Methods of treating myocarditis

Abstract

Methods of treating myocarditis are provided. In particular, antibodies that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) are useful for treating myocarditis and inhibiting infiltration of T cells and macrophages into cardiac tissue.

Description

METHODS OF TREATING MYOCARDITISCROSS-REFERENCE To ELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional applications 63 / 748,306, filed January 22, 2025, which applications are hereby incorporated by reference in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. R01 HL156021 , Grant No. R01 HL155990, and Grant No. R01 HL141466 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Immune checkpoint inhibitors (ICIs), antibodies against T-cell inhibiting cellsurface proteins such as CTLA-4, PD-1 , and LAG-3, have dynamically changed the landscape of cancer treatment. Anti-PD-1 therapy has emerged as the backbone of treatment in cancers that were previously associated with poor survival, such as metastatic melanoma1. Combination therapy (e g., anti-CTLA-4 and anti-PD-1) have additional benefits. However, ICIs (especially in combination) can lead to untoward immunological responses in healthy tissues, termed immune related adverse events (irAEs)2. ICI-myocarditis is a rare (<1-2% of ICI-treated patients) but highly morbid irAE, with a mortality rate of up to 46%3. The current mainstay of ICI-myocarditis treatment are corticosteroids for non-specific immunosuppression which has limited benefit and has a variety of adverse side effects4. As such, there is an unmet clinical need to devise more targeted therapies for the disease.SUMMARY

[0004] Methods of treating myocarditis are provided. In particular, antibodies that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) are useful for treating myocarditis and inhibiting infiltration of T cells and macrophages into cardiac tissue.

[0005] In one aspect, a method of treating myocarditis is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds to CXCR6.

[0006] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, and a scFv fragment.

[0007] In certain embodiments, the method further comprises administering an angiotensin- converting-enzyme (ACE) inhibitor, a beta blocker, a diuretic, a corticosteroid, an immunosuppressant, intravenous immunoglobulin (MG), an immune checkpoint inhibitor, or any combination thereof.

[0008] In certain embodiments, the antibody is administered intravenously, subcutaneously, or intraperitoneally. In some embodiments, the antibody is administered locally to the heart.

[0009] In certain embodiments, the antibody is administered before, during, or after treatment of the subject with an immune checkpoint inhibitor.

[0010] In certain embodiments, the antibody is administered before, during, or after treatment of the subject with anti-LAG-3 / anti-PD-1 combination therapy.

[0011] In certain embodiments, multiple cycles of treatment are administered to the subject.In some embodiments, the antibody is administered according to a daily dosing regimen or intermittently.

[0012] In another aspect, a method of inhibiting infiltration of T cells and macrophages into cardiac tissue of a subject is provided, the method comprising administering an effective amount of an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) to the subject. In some embodiments, the antibody is administered intravenously, subcutaneously, or intraperitoneally. In some embodiments, the antibody is administered locally to the heart.

[0013] In another aspect, a composition comprising an antibody that specifically binds to CXCR6 for use in treating myocarditis is provided. In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, and a scFv fragment. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient or carrier.

[0014] In another aspect, the use of an antibody that specifically binds to CXCR6 in the manufacture of a medicament or pharmaceutical composition for treating myocarditis is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0016] FIGS. 1A-1F. Lag3- / -,Pdcd1- / - mice exhibit pre-mature lethality with cardiac-enriched inflammation and arrhythmias. FIG.1A) Survival curves of Lag3- / -,Pdcd1- / - mice as compared to Pdcdl- / - mice. FIG. 1 B) Representative Histological Images of Lag3- / -,Pdcd1- / - and Pdcdl- / - mice FIG. 1C) Histological grading of Lag3- / -, Pdcdl- / - and Pdcdl- / - mice. FIG. 1D) Serum chemistry analysis of Lag3- / -, Pdcdl- / -, Pdcdl- / -, and healthy Wildtype mice. FIG. 1E) Representative arrhythmias seen on EKG of Lag3- / -, Pdcdl- / - mice. FIG. 1F) Arrhythmia burden (AV block, ventricular escape, PVCs) overtime in Lag3- / -, Pdcdl- / - mice.

[0017] FIGS. 2A-2F. Lag3- / -, Pdcdl- / - mice have dynamic changes in cardiac immune populations, with robust expansion of infiltrating T-cells FIG. 2A) Flow cytometry of CD3+ T- cells of 6 week old Lag3- / -, Pdcdl- / - mice and associated controls. FIG. 2B) UMAP of cardiac CD45+ sorted scRNA-seq, split by genotype (Lag3- / -, Pdcdl- / - mice n=4, WT mice n=6). FIG.20) Heatmap of markers defining each annotated cluster. FIG. 2D) Feature Plot representing Activated T-cells Markers. FIG. 2E) UMAP of cardiac T-cell clones in WT and Lag3- / -, Pdcdl - / - mice. FIG. 2F) Distribution of clone size in WT and Lag3- / -, Pdcdl- / - mice.

[0018] FIGS. 3A-3F. CXCR6+ T-cells are upregulated in mouse models and human ICI- myocarditis. FIG.3A) Feature Plot of Cxcr6 expression in WT and Lag3- / -, Pdcdl- / - mice. FIG.3B) Flow cytometry of CXCR6+ T-cells in different inflamed tissues in Lag3- / -, Pdcdl- / - mice.FIG. 3C) UMAP of publicly available human ICI-myocarditis and control, with Feature Plot for CXCR6. FIG. 3D) Subclusters of Cxcr6 expressing T-cells defines 5 unique clusters. FIG. 3E) Heatmap of Cxcr6 T-cell subcluster markers. FIG. 3F) Volcano Plot of markers Hyperexpanded T-cell clones.

[0019] FIGS. 4A-4F. CXCL16, the cognate ligand of CXCR6, is dynamically increased in cardiac macrophages. FIG. 4A) UMAP of Macrophage clusters demonstrating distinct populations. FIG. 4B) Heatmap of the defining markers of macrophage subsets. FIG. 4C) Feature Plot of Cxcl16 expression in WT and Lag3- / -, Pdcdl- / - mouse hearts. FIG. 4D) Violin Plot, split by cluster and sample, of Cxcl16 demonstrating expansion of Cxcl16 expressing cardiac macrophages. FIG.4E) Flow Cytometry on relative CXCL16+ Macrophage population (gated on Live / CD45+ / CD11b+ / F4-80+). FIG. 4F) Chemotaxis Assay of sorted T-cells from Lag3- / -, Pdcdl- / - blood and heart to recombinant mouse CXCL16 (rmCXCL16).

[0020] FIGS. 5A-5E. CXCR6+ T-cells are necessary for Lag3- / -, Pdcdl- / - myocarditis. FIG.5A) Survival curves of Lag3- / -, Pdcdl- / - treated with 250 pg twice weekly isotype control (n=8) and 250 pg twice weekly anti-CXCR6 (n=8). FIG. 5B) Representative histology of isotype control and anti-CXCR6 treated mice (Scale Bars: 500 pm and 100 pm). FIG.5C) Myocarditis Lesional area in isotype and anti-CXCR6 treated mice. FIG. 5D) Representative EKGs from isotype and anti-CXCR6 treated mice. FIG.5E) Burden of arrhythmias based on weekly EKGs of isotype and anti-CXCR6 treated mice.

[0021] FIGS. 6A-6C. VigiBase analysis of anti-LAG-3 therapy and risk for irAEs. FIG. 6A) Schematic of methodology for Vigibase analysis. FIG. 6B) Multivariate analysis of anti-PD-1 therapy associated myocarditis. FIG. 6C) anti-LAG-3 / anti-PD-1 therapy univariate and multivariate analysis across difference irAEs.

[0022] FIGS. 7A-7D. Left Ventricular (LV) Echocardiographic measurements on Lag3- / - ,Pdcd1- / - mice (n=8) in comparison with Wild Type (n=8) at 4 weeks and 6 weeks of age. FIG.7A. LV end diastolic volume (EDV) at 4 weeks. FIG. 7B. Ejection fraction (EF) at 4 weeks.FIG. 7C. LV end diastolic volume (EDV) at 6 weeks. FIG. 7D. Ejection fraction (EF) at 6 weeks.

[0023] FIG. 8A. Flow Cytometry of relative CD4 and CD8 T-cell populations in WT, Pdcdl- / -, and Lag3- / -, Pdcdl- / - models. FIG. 8B. FeaturePlot of Cd4 and Cd8a split by WT or Lag3- / - , Pdcdl- / - from CD45+ sorted scRNA-seq data.

[0024] FIG. 9A. Cxcr6 Feature Plot from Ctla4+ / -, Pdcdl- / - cardiac CD45+ scRNA-seq data.FIG. 9B. Violin Plot of Cxcr6 expression based on cell type cluster from Clta4+ / -, Pdcdl - / - cardiac CD45+ scRNA-seq data.

[0025] FIG. 10A. Relative CXCR3+ expression on T-cells from different tissues in Lag3- / - .Pdcdl- / - mice. FIG. 10B. FeaturePlot of Cxcr6 and Cxcr3 expression split by T-cell clone size.FIG. 10C. anti-CXCR3 vs Isotype control of Lag3- / -, Pdcdl- / - mice. FIG. 10D. Arrhythmia burden in anti-CXCR3 vs Isotype control. FIG. 10E. Representative histology of Isotype and anti-CXCR3 treated Lag3- / -, Pdcdl- / - mice.

[0026] FIG. 11. Gating Strategy of cardiac immune populations from mouse heart digests.DETAILED DESCRIPTION OF EMBODIMENTS

[0027] Methods of treating myocarditis are provided. In particular, anti-CXCR6 antibodies are useful for treating myocarditis and inhibiting infiltration ofT cells and macrophages into cardiac tissue.

[0028] Before the methods of treating myocarditis with anti-CXCR6 antibodies are described, it is to be understood that this invention is not limited to a particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range,and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0031] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies and reference to "the inhibitor" includes reference to one or more inhibitors and equivalents thereof, such as antagonists, receptor blockers, and the like, known to those skilled in the art, and so forth.

[0033] The term "antibody" encompasses monoclonal antibodies, polyclonal antibodies, as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies. The term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); bispecific antibodies, bispecific T cell engager antibodies (BiTE), trispecific antibodies, and other multispecific antibodies (see, e.g., Fan et al. (2015) J. Hematol. Oncol. 8:130, Krishnamurthy et al. (2018) Pharmacol Ther.185:122-134), F(ab')2and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11 :3287-3303, Vincke et al. (2012) Methods Mol B / o / 911 :15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126);humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific antigen-binding properties of the parent antibody molecule.

[0034] The phrase "specifically (or selectively) binds" with reference to binding of an antibody to an antigen (e.g., CXCR6) refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular antigen at least two times over the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, antibodies raised to an antigen from specific species such as rat, mouse, or human can be selected to obtain only those antibodies that are specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

[0035] "Antibody fragment", and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety and (4) nanobodiescomprising single Ig domains from non-human species or other specific single-domain binding modules; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and / or can contain any hinge region sequence found in an intact antibody, and / or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).

[0036] A "humanized antibody" is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0037] As used in this disclosure, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0038] By "isolated" is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0039] The term "conjugated" refers to the joining by covalent or noncovalent means of two compounds or agents.

[0040] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e. , arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted (e.g., those with myocarditis) as well as those in which prevention is desired (e.g., those with increased susceptibility to myocarditis, those with a risk of developing myocarditis, etc.).

[0041] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0042] By "therapeutically effective dose or amount" of an anti-CXCR6 antibody or antigenbinding fragment thereof that specifically binds to CXCR6 is intended an amount that, when administered, as described herein, brings about a positive therapeutic response in the treatment of myocarditis, such as an amount that reduces heart inflammation and arrhythmias and / or improves cardiac function and increases survival. Additionally, a therapeutically effective dose or amount of an anti-CXCR6 antibody or antigen-binding fragment thereof may inhibit infiltration of T cells and macrophages into cardiac tissue. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0043] The terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally- occurring amino acids.

[0044] "Substantially purified" generally refers to isolation of a substance (e.g., antibody, conjugate, compound, drug, nucleic acid, polynucleotide, protein, polypeptide, or peptide) such that the substance comprises the majority of the sample in which it resides and be defined as a percentage of the total sample. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying substances of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0045] By "subject" is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.

[0046] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0047] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0048] As used herein, the terms "detectable label", "detection agent", "imaging agent", "diagnostic agent", and “detectable moiety” are used interchangeably and refer to a molecule or substance capable of detection, including, but not limited to, fluorescers, chemiluminescers, chromophores, bioluminescent proteins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, isotopic labels, semiconductor nanoparticles, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of detectable labels which may be used in the practice of the invention include isotopic labels, including radioactive and non-radioactive isotopes, such as,3H,2H,12°I,123l,124l,125l,131l,35S,11C,13C,14C,32P,15N,13N,11°ln,111ln,177Lu,18F,52Fe,62Cu,64Cu,67Cu,67Ga,68Ga,86Y,90Y,89Zr,94mTc,94Tc,99mTc,154Gd,155Gd,156Gd,157Gd,158Gd,15O,186Re,188Re,51M,52mMn,55Co,72As,75Br,76Br,82mRb, and83Sr. In particular, detectable labels may comprise positron-emitting radionuclides suitable for PET imaging such as, but not limited to,64Cu,89Zr,68Ga,177Lu,82Rb,11C,13N,15O, and18F; or gamma-emitting radionuclides suitable for single photon emission computed tomography (SPECT) imaging such as, but not limited to,67Ga,99mTc,123l, and131l. Detectable labels may also include non-radioactive, paramagnetic metal ions suitable for MRI imaging such as, but not limited to, Mn2+, Fe3+, Fe2+, Gd3+, Ti2+, Cr3+, Co2+, Ni2+, and Cu2+. Detectable labels may also include fluorophores including without limitation, SYBR green, SYBR gold, a CAL Fluor dye such as CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, and CAL Fluor Red 635, a Quasar dye such as Quasar 570, Quasar 670, and Quasar 705, an Alexa Fluor such as Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 784, a cyanine dye such as Cy 3, Cy3.5, Cy5, Cy5.5, and Cy7, fluorescein, 2', 4', 5', 7'-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein (FAM), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), hexachlorofluorescein (HEX), rhodamine, carboxy-X-rhodamine (ROX), tetramethyl rhodamine (TAMRA), FITC, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, and quantum dots, enzymes such as alkaline phosphatase (AP), beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r) dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), p-galactosidase (lacZ), and xanthine guanine phosphoribosyltransferase (XGPRT), beta-glucuronidase (gus), placental alkaline phosphatase (PLAP), and secreted embryonic alkaline phosphatase (SEAP). Enzyme tags are used with their cognate substrate. The terms also include chemiluminescent labels such as luminol, isoluminol, acridinium esters, and peroxyoxalate and bioluminescent proteins such as firefly luciferase, bacterial luciferase, Renilla luciferase, and aequorin. The terms also include color-coded microspheres of known fluorescent light intensities (see e.g., microspheres with xMAP technology produced by Luminex (Austin, TX); microspheres containing quantum dot nanocrystals, for example, containing different ratios and combinations of quantum dot colors (e.g., Qdot nanocrystals produced by Life Technologies (Carlsbad, CA); glass coated metal nanoparticles (see e.g., SERS nanotags produced by Nanoplex Technologies, Inc. (Mountain View, CA); barcode materials (see e.g., sub-micron sized striped metallic rods such as Nanobarcodes produced by Nanoplex Technologies, Inc.), encoded microparticles with colored bar codes (see e.g., CellCard produced by Vitra Bioscience, vitrabio.com), glass microparticles with digital holographic code images (see e.g., CyVera microbeads produced by Illumina (San Diego, CA), near infrared (NIR) probes, andnanoshells. The terms also include contrast agents such as ultrasound contrast agents (e.g. SonoVue microbubbles comprising sulfur hexafluoride, Optison microbubbles comprising an albumin shell and octafluoropropane gas core, Levovist microbubbles comprising a lipid / galactose shell and an air core, Perflexane lipid microspheres comprising perfluorocarbon microbubbles, and Perflutren lipid microspheres comprising octafluoropropane encapsulated in an outer lipid shell), magnetic resonance imaging (MRI) contrast agents (e.g., gadodiamide, gadobenic acid, gadopentetic acid, gadoteridol, gadofosveset, gadoversetamide, gadoxetic acid), and radiocontrast agents, such as for computed tomography (CT), radiography, or fluoroscopy (e.g., diatrizoic acid, metrizoic acid, iodamide, iotalamic acid, ioxitalamic acid, ioglicic acid, acetrizoic acid, iocarmic acid, methiodal, diodone, metrizamide, iohexol, ioxaglic acid, iopamidol, iopromide, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipiodone, iobenzamic acid, iopanoic acid, iocetamic acid, sodium iopodate, tyropanoic acid, and calcium iopodate). The detectable label or imaging agent may be attached indirectly or directly to an antibody, wherein the label or contrast agent facilitates the use of the antibody in imaging.

[0049] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math.2:482489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0050] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0051] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined forthat particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0052] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0053] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0054] "Recombinant host cells", "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0055] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0056] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0057] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0058] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a donor polynucleotide, plasmid, or viral vector construct. In addition to the components of the expression cassette, the construct may also include, one or more selectable markers, a signal which allows theconstruct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0059] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0060] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0061] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as plasmid and viral vectors.

[0062] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0063] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.Anti-CXCR6 Antibodies

[0064] Anti-CXCR6 antibodies are provided that specifically bind to one or more epitopes of CXCR6. The term "anti-CXCR6 antibody" as used herein encompasses full-length antibodies as well as antigen-binding fragments thereof that include the antigen-binding region of an antibody, e.g., a molecule that includes the CDRs of an anti-CXCR6 antibody such as a Fab, F(ab')2, or Fvfragment, a single-chain variable fragment (scFv), or any other type of antibody fragment of interest (see definition of the term "antibody" above). Anti-CXCR6 antibodies that may be used in the practice of the subject methods include, without limitation, monoclonal antibodies, polyclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab’)2and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sei USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); singlechain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879- 5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11 :3287-3303, Vincke et al. (2012) Methods Mol Biol 911 : 15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep.1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.

[0065] Antibodies that specifically bind to CXCR6 can be prepared using any suitable methods known in the art. See, e.g., Antibodies: A Laboratory Manual, (second edition (2014), edited by Edward A. Greenfield, Dana-Farber, Cancer Institute), Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (second edition, 1986); and Kohler & Milstein, Nature 256:495-497 (1975). For example, an antigen comprising a peptide fragment of CXCR6 can be used to elicit an immune response in a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, an antigen can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface-active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.

[0066] Monoclonal antibodies which specifically bind to a CXCR6 antigen can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (Kohler et al., Nature 256, 495-97, 1985; Kozbor et al., J. Immunol. Methods 81, 31 42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-30, 1983; Cole et al., Mol. Cell Biol. 62, 109-20, 1984).

[0067] In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-55, 1984; Neuberger et al., Nature 312, 604-08, 1984; Takeda et al., Nature 314, 452-54, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.

[0068] Alternatively, humanized antibodies can be produced using recombinant methods, as described below. Antibodies which specifically bind to a particular antigen can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No.5,565,332. Human monoclonal antibodies can be prepared in vitro as described in Simmons et al., PLoS Medicine 4(5), 928-36, 2007.

[0069] Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to a particular antigen. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991). Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., Eur. J. Cancer Prev. 5, 507-11, 1996). Singlechain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, Nat. Biotechnol. 15, 159-63, 1997. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, J. Biol. Chem. 269, 199-206, 1994.

[0070] A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., Int. J Cancer 61 , 497-501 , 1995; Nicholls et al., J. Immunol. Meth. 165, 81-91 , 1993).

[0071] Antibodies which specifically bind to a CXCR6 antigen also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833 3837, 1989; Winter et al., Nature 349, 293299, 1991).

[0072] Chimeric antibodies can be constructed as disclosed in WO 93 / 03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94 / 13804, also can be prepared.

[0073] In some embodiments, anti-CXCR6 antibodies from B lymphocytes are obtained from blood donors and cloned. Nucleic acids encoding antibody light and heavy chains or fragments thereof containing variable domain complementarity-determining regions (e.g., Fab) can be amplified by PCR and cloned into vectors. ScFv antibodies can be generated by cloning into a vector a construct that connects the light and heavy chains via a linker in one open reading frame. The blood donor can be of any species. In some embodiments, human blood donors are used for generation of human antibodies. In other embodiments, camelid blood donors are used for generation of camelid antibodies. Camelid antibodies may be derived, for example, from Dromedary camels, bactrian camels, llamas and alpacas. Such camelids produce a unique type of antibody that lacks a light chain. Heavy-chain antibodies (HCAbs) or variable domain fragments thereof (e.g., single-domain antibodies or nanobodies) can also be used in the subject methods (see, e.g., Vincke et al. (2012) Methods Mol. Biol. 911 : 15-26, Krah et al. (2016) Immunopharmacol. Immunotoxicol. 38(1):21-8; herein incorporated by reference).

[0074] Antibodies can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which the relevant antigen is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.Chimeric and Humanized Antibodies

[0075] Suitable anti-CXCR6 antibodies include fully human, humanized or chimeric versions of such antibodies. For example, humanized antibodies are useful for in vivo applications in humans due to their low antigenicity. Similarly, caninized, felinized, etc. antibodies are useful for applications in dogs, cats, and other species respectively. Antibodies of interest includehumanized antibodies, or caninized, felinized, equinized, bovinized, porcinized, etc., antibodies, and variants thereof.

[0076] Chimeric monoclonal antibodies, in which the variable Ig domains of a non-human (e.g., mouse) monoclonal antibody are fused to human constant Ig domains, can be generated using standard procedures known in the art (See Morrison et al., Proc. Natl. Acad. Sci. USA 81 , 6841-6855 (1984); and, Boulianne et al, Nature 312, 643-646, (1984)).

[0077] Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as humanizing through "CDR grafting"); (2) transplanting the entire non-human variable domains, but "cloaking" them with a human-like surface by replacement of surface residues (a process referred to in the art as "veneering"); or (3) substituting human amino acids at positions determined to be unlikely to adversely effect either antigen binding or protein folding, but likely to reduce immunogenicity in a human environment (e.g., HUMAN ENGINEERING). In the present disclosure, humanized antibodies may include "humanized," "veneered," and / or "HUMAN ENGINEERED" antibodies. These methods are disclosed in, e.g., Jones et al., Nature 321 :522 525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.28:489-498 (1991); Padlan, Molec. Immunol. 31 :169-217 (1994); Studnicka et al. U.S. Pat. No. 5,766,886; Studnicka et al., (Protein Engineering 7: 805-814, 1994; Co et al., J. Immunol.152, 2968-2976 (1994); Riechmann, et al., Nature 332:323-27 (1988); and Kettleborough et al., Protein Eng. 4:773-783 (1991) each of which is incorporated herein by reference.

[0078] CDR grafting involves introducing one or more of the six CDRs from the mouse heavy and light chain variable Ig domains into the appropriate four framework regions of human variable Ig domains. This technique (Riechmann, et al., Nature 332:323-27 (1988)), utilizes the conserved framework regions (FR1-FR4) as a scaffold to support the CDR loops which are the primary contacts with antigen. A disadvantage of CDR grafting, however, is that it can result in a humanized antibody that has a substantially lower binding affinity than the original mouse antibody, because amino acids of the framework regions can contribute to antigen binding, and because amino acids of the CDR loops can influence the association of the two variable Ig domains. To maintain the affinity of the humanized monoclonal antibody, the CDR grafting technique can be improved by choosing human framework regions that most closely resemble the framework regions of the original mouse antibody, and by site-directed mutagenesis of single amino acids within the framework or CDRs aided by computer modeling of the antigen binding site (e.g., Co et al., J. Immunol. 152, 2968-2976 (1994)).Antibody Fragments

[0079] Anti-CXCR6 antibodies may be in the form of an antibody fragment. Antibody fragments comprise a portion of an intact full-length antibody and can include an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab; Fab'; F(ab')2; Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecfic, trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies); minibody; chelating recombinant antibody; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), bindingdomain immunoglobulin fusion proteins; camelized antibodies; VHH containing antibodies; and other polypeptides formed from antibody fragments. See, e.g., Holliger & Hudson (Nat. Biotech. 23:1126-36 (2005)).

[0080] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, monovalent fragments consisting of the VL, VH, CL and CH domains each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields a F(ab')2fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, that has two "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding, resulting in a single-chain antibody (scFv), in which a VL and VH region are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragment consists of the VH and CH1 domains.

[0081] Additional antibody fragments include a domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain. Diabodies are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., EP 404,097; WO 93 / 11161 ; Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljaket al., Structure 2:1121-1123, 1994). Diabodies can be bispecific or monospecific.Methods of Production of Anti-CXCR6 Antibodies

[0082] As discussed above, the present disclosure provides antibodies that specifically bind to CXCR6. Exemplary methods of making an anti-CXCR6 antibody are presented below.

[0083] Antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, antibodies may be made and isolated using methods of phage display. The antibody may also be isolated from sera of an animal host immunized with an immunogenic composition comprising CXCR6, which encompasses whole proteins and fragments thereof. Exemplary antibodies include an isolated antibody capable of specifically binding to CXCR6.

[0084] The antigen that coats the wells for phage display panning or the immunogenic composition used to elicit the antibody of the present disclosure may comprise an aggregate of one or more antigens. The method may involve exposing antigens to an aggregating condition so as to form an aggregate. Thus, the methods of production described above may further include a step of forming an aggregate of the isolated antigens. Examples of the aggregating conditions include heating, addition of an excipient that facilitates aggregation, and the like.

[0085] Antigens used to coat the wells for phage panning or to elicit antibodies of the present disclosure may be conjugated to another molecule. For example, the antigen can be conjugated to a second molecule such as a peptide, polypeptide, lipid, carbohydrate and the like that aids in solubility, storage or other handling properties, cell permeability, half-life, controls release and / or distribution such as by targeting a particular cell or cellular location (e.g., plasma membrane, lysosome, endosome, mitochondria etc.), tissue, or other bodily location (e.g., blood, particular organs, etc.).

[0086] A particular embodiment of an antigen conjugated to a second molecule is where the second molecule is an immunomodulator. "Immunomodulator" is a molecule that directly or indirectly modifies an immune response. A specific class of immunomodulators includes those that stimulate or aid in the stimulation of an immunological response. Examples include antigens and antigen carriers such as a toxin or derivative thereof, including tetanus toxoid.Phage Display

[0087] Phage display is used for the high-throughput screening of protein interactions.Phages may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds CXCR6 can be selected or identified, e.g., using labeled CXCR6 or CXCR6 bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv(individual Fv region from light or heavy chains) or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Exemplary methods are set forth, for example, in EP 368684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R. and Chames, Immunol. Today 2000, 21:371; Nagy et al. Nat. Med. 2002, 8:801; Huie et al., Proc. Natl. Acad. Sci. USA 2001, 98:2682; Lui et al., J. Mol. Biol. 2002, 315:1063, each of which is incorporated herein by reference. Several publications (e.g., Marks et al., Bio / Technology 1992, 10:779-783) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al., Nat. Biotechnol. 2000, 18:1287; Wilson et al., Proc. Natl. Acad. Sci. USA 2001, 98:3750; or Irving et al., J. Immunol. Methods 2001, 248:31). Cell surface libraries may be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 2000, 97:10701; Daugherty et al., J. Immunol. Methods 2000, 243:211). Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.

[0088] In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. For example, DNA sequences encoding heavy chain variable (VH) and light chain variable (VL) regions are amplified or otherwise isolated from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL regions may be joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. The VH or VL regions are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (e.g., the serine protease domain of CXCR6) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.

[0089] Additional examples of phage display methods that may be used to make the antibodies include those disclosed in PCT Application No. PCT / GB91 / 01134; PCT publications WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0090] As described in the references listed above, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in anydesired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92 / 22324; Mullinax et al., BioTechniques 1992, 12:864-869; and Sawai et al., AJRI 1995, 34:26-34; and Better et al., Science 1988, 240:1041-1043 (said references incorporated by reference in their entireties).Immunization and Antibody Production

[0091] Antibodies can be elicited in a host animal by administering an effective amount of CXCR6 or a fragment thereof as antigens to the host animal (i.e., a suitable mammal such as a mouse, rabbit or guinea pig, suitable avian, such as a chicken, or camelid) to elicit production of an antibody that specifically binds to CXCR6. Methods of immunizing an animal, including the adjuvants used, booster schedules, sites of injection, suitable animals, etc. are well understood in the art, e.g., Harlow et al. (Antibodies: A Laboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.), and administration of living cells to animals has been described for several mammals and birds, e.g., McKenzie et al (Oncogene 4:543-8, 1989), Scuderi et al (Med. Oncol. Tumor Pharmacother 2:233-42, 1985), Roth et al (Surgery 96:264-72, 1984) and Drebin et al (Nature 312:545-8, 1984). Next, a population of antibody producing cells is generated. In one embodiment, the population of cells is produced using hybridoma methods that well known to one of skill in the art (see, e.g., Harlow Antibodies: A Laboratory Manual, First Edition (1988) Cold Spring Harbor, N.Y.). Cells are fused to immortalized cells, such as myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The immortal cell line utilized can be selected to be deficient in enzymes necessary for the utilization of certain nutrients. Many such cell lines (such as myelomas) are known to those skilled in the art, and include, for example: thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyl transferase (HGPRT). These deficiencies allow selection for fused cells according to their ability to grow on, for example, hypoxanthine aminopterinthymidine medium (HAT). In alterative embodiments, populations of cells expressing monoclonal antibodies may be made using phage display methods.

[0092] Anti-CXCR6 antibodies, including antigen binding fragments of anti-CXCR6 antibodies, may also be produced by genetic engineering. In this technique, as with the standard hybridoma procedure, antibody-producing cells are sensitized to the desired antigen or immunogen. The messenger RNA isolated from the immune spleen cells or hybridomas is used as a template to make cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigenspecificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors. A combinatorial library can be constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones which co-express a heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody molecule). The vectors that carry these genes are co-transfected into a host (e.g. bacteria, insect cells, mammalian cells, or other suitable protein production host cell). When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.Phage Panning and Screening

[0093] Once the population of antibody-producing cells or phages is produced, the antibodies are screened using one or a combination of a variety of assays. In general, these assays are functional assays, and may be grouped as follows: assays that detect an antibody's binding affinity or specificity, and assays that detect the ability of an antibody to initialize or inhibit a process.

[0094] For example, the antigen is coupled to beads or wells or other solid support and incubated with phage displaying the antibody of interest. After washings, bound phage is then recovered by inoculation of log phase E. coli cells. The cells are grown and expanded with helper phage. Steps are repeated for the amplification of tightly bound phages. The phage- infected E. coli colonies after several round of enrichment are harvested and Fab antibodies are purified from the periplasmic fractions. The purified antibodies are then analyzed in accordance with methods known in the art. Certain exemplary examples are detailed below.

[0095] The population of antibody isolated from phage-infected cells or hybridomas is further analyzed and / or screened for binding to a single antigen (i.e., antigens that are not mixed with other antigens of the plurality of antigens) of the plurality of antigens in vitro or in situ (e.g. on cells). Immunospecific binding may be carried out according to methods routine and known in the art. The immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. See, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety.

[0096] Antibodies of the present disclosure may also be screened in vivo. The method involves administering an anti-CXCR6 antibody to an animal model for a disease or condition (e.g., myocarditis) and determining the effect of the antibody on the disease or condition of the model animal. In vivo assays of the invention include controls, where suitable controls include a sample in the absence of the antibody. Generally, a plurality of assay mixtures is run in parallel with different antibody concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

[0097] A monoclonal antibody of interest is one that modulates, i.e., reduces or increases a symptom of the animal model disease or condition by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or more, when compared to a control in the absence of the antibody. In general, a monoclonal antibody of interest will cause a subject animal to be more similar to an equivalent animal that is not suffering from the disease or condition. Antibodies that have therapeutic value that have been identified using the methods and compositions of the invention are termed "therapeutic" antibodies.

[0098] Selected monoclonal antibodies of interest can be expanded in vitro, using routine tissue culture methods, or in vivo, using mammalian subjects. For example, pristane-primed mice can be inoculated with log phase hybridoma cells in PBS for ascites production. Ascites fluid can be stored at -70° C. prior to further purification.Methods of Screening

[0099] A screening method provided by the present disclosure may involve the use of a phage library to screen for an antibody that specifically binds to CXCR6, and optionally having any of the additional features described herein. The binding agent may be selected for its potent inhibition of CXCR6 and / or its specific binding affinity. The method may be executed according to the phage display method described above.

[0100] Briefly, CXCR6 or a fragment thereof may be immobilized on an ELISA plate or on beads through a covalent or non-covalent interaction, such as hydrophobic adsorption, biotinavidin interaction, and Ni2+-6xHis interaction. The phage library is then incubated with the immobilized antigen / protease, washed, and recovered. During panning and selection, the bound phage is recovered and amplified in E. coli. Multiple successive selection rounds ensure a selection of a phage displaying a polypeptide that acts as an antibody specific for CXCR6. The stringency of the washes increases over a number of rounds (e.g. three). Many techniques well known in the art may be employed to increase the specificity of the recoveredphage. Examples include increased wash times, increased detergent concentrations, increased salt concentrations, and inclusion of known macromolecular inhibitors (e.g., small peptidic substrates, BPTI, Ecotin, and / or previously identified antibody inhibitors). Identification of inhibitory antibodies may include ELISAs and inhibition assays. Details on the assays to be performed in the method for selecting and isolating an anti-CXCR6 antibody are discussed above.

[0101] Also contemplated by the present disclosure is a library of nucleic acid constructs encoding the candidate anti-CXCR6 antibodies described herein. The library encodes a plurality of candidate anti-CXCR6 antibodies that may have one or more polypeptide regions in common (e.g. at least one heavy chain or light chain CDR) and at least one other polypeptide region that varies among the population.Nucleic Acids, Vectors, and Vector Systems Encoding Anti-CXCR6 Antibodies

[0102] In certain embodiments, anti-CXCR6 antibody heavy chains and light chains or antibody fragments thereof (e.g., a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, or a scFv fragment) are produced from a vector. A "vector" is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Polynucleotides encoding an antibody light chain and / or heavy chain, or fragments thereof, can be introduced into a cell with a single vector or in separate vectors (i.e., vector system). The ability of constructs to produce anti-CXCR6 antibodies can be empirically determined.

[0103] Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. An expression construct can be replicated in a living cell, or it can be made synthetically. For purposes of this application, the terms "expression construct," "expression vector," and "vector," are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.

[0104] In one embodiment, an expression vector for expressing an anti-CXCR6 antibody comprises a promoter "operably linked" to a polynucleotide encoding an anti-CXCR6 antibody heavy chain and / or light chain, or fragments thereof (e.g., comprising their variable domain CDRs). The phrase "operably linked" or "under transcriptional control" as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0105] In certain embodiments, the nucleic acid encoding a polynucleotide of interest is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I, II, or III. Typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (see, U.S. Patent Nos. 5,168,062 and 5,385,839, incorporated herein by reference in their entireties), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. These and other promoters can be obtained from commercially available plasmids, using techniques well known in the art. See, e.g., Sambrook et al., supra. Enhancer elements may be used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761 , the enhancer / promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521. such as elements included in the CMV intron A sequence.

[0106] Typically, transcription terminator / polyadenylation signals will also be present in the expression construct. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Patent No. 5,122,458). Additionally, 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same. Such sequences may include UTRs comprising an internal ribosome entry site (IRES).

[0107] Inclusion of an IRES permits the translation of one or more open reading frames from a vector. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485- 4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298: Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997 22 150-161. A multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003)100(25): 15125-15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like. A variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol.18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074- 1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1):37-44). These elements are readily commercially available in plasmids sold, e.g., by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA) and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). An IRES sequence may be included in a vector, for example, to express an anti-CXCR6 antibody heavy chain or fragment thereof in combination with an anti-CXCR6 antibody light chain or fragment thereof from an expression cassette.

[0108] Alternatively, a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products (e.g., an anti-CXCR6 antibody heavy chain orfragment thereof in combination with an anti-CXCR6 antibody light chain or fragment thereof) from a single vector. 2A linker peptides are inserted between the coding sequences in the multicistronic construct. The 2A peptide, which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels. 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1. See, e.g., Kim et al. (2011) PLoS One 6(4):e18556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625-629, Furler et al. (2001) Gene Ther. 8(11):864-873; herein incorporated by reference in their entireties.

[0109] An expression vector or vector system comprising one or more vectors can be used to transform an isolated cell, a cell line, or a population of cells, wherein the gene products of interest (e.g., antibody light chain and heavy chain, or fragments thereof) are selectively expressed by the cell or cells. In some embodiments, anti-CXCR6 antibodies are produced and secreted. The transformed cells secrete the antibodies into the surrounding media. Certain regulatory sequences can be included in the vector to enhance secretion, for example using a tissue plasminogen activator (TPA) leader sequence, an interferon (y or a) signal sequence or other signal peptide sequences from known secretory proteins. The secreted antibodies can then be isolated by various techniques described herein, for example, usingstandard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0110] Alternatively, the proteins are not secreted, and transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant proteins substantially intact. Intracellular proteins can also be obtained by removing components from the cell membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0111] For example, methods of disrupting cells include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulfate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which a polypeptide is expressed, culture conditions and any pre-treatment used.

[0112] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0113] Antibodies including heavy and light chains, or fragments thereof can be purified using affinity purification, such as by Protein A affinity chromatography, immunoaffinity chromatography using antibodies that bind to an anti-CXCR6 heavy chain or light chain epitope), or antigen-specific affinity chromatography with an immobilized CXCR6 antigen. The choice of a suitable affinity resin is within the skill in the art. After affinity purification, expressed antibodies can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.Antibody Conjugates

[0114] Anti-CXCR6 antibodies also find use in therapeutic and diagnostic (e.g., in vivo imaging, etc.) applications. For example, such an antibody may be conjugated to a payload, such as a therapeutic agent (e.g., for treating myocarditis) or labeling agent (e.g., an in vivo imaging agent), where upon binding of the antibody to CXCR6, the therapeutic or labelingagent is delivered selectively to a target immune cell (e.g., T cell or macrophage expressing CXCR6). The selective targeting of the therapeutic or imaging agent to immune cells expressing CXCR6 reduces unwanted exposure of non-target cells to the therapeutic agent, and can reduce toxicity upon administration. Moreover, in imaging applications (e.g., in vivo imaging for diagnostic, prognostic, and / or any other purpose), selective binding of the antibody to immune cells expressing CXCR6 concentrates the imaging agent in such cells (e.g., for detecting infiltration of immune cells into cardiac tissue), thereby increasing the signal-to-noise ratio and diagnostic / prognostic value of the resulting images.

[0115] Accordingly, any anti-CXCR6 antibody described herein may be in unconjugated form, or may be conjugated directly to an agent, such as a therapeutic and / or imaging (e.g., diagnostic) agent, or may be conjugated indirectly to carrier polymers comprising such other therapeutic or imaging agents.

[0116] Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent or luminescent or bioluminescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are known; for example, see (Stemberger, L. A. et al., J. Histochem. Cytochem.18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol.109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

[0117] In certain aspects, the imaging agent of an anti-CXCR6 antibody conjugate of the present disclosure is an imaging agent that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, single-photon emission computed tomography (SPECT)ZCT imaging, or the like. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels and radioisotopes, or the like. In certain aspects, the labeling agent is a multi-modal in vivo imaging agent that permits in vivo imaging using two or more imaging approaches (e.g., see Thorp-Greenwood and Coogan (2011) Dalton Trans. 40:6129-6143).

[0118] Conjugation of antibody moieties is described in U.S. Pat. No. 6,306,393. General techniques are also described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. This general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic agents. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0119] The carrier polymer may be, for example, an aminodextran or polypeptide of at least 50 amino acid residues. Various techniques for conjugating a drug or other agent to the carrierpolymer are known in the art. A polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, and can be about 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agents. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier and conjugate. Examples of agents to which the antibody can be conjugated include any of the cytotoxic, chemotherapeutic agents described herein.

[0120] Conjugated antibodies can be prepared by directly conjugating an antibody component with a therapeutic agent or labeling agent. The general procedure is analogous to the indirect method of conjugation except that a therapeutic or labeling agent is directly attached to an oxidized antibody component. For example, a carbohydrate moiety of an antibody can be attached to polyethylene glycol to extend half-life.

[0121] A therapeutic or labeling agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation, or using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991); Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995). A variety of bifunctional protein coupling agents are known in the art, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene).Pharmaceutical Compositions

[0122] An anti-CXCR6 antibody or antigen-binding fragment thereof (or conjugate including the same) can be formulated into pharmaceutical compositions optionally comprising one ormore pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0123] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0124] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the antibodies or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0125] A surfactant can be present as an excipient. Exemplary surfactants include:polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0126] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selectedfrom the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0127] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0128] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include those for oral or localized delivery.

[0129] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising anti-CXCR6 antibodies or antigen-binding fragments thereof (or conjugates including the same) described herein are in unit dosage form, meaning an amount of the anti-CXCR6 antibodies or antigen-binding fragments thereof (or conjugates including the same) appropriate for a single dose, in a premeasured or pre-packaged form.

[0130] The compositions herein may optionally include one or more additional agents, such as drugs for treating myocarditis, or other medications used to treat a subject for a condition or disease. For example, compounded preparations may include anti-CXCR6 antibodies orantigen-binding fragments thereof (or conjugates including the same) and one or more drugs for treating myocarditis, such as, but not limited to, angiotensin-converting-enzyme (ACE) inhibitors such as captopril, enalapril, lisinopril, benazepril, fosinopril, quinapril, ramipril, perindopril, moexipril, and trandolapril; beta blockers such as esmolol, nebivolol, bisoprolol, butaxamine, ICI-118,551 , SR 59230A; diuretics such as amphotericin B, tolvaptan, conivaptan, acetazolamide, dorzolamide, bumetanide, ethacrynic acid, furosemide, torsemide, amiloride, spironolactone, eplerenone, triamterene, potassium canrenoate, bendroflumethiazide, hydrochlorothiazide, caffeine, theophylline, and theobromine; intravenous immunoglobulin (MG); corticosteroids such as methylprednisolone, hydrocortisone, prednisone, and prednisolone; immunosuppressants such as azathioprine and cyclosporine; analgesics such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and naproxen, and diclofenac; C0X2 inhibitors such as rofecoxib, celecoxib, and etoricoxib; and opioids such as morphine, codeine, oxycodone, hydrocodone, dihydromorphine, and pethidine; immune checkpoint inhibitors such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, dostarlimab, and relatlimab; and other drugs for treating an inflammatory cardiac condition, or other medications used to treat a subject for a condition or disease. Alternatively, such agents can be contained in a separate composition from the composition comprising the anti-CXCR6 antibodies or antigen-binding fragments thereof (or conjugates including the same) and co-administered concurrently, before, or after the composition comprising the anti-CXCR6 antibodies or antigen-binding fragments thereof (or conjugates including the same).Dosage

[0131] In the methods of the present disclosure, an effective amount of anti-CXCR6 antibody or antigen-binding fragment thereof (or conjugate including the same) is administered to a subject in need thereof. The amount administered varies depending upon the goal of the administration, the particular myocarditis being treated, the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., human, non-human primate, primate, etc.), the degree of resolution desired, the formulation of the anti-CXCR6 antibody or conjugate, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. For example, the amount of the anti-CXCR6 antibody or conjugate employed to reduce cardiac inflammation and arrhythmia, and inhibit infiltration of T cells and macrophages into cardiac tissue is not more than about the amount that could otherwise be irreversibly toxic to the subject (i.e., maximum tolerated dose). In othercases, the amount is around or even well below the toxic threshold, but still in an effective concentration range, or even as low as threshold dose.

[0132] Individual doses are typically not less than an amount required to produce a measurable effect on the subject, and may be determined based on the pharmacokinetics and pharmacology for absorption, distribution, metabolism, and excretion ("ADME") of the antibody or conjugate, and thus based on the disposition of the composition within the subject. This includes consideration of the route of administration as well as dosage amount, which can be adjusted for, e.g., parenteral (applied by routes other than the digestive tract for systemic or local effects) applications. For instance, administration of an anti-CXCR6 antibody or conjugate is typically via injection (e.g., intravenous, intramuscular, or intracardiac), or a combination thereof.

[0133] Disposition of the antibody or conjugate and its corresponding biological activity within a subject is typically gauged against the fraction of antibody present at a target of interest. For example, an antibody, once administered, can accumulate at a biological target (e.g., CXCR6) that concentrates the material in inflamed heart tissue where immune cells (T cells and macrophages) have infiltrated. Thus, dosing regimens in which the antibody is administered so as to accumulate in a target of interest over time can be part of a strategy to allow for lower individual doses. This can also mean, for example, that a dose of an antibody that is cleared more slowly in vivo can be lowered relative to the effective concentration calculated from in vitro assays (e.g., effective amount in vitro approximates mM concentration, versus less than mM concentrations in vivo).

[0134] As an example, the effective amount of a dose or dosing regimen can be gauged from the IC50 of a given antibody for inhibiting the CXCR6 ligand, CXCL16, from binding to CXCR6. By "IC50" is intended the concentration required for 50% inhibition in vitro. Alternatively, the effective amount can be gauged from the EC50 of a given antibody concentration. By "EC50" is intended the plasma concentration required for obtaining 50% of a maximum effect in vivo.

[0135] In general, with respect to the anti-CXCR6 antibody or conjugate of the present disclosure, an effective amount is usually not more than 200* the calculated IC50. Typically, the amount of an antibody or conjugate that is administered is less than about 200*, less than about 150x, less than about 100* and many embodiments less than about 75x, less than about 60x, 50x, 45x, 40x, 35x, 30x, 25x, 20x, 15x, 10x and even less than about 8x or 2x than the calculated IC50. In one embodiment, the effective amount is about 1x to 50x of the calculated IC50, and sometimes about 2x to 40x, about 3x to 30x or about 4x to 20x of the calculated IC50. In other embodiments, the effective amount is the same as the calculated IC50, and in certain embodiments the effective amount is an amount that is more than the calculated IC50.

[0136] An effective amount may not be more than 100* the calculated EC50. For instance, the amount of antibody or conjugate that is administered is less than about 100*, less than about 50*, less than about 40*, 35*. 30x, or 25x and many embodiments less than about 20x, less than about 15x and even less than about 10x, 9.times., 9x, 7x, 6.times., 5x, 4x, 3x, 2x or 1x than the calculated EC50. In one embodiment, the effective amount is about 1 x to 30x of the calculated EC50, and sometimes about 1x to 20x, or about 1x to 10x of the calculated EC50. In other embodiments, the effective amount is the same as the calculated EC50, and in certain embodiments the effective amount is an amount that is more than the calculated EC50.

[0137] Effective amounts can readily be determined empirically from assays, from safety and escalation and dose range trials, individual clinician-patient relationships, as well as in vitro and in vivo assays.Administration

[0138] At least one therapeutically effective cycle of treatment with an anti-CXCR6 antibody or antigen-binding fragment thereof (or conjugate thereof) will be administered to a subject for treatment of myocarditis. By "therapeutically effective dose or amount" of an anti-CXCR6 antibody or antigen-binding fragment thereof that specifically binds to CXCR6 is intended an amount that, when administered, as described herein, brings about a positive therapeutic response in the treatment of myocarditis, such as an amount that reduces heart inflammation and arrhythmias and / or improves cardiac function and increases survival. Additionally, a therapeutically effective dose or amount of an anti-CXCR6 antibody or antigen-binding fragment thereof may inhibit infiltration of T cells and macrophages into cardiac tissue.

[0139] In certain embodiments, multiple therapeutically effective doses of compositions comprising an anti-CXCR6 antibody or antigen-binding fragment (or conjugate thereof), and / or one or more other therapeutic agents, such as other drugs for treating an inflammatory cardiac condition, or other medications will be administered. The compositions of the present invention are typically, although not necessarily, administered orally, via injection (subcutaneously, intravenously, intramuscularly, or intraperitoneally), by infusion, or locally. Additional modes of administration are also contemplated, such as intrahepatic, pulmonary, rectal, transdermal, transmucosal, intrathecal, pericardial, intra-arterial, and so forth.

[0140] The preparations are also suitable for local treatment. In a particular embodiment, a composition is used for localized delivery of an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof to the heart for the treatment of myocarditis. For example, compositions may be administered locally in the vicinity of heart inflammation or immune cell infiltration.

[0141] The pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like. The pharmaceutical compositions comprising an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof and / or other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.

[0142] In another embodiment, the pharmaceutical compositions comprising an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof and / or other agents are in a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non- sustained-release pharmaceutical composition.

[0143] The disclosure also provides a method for administering a conjugate comprising an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof to a patient suffering from myocarditis or an inflammatory cardiac condition that is responsive to treatment with an anti-CXCR6 antibody contained in the conjugate or composition. The method comprises administering, via any of the herein described modes, a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition. The method of administering may be used to treat any condition that is responsive to treatment with an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof.

[0144] Those of ordinary skill in the art will appreciate which conditions a specific anti-CXCR6 antibody can effectively treat. The actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the particular inflammatory cardiac condition being treated, the severity of the condition being treated, the judgment of the health care professional, and the particular anti-CXCR6 antibody or conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.

[0145] In certain embodiments, multiple therapeutically effective doses of an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered, once per day, twice per day, three times per day, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, every other week, and soforth. For example, in some embodiments, a composition comprising an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof will be administered once-weekly, twice- weekly or thrice-weekly for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present disclosure, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods described herein, a subject can receive intermittent therapy (i.e., once-weekly, twice-weekly or thrice- weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below. The amount administered will depend on the potency of the specific anti-CXCR6 antibody, the particular inflammatory cardiac condition that is treated, the magnitude of the effect desired, and the route of administration.

[0146] A purified anti-CXCR6 antibody (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents such as one or more other drugs for treating myocarditis such as angiotensin- converting-enzyme (ACE) inhibitors such as captopril, enalapril, lisinopril, benazepril, fosinopril, quinapril, ramipril, perindopril, moexipril, and trandolapril; beta blockers such as esmolol, nebivolol, bisoprolol, butaxamine, ICI-118,551 , SR 59230A; diuretics such as amphotericin B, tolvaptan, conivaptan, acetazolamide, dorzolamide, bumetanide, ethacrynic acid, furosemide, torsemide, amiloride, spironolactone, eplerenone, triamterene, potassium canrenoate, bendroflumethiazide, hydrochlorothiazide, caffeine, theophylline, and theobromine; intravenous immunoglobulin (IVIG); corticosteroids such as methylprednisolone, hydrocortisone, prednisone, and prednisolone; immunosuppressants such as azathioprine and cyclosporine; analgesics such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and naproxen, and diclofenac; COX2 inhibitors such as rofecoxib, celecoxib, and etoricoxib; opioids such as morphine, codeine, oxycodone, hydrocodone, dihydromorphine, and pethidine, immune checkpoint inhibitors such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, dostarlimab, and relatlimab; and other drugs for treating an inflammatory cardiac condition, or other medications used to treat a subject for a condition ordisease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[0147] An anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof can be provided in the same or in a different composition. Thus, an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof and / or other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising an anti- CXCR6 antibody or antigen-binding fragment or conjugate thereof and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating an inflammatory cardiac condition, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, an anti-CXCR6 antibody or antigen-binding fragment or conjugate thereof and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.Kits

[0148] Any of the compositions described herein may be included in a kit. For example, kits may include compositions comprising anti-CXCR6 antibodies, antigen-binding fragments thereof, conjugates of anti-CXCR6 antibodies, pharmaceutical formulations comprising anti- CXCR6 antibodies and / or conjugates thereof, recombinant nucleic acids or vector systems encoding the anti-CXCR6 antibodies and / or host cells (either transfected with the recombinant nucleic acids or vector systems encoding the anti-CXCR6 antibodies or separate).

[0149] In some embodiments, the kit comprises an anti-CXCR6 antibody or an antigenbinding fragment thereof that specifically binds to CXCR6. In certain embodiments, the kitcomprises anti-CXCR6 antibody conjugates or formulations including the antibodies and / or conjugates.

[0150] In certain embodiments, the anti-CXCR6 antibody included in the kit is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a bispecific antibody, a bispecific T cell engager antibody, a trispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, or a scFv fragment.

[0151] The kit may include one or more pharmaceutical formulations that include the antibody compositions described herein. As such, the kits may include a single pharmaceutical composition present as one or more unit dosages. In yet other embodiments, the kits may include two or more separate pharmaceutical compositions. In some embodiments, the pharmaceutical composition is suitable for topical delivery (e.g., a gel or cream comprising an antibody or conjugate for treatment of myocarditis).

[0152] Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device. The kit may also provide a delivery device pre-filled with the anti-CXCR6 antibody or antigen-binding fragment thereof (or conjugate thereof).

[0153] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods (i.e., instructions for treating an myocarditis as described herein). These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.Utility

[0154] The anti-CXCR6 antibodies or antigen-binding fragments thereof (or conjugates thereof), described herein, are useful for treating myocarditis, including myocarditis caused by treatment with immune checkpoint inhibitors (e.g., adverse effect of anti-LAG-3 / anti-PD-1 combination therapy). Treatment of myocarditis with anti-CXCR6 antibodies reduces heart inflammation and arrhythmias and inhibits infiltration of T cells and macrophages into cardiac tissue.Examples of Non-Limiting Aspects of the Disclosure

[0155] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1- 16 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A method of treating myocarditis, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds to a C-X-C motif chemokine receptor s (CXCR6).2. The method of aspect 1 , wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, and a scFv fragment.3. The method of aspect 1 or 2, further comprising administering an angiotensin- converting-enzyme (ACE) inhibitor, a beta blocker, a diuretic, a corticosteroid, an immunosuppressant, intravenous immunoglobulin (IVIG), an immune checkpoint inhibitor, or any combination thereof.4. The method of any one of aspects 1-3, wherein said administering comprises administering the antibody intravenously, subcutaneously, or intraperitoneally.5. The method of any one of aspects 1-3, wherein said administering comprises administering the antibody locally to the heart.6. The method of any one of aspects 1-5, wherein said administering comprises administering the antibody before, during, or after treatment of the subject with an immune checkpoint inhibitor.7. The method of any one of aspects 1-6, wherein said administering comprises administering the antibody before, during, or after treatment of the subject with anti-LAG-3 / anti-PD-1 combination therapy.8. The method of any one of aspects 1-7, wherein multiple cycles of treatment are administered to the subject.9. The method of aspect 8, wherein said administering comprises administering the antibody according to a daily dosing regimen or intermittently.10. Amethod of inhibiting infiltration ofT cells and macrophages into cardiac tissue of a subject, the method comprising administering an effective amount of an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) to the subject.11. The method of aspect 10, wherein said administering comprises administering the antibody intravenously, subcutaneously, or intraperitoneally.12. The method of aspect 10, wherein said administering comprises administering the antibody locally to the heart.13. A composition comprising an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) for use in treating myocarditis.14. The composition of aspect 13, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.15. The composition of aspect 13 or 14, further comprising a pharmaceutically acceptable excipient or carrier.16. Use of an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) in the manufacture of a medicament or pharmaceutical composition for treating myocarditis.EXPERIMENTAL

[0156] 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 to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0157] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.The present invention has been described in terms of particular embodiments found or proposed by the present inventors to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.Example 1CXCR6+ T-Cells Drive Immune Checkpoint Inhibitor MyocarditisIntroduction:

[0158] Lymphocyte activation gene 3 (LAG-3 ) is a newly targeted T-cell immune checkpoint.Combination of relatlimab (anti-LAG-3) with nivolumab (anti-PD-1) was FDA-approved for metastatic melanoma in 20225. In the seminal clinical trial that led to the approval of relatlimab, there was a nearly three-fold increase risk of myocarditis with the addition of anti-LAG-3 totreatment5. This observation underscores the importance of preclinical models of ICI- myocarditis that involve LAG-3. This is especially important as there are numerous clinical trials using LAG-3 inhibition in various cancer types and stages.

[0159] Growing data from our group and others have identified that T-cells are the drivers of ICI-myocarditis. Using a preclinical model of CTLA-4 / PD-1 ICI-myocarditis, we previously identified the expansion of clonal, CD8+ T-cells, with CD8+ T-cells being necessary for development of myocarditis and alpha-myosin heavy chain (a-MHC) being one possible target of these T lymphocytes6 7. Other investigations have found expansion of terminal effector memory CD8+ T-cells (TEMRA) expanded in the blood of patients with ICI-myocarditis8. However, several outstanding questions remain including identifying the granular cardiac T- cell subsets and the key markers of these pathogenic T-cells. The signals that drive these activated cardiac T-cells to expand and position within the cardiac environment also remain understudied.

[0160] More recently, the chemokine signaling axis between CXCL9 / CXCL10+ macrophages and CXCR3+ T-cells was demonstrated to play a significant role in recruitment of peripheral T-cells to the heart9 10. Indeed, macrophage depletion attenuated the myocarditis in multiple pre-clinical models, suggesting that specific groups of cardiac macrophages play supporting role in the pathogenesis of ICI-myocarditis9 10. The CCL5-CCR5 chemokine signaling axis has been implicated in other models of myocarditis - however chemokine biology within the context of ICI-myocarditis is still relatively undefined11 12.

[0161] Here, we performed deep cardiac phenotyping in mice lacking LAG-3 and PD-1 (Lag3- / -,Pdcd1- / -) and identified marked cardiac inflammation out of proportion to other organs. Through cardiac immune scRNA-seq and flow cytometry we uncover cardiac enrichment of CXCR6+ T-cells and subsequently validated this finding in other preclinical models and patient data. We then demonstrate that the CXCR6 ligand, CXCL16, was upregulated in cardiac macrophages. Treatment with anti-CXCR6 prevented pre-mature lethality, attenuated arrhythmias, and reduced the histologic severity of myocarditis. These data highlight the necessity of CXCR6+ T-cells for disease pathogenesis and give biologic plausibility to the use of anti-CXCR6 treatment in ICI-myocarditis.Methods:Animals

[0162] Pdcdl null and Lag3 null mice were purchased from the Jackson Laboratory (Strain 026644 and 028276). Lines were crossed to generate Lag3 / Pdcd1 null animals. All mice were housed at the UCSF Cardiovascular Research Institute Barrier, an animal facility accreditedby the Associated for Assessment and Accreditation of Laboratory Animal Care International. Animals were maintained in a controlled environment with a 12-hour light-dark cycle, with access to water and a standard chow diet at all times. All experiments were performed in accordance with the IACUC protocol. Mouse survival was assessed via Kaplan-Meier Survival curve with log rank statistical analysis performed.Preparation of Cardiac Dissociates for scRNA-TCR Seq

[0163] Mice were sacrificed and hearts were harvested and washed thoroughly with PBS.Hearts were subsequently minced finely and digested in mixture of 250U / mL Collagenase 3 (Worthington, LS004182), 125U / mL DNAse I (Worthington, LS002138), 170U / mL Hyaluronidase (Sigma, H3506) in RPMI for 50 minutes on GentleMACS system. Red blood cells were lysed using ACK lysing buffer (KD Medical / MediaTech, NC0274127). Cells were stained with PerCP / Cyanine5.5 anti-mouse CD45 (BioLegend, clone QA17A26, 157612) for 30 minutes at 4C. Following staining and washing with PBS, cells were resuspended in PBS with DAPI (1 :10,000). Live CD45+ immune cells were sorted by FACS on PerCP / Cy5.5 positive, DAPI negative events. The wildtype control sample consistent of pooled cardiac immune infiltrates from 6 mice, as the healthy heart has a low frequency of cardiac immune cells. The myocarditis sample consisted of four inflamed hearts from 4 Lag3- / -,Pdcd1 - / - mice. Mice were approximately 6 weeks of age.scRNA-TCR-seq

[0164] Each sample (targeting 5,000-15,000 cells per sample) was processed for single-cell 5' RNA and TCR sequencing utilizing the 10x Chromium system. Libraries were prepared following the manufacturer’s protocol through the assistance of Gladstone Genomics Core. The libraries were sequenced using NovaSeq X and analysis was completed using 10x Genomics Cell Ranger software. We identified 13,741 cells from pooled Lag3- / -,Pdcd1- / - mice (1 ,126 median genes per cell), and 4,014 cells from pooled WT mice (536 median genes per cell). Data were analyzed in R using the filtered h5 gene matrices in the Seurat. In brief, samples were subset to include cells with >200 but <3,000 unique transcripts to exclude probable non-cellular RNA reads and doublets. Cells with >5% of reads coming from mitochondrial transcripts were also excluded as probable dying cells. Seven clusters were identified using a resolution of 0.1. UMAP was used for dimensionality reduction with 15 nearest neighbors and minimum distance of 0.5.Histology

[0165] Organs were dissected from mice and fixed in 10% formalin for48h, and subsequently transferred to 70% ethanol. Samples were grossed, embedded, sectioned and H&E stained at AML Laboratories. Inflammation severity was scored by an independent, blinded pathologist. Lesional area for anti-CXCR6 vs Isotype depletion was similarly performed by an independent, blinded pathologist and defined as myocardial area with active or healing myocarditis based on histological appearance divided by the total histological area of the sample.Chemistries

[0166] Blood was obtained upon sacrifice of animals and spun at 10,000g for 10 mins, from which serum was isolated. Analysis of serum chemistries performed at University of Michigan ULAM Pathology Core. For cardiac troponin I analysis, serum was harvest from mice and serum cardiac troponin-l measurement was performed using ELISA Kit (Life Diagnostics, CTNI-1-US).Antibody Depletion

[0167] Lag3- / -,Pdcd1 - / - mice were randomly assigned to control, anti-CXCR6 treatment or anti-CXCR3 at 21 days of age. Experiments were concluded when mice reached 70 days of age. Mice were treated with anti-CXCR6 antibody courtesy of Edelweiss Immune, anti-CXCR3 antibody (BioXCell, BE0249) or rat IgG 1 isotype control (BioXCell, BE0088) at 250ug i.p. twice weekly, in a maximum volume of 100uL. Weekly EKGs were performed until time of death or experimental endpoint. Organs were harvested for histology following death. To detect an anticipated mortality difference of 100% (for control) to 40% (for an intervention that reduces mortality) with an a of 0.05 and 80% power, a sample size of 8 mice per group was needed.Flow Cytometry

[0168] Samples were run on an Attune NxT Acoustic Focusing cytometer (Life Technologies).Data were collected using Attune NxT software v.3.2.1. Analysis was performed in FlowJo v.10.10. Gating was first done on forward scatter and side scatter to exclude debris. Doublets were excluded by gating on FSC area versus FSC height. Zombie Violet Viability was used to assess live cells (VWR, 10761-308). Gating strategy demonstrated in FIG. 11. The following antibodies were used: CD45-PerCP / Cy5.5 (BioLegend, 103132, clone 30-F11; dilution 1:400); CD3-AF488 (BioLegend, 100210, clone 17A2; dilution 1 :200); CD4-APC (BioLegend, 100412, clone GK1.5; dilution 1:100); CD8a-PE / Cy7 (BioLegend, 100722, clone 53-6.7, dilution 1:400), CD11b-APC / Cy7 (BioLegend, 101225, clone M1 / 70, dilution 1 :400), F4 / 80- PE-eFluor601 (Fischer Scientific, 50-112-9765, clone BM8, 1:100).Electrocardiography

[0169] Mice were anesthetized at 2% isofluorane in accordance with IACUC protocol and one-lead electrocardiogram was performed using AD Instruments Power Lab-C and acquired and analyzed with LabChart Pro 8. Arrhythmias were defined as high degree atrioventricular block, ventricular arrhythmias such as premature ventricular contractions, ventricular tachycardia or ventricular escape rhythms.Echocardiography

[0170] Mice were anesthetized at 2% isofluorane and echocardiography was performed using Vevo 3100 Ultrasound Machine. Parasternal long axis images of the left ventricle were analyzed and calculated using Vevo analysis software.Chemotaxis Assay

[0171] Lag3- / -,Pdcd1- / - mice were harvested and cardiac immune cells and PBMCs were isolated as described above. Both PBMCs and cardiac immune cells were stained with DAPI, CD45-PerCP / Cy5.5 (BioLegend, 103132, clone 30-F11 ; dilution 1:400), and CD3-AF488 (BioLegend, 100210, clone 17A2; dilution 1:200). Cells were subsequently sorted via FACS for DAPI negative / PerCP-Cy5.5 positive / AF488 positive cells. 20,000 blood T-cells and 10,000 cardiac T-cells were plated in replicate on QCM Chemotaxis Cell Migration Assay, 24-well (5 pm), colorimetric (Millipore Sigma, ECM506) with 10% Fetal Bovine Serum (FBS), RPMI only and RPMI + 0.15ug / mL of recombinant mouse CXCL16 (Peprotech, 250-28) in bottom chamber and placed for 4h in 37C incubator. Per kit instructions, colorimetric analysis was performed on bottom chamber at 560nm.Pharmacovigilance Analysis

[0172] We queried VigiBase, the international World Health Organization pharmacovigilance database encompassing over 32 million reports from over 130 countries since 1967 to January 1st, 2024 (vigiaccess.org / )13 14. We searched for the following ICI: (anti-CTLA4: ipilimumab, tremelimumab; anti-PD1: nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, tislelizumab; anti-LAG3: relatlimab) and specifically, myocarditis within 24 immune related adverse events (irAE) established to cover all organs potentially affected by ICI-induced auto-immunity using the Medical Dictionary for Regulatory Activities dictionary14. The combination of preferred term levels used to identify these irAE were recently published and made available14. In VigiBase, we compared the rate of each specific irAE overall adverse drug reactions reported in ICI-treated cases including anti-LAG-3 combined with anti-PD-1 tothose reported in cases on anti-PD-1 without anti-LAG-3 using multivariate logistic regression adjusted on cancer types, age, sex, period of reporting and concomitant use of anti-CTLA4. This study was registered (clinicaltrials.gov: NCT05934214).Results:Anti-LAG-3 / anti-PD-1 combination therapy is associated with an increased risk of ICI- myocarditis.

[0173] In VigiBase, the international pharmacovigilance database (accessed in January 2024), we found 130,113 reports of adverse drug reactions associated with anti-PD-1 , of which 1,702 were ICI-myocarditis. Among these 130,113 cases, 365 (1.9%) had concomitant anti- LAG-3 therapy, and 15.3% (19,923 / 130,113) had anti-CTLA-4 therapy. Anti-PD-1 therapy was the only ICI used in 109,869 / 130,113 (84.4%) and combined with anti-CTLA-4 in 19,879 / 130,113 (15.3%). ICI-myocarditis (and other ICI-induced myotoxicities) were more frequently and specifically reported in cases treated with anti-LAG-3 (28 / 365, 7.7%) than in cases treated with anti-PD-1 without anti-LAG-3 (1 ,674 / 129,748, 1.3%; after adjustment for sex, age, cancer type, combination with anti-CTLA-4: adjusted OR=4.0, 95%CI=2.6-5.8, p=8x10-12, FIG. 6)4 15 16. Notably, in a multivariate analysis, anti-LAG-3 combination therapy portended a higher risk of myocarditis than anti-CTLA-4 combination therapy (OR=1.7, 95%CI=1.5-1.9, p=1x1019).LAG-3 / PD-1 KO mice die prematurely of cardiac inflammation and arrhythmias

[0174] Guided by initial studies performed in the context of anti-tumor immunity, we crossed Lag3- / - and Pdcdl- / - mice to generate Lag3- / -, Pdcdl- / - mice on a C57 / BL6 background17 18. Consistent with previous studies, Lag3- / -, Pdcdl- / - mice demonstrated pre-mature lethality, with most mice dying by 8-10 weeks of age (FIG. 1A). As we globally disrupted T-cell signaling, we methodically assessed cause of death in Lag3- / -, Pdcdl- / - mice and harvested organs of 5-6 week old Lag3- / -, Pdcdl- / - mice, including heart, lungs, stomach, kidney, liver and pancreas. Lag3- / -, Pdcdl- / - mice had robust cardiac-enriched inflammation as compared to Pdcdl- / - mice (FIG. 1B). Notably, we also observed a trend towards significance in inflammation within the pancreas and lungs of Lag3- / -, Pdcdl- / - mice. (FIG. 1 C). However, there was no significantly enriched inflammation in other tissues examined. We performed serum chemistry analysis of wildtype (WT), Pdcdl- / -, and Lag3- / -, Pdcdl- / - mice. While we saw a modest increase in Lag3- / -, Pdcdl- / - alanine aminotransferase (ALT) and creatinine (Or) as compared to WT mice, but not compared to Pdcdl- / - mice, there was a robust increase in serum cardiac troponin I levels, indicating extensive cardiac injury (FIG. 1D).

[0175] We performed detailed cardiac phenotyping in Lag3- / -,Pdcd1- / - mice. Using electrocardiography (ECG), we identified significant electrocardiographic disturbances, including atrioventricular (AV) block and ventricular arrhythmias in Lag3- / -,Pdcd1- / - mice. These ECG changes occurred prior to death (FIG. 1E), with more arrhythmias as quantified by weekly ECG. (FIG. 1F). To determine left ventricular systolic function and volume, we performed echocardiography on Lag3- / -,Pdcd1- / - mice. Similar to other preclinical models of ICI-myocarditis and human data, we did not observe changes in left ventricular ejection fraction (LVEF) or left ventricular end diastolic volume (LVEDV) (FIG. 7) {Johnson, 2016 #1;Axelrod, 2022 #9}.LAG-3 / PD-1 KO mice have robust expansion of activated clonal cardiac T-cells

[0176] With growing evidence that Lag3- / -,Pdcd1- / - mice had a robust cardiac-enriched phenotype, we next sought to define the cardiac immune cell populations present. Flow cytometry on the cardiac isolates revealed T-cell enrichment within cardiac immune population of Lag3- / -,Pdcd1- / - mice as compared to Pdcdl- / - and WT mice (FIG. 2A). Specifically, we observed an enrichment of CD8+ T-cells, without significant changes in relative abundance of CD4+ T-cells (FIG. 8).

[0177] To understand granular immune cell populations and their specific transcriptional programs, we performed scRNA-seq on sorted cardiac CD45+ immune cells from Lag3- / - , Pdcdl- / - and WT mice. We were able to resolve all expected immune cell populations and cluster cells into (1) Dendritic Cells, (2) Dividing T-cells, (3) Natural Killer (NK) Cells, (4) B- cells, (5) Naive T-cells, (6) Macrophages and (7) Activated T-cells (FIG. 2B). Marker genes were used to define both the immune cell type (e.g. T-cell) along with their specific cell state (e.g. Mki67 expression within Dividing T-cells). Within our Lag3- / -, Pdcdl- / - mice, we specifically observed upregulation of Activated T-cells, Dividing T-cells and Macrophages. Markers of our Activated T-cell cluster included Cd8a and canonical cytotoxicity genes such as Nkg7, both the chemokine Ccl5 and chemokine receptor Cxcr6, co-stimulatory genes (Jcos) and markers of T-cell exhaustion (Ctla4. Tigit) (FIGS. 2C-2D).

[0178] We performed simultaneous TCR-Seq to determine clonality of cardiac T-cells within our Lag3- / -, Pdcdl- / - mice. Interestingly, we observed 41 % of Lag3- / -, Pdcdl- / - cardiac T-cells were clonal (defined as >2 cells with same TCR) as compared to no clonality seen within WT cardiac T-cells. Further, there was a marked expansion of clones in our Lag3- / -, Pdcdl- / - as compared to WT mice (Supplementary Table). Indeed, over 20% of cardiac T-cells in Lag3- / - , Pdcdl- / - mice were noted to be either “Large” or “Hyperexpanded” clones of >10 T-cells (FIGS. 2E-2F).Cardiac CXCR6+ T-cells dynamically uprequlate in mouse and human ICI-myocarditis tissues

[0179] To further understand the role of recruitment and positioning of T-cells in the cardiac environment we focused our attention on CXCR6. From our CD45+ scRNA-seq data, CxcrG was both a marker of activated cardiac T-cells and robustly expanded in Lag3- / -,Pdcd1- / - mice as compared to WT controls (FIG. 3A). CxcrG expression was similarly increased in activated T-cells in a previously reported preclinical model of CTLA4 / PD-1 myocarditis from our lab (FIG.9). While CXCR3 has been reported to be a mechanism of recruitment in anti-tumor immunity, myocarditis and other iRAEs - the role of CXCR6 T-cells has been understudied9 10 19 20. To confirm our cardiac CD45+ scRNA-seq findings and determine if we saw similar changes in other inflamed organs in Lag3- / -,Pdcd1- / - mice - we isolated and performed flow cytometry on immune cells from the pancreas, liver and blood. While there was a marked expansion of CXCR6+ T-cells in comparison to total T-cell population in the heart, there was no significant differences in other assayed tissues - demonstrating CXCR6+ T-cells are not a pan-marker of inflammation in this model (FIG. 3B). To assess for human relevance, we probed a publicly accessible repository and analysis of scRNA-seq on ICI treated patients with and without myocarditis - and saw a pronounced increase in CXCR6 expression in ICI-myocarditis patient samples (FIG. 3C)19.

[0180] To characterize the different populations of CXCR6+ T-cells in Lag3- / -,Pdcd1- / - mice - we selected CxcrG expressing T-cells and performed downstream clustering and analysis. We defined 5 groups of T-cells (FIG. 3D) including Cluster 0 (Gzmk), Cluster 1 (Gzmb, Prf1), Cluster 2 (Gm29617), Cluster 3 (Cd4), Cluster 4 (Pclaf, Birc5). The dichotomy of Gzmk and Gzmb CD8+ T-cells has been observed in other T-cell inflammation - however the functional relevance of the two subsets is still an area of active investigation21. We observed that CxcrG expression overlapped with clonal T-cells graphically - and therefore sought to characterize the marker genes of hyperexpanded T-cell clones as compared to all cells within the Activated T-cell cluster. Indeed, CxcrG, along with Prf1 and Nkg7vjere markers of hyperexpanded clonal T-cells (FIG. 3E).CXCL16+ cardiac macrophages expand and position CXCR6+ T-cells in LAG3 / PD-1 myocarditis

[0181] As both cardiac T-cells and macrophages were particularly expanded in Lag3- / - ,Pdcd1- / - mice, we then turned our attention to defining our macrophage populations. Reclustering of the cardiac macrophage population aided in defining 5 unique clusters (FIG. 4A). We and others have previously reported on the role of CXCL9 / CXCL10 macrophages in the recruitment of CXCR3+ T-cells in ICI-myocarditis, with Cluster 0 marked by Cxcl10 and Interferon inducible genes (Jfi209, Ifi211) expression. Interestingly Cluster 1 had robustexpression of antigen presentation genes (H2-Aa, H2-Ab1, H2-Eb1), Complement (C1qa, C1qb, C1qc), and the chemokine Cxcl16 (FIG. 4B). As mentioned earlier, CXCL16 is the only known ligand of CXCR6. While there were modest levels of Cxcl16 in WT cardiac macrophages, there was an increase in expression in Lag3- / -,Pdcd1- / - mice (FIGS. 4C-4D). To validate our gene expression findings at the protein level, we performed flow cytometry which similarly demonstrated an expansion of CXCL16+ macrophages (CD11b+, F4 / 80+) in Lag3- / -,Pdcd1- / - hearts (FIG. 4E).

[0182] To test the relevance of CXCL16 within the cardiac T-cell population, we sorted T-cells from Lag3- / -,Pdcd1- / - mice heart and blood. While peripheral blood T-cells were chemoattracted to FBS as a positive control - they did not appear to respond to recombinant mouse CXCL16. In contrast, cardiac T-cells demonstrated chemotaxis towards CXCL16 (FIG.4F). This suggests that CXCL16 production from cardiac macrophages likely plays a role in the positioning of cardiac T-cells - presumably through the CXCL16-CXCR6 axis.CXCR6+ T-cells are necessary for LAG3 / PD-1 myocarditis

[0183] To this point, we had identified cardiac-specific enrichment of CXCR6+ T-cells that responded. The CXCL16, and CXCL16+ cardiac macrophages were dynamically upregulated in Lag3- / -,Pdcd1- / - mice. We next sought to determine the functional relevance of CXCR6+ T- cells. To answer this, we treated Lag3- / -,Pdcd1- / - mice with anti-CXCR6 antibody or isotype control from 3 weeks of age to 10 weeks of age. With anti-CXCR6 treatment, we observed total rescue of pre-mature lethality, with 100% of treated mice living until experimental time endpoint, in comparison with 100% lethality of isotype control mice (FIG. 5A). Histology revealed a marked reduction in inflammation within anti-CXCR6 treated hearts (FIGS. 5B-5C). To add, weekly ECGs of anti-CXCR6 treated mice or isotype control mice demonstrated a reduction in arrhythmias, with only 1 / 8 anti-CXCR6 treated mice with arrhythmias upon reaching experimental endpoint as compared to 100% arrhythmia burden in isotype-treated controls (FIGS. 5D-5E). Overall, anti-CXCR6 treatment rescued the myocarditis phenotype, demonstrating the necessity of CXCR6+ T-cells in the propagation of LAG-3 / PD-1 myocarditis.

[0184] We next investigated the differences between CXCR3+ T-cells in comparison to CXCR6+ T-cells. Flow cytometry of CXCR3+ T-cells in our Lag3- / -,Pdcd1- / - mice demonstrated an expansion within the T-cell population in blood, pancreas and heart (FIG. IOA). Notably, CXCR3 marked around one-third of cardiac T-cells. We did not identify CXCR3 as a marker of hyperexpanded clones in our scRNA / TCR-Seq, in contrast to CXCR6 (FIG. IOB). We similarly treated Lag3- / -,Pdcd1- / - mice with anti-CXCR3. anti-CXCR3 treatment rescued pre-mature lethality like anti-CXCR6 (FIG. 10C). While survival was prolonged, a larger portion of anti-CXCR3 treated mice (4 / 8) developed arrhythmias (FIG. 10D). Histologyof anti-CXCR3 treated mice did demonstrate heterogeneous improvement in myocarditis as compared to isotype controls (FIG. 10E).Discussion:

[0185] Herein, we have defined a new model of ICI-myocarditis focused on the targeted combination of LAG-3 and PD-1. We identified that Lag3- / -,Pdcd1- / - mice manifest cardiac enriched inflammation and pre-mature lethality due to myocarditis. Through scRNA-seq, we demonstrated a dynamic increase in Cxcr6 expressing T-cells that was not seen in other organs - and recapitulated in ICI myocarditis data. The cognate ligand, Cxcl16, was upregulated on a specific group of cardiac macrophages. Most notably, loss of CXCR6+ T- cells rescued myocarditis in our mice, suggesting that cardiac CXCR6+ T-cells are key facilitators of ICI-myocarditis.

[0186] With the emergence of I C I s (especially used in combination) as a core cancer therapy, we anticipate a growing burden of ICI-myocarditis22. As such, it is imperative to generate working pre-clinical models of ICI-myocarditis to elucidate molecular mechanisms to aid in the diagnosis and treatment of this highly morbid adverse event. Moreover, there is an increasing selection of new IC Is and combinations, and anti-LAG-3 / PD-1 is poised to become a mainstay of ICI therapy over the coming years due to its favorable irAE profile outside of ICI- myocarditis5. Therefore, having preclinical models that effectively recapitulate cardiotoxicity from anti-LAG-3 / PD-1 therapy is paramount to the delineating the disease pathogenesis. Our results from Lag3- / -,Pdcd1- / - mice highlight their utility as a model of ICI-myocarditis in this setting.

[0187] One of the major challenges of studying ICI-myocarditis in a pre-clinical model is the more subtle phenotypes seen in some pharmacological treatment models. In contrast, deletion of LAG-3 and PD-1 leads to 100% pre-mature lethality due to myocarditis characterized by activated, clonal T-cells within 10 weeks of age. As such, we contend it allows us to investigate immune cell subsets, necessity and rescue in a more apparent way. However, we also recognize that global deletion of genes may lead to a more severe, or even different, mechanisms of injury - as such, orthogonal validation of pre-clinical models in human tissue samples is critical. For example, we do not observe that Lag3- / -,Pdcd1- / - mice develop a concomitant myositis that has been reported in patient cases of ICI-myocarditis.

[0188] While our mouse model of ICI-myocarditis focused on LAG-3 and PD-1 , the finding of robust upregulation of CXCR6+ on cardiac T-cells appears to be a common thread between other pre-clinical ICI-myocarditis models as well as patients on different ICI-therapies6. In particular, our results identify the cardiac enrichment of a heterogenous group of CXCR6+ T- cells in Lag3- / -,Pdcd1- / - mice. These CXCR6+ T-cell clusters included distinct expression ofGranzyme K, Granzyme B, Helper CD4+ T-cells, and Actively Dividing T-cells, suggesting that in the context of our preclinical model of ICI-myocarditis, CXCR6 is not an exclusive marker of one unique T-cell population.

[0189] The mechanisms that drive higher rates of myocarditis in anti-LAG-3 / anti-PD-1 treated patients remain unclear, but may relate to LAG-3 activity on CD4+ T-cells. LAG-3 is canonically thought to be expressed on CD4+ T-cells and similarly bind peptide-MHC class II complexes. However, further studies are needed to clearly elucidate the role of CD4+ T-cells in anti-LAG-3 associated myocarditis.

[0190] Unlike other chemokine-chemokine receptor pathways with multiple ligand / receptors, the sole chemokine for CXCR6 is CXCL16. As such, we identified CXCL16 upregulation, at both the RNA and protein level, in a group of cardiac macrophages in our preclinical model. CXCL16, unlike other CXCL chemokines can exist in a membrane-bound state and has been previously described mediate the direct interaction between CXCR6+ T-cells and CXCL16+ myeloid cells in other microenvironments. Interestingly, CXCL16 macrophages appear to segregate from CXCL10 expressing macrophages - where we and others have shown the CXCL9 / 10-CXCR3 axis as critical in the recruitment of peripheral T-cells to the heart in ICI- myocarditis9 10. From the data generated in this paper, CXCR6+ T-cells represent a more clonal population of T-cells as compared to CXCR3+. Anti-CXCR3 treatment attenuated the myocarditis phenotype, though a portion of these mice still developed arrhythmias and significant cardiac immune infiltration.

[0191] Ultimately, the functional relevance of CXCR6+ T-cells was our largest outstanding question. Treatment with anti-CXCR6 effectively rescued the myocarditis phenotype in several orthogonal ways (histology, arrhythmias and most importantly survival). These data strongly indicate that CXCR6+ T-cells are critical drivers of our preclinical ICI-myocarditis model. This study provides the biologic plausibility of anti-CXCR6 treatment as a potential therapeutic for ICI-myocarditis - though this will need to be rigorously tested in focused pre-clinical models.

[0192] CXCR6+ is best known as a marker of tissue resident memory cells, and while our preclinical model demonstrates fulminant, active inflammation -a population of these CXCR6+ T-cells may evolve into memory T-cells should these mice not succumb to their fatal disease. Recent investigations have demonstrated that existing tissue resident memory T-cells, in the setting of previous cardiac injury, play a role in ICI-myocarditis23. Based on the lack of expansion of CXCR6+ T-cells in circulating T-cells, we suspect that CXCL9 / 10-CXCR3 axis may be involved in recruitment of peripheral T-cells with subsequent expression of CXCR6 in the heart for positioning within the cardiac environment and establishing tissue residency. However, these hypotheses require further rigorous experiments for validation. Lastly, another outstanding question is the necessity of CXCR6 in the pathogenic role of CXCR6+ T-cells. Inthe tumor microenvironment, CXCL16+ Dendritic Cells recruit CXCR6+ T-cells through the CXCL16-CXCR6 axis in order to trans-present IL-15 and induce pro-survival signaling24. As such, further investigation into the loss of CXCR6 on cardiac T-cells and how this may affect cardiac T-cell retention, T-cell survival and overall cardiac phenotype is warranted.

[0193] In summary, we have characterized a mouse model of Lag3 and Pdcdl deletion that effectively recapitulates ICI-myocarditis and serves as a robust tool to study the immunological drivers of the disease. Like other pre-clinical models and clinical data, we observed clonal, T- cell predominant cardiac infiltration with specific upregulation of the chemokine receptor CXCR6. The cognate ligand of CXCR6, CXCL16, is upregulated in a distinct population of cardiac macrophages. Loss of CXCR6+ T-cells through anti-CXCR6 treatment rescues the myocarditis phenotype. Overall our study highlights cardiac CXCR6+ T-cells as key mediators in our pre-clinical model of ICI-myocarditis.References

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Claims

WHAT IS CLAIMED IS:

1. A method of treating myocarditis, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6).

2. The method of claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, and a scFv fragment.

3. The method of claim 1 or 2, further comprising administering an angiotensin-converting-enzyme (ACE) inhibitor, a beta blocker, a diuretic, a corticosteroid, an immunosuppressant, intravenous immunoglobulin (IVIG), an immune checkpoint inhibitor, or any combination thereof.

4. The method of any one of claims 1-3, wherein said administering comprises administering the antibody intravenously, subcutaneously, or intraperitoneally.

5. The method of any one of claims 1-3, wherein said administering comprises administering the antibody locally to the heart.

6. The method of any one of claims 1-5, wherein said administering comprises administering the antibody before, during, or after treatment of the subject with an immune checkpoint inhibitor.

7. The method of any one of claims 1-6, wherein said administering comprises administering the antibody before, during, or after treatment of the subject with anti-LAG-3 / anti-PD-1 combination therapy.

8. The method of any one of claims 1-7, wherein multiple cycles of treatment are administered to the subject.

9. The method of claim 8, wherein said administering comprises administering the antibody according to a daily dosing regimen or intermittently.

10. Amethod of inhibiting infiltration ofT cells and macrophages into cardiac tissue of a subject, the method comprising administering an effective amount of an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) to the subject.

11. The method of claim 10, wherein said administering comprises administering the antibody intravenously, subcutaneously, or intraperitoneally.

12. The method of claim 10, wherein said administering comprises administering the antibody locally to the heart.

13. A composition comprising an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) for use in treating myocarditis.

14. The composition of claim 13, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2fragment, a Fvfragment, and a scFv fragment.

15. The composition of claim 13 or 14, further comprising a pharmaceutically acceptable excipient or carrier.

16. Use of an antibody that specifically binds to a C-X-C motif chemokine receptor 6 (CXCR6) in the manufacture of a medicament or pharmaceutical composition for treating myocarditis.

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