"3z(n=4) and N0D.0ca2- / 7; CD4-Cre mice (n=3). FIG. 17G shows representative flow cytometry plots showing the percentage of PD-1 SLAMF6 CD39‘ CD8+TPEX and PD-1+SLAMF6 CD39+CD8+TEX in islets (pre-gated on PD-1+CD8+T cells). FIG. 17H shows quantification of flow cytometry data for the percentage of TPEX and TEX in islets from NOD.Ocabfl / fl(n=4) and NOD.Ocabfl / fl;CD4-Cre mice (n=3).

[0400] OCA-B deletion in T cells protects N OD mice from spontaneous and PD- 1 blockade-induced diabetes is shoyvn in FIG. 18A-F. FIG. 18A shows Kaplan-Meier plot of diabetes-free mice in 12-week-old female littermate control NOD.Ocabfl / fl(n=5) or experimental NOD.Ocabfl / fl;CD4-Cre (n=6) mice receiving 250 pg a-PD-1 antibody injections at day 0, 4, 8, 12, and 16. Spontaneous T1D was monitored in NOD. Ocab^ (n=5) and N0D.0caA" / 2’; CD4-Cre (n=5) mice receiving PBS. FIG. 18B shows blood glucose levels were plotted from individual mice receiving a-PD-1 antibody in (A). FIG. 18C shows flow cytometry plots showing percentages of CD8+PD-1+TIM-3+islet T cells collected from 12-week-old mice 3 days post-a-PD-1 treatment, FIG. 18D shows quantification of the flow cytometry data from Ocabfl / fl(n=4) and Ocabfl / fl;CD4-Cre mice (n=3). FIG. 18E shows 12 week-old NOD. Oca^2-22or OcoA" A; CD4-Cre mice were injected once with a-PD-1 antibodies. Islet CD8+T cells were assessed for SLAMF6 and CD39 by flow cytometry-. An example Ocabfl / fland example Ocabfl / fl;CD4-Cre mouse are shown. FIG. 18F shows quantification of flow cytometry data from Ocabfl / fl(n=4) and Ocabfl / fl;CD4-Cre mice (n=3).ii. OCA-B T CELL KNOCKOUT MAINTAINS ANTI-TUMOR RESPONSE AND TUMOR CONTROL BY ICIAttorney Docket No. 21101.0501P1

[0401] To determine if OCA-B T cell loss has a corresponding effect on anti-tumor immunity, C57BL / 6 and NOD mice OCA-B conditional knockout mice with syngeneic tumor lines were used. In the C57BL / 6 strain background two lines, B16F10 and MC38 were used. Compared to littermate controls lacking the CD4-Cre driver, OCA-B loss had no effect on tumor growth of B16F10 tumors (FIG. 19). This result was expected because B16 cells are “immune-cold” and do no elicit robust immune responses unless engineered to express specific antigens or unless multiple checkpoint-inhibiting antibodies are combined (Martinez- Usatorre, A., et al., (2018) J Immunol 201, 792-803). In contrast, “immune-hot” MC38 colon carcinoma cells exhibit significant control by T cells that can be augmented by a-PD- 1 treatment (Hornet Moreno, B., et al., (2016) Cancer Immunol Res 4, 845-857). MC38 tumor control was also preserved with OCA-B T cell loss either in the absence or presence of ct-PD-1 antibody treatment (FIG. 20 A and FIG. 20B). Flow cytometry of cells from the tumor microenvironment (TME) showed equivalent CD45.2” leukocyte infiltration and ratios of CD8+to CD4” T cells with a-PD-1 treatment (FIG. 20C and FIG. 20D). Similar percentages of CD4fand CD8 ’ SLAMF6+CD39‘ and SLAMF6 CD394cells w'ere also observed in the TME regardless of the status of OCA-B following a-PD-1 antibody treatment (FIG. 20E and FIG. 20F). TIM-3 expression in tumor-infiltrating CD4+and CD8+was also equivalent (FIG.20G and FIG. 20H). Despite the lack of difference in T cell infiltration, exhaustion and tumor control, within the tumors there were interesting differences in both the CD4+and CD8+TCM-like (CD62LhiCD44hi) and naive-like (CD62LhiCD44lo) compartments (FIG. 21 A and FIG. 21B). The significance of these differences for tumor control is currently unclear.

[0402] OCA-B deletion in T cells leaves B16F10 tumor growth and immune response unaltered is shown in FIG. 19. Growth of s.c. B16F10 tumors in C57BL / 6 mice sufficient (blue, Ocab’1'!1') and deficient (red, Ocaft^-CDd-Cre) for OCA-B in T cells.

[0403] OCA-B in T cells is dispensable for anti -tumor immunity in C57BL / 6 mice is show n in FIG. 20A-H. FIG. 20A shows 5×105MC38 tumor cells were s.c. implanted into the hind flanks of female C57BL / 6 Ocabfl / fland Ocabfl / fl;CD4-Cre mice. 300 µg α-PD-1 (or PBS) was administered i.p. at day 4, 7, 10. Tumor sizes were measured every 3-4 days, and mice were euthanized at end point (day 23) or upon tumor size reaching 2 cm in any direction.Ocabfl / fl+PBS (n=8), Ocabfl / fl;CD4-Cre+PBS (n=11), Ocabfl / fl+α-PD-1 (n=8), and Ocabfl / fl;CD4-Cre+α-PD-1 (n=8). FIG. 20B shows mean tumor sizes from (A) are shown Ocabfl / fl+PBS (n=8), Ocabfl / fl;CD4-Cre+PBS (n=11), Ocabfl / fl+α-PD-1 (n=8), and Ocabfl / fl;CD4-Cre+α-PD-1 (n=8). FIG. 20C shows quantification of flow cytometry data showing the percentage of CD45.2+tumor-infiltrating leukocytes (TILs) in MC38 TMEsAttorney Docket No. 21101.0501P1from a-PD-1 antibody -treated Ocabr‘ / fl(n:::6) and Oc6'5""; CD4-Cre (n:::6) mice on day 23 or upon tumor size reaching 2 cm in any direction. FIG. 20D shows quantification of flow cytometry data showing the ratio of CD8+to CD4+T cells in the TME. FIG. 20E shows representative flow cytometry plots of endpoint TME CD4fand CD8 ’ T cell SLAMF6 and CD39 expression. Ocab1(n=6) and Oco / f7 / jZZ; CD4-Cre (n=5) tumor-bearing mice w-ere treated with a-PD-1 antibodies, FIG. 20F shows quantification of flow cytometry data for endpoint TME CD4+and CD8+T cell SLAMF6 and CD39 expression from Ocabfl / fl(n=6) and Ocabfl / fl;CD4-Cre (n=5) mice. FIG. 20G shows representative flow cytometry data for endpoint TME CD8+and CD4+T cell TIM-3 expression. FIG. 20H shows quantification of flow cytometry data at endpoint showing TME CD4+and CD8+T cell TIM-3 expression from Ocabfl / fl(n=6) and Ocabfl / fl;CD4-Cre (n=6) mice.

[0404] OCA-B deletion in T cells decreases the TCM- and naïve-like CD4+and CD8+T cell populations in MC38 TME is shown in FIG. 21A and FIG. 21B. FIG. 21A shows representative plots of flow cytometric analysis performed on tumors harvested from 6-10-week-old female a-PD-1 -treated C57BL / 6.Ocabfl / fland C57BL / 6.Ocabfl / fl;CD4-Cre mice on day 23 post-tumor inoculation (or upon tumor size reaching 2 cm in any direction). CD4+and CD8+TCM-like (CD62L+CD44+) and TN-like (CD62L+CD44-) cells in the TME are shown. FIG. 21B shows quantification of the percentages of TcM-like (CD62L. CD44 ) and Tx-like (CD62L CD44 ) in TME CD4’ cells (n=5 Ocab^fland n=5 Ocari / Z-''Z; CD4-Cre) and CD8’ cells from Ocabf controls and OcaYz'ACD4-Cre (n=5 ()cab:!!!and n=5 Ocad / yCDd-Cre for T\- like and n=5 Ocabb'b and n=6 Occi,&z7 / ^; CD4-Cre for TcM-like).

[0405] Because the protection from PD-1 -induced diabetes was observed using NOD background mice, tumor responses was also assessed using a syngeneic “immune-hot"’ 9495 sarcoma cell line (Young, A., et al., (2025). in prep), to simultaneously measure diabetes incidence and tumor control in the same animals. a-PD-1 antibody treatment is effective in controlling 9495 tumor growth in syngeneic NOD mice. To minimize complications from induced diabetes and hyperglycemia, mice were administered insulin pellets when glucose levels reached >250 mg / dL for two consecutive days to minimize complications from induced diabetes and hyperglycemia. In these tumors, a-PD-1 antibody treatment was effective, as expected. Again, tumor control either with or without a-PD-1 treatment was equivalent regardless of OCA-B status (FIG. 22A and FIG. 22B). Simultaneously, in another cohort of mice with the same experiment procedures performed, the OCA-B deficient group were again fully protected against diabetes (FIG. 23A). Similar to published work (Young,Attorney Docket No. 21101.0501P1A., et al., (2025). in prep), control mice with induced diabetes exhibited superior tumor control compared to uninvolved mice (FIG. 23B and FIG. 23C). As with MC38 tumors in C57BL / 6 mice, CD45+leukocyte tumor infiltration, CD8+to CD4+T cell ratio, CD8+T cell activation, (FIG. 22B-E) and TPEX to TEX differentiation (FIG. 22-1) were equivalent.Without wishing to be bound by theory, these findings indicate minimal differences in anti¬ tumor immune responses to multiple syngeneic tumor lines in using mice with OCA-B T cell knockout, indicating a divergence between anti-tumor immunity and the precipitation of irAEs that could be leveraged therapeutically.

[0406] OCA-B in I' cells is dispensable for anti-tumor immunity in NOD mice is shown in FIG. 22A-I, FIG. 22A shows 1.5* 10“ 9495 tumor cells were s.c. implanted into the hind flanks of male NOD. Oca^A and NOD. Oca& / 7 / / 7; CD4-Cre mice. Three doses of 200 µg α-PD-1 (or PBS vehicle control) were i.p. administered at day 10, 14 and 18. Tumor sizes 'ere measured during different stages of tumor growth. Ocabfl / fl+PBS (n=3), Ocabfl / fl;CD4-Cre+PBS (n=4), Ocabfl / fl+α-PD-1 (n=8), and Ocabfl / fl;CD4-Cre+α-PD-1 (n=12). FIG. 22B show's quantification of flow cytometry data for the percentage of CD45 ’ infiltrating cells in the TME from a-PD-1 antibody-treated control Ocabfl / f!(n=10) and littermate experimental Oca&-fl / '-'; CD4-Cre (n=6) mice, FIG. 22C shows representative flow cytometry plots depicting CD4+and CD8+TME T cells from α-PD-1 antibody-treated control NOD.Ocabfl / fland littermate experimental NOD.Ocabfl / fl;CD4-Cre mice. Cells were pre-gated using a-CD3 staining. FIG. 22D shows quantification of flow cytometry’ data depicting the percentage of CD4+and CD8+T cells in the TME from NOD. Oc«fr’7 / j(n=9) and NOD. Oca fz / y / ; CD4-Cre mice (n===4). FIG. 22E show s quantification of flow cytometry data showing the percentage of activated (CD62LloCD44h‘) CD8+T cells in the TME of a-PD-1 -treated Ocabbfl (n=9) and litermate experimental ( cad" A; CD4-Cre (n=5) mice at endpoint. FIG. 22F shows Representative flow cytometry plots depicting relative percentages of SLAMF6- and CD39-expressing infiltrating CD8+PD-1+T cells. FIG. 22G shows quantification of PD-1+SLAMF6+CD39'CD8+TPEX and PD-1+SLAMF6 CD39+CD8+TE in the TME from NOD. Oca&'" A(n=9) and NOD. OcaA^ACD4-Cre mice (n=5). FIG. 22H show's representative flow cytometry plots depicting TIM-3 levels in infiltrating CD8+T cells. FIG. 221 show's quantification of TIM-3 cells in the TME from NOD. OcaV™ (n=9) and NOD. Oca / i"'ACD4- Cre mice (n=5).

[0407] NOD tumor additional data mice is shown in FIG. 23A-C. FIG. 23A show's Kaplan- Meier plot of another cohort of diabetes-free mice performed with the same experiment procedures as in FIG. 22. FIG. 23B and FIG. 23C show tumor growth curves depictingAttorney Docket No. 21101.0501P1individual anti-PD-1 treated control (FIG. 23B) or experimental (FIG. 23C) mice stratified based on whether or not they became diabetic during the course of the experiment.”iii. DEVELOPMENT OF SMALL MOLECULE INHIBITORS OF OCT1: OCA-B COMPLEXES

[0408] Ihe Octi: OCA-B: DNA co-crystal structure (Chasman, D., et al., (1999) Genes Dev.13, 2650-2657) suggests multiple possible targeting strategies, including rationally designed OCA-B peptide mimics (FIG. 24A), which are functional but associated with toxicity in cells and mice due to the membrane penetrating peptide. To identify novel small molecule inhibitors, another strategy was used in which DNA minor groove -bin ding compounds with specificity for the consensus octamer motif (5’ATGCAAAT (SEQ ID NO: 3)) would disrupt Octi: OCA-B complexes. These inhibitors would dock on the underside of the DNA in the structure shown in FIG. 24A (bottom lightning bolt) and would interfere with the unstructured Oct1 linker domain that also spans the minor groove. Nine candidate minor groove-binding compounds with specificity for the octamer motif were synthesized and tested. To test efficacy, a two cell-based assay system was developed. The first is a transcription-based platform in which a stably transfected the human T cell line SupT1 was made to express secreted nanoluciferase (nLuc) under the control of five Octl / OCA-B binding sites. SupT1 T cells express OCA-B at higher levels than, e.g. Jurkat cells, and at levels comparable to primary thymocytes (FIG. 24B). A stable-transfected SupT1 clone expressing nLuc under the control of five wild-type octamer sequences (“5 > (Oct”) was generated and a second using mutant sites incapable of binding Octi and OCA-B (“5xMut”, FIG. 24C). Tire 5xOct clone produced substantial levels of luciferase activity relative to empty vector-transduced controls, while the 5xMut clone expressed lower but still measurable activity (FIG. 24D), Time course experiments show that 12- to 18 -hr incubations have not yet saturated nLuc activity and capture a dynamic range allowing both activators and inhibitors to be detected (FIG. 24E). An 18-20 hr was therefore chosen for all further experiments. AlamarBlue readings are also taken to measure cell viability, using 2.5 pM staurosporine a positive control for maximum toxicity. The treatment kills >98% of SupTl cells within 18 hr and correspondingly inhibits luciferase expression (FIG. 25). Of the 9 candidate compounds two - DB1033 (IC50~25 mM, FIG. 24F) and DB2232 (IC50~20 mM, FIG. 26A) - inhibited luciferase activity without significantly affecting luciferase expression from the mutant cell line and without significant toxicity. Other compounds such as DB2658Attorney Docket No. 21101.0501 Plshowed significant toxicity in the same screen (FIG. 26B) or were less active (e.g., DB2385, FIG. 26C-H). The DB1033 structure is shown in FIG. 8G. Molecular docking indicated that DB1033 preferentially interacts with DNA minor groove in the AAAT portion of the consensus octamer sequence, overlapping with the location of the unstructured Oct1 linker domain (FIG. 24H and FIG. 24I).

[0409] OCA-B cellular inhibition by small molecule DNA minor groove binders is shown in FIG. 24A-I. FIG. 24A shows the Oct1:OCA-B:DNA co-crystal structure showing two targeting approaches: a peptide that mimics the key OCA-B alpha helix involved in downstream effector functions (top lightning bolt) 40, and a minor groove DNA binding molecule with potential to disrupt the unstructured Oct1 linker domain between the two DNA binding subdomains (bottom lightning bolt). FIG. 24B shows OCA-B immunoblots from SupT1 and Jurkat T cell lines (lanes 3-4). Isolated mouse primary thymocyte and 2.93T cell lysates were also used as positive and negative controls, respectively (lanes 1-2), Histone H3 is shown as a loading control. FIG. 24C show s schematic for OCA-B luciferase reporter cell lines. SupTl cells were stably transfected with reporter plasmids containing five tandem canonical Octl: OCA-B binding sites linked to a CMV promoter upstream of secreted nLuc (5 Oct) or an identical plasmid except containing mutant binding sites used to determine nonspecific effects (5*Mut). Cells ere selected with puromycin, single-cell cloned, and individual clones used subsequently. FIG. 24D show s luciferase activity of pNL2.3 empty vector (“EV”), 5xOct, or 5xMut reporter lines. Cells were incubated for 18 hr after plating. FIG. 24E show's luciferase expression kinetics of cultured 5xOct cells. A time course of 2-46 hr is shown. Averages are shown from n=5 biological replicate platings. Trendline show's logistic growth fit. FIG. 24F show's Inhibition of cellular OCA-B activity by an escalating dose of the small molecule DB1033. Values from 5xQct and 5xMut clones w'ere normalized to one another in the absence of DB 1033. Averages are shown across n=6 biological replicate platings. Red curve shows the “5 xQct” fit to the Hill equation. Asterisks denote results from two-tailed student T-test comparing the two lines at the highest concentration. FIG. 24G show's chemical structure of DB1033. FIG. 24H shows molecular docking of DB1033 to canonical octamer B-form DNA. Red shows water molecules. DB1033 is shown in purple. Red / underlined nucleotides show the minor groove bases contacted by DB1033 docked with the lowest free energy. FIG. 24I shows wireframe model showing DB1033 bound to the minor groove AAAT site. Hydrogen bond interactions formed between DB1033 and the minor groove AAAT site are shown with dashed lines.Attorney Docket No. 21101.0501 Pl

[0410] Effect of staurosporine on luciferase -based transcription activity in SupTl cells is shown in FIG. 25. FIG. 25 shows a dose curve of staurosporine is shown using both the 5xOct and 5xMut cell lines. Trendlines show the fit to the Hill equation.

[0411] OCA-B cellular inhibition by additional small molecule DNA minor groove binders is shown in FIG. 26A-H. FIG. 26A shows inhibition of cellular OCA-B activity by DB2232. Blue datapoints represent control measurements of nonspecific transcription measured in 5xMut cells. Red datapoints represent experimental measurements from the 5*0ct line. Luciferase values from both cells lines in were normalized to 1.0 in the absence of DB2232. Trendlines show the fit to the Hill equation. FIG. 26B-H shows similar data for additional molecules, except cytotoxicity was measured using AlamarBlue.

[0412] The reduce the possibility’ of off-target hits, DB1033 compound activity was tested using an orthogonal cell-based method. A nano-bioluminescence resonance energy transfer (NanoBRET) assay was developed that measures OCA-B ’s interaction with its cognate transcription factor Octi. In this assay, 293T cells are co-transfected with an OCA-B construct carrying a HaloTag and an Octi construct carrying an nLuc tag (FIG. 27A).DB1033 again showed dose-dependent inhibition (FIG. 27B), indicating that DB1033 disrupts the interaction between Octi and OCA-B. To test this prediction at a mechanism level, electrophoretic mobility shift assays (EMSA) was used with recombinant Octi DNA binding domain and recombinant full-length OCA-B to measure formation of Oct1:OCA-B:DNA complexes (FIG. 27C). Cy 5 -conjugated double-stranded DNA was used to visualize complexes with cognate DNA in native polyacrylamide gels. Labeled DNA was in excess relative to protein such that changes in the ability to form a complex can be readily observed. Because of “caging effects”, changes in off-rates are not efficiently captured within a running native gel. Instead, in this assay effects of an inhibitor on complex formation are likely generated during the incubation prior to gel loading. The protein, DNA and buffer components 'ere assembled on ice for 25 minutes in stringent conditions (150 mM KC1), and incubated the mixture for a further 5 min at room temperature ±compound prior to loading. Octi readily formed a complex with DNA that was augmented and slightly supershifted in the presence of OCA-B, indicating the formation of a ternary’ complex (FIG. 27D, lane 3). Inclusion of DB1033 efficiently disrupted complex formation in a dose -dependent manner (lanes 4-7).

[0413] A complimentary’ in vitro assay uses differential scanning fluorimetry (nanoDSF) to measure Octi: OCA-B: DNA complex thermal stability. NanoDSF measures the gradualAttorney Docket No. 21101.0501 Plexposure of UV-absorbing tyrosine side chains as proteins unfold with increasing temperature. A single-polypeptide chimeric molecule was developed containing the Octi DNA binding domain fused to the first 44 amino acids of OCA-B, encompassing the structured part of OCA-B that contacts Octi (FIG. 27E). The first 15 amino acids of OCA-B do not appear to be well-structured and presumably serve as a linker between the two molecules, AlphaFold 3 predicts that the chimeric molecule folds similarly to natural Octi and OCA-B on DNA (FIG. 27F). The protein was expressed in E. coli with N-terminal 6-His, consecutive C -terminal Twinstrep and FLAG tags. Following purification, the protein was found to be mostly pure as assessed by Coomassie staining SDS-PAGE (FIG. 28A). Size exclusion chromatography indicated that the purified protein was mostly monodisperse, and EMSA indicated that it was able to interact with consensus octamer DNA (FIG. 28B and FIG. 28C) The presence of target DNA strongly increased the stability of the chimeric protein in nanoDSF, as expected (FIG. 27G), while adding DB1033 in the presence of DNA destabilized the complex (FIG. 27H). These findings identify DB1033 as the best compound tested, indicate that DB1033 acts to disrupt Octl: OCA-B complexes on DNA, and establishes a pipeline for assessing molecules that modulate Octi and / or OCA-B" s interactions with one another and with DNA, their overall stability’, and their transcriptional functionality’.

[0414] Representative data showing that DB1033 inhibits OCA-B-Octl -DNA complex formation is shown in FIG. 27A-H. FIG. 27A shows schematics of full-length Octi tagged with N-terminal nLuc and full-length OCA-B with a C-terminal HaloTag used for nanoBRET. FIG. 27B shows quantification of nanoBRET signal obtained by co-transfecting the 293T cells with the constructs shown in A in the presence of increasing concentrations of DB1033. An average of n=3 biological replicate platings is shown. DB1033 was applied at the indicated concentrations 1 hr after seeding the cells and cells were collected after a further 24 hr. FIG. 27C shows schematics of the C-terminally Twinstep-tagged Oct1 DNA-binding domain (Oct1DBD) and full-length OCA-B with a C-terminal FLAG tag used in EMSA. FIG.27D shows Cy5-DNA scan of native PAGE EMSA showing DNA-OctlDBD complex formation with OCA-B, and concentration-dependent inhibition by DB1033. FIG. 27E shows A schematic of the Oct1DBD:OCA-B1-44 fusion protein engineered for nanoDSF. FIG.27F shows structure of the OCA-Bi-44-OctlDBD complex predicted using Alphafold 3. Cyan: OCA-B1-44 peptide. Green: Oct1DBD peptide. Dark blue shows the Octi: DNA structure derived from X-ray crystallography (Chasman, D., et al., (1999) Genes Dev. 73, 2650-2657). FIG. 27G shows nanoDSF measurement of the thermal stability of 10 μM Oct1DBD-OCA-B1-Attorney Docket No. 21101.0501 Pl44 chimeric protein with or without added canonical octamer DNA. First derivative is shown below. FIG. 27H shows first derivative nanoDSF data in the presence of DNA and the presence (above) or absence (below) of 100 μM DB1033.

[0415] DB1033 and DB2232 effect on PD-1 blockade-induced diabetes in NOD mice is shown in FIG. 28A-F. FIG. 28A shows an image of a 12% SDS-polyacrylamide gel loaded with the purified recombinant chimeric Octi DBD-OCA-B protein and stained with Coomassie blue. Molecular weight makers are shown at left as a standard, FIG. 28B shows Size exclusion chromatography elution profile using the Octl-OCA-B chimera. FIG. 28C shows EMSA using the Octl-OCA-B chimera and Cy5-labeled perfect octamer consensus DNA. FIG. 28D shows Kaplan-Meier plot of diabetes-free mice in 8-week-old male NOD. Ocab’"'!' mice treated with PBS (n=7), NOD. Ocab^ mice treated with DB2232 (n=4) and NOD. OcaA®^; CD4-Cre mice treated with PBS (n===7). Black arrows: 250 pg a-PD-1 and 15 mg / kg i.p. injections at day 0, 4, 8, 12. Grey arrow: an additional i.p. injection of 15 mg / kg DB2232 was administered on day 11. FIG. 28E shows Average blood glucose levels of mice in FIG. 28D. FIG. 28F shows Body weight of mice at day 24 of tire experiment described in FIG. 28Div. SMALL MOLECULE OCT1: OCA-B INHIBITORS BLOCK A-PD-1-INDCUED DIABETES AND OTHER ENDOCRINOPATHIES IN NOD MICE

[0416] 8-week-old male NOD mice were treated with five i.p. injections of 15 mg / kg DB1033 or DB2232 simultaneously with a-PD-1 antibody treatment. 8 week-old male NOD. Oca / y" A; CD4-Cre mice were used as a positive control for protection. As with genetic OCA-B T cell knockout. DB1033 treatment completely protected mice against diabetes emergence (FIG. 29A and FIG. 29B). These results indicate 1) that DB1033 is effective in protecting mice against a-PD-1 antibody-induced diabetes, and that 2) acute inhibition at the same time as a-PD-1 antibody delivery is sufficient to protect mice against diabetes development. DB1033 toxicity was negligible or low-level, as there was a trending but statistically nonsignificant loss of weight associated with treated animals at endpoint (FIG.29C). Protection was not only confined to the pancreatic islets but was also noted with salivary and lacrimal gland tissue, where leukocyte infiltration was strongly reduced (FIG.29D and FIG. 29E). Importantly, the structurally related DB2232 compound, which was inferior in cell-based assays, was also functionally inferior at preventing diabetes (FIG. 28D-Attorney Docket No. 21101.0501 PlF). These findings provide evidence of a structure-activity relationship linking compound activity to OCA-B complex inhibition. In summai}', the findings show that OCA-B promotes the development of autoimmunity in NOD mice administered TCI while being dispensable for anti-tumor immunity on multiple strain backgrounds, and identify small molecule inhibitors that can be administered acutely and phenocopy tire genetic knockout.

[0417] Data showing that DB1033 inhibits PD-1 blockade -induced diabetes in NOD mice is shown in FIG. 29A-E. FIG. 29A shows a Kaplan-Meier plot of diabetes-free mice in 8-week-old male NOD.Ocabfl / fltreated with PBS (n=4), NOD.Ocabfl / fltreated with DB1033 (n=4) and NOD.Ocabfl / fl;CD4-Cre mice treated with PBS (n=4). Black arrows: 250 pg a-PD-1 and 15 mg / kg i.p. injections at day 0, 4, 8, 12. Grey arrow: an additional i.p. injection of 15 mg / kg DB1033 was administered on day 11. FIG. 29BA shows the average blood glucose levels of mice in FIG. 29A. FIG. 29C shows mouse body weight on day 24 of the experiment described in FIG. 29A, FIG. 29D shows example H& E images of lacrimal and salivary gland sections harvested from 8-w'eek-old male NOD mice injected with a-PD-1 antibodies in FIG. 29A. FIG. 29E shows quantification of the histological scoring described in FIG. 29D. NOD.Ocabfl / fltreated with PBS (n=4), NOD.Ocabfl / fltreated with DB1033 (n=4) and NOD.Ocabfl / fl;CD4-Cre mice treated with PBS (n=4). Histologic scoring was conducted in a blinded fashion.c. DISCUSSION

[0418] Here it is shown that OCA-B expression in T cells promotes induced irAEs following ICI treatment while minimally affecting anti-tumor responses. Also, for the first time, it is shown that a small molecule Octi: OCA-B inhibitor phenocopies the protection against irAEs observed using the OCA-B T cell knockouts. Tire latter findings indicate that acute OCA-B inhibition is sufficient to protect against irAEs and suggest the existence of a therapeutic window in which OCA-B pharmacological targeting can be used to block autoimmunity while sparing baseline immune function, including immunity to tumors.

[0419] OCA-B is one of three tissue-restricted transcription coregulators known as OCA proteins: OCA-B, OCA-T1 and OCA-T2 (Wu, X. S., et al., (2022). Nature 607, 169-175. 10.1038 / s41586-022-04842-7). Like the other two proteins OCA-B docks with the sequence-specific DNA binding POU transcription factors to regulate transcription (Strubin, M., et al., (1995) Cell 80, 497-506; Gstaiger, M., et al., (1995) Nature 373, 360-362; Chasman, D, et al., (1999) Genes Dev. 13, 2650-2657). The OCA-Ts are master regulators of tuft cells, and aAttorney Docket No. 21101.0501 Pltherapeutic vulnerability in small cell lung cancers originating from this cell type (Wu, X. S, et al, (2022). Nature 607, 169-175. 10.1038 / s41586-022-04842-7). OCA-B has important B cell-intrinsic functions including in the formation of germinal centers (Betzler, A. C, et al, (2022). Eur J Immunol 52, 404-417; Shi, W, et al, (2015). Nat Immunol 16, 663-673) (Betzler, A. C, et al, (2022). Eur J Immunol 52, 404-417; Shi, W, et al, (2015). Nat Immunol 16, 663-673). In T cells, OCA-B docks with the POU factor Octi to regulate target genes encoding cytokines and other immunomodulatory proteins (Shakya, A et al, (2015) J Exp Med 212, 2115-2131). Because OCA-B expression is 50-100-fold higher in B compared to T cells (Sun, W, et al, (2024) Proc Natl Acad Sci U S A 121, e2309153121), a calibrated dose of competitive inhibitor could selectively block T cell functions while sparing B cell-intrinsic OCA-B functions. For all three proteins, whole-body null mice are healthy, viable and fertile (Wu, X. S, et al, (2022). Nature 607, 169-175. 10.1038 / s41586-022-04842-7; Kim, U, et al, (1996). Nature 383, 542-547; Schubart, D. B, et al, (1996). Nature 383, 538-542), suggesting that they a good candidate drug targets, however no small molecules targeting OCA proteins have been described.

[0420] Multiple lines of evidence show that pathogenic human and mouse progenitor-like T cells promote and sustain autoimmunity including TlD(Chee, J, et al, (2014) J Immunol 192, 572-580; Gearty, S. V., et al, (2022) Nature 602, 156-161; Li, Y, et al, (2024) Nat Immunol 25, 1231-1244; Hughes, E. P, et al, (2024) Trends Imm 45, 158-166). In mice, OCA-B promotes pathogenesis in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, through maturation of stem-like cells into encephalitogenic effectors (Hughes, E. P, et al, (2025) J Clin Invest). These properties are T cell intrinsic. In a relapsing-remitting NOD. EAE model, OCA-B T cell-deficiency specifically protects mice from relapse (Hughes, E. P, et al, (2025) J Clin Invest). On the NOD strain background, both OCA-B whole body and T cell-specific knockout block the onset of spontaneous T1D (Kim, H, et al, (2021). J Exp Med 218, e20200533). Using more aggressive T1D mouse models including CD25-mediated Tregdepletion and the CD8-restricted, autoreactive NY8.3 transgenic line revealed partial protection, while using the CD4-restricted BDC2.5 and CD8-restricted RIP-mOVA-OT-1 models showed no differences (Kim, H, et al, (2021). J Exp Med 218, e20200533). The protection conferred by OCA-B loss is therefore strongest in more chronic polyclonal models. These findings also reveal that OCA-B T cell loss does not result in global immunosuppression, which would result in broader protection. In female NOD mice with newly arisen T1D, intravenous administration of membrane-penetrating OCA-B peptide mimics briefly reverses elevated blood glucose, as well as pancreatic islet TAttorney Docket No. 21101.0501 Plcell infiltration and pro-inflammatory cytokine production. In contrast to the pancreas. pLN showed no change in T cell numbers or percentages, but did show reduced T cell cytokine responses following self-peptide stimulation. However, greater than three peptide injections was toxic, with mice exhibiting ruffled skin, moribund appearance and weight loss. The Tat membrane-penetrating peptide is associated with acute toxicity that can be mitigated by spacing the dose (Polo, J.M., et al., (2004) Nat Med 10, 1329-1335; Caron, N. J., et al., (2004) Biochem Biophys Res Commun 319, 12-20; Krautwald, S., et al., (2004) J Biol Chem 279, 44005-44011).

[0421] The ability to trigger endocrine irAEs using a-PD-1 treatment in NOD mice presented an opportunity to test tire OCA-B T cell loss of function model in different ways. First, to test the effect of OCA-B loss in an aggressive but physiologically relevant model of autoimmune diabetes. Second, to use diabetes and other endocrinopathies as models for ICI-triggered irAEs. Third, to determine the role of OCA-B in anti-tumor immunity. It was found that OCA-B T cell loss protected NOD mice against ICI-triggered diabetes. The effect of OCA-B loss was partially dependent on the age at which ICI was administered, with 8-week-old mice showing more complete protection compared to 12-week-old mice. This finding is consistent with a model in which OCA-B loss protects NOD mice at multiple levels, one of which spans the 8-week timepoint and is PD-1 independen t, and one of which spans the 12-week timepoint and is partially PD- 1 dependent. Profiling the immune cells in these mice flow cytometrically indicated significant reduction in TEX, and a concomitant increase in cells with a progenitor-like (SLAMF6hiCD39lo) phenotype.

[0422] In sharp contrast to the autoimmune protection conferred by OCA-B loss, immune responses and tumor control were preserved either with or without a-PD-1 treatment. No differences were observed using immune-cold B16F10 melanoma cells, as well as immune-hot MC38 colon carcinoma cells and 9495 basal cell carcinoma cells. Similar to the preservation of tumor control, loss of OCA-B in T cells preserves normal primary response and viral control following infection with either the acute strain of lymphocytic choriomeningitis virus (LCMV) or the glial-tropic β-coronavirus JHMV (Sun, W., et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121; Shakya, A et al., (2015) J Exp Med 212, 2115-2131; Hughes, E. P., et al., (2025) J Clin Invest.). The normal T cell primary immune response phenotypes observed with Octi and OCA-B genetic deletion suggest the existence of a therapeutic window in which an OCA-B pharmaceutical could be used not just to block autoimmunity, but could be co-administered with ICIs to block irAEs while sparingAttorney Docket No. 21101.0501 Plimmunity to viruses and tumors. A likely side effect of therapies targeting this pathway would be an inability of newly-generated memory CD4+T cells to mount robust recall responses (Sun, W., et al., (2024) Proc Natl Acad Sci U S A 121, e2309153121; Shakya, A et al., (2015) J Exp Med 212, 2115-2131). The findings indicate that OCA-B promotes a memory-like population of CD4 T cells important for immune responses to tumor re-challenge and relapse (manuscript in prep). An OCA-B inhibitor may therefore need to be withdrawn prior to any potential tumor relapse.

[0423] Despite the prevailing but erroneous idea that transcription factors are “undruggable,” there are multiple historical (Polo, J. M., et al., (2004) Nat Med 10, 1329-1335; Wright, H. M., et al., (2000) J Biol Chem 275, 1873-1877; Vassilev, L. T., et al., (2004) Science 303, 844- 848; Cerchietti, L. C., et al., (2009) Blood 113, 3397-3405), and recent (Green, D. R. (2016). Cell 165, 1560; Skwarczynska, M., and Ottmann, C. (2015) Future Med Chem 7, 2195- 2219; Lu, G., et al., (2014). The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305-309; Fischer, E. S., et al., (2014) Nature 512, 49-53; Fiskus, W., et al., (2021). Blood Cancer J 11, 98; Issa, G. C., et al., (2023). The menin inhibitor revumenib in KMT2A-rearranged or NPM1-mutant leukaemia. Nature 615, 920-924; Antony-Debre, I., et al., (2017) J Clin Invest 127, 4297-4313; Taylor, S. J., et al., (2024) Nat Genet 56, 2213-2227; Gibson, S. A., et al., (2018) J Immunol 201, 383-392; Wang, F., et al., (2018) Blood 131, 2929-2942; Klase, Z., et al., (2014). PLoS Pathog 10, e!003997; Zhu, J., et al., (2020). Sci Rep 10, 1049; Struntz, N. B., et al., (2019) Cell Chem Biol 26, 711-723. e714; Frangoul, H., et al., (2021). N Engl J Med 384, 252-260; Jonasch, E., et al., (2021). N Engl J Med 385, 2036-2046) counterexamples, the latter enabled by technologies such as PROTAC (Bushweller, J. H. (2019). Nat Rev Cancer 19, 611-624;Bekes, M., et al., (2023). bioRxiv). It was shown that systemic administration of a small molecule inhibitor, DB1033, into NOD mice blocks the emergence of diabetes following the administration of a-PD-1 antibodies. These molecules disrupt Oct 1: OCA-B: DNA complexes in cell culture and in vitro. The finding that DB1033 administration concomitant with a-PD-1 antibody treatment is sufficient to block the induction of diabetes and other endocrinopathies suggests that OCA-B actions close to the time of injection, and therefore spatially proximate to the pLNs and pancreatic islets, rather than development of auto-reactive T cells in an OCA-B deficient environment from the double-positive thymocyte stage onwards, mediates autoimmune protection. Moreover, it was shown that a structurally related compound, DB2232, that is inferior in attenuating OCA-B-dependent transcription in cell culture and in disrupting Octi: OCA-B complexes in vitro is also functionally inferior at protecting NODAttorney Docket No. 21101.0501 Plmice against ICI-triggered diabetes, strongly suggesting that these compounds act through OCA-B disruption. These reagents provide a conceptual milestone as a first-in-class small molecule Octi / OCA-B inhibitor that can reduce autoimmunity. More work is necessary' to develop superior and more drug-like small molecule inhibitors.3. IDENTIFICATION OF ADDITIONAL OCA-B INHIBITORS AND EVALUATION IN PRECLINICAL MS MODELS

[0424] Developing new treatments to block autoimmunity while preserving beneficial immune function is a major goal in the field. T cells are central drivers of autoimmune pathogenesis, including in the pathophysiology of multiple sclerosis (MS). It has been shown that the transcriptional coregulator OCA-B is induced in pathogenic CD4+ T cells, where it docks with its cognate transcription factor Octi (FIG. 30) to drive the expression of targets including Il2, Il17a, Ifng, and Csf2 (Gmcsf) leading to pathogenic maturation. OCA-B regulates these genes only under conditions of antigen reencounter, e.g., during memory’ T cell responses to antigenic rechallenge or repeated encounter with self-antigen, a necessary feature of autoimmunity. Consistently, OCA-B deletion in mouse T cells blocks clinical, cellular, and molecular manifestations of type-1 diabetes (T1 D) and experimental autoimmune encephalomyelitis (EAE, a model of MS) while maintaining immune responses to neurotropic viruses. In a relapsing / remitting EAE model, OCA-B deletion specifically protects against relapse. GWAS identifies human polymorphisms at the Ocab / Pou2afl locus, as well as in binding sites for OCA-B, as autoimmune drivers. These observations, and OCA-B's lymphoid-restricted expression, position it as a robust drug target that could be used to treat autoimmune disease while preserving primary immune responses. Herein, a druggable pocket in the Octi: OCA-B interface was identified (FIG. 30) and used with cell-based screening platforms to identify small molecule OCA-B activators / inhibitors.

[0425] A primary OCA-B targeting strategy is shown in FIG. 30. Right: Oct1 / OCA-B / DNA co-crystal structure PDBID:1CQT Oval highlights area to disrupt the OCA-B / Octi interaction or identify molecules that bind the pocket most visible at far left that could be linked to a degrader. This strategy has identified small molecule activators and inhibitors As detailed herein, it is hypothesized that OCA-B is a druggable factor that drives expression of pathogenic target genes in autoreactive CD4+T cells to promote MS and other autoimmune diseases. The objectives of the studies detailed here are to identify additional superior OCA-B inhibitors and to test them in preclinical MS models. These objectives will be realized byAttorney Docket No. 21101.0501P1focusing on human MS, advancing prior hits and expanding these successful screens, and using DNA encoded libraries to identify additional compounds. Each of these efforts is further detailed below.a. OVERARCHING APPROACH

[0426] The Octi: OCA-B: DNA co-crystal structure immediately suggests multiple possible targeting strategies, including an OCA-B peptide mimic described herein above (FIG. 31, top lightning bolt). Another strategy uses DMA minor groove -binding small molecules with specificity for the octamer sequence. These inhibitors dock on tire underside of the DNA in the structure shown in FIG. 31 (bottom lightning bolt). Eleven candidate compounds were synthesized and tested with specificity for the octamer sequence 5'ATGCAAAT. To test efficacy, a two cell-based reporter system was developed. The first is a transcription-based platform. To build this system, the human T cell line SupTl was stably transfected to express secreted nanoluciferase (nine) under the control of five Octl / OCA-B binding sites, SupTl T cells express OCA-B at similar levels to activated CD4 T cells (FIG. 32). Time course experiments show that 12- to 18-hr incubations have not yet saturated nluc in the media and efficiently capture a dynamic range of nluc production allowing for both activators and inhibitors to be detected (not shown). To reduce hits due to nonspecific inhibition and toxicity, a second SupTl line was generated in which nluc is driven by a promoter with five mutant binding sites. This line expresses lower but measurable nluc (FIG. 33A). Alamar Blue readings were taken to measure cell viability, with 2.5 pM staurosporine treatment as a control for maximum toxicity, killing >98% of SupTl cells within 18 hr (not shown). This assay is currently configured as medium throughput, using 24-well plates and N=6 biological replicates per condition. Should it be necessary, the assay can be converted to 96- or 384-well plate format (the later with robotics, see letter from Drug Screening core director Bai Liu).

[0427] Three OCA-B inhibitor strategies are shown in FIG. 31. At right is the OctkOCA- B DNA co-crystal structure showing approaches with a peptide that mimics the key OCA-B alpha helix that mediates downstream effector functions (top lightning bolt), a DNA binding molecule that blocks Octi and hence OCA-B binding to DNA (bottom lightning bolt), and a strategy that blocks OCA-B binding to Octi through a druggable pocket (middle lightning bolt, P4 on the detail at left). Bottom left shows output from F Pocket, which uses 9 descriptors of druggability to calculate a score, on four potential pockets. Values above 0.5 suggest potential druggability. P4 generates a perfect score.Attorney Docket No. 21101.0501 Pl

[0428] FIG. 32 shows SupTl cells express OCA-B at similar levels to activated primary mouse CD4 cells. β-actin is shown as a loading control

[0429] An optimal screening workflow uses an orthogonal secondary screening methodology to reduce off-target hits. The second assay (FIG. 33B) uses NanoBRET to directly measure OCA-B's interaction with its cognate transcription factor Octi. In this assay, 293T cells are co-transfected with an OCA-B construct carrying a HaloTag and an Octi construct carrying a nluc tag. Alamar Blue and staurosporine toxicity readouts are again used as controls. Of the eleven candidate compounds synthesized two - known as DB1033 (IC50~ 25 μM, FIG. 33C) and DB2232 (IC50~ 20 μM, FIG. 26A)- scored in the transcription assay. Both also showed dose-dependent inhibition in NanoBRET (not shown). While unlikely to be translated (there are many more AAA T than Octi sites in the genome), this reagent provides a conceptual milestone as a first-in-class small molecule Octl / OCA-B inhibitor. These compounds will serve as the main positive controls and benchmarks for inhibition.

[0430] Two compound screening assay systems are shown in FIG. 33A-C. FIG. 33A shows transcription-based activity assay in SupTl cells. FIG. 33B shows more complex NanoBRET assay used for counter screening. FIG. 33C shows dose-dependent inhibition of transcription activity by the minor groove binder DB1033 is shown in red. Blue shows the effect on a mutant cell line with OCA-B independent activity (IC50» 70 μM).

[0431] High-performance computing was used to conduct an in silico screen of 139,735 compounds from the NCI developmental therapeutics program (DTP) library. These compounds were molecularly docked with the Octi: OCA-B: DNA structure at the three best potential binding pockets (DOCK10 and HiPerGator software, FIG. 31). This analysis identified several hundred possible candidates, mostly robustly for pocket P4 (FIG. 18). 112 of tire strongest P4 hits have been tested for efficacy and toxicity using the transcription assay (e.g., FIG. 34A), and have followed up 7 of the most promising with NanoBRET (FIG. 34B). An array of activators and inhibitors were identified (FIG. 34C-E). Interestingly many activators contained hexamethylenetetramine moieties (e.g., NSC 172855 in FIG. 34C) Most likely these compounds have selective affinity for the Octi OCA-B pocket and dock with Octi OCA-B complexes at P4. Hydrolysis of the hexamethylenetetramine group then allows the resulting aldehydes to crosslink the complex. This mechanism would explain both the activation and the toxicity observed at higher concentrations (FIG. 34C). The reactivity of compounds like NSC 172855 makes them suboptimal starting points for medicinal chemistry but their ability to stabilize Octl: OCA-B complexes makes them novel tools forAttorney Docket No. 21101.0501 Pltesting mechanism of action. Without wishing to be bound by theory, these findings indicate that this pipeline effectively identifies small molecule candidate compounds.

[0432] More drug -like active activators and inhibitors were also identified (FIG. 34E). These can be used as starting points for medicinal chemistry and SAR. lire larger Hit locator library (460,160 compounds can also be computationally screened, which is more enriched for druglike molecules. The top-scoring candidates can again be tested using the transcription-based and NanoBRET assays. Hits can then be tested for mechanism of action using a novel recombinant chimeric Octl-OCA-B protein. The same purified chimeric proteins can be used to screen for OCA-B binders using both DNA-encoded chemical libraries and libraries of natural metabolites.

[0433] Hits from a computation chemical screen of 139,735 compounds are shown in FIG.34A-E. FIG. 34A shows Octi: OCA-B crystal structure with docked top-scoring compound for P4 (yellow) shown as sticks. FIG. 34B shows detail from FIG. 34A. FIG. 34C shows activity of a hit from the screen (NSC 172855) is shown in the transcription assay. Tire compound is toxic at higher concentrations. FIG. 34D shows the same compound in the NanoBRET assay. FIG. 34E shows three additional compounds, two agonists and one antagonist, are shown. Note that limited toxicity results in “leakiness” that can increase baseline luciferase release. Data show mean for ≥n=5 replicates. Error bars denote ^standard deviation.b. AIM 1: DETERMINE IF CNS-INFILTRATING T CELLS IN HUMAN MS LESIONS EXPRESS ELEVATED OCA-B

[0434] CD4 + T cells expressing OCA-B at high levels confer disease in mouse EAE models. T cell OCA-B knockouts significantly blunt disease, and completely eliminate disease in relapsing / remitting mouse models. These results indicate a dominant role for this transcription regulator in preclinical models. However, nothing is known about OCA-B in human T cells. Determining the OCA-B pattern of expression, especially in pathogenic T cells, is a critical steppingstone in advancing this research.

[0435] To determine if OCAB (POU2AF1) is elevated in human MS samples, high-throughput single nucleus RNA-seq data of glial and immune cells (including T cells) were mined from 156 human MS patient brain lesions (Macnair W, et al., Neuron. 2025 Feb 5;113(3):396-410.e9). This paper focuses on glial cell responses, however by reclustering T cell nuclei in the absence of B cells, whose expression could complicate the analysis,Attorney Docket No. 21101.0501 Plpotentially pathogenic cells and the expression of P0U2AF1 were able to identified. The number of control (CTR) and relapsing-remitting (RRMS) samples was small, as expected given their lower mortality rates. The N=1 RRMS sample showed far higher P0U2AF1 expression compared to CTR (FIG. 35A). There were more primary-progressive (PPMS) samples, but interestingly only one sample showed significant expression, perhaps reflecting imperfections in diagnoses. Most dramatically, a subset of secondary-progressive (SPMS) samples, which would include many RRMS patients whose therapy had eventually failed, show elevated OCA-B expression (p<0.01, FIG. 35A). Because the datapoints were asymmetrically distributed around means, significance was ascribed using a one-way ANOVA with Tukey correction test. Similar results were obtained using a Wilcoxon test. UMAP feature plots of the aggregated single nucleus data also show higher P0U2AF1 expression in SPMS samples in one specific cluster that expresses the pathogenic marker IL 1 R1 (FIG. 35B), These results, from an external dataset, support the hypothesis that OCA-B is elevated in pathogenic CD4+ T cells in humans.

[0436] OCA-B expression in CD4 + T cells in human MS lesions is shown in FIG. 35A and FIG. 35B. FIG. 35A shows T cell nuclei from single-nucleus RNAseq data of MS patients and controls (ref.58) were reclustered, and POU2AFi (OCAB) expression plotted. Expression in control (CTR) tissue was compared to primary-progressive (PPMS), relapsing-remitting (RRMS) and secondary-progressive MS (SPMS). Significance was ascribed by one way ANOVA. Other comparisons were nonsignificant. FIG. 35B shows feature plots of T cell clustering by UMAP. Arrow identifies a pathogenic cluster marked by strong expression of proinflammatory genes (e.g., IL1R1) in SPMS. Several clusters also showed expression of, e.g., oligodendrocyte markers (not shown), suggesting doublets, however no such contamination was noted in the highlighted cluster.i. DETERMINE OCA-B PROTEIN EXPRESSION LEVELS AND LOCALIZATION IN T CELLS OF MS PATIENT LESIONS

[0437] To study OCA-B expression in patient CNS directly, FFPE-embedded tissue blocks from brains of patients diagnosed with MS can be used, for the most part PPMS and SPMS. The blocks are de-identified but maintain basic patient information and clinical reports via shadow IDs. Direct immunofluorescence in tissue sections of MS lesions with OCA-B, CD4 and CD 19 antibodies were attempted to detect OCA-B in T and B cells, which was difficult to interpret because of the weak intensity of OCA-B antibody staining (not shown). AAttorney Docket No. 21101.0501 Plsuperior way to test this samples is with RNAscope (ACD / Biotechne), a technology that combines antibody staining and modified in situ hybridization. RNAscope has the advantages of using fluorescent (or if needed chromogenic) dyes to identify signal from multiple mRNAs with high signal / noise and high dynamic range in a way that can be combined with antibodies (dual RNAscope in situ hybridization / IF). Probes are available for P0U2AF1 as well as CD4, CDBA and CD19. Using RNAscope, P0U2AF1 expression levels can be systematically assessed across the current samples, testing the specific hypothesis that P0U2AF1 will be detectable in lesion-proximal patient CD4+T cells. Without wishing to be bound by theory, it is expected that P0U2AF1 levels will be even higher in B cells, which are known to strongly and constitutively express OCA-B in all subtypes until the plasma cell stage. No expression in all other cell types (astrocytes, oligodendrocytes, neurons, microglia, and non-lymphoid infiltrating blood cells) is expected to be observed. Depending on the nature of the output data, ANOVA or similar assays can be used to assess significance. Assuming a failure rate of -10% (4 non-results and 36 samples with high quality present / absent staining), based on FIG. 22 it is expected that -20% or 6-8 human samples will show robust staining.ii. DETERMINE THE LOCALIZATION AND DEGREE OF EXPRESSION OF OCA-B+ CD4+ T CELLS IN CNS LESIONS FROM AN INDEPENDENT COHORT OF ~40 MS PATIENTS

[0438] OCA-B expression can be assessed in an independent cohort available from the Netherlands Brain Bank, part of the Netherlands Institute for Neuroscience. This nonprofit agency provides tissue samples, together with basic patient and pathological information (type of MS anatomic location within the brain of the lesion, medication / intake, etc.). Blood cells and CSF is also available for a subset of patients. More than 1500 specimens of cerebellar, DRG and hippocampal MS lesions are available. Tissue transfer occurs via simple MTA and tissue is deidentified. HIPM compliance is preserved because no patient identifiers are provided. Focusing on secondary-progressive MS (SPMS), OCA-B expression levels can be systematically assessed using RNAscope within patient and control tissue.c. AIM 2: ADVANCE OCA-B SMALL MOLECULE INHIBITORS IDENTIFIED BY COMPUTATIONAL CHEMISTRY

[0439] A library7of 140,000 compounds was screened in silica, focusing on one potential druggable pocket in the Octi: OCA-B interface. Using a cell-based transcription assay a setAttorney Docket No. 21101.0501 Plof potential OCA-B activators and inhibitors were identified. Next, an orthogonal NanoBRET screen was used to show that many of these molecules modulate Octl: OCA-B complex formation in cells. Using a hit advancement strategy (FIG. 36), it was shown that the first-in-class DB2033 inhibitor disrupts the protein:DNA complex in vitro (below). Without wishing to be bound by theory, these findings show that the entire pipeline from screening to mechanism of action (FIG. 36, top) works. Here, similar strategies can be used to identify and refine candidate activators and inhibitors of OCA-B function and to determine their mechanisms of action. Knowing that agonists can be easily turned into antagonists, the ultimate goal is to identify robust inhibitors that are efficacious in vivo both as lab tools and as lead compounds to that can ultimately be tested for pharmacokinetics, pharmacodynamics, and in vivo toxicity / efficacy.

[0440] The hit advancement strategy is shown in FIG. 36.i. OPTIMIZE AND TEST EXISTING HITS FROM THE NCI DTP LIBRARY.

[0441] The initial round of computational screening and in vitro evaluation identified compounds containing potentially reactive functional groups that are likely responsible for the observed activity (e.g. hexamethylenetetramines and the bis-aminal NSC 303535).Expanding the screening platform, activity in a more diverse set of scaffolds was identified (FIG. 34C-E and not shown). Both activators (e.g. NSC 351358) and inhibitors (e.g. NSC 328487) of OCA-B-dependent transcription activity were identified. These hits are significantly more tractable for development. Medicinal chemistry can be performed around these and other hits. Given the symmetric nature of these hit compounds, it can be straightforward to prepare focused chemical libraries (~20 compounds) based on each hit scaffold. Evaluation of these libraries can identify scaffolds with or without defined structureactivity relationships. Scaffolds with observable potency trends will be subject to subsequent rounds of molecular modeling and medicinal chemistry.

[0442] These molecules will then be inserted back into the pipeline starting with the transcriptiona...

Claims

Attorney Docket No. 21101.0501 PlCLAIMSWhat is claimed is:

1. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a compound having a structure selected from:or a pharmaceutically acceptable salt thereof.

2. Tire method of claim L wherein the compound is:or a pharmaceutically acceptable salt thereof.The method of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof.The method of any one of claims 1 to 3, wherein the subject is a mammal.Tire method of any one of claims 1 to 3, wherein the subject is a human.Attorney Docket No. 21101.0501 Pl6. The method of any one of claims 1 to 5, wherein the subject has been diagnosed with a need for treatment of the autoimmune disease prior to the administering step.

7. The method of any one of claims 1 to 6, further comprising the step of identifying a subject in need of treatment of the autoimmune disease.

8. The method of any one of claims 1 to 7, wherein the effective amount is a therapeutically effective amount.

9. The method of any one of claims 1 to 7, wherein the effective amount is a prophylactically effective amount.

10. The method of any one of claims 1 to 9, wherein the autoimmune disease is selected from primary biliary cirrhosis, psoriasis, Addison disease. Celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory bowel disease, multiple sclerosis (MS), myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, systemic lupus erythematosus, and type I diabetes.

11. The method of any one of claims 1 to 9, wherein the autoimmune disease is MA, 12. The method of any one of claims 1 to 9, wherein the autoimmune di sease is type I diabetes.

13. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a compound having a structure represented by a formula:wherein X1is selected from O and S;wherein R1is selected from hydrogen and halogen; andwherein each of R2and R3is independently selected from –C(=NH)NHR10and Cy1; wherein each occurrence of R10is independently selected from hydrogen and Cl- C4 alkyl; andAttorney Docket No. 21101.0501 Plwherein each occurrence of Cy1is independently a structure:p20owherein n is selected from 0 and 1;wherein each of R20aand R20bis independently selected from hydrogen and halogen,or a pharmaceutically acceptable salt thereof,provided that when each of R2and R3is –C(=NH)NHR10, then either R1is halogen, R10is hydrogen, or R1is halogen and R10is hydrogen.

14. The method of claim 13, wherein X1is S,15. The method of claim 13 or claim 14, wherein R1is hydrogen.

16. The method of claim 13 or claim 14, wherein R1is halogen.

17. The method of claim 13 or claim 14, wherein R1is -F.

18. The method of any one of claims 13 to 17, wherein each of R2and R3is – C(=NH)NHR10.

19. The method of any one of claims 13 to 17, wherein each of R2and R3is –C(=NH)NH2.

20. The method of any one of claims 13 to 17, wherein each of R2and R3is -C(=NH)NH(C1-C4 alkyl).

21. The method of any one of claims 13 to 17, wherein each of R2and R’ is Cy!.

22. The method of any one of claims 13 to 21, wherein Cy1is a structure:

23. The method of any one of claims 13 to 21, wherein Cy1is a structure:Attorney Docket No. 21101.0501 Pl24. The method of any one of claims 13 to 21, wherein Cy1is a structure:

25. The method of any one of claims 13 to 21, wherein Cy1is a structure:F26. The method of claim 13, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

27. The method of claim 13, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

28. The method of claim 13, wherein the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

29. The method of claim 13, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

30. The method of claim 13, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

31. The method of claim 13, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.Attorney Docket No. 21101.0501 Pl 32. The method of claim 13. wherein the compound is selected from:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

33. The method of any one of claims 13 to 32, wherein the subject is a mammal.

34. The method of any one of claims 13 to 32, wherein the subject is a human.

35. The method of any one of claims 13 to 34, wherein the subject has been diagnosed with a need for treatment of the autoimmune disease prior to the administering step.

36. The method of any one of claims 13 to 35, further comprising the step of identifying a subject in need of treatment of the autoimmune disease.

37. The method of any one of claims 13 to 36, wherein the effective amount is a therapeutically effective amount.Attorney Docket No. 21101.0501 Pl38. The method of any one of claims 13 to 36, wherein the effective amount is a prophylactically effective amount.

39. The method of any one of claims 13 to 38, wherein the autoimmune disease is selected from primary’ biliary cirrhosis, psoriasis, Addison disease, Celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory’ bowel disease, multiple sclerosis (MS), myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, systemic lupus erythematosus, and type I diabetes.

40. The method of any one of claims 13 to 38, wherein the autoimmune disease is MS.

41. The method of any one of claims 13 to 38, wherein the autoimmune disease is type 1 diabetes.

42. A compound having a structure represented by a formula:wherein X2is selected from O, S, and Se;wherein R1is selected from hydrogen and halogen; andwherein each occurrence ofn is independently selected from 0 and 1;wherein each occurrence of R20aand R20bis independently selected from hydrogen and halogen,or a pharmaceutically acceptable salt thereof,provided that either R1is halogen, each occurrence of R20aand R20bis independently halogen, or R1is halogen and each occurrence of R20aand R20bis independently halogen.

43. The compound of claim 42, wherein X2is S.

44. The compound of claim 42 or claim 43, wherein R1is hydrogen.

45. The compound of claim 42 or claim 43, wherein R1is halogen.Attorney Docket No. 21101.0501 Pl46. The compound of any one of claims 42 to 45, wherein each occurrence of n is 0.

47. The compound of any one of claims 42 to 45, wherein each occurrence ofn is 1.

48. The compound of claim 47, wherein each occurrence of R20aand R20bis hydrogen.

49. The compound of claim 47, wherein each occurrence of R20aand R20bis halogen.

50. The compound of claim 47, wherein each occurrence of R20aand R20bis -F.

51. The compound of claim 42, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

52. The compound of claim 42, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

53. The compound of claim 42, wherein the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

54. The compound of claim 42 wherein the compound is selected from:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

55. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of claims 42 to 54, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

56. A method of inhibiting OCA-B and / or OCA-T in a cell, the method comprising contacting the cell with an effective amount of the compound of any one of claims 42 to 54, or a pharmaceutically acceptable salt thereof.

57. The method of claim 56, wherein the method inhibits OCA-B.

58. The method of claim 56, wherein the method inhibits OCA-T59. The method of claim 56, wherein the method inhibits OCA-B and OCA-T.

60. The method of any one of claims 56 to 59, wherein the cell is mammalian.

61. The method of any one of claims 56 to 59, wherein the cell is human.

62. The method of any one of claims 56 to 61, wherein the cell has been isolated from a human prior to the administering step.

63. The method of any one of claims 56 to 62, wherein con tacting is via administration to a subject.

64. The method of any one of claims 56 to 63, wherein the subject has been diagnosed with a need for inhibition of OCA-B and / or OCA-T activity prior to the administering step.

65. The method of any one of claims 56 to 64, wherein the subject has been diagnosed with a need for treatment of disease or disorder associated with OCA-B and / or OCA-T activity.Attorney Docket No. 21101.0501 Pl66. A method of inhibiting OCA-B and / or OCA-T in a subject, the method comprising administering to the subject an effective amount of the compound of any one of claims 42 to 54, or a pharmaceutically acceptable salt thereof.

67. The method of claim 66, wherein the method inhibits OCA-B.

68. The method of claim 66, wherein the method inhibits OCA-T69. The method of claim 66, wherein the method inhibits OCA-B and OCA-T.

70. The method of any one of claims 66 to 69, wherein the subject has been diagnosed with a need for inhibition of OCA-B and / or OCA-T activity prior to the administering step.

71. The method of any one of claims 66 to 70, wherein the subject has been diagnosed with a need for treatment of a disease or disorder associated with OCA-B and / or OCA-T activity prior to the administering step.

72. The method of any one of claims 66 to 71, further comprising the step of identifying a subject in need of treatment of a disease or disorder associated with OCA-B and / or OCA-T activity.

73. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the compound of any one of claims 42 to 54, or a pharmaceutically acceptable salt thereof,74. The method of claim 56, wherein the subject is a mammal.

75. The method of claim 56, wherein the subject is a human.

76. The method of any one of claims 56 to 75, wherein the subject has been diagnosed with a need for treatment of cancer prior to the administering step,77. The method of any one of claims 56 to 76, further comprising the step of identifying a subject in need of treatment of cancer.

78. The method of any one of claims 56 to 77, wherein the effective amount is a therapeutically effective amount.Attorney Docket No. 21101.0501 Pl79. The method of any one of claims 56 to 77, wherein the effective amount is a prophylactically effective amount.

80. The method of any one of claims 56 to 79, wherein the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, kidney cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, lung cancer, a glioma, and plasma cell neoplasm (myeloma).

81. The method of any one of claims 56 to 79, wherein the cancer is lung cancer.

82. The method of any one of claims 56 to 79, wherein the cancer is non-small cell lung cancer (NSCLC).

83. The method of any one of claims 56 to 79, wherein the cancer is a lymphoma.

84. The method of claim 83, wherein the lymphoma is diffuse large B cell lymphoma.

85. A kit comprising an effective amount of the compound of any one of claims 42 to 54, or a pharmaceutically acceptable salt thereof, and one or more selected from:(a) an agent known to treat an autoimmune disease;(b) a chemotherapeutic agent;(c) instructions for administering the compound in connection with treating an autoimmune disease, cancer, and / or an immune-related adverse event (irAE); and(d) instructions for treating an autoimmune disease, cancer, and / or an immune- related adverse event (irAE).

86. The kit of claim 85, wherein the agent known to treat an autoimmune disease is selected from a corticosteroid (e.g., prednisone, methylprednisolone)), a disease-modifying antirheumatic drug (DMARD) (e.g., methotrexate, sulfasalazine, leflunomide), a biologic (e.g., adalimumab, infliximab, secukinumab), an immunosuppressant (azathioprine,Attorney Docket No. 21101.0501 Plcyclosporine, mycophenolate mofetil), a j anus kinase (JAK) inhibitor e.g, tofacitinib), and a nonsteroidal aiiti-inflammatory drug (NSAID) (ibuprofen, naproxen).

87. The kit of claim 85, wherein the chemotherapeutic agent is selected from an alkylating or cross-linking agent, an antimetabolite agent, an antineoplastic antibiotic agent, a mitotic inhibitor agent, an mTor inhibitor agent, a kinase inhibitor, and an immune checkpoint inhibitor.

88. The kit of claim 87, wherein the antineoplastic antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt thereof.

89. Tire kit of claim 87, wherein the antimetabolite agent is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof.

90. The kit of claim 87, wherein the alkylating or cross-linking agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharmaceutically acceptable salt thereof.

91. The kit of claim 87, wherein the mitotic inhibitor agent is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etoposide, vincristine, ixabepilone, vinorelbine, vinblastine, mitoxantrone, and teniposide, or a pharmaceutically acceptable salt thereof.

92. The kit of claim 87, wherein the mTor inhibitor agent is selected from everolimus, sirolimus, and temsirolimus, or a pharmaceutically acceptable salt thereof.

93. The kit of claim 87, wherein the kinase inhibitor is selected from BCR-ABL inhibitors (e.g., imatinib, dasatinib, nilotinib, bosutinib, ponatinib), EGFR inhibitors (e.g., erlotinib, gefitinib, afatinib, osimertinib, dacomitinib), ALK Inhibitors (e.g., crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, VEGFR inhibitors (e.g., sunitinib, sorafenib, pazopanib, axitinib, regorafenib, lenvatinib, cabozantinib), BRAF inhibitors (e.g., vemurafenib,Attorney Docket No. 21101.0501 Pldabrafenib, encorafenib), MEK inhibitors (e.g., trametinib, cobimetinib, binimetinib), HER2 inhibitors (e.g., lapatinib, neratinib, tucatinib), CDK4 / 6 inhibitors (e.g., palbociclib, ribociclib, abemaciclib), BTK inhibitors (e.g., ibrutinib, acalabrutinib, zanubrutinib, pirtobrutinib), PI3K inhibitors (e.g., idelalisib, copanlisib, duvelisib, alpelisib), FLT3 inhibitors (e.g., midostaurin, gilteritinib, quizartinib), and JAK inhibitors (e.g., ruxolitinib, fedratinib, pacritinib, momelolitinib), or a pharmaceutically acceptable salt thereof, 94. The kit of claim 87, wherein the immune checkpoint inhibitor is selected from pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, relatlimab. cemiplimab, dostarlimab, tremelimumab, retifanlimab, and toripalimab.

95. The kit of any one of claims 85 to 94, wherein the compound and the agent known to treat an autoimmune disease are co-packaged.

96. The kit of any one of claims 85 to 94, wherein the compound and the agent known to treat an autoimmune disease are co-formulated.

97. The kit of any one of claims 85 to 94, wherein the compound and the chemotherapeutic agent are co-packaged.

98. The kit of any one of claims 85 to 94, wherein the compound and the chemotherapeutic agent are co-formulated.

99. A method of treating or preventing an immune-related adverse event (irAE) in a cancer patient undergoing immunotherapy, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula:wherein X3is selected from O, S, and Se;wherein R1is selected from hydrogen and halogen; andwherein each of R2and R3is independently selected from –C(=NH)NHR10or Cy1;wherein each occurrence of R10is independently selected from hydrogen or Cl- C4 alkyl; andAttorney Docket No. 21101.0501 Plwherein each occurrence of Cy1is independently selected from a structure:wherein each occurrence of n is independently selected from 0 or 1; and wherein each occurrence of R20ais independently selected from hydrogen or halogen and each occurrence of R20bis independently selected from hydrogen or halogen,or a pharmaceutically acceptable salt thereof.

100. The method of claim 99, wherein X3is selected from O and S and wherein when each of R2and R’ is independently selected from -C(:::NH)NHR10, then either R1is halogen, R!0is hydrogen, or R1is halogen and R'° is hydrogen.

101. The method of claim 99, wherein the compound has a structure represented by a formula:provided that either R1is halogen, each occurrence of R20aand R20bis independently selected from halogen, or R1is halogen and each occurrence of R20aand R20bis independently selected from halogen.

102. The method of claim 99, wherein X3is S.

103. The method of claim 99 or claim 102, wherein R1is hydrogen.

104. The method of claim 99 or claim 102, wherein R1is halogen.

105. The method of any one of claims 99 to 104, wherein each of R2and R3is independently selected from -C(=NH)NHR10.Attorney Docket No. 21101.0501 Pl106. The method of any one of claims 99 to 104, wherein each of R2and R3is independently selected from Cy1.

107. The method of claim 99, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

108. The method of claim 99, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

109. The method of claim 99, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

110. The method of claim 99, wherein the compound has a structure represented by a formula:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

111. The method of claim 99, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

112. The method of claim 99, wherein the compound has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

113. The method of claim 99, wherein the compound is selected from:Attorney Docket No. 21101.0501 PlAttorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

114. The method of claim 99, wherein the compound is selected from:Attorney Docket No. 21101.0501 Plor a pharmaceutically acceptable salt thereof.

115. The method of any one of claims 99 to 114 wherein the subject is a mammal.

116. The method of any one of claims 99 to 114, wherein the subject is a human.

117. The method of any one of claims 99 to 116 wherein the subject has been diagnosed with a need for treatment of the autoimmune disease prior to the administering step.

118. The method of any one of claims 99 to 117, further comprising the step of identifying a subject in need of treatment of the autoimmune disease.

119. The method of any one of claims 99 to 118, wherein the effective amount is a therapeutically effective amount.

120. The method of any one of claims 99 to 118, wherein the effective amount is a prophylactically effective amount.

121. The method of any one of claims 99 to 120, wherein the irAE is selected from a rash, diarrhea, colitis, hypothyroidism, and pneumonitis.

122. The method of any one of claims 99 to 121, wherein the cancer patient is undergoing immunotherapy with an immune checkpoint inhibitor.

123. The method of claim 122, wherein the immune checkpoint inhibitor is selected from pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, relatlimab. cemiplimab, dostarlimab, tremelimumab, retifanlimab, and toripalimab.

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