RECEPTOR FOR VISTA

INTRODUCTION

Immunotherapy has been a game-changer in the field of cancer therapy. Developments in immune checkpoint- based therapy are progressing at a breathtaking pace. Nevertheless, only a fraction of patients respond to immunotherapies. A particular challenge in cancer immunotherapy has been the identification of mechanism-based biomarkers that could be used to identify candidates for such treatment and guide disease-management decisions (Topalian et al. , N Engl J Med, 366(26): 2443-54 (2012)). Therefore, patient selection is an important issue as it will avoid treatment-related toxicity and cost in patients who are unlikely to benefit.

In order to ensure that an immune inflammatory response is not constantly activated once tumor antigens have stimulated a response, multiple controls or “checkpoints” are in place or activated. These checkpoints are mostly represented by T-cell receptor biding to ligands on cells in the surrounding tumor microenvironment, forming immunological synapses which then regulate the function of the T cell.

VISTA (V-Domain Ig Suppressor of T Cell Activation) is a negative checkpoint control protein that regulates T cell activation and immune responses. It is a type I transmembrane protein which contains a single Ig-like V-type domain with homology to similar domains of both the B7 and CD28 families and an intracellular domain. VISTA cytoplasmic tail domain contains two potential protein kinase C binding sites as well as proline residues that could function as docking sites, suggesting that VISTA could potentially function as both a receptor and a ligand.

VISTA is homologous to PDL-1 but displays a unique expression pattern that is restricted to the hematopoietic compartment. VISTA is most highly expressed on myeloid and granulocytic cells, expressed at lower levels on T cells but not present on B cells (Wang et al. , JEM 208(3):577-592 (2011 ); Flies et al. , J. Immunology 187(4): 1537-1541 (2011 )). VISTA is induced on T cells and myeloid cell populations upon activation or immunization, suggesting that inflammation induces its expression (Wang et al. , supra).

On the other hand, no VISTA expression was detected in tumor cells (Le Mercier et al. , Cancer Res; 74:1933-44 (2014)), although it was reported that human gastric cancer cells express VISTA at a low frequency (Boger et al. , Oncolmmunology, 6:4, e1293215 (2017)). When present, VISTA expression appears restricted to the infiltrating CD11 b+ cells in the tumor microenvironment of colon or lung cancers. However, it was noted that further studies were required to identify tumor characteristics that may be associated with VISTA expression in the tumor microenvironment (Lines et al. , Cancer Immunol Res; 2(6):510-7 (2014)).

VISTA appears to function both as a negative receptor on T cells and as a ligand expressed on APCs interacting with an unknown receptor on T cells.

Several findings suggest that VISTA negatively regulates T cell responses by acting as a ligand that interacts with an unknown receptor on T cells. Like PD-L1 , VISTA is a ligand that profoundly suppresses immunity (Lines et al. , Cancer Res; 74:1924-32 (2014)), and like PD-L1 , blocking VISTA allows for the development of therapeutic immunity to cancer in pre-clinical oncology models (see Le Mercier et al. , supra). Whereas blocking VISTA enhances immunity, especially CD8+ and CD4+ mediated T cell immunity, treatment with a soluble Ig fusion protein of the extracellular domain of VISTA (VISTA-lg) inhibits T cell proliferation and cytokine production in vitro and overexpression of VISTA on MCA105 tumor cells interferes with the protective antitumor immunity in mice (Wang et al. , supra). Moreover, administration of a VISTA-specific monoclonal antibody enhanced CD4+ T cell response in vivo and the development of autoimmunity in mice (Wang et al. , supra). On the other hand, VISTA appears to have functional activities that are non-redundant with other Ig superfamily members and may play a role in the development of autoimmunity and immune surveillance in cancer. Specifically, although studies using Fc fusion proteins clearly show that VISTA has ligand activity (Wang et al. , supra, Lines et al. , supra), receptor-like signaling activity has also been described (Flies et al. , J Clin Invest; 124:1966-75 (2014)). Indeed, a direct negative role of VISTA as a receptor on T cells is supported by a number of studies.

It is well known that the composition of the immune cell infiltrates varies not only between different tumor entities, but also within tumors of the same anatomic site. Authors have speculated that the response to different immunotherapeutic combinations

will probably rely on the patient’s immune milieu (Farkona et al. , BMC Medicine 14: 73 (2016)). In this regard, PDL-1 expression is known to be induced to evade immune attack (Sharma et al. , Cell, 168: 707-23 (2017)). PDL-1 expression shows intratumoral and intertumoral variations (Mino-Kenudson, Cancer Biol Med, 13(2): 157-70 (2016)), but is associated with an objective response to an anti-PD-1 antibody (Topalian et al. , supra). On the other hand, the VISTA binding partners that mediate the protein’s effects have not been identified yet (Le Mercier et al. , Frontiers in Immunology, 6:418 (2015)). Although two phase-l clinical trials with anti-VISTA molecule have been initiated, there is no biomarker capable of predicting a patient’s response to these treatments. Thus, there is a need for identifying VISTA’s binding partner, as it would facilitate therapeutic development and enable selection of patients susceptible to treatment with anti-VISTA therapeutic agent.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. The practice of the invention employs, unless other otherwise indicated, conventional techniques or protein chemistry, molecular virology, microbiology, recombinant DNA technology, and pharmacology, which are within the skill of the art. Such techniques are explained fully in the literature (see e.g. , Ausubel et al. , Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985; and Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001 ). The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

FIGURE LEGENDS

FIG. 1 shows a flow chart of the CAPTIREC™ screening procedure using TRICEPS reagents. The Ligand of Interest was a VISTA-Fc fusion protein. The Control ligand was an anti-CD28 antibody.

FIG. 2 shows a Protter illustration of PSGL-1 . N-glycosylation sites are represented by residues surrounded by squares and the experimentally observed peptides are represented by filled in circles.

FIG. 3 shows the results of exemplary binding assays for VISTA-Fc to the extracellular domain of PSGL-1 construct A.

FIG. 4 shows the results of exemplary binding assays for VISTA-Fc to the extracellular domain of PSGL-1 construct B.

FIG. 5 shows an exemplary histogram of the detection of PSGL-1 in HL-60 cells by flow cytometry. The isotype and background are represented by the gray shaded peak and the PSGL-1 expressing cells are represented by the white shaded peak.

FIG 6 shows an exemplary Western Blot detecting the interaction between VISTA and PSGL-1 . PSGL-1 is indicated by arrows; incomplete reduction of PSGL-1 is known to result in more than one band.

FIG. 7 shows a bar chart of an anti-VISTA antibody attenuating the interaction between VISTA and PSGL-1 . Each bar represents the band intensities corresponding to the PSGL-1 protein. symbol represents no anti-VISTA antibody added. “+” symbol represents pre incubation with anti-VISTA antibody.

FIG. 8 shows an exemplary histogram of the detection of PSGL-1 in PBMCs by flow cytometry. The isotype and background are represented by the gray shaded peak and the PSGL-1 expressing cells are represented by the white shaded peak.

FIG. 9 shows an exemplary Western Blot showing the co-immunoprecipitation of PSGL-1 using anti-VISTA and anti-PSGL-1 antibodies. PSGL-1 is indicated by an arrow.

FIG. 10 shows PSGL-1 expression in exemplary flow cytometry assays of naive 6t resting, effector and exhausted effector T cell subsets.

FIG. 1 1 shows PSGL-1 expression in exemplary flow cytometry assays of circulating central memory and circulating effector memory T cell subsets.

FIG. 12 shows an example of multiplex staining of mRNA for PSGL1 , VISTA and PDUon a squamous lung tumor

FIG. 13 shows a bar chart of PSGL-1 inhibiting VISTA -dependent IL-2 release from CD4+ T cells.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

The term“about” or“approximately” refers to the normal range of error for a given value or range known to the person of skills in the art. It usually means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range.

As used herein,“administer” or“administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. , an anti-PSGL-1 antibody and/or anti-VISTA antibody provided herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

As used herein, an“antagonist” or“inhibitor” refers to a molecule that is capable of inhibiting or otherwise decreasing one or more of the biological activities of a target protein, such as PSGL-1 , VISTA or a different co-inhibitory molecule described herein. In some embodiments, an antagonist of PSGL-1 (e.g. , an antagonistic antibody provided herein) can, for example, act by inhibiting or otherwise decreasing the activation and/or cell signaling pathways of the cell expressing PSGL-1 ( e.g . , a T cell) and/or the cell expressing VISTA (e.g. , a VISTA-bearing tumor cell, a regulatory T cell, a myeloid-derived suppressor cell or a suppressive dendritic cell), thereby inhibiting a biological activity of the cell relative to the biological activity in the absence of the antagonist. In some embodiments the antibodies provided herein are antagonistic anti-PSGL-1 antibodies. In some embodiments, an antagonist of a co-inhibitory molecule (e.g. , an antagonistic antibody against VISTA, CD86, CD80, PDL-1 , PDL-2, CTLA-4, PD1 , LAG 3, BTNL2, B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA, HVEM, LAIR1 , TIM1 , Galectin 9, TIM3, CD48, 2B4, CD155, CD112, CD113 or TIGIT) can, for example, act by inhibiting or otherwise decreasing the activation and/or cell signaling pathways of the cell expressing the co-inhibitory molecule (e.g. , a T cell or an antigen-presenting cell), thereby inhibiting a biological activity of the cell relative to the biological activity in the absence of the antagonist. In some embodiment, the antagonist molecule is an antagonistic antibody, i.e. an antibody that inhibits or reduces one or more of the biological activities of an antigen, such as PSGL-1 , VISTA or a different co-inhibitory molecule described herein. Certain antagonistic antibodies substantially or completely inhibit one or more of the biological activities of said antigen.

As used herein, an“agonist” or“activator” refers to a molecule that is capable of activating or otherwise increasing one or more of the biological activities of a target protein, such as a co-stimulatory molecule. In some embodiments, an agonist of a co stimulatory molecule (e.g. , an agonistic antibody of CD154, TNFRSF25, GITR, 4-1 BB, 0X40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41 BBL, OX40L, CD70, HHLA2, ICOSL, a cytokine, LIGHT, HVEM, CD30, CD30L, B7-H2, CD80, CD86, CD40L, TIM4, TIM1 , SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2, and CD226) may, for example, act by activating or otherwise increasing the activation and/or cell signaling pathways of the cell expressing the co-stimulatory molecule (e.g. , a T cell or an antigen-presenting cell), thereby increasing a biological activity of the cell relative to the biological activity in the absence of the agonist. In some embodiment, the agonist molecule is an agonistic antibody, i.e. an antibody that activates or increases one or more of the biological activities of an antigen, such as PSGL-1 , VISTA or a different co-inhibitory molecule described herein. Certain agonistic antibodies substantially or completely activate one or more of the biological activities of said antigen.

The terms “antibody” and “immunoglobulin” or “Ig” are used interchangeably herein. These terms are used herein in the broadest sense and specifically cover monoclonal antibodies (including full length monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies, chimeric antibodies, and antibody fragments, provided that said fragments retain the desired biological function. An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or an antigen-encoding nucleic acid. These terms are intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed., Oxford University Press.; Kuby (1997) Immunology, Third Ed., W.H. Freeman and Company, New York). In some embodiments, the specific molecular antigen can be bound by an antibody provided herein includes the target PSGL-1 polypeptide, fragment or epitope.

Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti -idiotypic (anti-ld) antibodies, and functional fragments of any of the above, which refers a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g. , including monospecific, bispecific, etc. ), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g. , antigen binding domains or molecules that contain an antigen-binding site that binds to a VISTA antigen (e.g. , one or more complementarity determining regions (CDRs) of an anti-VISTA antibody). Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al. , Cell Biophysics, 22:189-224 (1993); Pliickthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein can be of any type (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g. , lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2), or any subclass (e.g. , lgG2a and lgG2b) of immunoglobulin molecule. Anti-PSGL-1 antibodies or anti-VISTA antibodies provided herein can be agonistic antibodies or antagonistic antibodies.

The terms“anti-PSGL-1 antibodies,”“antibodies that bind to PSGL-1 “antibodies that bind to a PSGL-1 epitope,” and analogous terms are used interchangeably herein and refer to antibodies that bind to a PSGL-1 polypeptide, such as a PSGL-1 antigen or epitope. Such antibodies include humanized antibodies. An antibody that binds to a PSGL-1 antigen may be cross-reactive with related antigens. In some embodiments, an antibody that binds to PSGL-1 does not cross-react with other antigens. In some embodiments, an anti-PSGL-1 antibody described herein does not block or inhibit the binding of PSGL-1 to P-selectin, L-selectin or E-selectin. An antibody that binds to PSGL-1 can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art. An antibody binds to PSGL-1 , for example, when it binds to PSGL-1 with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs), for example, an antibody that specifically binds to PSGL-1 . Typically, a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g. , Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. In some embodiments, an antibody“which binds” an antigen of interest is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a“non-target” protein will be less than about 10% of the binding of the antibody to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a target molecule, the term“specific binding” or“specifically binds to” or is“specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or“specifically binds to” or is“specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 104 M, alternatively at least about 10 5 M, alternatively at least about 106 M, alternatively at least about 107 M, alternatively at least about 108 M, alternatively at least about 109 M, alternatively at least about 10 1° M, alternatively at least about 10 11 M, alternatively at least about 10 12 M, or greater. In some embodiments, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, an antibody that binds to PSGL-1 or VISTA has a dissociation constant (KD) of < 1 mM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In some embodiments, anti-PSGL-1 antibody or anti-VISTA antibody binds to an epitope of PSGL-1 or VISTA that is conserved among PSGL-1 or VISTA from different species.

An“antigen” is a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide, including, for example, a PSGL-1 polypeptide.

The term“antigen binding fragment,”“antigen binding domain,”“antigen binding region,” and similar terms refer to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g. , the complementarity determining regions (CDRs)).

The term“anti gen -presenting cell” or“APC” refers to a heterogeneous group of immune cells that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes, such as T cells. APCs include, but are not limited to, dendritic cells, macrophages, Langerhans cells and B cells.

The terms“binds” or“binding” as used herein refer to an interaction between molecules to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as PSGL-1 , is the affinity of the antibody or functional fragment for that epitope. The ratio of association (ki) to dissociation (/c ?) of an antibody to a monovalent antigen ( ki / k i) is the association constant K, which is a measure of affinity. The value of K varies for different complexes of antibody and antigen and depends on both ki and k The association constant K for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent PSGL-1 , come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

The term“biological sample” refers to a sample that has been obtained from a biological source, such as a patient or subject. In some embodiments, a biological sample includes, but is not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, and the like. Preferably, a biological sample is a tumor sample. In some

preferred embodiments, a biological sample is obtained by a tissue biopsy ( e. tumor biopsy, which can include immune infiltrates) .

The term“block,” or a grammatical equivalent thereof, when used in the context of an antibody refers to an antibody that prevents or stops a biological activity of the antigen to which the antibody binds. A blocking antibody includes an antibody that combines with an antigen without eliciting a reaction, but that blocks another protein from later combining or complexing with that antigen. The blocking effect of an antibody can be one which results in a measurable change in the antigen’s biological activity. In some embodiments, an anti-PSGL-1 antibody described herein blocks the ability of VISTA to bind PSGL-1 , which can result in inhibiting or blocking suppressive signals of VISTA. Certain anti-PSGL-1 antibodies described herein inhibit or block suppressive signals of VISTA on VISTA-expressing cells, including by about 98% to about 100% as compared to the appropriate control (e.g. , the control being cells not treated with the antibody being tested). In some embodiments, the anti-PSGL-1 antibody described herein blocks the binding of PSGL-1 to the extracellular domain VISTA and/or blocks the binding of a VISTA-expressing cell to a PSGL-1 -expressing cell. In some embodiments, the anti-PSGL-1 antibody described herein does not block the binding of PSGL-1 to a protein other than VISTA, such as P-selectin, L-selectin, and/or E-selectin.

The term “VISTA” or “VISTA polypeptide” and similar terms refers to the polypeptide (“polypeptide,” “peptide” and “protein” are used interchangeably herein) encoded by the human Chromosome 10 Open Reading Frame 54 (VISTA) gene, which is also known in the art as B7-H5, platelet receptor Gi24, GI24, Stress Induced Secreted Proteinl , SISP1 , and PP2135, for example, comprising the amino acid sequence of:

1 mgvptaleag swrwgsllfa lflaaslgpv aafkvatpys lyvcpeggnv tltcrllgpv

61 dkghdvtfyk twyrssrgev gtcserrpir nltfqdlhlh hgghqaants hdlaqrhgle

121 sasdhhgnfs itmrnltlld sglycclwe irhhhsehrv hgamelqvqt gkdapsncw

181 ypsssqdsen itaaalatga civgilclpl illlvykqrq aasnrraqel vrmdsniqgi

241 enpgfeaspp aqgipeakvr hplsyvaqrq psesgrhlls epstplsppg pgdvffpsld

301 pvpdspnfev i (SEQ ID NO: 1)

and related polypeptides, including SNP variants thereof. The VISTA polypeptide has been shown to or is predicted to comprise several distinct regions within the amino acid sequence including: a signal sequence (residues 1 -32; see Zhang et al. , Protein Sci. 13:2819-2824 (2004)); an immunoglobulin domain - IgV-like (residues 33-162); and a transmembrane region (residues 195-215). The mature VISTA protein includes amino acid residues 33-311 of SEQ ID NO: 1 . The extracellular domain of the VISTA protein includes amino acid residues 33-194 of SEQ ID NO: 1. Related polypeptides include allelic variants (e.g. , SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, preferably, which retain VISTA activity and/or are sufficient to generate an anti-VISTA immune response. VISTA can exist in a native or denatured form. The VISTA polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A "native sequence VISTA polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding VISTA polypeptide derived from nature. Such native sequence VISTA polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence VISTA polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific VISTA polypeptide (e.g. , an extracellular domain sequence), naturally-occurring variant forms (e.g. , alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.

A cDNA nucleic acid sequence encoding the VISTA polypeptide, for example, comprises:

1 atgggcgtcc ccacggccct ggaggccggc agctggcgct ggggatccct gctcttcgct

61 ctcttcctgg ctgcgtccct aggtccggtg gcagccttca aggtcgccac gccgtattcc

121 ctgtatgtct gtcccgaggg gcagaacgtc accctcacct gcaggctctt gggccctgtg

181 gacaaagggc acgatgtgac cttctacaag acgtggtacc gcagctcgag gggcgaggtg

241 cagacctgct cagagcgccg gcccatccgc aacctcacgt tccaggacct tcacctgcac

301 catggaggcc accaggctgc caacaccagc cacgacctgg ctcagcgcca cgggctggag

361 tcggcctccg accaccatgg caacttctcc atcaccatgc gcaacctgac cctgctggat

421 agcggcctct actgctgcct ggtggtggag atcaggcacc accactcgga gcacagggtc

481 catggtgcca tggagctgca ggtgcagaca ggcaaagatg caccatccaa ctgtgtggtg

541 tacccatcct cctcccagga tagtgaaaac atcacggctg cagccctggc tacgggtgcc

601 tgcatcgtag gaatcctctg cctccccctc atcctgctcc tggtctacaa gcaaaggcag

661 gcagcctcca accgccgtgc ccaggagctg gtgcggatgg acagcaacat tcaagggatt

721 gaaaaccccg gctttgaagc ctcaccacct gcccagggga tacccgaggc caaagtcagg

781 caccccctgt cctatgtggc ccagcggcag ccttctgagt ctgggcggca tctgctttcg

841 gagcccagca cccccctgtc tcctccaggc cccggagacg tcttcttccc atccctggac

901 cctgtccctg actctccaaa ctttgaggtc atctag (SEQ ID NO: 2)

As described herein, VISTA is an immunomodulator, that is a negative checkpoint regulator of immune responses (e.g. , inhibits or suppresses immune responses). As also described herein, PSGL-1 is a receptor of VISTA. As also described herein, methods for modulating (e.g. , preventing, inhibiting, blocking) the interaction of PSGL-1 and VISTA with agents (e.g. , antibodies) that bind to PSGL-1 and/or VISTA, are useful, including, for example, for inhibiting or blocking suppressive signals of VISTA. Modulating the interaction of VISTA and PSGL-1 can result in an increased immune response, including an increase in immune activation (e.g. , T cell activation such as T cell proliferation). Antibodies that bind to VISTA, useful in methods as described herein, include those disclosed in W02014/197849 (PCT/US2014/041388).

Orthologs to the VISTA polypeptide are also well known in the art. For example, the mouse ortholog to the VISTA polypeptide is V-region Immunoglobulin-containing Suppressor of T cell Activation (VISTA) (also known as PD-L3, PD-1 H, PD-XL, Pro1412 and UNQ730), which shares approximately 70% sequence identity to the human polypeptide. Orthologs of VISTA can also be found in additional organisms including chimpanzee, cow, rat and zebrafish.

A“VISTA-expressing cell,” “a cell having expression of VISTA” or a grammatical equivalent thereof refers to a cell that expresses endogenous or transfected VISTA on the cell surface. VISTA expressing cells include VISTA-bearing tumor cells, regulatory T cells ( e.g . , CD4+ Foxp3+ regulatory T cells), myeloid-derived suppressor cells ( e.g . , CD11 b+ or CD11 bh,gh myeloid-derived suppressor cells) and/or suppressive dendritic cells (e.g. , CD11 b+ or CD11 bh,gh dendritic cells). A cell expressing VISTA produces sufficient levels of VISTA on its surface, such that an anti -VISTA antibody can bind thereto and/or PSGL-1 or a cell expressing PSGL-1 can bind thereto. In some aspects, inhibition or blocking of such binding may have a therapeutic effect. A cell that "overexpresses" VISTA is one that has significantly higher levels of VISTA at the cell surface thereof, compared to a cell of the same tissue type that is known to express VISTA. Such overexpression may be caused by gene amplification or by increased transcription or translation. VISTA overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the VISTA protein present on the surface of a cell (e.g. via an immunohistochemistry assay; FACS analysis). Alternatively, or additionally, one may measure levels of VISTA-encoding nucleic acid or mRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; see W098/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable agent, and binding of the antibody to cells in the patient can be evaluated, e.g. by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. A VISTA-expressing tumor cell includes, but is not limited to, acute myeloid leukemia (AML) tumor cells.

A “VISTA-mediated disease,” “VISTA-mediated disorder” and “VISTA-mediated condition” are used interchangeably and refer to any disease, disorder or condition that is completely or partially caused by or is the result of VISTA. Such diseases, disorders or conditions include those caused by or otherwise associated with VISTA, including by or associated with VISTA-expressing cells (e.g. , tumor cells, myeloid-derived suppressor cells (MDSC), suppressive dendritic cells (suppressive DC), and/or regulatory T cells (T-regs)). In some embodiments, VISTA is aberrantly (e.g. , highly) expressed on the surface of a cell. In some embodiments, VISTA may be aberrantly upregulated on a particular cell type. In other embodiments, normal, aberrant or excessive cell signaling is caused by binding of VISTA to a VISTA receptor (e.g. , PSGL-1 ), which can bind or otherwise interact with VISTA.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a tumor or cancer. "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer ( e.g . epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain cancer, as well as head and neck cancer, and associated metastases. In some embodiments, the cancer is a hematological cancer, which refers to cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of a hematologic cancer are leukemia (e.g. , acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), or acute monocytic leukemia (AMoL)), lymphoma (Hodgkin lymphoma or non-Hodgkin lymphoma), and myeloma (multiple myeloma, plasmacytoma, localized myeloma or extramedullary myeloma).

A“co-inhibitory molecule” (also known as a“negative checkpoint regulator” or “NCR”) refers to a molecule that down -regulates immune responses (e.g. , T cell activation) by delivery of a negative signal to T cells following their engagement by ligands or counter-receptors. Exemplary functions of a co-inhibitory molecule is to prevent out-of-proportion immune activation, minimize collateral damage, and/or maintain peripheral

self- tolerance. In some embodiments, a co-inhibitory molecule is a ligand or receptor expressed by an antigen presenting cell. In some embodiments, a co-inhibitory molecule is a ligand or receptor expressed by a T cell. In some embodiments, a co-inhibitory molecule is a ligand or receptor expressed by both an antigen presenting cell and a T cell.

A “co-stimulatory molecule” refers to a molecule that up-regulates immune responses (e.g. , T cell activation) by delivery of a positive signal to T cells following their engagement by ligands or counter-receptors. For a T cell to become fully activated, two signals are required: 1 ) an antigen-specific signal is provided through the T cell receptor interacting with peptide-MHC molecules on an antigen presenting cell; and 2) a co stimulatory signal, which is antigen nonspecific, and is provided by the interaction between co-stimulatory molecules expressed on the membrane of an antigen presenting cell and the T cell. T cell co-stimulation provides for T cell proliferation, differentiation and survival. In some embodiments, a co-stimulatory molecule is a ligand or receptor expressed by an antigen presenting cell. In some embodiments, a co-stimulatory molecule is a ligand or receptor expressed by a T cell. In some embodiments, a co-stimulatory molecule is a ligand or receptor expressed by both an antigen presenting cell and a T cell.

A "chemotherapeutic agent" is a chemical or biological agent (e.g. , an agent, including a small molecule drug or biologic, such as an antibody or cell) useful in the treatment of cancer, regardless of mechanism of action. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, AR1 NOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide;

cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1 -TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammal I and calicheamicin omega II (see, e.g., Agnew, Chem Inti. Ed. Engl. 33:183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzi nostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholino-doxorubicin, cyanomorpholi no doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti -metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;

triaziquone; 2,2',2 '-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C "); thiotepa; taxoids, e.g. , TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin. Additional chemotherapeutic agents include cytotoxic agents useful as antibody drug conjugates, such as maytansinoids (DM1 and DM4, for example) and auristatins (MMAE and MMAF, for example).

Also included in the definition of chemotherapeutic agent are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti -estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, Rl VISor® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as ME inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g. , ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAX1 D®; PROLEUKIN® rlL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti -angiogenic agents such as bevacizumab (AVASTIN®, Genentech) (x) immunmodulatory agents such as Bispecific T Cell Engager (BITE) antibodies and chimeric antigen receptor (CAR) T cells; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

A“CDR” refers to one of three hypervariable regions (H1 , H2 or H3) within the non framework region of the immunoglobulin (Ig or antibody) VH B-sheet framework, or one of three hypervariable regions (L1 , L2 or L3) within the non-framework region of the antibody VL b-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al. , J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1 -75 (1978)). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved b-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901 -917 (1987)). Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al. , J. Mol. Biol. 273:927-948 (1997); Morea et al. , Methods 20:267-279 (2000)). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al. , supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

The term "hypervariable region", "HVR", or "HV", when used herein refers to the regions of an antibody variable domain that are hypervariable in sequence and/or form

structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H 1 , H2, H3), and three in the VL (LI, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat CDRs are based on sequence variability and are the most commonly used (Kabat eta/. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )). Chothia refers instead to the location of the structural loops (Chothia and Lesk J Mol. Bioi. 196:901 -917 (1987)). The end of the Chothia CDR-HI loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below.

Recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lafranc et al. , Dev. Comp. Immunol. 27(1 ):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the "location" of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues and are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. Correspondence between the Kabat numbering and the IMGT unique numbering system is also well known to one skilled in the art ( e.g . Lefranc et al. , supra). An Exemplary system, shown herein, combines Kabat and Chothia.

Table 1 : CDR Definitions

Hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26-35A (H1 ), 50-65 or 49-65 (H2) and 93-102, 94-1 02, or 95-102 (H3) in the VH. The variable domain residues are 25 numbered according to Kabat et al. , supra, for each of these definitions. As used herein, the terms "HVR" and "CDR" are used interchangeably.

The term“constant region” or“constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CH1 , CH2 and CH3 domains of the heavy chain and the CL domain of the light chain.

In the context of a polypeptide, the term“derivative” as used herein refers to a polypeptide that comprises an amino acid sequence of a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or an antibody that binds to a PSGL-1 polypeptide which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term“derivative” as used herein also refers to a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or an antibody that binds to a PSGL-1 polypeptide which has been chemically modified, e.g. , by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or a PSGL-1 antibody may be chemically modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. A derivative of a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or a PSGL-1 antibody may be chemically modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or a PSGL-1 antibody may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or a PSGL-1 antibody described herein.

The term“detectable probe,” as used herein, refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

The term“diagnostic agent” refers to a substance administered to a subject that aids in the diagnosis of a disease. Such substances can be used to reveal, pinpoint, and/or define the localization of a disease-causing process. In some embodiments, a diagnostic agent includes a substance that is conjugated to an antibody provided herein, that when administered to a subject or contacted to a sample from a subject, aids in the diagnosis of cancer, tumor formation, or any other VISTA-mediated disease, disorder or condition.

The term“detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an antibody provided herein, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g. , by quantitation).

The term “detecting” as used herein encompasses quantitative or qualitative detection.

The term“encode” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

The term“epitope” as used herein refers to the region of an antigen, such as PSGL-1 polypeptide or PSGL-1 polypeptide fragment, to which an antibody binds. Preferably, an epitope as used herein is a localized region on the surface of an antigen, such as PSGL-1 polypeptide or PSGL-1 polypeptide fragment, that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g. , a human), that is capable of eliciting an immune response. An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope can be formed by contiguous residues or by non-contiguous residues brought into close proximity by the folding of an antigenic protein. Epitopes formed by contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by non-contiguous amino acids are typically lost under said exposure. In some embodiments, a PSGL-1 epitope is a three-dimensional surface feature of a PSGL-1

polypeptide. In other embodiments, a PSGL-1 epitope is linear feature of a PSGL-1 polypeptide. Generally, an antigen has several or many different epitopes and reacts with many different antibodies.

The term“excipient” as used herein refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but not limited to, proteins (e.g. , serum albumin, etc. ), amino acids (e.g. , aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc. ), fatty acids and phospholipids (e.g. , alkyl sulfonates, caprylate, etc. ), surfactants (e.g. , SDS, polysorbate, nonionic surfactant, etc. ), saccharides (e.g. , sucrose, maltose, trehalose, etc. ) and polyols (e.g. , mannitol, sorbitol, etc. ). See, also, Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, which is hereby incorporated by reference in its entirety.

In the context of a peptide or polypeptide, the term“fragment” as used herein refers to a peptide or polypeptide that comprises less than the full length amino acid sequence. Such a fragment may arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue(s) from the amino acid sequence. Fragments may, for example, result from alternative RNA splicing or from in vivo protease activity. In some embodiments, PSGL-1 or VISTA fragments include polypeptides comprising 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 contiguous 100 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 of the amino acid sequence of a PSGL-1 or VISTA polypeptide or an antibody that binds to a PSGL-1 or VISTA polypeptide. In some embodiments, a fragment of a PSGL-1 or VISTA polypeptide or an antibody that binds to a PSGL-1 or VISTA antigen retains at least 1 , at least 2, or at least 3 functions of the polypeptide or antibody.

The term“framework” or“FR” residues refers to those variable domain residues other than the hypervariable region residues herein defined. FR residues are those variable domain residues flanking the CDRs. FR residues are present, e.g. , in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies.

A“functional fragment” of an antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least specific binding to the target antigen.

The term“fusion protein” as used herein refers to a polypeptide that comprises an amino acid sequence of an antibody and an amino acid sequence of a heterologous polypeptide or protein (e.g. , a polypeptide or protein not normally a part of the antibody (e.g. , a non-anti- PSGL-1 antibody or a non-anti -VISTA antibody)). The term“fusion” when used in relation to PSGL-1 , VISTA, an anti-PSGL-1 antibody, or an anti-VISTA antibody refers to the joining of a peptide or polypeptide, or fragment, variant and/or derivative thereof, with a heterologous peptide or polypeptide. In some embodiments, the fusion protein retains the biological activity of the PSGL-1 , the VISTA, the anti-PSGL-1 antibody, or the anti-VISTA antibody. In some embodiments, the fusion protein comprises an anti-PSGL-1 antibody or an anti-VISTA antibody VH domain, VL domain, VH CDR (one, two or three VH CDRs), and/or VL CDR (one, two or three VL CDRs), wherein the fusion protein binds to a PSGL-1 or VISTA epitope.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (a), delta (5), epsilon ( ), gamma (y) and mu (m), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: a, d and y contain approximately 450 amino acids, while m and e contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely lgG1 , lgG2, lgG3 and lgG4. A heavy chain can be a human heavy chain.

The term“hinge region” refers herein to a flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links these 2 chains by disulfide bonds. The hinge region is generally defined as stretching from Glu216 to Pro230 of human lgG1 (Burton, Mol Immunol, 22: 161 -206, 1985). Hinge regions of other IgG isotypes may be aligned with the lgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions. The "CH2 domain" of a human IgG Fc portion (also referred to as "Cy2" domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, Mol Immunol, 22: 161 -206, 1985). The "CH3 domain" comprises the stretch of residues C- terminal to a CH2 domain in an Fc portion (i.e. , from about amino acid residue 341 to about amino acid residue 447 of an IgG) .

The term“host” as used herein refers to an animal, such as a mammal (e.g. , a human).

The term“host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

“Humanized” forms of nonhuman (e.g. , murine) antibodies are chimeric antibodies that include human immunoglobulins (recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable domains, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In some embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al. , Nature, 321 :522-525 (1986); Riechmann et al. , Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. , 2:593-596 (1992); Carter et al. , Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992); and U.S. Patent Nos: 6,800,738 (issued Oct. 5, 2004), 6,719,971 (issued Sept. 27, 2005), 6,639,055 (issued Oct. 28, 2003), 6,407,213 (issued June 18, 2002), and 6,054,297 (issued April 25, 2000).

An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc. In some embodiments, effective amount also refers to the amount of an antibody provided herein to achieve a specified result (e.g. , inhibition of a PSGL-1 or VISTA biological activity of a cell, such as modulating T cell activation). In some embodiments, this term refers to the amount of a therapy (e.g. , an antibody provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition and/or a symptom related thereto. This term also encompasses an amount necessary for the reduction or amelioration of the advancement or progression of a given disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given disease, disorder or condition, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g. , a therapy other than anti-PSGL-1 antibody provided herein). In some embodiments, the effective amount of an antibody is from about 0.1 mg/kg (mg of antibody per kg weight of the subject) to about 100 mg/kg. In some embodiments, an effective amount of an antibody provided therein is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg about 90 mg/kg or about 100 mg/kg (or a range therein).

The term“inhibit,” or a grammatical equivalent thereof, when used in the context of an antibody refers to an antibody that suppresses, restrains or decreases a biological activity of the antigen to which the antibody binds. The inhibitory effect of an antibody can be one which results in a measurable change in the antigen’s biological activity. In some embodiments, an anti-PSGL-1 antibody described herein inhibits the ability of VISTA to bind PSGL-1 , which can result in inhibiting the co-inhibitory activity of VISTA. Certain anti-PSGL-1 antibodies described herein inhibit or block suppressive signals of VISTA on VISTA-expressing cells by greater than 5%, such as from about 5% to about 50%, or by greater than 50% (e.g. , from about 50% to about 98%) as compared to the appropriate control (e.g., the control being cells not treated with the antibody being tested). In some embodiments, the anti-PSGL-1 antibody described herein inhibit the binding of PSGL-1 to the extracellular domain VISTA and/or inhibit the binding of a VISTA-expressing cell to a PSGL-1 -expressing cell. Additionally, in some embodiments, the anti-PSGL-1 antibody described herein does not inhibit the binding of PSGL-1 to a protein other than VISTA, such as P-selectin, L-selectin, and/or E-selectin.

The term “immune infiltrate” or “tumor immune cells” refers to cells that infiltrate the microenvironment of a tumor, including, but not limited to, lymphocytes (e.g. , T cells, B-cells, natural killer (NK) cells), dendritic cells, mast cells, and macrophages.

As used herein, the term“in combination” in the context of the administration of other therapies refers to the use of more than one therapy (e.g. , an anti-PSGL-1 antibody and an anti-VISTA antibody). The use of the term“in combination” does not restrict the order or the time in which therapies are administered to a subject (e.g. , one therapy before, concurrent with, or after another therapy). A first therapy can be administered before (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently, or after (e.g. , 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the administration of a second therapy to a subject which had, has, or is susceptible to a VISTA- mediated disease, disorder or condition. Any additional

therapy can be administered in any order or time with the other additional therapies ( e.g . , an anti-PSGL-1 antibody and an anti-VISTA antibody). In some embodiments, the antibodies can be administered in combination with one or more therapies (e.g. , therapies that are not the antibodies that are currently administered to prevent, treat, manage, and/or ameliorate a VISTA-mediated disease, disorder or condition). Non-limiting examples of therapies that can be administered in combination with an antibody include an antagonist to a co-inhibitory molecule, an agonist to a co-stimulatory molecule, a chemotherapeutic agent, radiation, analgesic agents, anesthetic agents, antibiotics, or immunomodulatory agents or any other agent listed in the U.S. Pharmacopoeia and/or Physician’s Desk Reference.

An “isolated” antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source and/or other contaminant components from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”) . In some embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g. , culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. In some embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, e.g. , it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components can also include, but are not limited to, materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method (Lowry et al. J. Bio. Chem. 193: 265-275, 1951 ), such as 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In some embodiments, antibodies provided herein are isolated.

An“isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In some embodiments, a nucleic acid molecule(s) encoding an antibody provided herein is isolated or purified.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy- terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (K) of lambda (l) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

As used herein, the terms“manage,”“managing,” and“management” refer to the beneficial effects that a subject derives from a therapy (e.g. , a prophylactic or therapeutic agent), which does not result in a cure of the disease. In some embodiments, a subject is administered one or more therapies (e.g. , prophylactic or therapeutic agents, such as an antibody provided herein) to“manage” a VISTA-mediated disease, disorder or condition, including one or more symptoms thereof, so as to prevent the progression or worsening of the disease, disorder or condition.

The term“monoclonal antibody” refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies, i.e. the antibodies forming this population are essentially identical except for possible naturally occurring mutations which might be present in minor amounts. In other words, a monoclonal antibody is a homogeneous antibody arising from the growth of a single cell clone (for example a hybridoma, a eukaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, etc.) and is generally characterized by heavy chains of one and only one class and subclass, and light chains of only one type. These antibodies are highly specific and are directed against a single antigen. In addition, in contrast with preparations of polyclonal antibodies which typically include various antibodies directed against various determinants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In some embodiments, a“monoclonal antibody,” as used herein, is an antibody produced by a single hybridoma or other cell, wherein the antibody binds to only a VISTA epitope as determined, e.g. , by ELISA or other antigen-binding or competitive binding assay known in the art. The term“monoclonal” is not limited to any particular method for making the antibody. For example, monoclonal antibodies provided herein may be made by the hybridoma method as described in Kohler et al . ; Nature, 256:495 (1975) or may be isolated from phage libraries using the techniques. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al. , eds., John Wiley and Sons, New York). Other exemplary methods of producing other monoclonal antibodies are provided in the Examples herein.

The term “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not manipulated by a human being.

The term“pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

“Polyclonal antibodies” as used herein refers to an antibody population generated in an immunogenic response to a protein having many epitopes and thus includes a variety of different antibodies directed to the same and to different epitopes within the protein.

Methods for producing polyclonal antibodies are known in the art (See, e.g. , see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al. , eds., John Wiley and Sons, New York).

As used herein, the term“polynucleotide,”“nucleotide,” nucleic acid”“nucleic acid molecule” and other similar terms are used interchangeable and include DNA, RNA, mRNA and the like.

As used herein, the terms“prevent,”“preventing,” and“prevention” refer to the total or partial inhibition of the development, recurrence, onset or spread of a VISTA-mediated disease, disorder or condition and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g. , a combination of prophylactic or therapeutic agents, such as an antibody provided herein).

As used herein, the term“prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a VISTA-mediated disease, disorder or condition, and/or symptom related thereto in a subject. In some embodiments, the term“prophylactic agent” refers to an anti-PSGL-1 antibody provided herein. In some other embodiments, the term“prophylactic agent” refers to an agent other than an anti- PSGL-1 antibody provided herein. In some embodiments, a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to prevent a VISTA-mediated disease, disorder or condition, and/or a symptom related thereto or impede the onset, development, progression and/or severity of a VISTA-mediated disease, disorder or condition, and/or a symptom related thereto. In some embodiments, the prophylactic agent is a humanized anti-PSGL-1 antibody, such as a humanized anti-PSGL-1 monoclonal antibody.

The term“P-selectin glycoprotein ligand 1” (also known as PSGL-1 , PSGL1 , selectin P ligand, SELPLG, CLA, and CD162,) refers to a polypeptide (“polypeptide,”“peptide” and “protein” are used interchangeably herein) encoded by the SELPLG gene, for example, comprising the amino acid sequence:

1 mplqllllli llgpgnslql wdtwadeaek algpllardr rqateyeyld ydflpetepp

61 emlrnstdtt pltgpgtpes ttvepaarrs tgldaggavt elttelanmg nlstdsaame

121 iqttqpaate aqttplaate aqttrltate aqttplaate aqttppaate aqttqptqle

181 aqttapaame aqttapaame aqttppaame aqttqttame aqttapeate aqttqptate

241 aqttplaame alstepsate alsmepttkr qlfipfsvss vthkqipmaa snlsvnypvq

301 apdhisvkqc llaililalv atiffvctw lavrlsrkqh mypvrnyspt emvcissllp

361 dqqeqpsata nqqlskaksp qltpepredr eqddltlhsf lp (SEQ ID NO: 3)

and related polypeptides, including SNP variants thereof. PSLG-1 is a human mucin-type glycoprotein ligand that is known to bind all three selectins (P-selectin, E-selectin, and L-selectin), but it binds P-selectin with the highest affinity (McEver et al. , J. Clin. Invest., 100(3):485-492 (1997) and Carlow et al. , Immunological Reviews, 230:75-96 (2009)). PSGL-1 is a disulfide-bonded homodimer with two 120-kD subunits and is expressed on the surface of monocytes, lymphocytes, granulocytes, and in some CD34+ stem cells. As such, this protein has been known to play a role in leukocyte trafficking during inflammation by tethering of leukocytes to activated platelets or endothelia expressing selectins. PSGL-1 typically has two post-translational modifications, tyrosine sulfation and the addition of the sialyl Lewis x tetrasaccharide (sLex) to its O-linked glycans, for its high-affinity binding activity. Aberrant expression of the SELPLG gene and polymorphisms in this gene are associated with defects in innate and adaptive immune responses.

As those skilled in the art will appreciate, an anti-PSGL-1 antibody provided herein can bind to a PSGL-1 polypeptide, polypeptide fragment, antigen, and/or epitope, as an epitope is part of the larger antigen, for example, which is part of the larger polypeptide fragment, which, in turn, for example, is part of the larger polypeptide. PSGL-1 can exist in a native or denatured form. The PSGL-1 polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A "native sequence PSGL-1 polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PSGL-1 polypeptide derived from nature. Such native sequence PSGL-1 polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PSGL-1 polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific PSGL-1 polypeptide (e.g. , an extracellular

domain sequence), naturally-occurring variant forms (e.g. , alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.

A cDNA nucleic acid sequence encoding a PSGL-1 polypeptide, for example, comprises:

1 atggggtgtg ggctgtcaca tggccctgcc taagtaacca cattctcgct tcctccttcc

61 acacacagcc attgggggtt gctcggatcc gggactgccg cagggggtgc cacagcagtg

121 cctggcagcg tgggctggga ccttgtcact aaagcagaga agccacttct tctgggccca

181 cgaggcagct gtcccatgct ctgctgagca cggtggtgcc atgcctctgc aactcctcct

241 gttgctgatc ctactgggcc ctggcaacag cttgcagctg tgggacacct gggcagatga

301 agccgagaaa gccttgggtc ccctgcttgc ccgggaccgg agacaggcca ccgaatatga

361 gtacctagat tatgatttcc tgccagaaac ggagcctcca gaaatgctga ggaacagcac

421 tgacaccact cctctgactg ggcctggaac ccctgagtct accactgtgg agcctgctgc

481 aaggcgttct actggcctgg atgcaggagg ggcagtcaca gagctgacca cggagctggc

541 caacatgggg aacctgtcca cggattcagc agctatggag atacagacca ctcaaccagc

601 agccacggag gcacagacca ctccactggc agccacagag gcacagacaa ctcgactgac

661 ggccacggag gcacagacca ctccactggc agccacagag gcacagacca ctccaccagc

721 agccacggaa gcacagacca ctcaacccac aggcctggag gcacagacca ctgcaccagc

781 agccatggag gcacagacca ctgcaccagc agccatggaa gcacagacca ctccaccagc

841 agccatggag gcacagacca ctcaaaccac agccatggag gcacagacca ctgcaccaga

901 agccacggag gcacagacca ctcaacccac agccacggag gcacagacca ctccactggc

961 agccatggag gccctgtcca cagaacccag tgccacagag gccctgtcca tggaacctac

1021 taccaaaaga ggtctgttca tacccttttc tgtgtcctct gttactcaca agggcattcc

1081 catggcagcc agcaatttgt ccgtcaacta cccagtgggg gccccagacc acatctctgt

1141 gaagcagtgc ctgctggcca tcctaatctt ggcgctggtg gccactatct tcttcgtgtg

1201 cactgtggtg ctggcggtcc gcctctcccg caagggccac atgtaccccg tgcgtaatta

1261 ctcccccacc gagatggtct gcatctcatc cctgttgcct gatgggggtg aggggccctc 1321 tgccacagcc aatgggggcc tgtccaaggc caagagcccg ggcctgacgc cagagcccag 1381 ggaggaccgt gagggggatg acctcaccct gcacagcttc ctcccttagc tcactctgcc 1441 atctgttttg gcaagacccc acctccacgg gctctcctgg gccacccctg agtgcccaga 1501 ccccattcca cagctctggg cttcctcgga gacccctggg gatggggatc ttcagggaag 1561 gaactctggc cacccaaaca ggacaagagc agcctggggc caagcagacg ggcaagtgga 1621 gccacctctt tcctccctcc gcggatgaag cccagccaca tttcagccga ggtccaaggc 1681 aggaggccat ttacttgaga cagattctct cctttttcct gtcccccatc ttctctgggt 1741 ccctctaaca tctcccatgg ctctccccgc ttctcctggt cactggagtc tcctccccat 1801 gtacccaagg aagatggagc tcccccatcc cacacgcact gcactgccat tgtcttttgg 1861 ttgccatggt caccaaacag gaagtggaca ttctaaggga ggagtactga agagtgacgg 1921 acttctgagg ctgtttcctg ctgctcctct gacttggggc agcttgggtc ttcttgggca 1981 cctctctggg aaaacccagg gtgaggttca gcctgtgagg gctgggatgg gtttcgtggg 2041 cccaagggca gacctttctt tgggactgtg tggaccaagg agcttccatc tagtgacaag 2101 tgacccccag ctatcgcctc ttgccttccc ctgtggccac tttccagggt ggactctgtc 2161 ttgttcactg cagtatccca actgcaggtc cagtgcaggc aataaatatg tgatggacaa 2221 acgata (SEQ ID NO: 4)

Orthologs to the human PSGL-1 polypeptide are also well known in the art. For example, orthologs of PSGL-1 can be found in organisms such as mouse (Mus musculus), rat (Rattus norvegicus), dog (Canis lupus familiaris) , cattle (Bos Taurus), zebrafish ( Danio rerio ), horse (Equus caballus), chimpanzee (Pan troglodytes), etc.

A“PSGL-1 -mediated disease,”“PSGL-1 -mediated disorder” and“PSGL-1 -mediated condition” are used interchangeably and refer to any disease, disorder or condition that is completely or partially caused by or is the result of PSGL-1 . Such diseases, disorders or conditions include those caused by or otherwise associated with PSGL-1 , including by or associated with PSGL-1 -expressing cells (e.g. , tumor cells, myeloid-derived suppressor cells (MDSC), suppressive dendritic cells (suppressive DC), and/or regulatory T cells (T-regs)). In some embodiments, PSGL-1 is aberrantly (e.g. , highly) expressed on the surface of a cell. In some embodiments, PSGL-1 may be aberrantly upregulated on a particular cell type. In other embodiments, normal, aberrant or excessive cell signaling is caused by binding of PSGL-1 to a PSGL-1 ligand (e.g. , VISTA), which can bind or otherwise interact with PSGL-1 . In preferred embodiment, the PSGL-1 -mediated disease is caused by binding of PSGL-1 to a specific PSGL-1 ligand (e.g., VISTA) but not to the other ligands (e.g. , the selectins).

The term“radiation,” when used in the therapeutic context refers to a type of treatment that uses a beam of intense energy to kill target cells (e.g. , cancer cells). Radiation therapy includes the use of X-rays, protons or other forms of energy that are administered through an external beam. Radiation therapy also includes radiation treatment that is placed into a patient’s body (e.g. , brachytherapy) whereby a small container of radioactive material is implanted directly into or near a tumor.

The terms“relative expression level” refers to a quantification of the expression level of a protein in a given sample relative to another reference protein in the same sample and/or to another reference sample. In the context of the methods described herein, the level of expression of PSGL-1 can be expressed in absolute numbers, such as based on a standard curve, or can be expressed in relative expression levels against one or more other proteins that are assayed in the sample (e.g. , VISTA, CD11 b, CD33, CD4, or CD8).

The term “recombinant antibody” refers to an antibody that is prepared, expressed, created or isolated by recombinant means. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g. , a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see e.g. , Taylor, L. D. et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242). In some embodiments, however, such recombinant antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, the term“side effects” encompasses unwanted and adverse effects of a therapy (e.g. , a prophylactic or therapeutic agent). Unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g. , a prophylactic or therapeutic agent) might be harmful or uncomfortable or risky. Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, and loss of appetite, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Additional undesired effects experienced by patients are numerous and known in the art. Many are described in the Physician’s Desk Reference (67th ed., 2013).

As used herein, the terms“subject” and“patient” are used interchangeably. As used herein, in some embodiments, a subject is a mammal, such as a non-primate (e.g. , cows, pigs, horses, cats, dogs, rats, etc. ) or a primate (e.g. , monkey and human). In some embodiments, the subject is a human. In some embodiments, the subject is a mammal (e.g. , a human) having a VISTA- mediated disease, disorder or condition and/or a symptom related thereto. In another embodiment, the subject is a mammal (e.g. , a human) at risk of developing a VISTA- mediated disease, disorder or condition and/or a symptom related thereto.

As used herein“substantially all” refers to refers to at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

As used herein, the term“therapeutic agent” refers to any agent that can be used in treating, preventing or alleviating a disease, disorder or condition, including in the treatment, prevention or alleviation of one or more symptoms of a VISTA-mediated disease, disorder, or condition and/or a symptom related thereto. In some embodiments, a therapeutic agent refers to an anti-PSGL-1 antibody provided herein. In some embodiments, a therapeutic agent refers to an agent other than an anti-PSGL-1 antibody provided herein. In some embodiments, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment, prevention or alleviation of one or more symptoms of a VISTA-mediated disease, disorder, condition, and/or a symptom related thereto.

The combination of therapies (e.g. , use of therapeutic agents) can be more effective than the additive effects of any two or more single therapies. For example, a synergistic effect of a combination of therapeutic agents permits the use of lower dosages of one or more of the agents and/or less frequent administration of the agents to a subject with a VISTA-mediated disease, disorder or condition and/or a symptom related thereto. The ability to utilize lower dosages of therapeutic therapies and/or to administer the therapies less frequently reduces the toxicity associated with the administration of the therapies to a subject without reducing the efficacy of the therapies in the prevention, treatment or alleviation of one or more symptoms of a VISTA-mediated disease, disorder or condition and/or a symptom related thereto. In addition, a synergistic effect can result in improved efficacy of therapies in the prevention, treatment or alleviation of one or more symptoms of a VISTA-mediated disease, disorder or condition and/or a symptom related thereto. Finally, synergistic effect of a combination of therapies (e.g., therapeutic agents) may avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

The term“therapeutically effective amount” as used herein refers to the amount of a therapeutic agent (e.g. , an anti-PSGL antibody or any other therapeutic agent, including as described herein, including, for example, an anti-VISTA antibody) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition and/or a symptom related thereto. A therapeutically effective amount of a therapeutic agent can be an amount necessary for the reduction or

amelioration of the advancement or progression of a given disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given disease, disorder or condition and/or to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g. , a therapy other than the administration of an anti-PSGL-1 antibody, including as described herein).

As used herein, the term“therapy” refers to any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of a VISTA-mediated disease, disorder or condition. In some embodiments, the terms “therapies” and“therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the treatment, prevention and/or amelioration of a VISTA-mediated disease, disorder or condition known to one of skill in the art such as medical personnel.

As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a VISTA-mediated disease, disorder or condition, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more therapeutic agents, such as an anti-PSGL-1 antibody, including as described herein). In some embodiments, such terms refer to the reduction or inhibition of cancer (e.g. , a hematological cancer). In some embodiments, such terms refer to the reduction or amelioration of the progression, severity, and/or duration of a disease, disorder or condition, that is responsive to immune modulation, such modulation resulting from increasing T cell activation.

The term“tumor microenvironment” refers to the cellular environment in which a tumor exists. A tumor microenvironment can include surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix.

The term“variable domain” or“variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR). Each variable region comprises three CDRs which are connected to four FR. The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Although not directly involved in antigen binding, the FR determines the folding of the molecules and thus the amount of CDR that is presented on the surface of the variable region for interaction with the antigen. In some embodiments, the variable region is a human variable region.

The term“variable domain residue numbering as in Kabat” or“amino acid position numbering as in Kabat”, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 ). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues ( e.g . residues 82a, 82b, and 82c, etc., according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 -107 of the light chain and residues 1 -113 of the heavy chain) (e.g. , Kabat et al. , Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )). The“EU numbering system” or“EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g. , the EU index reported in Kabat et al. , supra). The“EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Other numbering systems have been described, including, for example, by AbM, Chothia, Contact and IMGT.

The term“variant” when used in relation to PSGL-1 , VISTA or to an anti-PSGL-1 antibody or an anti-VISTA antibody refers to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence. For example, a PSGL-1 or VISTA variant may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of native PSGL-1 or VISTA, respectively. Also by way of example, a variant of an anti-PSGL-1 antibody or anti-VISTA antibody may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified anti-PSGL-1 antibody or anti-VISTA antibody. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding the variants. In some embodiments, the PSGL-1 variant, VISTA variant or anti-PSGL-1 antibody or an anti-VISTA antibody variant at least retains PSGL-1 , VISTA, anti-PSGL-1 antibody or anti-VISTA antibody functional activity, respectively. In some embodiments, an anti-PSGL-1 antibody variant binds PSGL-1 and/or is antagonistic to PSGL-1 activity. In some embodiments, an anti-VISTA antibody variant binds VISTA and/or is antagonistic to VISTA activity. In some embodiments, the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes PSGL-1 , VISTA, anti-PSGL-1 antibody or anti-VISTA antibody VH or VL regions or subregions.

The term“vector” refers to a substance that is used to introduce a nucleic acid molecule into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription

enhancers, transcription terminators, and the like which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g. both an antibody heavy and light chain), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecule is expressed in a sufficient amount to produce the desired product (e.g. an anti-PSGL-1 antibody provided herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

DETAILED DESCRIPTION

The practice of the disclosure employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g. , Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001 ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al. , Current Protocols in Molecular Biology, John Wiley 6t Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley 6t Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991 ) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

PSGL-1 IS A RECEPTOR FOR VISTA

During carcinogenesis, tumor cells interact with a complex microenvironment which is composed of extracellular matrix and non-neoplastic host cells, including mesenchymal cells, vascular endothelial cells and inflammatory or immune cells. The microenvironment plays a crucial role in suppressing tumor-specific T-cell responses. In order to ensure that an immune inflammatory response is not constantly activated once tumor antigens have stimulated a response, multiple checkpoints are in place or activated. These checkpoints are mostly represented by T-cell receptor binding to ligands on cells in the surrounding microenvironment, forming immunological synapses which then regulate the functions of the T cell.

VISTA is one these immune checkpoints. The protein is hematopoietically restricted and in multiple cancer models, it was only detected on tumor infiltrating leukocytes and not on tumor cells. VISTA negatively regulates T cell immunity via direct impact on T cells by engaging different receptor/ligand, as, unique among immune checkpoint proteins, it acts both as a ligand and a receptor (Le Mercier, supra).

The present inventors have now identified PSGL-1 as a binding partner (e.g. , a ligand or a receptor) of the VISTA protein. PSGL-1 is a homodimeric 120-kDa transmembrane glycoprotein bearing 0- and N-linked glycans whose best-known role is in immune cell trafficking via selectin binding. PSGL-1 is expressed in cells of lymphoid, myeloid cells, and dendritic lineages (Laszik et al. , Blood, 88(8): 3010-21 (1996)). Naive T cells express the non-selectin-binding form of PSGL-1 , which can engage potentially other currently unknown binding partners (Veerman et al. , Nat. Immunol. 8(5), 532-539 (2007)). Expression in tumor cells has also been observed. It has been recently demonstrated that PSGL-1 promotes T cell exhaustion, thus facilitating melanoma tumor growth, through the induction of an unknown partner (Tinoco et al. , Immunity, 44: 1 190-03 (2016)) .

The inventors have demonstrated direct binding between the two proteins and shown that PSGL-1 and VISTA cooperate in preventing T cell activation. Indeed, the physical interaction between VISTA and PSGL-1 underlies a functional one, as both genes are co-expressed in a number of tumors. None of the other putative VISTA receptors

display such co-localization, emphasizing the specificity of the relationship. Moreover, VISTA and PSGL-1 are expressed in tumor cell microenvironment. More specifically, in situ hybridization revealed that both genes are expressed in within adjacent cells in tumor microenvironment. Every PSGL-1 expressing cell is adjacent to a VISTA-expressing cell in immune infiltrates, indicating that PSGL-1 is a reliable proxy for activated VISTA.

DIAGNOSIS OF VISTA-MEDIATED DISORDERS

The above data indicate that PSGL-1 is dependable biomarker for diagnosing a VISTA-mediated disorder, such as a VISTA-mediated cancer. Reagents such as labeled nucleic acid probes or antibodies provided herein, which bind to PSGL-1 nucleic acid or protein can thus be used for diagnostic purposes to detect, diagnose, or monitor a VISTA-mediated disease, disorder or condition.

Thus, in a first aspect, the invention relates to an in vitro method for detecting a VISTA-mediated cancer in a subject, said method comprising the steps of:

a) contacting a biological sample of said subject with a reagent capable of binding

PSGL-1 protein or nucleic acid; and

b) detecting the binding of said reagent with said biological sample.

According to the present method, the binding of PSGL-1 indicates the presence of VISTA-mediated cancer. Preferably, the binding of PSGL-1 in immune infiltrates of the tumor microenvironment indicates the presence of VISTA-mediated cancer.

The reagent capable of binding PSGL-1 protein or nucleic acid may be any reagent or compound known to the person of skills in the art which is capable of binding specifically to PSGL-1 . For example, the skilled person will immediately realize that a DNA or RNA probe which hybridizes specifically with PSGL-1 binds specifically to PSGL-1 . Likewise, the skilled person will immediately realize that an anti-PSGL-1 antibody such as those described herein binds specifically to PSGL-1 .

The invention also relates to an in vitro method for detecting a VISTA-mediated cancer in a subject, said method comprising the steps of:

a) contacting a biological sample of said subject with a reagent capable of binding

PSGL-1 protein or nucleic acid; and

b) quantifying the binding of said reagent with said biological sample.

According to the present method, the binding of PSGL-1 indicates the presence of VISTA-mediated cancer. Preferably, the binding of PSGL-1 in immune infiltrates of the tumor microenvironment indicates the presence of VISTA-mediated cancer.

As will be apparent to the skilled artisan, the level of reagent binding to PSGL-1 may be quantified by any means known to the person of skills in the art, as detailed hereafter. Preferred methods include the use of immunoenzymatic assays, such as ELISA or ELISPOT, immunofluorescence, immunohistochemistry (IHC), radio-immunoassay (RIA), or FACS.

The quantification of step b) of the present method is a direct reflection of the level of PSGL-1 expression in the sample, notably in immune infiltrates of the tumor microenvironment. The present method thus allows for identifying a VISTA-mediated cancer by determining the level of expression of PSGL-1 , as described above. In a preferred embodiment, the level of expression of PSGL-1 in said sample, notably in immune infiltrates of the tumor microenvironment, is compared to a reference level.

According to a further preferred embodiment, the invention relates to an in vitro method for detecting a VISTA-mediated cancer in a subject, said method comprising the steps of:

a) determining the level of expression of PSGL-1 in a biological sample of said subject; and

b) comparing the level of expression of step a) with a reference level;

wherein an increase in the assayed level of PSGL-1 in step a) compared to the reference level is indicative of a VISTA-mediated disease, disorder or condition.

The invention also relates to an in vitro method for diagnosing a VISTA-mediated cancer in a subject, said method comprising the steps of:

a) determining the level of expression of PSGL-1 in a biological sample of said subject; and

b) comparing the level of expression of step a) with a reference level;

wherein an increase in the assayed level of PSGL-1 in step (b) compared to the reference level is indicative of a VISTA-mediated disease, disorder or condition.

The expression level of PSGL-1 is advantageously compared or measured in relation to levels in a control cell or sample also referred to as a“reference level” or“reference expression level”. “Reference level”, “reference expression level”,“control level” and “control” are used interchangeably in the specification. A “control level” means a separate baseline level measured in a comparable control cell, which is generally disease or cancer free. The said control cell may be from the same individual, since, even in a cancerous patient, the tissue which is the site of the tumor still comprises non -tumor healthy tissue. It may also originate from another individual who is normal or does not present with the same disease from which the diseased or test sample is obtained. Within the context of the present invention, the term“reference level” refers to a“control level” of expression of PSGL-1 used to evaluate a test level of expression of PSGL-1 in a cancer cell-containing sample of a patient. For example, when the level of PSGL-1 in the biological sample of a patient is higher than the reference level of PSGL-1 , the cells will be considered to have a high level of expression, or overexpression, of PSGL-1 . The reference level can be determined by a plurality of methods. Expression levels may thus define PSGL-1 bearing cells or alternatively the level of expression of PSGL-1 independent of the number of cells expressing PSGL-1 . Thus, the reference level for each patient can be prescribed by a reference ratio of PSGL-1 , wherein the reference ratio can be determined by any of the methods for determining the reference levels described herein.

For example, the control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. The“reference level” can be a single number, equally applicable to every patient individually, or the reference level can vary, according to specific subpopulations of patients. Thus, for example, older men might have a different reference level than younger men for the same cancer, and women might have a different reference level than men for the same cancer. Alternatively, the “reference level” can be determined by measuring the level of

expression of PSGL-1 in non-oncogenic cancer cells from the same tissue as the tissue of the neoplastic cells to be tested. As well, the“reference level” might be a certain ratio of PSGL-1 in the neoplastic cells of a patient relative to the PSGL-1 levels in non-tumor cells within the same patient. The“reference level” can also be a level of PSGL-1 of in vitro cultured cells, which can be manipulated to simulate tumor cells, or can be manipulated in any other manner which yields expression levels which accurately determine the reference level. On the other hand, the “reference level” can be established based upon comparative groups, such as in groups not having elevated PSGL-1 levels and groups having elevated PSGL-1 levels. Another example of comparative groups would be groups having a particular disease, condition or symptoms and groups without the disease. The predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group.

The reference level can also be determined by comparison of the level of PSGL-1 in populations of patients having the same cancer. This can be accomplished, for example, by histogram analysis, in which an entire cohort of patients is graphically presented, wherein a first axis represents the level of PSGL-1 , and a second axis represents the number of patients in the cohort whose tumor cells express PSGL-1 at a given level. Two or more separate groups of patients can be determined by identification of subsets populations of the cohort which have the same or similar levels of PSGL-1. Determination of the reference level can then be made based on a level which best distinguishes these separate groups. A reference level also can represent the levels of two or more markers, one of which is PSGL-1. Two or more markers can be represented, for example, by a ratio of values for levels of each marker.

Likewise, an apparently healthy population will have a different‘normal’ range than will have a population which is known to have a condition associated with expression of PSGL-1. Accordingly, the predetermined value selected may take into account the category in which an individual falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. By “elevated”“increased” it is meant high relative to a selected control. Typically, the

control will be based on apparently healthy normal individuals in an appropriate age bracket.

It will also be understood that the controls according to the invention may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include tissue or cells obtained at the same time from the same subject, for example, parts of a single biopsy, or parts of a single cell sample from the subject.

Preferably, the reference level of PSGL-1 is the level of expression of PSGL-1 in normal tissue samples (e.g. , from a patient not having a VISTA-mediated disease, disorder or condition, or from the same patient before disease onset).

In some embodiments, expression of a given protein is an indication of the presence of a certain type of cell in a sample. For example, the expression of PSGL-1 , CD4 and/or CD8 by cells in the sample can indicate the presence of T cells in the sample. Likewise, expression of VISTA alone or in combination with CD11 b or CD33 by cells in the sample can indicate the presence of VISTA-bearing tumor cells, regulatory T cells (e.g. , CD4+ Foxp3+ regulatory T cells), myeloid-derived suppressor cells (e.g. , CD11 b+ or CD11 bh,gh and/or CD33+ myeloid-derived suppressor cells) and/or suppressive dendritic cells (e.g. , CD1 1 b+ or CD11 bh,gh dendritic cells). Preferably, expression of VISTA, CD11 b, CD33, CD4, and CD8, notably in immune infiltrates of the tumor microenvironment, indicates the presence of a VISTA-mediated cancer in a subject.

According to these specific embodiment, the in vitro method for detecting a VISTA-mediated cancer in a subject comprises the steps of:

a) determining the level of expression of PSGL-1 and at least one of VISTA, CD11 b,

CD33, CD4, and CD8 in a biological sample of said subject; and

b) comparing the level of expression of PSGL-1 and at least one of VISTA, CD11 b,

CD33, CD4, and CD8 of step a) with a reference level;

wherein an increase in the assayed level of PSGL-1 in step (b) compared to the reference level is indicative of a VISTA-mediated disease, disorder or condition.

The invention also relates to an in vitro method for diagnosing a VISTA-mediated cancer in a subject, said method comprising the steps of:

a) determining the level of expression of PSGL-1 and at least one of VISTA, CD11 b, CD33, CD4, and CD8 in a biological sample of said subject; and

b) comparing the level of expression of PSGL-1 and at least one of VISTA, CD11 b, CD33, CD4, and CD8 of step a) with a reference level;

wherein an increase in the assayed level of PSGL-1 in step (b) compared to the reference level is indicative of a VISTA-mediated disease, disorder or condition.

A more definitive diagnosis of a VISTA-mediated disease, disorder or condition may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the VISTA-mediated disease, disorder or condition.

IDENTIFICATION OF PATIENTS SUSCEPTIBLE TO RESPOND TO ANTI-VISTA THERAPEUTIC AGENTS

The above data indicate that PSGL-1 is a dependable biomarker for diagnosing a VISTA-mediated disorder, such as a VISTA-mediated cancer. Patients thus identified are susceptible to respond to anti-VISTA therapeutic agents.

In another aspect, the invention relates to an in vitro method for identifying tumor patients which are susceptible to be treated by an anti-VISTA therapeutic agent. Advantageously, said patients express PSGL-1 , notably in immune infiltrates, and the expression of PSGL-1 indicates that said patients are susceptible to be treated by an anti-VISTA therapeutic agent.

In a first embodiment, the present invention relates to an in vitro method of diagnosing in a patient a cancer which is susceptible to treatment with a VISTA-blocking agent, said method comprising the steps of:

a) determining the level of expression of PSGL-1 in a biological sample of said patient; and

b) comparing the level of expression of step a) with a reference level; and

c) diagnosing that the cancer is susceptible to treatment with a VISTA-blocking agent from said comparison.

In another embodiment, said reference level is the level of expression of PSGL-1 in a second biological sample from a second patient having the same VISTA-mediated cancer as the first patient, wherein the second patient is responsive to the treatment. In a preferred embodiment, a similar level of expression of PSGL-1 in the first biological sample compared to the level of expression of PSGL-1 in the second biological sample indicates that the first patient will be responsive to treatment.

In another embodiment, step a) comprises determining the level of expression of PSGL-1 and at least one of VISTA, CD11 b, CD33, CD4, and CD8 in said biological sample, preferably by immune infiltrates. Advantageously, the level of expression of PSGL-1 and at least one of VISTA, CD11 b, CD33, CD4, and CD8, or the relative expression levels thereof, in the first biological sample with the level of expression of PSGL-1 is compared with at least one of VISTA, CD11 b, CD33, CD4, and CD8, or the relative expression levels thereof, in a second biological sample from a second patient having the same VISTA-mediated cancer as the first patient, wherein the second patient is responsive to the treatment. In a preferred embodiment, a similar level of expression of PSGL-1 and at least one of VISTA, CD11 b, CD33, CD4, and CD8, or the relative expression levels thereof, in the first biological sample compared to the level of expression of PSGL-1 and at least one of VISTA, CD11 b, CD33, CD4, and CD8, or the relative expression levels thereof, in the second biological sample indicates that the first patient will be responsive to treatment.

MEASURING PSGL- 1 EXPRESSION

PSGL-1 expression can be measured by any means available to the person of skills in the art. The expression of PSGL-1 can thus be measured by measuring the level of a PSGL-1 nucleic acid (e.g. , PSGL-1 mRNA or the corresponding cDNA) or by measuring the level of the PSGL-1 protein.

In this case, the method according to the invention may comprise one or a plurality of intermediate steps between sampling the biological sample and measuring the expression of PSGL-1 , said steps corresponding to the extraction from said biological sample of an mRNA sample (or of the corresponding cDNA) or a protein sample. The

preparation or extraction of mRNA (and the retrotranscription thereof to cDNA) or proteins from a cell sample are merely routine procedures well-known to those skilled in the art.

Once the mRNA (or corresponding cDNA) or protein sample is obtained, the expression of PSGL-1 , in respect of either the mRNA (i.e. , in all the mRNA or cDNA present in the sample), or proteins (i.e. , in all the proteins present in the sample), may be measured. The method used for this purpose is then dependent on the type of transformation (mRNA, cDNA or protein) and the type of sample available.

When the expression of the marker is measured in respect of mRNA (or corresponding cDNA), any method commonly used by those skilled in the art may be applied. These technologies for analyzing the level of gene expression, such as for example transcriptome analysis, include well-known methods such as PCR (Polymerase Chain Reaction, if using DNA), RT-PCR (Reverse Transcription-PCR, if using RNA) or quantitative RT-PCR or nucleic acid arrays (including DNA arrays and oligonucleotide arrays) for a greater throughput.

The term“nucleic acid arrays” as used herein refers to a plurality of different nucleic acid probes attached to a substrate, which may be a microchip, a glass slide, or a bead having the size of a microsphere. The microchip may consist of polymers, plastics, resins, polysaccharides, silica or a material based on silica, carbon, metals, inorganic glass, or nitrocellulose.

The probes may be nucleic acids such as cDNA ("cDNA array"), mRNA ("mRNA array") or oligonucleotides ("oligonucleotide array"), said oligonucleotides typically being suitable for having a length between approximately 25 and 60 nucleotides.

To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of said gene is labelled, then placed in contact with the array under hybridization conditions, resulting in the formation of complexes between said labelled target nucleic acid and the probes attached to the chip surface which are complementary to this nucleic acid. The presence of labelled hybridized complexes is then detected.

These technologies are suitable for monitoring the level of expression of one gene in particular or of a plurality of genes or even all the genes of the genome (full genome or full transcriptome) in a biological sample (cells, tissues, etc.).

In a preferred embodiment, the expression profile is determined using quantitative PCR. Quantitative, or real-time, PCR is a well-known and easily available technology for those skilled in the art and does not need a precise description.

In a particular embodiment, which should not be considered as limiting the scope of the invention, the determination of the expression profile using quantitative PCR may be performed as follows. Briefly, the real-time PCR reactions are carried out using the TaqMan Universal PCR Master Mix (Applied Biosystems). 6 pL cDNA is added to a 9 pL PCR mixture containing 7.5 pL TaqMan Universal PCR Master Mix, 0.75 pL of a 20X mixture of probe and primers and 0.75pl water. The reaction consisted of one initiating step of 2 min at 50 deg. C, followed by 10 min at 95 deg. C, and 40 cycles of amplification including 15 sec at 95 deg. C and 1 min at 60 deg. C. The reaction and data acquisition can be performed using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems). The number of template transcript molecules in a sample is determined by recording the amplification cycle in the exponential phase (cycle threshold or CT) , at which time the fluorescence signal can be detected above background fluorescence. Thus, the starting number of template transcript molecules is inversely related to CT.

In another preferred embodiment, the expression profile is determined by the use of a nucleic microarray.

The present invention further relates to a microarray dedicated to the implementation of the methods according to the invention, comprising at most 500, preferably at most 300, at most 200, more preferably at most 150, at most 100, even more preferably at most 75, at most 50, at most 40, at most 30, at most 20, at most 10 distinct probes, at least 1 of which specifically binds to PSGL-1 mRNA (or corresponding cDNA) or protein. In a preferred embodiment, said microarray is a nucleic acid microarray, comprising at most 500, preferably at most 300, at most 200, more preferably at most 150, at most 100, even more preferably at most 75, at most 50, at most 40, at most 30, at most 20, at most 10 distinct probes (thus excluding for instance pangenomic

microarrays), at least 1 of which specifically hybridizes to PSGL-1 mRNA (or corresponding cDNA). Said microarray may also contain at least one probe which specifically hybridizes to a housekeeping gene in addition to the probe specifically hybridizing to PSGL-1 . For example, the housekeeping gene is the beta-2-microglobulin gene.

Alternatively, it is possible to use any current or future technology suitable for determining gene expression on the basis of the quantity of mRNA in the sample. For example, those skilled in the art can measure the expression of a gene by hybridization with a labelled nucleic acid probe, such as for example by means of Northern Blot (for mRNA) or Southern Blot (for cDNA), but also using techniques such as the serial analysis of gene expression (SAGE) method and the derivatives thereof, such as LongSAGE, SuperSAGE, DeepSAGE, etc.
CLAIMS

1 ) An in vitro method for diagnosing a VISTA- mediated tumor in a subject, said method comprising the steps of:

a) Contacting a biological sample of said subject with a reagent capable of binding specifically to PSGL-1 nucleic acid or protein; and

b) quantifying the binding of said reagent with said biological sample, thus determining the level of expression of PSGL-1 in said sample.

2) The method of claim 1 , wherein said reagent is chosen between a DNA probe, an RNA probe, and an anti-PSGL-1 antibody.

3) The method of any one of claims 1 or 2, wherein the binding of PSGL-1 in immune infiltrates of the tumor microenvironment is quantified.

4) The method of any one of claims 1 to 3, further comprising a step of scoring the tumor by comparing the level of step (B) to an appropriate scale based on two parameters which are the intensity of staining and the percentage of positive cells.

5) The method of any one of claims 1 to 4, further comprising a step of comparing the level of expression of step b) with a reference level, wherein an increase in the assayed level of PSGL-1 in step (b) compared to the reference level is indicative of a VISTA- mediated tumor.

6) The method of claim 5, wherein said reference level is the level of expression of PSGL- 1 in normal tissue samples.

7) The method of any one of claims 5 or 6, wherein:

• step a) further comprises measuring the level of expression of at least one of VISTA, CD11 b, CD33, CD4, and CD8 by said immune infiltrates in said biological sample; and

· step b) comprises comparing the level of expression of step a) with a control level,

whereby an increase in the assayed level of PSGL-1 and/or VISTA, CD11 b, CD33, CD4, or CD8 compared to the control level of the PSGL-1 and/or VISTA, CD11 b, CD33, CD4, or CD8, is indicative of a VISTA-mediated cancer.

8) The method of any one of claims 5 to 7, wherein the VISTA- mediated tumor is selected from the group consisting of hematological cancers (e.g., leukemias, lymphomas, or myelomas), bladder, breast, colon, connective tissue, rectal, gastric, esophageal, lung, larynx, kidney, oral, ovarian, or prostate cancers, or sarcomas, melanomas, or gliomas, or metastases of any of these cancers.

9) An anti-VISTA therapeutic agent for use in treatment of a VISTA-mediated cancer in a patient, said use comprising a prior step of diagnosing said VISTA-mediated cancer in said patient according to claims 1 to 8.

10) The anti-VISTA therapeutic agent for the use of claim 9, wherein said agent is an anti- VISTA antibody.

11 ) The anti-VISTA therapeutic agent for the use of claim 10, wherein said anti-VISTA antibody is selected in the group consisting of:

a) an anti-VISTA antibody, said antibody comprising a heavy chain comprising 3 CDRs of sequences SEQ IS NOs: 1296, 1354, and 1393, as defined by Kabat; and a light chain comprising 3 CDRs of sequences SEQ IS NOs: 1432, 1477, and 1499, as defined by Kabat; and

b) an anti-VISTA antibody, said antibody comprising a heavy chain comprising 3 CDRs of sequences SEQ IS NOs: 1296, 1559, and 1393, as defined by Kabat; and a light chain comprising 3 CDRs of sequences SEQ IS NOs: 1432, 1633, and 1499, as defined by Kabat.

12) The anti-VISTA therapeutic agent for the use of claim 10, wherein said anti-VISTA antibody is a humanized antibody.

13) The anti-VISTA therapeutic agent for the use of any one of claims 9 to 12, further comprising a step of adapting the treatment with the anti-VISTA therapeutic agent, wherein said adaptation of treatment is:

a reduction or suppression of said anti-VISTA therapeutic agent treatment if the patient has been diagnosed as non -responding to the anti-VISTA therapeutic agent, or

the continuation of said anti -VISTA therapeutic agent treatment if the patient has been diagnosed as responding to the anti -VISTA therapeutic agent.

14) An antibody which agonizes or antagonizes the interaction of VISTA and PSGL-1.

15) The antibody of claim 14, which is an agonistic anti-PSGL-1 antibody or antibody fragment.

16) The antibody of claim 14, which is an antagonistic anti-PSGL-1 antibody or antibody fragment.

17) The antibody of claim 16, said antibody being capable of inhibiting or blocking the binding of PSGL-1 to the extracellular domain of VISTA.

18) The antibody of claim 16, said antibody being capable of inhibiting or blocking the binding of a VISTA-expressing cell to a PSGL-1 -expressing T cell.

19) The antibody of claim 18, wherein the VISTA-expressing cell is a myeloid cell, a dendritic cell, a macrophage or a T cell.

20) The antibody of any one of claims 18 or 19, wherein the VISTA-expressing cell is a tumor cell.

21 ) The antibody of any one of claims 18 to 20, wherein the PSGL-1 -expressing cell is a T cell.

22) The antibody of any one of claims 14 to 21 , wherein said antibody does not block or inhibit the binding of PSGL-1 to P-selectin, L-selectin or E-selectin.

23) A pharmaceutical composition comprising the antibody of any one of claims 14 to 22 and physiologically acceptable carriers, excipients and/or stabilizers.

24) The pharmaceutical composition of claim 23, further comprising an antagonist to a co- inhibitory molecule or an agonist to a co-stimulatory molecule.

25) The pharmaceutical composition of claim 24, wherein the antagonist is an antibody against the co-inhibitory molecule.

26) The pharmaceutical composition of any one of claims 24 or 25, wherein the co- inhibitory molecule is selected from the group consisting of CD86, CD80, PDL-1 , PDL- 2, CTLA-4, PD1 , LAG 3, BTNL2, B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA, HVEM, LAIR1 , TIM1 , Galectin 9, TIM3, CD48, 2B4, CD155, CD112, CD113 and TIGIT.