Compositions and methods for modulating VSTM-1 mediated signal transduction

Abstract

Compositions and methods of use thereof for modulating VSTM-1 mediated signaling are provided. For example, immunomodulatory agents are provided that reduce VSTM-1 expression, ligand binding, crosslinking, VSTM-1 mediated signaling, or a combination thereof. In another embodiment, immunomodulatory agents are provided that enhance or promote VSTM-1 expression, ligand binding, crosslinking, VSTM-1 mediated signaling, or a combination thereof. Such agents can be used to modulate an immune response in a subject in need thereof.

Description

COMPOSITIONS AND METHODS FOR MODULATING VSTM-1 MEDIATEDSIGNAL TRANSDUCTIONREFERENCE TO RELATED APPLICATIONS

[0001] This invention claims the benefit of priority to United States Provisional Application Serial Number 63 / 622,404, filed on January 18, 2024, which is hereby incorporated by this reference in its entirety.FIELD OF THE INVENTION

[0002] The invention is generally related to the field of immunomodulation, and more particularly to compositions and methods for modulating immune responses in a subject.BACKGROUND OF THE INVENTION

[0003] Immunotherapies have made significant advances in the treatment of diseases such as cancer (Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017).Early immunotherapies accelerated T-cell activity. Current immune-checkpoint inhibitors take the brakes off the anti-tumor immune responses. Successful clinical trials with programmed cell death protein 1 (PI)-l ) monoclonal antibodies and other immune- checkpoint inhibitors have opened new avenues in cancer immunology. The failure of a large subset of cancer patients to respond to these new immunotherapies has led to intensified research to find new therapies.

[0004] Therefore, it is an object of the invention to provide compositions that V-set and transmembrane domain-containing protein 1 (VSTM-1) mediated signal transduction thereby promoting a suppressive immunological response. Such compositions are useful for the treatment of inflammatory diseases and disorders and autoimmune diseases.

[0005] It is also an object of the invention to provide compositions that modulate VSTM-1 mediated signal transduction to thereby enhance or promote an activating immunological response. Such compositions are useful for the treatment of cancer and infectious diseases.SUMMARY OF THE INVENTION

[0006] Compositions and methods of their use for modulating VSTM-1 mediated signal transduction are provided. One aspect provides compositions and methods that induce, promote, or enhance VSTM-1 mediated signal transduction. For example, immunomodulatory agents are provided that induce, promote, or enhance VSTM-1 expression, ligand binding, crosslinking, signal transduction, or a combination thereof.

[0007] Another embodiment provides compositions and methods for inhibiting, reducing, or blocking VSTM-1 mediated signal transduction. For example, immunomodulatory agents are provided that inhibit, reduce, or block VSTM-1 expression, ligand binding, crosslinking, signal transduction, or a combination thereof.

[0008] Immunostimulatory agents (also referred to as binding moieties) specifically bind to VSTM-1 and modulate VSTM-1 mediated signal transduction, for example VSTM-1 signal transduction in immune cells.

[0009] In one embodiment the disclosed immunomodulatory agents can be used to modulate an immune response in a subject in need thereof by inducing, promoting, or enhancing VSTM-1 mediated signaling. An immune response can be, for example, inducing, promoting, or enhancing Tregs activation or proliferation.

[0010] In another embodiment the disclosed immunomodulatory agents can be used to modulate an immune response in a subject in need thereof by inhibiting VSTM-1 mediated signaling. An immune response can be, for example, inhibiting, reducing, or blocking migration of immunosuppressive regulatory T cells (Tregs) and / or tumor- associated macrophages (TAMs) to the tumor microenvironment, depletion of tumorresident Tregs, depletion of tumor-resident TAMs, depletion of tumor cells expressing VSTM-1, or inhibiting, reducing, or blocking trafficking of Tregs, TAMs, or tumor cells.

[0011] In one embodiment the disclosed immunomodulatory agents can be used to modulate an immune response in a subject in need thereof by inhibiting VSTM-1 mediated signaling to inhibit, reduce, or block tumor metastasis.

[0012] One embodiment provides an antibody or antigen binding fragment thereof that immunospecifically binds to VSTM-1 (any one of SEQ ID NO: 1-5) and induces, promotes, or enhances VSTM-1 mediated signal transduction. Another embodiment provides an anti- VSTM-1 antibody or antigen binding fragment thereof that induces, promotes, or enhances the interaction between VSTM-1 and its ligands.

[0013] Another embodiment provides an antibody or antigen binding fragment thereof that immunospecifically binds to VSTM-1 (any one of SEQ ID NO: 1-5) and inhibits, reduces, or blocks VSTM-1 mediated signal transduction. Another embodiment provides an anti- VSTM-1 antibody or antigen-binding fragment thereof that inhibits, interferes, or blocks the interaction between VSTM-1 and its ligands.

[0014] In another embodiment the agent is a VSTM-1 fusion protein, for example a fusion protein that includes one or more extracellular domains of VSTM-1 or functional variant thereof linked to an immunoglobulin domain.

[0015] In other embodiments the immunomodulatory agent is an VSTM-1 protein (any one of SEQ ID NO:2-5) or a functional fragment or variant thereof. For example, the VSTM-1 protein or functional fragment or variant thereof can have at least 80%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NO: 1-5).

[0016] In other embodiments the immunomodulatory agent is a soluble VSTM-1 protein or a functional fragment or variant thereof. For example, the soluble VSTM-1 protein can consist of one or more extracellular domains of VSTM-1 or a functional fragment or variant thereof including, but not limited to, SEQ ID NO: 3.

[0017] One embodiment provides an immunomodulatory agent that specifically binds any one of SEQ ID NO: 1-5 and modulates VSTM-1 mediated signal transduction.

[0018] In one embodiment, the antibody or antigen binding fragment thereof is an agonist antibody having least 99% sequence identity to SEQ ID NO: 18, 19, 20, or 21.

[0019] In another embodiment, the antibody or antigen binding fragment thereof comprises a variable light chain having at least 99% sequence identity to SEQ ID NO:22, 23, 46, 47, and a variable heavy chain having an amino acid sequence according to SEQ ID NO:24, 25, 26, 27, 28, 29, 30, 31, 32, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or combinations thereof.

[0020] In particular embodiments, the antibody or antigen binding fragment thereof comprises a) a variable light chain of SEQ ID NO:22 and a variable heavy chain of SEQ ID NO:24; b) a variable light chain of SEQ ID NO:22 and a variable heavy chain of SEQ ID NO:25; c) a variable light chain of SEQ ID NO:22 and a variable heavy chain of SEQ ID NO:26; d) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO:24; e) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO:25; f) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO:26; g) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO:27; h) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO:28; i) a variable light chain of SEQ ID NO:22 and a variable heavy chain of SEQ ID NO:29; j) a variable light chain of SEQ ID NO:22 and a variable heavy chain of SEQ ID NO:30; k) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO:29; 1) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQ ID NO: 30; m) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:31; n) a variable light chain of SEQ ID NO:22 and a variable heavy chain of SEQ ID NO:32; o) a variable light chain of SEQ ID NO:23 and a variable heavy chain of SEQID NO: 31; or p) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:32.

[0021] In yet another embodiment, the antibody or antigen binding fragment thereof comprises a variable light chain having at least 99% sequence identity to SEQ ID NO:33, 34, 66, 67, and a variable heavy chain having an amino acid sequence according to SEQ ID NO 35, 36, 37, 38, 39, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or combinations thereof

[0022] In particular embodiments, the antibody or antigen binding fragment thereof comprises a) a variable light chain of SEQ ID NO:33 and a variable heavy chain of SEQ ID NO:35; b) a variable light chain of SEQ ID NO:33 and a variable heavy chain of SEQ ID NO:36; c) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:35; d) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:36; e) a variable light chain of SEQ ID NO:33 and a variable heavy chain of SEQ ID NO:37; f) a variable light chain of SEQ ID NO:33 and a variable heavy chain of SEQ ID NO:38; g) a variable light chain of SEQ ID NO:33 and a variable heavy chain of SEQ ID NO:39; h) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:37; i) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:38; or j) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:39. In some embodiments, the antibody or antigen binding fragment thereof induces, promotes, or enhances the interaction between VSTM-1 and its ligands. In other embodiments, the antibody or antigen binding fragment thereof inhibits, interferes, or blocks the interaction between VSTM-1 and its ligands.

[0023] In some embodiments, the antibody or antigen binding fragment thereof induces, promotes, or enhances the interaction between VSTM-1 and its ligands. In other embodiments, the antibody or antigen binding fragment thereof inhibits, interferes, or blocks the interaction between VSTM-1 and its ligands.

[0024] Another embodiment provides a fusion protein or an antigen binding fragment, wherein the fusion protein or an antigen binding fragment thereof comprises one or more extracellular domains of VSTM-1 or functional variant thereof linked to an immunoglobulin domain, the fusion protein or antigen binding fragment thereof having 80%, 85%, 90%, 95%, 99%, or 100% to any one of SEQ ID NO:6-17. Other embodiments provide a humanized fusion protein or an antigen binding fragment thereof comprising the immunoglobulin domain having at least 99% sequence identity to SEQ ID NO:40, 41, 42, 43, 44, 45 or combinations thereof. In particular embodiments, the humanized fusion protein of antigen binding fragment thereof has 80%, 85%, 90%, 95%,99%, or 100% to any one of SEQ ID NO: The fusion protein or an antigen binding fragment thereof inhibits, reduces, or blocks VSTM-1 mediated signal transduction.

[0025] Another aspect provides a pharmaceutical composition comprising an effective amount of the VSTM-1 immunomodulatory agent of claim 1 to modulate VSTM-1 expression, ligand binding, crosslinking, VSTM-1 mediated signaling, or a combination thereof. In one embodiment, the composition modulates hyperinflammatory conditions, restores homeostasis and prevents VSTM-1 mediated disease in a subject in need thereof. In another embodiment, the composition further comprises one or more additional therapeutic agents selected from cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals, anti-parasitics, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof, antigen and vaccine formulations, peptide drugs, antiinflammatories, ligands that bind to Toll-Like Receptors to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, other molecules that deactivate or down-regulate suppressor or regulatory T-cells, or combinations thereof. In other embodiments, the composition is administered with other immunomodulatory agents selected from PD-1 antagonists, CTLA4 antagonists, potentiating agents or other VSTM-1 antagonists.

[0026] Another aspect provides methods of promoting a suppressive immune response in a subject typically include administering to a subject in need thereof an effective amount of an immunomodulatory agent that induces, promotes, or enhances VSTM-1 expression, ligand binding, crosslinking, signal transduction, or a combination thereof. The subject can have, for example, inflammatory disease including, but not limited to autoimmune disease and transplant rejection disease. In one embodiment the subject is treated by administering one or more fusion proteins described above.

[0027] Another embodiment provides the VSTM-1 immunomodulatory agents can be used to treat hyperinflammatory disease or condition characterized by increased expression of VSTM-1 relative to expression of VSTM-1 in subjects without the disease or condition. In one embodiment the subject has a pulmonary inflammatory disease or condition. In another embodiment, the pharmaceutical composition blocks neutrophil- mediated inflammation and cytokine production associated with pulmonary tissue damage to reduce pulmonary pathology and prevent or treat pulmonary inflammatory disease. Methods of increasing an immune response in a subject typically includeadministering to a subject in need thereof an effective amount of an immunomodulatory agent that inhibits, reduces, or blocks VSTM-1 expression, ligand binding, crosslinking, signal transduction, or a combination thereof. The subject can have, for example, a hyperproliferative disease, cancer, or an infectious disease. In one embodiment the subject is treated by administering one or more fusion proteins described above.

[0028] In some embodiments, the subject, or the disease is characterized by increased expression of VSTM-1, increased expression of a VSTM-1 ligand, or a combination thereof. In particular embodiments, the disease is a cancer of a variety of cancers or abnormal proliferative growths as listed within this application. The agent can be administered contemporaneously, in alternation, or in combination formulated as a unit dose, optionally with a one or more additional therapeutic agents including but not limited to a vaccine or a component thereof.

[0029] In some embodiments, the subject or the disease or condition is characterized by increased expression of VSTM-1 relative to expression of VSTM-1 in subjects without the disease or condition.

[0030] One embodiment provides a method for treating cancer or an infection in a subject in need thereof by administering to the subject an effective amount of a pharmaceutical composition including an VSTM-1 monoclonal antibody, soluble VSTM- 1 polypeptide, VSTM-1 fusion protein, or combinations thereof in an amount effective to inhibit, reduce, or block VSTM-1 signal transduction in immune cells.

[0031] One embodiment the VSTM-1 immunomodulatory agents can be used to treat inflammation and or autoimmune disorders, including but not limited VSTM-1 fusion proteins.

[0032] In one embodiment anti-VTSMl antibodies that bind to ligands of VSTM-1 can be used to treat cancer.

[0033] One embodiment provides a method for assessing or predicting the efficacy of a treatment using an anti-VSTM-1 binding moiety by assaying the cells of a subject in need of treatment to determine whether the cells express VSTM-1, binding partners of VSTM-1, or both. In one embodiment, a treatment is effective if it inhibits, reduces, or blocks VSTM-1 expression, ligand binding, crosslinking, signal transduction, or a combination thereof. Exemplary cells to be assayed include but are not limited to cancer cells obtained from the subjected.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0034] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

[0035] Figures 1 A-1H are representative PMNs from fresh whole blood (FIGs 1 A- 1B) and bar graphs show blood PMNs from 5 whole blood donors showing that VSTM-1 is highly expressed in neutrophils.

[0036] Figures 2A-2I are bar graphs and flow cytometry data showing that VSTM-1 is expressed on some monocyte populations but not on lymphocytes.

[0037] Figures 3A-3C are stained lung tissue sections and bar graphs showing that VSTM-1 expression is retained inflamed lung tissue.

[0038] Figures 4A-4F are bar graphs showing that VSTM-1 binds to amphipathic, alpha-helical peptides.

[0039] Figure 5 is a bar graph showing the primary screening of anti-VSTM-1 hybridoma supernatants for binding to VSTM-1 -Fc.

[0040] Figures 6A-6B are bar graphs showing the secondary screening of anti- VSTM-1 hybridoma supernatants for binding to VSTM-1. FIG. 6A shows the second round of ELISA while FIG. 6B shows the second round of cell binding.

[0041] Figures 7A-7H show the screening of anti-VSTM-1 hybridoma supernatants to VSTM-1+ cells.

[0042] Figures 8A-8F are binding screens showing that anti-VSTM-1 mAbs bind to cell surface expressed VSTM-1.

[0043] Figure 9 is a cell-free binding screen showing the relative binding of Anti- VSTM-1 mAbs to VSTM-1 by ELISA.

[0044] Figures 10A-10B cell surface binding (FIG. 10 A) and affinity and binding data (FIG. 10B) showing that anti-VSTM-1 clones NP690 and NP693 bind to human VSTM-1.

[0045] Figures 11 A-l IB are bar graphs showing that NP690. IgGl and NP693.IgGl bind to both cell surface and secreted isoforms of VSTM-1.

[0046] Figures 12A-12B are flow cytometry data showing that neither NP690 nor NP693 bind to Cynomolgus Macaque VSTM-1.

[0047] Figures 13A-13C show that Anti-VSTM-1 mAb NP693 suppresses the ERK / MAPK pathway.

[0048] Figures 14A-14D are bar graphs showing that anti-VSTM-1 mAbs suppress Inflammatory cytokine production.

[0049] Figures 15A-15C are bar graphs showing that anti-VSTM-1 mAbs suppressed inflammatory cytokines production from neutrophils under various stimulants.

[0050] Figures 16A-16C show that anti-VSTM-1 antibodies NP690 and NP693 block IL- 17 from TH17 polarized cells.

[0051] Figures 17A-17B are bar graphs showing that anti-VSTM-1 antibodies suppress myeloid cell help during TH17 polarization.

[0052] Figures 18A-18B are bar graphs showing that NP690 and NP693 agonist mAbs attenuate production of ROS in myeloid cells.

[0053] Figures 19A-19E show that anti-VSTM-1 antibodies NP690 and NP693 agonist mAbs inhibit NETosis of stimulated neutrophils.

[0054] Figures 20A-20E shows that NP690 and NP693 reduce pulmonary PMN influx in a humanized acute pulmonary inflammation model.

[0055] Figures 21A-21D are illustrative of how granulocytic inflammation drives pulmonary inflammatory disease.

[0056] Figures 22A-22B show the affinity of top mAb candidates by Octet and cell binding.

[0057] Figures 23 A-23G show the effects of aVSTM-1 agonist mAb on TNFa, IL-6, and IL-ip.

[0058] Figures 24A-24G show that the VSTM-1 agonist mAb suppresses ROS and NETosis.

[0059] Figures 25A-25E show that the construct used to KI human VSTM-1 into mice is functional and can be agonized by anti-VSTM-1 agonist mAb. FIG 25C-25E verifies gene knock-in of human VSTM-1, which results in expression of VSTM-1 protein in KI mice.

[0060] Figures 26A-26B show VSTM-1 knock-in mice adoptively transferred human PMNs, followed by challenge with 1 mg / kg LPS via intratracheal aspiration. Anti- VSTM-1 agonist mAb reduced lung infiltration of PMNs in this model.

[0061] Figures 27 is a schematic of pulmonary disease modeling in human myeloid cell-replete mice.

[0062] Figure 28 shows TNFa suppression by NP690 / 693 variant mAb cell supes.

[0063] Figures 29A-29B show binding vs activity of NP690 / 693 variants.

[0064] Figures 30A-30B show ROS suppression by humanized anti-VSTM-1 variants.

[0065] Figures 31 A- 3 IB show TNFa suppression by humanized anti-VSTM-1 variants (mAbs).

[0066] Figures 32A-32B show percent TNFa suppression by humanized anti- VSTM-1 variants (purified mAbs).

[0067] Figures 33A-33B show IL-6 suppression by humanized anti-VSTM-1 variants (purified mAbs).

[0068] Figure 34A- 34B-show a multivariable comparison of humanized variants.

[0069] Figures 35A-35B show IFNy suppression by humanized anti-VSTM-1 variants (purified mAbs).

[0070] Figures 36A-36F show Legendplex analysis of supernatants from PBMCs treated with humanized purified anti-VSTM-1 variants.

[0071] Figures 37A-37E show Legendplex analysis of supernatants from PBMCs treated with humanized purified anti-VSTM-1 variants.DETAILED DESCRIPTION OF THE INVENTIONI. Definitions

[0072] As used herein, the terms “immunomodulatory agent” and “binding moiety” are used interchangeably.

[0073] As used herein, the term “VSTM-1 immunomodulatory agent” refers to V- set and transmembrane domain-containing protein 1 (VSTM-1) binding moi eties including, but not limited to antibodies and antigen binding fragments thereof, and VSTM-1 fusion proteins and binding fragments thereof. In one embodiment, VSTM-1 has an amino acid sequence according to UniProtKB - Q6UX27 (VSTM-1 HUMAN) which is incorporated by reference in its entirety.

[0074] As used herein, a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecifically binding to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e. g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totallyunrelated antigen. In some embodiments, however, antibodies (and their antigen binding fragments) will not cross-react with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

[0075] As used herein, a molecule is said to “physiospecifically bind” a second molecule if such binding exhibits the specificity and affinity of a receptor to its cognate binding ligand. A molecule can be capable of physiospecifically binding to more than one other molecule.

[0076] As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain).The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from a “Complementarity Determining Region” or “CDR” (z. e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1 :253;Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231 :25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U. S. Patent No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies). In particular, suchantibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG?, IgGs, IgG4, IgAi and IgA?) or subclass.

[0077] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody’s “variable region” antigen recognition site and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')?, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins including the antibody’s “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.).

[0078] As used herein, the term “fragment” refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0079] As used herein the term “modulate” relates to a capacity to alter an effect, result, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating, but not abolishing) or it can completely abolish such activity (e.g., neutralizing). Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In a still further embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction. For example, the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments, such modulation will provide at least a 10% change in a measurableimmune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100-fold change in such activity.

[0080] The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is able to substantially block an activity of a ligand or receptor if the extent of blockage is physiologically or therapeutically relevant (for example if such extent is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).

[0081] As used herein, the “activating” or “stimulatory” signals encompass signals that result in enhancing an activity or enhancing signal transduction.

[0082] As used herein, “suppressive” signals refer to signals that suppress immune activity.

[0083] The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of a parent or reference antibody but which differs in amino acid sequence from the parent or reference antibody or antigen binding fragment thereof by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or antigen binding fragment thereof. In some embodiments, such derivatives will have substantially the same immunospecificity and / or characteristics, or the same immunospecificity and characteristics as the parent or reference antibody or antigen binding fragment thereof. The amino acid substitutions or additions of such derivatives can include naturally occurring (z.e., DNA-encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, chimeric or humanized variants, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

[0084] As used herein, a “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0085] As used herein, the term “humanized antibody” refers to an immunoglobulin including a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor. ” Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85- 99%, or about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody including a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human.

[0086] The term “endogenous concentration” refers to the level at which a molecule is natively expressed (i.e., in the absence of expression vectors or recombinant promoters) by a cell (which cell can be a normal cell, a cancer cell or an infected cell).

[0087] As used herein, the terms “treat,” “treating,” “treatment” and “therapeutic use” refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder. As used herein, a “therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0088] As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease prior to the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0089] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. The term “cancer” refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or weblike matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct spherelike structures in three-dimensional basement membrane or extracellular matrix preparations.

[0090] As used herein, an “immune cell” refers to any cell from the hemopoietic origin including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0091] As used herein, “inflammatory molecules” refer to molecules that result in inflammatory responses including, but not limited to, cytokines and metalloproteases such as including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL-18, IL-17, IL-6, IL- 23, IL-22, IL-21, and MMPs.

[0092] As used herein, “valency” refers to the number of binding sites available per molecule.

[0093] As used herein, the terms “immunologic,” “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and / or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+T helper cells and / or CD8+cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

[0094] An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

[0095] As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.

[0096] As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (c.g, phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. The polypeptides can be “exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0097] As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0098] Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss ofactivity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0099] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4. 5); valine (+4. 2); leucine (+3. 8); phenylalanine (+2. 8); cysteine / cystine (+2. 5); methionine (+1. 9); alanine (+1. 8); glycine (-0. 4); threonine (-0. 7); serine (-0. 8); tryptophan (-0. 9); tyrosine (-1. 3); proline (-1. 6); histidine (-3. 2); glutamate (-3. 5); glutamine (-3. 5); aspartate (-3. 5); asparagine (-3. 5); lysine (-3. 9); and arginine (-4. 5).

[0100] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0. 5 are even more particularly preferred.

[0101] Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3. 0); lysine (+3. 0); aspartate (+3. 0 ± 1); glutamate (+3. 0 ± 1); serine (+0. 3); asparagine (+0. 2); glutamine (+0. 2); glycine (0); proline (-0. 5 ± 1); threonine (-0. 4); alanine (-0. 5); histidine (-0. 5); cysteine (-1. 0); methionine (-1. 3); valine (-1. 5); leucine (-1. 8); isoleucine (-1. 8); tyrosine (-2. 3); phenylalanine (-2. 5); tryptophan (-3. 4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular an immunologically equivalent polypeptide. In such changes, the substitution of aminoacids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0. 5 are even more particularly preferred.

[0102] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Vai: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

[0103] The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0104] For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:100 times the fraction W / Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

[0105] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.II. Compositions

[0106] One embodiment provides compositions that specifically bind to VSTM-1 and modulate signal transduction through VSTM-1, for example to reduce, inhibit, or block VSTM-1 mediated signal transduction in immune cells. Representative immune cells include but are not limited to monocytes and T cells. Another embodiment provides compositions that inhibit, reduce, or block the interaction with VSTM-1 with one or more ligands of VSTM-1, and thereby reduce, inhibit, or block VSTM-1 mediated signal transduction in immune cells. The binding moieties can bind directly to VSTM-1 and inhibit, reduce or block the interaction of VSTM-1 with one or more of its ligands. In still another embodiment, the binding moieties specifically bind or to a complex of VSTM-1 with one or more ligands.

[0107] Another embodiment provides immunomodulatory compositions that specifically bind to VSTM-1 and enhance or promote VSTM-1 mediated signal transduction. In one embodiment, immunomodulatory compositions enhance VSTM-1 mediated signal transduction and thereby induce or enhance an immune response in a subject. In another embodiment the immunomodulatory agent binds to VSTM-1, an extracellular domain of VSTM-1, or to a ligand of VSTM-1 and promotes signal transduction though VSTM-1 to induce, enhance, or promote and immune response in a subject in need thereof.A. VSTM-1 Polypeptides

[0108] VSTM-1 polypeptides are disclosed as well as binding moieties that specifically bind to VSTM-1, for example bind immunospecifically to VSTM-1, and modulate VSTM-1 mediated signal transduction. Modulating VSTM-1 mediated signal transduction includes agonizing or antagonizing VSTM-1 mediated signal transduction.

[0109] Inhibiting, reducing, or blocking VSTM-1 signal transduction in immune cells can occur as a result of the binding moiety binding directly to VSTM-1. In some embodiments the VSTM-1 binding moiety binds to VSTM-1 and inhibits, reduces or blocks the interaction or association of VSTM-1 and one or more of ligands of VSTM-1. VSTM-1 binding moieties can include VSTM-1 polypeptides including the amino acidsequence of full-length VSTM-1, or a fragment or variant thereof, or a fusion protein thereof.

[0110] In other embodiments, enhancing or promoting VSTM-1 signal transduction in immune cells can occur as a result of the binding moiety binding directly to VSTM-1.

[0111] VSTM-1 (V-set and transmembrane domain containing 1) is a membrane molecule identified from immunogenomics, which has two main isoforms, VSTM-l-vl and VSTM-l-v2. VSTM-l-vl is a type I transmembrane protein, and VSTM-l-v2 is a classical secretory protein, lacking only the transmembrane domain compared with VSTM-l-vl (Li, T., et al., Monoclon Antib Immunodiagn Immunother . ; 32(4):283-9 (2013)). VSTM-l-vl is thought to be an inhibitory immune receptor in the regulation of phagocytes. VSTM-l-v2 is thought to behave as a cytokine promoting IL17A secretion by CD4+ T-cells, and differentiation and activation of IL17 producing helper T-cells (TH17).1. Human SIRLlvl (VSTM-1)

[0112] Sequences for human VSTM-1 are known in the art. For example, the nucleic acid sequence for human VSTM-1 transcript variant 1 is as follows: CTCTATCTGC ACATCCTGGG GACGAACCGG GCAGCCGGAG AGCTGCGGCC GGCCCAGTCC CGCTCCGCCT TTGAAGGGTA AAACCCAAGG CGGGGCCTTG GTTCTGGCAG AAGGGACGCT ATGACCGCAG AATTCCTCTC CCTGCTTTGC CTCGGGCTGT GTCTGGGCTA CGAAGATGAG AAAAAGAATG AGAAACCGCC CAAGCCCTCC CTCCACGCCT GGCCCAGCTC GGTGGTTGAA GCCGAGAGCA ATGTGACCCT GAAGTGTCAG GCTCATTCCC AGAATGTGAC ATTTGTGCTG CGCAAGGTGA ACGACTCTGG GTACAAGCAG GAACAGAGCT CGGCAGAAAA CGAAGCTGAA TTCCC CTTCA CGGACCTGAA GCCTAAGGAT GCTGGGAGGT ACTTTTGTGC CTACAAGACA ACAGCCTCCC ATGAGTGGTC AGAAAGCAGT GAACACTTGC AGCTGGTGGT CACAGATAAA CACGATGAAC TTGAAGCTCC CTCAATGAAA ACAGACACCA GAACCATCTT TGTCGCCATC TTCAGCTGCA TCTCCATCCT TCTCCTCTTC CTCTCAGTCT TCATCATCTA CAGATGCAGC CAGCACAGTT CATCATCTGA GGAATCCACC AAGAGAACCA GCCATTCCAA ACTTCCGGAG CAGGAGGCTG CCGAGGCAGA TTTATCCAAT ATGGAAAGGG TATCTCTCTC GACGGCAGAC CCCCAAGGAG TGACCTATGC TGAGCTAAGC ACCAGCGCCC TGTCTGAGGC AGCTTCAGAC ACCACCCAGG AGCCCCCAGG ATCTCATGAA TATGCGGCAC TGAAAGTGTA GCAAGAAGAC AGCCCTGGCC ACTAAAGGAG GGGGGATCGT GCTGGCCAAG GTTATCGGAA ATCTGGAGATGCAGATACTG TGTTTCCTTG CTCTTCGTCC ATATCAATAA AATTAAGTTT CTCGTCTTAA AAAGAAA (SEQ ID NO: 1, NCBI Reference Sequence: NM 198481.4 which is incorporated by reference in its entirety.)

[0113] The consensus amino acid sequence for VSTM-1 is:MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQ NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLOLVVTDKHDELEAPSMKTDTRTIFVAIFSCISILLLFLSVFIIYRCSOHSSSS EESTKRTSHSKLPEOEAAEADLSNMERVSLSTADPOGVTYAELSTSALSEAASDT TOEPPGSHEYAALKV* (SEQ ID NO:2).

[0114] The underlined sequence is the signal sequence. The bolded sequence is the transmembrane sequence, and the double underlined sequence is the intracellular domain. The unmarked text is the extracellular domain. In one embodiment, the VSTM-1 protein does not contain the signal sequence.

[0115] One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:2 or a functional fragment thereof and modulates VSTM-1 mediated signal transduction.

[0116] The extracellular domain of human VSTM-1 and has the following sequence:

[0117] YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRK VNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLV VTDKHDELEAPSMKTDTRTI (SEQ ID NO:3).

[0118] One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO: 3 or a functional fragment thereof and modulates VSTM-1 mediated signal transduction including but not limited to a monoclonal antibody.2. VSTM variant 2

[0119] The consensus sequence for VSTM variant 2 is:SIRLlv2 (VSTM-1) Amino Acid SequenceMTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHS QNVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHE WSESSEHLQLVVTDKHDELEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSN MERVSLSTADPQGVTYAELSTSALSEAASDTTQEPPGSHEYAALKV* (SEQ ID NO:4).

[0120] The underlined text is the signal sequence.

[0121] The amino acid sequence without the signal peptide is:YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGY KQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKH DELEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERVSLSTADPQGVTY AELSTSALSEAASDTTQEPPGSHEYAALKV* (SEQ ID N0:5).

[0122] One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO: 5 or a functional fragment thereof and modulates VSTM-1 mediated signal transduction including but not limited to a monoclonal antibody.B. Binding Partners

[0123] One embodiment provides immunomodulatory agents that specifically bind to a binding partner of VSTM-1 and modulates VSTM-1 mediated signal transduction. In one embodiment, the binding partner a ligand of MPXL3.C. Immunomodulatory Agents or Binding Moieties

[0124] Immunomodulatory agents or binding moieties including agonists and antagonists of VSTM-1 are provided. An agonist of VSTM-1 typically induces, promotes, or enhances VSTM-1 mediated signaling. An antagonist of VSTM-1 typically inhibits, reduces, or blocks VSTM-1 mediated signaling. The disclosed compositions and methods can be used to modulate VSTM-1 and / or counter-receptor signaling on, for example, immune cells including but not limited to monocytes, Tregs, tumor-associated macrophages (TAMs), Myeloid Derived Suppressor Cells (MDSC), T cells, Th2 cells, myeloid cells including antigen-presenting cells (e.g., monocyte, macrophage, or dendritic cell), T cells, Natural Killer (NK) cells, or a combination thereof. In some embodiments, the compositions are specifically targeted to one or more cell types. In some embodiments, the disclosed compositions can be used on tumor cells.

[0125] In some embodiments, the anti-VSTM-1 agonists induce, promote, or enhance VSTM-1 mediated signaling through a known ligand or unknown counterreceptor through VSTM-1 interaction with said known or unknown counter-receptor. For example, in some embodiments, the VSTM-1 agonist binds to, induces, promotes or creates a conformation change, or otherwise promotes VSTM-1 mediated signal transduction.

[0126] In some embodiments, the anti-VSTM-1 antagonists inhibit, reduce, block, or otherwise disrupt signaling through a known or unknown counter-receptor through blockade of VSTM-1 interaction with said known or unknown counter-receptor. For example, in some embodiments, the VSTM-1 antagonist binds to, inhibits, blocks, createsa conformation change, or otherwise interferes with VSTM-1 mediated signal transduction.1. Antibodies

[0127] In one embodiment the immunomodulatory agent or binding moiety is an antibody. Suitable antibodies can be prepared by one of skill in the art. Nucleic acid and polypeptide sequences for VSTM-1 are known in the art and exemplary sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare an antibody or antigen binding fragment thereof specific for VSTM-1. The antibody or antigen binding fragment, therefore, can be an agonist or antagonist of VSTM-1 mediated signaling.

[0128] The activity of an antibody or antigen binding fragment thereof that is specific for VSTM-1 can be determined using functional assays that are known in the art, and include the assays discussed below. Typically, the assays include determining if the antibody or antigen binding fragment thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through VSTM-1.

[0129] In some embodiments, the disclosed antibodies and antigen binding fragments thereof immunospecifically bind to human or mouse VSTM-1. In some embodiments, the antibody binds to an extracellular domain of human or mouse VSTM-1.

[0130] For example, molecules are provided that can immunospecifically bind to VSTM-1 :(I) arrayed on the surface of a cell (especially a live cell);(II) arrayed on the surface of a cell (especially a live cell) at an endogenous concentration;(III) arrayed on the surface of a live cell, and modulates binding between VSTM-1 and a ligand thereof;(IV) arrayed on the surface of a live cell, and reduces or inhibits immune response by VSTM-1;(V) arrayed on the surface of a live cell, wherein the cell is a tumor cell;(VI) combinations of I- IV and V;(VII) combinations of I- III and V; and(VIII) arrayed on the surface of a live myeloid or lymphoid derived cancer cells (AML or ALL) and enhances apoptosis and differentiation resulting in reduced selfrenewal of cancer stem cells.

[0131] To prepare an antibody or antigen binding fragment thereof that specifically binds to VSTM-1 purified proteins, polypeptides, fragments, fusions, or epitopes to VSTM-1 or polypeptides expressed from nucleic acid sequences thereof, can be used. The antibodies or antigen binding fragments thereof can be prepared using any suitable methods known in the art such as those discussed in more detail below. a. Human and Humanized Antibodies

[0132] In some embodiments, the antibodies are humanized antibodies. Many nonhuman antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

[0133] Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.

[0134] Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody 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 contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all, of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0135] Antibodies, including human and humanized antibodies, are comprised of light and heavy chains. Each chain is comprised of a variable domain and a constant domain. The constant domain of the light chain can be kappa or lamba sequences as are well known in the art. The heavy chain constant domain is comprised of a first constant domain, a hinge and an Fc domain. Antibodies come in 5 classes, as determined by their sequences. The classes are IgA, IgD, IgE, IgG and IgM. The IgG antibodies comprise 4 subclasses, including IgGl, IgG2, IgG3 and IgG4. The antibody-drug conjugates of the invention are typically of the IgG class and more typically either IgGl or IgG4. If the IgGl classes are utilized, the constant domain sequence can be wild type or contain specific mutations to increase or reduce the binding to Fc gamma receptors. These mutations are well known in the art, but include mutating the glycosylation site (e.g., N297Q), mutating the Fc gamma receptor binding site (L234A and L235A or L234F, L235E and P331S). If the IgG4 class is utilized, the constant domain sequence can be wild type or contain a specific mutation (S228P) to reduce chain exchange as is well known in the art.

[0136] For the VSTM-1 antibodies of the invention, it is preferrable that the Fc be an IgGl class that has increased binding to Fc gamma receptor 2B. Such mutations are well known in the art and include (i) S267E (Li et al., PNAS 109(27), 2012, (ii) S239D and I332E, (iii) S239D, A330L and I332E, (iv) G236A, S239D and I332E, (v) S267E and L328F, (vi) P238D and (vii) E233D, G237D, P238D, H268D, P271G and A330R ((ii) - (vii) Liu et al., Antibodies 9(4), 2020).

[0137] Exemplary human IgGl Fc sequences are well known in the art but include the following.

[0138] IgGl Wild Type: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G* (SEQ ID NO:40).

[0139] IgGl EF (S267E / L328F):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G* (SEQ ID NO:41).

[0140] IgGl DE (S239D / I332E):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG* (SEQ ID NO:42).

[0141] IgGl DLE (S239D / A330L / I332E):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG* (SEQ ID NO:43).

[0142] IgGl ADE (G236A / S239D / I332E):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO:44).

[0143] Exemplary human light chain constant domains are well known in the art but include the following sequence: RTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGEC (SEO ID NO:45),

[0144] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a humanized form of a nonhuman antibody (or a fragment thereof) is a chimeric antibody or fragment, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0145] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.

[0146] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs areavailable which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0147] The antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the present compositions include fluorescent, enzymatic and radioactive markers. b. Single-Chain Antibodies

[0148] In some embodiments, the antibodies are single-chain antibodies. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C- terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. c. Monovalent Antibodies

[0149] In some embodiments, the antibodies are monovalent antibodies. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen.

[0150] The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. d. Hybrid Antibodies

[0151] In some embodiments, the antibodies are hybrid antibodies. In hybrid antibodies, one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques. Such hybrids may, of course, also be formed using chimeric chains. e. Conjugates or Fusions of Antibody Fragments

[0152] In some embodiments, the antibodies are conjugates or fusions of antibody fragments. The targeting function of the antibody can be used therapeutically by coupling the antibody or a fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent.

[0153] Such coupling of the antibody or fragment with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, or by linking the antibody or fragment to a nucleic acid such as an siRNA, comprising the antibody or antibody fragment and the therapeutic agent.

[0154] In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the site of treatment for longer periods of time. For example, it may be desirable to maintain titers of the antibody in the circulation or in the location to betreated for extended periods of time. Antibodies can be engineered with Fc variants that extend half-life, e.g., using Xtend™ antibody half-life prolongation technology (Xencor, Monrovia, CA). In other embodiments, the half-life of the anti-DNA antibody is decreased to reduce potential side effects. The conjugates disclosed can be used for modifying a given biological response. The drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.2. NP690 and NP693 agonist monoclonal antibodies

[0155] In one embodiment, the disclosure provides agonist antibodies that binds to VSTM-1 to induce signaling and suppress myeloid cell inflammatory functions. In another embodiment, the agonizing VSTM-1 antibodies are used to treat autoimmune and inflammatory disease, wherein the agonizing VSTM-1 on inflammatory monocyte and granulocyte results in inhibitory signaling that dampens proinflammatory functions and alleviates dysregulated immunopathology. In yet another embodiment, the agonizing VSTM-1 antibodies are used for cancer treatment, wherein the agonizing VSTM-1 reduces NETosis, which has been shown to promote metastasis and cancer progression; may also suppress myeloid-derived suppressor cells to remodulate the tumor microenvironment and promote T cell effector functions.

[0156] In one embodiment, the disclosure provides NP690 monoclonal antibodies that are agonists of VSTM-1.

[0157] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to the NP690 (anti-VSTMvl VM3F4 hGl) heavy chain with the following sequence: MEWSWVFLFFLSVTTGVHSEVQLQQSGPELVKPGASVKISCKASGYTFTDYSME W VKQNHGK SLEW VGYVYPNNGGT GYNQKFKSKATMT VDK S S ST AYMELHSLT SEDSAVYYCARRDYYVNRGYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18).

[0158] In another embodiment, the disclosure provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to the NP690 (anti-VSTMvl VM3F4 hGl) light chain with the following sequence:MS VPTQVLGLLLLWLTDARCQIVLTQSP AIMS ASLGERVTMTCT AS S S VS SNYLH WYQQKPGSSPKLWIYSTSNLPSGVPARFSGSGSGTSYSLTIISMEAEDAATYYCH QYHRFPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC (SEQ ID NO: 19).

[0159] One other embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to the NP693 (anti-VSTMvl VM10H11 hGl) heavy chain with the following sequence:MEWSWVFLFFLSVTTGVHSQVQVQQPGPELVKPGASVKISCKASGYAFSNYWM NWVKQRPGEGLEWIGRIYPGDGDTNYNGNFKDK AILTADTS S ST ASIQLNSLTSQ DSAVYFCARWEGGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:20).

[0160] Another embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to the NP693 (anti-VSTMvl VM10H11 hGl) light chain with the following sequence:MSVPTQVLGLLLLWLTDARCDIVMTQSHKFMSTSVGDRVSITCKASQDVEAAVA WYQQRPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTLSNVQSEDLADYFC QQYSSYPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC (SEQ ID NO:21). a. NP690 light chain variable domain sequences i. NP690 VL

[0161] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VL with the following sequence:QIVLTQSPAIMSASLGERVTMTCTASSSVSSNYLHWYQQKPGSSPKLWIYSTSNLP SGVPARFSGSGSGTSYSLTIISMEAEDAATYYCHQYHRFPPTFGSGTKLEIK (SEQ ID NO:22).(a) NP690 light chain NP690 VL, human kappa wild type constant domain

[0162] One embodiment provides a NP690 NP690 VL, human kappa wild type constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QIVLTQSPAIMSASLGERVTMTCTASSSVSSNYLHWYQQKPGSSPKLWIYSTSNLP SGVPARFSGSGSGTSYSLTIISMEAEDAATYYCHQYHRFPPTFGSGTKLEIKRTVA APSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEO DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:46). ii. NP690 humanized light chain variable region NP690 VL 1

[0163] Another embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VL_l with the following sequence: DIQLTQSPSFLSASVGDRVTITCTASSSVSSNYLHWYQQKPGKAPKLLIYSTSNLPS GVPSRFSGSGSGTEYTLTISSLQPEDFATYYCHQYHRFPPTFGSGTKLEIK (SEQ ID NO:23).(a). NP690 humanized light chain NP690 VL 1, human kappa wild type constant domain

[0164] One embodiment provides a NP690 humanized NP690 VL 1, human kappa wild type constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIQLTQSPSFLSASVGDRVTITCTASSSVSSNYLHWYQQKPGKAPKLLIYSTSNLPS GVPSRFSGSGSGTEYTLTISSLQPEDFATYYCHQYHRFPPTFGSGTKLEIKRTVAAP SVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:47). b. NP690 heavy chain variable domain sequences i. NP690 VH

[0165] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH with the following sequence:EVQLQQSGPELVKPGASVKISCKASGYTFTDYSMEWVKQNHGKSLEWVGYVYP NNGGTGYNQKFKSKATMTVDKSSSTAYMELHSLTSEDSAVYYCARRDYYVNRG YVMDYWGQGTSVTVSS (SEQ ID NO:24).(a). NP690 heavy chain NP690 VH, human wild type IgGl constant domains

[0166] One embodiment provides a NP690 humanized NP690 VH, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGPELVKPGASVKISCKASGYTFTDYSMEWVKQNHGKSLEWVGYVYP NNGGTGYNQKFKSKATMTVDKSSSTAYMELHSLTSEDSAVYYCARRDYYVNRG YVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI< EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:48).(b). NP690 heavy chain NP690 VH, human IgGl constant domains (S267E / L328F)

[0167] One embodiment provides a NP690 humanized NP690 VH, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGPELVKPGASVKISCKASGYTFTDYSMEWVKQNHGKSLEWVGYVYP NNGGTGYNQKFKSKATMTVDKSSSTAYMELHSLTSEDSAVYYCARRDYYVNRG YVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI< EYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:49).ii. NP690 VH 1

[0168] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_l with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNNGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO:25).(a). NP690 humanized heavy chain NP690 VH 1, human wild type IgGl constant domains

[0169] One embodiment provides a NP690 humanized NP690 VH 1, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNNGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:50).(b). NP690 humanized heavy chain NP690 VH 1, human IgGl constant domains (S267E / L328F)

[0170] One embodiment provides aNP690 humanized NP690 VH 1, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNNGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID N0:51). iii. NP690 VH 2

[0171] One embodiment provides an agonist antibody having 85%, 90%, 95%, or100% sequence identity to NP690_VH_2 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNNGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO:26).(a). NP690 humanized heavy chain NP690 VH 2, human wild type IgGl constant domains

[0172] One embodiment provides a NP690 humanized NP690 VH 2, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNNGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:52).(b). NP690 humanized heavy chain NP690 VH 2, human IgGl constant domains (S267E / L328F)

[0173] One embodiment provides a NP690 humanized NP690 VH 2, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNNGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:53). iv. NP690 VH 3

[0174] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_3 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNNGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO:27).(a). NP690 humanized heavy chain NP690 VH 3, human wild type IgGl constant domains

[0175] One embodiment provides a NP690 humanized NP690 VH 3, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNNGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:54).(b). NP690 humanized heavy chain NP690 VH 3, human IgGl constant domains (S267E / L328F)

[0176] One embodiment provides a NP690 humanized NP690 VH 3, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNNGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:55). v. NP690 VH 4

[0177] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_4 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNNGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO:28).(a). NP690 humanized heavy chain NP690 VH 4, human wild type IgGl constant domains

[0178] One embodiment provides a NP690 humanized NP690 VH 4, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNNGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:56).(b). NP690 humanized heavy chain NP690 VH 4, human IgGl constant domains (S267E / L328F)

[0179] One embodiment provides a NP690 humanized NP690 VH 4, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNNGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:57). vi. NP690 VH 5

[0180] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_5 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNQGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO:29).(a). NP690 humanized heavy chain NP690 VH 5, human wild type IgGl constant domains

[0181] One embodiment provides a NP690 humanized NP690 VH 5, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNQGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:58).(b). NP690 humanized heavy chain NP690 VH 5, human IgGl constant domains (S267E / L328F)

[0182] One embodiment provides a NP690 humanized NP690 VH 5, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNQGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:59). vii. NP690 VH 6

[0183] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_6 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNQGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO: 30).(a). NP690 humanized heavy chain NP690 VH 6, human wild type IgGl constant domains

[0184] One embodiment provides a NP690 humanized NP690 VH 6, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:

[0185] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYVYPNQGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCA RRDYYVNRGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO:60).(b). NP690 humanized heavy chain NP690 VH 6, human IgGl constant domains (S267E / L328F)

[0186] One embodiment provides a NP690 humanized NP690 VH 6, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVKQAPGQGLEWMGYV YPNQGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID N0:61). viii. NP690 VH 7

[0187] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_7 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNQGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO:31).(a). NP690 humanized heavy chain NP690 VH 7, human wild type IgGl constant domains

[0188] One embodiment provides a NP690 humanized NP690 VH 7, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNQGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:62).(b). NP690 humanized heavy chain NP690 VH 7, human IgGl constant domains (S267E / L328F)

[0189] One embodiment provides a NP690 humanized NP690 VH 7, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNQGGTGYAQKFQGRATMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:63). ix. NP690 VH 8

[0190] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP690_VH_8 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNQGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSS (SEQ ID NO: 32).(a). NP690 humanized heavy chain NP690 VH 8, human wild type IgGl constant domains

[0191] One embodiment provides a NP690 humanized NP690 VH 8, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNQGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:64).(b). NP690 humanized heavy chain NP690 VH 8, human IgGl constant domains (S267E / L328F)

[0192] One embodiment provides a NP690 humanized NP690 VH 8, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMEWVRQAPGQGLEWMGYV YPNQGGTGYAQKFQGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRDYYVN RGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT VSWNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG* (SEQ ID NO:65). c. Humanization and Dehumanization of NP690

[0193] Provided herein are humanized and dehumanized NP690 antibodies. Table 1 the NP690 light and heavy variable domains and the positions that differ between the engineered chains.

[0194] Table 1: Humanization and Deimmunization of NP690

[0195] Table 2 shows the combinations of the variable domains to create the NP690 antibodies.

[0196] Table 2: NP690 light chain and heavy chain combinations.d. NP693 light chain variable domain sequences i. NP693 VL

[0197] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP693 VL with the following sequence:DIVMTQSHKFMSTSVGDRVSITCKASQDVEAAVAWYQQRPGQSPKLLIYWASTR HTGVPDRFTGSGSGTDFTLTLSNVQSEDLADYFCQQYSSYPLTFGSGTKLEIK (SEQ ID NO:33). a) NP690 light chain NP693 VL, human kappa wild type constant domain

[0198] One embodiment provides a NP693 VL, human kappa wild type constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIVMTQSHKFMSTSVGDRVSITCKASQDVEAAVAWYQQRPGQSPKLLIYWASTR HTGVPDRFTGSGSGTDFTLTLSNVQSEDLADYFCQQYSSYPLTFGSGTKLEIKRTy AAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:66). ii. NP690 humanized light chain variable region NP693 VL 1

[0199] Another embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP693_VL_1 with the following sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVEAAVAWYQQKPGKAPKLLIYWASTR HTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPLTFGQGTKLEIK (SEQ ID NO:34).(a). NP693 humanized light chain NP690 VL 1, human kappa wild type constant domain

[0200] One embodiment provides aNP693 humanized NP690 VL 1, human kappa wild type constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIQMTQSPSSLSASVGDRVTITCKASQDVEAAVAWYQQKPGKAPKLLIYWASTR HTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPLTFGQGTKLEIKRTy AAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTE ODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:67). e. NP693 heavy chain variable domain sequences i. NP693 VH

[0201] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP693_VH with the following sequence:QVQVQQPGPELVKPGASVKISCKASGYAFSNYWMNWVKQRPGEGLEWIGRIYP GDGDTNYNGNFKDKAILTADTSSSTASIQLNSLTSQDSAVYFCARWEGGFAYWG QGTLVTVSS (SEQ ID NO:35).(a). NP693 heavy chain NP690 VH, human wild type IgGl constant domains

[0202] One embodiment provides a NP693 humanized NP693 VH, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQVQQPGPELVKPGASVKISCKASGYAFSNYWMNWVKQRPGEGLEWIGRIYP GDGDTNYNGNFKDKAILTADTSSSTASIQLNSLTSQDSAVYFCARWEGGFAYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* (SEQ ID NO: 68).(b). NP693 heavy chain NP693 VH, human IgGl constant domains (S267E / L328F)

[0203] One embodiment provides a NP693 humanized NP693 VH, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQVQQPGPELVKPGASVKISCKASGYAFSNYWMNWVKQRPGEGLEWIGRIYP GDGDTNYNGNFKDKAILTADTSSSTASIQLNSLTSQDSAVYFCARWEGGFAYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVK FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI< AFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* (SEQ ID NO:69). ii. NP693 VH1

[0204] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP693_VH1 with the following sequence:QVQLVQSGAEVVKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGDGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSS (SEQ ID NO:36).(a). NP693 humanized heavy chain NP693 VH 1, human wild type IgGl constant domains

[0205] One embodiment provides a NP693 humanized NP693 VH 1, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVVKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGDGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:70).(b). NP693 humanized heavy chain NP693 VH 1, human IgGl constant domains (S267E / L328F)

[0206] One embodiment provides aNP693 humanized NP693 VH 1, human IgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVVKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGDGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:71). iii. NP693 VH2

[0207] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP693_VH2 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGDGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSS (SEQ ID NO:37).(a). NP693 humanized heavy chain NP693 VH 2, human wild type IgGl constant domains

[0208] One embodiment provides a NP693 humanized NP693 VH 2, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGDGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:72).(b). NP693 humanized heavy chain NP693 VH 2, human IgGl constant domains (S267E / L328F)

[0209] One embodiment provides a NP693 humanized NP693 VH 2, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGDGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:73). iv. NP693 VH3

[0210] One embodiment provides an agonist antibody having 85%, 90%, 95%, or 100% sequence identity to NP693_VH3 with the following sequence:QVQLVQSGAEVVKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGEGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSS (SEQ ID NO:38).(a). NP693 humanized heavy chain NP693 VH 3, human wild type IgGl constant domains

[0211] One embodiment provides a NP693 humanized NP693 VH 3, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVVKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGEGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:74).(b). NP693 humanized heavy chain NP693 VH 3, human IgGl constant domains (S267E / L328F)

[0212] One embodiment provides aNP693 humanized NP693 VH 3, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVVKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGEGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:75). v. NP693 VH4

[0213] One embodiment provides an agonist antibody having 85%, 90%, 95%, or100% sequence identity to NP693_VH4 with the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGEGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSS (SEQ ID NO:39).(a). NP693 humanized heavy chain NP693 VH 4, human wild type IgGl constant domains

[0214] One embodiment provides a NP693 humanized NP693 VH 4, human wild type IgGl constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGEGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:76).(b). NP693 humanized heavy chain NP693 VH 4, human IgGl constant domains (S267E / L328F)

[0215] One embodiment provides a NP693 humanized NP693 VH 4, humanIgGl constant domain (S267E / L328F) having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGRI YPGEGDTNYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCARWEGGFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG* (SEQ ID NO:77). f. Humanization and Dehumanization of NP693

[0216] Provided herein are humanized and dehumanized NP693 antibodies. Table 3 the NP693 light and heavy variable domains and the positions that differ between the engineered chains.

[0217] Table 3: Humanization and Deimmunization of NP693.

[0218] Table 4 shows the combinations of the variable domains to create the NP693 antibodies.

[0219] Table 4: NP693 light chain and heavy chain combinations.3. Proteins and Polypeptides a. Protein and Polypeptide Compositions

[0220] The immunomodulatory or binding agent can be a VSTM-1 protein, polypeptide, or fusion protein. For example, the immunomodulatory agent or binding moiety can be an isolated or recombinant protein or polypeptide, or functional fragment, variant, or fusion protein thereof of VSTM-1.

[0221] The VSTM-1 protein or polypeptide, or functional fragment, variant, or fusion protein thereof can be an agonist or an antagonist. For example, in some embodiments an antagonist of VSTM-1 is a VSTM-1 polypeptide or a fragment or fusion protein thereof that binds to a ligand of VSTM-1. The polypeptide can be a soluble fragment, for example the extracellular domain of VSTM-1, or a functional fragment thereof, or a fusion protein thereof. In some embodiments, a soluble ligand of VSTM-1 may serve as an antagonist, decreasing VSTM-1 mediated signal transduction.

[0222] The activity of a protein or polypeptide of VSTM-1, or any fragment, variant or fusion protein thereof can be determined using functional assays that are known in the art, and include the assays discussed below. Typically, the assays include determining if the protein, polypeptide or fragment, variant or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through the VSTM-1 receptor. In some embodiments, the assay includes determining if the protein, polypeptide or fragment,variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) the immune response associated with VSTM-1. Typically, the assays include determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases i.e., agonist) or decreases (i.e., antagonist) signaling through VSTM-1. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof decreases (i.e., agonist) or increases (i.e., antagonist) an immune response regulated by VSTM-1. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., antagonist) the apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells resulting in reduced self-renewal capacity of AML and ALL stem cells.

[0223] Nucleic acid and polypeptide sequences for VSTM-1 are known in the art and exemplary protein and peptide sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare any protein or polypeptide of VSTM-1, or any fragment, variant, or fusion protein thereof. Generally, the proteins, polypeptides, fragments, variants, and fusions thereof of VSTM-1 are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide VSTM-1 proteins with and without a signal sequence are disclosed. It is understood that in some cases, the mature protein as it is known or described in the art, i.e., the protein sequence without the signal sequence, is a putative mature protein. During normal cell expression, a signal sequence can be removed by a cellular peptidase to yield a mature protein. The sequence of the mature protein can be determined or confirmed using methods that are known in the art. i. Fragments

[0224] As used herein, a fragment of VSTM-1 refers to any subset of the polypeptide that is at least one amino acid shorter than full length protein. Useful fragments include those that retain the ability to bind to their natural ligand or ligands. A polypeptide that is a fragment of any full-length VSTM-1 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent,95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural ligand respectively as compared to the full-length protein.

[0225] Fragments of VSTM-1 include cell free fragments. Cell free polypeptides can be fragments of full-length, transmembrane, polypeptides that may be shed, secreted or otherwise extracted from the producing cells. Cell free fragments of polypeptides can include some or all of the extracellular domain of the polypeptide and lack some or all of the intracellular and / or transmembrane domains of the full-length protein. In one embodiment, polypeptide fragments include the entire extracellular domain of the full- length protein. In other embodiments, the cell free fragments of the polypeptides include fragments of the extracellular domain that retain biological activity of full-length protein. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and / or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both. In some embodiments the extracellular domain is the only functional domain of the fragment (e.g., the ligand binding domain). ii. Variants

[0226] Variants of VSTM-1, and fragments thereof are also provided. In some embodiments, the variant is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any one of SEQ ID NO:2-5. Useful variants include those that increase biological activity, as indicated by any of the assays described herein, or that increase half-life or stability of the protein. The protein and polypeptides of VSTM-1, and fragments, variants, and fusion proteins thereof can be engineered to increase biological activity. For example, in some embodiments, a VSTM-1 polypeptide, protein, or fragment, variant or fusion thereof has been modified with at least one amino acid substitution, deletion, or insertion that increases a function thereof.

[0227] Finally, variant polypeptides can be engineered to have an increased half-life relative to wild type. These variants typically are modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. The variants can be modified to adjust for effects of affinity for the receptor on the half-life of proteins, polypeptides, fragments, or fusions thereof at serum and endosomal pH. iii. Fusion Proteins

[0228] Fusion polypeptides have a first fusion partner including all or a part of a human or mouse VSTM-1 polypeptide fused to a second polypeptide directly or via a linker peptide sequence that is fused to the second polypeptide. In one embodiment, the ECD of human or mouse VSTM-1 or a fragment thereof is fused to a second polypeptide. The fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker domain can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide or second polypeptide) of the fusion protein. Similarly, the domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide, second polypeptide or peptide / polypeptide linker domain) of the fusion protein. In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0229] Fusion proteins disclosed herein are of formula I:N-R1-R2-R3-C wherein “N” represents the N-terminus of the fusion protein, “C” represents the C- terminus of the fusion protein. In some embodiments, “Ri” is a polypeptide or protein of VSTM-1 or fragment or variant thereof, “R2” is an optional peptide / polypeptide linker domain, and “R3” is a second polypeptide. Alternatively, R3 may be a polypeptide or protein of VSTM-1, or fragment or variant thereof and Ri may be a second polypeptide. In some embodiments, the VSTM-1 polypeptide is the extracellular domain.

[0230] Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric / homomultimeric or heterodimeric / heteromultimeric.

[0231] In some embodiments, the fusion protein includes the extracellular domain of VSTM-1, or a fragment or variant thereof, fused to an Ig Fc region. Recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain or a fragment or variant thereof to the Fc region of human IgGl, IgG2, IgG3 or IgG4 or mouse IgG2a, or other suitable Ig domains, as described previously (Chapoval, et al., Methods Mol. Med. , 45:247-255 (2000)).

[0232] One embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to VSTM-1 Fc (IgGl Fc; wild type) with the following sequence:MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQ NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLOLVVTDKHDELEAPSMKTDTRTLVPRGSDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOY NSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOVYT LPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPG* (SEQ ID NO:6).

[0233] The underlined sequence is the signal sequence. The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl wild type).

[0234] One embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to VSTM-1 Fc (IgGl Fc; wild type) with the following sequence: YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGYK QEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKHDE LEAPSMKTDTRTLVPRGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLH ODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSL TCLVI<GFYPSDIAVEWESNGOPENNYI<TTPPVLDSDGSFFLYSI<LTVDI<SRWOO GNVFSCSVMHEALHNHYTOKSLSLSPG* (SEQ ID NO:7).

[0235] The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl wild type).

[0236] One embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to VSTM-1 Fc (IgGl Fc; FES) having the following sequence:MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCOAHSO NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLOLVVTDKHDELEAPSMKTDTRTLVPRGSDKTHTCPPCPAPEFEGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOY NSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPASIEKTISKAKGOPREPOVYT LPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPG (SEQ ID NO:8).

[0237] The underlined sequence is the signal sequence. The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl FES).

[0238] Another embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to VSTM-1 Fc (IgGl Fc; FES) having the following sequence: YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGYK QEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKHDE LEAPSMKTDTRTLVPRGSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEOYNSTYRVVSVLTVLH ODWLNGKEYKCKVSNKALPASIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSL TCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOO GNVFSCSVMHEALHNHYTOKSLSLSPG (SEQ ID NO: 9).

[0239] The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl FES).

[0240] Another embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to VSTM-1 Fc (IgG4 Fc; G4P) having the following sequence: MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCOAHSO NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLOLVVTDKHDELEAPSMKTDTRTESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSOEDPEVOFNWYVDGVEVHNAKTKPREEOFNSTY RVVSVLTVLHODWLNGKEYKCKVSNKGLPSSTEKTISKAKGOPREPOVYTLPPSO EEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSLSLG* (SEQ ID NO: 10).

[0241] The underlined sequence is the signal sequence. The unmarked text is the extracellular domain. The double underlined sequence is the hG4P Fc (G4P sequence.

[0242] Another embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to VSTM-1 Fc (IgG4 Fc; G4P) having the following sequence: YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGYK QEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKHDE LEAPSMKTDTRTESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSOEDPEVOFNWYVDGVEVHNAKTKPREEOFNSTYRVVSVLTVLHODWL NGKEYKCKVSNKGLPSSIEKTISKAKGOPREPOVYTLPPSOEEMTKNOVSLTCLV KGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWOEGNVFS CSVMHEALHNHYTOKSLSLSLG (SEQ ID NO: 11).

[0243] The unmarked text is the extracellular domain. The double underlined sequence is the hG4P Fc (G4P sequence).

[0244] One embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to SIRLlv2 Fc (IgGl Fc; wild type) with the following sequence: MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCOAHSO NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLQLVVTDKHDELEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERV SI ST A DPOG VT Y A F.I .STS ALSEA A SDTTQEPPGSFTEY A Al K V I . VP RGSDKTHTC P PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTI SKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEAI.HNHYTOKSLSLSP * (SEQ ID NO: 12).

[0245] The underlined sequence is the signal sequence. The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl wild type).

[0246] One embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to SIRLlv2 Fc (IgGl Fc; wild type) with the following sequence: YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGYK QEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKHDE LEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERVSLSTADPQGVTYAEL STSALSEAASDTTQEPPGSHEYAALKVLVPRGSDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO: 13).

[0247] The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl wild type).

[0248] One embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to SIRLlv2 Fc (IgGl Fc; FES) having the following sequence: MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCOAHSO NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLQLVVTDKHDELEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERV SLSTADPOGVTYAELSTSALSEAASDTTOEPPGSHEYAALKVLVPRGSDKTHTCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPASIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEAEHNHYTOKSLSLSP i (SEQ ID NO: 14).

[0249] The underlined sequence is the signal sequence. The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl FES).

[0250] Another embodiment provides a fusion protein having 85%, 90%, 95%, or100% sequence identity to SIRLlv2 Fc (IgGl Fc; FES) having the following sequence: YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGYK QEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKHDE LEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERVSLSTADPQGVTYAELSTSALSEAASDTTOEPPGSHEYAALKVLVPRGSDKTHTCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOY NSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPASIEKTISKAKGOPREPOVYT LPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPG (SEQ ID NO: 15).

[0251] The unmarked sequence is the extracellular sequence. The bolded sequence is a linker. The double underlined sequence is Fc Domain (IgGl FES).

[0252] Another embodiment provides a fusion protein having 85%, 90%, 95%, or100% sequence identity to SIRLlv2 Fc (IgG4 Fc; G4P) having the following sequence: MTAEFLSLLCLGLCLGYEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCOAHSO NVTFVLRKVNDSGYKQEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSE SSEHLQLVVTDKHDELEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERVSLSTADPOGVTYAELSTSALSEAASDTTOEPPGSHEYAALKVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSOEDPEVOFNWYVDGVEVHN AKTKPREEOFNSTYRVVSVLTVLHODWLNGKEYKCKVSNKGLPSSIEKTISKAK GOPREPOVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSLSLG* (SEQ ID NO: 16).

[0253] The underlined sequence is the signal sequence. The unmarked text is the extracellular domain. The double underlined sequence is the hG4P Fc (G4P sequence.

[0254] Another embodiment provides a fusion protein having 85%, 90%, 95%, or 100% sequence identity to SIRLlv2 Fc (IgG4 Fc; G4P) having the following sequence:YEDEKKNEKPPKPSLHAWPSSVVEAESNVTLKCQAHSQNVTFVLRKVNDSGYK QEQSSAENEAEFPFTDLKPKDAGRYFCAYKTTASHEWSESSEHLQLVVTDKHDE LEAPSMKTGSSSEESTKRTSHSKLPEQEAAEADLSNMERVSLSTADPQGVTYAEL STSALSEAASDTTOEPPGSHEYAALKVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSOEDPEVOFNWYVDGVEVHNAKTKPREEOFNSTY RVVSVLTVLHODWLNGI<EYI<CT<VSNI<GLPSSIEI<TISI<AI<GOPREPOVYTLPPSO EEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSLSLG (SEQ ID NO: 17).

[0255] The unmarked text is the extracellular domain. The double underlined sequence is the hG4P Fc (G4P sequence). iv. Polypeptide Modifications

[0256] The polypeptides and fusion proteins may be modified by chemical moieties that may be present in polypeptides in a normal cellular environment, for example, phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, sumoylation and ubiquitylation. Fusion proteins may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

[0257] The polypeptides and fusion proteins may also be modified by chemical moieties that are not normally added to polypeptides in a cellular environment. For example, the disclosed fusion proteins may also be modified by covalent attachment of polymer chains, including, but not limited to, polyethylene glycol polymer (PEG) chains (i.e., pegylation). Conjugation of macromolecules to PEG has emerged recently as an effective strategy to alter the pharmacokinetic (PK) profiles of a variety of drugs, and thereby to improve their therapeutic potential. PEG conjugation increases retention of drugs in the circulation by protecting against enzymatic digestion, slowing filtration by the kidneys and reducing the generation of neutralizing antibodies. In addition, PEG conjugates can be used to allow multimerization of the fusion proteins.

[0258] Modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is cyclization of the protein.

[0259] Examples of chemical derivatives of the polypeptides include lysinyl and amino terminal residues derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge ofthe lysinyl residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O -methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may be selectively modified by reaction with carbodiimides (R — N=C=N— R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia. Fusion proteins may also include one or more D-amino acids that are substituted for one or more L-amino acids. v. Modified Binding Properties

[0260] Binding properties of the proteins, polypeptides, fragments, variants and fusions thereof are relevant to the dose and dose regimen to be administered. In one embodiment the disclosed proteins, polypeptides, fragments, variants and fusions thereof have binding properties to VSTM-1 or an VSTM-1 ligand that demonstrate a higher term, or higher percentage, of occupancy of a binding site (e.g., on the ligand) relative to other receptor molecules that bind thereto. In other embodiments, the disclosed proteins, polypeptides, fragments, variants and fusions thereof have reduced binding affinity to VSTM-1 relative to wild type protein.

[0261] In some embodiments the proteins, polypeptides, fragments, variants and fusions thereof have a relatively high affinity for VSTM-1 and may therefore have a relatively slow off rate. In other embodiments, the proteins polypeptides, fragments, variants and fusions thereof are administered intermittently over a period of days, weeks or months to dampen immune responses which are allowed to recover prior to the next administration, which may serve to alter the immune response without completely turning the immune response on or off and may avoid long term side effects.4. Isolated Nucleic Acid Molecules

[0262] Isolated nucleic acid sequences encoding the VSTM-1 proteins, polypeptides, fragments, variants and fusions thereof are disclosed herein. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally occurring nucleic acid sequence, since such non-naturally occurringsequences are not found in nature and do not have immediately contiguous sequences in a naturally occurring genome.

[0263] An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

[0264] Nucleic acids encoding the proteins, polypeptides, fragments, variants and fusions thereof may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the mammal from which the nucleic acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.

[0265] Nucleic acids can be in sense or antisense orientation or can be complementary to a reference sequence encoding a polypeptide or protein of VSTM-1. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5- methyl-2’ -deoxy cytidine or 5 -bromo-2’ -deoxy cytidine for deoxy cytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See,for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187- 195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

[0266] Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N. R., ed. , BIOS Scientific Publishers, Oxford, 2008).5. Vectors and Host Cells

[0267] Vectors encoding the proteins, polypeptides, fragments, variants and fusions thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0268] Nucleic acids in vectors can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe thecoding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0269] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0270] An expression vector can include a tag sequence. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A. In one embodiment, a nucleic acid molecule encoding one of the disclosed polypeptides is present in a vector containing nucleic acids that encode one or more domains of an Ig heavy chain constant region, for example, having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl chain.

[0271] Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE- dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (c.g, a prokaryotic cell or a eukaryotic cell such as a CHO cell) can be used to, for example, produce the proteins, polypeptides, fragments, variants and fusions thereof described herein.

[0272] The vectors described can be used to express the proteins, polypeptides, fragments, variants and fusions thereof in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primarycells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one embodiment, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.

[0273] In vivo nucleic acid therapy can be accomplished by direct transfer of a functionally active DNA into mammalian somatic tissue or organ in vivo. For example, nucleic acids encoding polypeptides disclosed herein can be administered directly to lymphoid tissues. Alternatively, lymphoid tissue specific targeting can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs) such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue specific TREs are known in the art.

[0274] Nucleic acids may also be administered in vivo by viral means. Nucleic acid molecules encoding fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art. Other virus vectors may also be used, including recombinant adenoviruses and vaccinia virus, which can be rendered non-replicating. In addition to naked DNA or RNA, or viral vectors, engineered bacteria may be used as vectors.

[0275] Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles and polycations such as asialoglycoprotein / polylysine.

[0276] In addition to virus- and carrier-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA and particle-bombardment mediated gene transfer.6. Small Molecules

[0277] The immunomodulatory agent can be a small molecule. Small molecules agonists and antagonists VSTM-1 are known in the art or can be identified using routine screening methods.

[0278] In some embodiments, screening assays can include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on particular classes of compounds suspected of modulating the level of VSTM-1. Assays can include determinations of VSTM-1 mediated signaling activity. Other assays can include determinations of nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.D. Pharmaceutical Compositions

[0279] Pharmaceutical compositions including the disclosed immunomodulatory agents are provided. Pharmaceutical compositions containing the immunomodulatory agent can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0280] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0281] For the disclosed immunomodulatory agents, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. For the disclosed immunomodulatory agents, generally dosage levels of 0. 001 to 20 mg / kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.

[0282] In certain embodiments, the immunomodulatory agent is administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the immunomodulatory agent composition which is greater than that which can be achieved by systemic administration. The immunomodulatory agent compositions can be combined with a matrix as described above to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.1. Formulations for Parenteral Administration

[0283] In some embodiments, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.2. Formulations for Oral Administration

[0284] In some embodiments the compositions are formulated for oral delivery. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Suchcompositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co. , Easton, Pa. 18042) pages 1435-1712, which are incorporated herein by reference. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the compositions. Liposomal encapsulation may be used, and the liposomes may be derivatized with various polymers (e.g., U. S. Patent No. 5,013,556). See also Marshall, K. In: Modem Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or chemically modified forms thereof) and inert ingredients which protect peptide in the stomach environment, and release of the biologically active material in the intestine.

[0285] The agents can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety permits uptake into the blood stream from the stomach or intestine, or uptake directly into the intestinal mucosa. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical usage. Other moieties that may be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly- 1,3- dioxolane and poly-1, 3, 6-tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley- Interscience: New York, N. Y.) pp. 367-383; and Newmark, et al. (1982) J. AppL Biochem. 4: 185-189],

[0286] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

[0287] Controlled release oral formulations may be desirable. The agent can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release is based on the Oros therapeutic system (Alza Corp. ), i.e., the drug is enclosed in a semipermeable membrane whichallows water to enter and push drug out through a single small opening due to osmotic effects.

[0288] For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. In some embodiments, the release will avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5. 0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.3. Formulations for Topical Administration

[0289] The disclosed immunomodulatory agents can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.

[0290] Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

[0291] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc. , St. Louis, Mo. ); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo. ); the Ventolin metered dose inhaler (Glaxo Inc. , Research Triangle Park, N. C. ); and the Spinhaler powder inhaler (Fisons Corp. , Bedford, Mass. ). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

[0292] Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.

[0293] Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations may require the inclusion of penetration enhancers.4. Controlled Delivery Polymeric Matrices

[0294] The immunomodulatory agents disclosed herein can also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

[0295] Either non-biodegradable or biodegradable matrices can be used for delivery of fusion polypeptides or nucleic acids encoding the fusion polypeptides, although in some embodiments biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases, linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water) and can optionally be crosslinked with multivalent ions or polymers.

[0296] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5: 13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0297] The devices can be formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than thedosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.III. Methods of ManufactureA. Methods of Making Antibodies

[0298] The disclosed antibodies can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, an antibody is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL and one VH region in the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, most recent edition).

[0299] VSTM-1 deficient (“knockout) mice or wild type mice can be utilized for the generation of high affinity mAbs against VSTM-1 using proprietary immunization techniques.

[0300] The disclosed antibodies can be modified by recombinant means to increase greater efficacy of the antibody in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U. S. Pat. No. 5,624,821, U. S. Pat. No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). The modification in amino acids includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesired activities, e.g., complement-dependent cytotoxicity. Frequently, the antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, or fusion proteins of VSTM-1. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0301] For example, suitable antibodies with the desired biologic activities can be identified using in vitro assays including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as the inhibition of tumor growth. The antibodies provided herein can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.

[0302] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0303] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0304] Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic peptide. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created byfusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0305] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0306] A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0307] Monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0308] Antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

[0309] Methods of making antibodies using protein chemistry are also known in the art. One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc. , Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or antigen binding fragment thereof via similar peptide condensation reactions.

[0310] For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-alpha- thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.B. Methods for Producing Proteins

[0311] The disclosed proteins, polypeptides, fragments, variants and fusions thereof can be manufactured using conventional techniques that are known in the art. Isolatedfusion proteins can be obtained by, for example, chemical synthesis or by recombinant production in a host cell. To recombinantly produce a protein, polypeptide, fragment, variant or fusion thereof, a nucleic acid containing a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked.

[0312] Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

[0313] In eukaryotic host cells, a number of viral-based expression systems can be utilized to express fusion proteins. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.

[0314] Mammalian cell lines that stably express proteins, polypeptides, fragments, variants or fusions thereof, can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3. 1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of proteins, polypeptides, fragments, variants or fusions thereof, in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Additional suitable expression systems include the GS Gene Expression System™ available through Lonza Group Ltd.

[0315] Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by metabolic selection, or antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells. Alternatively, a protein, polypeptide, fragment, variant or fusion thereof, can be produced by (a) ligating amplified sequences into a mammalian expression vector suchas pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.

[0316] Proteins, polypeptides, fragments, variants or fusions thereof, can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, proteins, polypeptides, fragments, variants or fusions thereof can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, an Fc-fusion polypeptide in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify polypeptides. Fusion proteins can additionally be engineered to contain a secretory signal (if there is not a secretory signal already present) that causes the Proteins, polypeptides, fragments, variants or fusions thereof to be secreted by the cells in which it is produced. The secreted Proteins, polypeptides, fragments, variants or fusions thereof can then conveniently be isolated from the cell media.C. Methods for Producing Isolated Nucleic Acid Molecules

[0317] Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptasecan be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli el al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878; and Weiss (1991) Science 254: 1292-1293.

[0318] Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3’ to 5’ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also be obtained by mutagenesis. Protein-encoding nucleic acids can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992.IV. Assays and Antibody Screening

[0319] One embodiment provides assays for antibody screening. Assays for antibody screening include:1. Analysis of binding affinity of VSTM-1 -Fc to ligands in comparison to VSTM-1.2. Functional assays to confirm VSTM-1 -Fc prevents signaling by VSTM-1 expressing cells. Reporter cells may be utilized for these assays, or primary VSTM-1 + cells are another option.A. Phase screening1. Phase I screening: screen for mAb binding to cell lines transfected to express cell surface VSTM-1. Additionally, mAbs should have the capacity to bind endogenously expressed VSTM-1 on the surface of primary human cell subsets, or endogenously expressed. These mAbs should be highly specific for VSTM-1.2. Phase II screening: Functional assays to confirm that VSTM-1 mAbs or combination of mAbs modulate VSTM-1 mediated signaling. These assays will utilize cell lines that express endogenous VSTM-1, or primary cells such as humanmonocytes, macrophages and dendritic cell subsets or any other leukocyte populations that express VSTM-1 to assess function in the presence of VSTM-1 mAbs. Additionally, reporter cells lines may be used to determine if signaling pathways such as NF-kB (NF- kB reporter) or NF AT (NF AT reporter) are altered following culture with VSTM-1 mAbs.3. Phase III screening: Functional assays to determine if VSTM-1 mAbs are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of VSTM- 1 expressing cell lines. In particular, VSTM-1 mAbs will be tested for the ability to deplete through one of these methods leukemia cell lines, known to express VSTM-1 on the cell surface. VSTM-1 mAbs may also be engineered to deplete VSTM-1 expressing cells and tested as described later in this document through known methods.Phase II and III assays can be used to predict the concentrations of VSTM- 1 mAb(s) required to block physiological levels of ligands in vivo.V. Method of Use

[0320] Antagonists or agonists of VSTM-1 mediated signaling can be used to modulate immune responses in subjects in need of such treatment.

[0321] In one embodiment, the VSTM-1 binding moi eties induce, promote, or enhance ligand binding to VSTM-1 and induce, promote, or enhance proliferation or activation of VSTM-1+ immunosuppressive cells or cause depletion of these cells.

[0322] In one embodiment, the VSTM-1 binding moi eties inhibit, reduce or block ligand binding to VSTM-1 and inhibit, reduce, or block VSTM-1 + immunosuppressive cells or cause depletion of these cells.

[0323] Exemplary methods are discussed in more detail below.A. Immune Response Stimulation1. Therapeutic Strategies

[0324] Methods of inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering a subject an effective amount of a VSTM-1 immunomodulatory agent or binding moiety, or cells primed ex vivo with the VSTM-1 immunomodulatory agent or binding moiety. The immune response can be, for example, inhibition of suppressive immune signals from for example, Treg and MDSC at a tumor sites.

[0325] Alternatively, the immunomodulatory agent can stimulate signal transduction through VSTM-1 and promote or enhance an immune response.

[0326] In some embodiments, the VSTM-1 immunomodulatory agents or binding moieties can be used to block suppressive immune cells to tumor microenvironments. In another embodiment, the VSTM-1 immunomodulatory agent or binding moieties can be used to inhibit, reduce, or block tumor metastasis. In some embodiments, the agent can reduce or inhibit the activity of Tregs, reduce the production of cytokines such as IL- 10 from Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the ratio of Tregs within an immune cell population, or reduce the survival of Tregs. The immunomodulatory agent or binding moiety can be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and / or T cell anergy. Overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.

[0327] The methods can be used in vivo or ex vivo to inhibit, reduce, or block suppressive immune responses and thereby have a stimulating therapeutic effect.

[0328] In some embodiments, the agent, or nucleic acid encoding the agent, is administered directly to the subject. In some embodiments, the agent or nucleic acid encoding the agent, is contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to the subject (e.g., adoptive transfer). The agents can enable a more robust immune response to be possible. The disclosed compositions are useful to stimulate or enhance immune responses involving T cells by inhibiting, reducing or blocking suppressive immune signal transduction through VSTM-1.

[0329] The immunomodulatory agents utilized for increasing an immune response are typically those that reduce VSTM-1 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. For example, the agent can be an antagonist of VSTM-1, such as an antagonist (blocking) anti- VSTM-1 antibody or antigen binding fragment thereof. The agent can also be a VSTM-1 polypeptide, for example, a soluble polypeptide, or fusion protein thereof that can serve as a decoy receptor for one or more VSTM-1 ligands or receptors.

[0330] VSTM-1 blockade, for example using function blocking anti- VSTM-1 antibodies, can be an alternative agent or complementary agent to soluble VSTM-1 polypeptides and fusion proteins. For example, in some embodiments, VSTM-1 blockade is combined with a decoy receptor such as soluble VSTM-1 or fusion protein thereof. The combined treatment (e.g., VSTM-1 -Fc and VSTM-1 blockade) may be complementary.

[0331] In some embodiments, immune response stimulating therapy (e.g., in the treatment of cancer or infections) includes depletion of VSTM-1 + cells.

[0332] Development and identification of VSTM-1 depleting mAbs can be carried out according to known construction and screening methods including those discussed herein. See, for example, Reff, et al, Blood. Vol83, No 2, 1994: pp 435-445, which describes preparation of an anti-CD20 chimeric antibody that binds to human Clq, and mediates complement-dependent cell lysis (CDCC) in the presence of human complement, and anti-body-dependent cellular cytotoxicity (ADCC) with human effector cells. Rituximab destroys B cells and is therefore used to treat diseases which are characterized by overactive, dysfunctional, or excessive numbers of B cells. Other B celldepleting antibodies include ocrelizumab and ofatumumab. In another example, CD3 Abs can preferentially target and deplete activated effector T cells while preserving CD4+Foxp3+Tregs. The antibodies transiently deplete T cells although they display no or little complement-dependent and antibody-dependent cellular cytotoxicity. Redirected cell lysis due to the ability to crosslink CD3 molecules expressed by two different cells (cytotoxic CD8+ T cells on one side and other target T cells on the other side) has been shown, however, T cell depletion mostly results from AICD (reviewed in You, Front Immunol. 2015; 6: 242).2. Subjects to be Treated a. Treatment of Cancer

[0333] The disclosed compositions and methods can be used to treat cancer. Generally, the agents are used to stimulate or enhance an immune response to cancer in the subject by administering to the subject an amount of an immunomodulatory agent, for example an immunomodulatory agent that inhibits, reduces, or blocks VSTM-1 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. The immunomodulatory agent can bind VSTM-1 and promote or enhance an immune response by stimulating signal transduction through VSTM-1. The method can reduce or more symptoms of the cancer.

[0334] In one embodiment the VSTM-1 immunomodulatory agents or binding moi eties inhibit, reduce, or block VSTM-1 and ligand binding and thereby inhibit, reduce, or block Treg and MDSC suppressive functions at a tumor site.

[0335] In another embodiment, the VSTM-1 immunomodulatory agents or binding moieties inhibit, reduce, or block VSTM-1 and deplete the suppressive immune cells, for example in a tumor microenvironment.

[0336] In another embodiment, the VSTM-1 immunomodulatory agents or binding moi eties inhibit, reduce, or block VSTM-1 and ligand binding and thereby inhibit, reduce, or block trafficking of suppressive immune cells to a tumor microenvironment and thereby inhibit, reduce, or block tumor metastasis.

[0337] Cancer cells acquire a characteristic set of functional capabilities during their development through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, limitless replicative potential, and sustained angiogenesis. The term “cancer cell” is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1 / 4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD 19, CD20, HER2 / neu, etc. ).

[0338] The methods and compositions disclosed herein are useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma.

[0339] Cancers caused by aberrations in apoptosis can also be treated by the disclosed methods and compositions. Such cancers may include, but are not be limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatouspolyposis, and myelodysplastic syndromes. In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented by the methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented by the methods and compositions.

[0340] The disclosed compositions and methods are particularly useful for the treatment of cancers that are associated with cells that express abnormally high levels of VSTM-1 or VSTM-1 specific binding partner, including a ligand or counter-receptor.

[0341] Specific cancers and related disorders that can be treated or prevented by methods and compositions disclosed herein include, but are not limited to, leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large-cell lymphoma (ALCL), acute myeloid lymphoma (AML)); multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including but not limited to, nonsmall cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma,squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer); Wilms' tumor; bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed. , J. B. Lippincott Co. , Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U. S. A. , Inc. , United States of America). b. Treatment of Infections

[0342] The disclosed compositions and methods can be used to treat infections and infectious diseases. Generally, the agents are used to stimulate or enhance an immune response to infection in the subject by administering to the subject an amount of an immunomodulatory agent that modulates VSTM-1 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. In one embodiment, the immunomodulatory agent inhibits, reduces, or blocks a suppressive immune signal transduction through VSTM-1. In another embodiment, the immunomodulatory agent induces, promotes, or enhances an immune response by inducing, promoting, or enhancing signal transduction through VSTM-1. The method can reduce one or more symptoms of the infection.

[0343] The infection or disease can be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, i.e., by cytotoxic T lymphocytes.

[0344] The infection or disease can be acute or chronic. An acute infection is typically an infection of short duration. During an acute microbial infection, immune cells begin expressing immunomodulatory receptors. Accordingly, in some embodiments, the method includes increasing an immune stimulatory response against an acute infection.

[0345] The infection can be caused by, for example, but not limited to Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, o Mycobacterium.

[0346] In some embodiments, the disclosed compositions are used to treat chronic infections, for example infections in which T cell exhaustion or T cell anergy has occurred causing the infection to remain with the host over a prolonged period of time.

[0347] Exemplary infections to be treated are chronic infections cause by a hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus, or a human papilloma virus.

[0348] Because viral infections are cleared primarily by T cells, an increase in T- cell activity would be therapeutically useful in situations where more rapid or thorough clearance of an infective viral agent would be beneficial to an animal or human subject. Thus, the disclosed compositions can be administered for the treatment of local or systemic viral infections, including, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and common cold (e.g., human rhinovirus) and other viral infections, caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus. The molecules can be administered topically to treat viral skin diseases such as herpes lesions or shingles, or genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.

[0349] Representative infections that can be treated, include but are not limited to infections cause by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei,Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

[0350] Other microorganisms that can be treated using the disclosed compositions and methods include, bacteria, such as those of Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (albicans, krusei, glabrata, tropicalis, etc. ), Cryptococcus, Aspergillus (fumigatus, niger, etc. ), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp. , Giardia lambia, Cryptosporidium sp. , Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, etc. ), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma, etc.B. Immune Response Inhibiting1. Therapeutic Strategies

[0351] Methods of reducing or inhibiting an immune response in a subject are provided. Typically, the methods include administering a subject an effective amount of a VSTM-1 immunomodulatory agent or combinations thereof, or cells primed ex vivo with these immunomodulatory agents. The immune response can be, for example, promoting or enhancing a suppressive immune response. In one embodiment, the disclosed compositions promote, enhance or activate Tregs, increase the production of cytokines such as IL-10 from Tregs, increase the differentiation of Tregs, increase the number of Tregs, increase the ratio of Tregs within an immune cell population, or increase the survival of Tregs. to provide an immune suppressive response.

[0352] In another embodiment, the immunomodulatory agent promotes a suppressive immune response by inducing, promoting, or enhancing signal transduction through VSTM-1.

[0353] The methods can be used in vivo or ex vivo as immune response-inhibiting therapeutic applications. Thus, in some embodiments, the agent, or nucleic acid encoding the agent, is administered directly to the subject. In some embodiments, the agent or nucleic acid encoding the agent, is contacted with cells (e.g., immune cells) ex vivo, and the treat cells are administered to the subject (e. g. adoptive transfer). In general, thedisclosed immunomodulatory agents can be used for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an overactive or inappropriate immune response. The agents can enable a less robust immune response to be possible. The disclosed compositions are useful to reduce or inhibit immune responses involving T cells.

[0354] The immunomodulatory agents utilized for reducing an immune response are typically those that increase VSTM-1 expression, ligand binding, crosslinking, VSTM-1 mediated signaling, or a combination thereof. For example, the agent can be an agonist of VSTM-1, such as an agonist (stimulating) anti- VSTM-1 antibody or antigen binding fragment thereof. a. Inflammatory Responses

[0355] The disclosed compositions and methods can be used to treat inflammation. Generally, the agents are used to reduce or inhibit an immune response in the subject by administering to the subject an amount of an immunomodulatory agent that modulates VSTM-1 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. The method can reduce or more symptoms of the inflammation. In inflammation can be acute, chronic, or persistent inflammation.

[0356] In some embodiments, the immunomodulatory agents slow down the immune system. For example, agent can be used to control hyper-inflammatory response causing damage healthy tissues. Accordingly, in some embodiments, the agents are administered to a subject undergoing a hyper-inflammatory response. In such cases, controlling excessive immune responses can be beneficial to the subject. b. Inflammatory and Autoimmune Diseases / disorders

[0357] Agents that modulate VSTM-1 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof can also be used to treat inflammatory or autoimmune diseases and disorders. Representative inflammatory or autoimmune diseases / disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet’s disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn’s disease, Dego’s disease,dermatomyositis, dermatomyositis - juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, grave’s disease, guillain-barre, hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud’s phenomenon, Reiter’s syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren’s syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener’s granulomatosis.

[0358] In some embodiments the inflammation or autoimmune disease is caused by a pathogen or is the result of an infection. c. Inflammatory Airway Disorders

[0359] There is a significant unmet need for novel therapeutics to treat patients with progressive inflammatory airway disorders such as chronic obstructive pulmonary disease (COPD) where sustained granulocytic inflammation promotes a gradual decline in lung function even during corticosteroid or p2-agonist intervention. VSTM-1 is a cell-surface inhibitory receptor highly expressed on granulocytes and pulmonary monocytes. VSTM-1 inhibitory signaling is induced when it binds to amphipathic alpha-helical damage- associated molecular pattern (DAMP) motifs on ligands such as cathelici din and the SI 00 proteins. VSTM-1 thus functions as a regulator of myeloid cell-driven inflammatory cascades. This immunosuppressive function, combined with the strong expression profile of VSTM-1 on pulmonary myeloid cells — particularly neutrophils — coupled with the prominent role of neutrophils as inflammatory mediators of lung immunopathology, makes VSTM-1 a promising novel therapeutic target for COPD.

[0360] Provided herein is an agonist monoclonal antibody (mAb) against VSTM-1 to evaluate the therapeutic potential of VSTM-1 engagement and downstream immunosuppressive signaling under hyperinfl ammatory conditions. Augmentation of VSTM-1 signal transduction by an agonist mAb suppressed NETosis and the production of reactive oxygen species (ROS) in inflammatory granulocytes. Moreover, agonism of VSTM-1 by a therapeutic mAb regulated TNFa, IL-6, and IL-ip cytokine production in myeloid cells in response to danger stimuli. A limitation of testing the potential of VSTM-1 as a therapeutic target is that the receptor is not expressed in mice. We thereforeused CRISPR / Cas technology to construct VSTM-1 knock-in C57BL / 6 mice where human VSTM-1 is expressed under the neutrophil-specific mouse Ly6G promoter. In this novel system, engagement of VSTM-1 by an agonist mAh during LPS lung challenge reduced pulmonary neutrophilia and overall disease score. These preclinical data support targeting of VSTM-1 as a novel therapeutic intervention for chronic inflammatory diseases of the lung.VI. Combination Therapies

[0361] The disclosed immunomodulatory agents can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the immunomodulatory agent and the additional therapeutic agent are administered separately, but simultaneously. The immunomodulatory agent and the additional therapeutic agent can also be administered as part of the same composition. In other embodiments, the immunomodulatory agent and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regime.

[0362] The subject can be administered a first therapeutic agent 1, 2, 3, 4, 5, 6, or more hours, or 1, 2, 3, 4, 5, 6, 7, or more days before administration of a second therapeutic agent. In some embodiments, the subject can be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days prior to a first administration of second agent. The immunomodulatory agent can be the first or the second therapeutic agent.

[0363] The immunomodulatory agent and the additional therapeutic agent can be administered as part of a therapeutic regimen. For example, if a first therapeutic agent can be administered to a subject every fourth day, the second therapeutic agent can be administered on the first, second, third, or fourth day, or combinations thereof. The first therapeutic agent or second therapeutic agent may be repeatedly administered throughout the entire treatment regimen.

[0364] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for treatment of HIV or Hepatitis B or C), anti-parasites (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatories, ligands that bind to Toll-Like Receptors (including but not limited to CpGoligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, and other molecules that deactivate or down-regulate suppressor or regulatory T-cells.

[0365] The additional therapeutic agents are selected based on the condition, disorder or disease to be treated. For example, the immunomodulatory agent can be coadministered with one or more additional agents that function to enhance or promote an immune response or reduce or inhibit an immune response.A. Increasing Immune Responses1. Antimicrobials

[0366] For example, VSTM-1 immunomodulatory agents can be used in a preventive or prophylactic role in the treatment and prevention of disease as discussed above, and also in the context of severe trauma injuries like major burn, open bone fracture, accidental amputation or other wounds. Therefore, the VSTM-1 immunomodulatory agents can be administered to the subject in combination with an antimicrobial such as an antibiotic, an antifungal, an antiviral, an antiparasitic, or essential oil.

[0367] In some embodiments, the subject is administered the VSTM-1 immunomodulatory agent and / or the antimicrobial at time of admission to the hospital to prevent further bacterial, fungal or viral complications. The antibiotic can target pathogens and the VSTM-1 immunomodulatory agent can stimulate the immune system to provide an enhanced response to treat or prevent further infection or disease.2. Chemotherapeutic Agents

[0368] The VSTM-1 immunomodulatory agents can be combined with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include, but are not limited to amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or acombination thereof. Representative pro-apoptotic agents include, but are not limited to fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and combinations thereof.3. Other Immunomodulators a. PD-1 antagonists

[0369] In some embodiments, VSTM-1 immunomodulatory agents are coadministered with a PD-1 antagonist. Programmed Death-1 (PD-1) is a member of the CD28 family of receptors that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and / or the strength and / or duration of a T cell response. Suitable PD-1 antagonists are described in U. S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated by reference herein in their entities, and include compounds or agents that either bind to and block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[0370] In some embodiments, the PD-1 receptor antagonist binds directly to the PD- 1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to PD-1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

[0371] It is believed that PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. U. S. A, 105: 10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0372] In some embodiments, the PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.

[0373] Other PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or ligands of PD-1, and other antibodies.

[0374] Suitable anti -PD-1 antibodies include, but are not limited to, those described in the following US Patent Nos: 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,580,247, 8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776, 9,205,148, 9,358,289, 9,387,247, 9,492,539, 9,492,540, all of which are incorporated by reference in their entireties.

[0375] See also Berger et al., Clin. Cancer Res. , 14:3044-3051 (2008).

[0376] Exemplary anti-B7-Hl (also referred to as anti-PD-Ll) antibodies include, but are not limited to, those described in the following US Pat Nos: 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507 all of which are specifically incorporated by reference herein in their entirety.

[0377] For anti-B7-DC (also referred to as anti-PD-L2) antibodies see US Pat. Nos. : 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147 all of which are specifically incorporated by reference herein in their entirety.

[0378] Other exemplary PD-1 receptor antagonists include, but are not limited to B7-DC polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these. In some embodiments, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0379] The PD-1 antagonist can also be a fragment of a mammalian B7-H1, for example from mouse or primate, such as a human, wherein the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example an Ig fusion protein.

[0380] Other useful polypeptides PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof, which can bind to the PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the protein B7. 1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Molnar et al., PNAS, 105: 10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligand and preventbinding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.

[0381] PD -1 and B7-H1 anti-sense nucleic acids, both DNA and RNA, as well as siRNA molecules can also be PD-1 antagonists. Such anti-sense molecules prevent expression of PD-1 on T cells as well as production of T cell ligands, such as B7-H1, PD- L1 and / or PD-L2. For example, siRNA (for example, of about 21 nucleotides in length, which is specific for the gene encoding PD-1, or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially) complexed with carriers, such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009), are readily taken up by cells that express PD-1 as well as ligands of PD-1 and reduce expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thereby activating T cells. b. CTLA4 antagonists

[0382] Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as additional therapeutic agents. In some embodiments, the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibody contemplated for use in the methods of the invention includes an antibody as described in PCT7US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0383] Dosages for anti-PD-1, anti-B7-Hl, and anti-CTLA4 antibody, are known in the art and can be in the range of, for example, 0. 1 to 100 mg / kg, or with shorter ranges of 1 to 50 mg / kg, or 10 to 20 mg / kg. An appropriate dose for a human subject can be between 5 and 15 mg / kg, with 10 mg / kg of antibody (for example, human anti-PD-1 antibody) being a specific embodiment.

[0384] Specific examples of an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg / kg, and Tremelimumab a human anti-CTLA4 antibody, administered at a dose of, for example, about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135-137 (2010), published online December 2009.

[0385] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T cells.4. Potentiating Agents

[0386] In some embodiments, additional therapeutic agents include a potentiating agent. The potentiating agent acts to increase efficacy the immune response up-regulator, possibly by more than one mechanism, although the precise mechanism of action is not essential to the broad practice of the present invention.

[0387] In some embodiments, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazahosphorine drug and analogs include ifosfamide (IFO, If ex), perfosfamide, trophosphamide (trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates, prodrugs and metabolites thereof (US patent application 20070202077 which is incorporated in its entirety). Ifosfamide (MITOXANA®) is a structural analog of cyclophosphamide and its mechanism of action is considered to be identical or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxy cyclophosphamide) and trophosphamide are also alkylating agents, which are structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorines derivatives have been designed and evaluated with an attempt to improve the selectivity and response with reduced host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a chemically stable 4- thioethane sulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is IFO derivative in which the isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogs are described in US patent 5,190,929 entitled “Cyclophosphamide analogs useful as anti-tumor agents” which is incorporated herein by reference in its entirety.

[0388] While CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that induce DNA crosslinking and, at higher doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes, as lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, and cycling cells are most sensitive to DNA alkylation agents.

[0389] Low doses of CTX (< 200 mg / kg) can have immune stimulatory effects, including stimulation of anti-tumor immune responses in humans and mouse models of cancer (Brode & Cooke Crit Rev. Immunol. 28: 109-126 (2008)). These low doses are sub-therapeutic and do not have a direct anti -turn or activity. In contrast, high doses of CTX inhibit the anti-tumor response. Several mechanisms may explain the role of CTX in potentiation of anti-tumor immune response: (a) depletion of CD4+CD25+FoxP3+ Treg (and specifically proliferating Treg, which may be especially suppressive), (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), resulting in suppression of tumor cell growth; (d) mobilization and expansion of CD1 lb+Gr-l+ MDSC. These primary effects have numerous secondary effects; for example, following Treg depletion macrophages produce more IFN-y and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic proliferation of lymphocytes.

[0390] Treg depletion is most often cited as the mechanism by which CTX potentiates the anti-tumor immune response. This conclusion is based in part by the results of adoptive transfer experiments. In the AB 1-HA tumor model, CTX treatment at Day 9 gives a 75% cure rate. Transfer of purified Treg at Day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58: 1219- 1228 (2009). A similar result was observed in the HHD2 tumor model: adoptive transfer of CD4+CD25+ Treg after CTX pretreatment eliminated therapeutic response to vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

[0391] Numerous human clinical trials have demonstrated that low dose CTX is a safe, well-tolerated, and effective agent for promoting anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47: 1-12 (1998)).

[0392] The optimal dose for CTX to potentiate an anti-tumor immune response, is one that lowers overall T cell counts by lowering Treg levels below the normal range but is subtherapeutic (see Machiels et al., Cancer Res. 61 :3689-3697 (2001)).

[0393] In human clinical trials where CTX has been used as an immunopotentiating agent, a dose of 300 mg / m2has usually been used. For an average male (6 ft, 170 pound (78 kg) with a body surface area of 1.98 m2), 300 mg / m2is 8 mg / kg, or 624 mg of total protein. In mouse models of cancer, efficacy has been seen at doses ranging from 15 - 150 mg / kg, which relates to 0.45 - 4.5 mg of total protein in a 30g mouse (Machiels et al. Cancer Res. 61 :3689-3697 (2001), Hengst et al Cancer Res. 41 :2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

[0394] For larger mammals, such as a primate, such as a human, patient, such mg / m2doses may be used but unit doses administered over a finite time interval may also be used. Such unit doses may be administered on a daily basis for a finite time period, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days or up to 20 days or up to 25 days, are all specifically contemplated by the invention. The same regimen may be applied for the other potentiating agents recited herein.

[0395] In other embodiments, the potentiating agent is an agent that reduces activity and / or number of regulatory T lymphocytes (T-regs), such as Sunitinib (SUTENT®), anti- TGFP or Imatinib (GLEEVAC®). The recited treatment regimen may also include administering an adjuvant.

[0396] Useful potentiating agents also include mitosis inhibitors, such as paclitaxol, aromatase inhibitors (e. g. Letrozole) and angiogenesis inhibitors (VEGF inhibitors e. g. Avastin, VEGF-Trap) (see, for example, Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF- secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15; 12(22):6808-16.), anthracy clines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL- 18 antagonists.B. Reducing Immune Responses1. Immunosuppressive Agents

[0397] In some embodiments, the immune response, or inflammatory / autoimmune disease / disorder is treated by administering to the subject a VSTM-1 immunomodulatory agent and a second agent that is an immune suppressant. Immunosuppressive agents include, but are not limited to antibodies against other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or against cytokines), fusion proteins (e.g., CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-a blockers such as Enbrel, Remicade, Cimzia and Humira, cyclophosphamide (CTX) (z.e., Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX) (i.e., Rheumatrex®, Trexall®), belimumab (i.e., Benlysta®), or other immunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds, or steroids), anti-proliferatives, cytotoxic agents, or other compounds that may assist in immunosuppression.

[0398] The therapeutic agent can be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteins compete with the co-stimulatory receptor, CD28, on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen presenting cells, and thus function to inhibit T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acidsubstitutions (L104E and A29Y) that markedly increase its avidity to CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

[0399] In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Cyclophosphamide (the generic name for Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), also known as cytophosphane, is a nitrogen mustard alkylating agent from the oxazophorines group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the primary drug used for diffuse proliferative glomerulonephritis in patients with renal lupus.

[0400] The therapeutic agent can be administered in an effective amount to reduce the blood or serum levels of anti-double stranded DNA (anti-ds DNA) auto antibodies and / or to reduce proteinuria in a patient in need thereof.

[0401] In another embodiment, the therapeutic agent increases the amount of adenosine in the serum, see for example, WO 08 / 147482. For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or another agent (e.g., a cytokine or monoclonal antibody or small molecule) that increases the expression of CD73, see for example WO 04 / 084933. In another embodiment the therapeutic agent is Interferon-beta.

[0402] The therapeutic agent can be a small molecule that inhibits or reduces differentiation, proliferation, activity, and / or cytokine production and / or secretion by Thl, Thl7, Th22, and / or other cells that secrete, or cause other cells to secrete, inflammatory molecules, including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL-18 IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In another embodiment, the therapeutic agent is a small molecule that interacts with Tregs, enhances Treg activity, promotes or enhances IL-10 secretion by Tregs, increases the number of Tregs, increases the suppressive capacity of Tregs, or combinations thereof.

[0403] In some embodiments, the composition increases Treg activity or production. Exemplary Treg enhancing agents include but are not limited to glucocorticoid fluticasone, salmeteroal, antibodies to IL-12, IFN-y, and IL-4; vitamin D3, and dexamethasone, and combinations thereof.

[0404] In some embodiments, the therapeutic agent is an antibody, for example, a functions blocking antibody against a proinflammatory molecule such as IL-6, IL-23, IL- 22 or IL-21.

[0405] As used herein the term “rapamycin compound” includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds which are thought to have the same mechanism of action as rapamycin (c.g,inhibition of cytokine function). The language “rapamycin compounds” includes compounds with structural similarity to rapamycin, e.g., compounds with a similar macrocyclic structure, which have been modified to enhance their therapeutic effectiveness. Exemplary Rapamycin compounds are known in the art (See, e. g. WO95122972, WO 95116691, WO 95104738, U. S. Patent No. 6,015,809; 5,989,591; U. S. Patent No. 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

[0406] The language “FK506-like compounds” includes FK506, and FK506 derivatives and analogs, e.g, compounds with structural similarity to FK506, e.g, compounds with a similar macrocyclic structure which have been modified to enhance their therapeutic effectiveness. Examples of FK506-like compounds include, for example, those described in WO 00101385. In some embodiments, the language “rapamycin compound” as used herein does not include FK506-like compounds.2. Anti-inflammatories

[0407] Other suitable therapeutic agents include, but are not limited to, antiinflammatory agents. The anti-inflammatory agent can be non-steroidal, steroidal, or a combination thereof. One embodiment provides oral compositions containing about 1% (w / w) to about 5% (w / w), typically about 2. 5 % (w / w) or an anti-inflammatory agent. Representative examples of non-steroidal anti-inflammatory agents include, without limitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids; propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these non-steroidal antiinflammatory agents may also be employed.

[0408] Representative examples of steroidal anti-inflammatory drugs include, without limitation, corticosteroids such as hydrocortisone, hydroxyl-triamcinolone, alphamethyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadr enol one, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylesters, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradr enol one, fludrocortisone, diflurosone diacetate, fluradr enol one acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.VII. Kits

[0409] The disclosed VSTM-1 immunomodulatory agents can be packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity. The agent can be supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. For example, the agent can be supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, or at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent can be stored at between 2 and 8°C in their original container and are typically administered within 12 hours, or within 6 hours, or within 5 hours, or within 3 hours, or within 1 hour after being reconstituted.

[0410] In an alternative embodiment, agent can be supplied in liquid form in a hermetically sealed container indicating the quantity and concentration. In some embodiments, the liquid form of the agent supplied in a hermetically sealed container including at least 1 mg / ml, or at least 2. 5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml of the agent.

[0411] Pharmaceutical packs and kits including one or more containers filled with agent are also provided. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceuticalpack or kit. The pharmaceutical pack or kit can also include one or more containers filled with one or more of the ingredients of the disclosed pharmaceutical compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0412] Kits designed for the above-described methods are also provided. Embodiments typically include one or more VSTM-1 immunomodulatory agents. In particular embodiments, a kit also includes one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers. In other embodiments, a kit also includes one or more anti-inflammatory agents useful for the treatment inflammatory and autoimmune diseases, in one or more containers.

[0413] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0414] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.EXAMPLESExample 1: Anti-VSTM-1 NP690 and NP693 agonists of inflammatory conditions

[0415] Figures 1 A-1H show that VSTM-1 is highly expressed on blood neutrophils. Whole blood polymorphonuclear cells (PMNS) were isolated from each of 5 healthy donors and stained for VSTM-1 expression with 1 ug / mL AF647-labeled anti-VSTM-1 mAb (black bars) or isotype (white bars) and analyzed by flow cytometry. gMFI = geometric Mean Fluorescent Intensity. N=3 technical replicates for each of 6 donors; error bars = SEM.

[0416] Figures 2A-2I show that VSTM-1 is expressed on some monocyte populations but not on lymphocytes. Frozen PBMCs from 5 separate healthy donors (D#l 17-120) were thawed and stained for VSTM-1 expression on gated immune cell subpopulations by flow cytometry. Cell populations were gated as shown. Within each gate, VSTM-1 expression (orange bars) was compared to isotype control (white bars) foreach donor. gMFI = geometric Mean Fluorescent Intensity. N=3 technical replicates for each of 5 donors; error bars = SEM.

[0417] Figures 3A-3C show that VSTM-1 expression is retained inflamed lung tissue. _FFPE lung tissue sections from 5 normal donors or 10 COPD patients were stained with 2 ug / mL anti-VSTM-1 mAh 0230-NP 1249- 163 (NextCure) or isotype control mAh 230-NP926-032 (NextCure). Images from patient matched samples were de-convoluted, background signal (isotype-staining) was subtracted from VSTM-1 signal, and mean VSTM-1 signal was quantified with Image J software. P value calculated by student’s t test; error bars = SEM.

[0418] Figures 4A-4F show that VSTM-1 binds to amphipathic, alpha-helical peptides. VSTM-1 transduced 293T cells, VSTM-1 transduced THP-1 cells, empty vector controls cells (EV), or primary blood neutrophils were probed with 5 ug / mL biotin-conjugated or his-tagged recombinant LL-37 or S100A8 and then stained with streptavidin-AF647 or anti-his-AF647 secondary, respectively. Control mAb = nonspecific IgGl. Sec. Only = secondary staining mAb only (no primary). Cell binding of recombinant protein was measured by flow cytometry. gMFI = geometric Mean Fluorescent Intensity. Where indicated, bar graphs of protein binding represent gMFI minus non-specific binding to empty vector (EV) control cells (VSTM-lneg293T cells). N=3 technical replicates; error bars = SEM.

[0419] Figure 5 shows the primary screening of anti-VSTM-1 hybridoma supernatants for binding to VSTM-l-Fc. NZBWF1 / J mice were immunized with VSTM- Ivl-human IgGl protein. Binding of hybridoma culture medium derived from human VSTM-1 -immunized mouse splenocytes to wells coated with recombinant human VSTM- l-Fc was quantified by ELISA via the addition of biotin conjugated rabbit anti -mouse IgG detection antibody. 15 hybridomas were subsequently selected for secondary screening.

[0420] Figures 6A-6B show the secondary screening of anti-VSTM-1 hybridoma supernatants for binding to VSTM-1. (FIG. 6A) Binding of select hybridoma culture media derived from human VSTM-1 -immunized mouse splenocytes to coated recombinant human VSTM-l-Fc as measured by ELISA via the addition of biotin conjugated rabbit anti-mouse IgG detection antibody, or (FIG. 6B) binding of hybridoma supernatant to HEK293T cells transduced to overexpress VSTM-1 as quantified by flow cytometry using a phycoerythrin conjugated rabbit anti-mouse IgG detection antibody. 6 hybridoma supes were subsequently selected for secondary screening.

[0421] Figures 7A-7B show the screening of anti-VSTM-1 hybridoma supernatants to VSTM-1+ cells. HEK293T-Wild-type (GFP-) and HEK293T-VSTM-1 transduced (GFP+) cells were mixed and stained with hybridoma supernatants. Binding of antibodies was detected using AF647 anti-mouse IgG on GFP+ (VSTM-1+) and GFP- (VSTM-1-) fractions.

[0422] Figures 8A-8F show that anti-VSTM-1 mAbs bind to cell surface expressed VSTM-1. HEK293T-OKT3 cells or HMC3-OKT3 cells were transduced with VSTM-1 or empty vector (EV), then incubated with CHO-cell supernatant corresponding to NP690. IgGl (3F4), NP691. IgGl (6E2), NP692. IgGl (1D4), or NP693. IgGl (10H11) secreting cells. mAb binding to 293T or HMC3 cells was detected by flow cytometry using AF647 conjugated anti -human IgG secondary. Percent cells = frequency of live cells. gMFI = geometric Mean Fluorescent Intensity. N = 3 technical replicates; error bars = SEM.

[0423] Figure 9 shows the binding of Anti-VSTM-1 mAbs to VSTM-1 by ELISA. Cell supernatants from CHO cells secreting NP690, NP692, NP693, or NP694 were coated on 96-wells plates and subsequent binding to VSTM-l-His tagged protein was detected by ELISA. Control media = non-specific CHO cell supernatant. N = 4 technical replicates; error bars = SEM.

[0424] Figures 10A-10B show that anti-VSTM-1 clones NP690 and NP693 bind to human VSTM-1. Purified anti-VSTM-1 clones were tested for binding to VSTM-1 by Octet affinity assay and cell binding assay. For cell binding assay, VSTM-1 transduced HEK293T cells were stained with titrated concentrations primary mAb for 45 mins on ice and secondary Goat a-Human IgG-AF647 for 15 mins on ice. Cell surface binding was analyzed by flow cytometry; MFI = Mean Fluorescent Intensity. N=3 technical replicates; error bars=SDM.Figures 11 A-l IB show that NP690. IgGl and NP693. IgGl bind to both cell surface and secreted isoforms of VSTM-1. 96-well plates were coated with VSTM-lvi-Fc (membrane bound isoform) or VSTM-lV2-Fc (secreted isoform), incubated with biotin-conjugated mAb at the indicated concentration, then quantified by ELISA. N=3 technical replicates; error bars = SEM.

[0425] Figures 12A-12B are flow cytometry data showing that neither NP690 nor NP693 bind to Cynomolgus Macaque VSTM-1. Cynomolgus VSTM-1 (cVSTM-1), human VSTM-1 (hVSTM-1), or empty vector (EV) was stably transduced into THP-1 cells and binding of NP690, NP693, or isotype control (iso) was tested by flowcytometry. N = 4 technical replicates; gMFI = geometric Mean Fluorescent Intensity; error bars = SEM.

[0426] Figures 13A-13C show that Anti -VS TM-1 mAh NP693 Suppresses ERK / MAPK. (FIG. 13A) VSTM-1 transduced THP-1 cells were stimulated for 2 hours with 4 pM ionomycin with the indicated concentration of soluble mAb, then lysed and probed for phospho-ERK and total ERK by Western blot. (FIG. 13B-13C) VSTM-1 transduced HL-60 cells were stimulated for 20 min with 4 pM ionomycin, then incubated on 5 ug / mL of plate bound mAb for 20 min. Phospho-ERK was quantified by intracellular staining and flow cytometry. gMFI = geometric Mean Fluorescent Intensity.

[0427] Figures 14A-14D are bar graphs showing that anti-VSTM-1 mAbs suppress Inflammatory cytokine production. Thawed PBMCs were activated with aCD3 / 28 (Immunocult, StemCell) and treated with 5 ug / mL soluble mAb for 24 hours. Cell supernatant was tested for cytokines by Legendplex (Biolegend). N=4 technical replicates; error bars = SEM; * = P<0. 05 by one-way ANOVA.

[0428] Figures 15A-15C are bar graphs showing that anti-VSTM-1 mAbs suppressed inflammatory cytokines production from neutrophils under various stimulants. Isolated blood PMNs from a healthy donor were with 100 ng / mL LPS, 4 pM ionomycin, or 20 ug / mL coated IgA for 17 hours with or without soluble anti-VSTM-1 or isotype mAb for 24 hours. Cell supernatants were tested for cytokines by Legendplex (Biolegend). N=4 technical replicates; error bars = SEM.

[0429] Figures 16A-16C show that anti-VSTM-1 antibodies NP690 and NP693 block IL-17 from TH17 polarized cells. Thawed PBMCS were polarized with IL-ip, IL- 6, IL-23, TGFP, afFNy, aIL-4, and anti-CD3 / 28 with soluble treatment mAb for 7 days. Cell supernatant was tested for IL-17A or IL-17F by ELISA (R&D Systems). Bar graphs N=2-4 technical replicates from a single donor; violin plot=6 biological replicates; error bars = SEM.

[0430] Figures 17A-17B are bar graphs showing that anti-VSTM-1 antibodies suppress myeloid cell help during TH17 polarization. Thawed PBMCS or isolated CD4+T cells plus soluble treatment mAb were polarized with IL-ip, IL-6, IL-23, TGFP, alFNy, aIL-4, and anti-CD3 / 28 or with anti-CD3 / 28 only for 7 days. Cell supernatant was tested for IL-17A by ELISA (R&D Systems). Bar graphs N=4-5 technical replicates from a single donor; error bars = SEM; *=P<0. 05 by one-way ANOVA; ns=not significant.

[0431] Figures 18A-18B are bar graphs showing that NP690 and NP693 agonist mAbs attenuate production of ROS in myeloid cells. VSTM-1 transduced THP-1 cells orprimary blood neutrophils were treated with 4 pM ionomycin plus soluble mAb and analyzed for reactive oxygen species (ROS) by CellROX Deep Red staining and flow cytometry. MFI = Mean Fluorescent Intensity of live cells. N=3-4 technical replicates; error bars = SEM; *=P<0. 05 by one-way ANOVA.

[0432] Figures 19A-19E show that anti-VSTM-1 antibodies NP690 and NP693 agonist mAbs inhibit NETosis of stimulated neutrophils. (FIG. 19A-19D) Primary blood neutrophils were stimulated with 4 pM ionomycin and treated with soluble mAb or GSK484 positive control (small molecule inhibitor of PAD4 enzyme), stained for extracellular DNA, and quantified by flow cytometry. Error bars=SEM; bar graphs N=4 technical replicates; Violin plots N=6 biological replicates; * = P<0. 05 by one way ANOVA (FIG. 19E) Primary blood neutrophils were stimulated with 4 pM A-23187 and treated with soluble mAb or GSK484 positive control and NET- associated elastase was quantified by colorimetric assay (Cayman Chemical Cat #600616). N=2 technical replicate; error bars = SEM.

[0433] Figures 20A-20E shows that NP690 and NP693 reduce pulmonary PMN influx in a humanized acute pulmonary inflammation model. 2e7 RBC-depleted whole blood cells were intravenously injected in NOG-EXL mice, followed by tracheal instillation of 3. 5U / mouse of porcine pancreatic elastase to induce lung inflammation. Mice were subsequently intraperitoneally injected with 10 mg / kg treatment mAb and 5 mg / kg mouse neutrophil depleting antibody 1A8. Lungs were harvested 17 hours later and immunophenotyped by flow cytometry. N=6 biological replicates; Error bars=SEM Example 2: VSTM-1 Agonist mAb Therapy Reduces Granulocytic Inflammation And COPD

[0434] An anti-VSTM-1 agonist mAb generated for therapeutic intervention of granulocytic inflammatory disorders of the lung suppressed ROS, NETosis, and inflammatory cytokines. HumanVSTM-1 expressing syngeneic mice were generated and characterized. Pilot in vivo lung disease models indicate that anti-VSTM-1 agonist mAb treatment can reduce pulmonary pathology.

[0435] Figure 21A is a schematic of granulocyte-mediated pathology during pulmonary inflammatory disease. Figure 21B is a schematic of VSTM-1 regulatory mechanism. VSTM-1 binds soluble amphipathic damage-associated molecular pattern (DAMP) motifs that are released during inflammatory insult. Ligand binding results in recruitment of SHP1 / 2 phosphatase, which dampens ERK-mediated signaling and results in suppression of reactive oxygen species (ROS) generation, NF-kB activity, and (inneutrophils) PAD4-mediated NETosis. Figure 21C is an Illustration of VSTM-1 function and regulation during hyper-inflammatory processes. Figure 21D shows (left) VSTM-1 staining of FFPE lung tissue from COPD patients. Quantitation performed by staining patient matched samples with anti-VSTM-1 or isotype control, deconvoluting images, then subtracting the background (isotype) signal from the VSTM-1 signal. N = 10 COPD patients. (Right) Illustration of anti-VSTM-1 agonist mAb therapeutic strategy. VSTM-1 is a myeloid cell restricted immune inhibitory receptor that is highly expressed on granulocytes.

[0436] Figure 22 A shows binding kinetics and affinity of top anti-VSTM-1 mAb candidate as measured by Octet™ (Sartorius). Figure 22B shows (left) cell surface binding or (right) competition binding between equimolar concentrations of recombinant LL-37 or aVSTM-1 mAb on VSTM-1 -transduced or empty vector HEK293T cells.

[0437] Figures 23A-23B show that TNFa is secreted by (left) HL-60 or (right) THP- 1 cells stably transduced with VSTM-1 and activated with 20 ng / mL LPS + 50 ng / mL IFNy for 24 hrs. Figures 23C-23G show cytokines secreted by aCD3 / 28 activated PBMCs (FIG. 23C-23D) or cytokines secreted by LPS activated blood neutrophils ex vivo (FIG. 23E-23G). All cells treated with 5 ug / mL coated mAb. N = 4-5 technical replicates.

[0438] Figures 24A-24G show reactive oxygen species (ROS) (FIG. 24A-24b) or NETosis (FIG. 24C-24G) in the indicated cell type after Ca++ ionophore stimulation in the presence of 5 ug / mL coated mAb or 10 pM GSK484 (PAD4 enzyme inhibitor). N = 2-4 technical replicates or (for DNA staining) 6 donor samples.

[0439] Figures 25A-25B show RAW264. 7 mouse cells stably transduced with VSTM-1 (left) express VSTM-1 on the cell surface and (right) respond to anti-VSTM-1 agonist mAb ROS suppression when under ionomycin stimulation. Figure 25C shows PCR amplification of human VSTM-1 in tail snips from wild type (WT), heterozygous (Het), or homozygous (Hom) knock-in mice. Figures 25D-25E show cell surface expression of VSTM-1 on circulating neutrophils from WT or VSTM-1 Het mice.

[0440] Figures 26A-26B show that VSTM-1 knock-in mice were challenged with 1 mg / kg LPS via intratracheal aspiration. 8 hours post-challenge mice were treated with 10 mg / kg mAb via intraperitoneal injection. 24 hours post-challenge, mouse lungs were harvested for (FIG. 26A) neutrophil influx and (FIG. 26B) pathology by H&E staining. N = 5 mice per treatment group.

[0441] Figure 27 is a schematic of pulmonary disease modeling in human myeloid cell-replete mice.

[0442] The modality of VSTM-1 agonist mAb will be optimized for maximal activity and therapeutic index. In vivo modeling will be expanded to more chronic models of inflammatory disease. Benchmarking and studies of combo treatments will be conducted to investigate potential synergy with current clinical therapeutics.Example 3: NP960 and 693 Binding studies

[0443] Table 5 shows Octet binding studies for NP690 variant monoclonal antibodies and Table 6 shows Octet binding studies for NP693. For both NP960 and 693, all variants have a slightly lower affinity than the parent mAbs though all are still high (< 3 nM). For NP690, the closest variant to the parent is 1382 which is distinct from the rest in terms of having a slower off-rate as the parent mAb. For NP693, within model fitting errors, all variants are very close in affinity, the closest being NP1378.

[0444] Table 5: Octet Binding of NP960 Variant mAbs.

[0445] Table 6: Octet Binding of NP693 Variant mAbsExample 4: NP960 and 693 Activity Studies

[0446] Figure 28 shows the NP690 and 693 variant antibodies suppressed TNFa. CHO cell supernatant was used to coat 96-well plates overnight (O / N). HL-60-VSTM-1 cells were subsequently plated O / N in IMDM containing 10 ng / mL LPS + 50 ng / mL ZFNy. HL-60-VSTM-1 cell supernatant was assayed for TNFa via ELISA.

[0447] Figures 29A-29B show binding vs activity of NP690 / 693 variants. CHO cell supernatant was used to coat 96-well plates O / N. HL-60-VSTM-1 cells were subsequently plated O / N in IMDM containing 10 ng / mL LPS + 50 ng / mL IFNy. HL-60- VSTM-1 cell supernatant was assayed for TNFa via ELISA, normalized to total supe protein, and plotted against KD or Kdis as determined by Octet.

[0448] Table 7 shows the humanized anti-VSTM-1 variants.

[0449] Table 7: Humanized Anti-VSTM-1 Variants

[0450] Figures 30A-30B show ROS suppression by humanized anti-VSTM-1 variants. 200k HL-60-VSTM-1 cells per well were incubated for min 15 with theindicated concentration of mAb + equal concentrations of anti-IgGl Xlinking Fab in media containing 4 uM ionomycin; subsequently added 5 uM CellROX Deep Red reagent and incubated an additional 30 min; washed 3x and quantified ROS by FACs.

[0451] Figures 31 A- 3 IB show TNFa suppression by humanized anti-VSTM-1 variants (mAbs). 75k HL-60-VSTM-1 cells or 100k PBMCs / well were cultured in 200 uL media containing 12. 5 ng / mL PMA + 500 ng / mL lono or 20 ng / mL LPS + 50 ng / mL IFNg, respectively, plus the indicated concentration of sol mAb with equal concentrations of anti-IgG crosslinking mAb.

[0452] Figures 32A-32B show percent TNFa suppression by humanized anti- VSTM-1 variants (purified mAbs).

[0453] Figures 33A-33B show IL-6 suppression by humanized anti-VSTM-1 variants (purified mAbs). 100k PBMCs / well were cultured for 72 hours in 200 uL media containing 20 ng / mL LPS + 50 ng / mL ZFNy, respectively, plus the indicated concentration of sol mAb with equal concentrations of anti-IgG crosslinking mAb; IL-6 in supe measured by ELISA.

[0454] Figures 34A-34B show a multivariable comparison of humanized variants to suppress TNFa Figure 34A shows PBMCs treated with IFNg and LPS, and Figure 34B shows HL-60 cell line treated with PMA+ionomycin.

[0455] Figures 35A-35B show IFNy suppression by humanized anti-VSTM-1 variants (purified mAbs).

[0456] Figures 36A-36F show Legendplex analysis of supernatants from PBMCs treated with humanized purified anti-VSTM-1 variants. 100k PBMCs / well were cultured for 72 hours in 200 uL media containing 20 ng / mL LPS + 50 ng / mL IFNg plus the indicated concentration of sol mAb with equal concentrations of anti-IgG crosslinking mAb.

[0457] Figures 37A-37E show Legendplex analysis of supernatants from PBMCs treated with humanized purified anti-VSTM-1 variants.

Claims

What is claimed is:

1. A immunomodulatory agent that modulates V-set and transmembrane domaincontaining protein 1 (VSTM-1) expression, ligand binding, crosslinking, VSTM-1 mediated signaling, or a combination thereof comprising:(i) an anti-VSTM-1 antibody or an antigen binding fragment thereof, ,(ii) an anti-VSTM-1 fusion protein or an antigen binding fragment, or(iii) combinations thereof; wherein the immunomodulatory agent immunospecifically binds to VSTM-1 having at least 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, or 5.

2. The VSTM-1 immunomodulatory agent of claim 1, wherein the antibody or antigen binding fragment thereof is an agonist antibody having least 99% sequence identity to SEQ ID NO: 18, 19, 20, or 21.

3. The VSTM-1 immunomodulatory agent of claim 1, wherein the antibody or antigen binding fragment thereof comprises: a. a variable light chain having at least 99% sequence identity to SEQ ID NO:22, 23, 46, 47, and b. a variable heavy chain having an amino acid sequence according to SEQ ID NO:24, 25, 26, 27, 28, 29, 30, 31, 32, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or combinations thereof.

4. The VSTM-1 immunomodulatory agent of claim 1, wherein the antibody or antigen binding fragment thereof comprises: a. a variable light chain having at least 99% sequence identity to SEQ ID NO:33, 34, 66, 67, and b. a variable heavy chain having an amino acid sequence according to SEQ ID NO 35, 36, 37, 38, 39, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or combinations thereof.I l l5. The VSTM-1 immunomodulatory agent of claim 3, wherein the antibody or antigen binding fragment thereof comprises: a) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:24; b) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:25; c) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:26; d) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:24; e) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:25; f) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:26; g) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:27; h) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:28; i) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:29; j) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:30; k) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:29; l) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:30; m) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID N0:31; n) a variable light chain of SEQ ID NO: 22 and a variable heavy chain of SEQ ID NO:32; o) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID N0:31; or p) a variable light chain of SEQ ID NO: 23 and a variable heavy chain of SEQ ID NO:32.

6. The VSTM-1 immunomodulatory agent of claim 4, wherein the antibody or antigen binding fragment thereof comprises: a) a variable light chain of SEQ ID NO: 33 and a variable heavy chain of SEQ ID NO:35; b) a variable light chain of SEQ ID NO: 33 and a variable heavy chain of SEQ ID NO:36; c) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:35; d) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:36; e) a variable light chain of SEQ ID NO: 33 and a variable heavy chain of SEQ ID NO:37; f) a variable light chain of SEQ ID NO: 33 and a variable heavy chain of SEQ ID NO:38; g) a variable light chain of SEQ ID NO: 33 and a variable heavy chain of SEQ ID NO:39; h) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:37; i) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:38; or j) a variable light chain of SEQ ID NO:34 and a variable heavy chain of SEQ ID NO:39.

7. The VSTM-1 immunomodulatory agent of claim 1, wherein the antibody or antigen binding fragment thereof induces, promotes, or enhances the interaction between VSTM-1 and its ligands.

8. The VSTM-1 immunomodulatory agent of claim 1, wherein the antibody or antigen binding fragment thereof inhibits, interferes, or blocks the interaction between VSTM-1 and its ligands.

9. The VSTM-1 immunomodulatory agent of claim 1, wherein the fusion protein or an antigen binding fragment thereof comprises one or more extracellular domains of VSTM-1 or functional variant thereof linked to an immunoglobulin domain.

10. The VSTM-1 immunomodulatory agent of claim 1, wherein the fusion protein or antigen binding fragment thereof comprises at least 99% sequence identity to SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or combinations thereof.

11. The VSTM-1 immunomodulatory agent of claim 9, wherein the fusion protein or antigen binding fragment thereof is a humanized fusion protein comprising an immunoglobulin domain having at least 99% sequence identity to SEQ ID NO: 40, 41, 42, 43, 44, 45.

12. The VSTM-1 immunomodulatory agent of claim 9, wherein the fusion protein or an antigen binding fragment thereof inhibits, reduces, or blocks VSTM-1 mediated signal transduction.

13. A pharmaceutical composition comprising an effective amount of the VSTM-1 immunomodulatory agent of claim 1 to modulate VSTM-1 expression, ligand binding, crosslinking, VSTM-1 mediated signaling, or a combination thereof.

14. The pharmaceutical composition of claim 13, wherein the composition modulates hyperinflammatory conditions, restores homeostasis and prevents VSTM-1 mediated disease in a subject in need thereof.

15. The pharmaceutical composition of claim 9, wherein the composition further comprises one or more additional therapeutic agents selected from cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals, anti-parasitics, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof, antigen and vaccine formulations, peptide drugs, anti-inflammatories, ligands that bind to Toll-Like Receptors to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, other molecules that deactivate or down-regulate suppressor or regulatory T-cells, or combinations thereof.

16. The pharmaceutical composition of claim 9, wherein the composition is administered with other immunomodulatory agents selected from PD-1 antagonists, CTLA4 antagonists, potentiating agents or other VSTM-1 antagonists.

17. A method for promoting an immune suppressive response in a subject in need thereof comprising administering an effective amount of the VSTM-1 immunomodulatory agent according to claim 1 to induce, promote, or enhance VSTM-1 mediated signal transduction18. The method of claim 17, wherein the subject has a hyperinflammatory disease or condition characterized by increased expression of VSTM-1 relative to expression of VSTM-1 in subjects without the disease or condition.

19. The method of claim 17, wherein the subject has a pulmonary inflammatory disease or condition.

20. The method of claim 17, wherein the pharmaceutical composition blocks neutrophil-mediated inflammation and cytokine production associated with pulmonary tissue damage to reduce pulmonary pathology and prevent or treat pulmonary inflammatory disease.