RU2812846C2 - Receptor for vista - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: a method of in vitro diagnosis of a tumor caused by or otherwise associated with cells expressing V-domain immunoglobulin suppressor of T-cell activation (VISTA) in a subject and the use of an anti-VISTA therapeutic agent for the preparation of a medicinal product for the treatment of cancer caused or otherwise associated with VISTA-expressing cells in a patient, wherein the said treatment includes the preliminary stage of diagnosing the said cancer in the said patient using the above in vitro tumor diagnostic method. The method includes the steps of contacting a biological sample from a specified subject with a reagent capable of specifically binding to P-selectin glycoprotein ligand 1 (PSGL-1) nucleic acid or PSGL-1 protein; and quantifying the binding of the said reagent to the said biological sample, thereby determining the level of expression of PSGL-1 in the said sample.

EFFECT: invention expands the range of diagnostic tools for tumors caused by or otherwise associated with cells expressing VISTA.

11 cl, 13 dwg, 12 tbl, 8 ex

Description

INTRODUCTION

Immunotherapy has revolutionized the landscape of cancer therapy. The development of immune checkpoint-based therapies is progressing at breakneck speed. However, only a proportion of patients are receptive to immunotherapy treatments. A particular challenge in cancer immunotherapy has been the identification of biomarkers associated with this mechanism 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)). Thus, patient selection is an important issue because it will avoid toxicity and treatment-related costs in patients who are unlikely to benefit.

To ensure that the immune inflammatory response is not continuously activated after tumor antigens have stimulated the response, several controls or “checkpoints” exist or are activated. These checkpoints are mainly represented by the T cell receptor, which binds to ligands on cells in the tumor microenvironment to form immunological synapses, which then regulate T cell function.

VISTA (V-domain immunoglobulin suppressor of T-cell activation) is a negative checkpoint regulator protein that regulates T-cell activation and immune responses. It is a type I transmembrane protein that contains one IgV-like domain with homology to similar domains of both the B7 and CD28 families, and an intracellular domain. The cytoplasmic tail domain of VISTA contains two potential binding sites for protein kinase C, as well as proline residues that may function as docking sites, suggesting that VISTA may function as both a receptor and a ligand.

VISTA is homologous to programmed cell death protein ligand 1 (PDL-1) but exhibits a unique expression pattern that is restricted to the hematopoietic cell compartment. VISTA is expressed highest 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. al., J. Immunology, 187(4): 1537-1541 (2011)). VISTA is induced on T cell and myeloid cell populations following activation or immunization, suggesting the involvement of inflammation in its expression (Wang et al., supra). On the other hand, no expression of VISTA is found in tumor cells (Le Mercier et al., Cancer Res; 74: 1933-44 (2014)), although there is a published report regarding the expression of VISTA at low frequency in human gastric cancer cells (Böger et al. ., OncoImmunology, 6: 4, e1293215 (2017)). If VISTA expression occurs, it appears to be restricted to CD11b ⁺ cells infiltrating the tumor microenvironment in colon or lung cancer. However, it was noted that further studies are needed to determine 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 act both as a negative receptor on T cells and as a ligand expressed on antigen presenting cells (APCs) interacting with an unknown receptor on T cells.

Some evidence suggests 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 significantly suppresses immunity (Lines et al., Cancer Res., 74: 1924-32 (2014)), and as with PD-L1 , blocking VISTA promotes immune development during cancer therapeutic treatment in preclinical cancer models (see Le Mercier et al., supra). While blocking VISTA enhances immunity, particularly CD8 ⁺ and CD4 ⁺ T cell-mediated immunity, treatment with a soluble Ig-extracellular domain fusion protein of VISTA (VISTA-Ig) results in inhibition of T cell proliferation and cytokine production in vitro , and overexpression of VISTA on MCA105 tumor cells impairs protective antitumor immunity in mice (Wang et al., supra). In addition, administration of a VISTA-specific monoclonal antibody enhanced CD4 ⁺ T cell responses in vivo and the development of an autoimmune response in mice (Wang et al., supra). On the other hand, VISTA appears to have functional activities that are not duplicated by other members of the Ig superfamily, and may play a role in the development of autoimmunity and immune surveillance in cancer. In particular, although studies using Fc (crystallizable fragment of immunoglobulin) fusion proteins clearly show that VISTA acts as a ligand (Wang et al., supra, Lines et al., supra), its action in signal transduction has also been described , similar to those involving receptors (Flies et al., J. Clin, Invest., 124: 1966-75 (2014)). Indeed, the 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 immune cell infiltrates varies not only between different tumor entities, but also within tumors of the same anatomical location. The authors suggested that the response to various immunotherapeutic combinations would likely depend on the immune environment of the patient's cells (Farkona et al., BMC Medicine, 14: 73 (2016)). In this regard, it is known that in order to avoid immune attack, it is necessary to induce the expression of PDL-1 (Sharma et al., Cell, 168: 707-23 (2017)). PDL-1 expression has been shown to vary within and outside the tumor (Mino-Kenudson, Cancer Biol. Med., 13(2): 157-70 (2016)), but is associated with an objective response to antibody to protein 1 programmed cell death (PD-1) (Topalian et al., supra). On the other hand, VISTA binding partners that mediate the effects of this protein have not yet been identified (Le Mercier et al., Frontiers in Immunology, 6: 418 (2015)). Although two phase I clinical trials have been initiated with the anti-VISTA molecule, there is no biomarker that can predict patient response to these therapies. Thus, there is a need to identify a VISTA binding partner as this would facilitate the development of therapeutics and allow the selection of patients susceptible to treatment with an anti-VISTA therapeutic agent.

All methods and materials similar or equivalent to those described in this application can be used in the practice or testing of the present invention, together with suitable methods and materials described in this application. The practice of this invention utilizes, unless otherwise indicated, conventional methods of protein chemistry, molecular virology, microbiology, recombinant DNA technology and pharmacology within the skill of the person skilled in the art. Such methods are fully explained in the literature (see, for example, 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 combination with laboratory techniques and methods of molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described in this application are nomenclatures 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. In addition, the materials, methods, and examples cited are illustrative only and are not intended to be limiting unless otherwise indicated.

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

CAPTIONS FOR THE FIGURES

In FIG. Figure 1 shows a flow diagram of the CAPTIREC™ Trifunctional Reagents for Chemistry and Proteomics (TRICEPS™) screening methodology. The VISTA-Fc fusion protein was used as the test ligand. Anti-CD28 antibody was used as a control ligand.

In FIG. Figure 2 shows an image for PSGL-1 (P-selectin glycoprotein ligand 1) using the Protter application. N-glycosylation sites are indicated by residues enclosed in squares, and experimentally detected peptides are indicated by filled circles.

In FIG. Figure 3 shows the results of representative binding assays for VISTA-Fc with the PSGL-1 extracellular domain of construct A.

In FIG. Figure 4 shows the results of representative binding assays for VISTA-Fc with the PSGL-1 extracellular domain of construct B.

In FIG. Figure 5 shows a typical histogram of PSGL-1 detection results in HL-60 cells by flow cytometry. Isotype control and background are represented by a gray shaded peak, and cells expressing PSGL-1 are represented by a white shaded peak.

In FIG. Figure 6 shows a typical Western blot showing the interaction between VISTA and PSGL-1. PSGL-1 is indicated by arrows; It is known that incomplete restoration of PSGL-1 leads to the formation of more than one zone.

In FIG. 7 shows a histogram showing the weakening of the interaction between VISTA and PSGL-1 in the presence of anti-VISTA antibody. The height of each bar reflects the intensity of the zones corresponding to the PSGL-1 protein. The symbol "-" indicates that no anti-VISTA antibody was added. The "+" symbol indicates preincubation with anti-VISTA antibody.

In FIG. 8 shows a typical histogram of PSGL-1 detection results in PBMCs (peripheral blood mononuclear cells) by flow cytometry. Isotype control and background are represented by a gray shaded peak, and cells expressing PSGL-1 are represented by a white shaded peak.

In FIG. Figure 9 shows a typical Western blot showing co-immunoprecipitation of PSGL-1 using anti-VISTA and anti-PSGL-1 antibodies. PSGL-1 is indicated by an arrow.

In FIG. 10 shows the results of PSGL-1 expression in representative flow cytometry assays in subsets of naïve and resting, effector and exhausted effector T cells.

In FIG. 11 shows the results of PSGL-1 expression in representative flow cytometry assays in subsets of circulating central memory T cells and circulating effector memory T cells.

In FIG. 12 shows an example of multiple messenger ribonucleic acid (mRNA) staining for PSGL-1, VISTA, and PDL-1 in a squamous cell lung cancer tumor.

In FIG. 13 is a bar graph demonstrating the inhibition of VISTA-dependent interleukin-2 (IL-2) release from CD4 ⁺ T cells in the presence of PSGL-1.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as 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 multiple definitions exist for a term used herein, those given in this section shall prevail unless otherwise noted.

The term "about" or "approximately" refers to a common range of error values for a given value or range known to those skilled in the art. Typically it means being within 20%, such as within 10% or within 5% (or 1% or less) of a given value or range.

As used herein, the term “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance that is outside the body (for example, the anti-PSGL-1 antibody and/or the anti-VISTA antibody of the present invention) to a patient, such as mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery set forth herein or known in the art. When a disease or symptom thereof is being treated, administration of the substance is usually carried out after the onset of the disease or the onset of its symptoms. In the case of preventing a disease or its symptoms, the administration of the substance is usually carried out before the onset of the disease or the onset of its symptoms.

As used herein, the term “antagonist” or “inhibitor” refers to a molecule that is capable of inhibiting or otherwise reducing one or more biological activities of a target protein, such as PSGL-1, VISTA, or other co-inhibitory molecule described herein. In some embodiments, a PSGL-1 antagonist (e.g., an antagonist antibody provided herein) may act, for example, by inhibiting or otherwise attenuating activation and/or cell signaling pathways in a cell expressing PSGL-1 (e.g., a T cell) , and/or in a cell expressing VISTA (eg, a VISTA-bearing tumor cell, a regulatory T cell, a myeloid suppressor cell, or a suppressive dendritic cell), thereby inhibiting the biological activity of the cell relative to the biological activity in the absence of the antagonist. In some embodiments, the antibodies provided herein are PSGL-1 antagonist antibodies. In some embodiments, an antagonist of a coinhibitory molecule (e.g., antagonistic antibody to VISTA, CD86, CD80, PDL-1, PDL-2, CTLA-4 (cytotoxic T-lymphocyte antigen 4), PD1 (programmed cell death protein 1), LAG3 (lymphocyte activation gene product), BTNL2 (butyrophilin-like protein 2), B7-H3, B7-H4, to butyrophilin, CD48, CD244, TIM-3 (molecule 3 containing T-cell immunoglobulin and mucin domains), CD200R ( CD200 receptor), CD200, CD160, BTLA (B and T lymphocyte attenuator), HVEM (herpes virus entry mediator), LAIR1 (leukocyte immunoglobulin-like receptor 1), TIM1, galectin 9, TIM3, CD48, 2B4, CD155, CD112 , CD113 or TIGIT (T cell immunoreceptor with Ig and ITIM domains; ITIM stands for immunoreceptor tyrosine inhibitory motif)) may act, for example, by inhibiting or otherwise attenuating activation and/or cell signaling pathways in a cell expressing the coinhibitory molecule (e.g. T cell or antigen presenting cell), thereby inhibiting the biological activity of the cell compared to the biological activity in the absence of the antagonist. In one embodiment, the antagonist molecule is an antagonistic antibody, i.e. an antibody that inhibits or reduces one or more biological activities of an antigen, such as PSGL-1, VISTA, or other coinhibitory molecule described herein. Certain antagonistic antibodies significantly or completely inhibit one or more biological activities of the specified antigen.

As used herein, the term “agonist” or “activator” refers to a molecule that is capable of activating or otherwise enhancing one or more biological activities of a target protein, such as a costimulatory molecule. In some embodiments, a costimulatory molecule agonist (e.g., anti-CD154 agonist antibody, TNFRSF25 (tumor necrosis factor receptor (TNFR) superfamily (SF) member 25), GITR (glucocorticoid-induced TNFR), 4-1BB, OX40, CD27, TMIGD2 (transmembrane and immunoglobulin domain containing protein 2), ICOS (inducible T cell costimulator), CD28, CD40, TL1A (TNF-like ligand 1A), GITRL (GITR ligand), 4-1BBL (4-1BB ligand), OX40L (OX40 ligand), CD70, HHLA2 (human endogenous retrovirus-H long terminal repeat associated protein 2), ICOSL (ICOS ligand), cytokine, LIGHT, HVEM, CD30, CD30L (CD30 ligand), B7-H2, CD80, CD86 , CD40L (CD40 ligand), TIM4, TIM1, SLAM (signaling lymphocyte-activating molecule), CD48, CD58, CD155, CD112, DR3 (death receptor 3), GITR, CD2 and CD226) may act, for example, by activating or otherwise enhancing the activation and/or cell signaling pathways of a cell expressing a given co-stimulatory molecule (eg, a T cell or an antigen presenting cell), thereby enhancing the biological activity of the cell compared to the biological activity in the absence of the agonist. In one embodiment, the agonist molecule is an agonist antibody, i.e. an antibody that activates or enhances one or more biological activities of an antigen, such as PSGL-1, VISTA, or other coinhibitory molecule described herein. Certain agonistic antibodies significantly or completely activate one or more biological activities of the specified 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 against a specific antigen can be generated by recombinant methods, such as by screening libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or a nucleic acid encoding the antigen. These terms are intended to include a B cell polypeptide product belonging to the immunoglobulin class of polypeptides that is capable of binding to a specific molecular antigen and consists of two identical pairs of polypeptide chains, each pair having one heavy chain (approximately 50-70 kilodaltons). (kDa)) and one light chain (about 25 kDa), and each amino-terminal portion of each chain includes a variable region containing from 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 that can bind to an antibody provided herein includes a target polypeptide, fragment, or epitope of PSGL-1.

Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, intraantibodies, anti-idiotypic (anti-Id) antibodies and functional fragments of any of the above, which refer to 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 Fv (scFv) (including, for example, monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab) ₂ fragments, F(ab') ₂ Fv disulfide bonded fragments (sdFv), Fd fragments, Fv fragments, diabody, tribody, tetrabody and minibody. In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains, or molecules containing an antigen binding site that binds to a VISTA antigen (e.g., containing one or more complementarity determining regions (CDRs) antibodies to VISTA). Descriptions of such antibody fragments can be found, for example, in 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); and Skerra, Meth. Enzymol, 178: 497-515 (1989) and in Day ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein may be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (eg IgG2a and IgG2b). The anti-PSGL-1 antibodies or anti-VISTA antibodies provided herein may 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 similar terms are used interchangeably herein to refer to antibodies that bind to the PSGL-1 polypeptide. 1, such as the PSGL-1 antigen or epitope. Such antibodies include humanized antibodies. An antibody that binds to PSGL-1 antigen may cross-react with related antigens. In some embodiments, an antibody that binds PSGL-1 does not cross-react with other antigens. In some embodiments, the 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, using immunoassays, BIAcore, or other methods known to those skilled in the art. An antibody binds to PSGL-1 when it binds to PSGL-1 with higher affinity than to any cross-reacting antigen, as determined using experimental methods such as radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs), for example, an antibody that specifically binds to PSGL-1. Typically, the signal from a specific or selective interaction will be at least twice the background signal or noise, and may be more than 10 times the background. See, for example, Paul, ed., 1989, Fundamental Immunology, Second Edition, Raven Press, New York, pages 332-336 for a discussion regarding antibody specificity. In some embodiments, an antibody “that binds to” an antigen of interest is an antibody that binds to the antigen with sufficient affinity to make the antibody useful as a diagnostic and/or therapeutic agent for targeting a cell or tissue expressing given antigen, and did not cross-react significantly with other proteins. In such embodiments, the extent of binding of the antibody to the "off-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) or radioimmunoprecipitation (RIA) analysis. . With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" a particular polypeptide or epitope or is "specific to" a particular polypeptide or epitope on a particular target polypeptide means binding that is different from non-specific when measured interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that has no binding activity. For example, specific binding can be determined using competition with a control molecule that is similar to the target, for example, in the presence of an excess of unlabeled target. In this case, specific binding is indicated by the fact that binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. The term "specific binding" or "specifically binds to" a particular polypeptide or epitope or is "specific to" a particular polypeptide or epitope on a particular target polypeptide as used herein can be applied, for example, to a molecule having a Kd for the target , being at least about 10 ^-4 M, alternatively at least about 10 ^-5 M, alternatively at least about 10 ^-6 M, alternatively at least about 10 ^-7 M, alternatively at least about 10 ^-8 M, alternatively at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, alternatively at least about 10 ^-11 M, alternatively at least about 10 ^-12 M or less . In some embodiments, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope of a particular polypeptide without substantially binding to any other polypeptide or epitope of a polypeptide. In some embodiments, an antibody that binds to PSGL-1 or VISTA has a dissociation constant (Kd) of no more than 1 μM, no more than 100 nM, no more than 10 nM, no more than 1 nM, or no more than 0.1 nM. In some embodiments, an anti-PSGL-1 antibody or an anti-VISTA antibody binds to an epitope of PSGL-1 or VISTA that is conserved among PSGL-1 or VISTA from different species.

The term "antigen" refers to a given antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural 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 part of an antibody that contains amino acid residues that interact with an antigen and give the binding agent its inherent specificity and affinity for that antigen (for example, complementarity determining residues). areas (CDR)).

The term "antigen presenting cell" or "APC" refers to a heterogeneous group of immune cells that mediate cellular immune responses 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 "bind" or "linking" as used herein refer to the interaction between molecules to form a complex. The interactions may be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions and/or van der Waals interactions. A complex can also be formed by the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of all noncovalent interactions between one antigen-binding site on an antibody and one epitope on a target molecule, such as PSGL-1, represents the affinity of the antibody or functional fragment for that epitope. The ratio of the rate constant of association (k ₁ ) to the rate constant of dissociation (k _-1 ) of an antibody with a monovalent antigen (k ₁ /k _-1 ) is the association constant K, which is a measure of affinity. The value of K varies for different antibody-antigen complexes and depends on both the constants k ₁ and k _-1 . The association constant K for the antibody provided herein can be determined using any method described herein or any other method well known to those skilled in the art. Affinity for a single binding site does not always reflect the true strength of the interaction between antibody and antigen. When complex antigens containing multiple, repetitive antigenic determinants, such as polyvalent PSGL-1, come into contact with antibodies containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the likelihood of interaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity. The avidity of an antibody may be a more appropriate measure of its binding ability than the affinity of its individual binding sites. For example, high avidity can compensate for low affinity, as is sometimes observed for pentameric IgM antibodies, which may have lower affinity than IgG, but the higher avidity of IgM, resulting from their multivalency, allows them to bind antigen efficiently.

The term "biological sample" refers to an image that is obtained from a biological source, such as a patient or subject. In some embodiments, the biological sample includes, but is not limited to, whole blood, partially purified blood, PBMC, tissue biopsies, and the like. Preferably, the biological sample is a tumor sample. In some preferred embodiments, the biological sample is obtained through a tissue biopsy (eg, a tumor biopsy is obtained, which may include immune infiltrates).

The term "block" or its grammatical equivalent, when used in the context of an antibody, refers to an antibody that prevents or abolishes the biological effect of the antigen to which the antibody binds. A blocking antibody includes an antibody that combines with an antigen without causing a reaction, but which blocks another protein if it subsequently combines or complexes with that antigen. The blocking effect of an antibody may be an effect that results in a change in the biological activity of the antigen, which can be measured. In some embodiments, the anti-PSGL-1 antibody described herein blocks the ability of VISTA to bind to PSGL-1, which may result in inhibition or blocking of inhibitory signals from VISTA. Certain anti-PSGL-1 antibodies described herein inhibit or block suppressive signals from VISTA on VISTA-expressing cells, including by about 98% to about 100% of the corresponding control (e.g., the control is cells not treated with the test antibody). In some embodiments, an anti-PSGL-1 antibody described herein blocks the binding of PSGL-1 to the extracellular domain of 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 refer to a polypeptide ("polypeptide", "peptide" and "protein" are used interchangeably herein) encoded by a gene from human chromosome 10 open reading frame 54 (VISTA) that also known in the art as B7-H5, platelet receptor Gi24, GI24, stress-inducible secreted protein 1, SISP1, and PP2135, containing the amino acid sequence:

and related polypeptides, including their SNP (single nucleotide polymorphism) variants. The VISTA polypeptide is shown or believed to contain several different regions within the amino acid sequence, including: a signal sequence (residues 1-32; see Zhang et al., Protein Sci, 13: 2819-2824 (2004)); immunoglobulin domain - IgV-like (residues 33-162); and a transmembrane region (residues 195-215). The mature VISTA protein includes amino acid residues 33-311 in SEQ ID NO: 1. The extracellular domain of the VISTA protein includes amino acid residues 33-194 in SEQ ID NO: 1. Related polypeptides include allelic variants (eg, SNP variants); splice variants; fragments; derivatives; variants with substitutions, deletions and insertions; fusion polypeptides; and cross-species homologues, preferably those that retain VISTA activity and/or are sufficient to generate an immune response to VISTA. VISTA can be in native or denatured form. The VISTA polypeptides described herein can be isolated from various sources, for example, from different types of human tissue or other source, or obtained by recombinant or synthetic methods. A “native sequence VISTA polypeptide” is a polypeptide having the same amino acid sequence as the corresponding naturally occurring VISTA polypeptide. Such native sequence VISTA polypeptides can be isolated from natural sources or can be produced by recombinant or synthetic methods. The term “native sequence VISTA polypeptide” specifically covers naturally occurring truncated or secreted forms of a particular VISTA polypeptide (e.g., extracellular domain sequence), naturally occurring variant forms (e.g., forms resulting from alternative splicing), and naturally occurring allelic variants of the polypeptide.

The nucleic acid sequence of the cDNA encoding the VISTA polypeptide, for example, contains:

As described herein, VISTA is an immunomodulator that is a negative regulator of immune checkpoint responses (eg, inhibits or suppresses immune responses). In addition, as described herein, PSGL-1 is a VISTA receptor. In addition, as described herein, methods of 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, to inhibit or block suppressive signals from VISTA. Modulation of the interaction of VISTA and PSGL-1 may cause an enhanced immune response, including increased activation of the immune system (eg, T cell activation, such as T cell proliferation). Antibodies that bind to VISTA used in the methods described in this application include those disclosed in WO 2014/197849 (PCT/US 2014/041388).

Orthologs of the VISTA polypeptide are also well known in the art. For example, the mouse orthologue of the VISTA polypeptide is a V-region immunoglobulin suppressor of T cell activation (VISTA) (also known as PD-L3, PD-1H, PD-XL, Pro 1412, and UNQ730), which is approximately 70% consistent in sequence identical to the sequence of this polypeptide in humans. VISTA orthologues can also be found in other organisms, including chimpanzee, cow, rat, and zebrafish.

The expression "VISTA-expressing cell", "cell having VISTA expression" or its grammatical equivalent refers to a cell that expresses endogenous or transfected VISTA on the cell surface. VISTA-expressing cells include VISTA-bearing tumor cells, regulatory tumor cells (eg, CD4 ⁺ Foxp3 ⁺ regulatory T cells), myeloid suppressor cells (eg, CD11b ⁺ or CD11b ^high myeloid suppressor cells), and/or suppressive dendritic cells (eg CD11b ⁺ or CD11b ^high dendritic cells). A cell expressing VISTA produces significant levels of VISTA on its surface such that anti-VISTA antibody can bind to it and/or PSGL-1, or the cell expressing PSGL-1 can bind to it. In some aspects, inhibiting or blocking such binding may have a therapeutic effect. A cell that "overexpresses" VISTA is a cell that has significantly higher levels of VISTA on its cell surface compared to a cell of the same tissue type that is known to express VISTA. Such overexpression may be due to gene amplification or increased transcription or translation. VISTA overexpression can be determined in a diagnostic or prognostic assay by assessing increased levels of VISTA protein present on the cell surface (eg, using immunohistochemical analysis; FACS analysis). Alternatively or in addition, the levels of VISTA-encoding nucleic acid or mRNA in a cell can be measured, for example, using fluorescence in situ hybridization (FISH; see application W098/45479 published October 1998), Southern blotting, Northern blotting or polymerase chain reaction (PCR), such as quantitative real-time PCR (RT-PCR). In addition to the assays described above, various in vivo assays are available to skilled practitioners. For example, the cells of a patient's body can be exposed to an antibody that can be labeled with a detectable agent, and the binding of the antibody to the patient's cells can be assessed, for example, 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.

The terms "VISTA-mediated disease", "VISTA-mediated disorder" and "VISTA-mediated condition" are used interchangeably and refer to any disease, disorder or condition that is wholly or partly caused by or which results from the presence of VISTA. Such diseases, disorders or conditions include those caused by or otherwise associated with VISTA, including those caused by or associated with VISTA-expressing cells (eg, tumor cells, myeloid-derived suppressor cells (MDSCs), suppressive dendritic cells (having suppressive effect of DC) and/or regulatory T cells (T _reg )). In some embodiments, VISTA is aberrantly (eg, highly) expressed on the cell surface. In some embodiments, VISTA may be aberrantly activated on a particular cell type. In other embodiments, normal, aberrant or excessive cell signaling is due to the binding of VISTA to a VISTA receptor (eg, PSGL-1), which can bind or otherwise interact with VISTA.

The terms "cell proliferation disorder" and "proliferative disorder" refer to disorders that are associated with some level of abnormal cell proliferation. In some embodiments, the cell proliferation disorder is a tumor or cancer. The term "tumor" as used herein refers to any growth and proliferation of tumor cells, whether malignant or benign, and any precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cellular proliferation disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as defined herein. The terms "cancer" and "cancerous" refer to or describe a 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 lymphocytic leukemia. More specific examples of such cancers include squamous cell carcinoma (eg, squamous cell carcinoma of epithelial origin), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastric or stomach cancer , including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer colon, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cell cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain cancer, skin cancer, esophageal cancer, and head and neck cancer and metastases associated with these types of cancer. In some embodiments, the cancer is a hematologic malignancy, referring to cancer that originates in hematopoietic tissue, such as bone marrow, or in cells of the immune system. Examples of hematologic malignancies include leukemia (eg, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), or acute monocytic leukemia (AMoL)), lymphoma (Hodgkin's lymphoma, or non-Hodgkin's lymphoma) and myeloma (multiple myeloma, plasmacytoma, localized myeloma or extramedullary myeloma).

A "coinhibitory molecule" (also known as a "negative checkpoint regulator" or "NCR") refers to a molecule that suppresses immune responses (eg, T cell activation) by transmitting a negative signal to T cells after they encounter ligands or counterreceptors . Typical functions of a coinhibitory molecule are to prevent disproportionate immune activation, minimize adverse adverse effects, and/or maintain peripheral autotolerance. In some embodiments, the coinhibitory molecule is a ligand or receptor expressed by an antigen presenting cell. In some embodiments, the coinhibitory molecule is a ligand or receptor expressed by a T cell. In some embodiments, the coinhibitory molecule is a ligand or receptor expressed by both the antigen presenting cell and the T cell.

"Co-stimulatory molecule" refers to a molecule that activates immune responses (eg, causes activation of T cells) by transmitting a positive signal to T cells after they encounter ligands or counterreceptors. A T cell requires two signals to become fully activated: 1) an antigen-specific signal, which is provided through the T cell receptor interacting with peptide-MHC (major histocompatibility complex) molecules on the antigen-presenting cell; and 2) a costimulatory signal, which is antigen-nonspecific and is provided through the interaction between costimulatory molecules expressed on the membrane of the antigen-presenting cell and the T cell. T cell costimulation promotes T cell proliferation, differentiation, and survival. In some embodiments, the costimulatory molecule is a ligand or receptor expressed by an antigen presenting cell. In some embodiments, the costimulatory molecule is a ligand or receptor expressed by a T cell. In some embodiments, the costimulatory molecule is a ligand or receptor expressed by both the antigen presenting cell and the T cell.

A "chemotherapeutic agent" is a chemical or biological agent (eg, an agent comprising a small molecule drug or a biological agent such as an antibody or a cell) useful for treating cancer, regardless of the mechanism of action. Chemotherapeutic agents include compounds used in targeted therapies and conventional chemotherapy. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (in particular bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetyl camptothecin, scopolectin and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, particularly calicheamicin gamma II and calicheamicin omega II (see, for example, Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dinemycin, including dinemycin A; esperamycin; as well as the neocarcinostatin chromophore and related chromophores of chromoproteins - enediin antibiotics), aclacinomysins, actinomycin, outramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, deto rubicin, 6 -diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C , mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as antsitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquon; elfornitine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2''-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurine A, roridin A and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, N.J.), the cremophor-free formulation of paclitaxel in the form of albumin-bound nanoparticles ABRAXANE™ (American Pharmaceutical Partners, Schaumberg, IL.), and docetaxel TAXOTERE® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); and pharmaceutically acceptable salts, acids or derivatives of any of the above compounds, as well as combinations of two or more of the above compounds, such as CHOP, the short name for combination therapy with cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, the short name for the regimen treatment with oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin. Additional chemotherapeutic agents include cytotoxic agents useful in antibody-drug conjugates, such as maytansinoids (DM1 and DM4, for example) and auristatins (MMAE and MMAF, for example).

Also included in the definition of a chemotherapeutic agent are: (1) antihormonal agents that act by regulating or inhibiting the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifene, trioxifene, keoxifene, LY 117018, onapristone and FARESTON® (toremifene citrate); (2) aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestane, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis) and ARIMIDEX® (anastrozole; AstraZeneca); (3) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); (4) protein kinase inhibitors, such as MEK inhibitors (WO 2007/044515); (5) lipid kinase inhibitors; (6) antisense oligonucleotides, in particular oligonucleotides that suppress the expression of genes in signaling pathways involved in aberrant cell proliferation, for example, PKC-alpha (protein kinase C) genes, Raf and H-Ras kinases, such as oblimersen (GENASENSE®, Genta Inc.); (7) ribozymes, such as inhibitors of vascular endothelial growth factor (VEGF) expression (eg, ANGIOZYME®) and inhibitors of HER2 (human epidermal growth factor receptor type 2) expression; (8) vaccines, such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN® and VAXID®; recombinant interleukin-2 (rIL-2) PROLEUKIN®; topoisomerase 1 inhibitors such as LURTOTECAN®; rmRH ABARELIX®; (9) antiangiogenic agents such as bevacizumab (AVASTIN®, Genentech); (10) immunomodulatory agents such as bispecific (BITE) T cell recruiter antibodies and chimeric antigen receptor (CAR) T cells; and pharmaceutically acceptable salts, acids or derivatives of any of the above compounds.

"CDR" refers to one of the three heavy chain hypervariable regions (H1, H2 or H3) within the non-framework region of the β-sheet structure of the heavy chain variable domain (VH) of an immunoglobulin (Ig or antibody) or to one of the three light chain hypervariable regions ( L1, L2 or L3) within the non-framework region of the β-sheet structure of the antibody light chain variable domain (VL). Accordingly, CDRs are sequences of variable regions interleaved within framework sequences. CDR regions are well known to those skilled in the art and have been identified, for example, by Kabat as regions of greatest hypervariability within the variable (V) domains of an antibody (Kabat et al., J. Biol, Chem., 252: 6609-6616 (1977); Kabat, Adv. Prot Chem., 32: 1-75 (1978)). In addition, the sequences of the CDR regions have been structurally defined by Chothia as those residues that are not part of the conserved β-sheet structure and are therefore capable of adopting different conformations (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987) ). Both nomenclatures are generally accepted nomenclatures in the art. The sequences of the CDR regions were also determined according to the AbM, contact and IMGT databases. CDR positions within the 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 in the hypervariable region varies from antibody to antibody, additional residues compared to residues at the canonical positions are traditionally numbered a, b, c, and so on, following the number of residues in the canonical variable domain numbering scheme (Al-Lazikani et al., above (1997)). Such nomenclature is also well known to those skilled in the art.

The term "hypervariable region", "HVR" or "HV", as used herein, refers to regions of the variable domain of an antibody that have hypervariability in sequence and/or form loops with a specific structure. Typically, antibodies contain six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). A number of methods for determining the boundaries of hypervariable regions are used and are included in this description. The Kabat definition of CDR is based on sequence variability and is the most commonly used (Kabat et. al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia, alternatively, refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol, 196: 901-917 (1987)). The end of the CDR-H1 no Chothia loop, when numbered using the Kabat numbering rule, varies between positions H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme allows for inserts at positions H35A and H35B; if neither in position 35A, nor in position 35B there is no insert, then the loop ends in position 32; if the insert is present only in position 35A, then the loop ends in position 33; if there are inserts in both positions 35A and 35B, then the loop ends in position 34) . AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM software to model antibody structure. The location of hypervariable regions by the “contact” method is based on the analysis of the available crystal structures of the complexes. The residues of each of these hypervariable regions are indicated below.

Recently, the ImMunoGeneTics (IMGT) Information ^System® universal numbering system was developed and widely implemented (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) in humans and other vertebrates. Here, CDRs are specified both in terms of amino acid sequence and location in the light or heavy chain. Because the “location” of the CDRs in the immunoglobulin variable domain structure is conserved across species and is present in structures called loops, using numbering systems that align variable domain sequences according to structural features, it is easy to identify CDR and framework residues. This information can be used in the case of grafting and replacement of CDR residues from immunoglobulins of one type into an acceptor framework, usually from a human antibody. The correspondence between Kabat numbering and the unified IMGT numbering system is also well known to one skilled in the art (eg, Lefranc et al., supra). The typical system shown here combines the Kabat and Chothia systems.

Hypervariable regions may contain "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26- 35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. The number of such variable domain residues is 25 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 the carboxy-terminal portion of the light and heavy chain that is not directly involved in antibody-antigen binding but exhibits various effector functions, such as interaction with the Fc receptor. These terms refer to that part of the immunoglobulin molecule that has a more conserved amino acid sequence compared to the other part of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the heavy chain constant CH1, CH2 and CH3 domains and the light chain constant CL domain.

In the context of a polypeptide, the term "derivative" as used herein refers to a polypeptide that contains the 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 that has been altered by substitution, deletion, or insertion amino acid residues. 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 that has been chemically modified, for example, by covalently attaching 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 an anti-PSGL-1 antibody may be chemically modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, by derivatization using known protective agents. /blocking groups, as a result of proteolytic cleavage, binding to a cellular ligand or other protein, and so on. These derivatives are modified in such a way that they differ from the natural or original peptide or polypeptides either in the type or arrangement of the attached molecules. Derivations also include the deletion of one or more chemical groups that are naturally present in the peptide or polypeptide. A derivative of a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or an anti-PSGL-1 antibody may be chemically modified by chemical modification using methods known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, processing regimen , the use of metabolic synthesis of tunicamycin and so on. In addition, a PSGL-1 polypeptide derivative, a PSGL-1 polypeptide fragment, or an anti-PSGL-1 antibody may contain one or more non-classical amino acids. The polypeptide derivative has a function similar or identical to the function of a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or an anti-PSGL-1 antibody described herein.

The term "detection probe" as used herein refers to a compound that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, which provides a detectable signal due to its activity.

The term "diagnostic agent" refers to a substance administered to a subject that assists in the diagnosis of a disease. Such substances can be used to identify, pinpoint and/or localize the disease-causing process. In some embodiments, the diagnostic agent includes a substance conjugated to an antibody provided herein that, when administered to a subject or in contact with a sample obtained from the subject, assists in the diagnosis of cancer, tumorigenesis, or any other VISTA-mediated disease, disorder, or condition.

The term "detectable agent" refers to a substance that can be used to determine the presence or presence of a desired molecule, such as an antibody provided herein, in a sample or body of a subject. The detectable agent may be a substance that can be visualized, or a substance that can be determined and/or otherwise measured (eg, by quantitation).

The term "detection" as used herein covers quantitative or qualitative detection.

The term "encode" or its grammatical equivalents when used in relation to a 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, which can be transcribed to produce mRNA and which is then translated into a polypeptide and/or fragment thereof. The antisense strand is complementary to such a nucleic acid molecule and a coding sequence can be derived from it.

The term "epitope" as used herein refers to the region of an antigen, such as a PSGL-1 polypeptide or a fragment of a PSGL-1 polypeptide, to which an antibody binds. Preferably, the term "epitope" as used herein refers to a localized region on the surface of an antigen, such as a PSGL-1 polypeptide or a fragment of a PSGL-1 polypeptide, that is capable of binding to one or more antigen-binding sites of an antibody and that in an animal such as a mammal (for example, a human) has antigenic or immunogenic activity, that is, it is capable of causing an immune response. An immunogenic epitope is the portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is the portion of a polypeptide to which an antibody binds, as determined by any method well known in the art, for example, using an immunoassay. Antigenic epitopes do not necessarily have to be immunogenic. Epitopes typically consist of chemically active surface moieties of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structure characteristics as well as specific charge characteristics. An epitope can be formed by contiguous residues or non-contiguous residues brought into close proximity by antigen-protein folding. Epitopes formed by contiguous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by non-contiguous amino acids are usually lost when exposed to denaturing solvents. In some embodiments, the PSGL-1 epitope is a spatial component of the surface of the PSGL-1 polypeptide. In other embodiments, the PSGL-1 epitope is a linear component of a PSGL-1 polypeptide. Typically, an antigen has several or many different epitopes and interacts with many different antibodies.

The term "excipient" as used herein refers to an inert substance that is typically used as a diluent, excipient, preservative, binder, or stabilizing agent, and includes, but is 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 (sodium dodecyl sulfate), polysorbate, non-ionic surfactant, etc.), saccharides (eg sucrose, maltose, trehalose, etc.) and polyols (eg mannitol, sorbitol, etc.) . See also Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, which is hereby incorporated by reference in its entirety.

When referring to a peptide or polypeptide, the term "fragment" as used herein refers to a peptide or polypeptide that contains an amino acid sequence less than full length. Such a fragment may arise, for example, as a result of amino-terminal truncation, carboxy-terminal truncation and/or deletion of residue(s) within the amino acid sequence. For example, fragments may result from alternative ribonucleic acid (RNA) splicing or protease activity in vivo. In some embodiments, PSGL-1 or VISTA fragments include polypeptides comprising an amino acid sequence consisting 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 acid residues, at least 70 contiguous amino acid residues, at least at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues residues of 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 PSGL-1 or VISTA polypeptide fragment or 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 region” or “FR” residues refers to those variable domain residues that differ from the hypervariable region residues as defined herein. FR residues are those variable domain residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, single domain antibodies, diabodies, linear antibodies and bispecific antibodies.

A "functional fragment" of an antibody will exhibit at least one biological function, if not some or all of the biological functions inherent in the intact antibody, which function is at least specific binding to a target antigen.

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

The term "heavy chain", when used in relation to an antibody, refers to a polypeptide chain of about 50-70 kDa in size, the amino-terminal part of which includes a variable region consisting of about 120-130 or more amino acids, and the carboxy-terminal part of which includes a constant region region. The constant region can be one of five different types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. These different heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with the light chain, these different 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 IgG1, IgG2, IgG3 and IgG4. The heavy chain may be a human immunoglobulin heavy chain.

The term “hinge region” refers herein to a flexible region of amino acids located in the central part of the heavy chains of immunoglobulins of the IgG and IgA classes, which links these 2 chains with disulfide bonds. Typically, the hinge region is defined as the region extending from Glu216 to Pro230 in the human IgG1 molecule (Burton, Mol. Immunol., 22: 161-206, 1985). The hinge regions of other IgG isotypes can be aligned to the IgG1 sequence by placing the first and last cysteine residues forming S-S bonds between the heavy chains in the same positions. The "CH2 domain" in the Fc portion of human IgG (also called the "Cγ2" domain) generally extends from about amino acid 231 to about amino acid 340. The uniqueness of the CH2 domain is that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are located between these two CH2 domains of the intact native IgG molecule. It has been suggested that the presence of a carbohydrate moiety may provide a substitute for domain-domain pairing and help stabilize the CH2 domain (Burton, Mol. Immunol., 22: 161-206, 1985). The "CH3 domain" contains a region of residues located C-terminal to the CH2 domain in the Fc portion (ie, from about amino acid residue 341 to about amino acid residue 447 in IgG).

The term "host" as used herein refers to an animal, such as a mammal (eg, human).

The term “host cell” as used herein refers to the specific affected cell transfected with the nucleic acid molecule and the progeny or possible progeny of such cell. The 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 subsequent generations, or due to the incorporation of the nucleic acid molecule into the genome of the host cell.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies comprising human immunoglobulins (recipient antibody) in which the native CDR residues are replaced with residues from the corresponding CDR of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and functional activity. In some cases, one or more residues of the FR region of a human immunoglobulin are replaced with corresponding immunoglobulin residues from a non-human species. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve the effectiveness of the antibodies. A humanized antibody heavy or light chain may 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 non-human immunoglobulin, and all or substantially all of the FRs are FRs from the sequence human immunoglobulin. In some embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. 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 US Patent Nos. 6,800,738 (Published Oct. 5, 2004), 6,719971 (Published Sept. 27, 2005), 6,639,055 (Published Oct. 28, 2003), 6407213 (Published June 18, 2002), and 6,054,297 (Published Apr. 25. 2000).

An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount may be administered in one or more than one administration, application or dosing procedure. Such delivery depends on a number of variable parameters, including the period of time over which a particular dosage unit should be used, the bioavailability of the agent, the route of administration, and so on. In some embodiments, an effective amount also refers to the amount of an antibody provided herein required to achieve a particular result (eg, inhibiting PSGL-1 or VISTA-associated biological activity of a cell, such as modulating T cell activation). In some embodiments, the term refers to a therapeutic dosage (eg, of an antibody provided herein) that is sufficient to reduce and/or ameliorate the severity and/or duration of a particular disease, disorder or condition and/or associated symptom. The term also covers an amount necessary to reduce or attenuate the development or progression of a particular disease, disorder or condition, reduce or reduce the likelihood of relapse, development or onset of a particular disease, disorder or condition and/or to improve or enhance prophylactic(s) or therapeutic(s). their) the effect(s) of other therapy (eg, therapy other than the anti-PSGL-1 antibody provided herein). In some embodiments, the effective amount of the antibody is from about 0.1 mg/kg (mg of antibody per kg of body weight of the subject) to about 100 mg/kg. In some embodiments, the effective amount of an antibody provided herein 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 , approximately 15 mg/kg, approximately 20 mg/kg, approximately 25 mg/kg, approximately 30 mg/kg, approximately 35 mg/kg, approximately 40 mg/kg, approximately 45 mg/kg, approximately 50 mg/kg, approximately 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg (or in this range).

The term "inhibit" or its grammatical equivalent, when used in the context of an antibody, refers to an antibody that inhibits, abolishes, or reduces the biological activity of the antigen to which the antibody binds. The inhibitory effect of an antibody may be an effect that results in a change in the biological activity of the antigen that can be measured. In some embodiments, the anti-PSGL-1 antibody described herein inhibits the ability of VISTA to bind to PSGL-1, which may result in suppression of the coinhibitory activity of VISTA. Certain anti-PSGL-1 antibodies described herein inhibit or block suppressive signals from VISTA on VISTA-expressing cells by an amount greater than 5%, such as from about 5% to about 50%, or by an amount greater than 50 % (eg, from about 50% to about 98%) compared to the corresponding control (eg, the control is cells not treated with the test antibody). In some embodiments, an anti-PSGL-1 antibody described herein inhibits the binding of PSGL-1 to the extracellular domain of VISTA and/or inhibits 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 tumor microenvironment, 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 administering other therapeutic agents refers to the use of more than one therapy (eg, the use of an anti-PSGL-1 antibody and an anti-VISTA antibody). Use of the term “in combination” does not limit the order in which the therapeutic agents are administered to a subject or the timing of their administration (eg, one therapy is administered before, concurrently with, or after another therapy). The first therapeutic agent may be administered prior to administration (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), simultaneously with or after administration (e.g., after 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) second therapeutic means to a subject who has had, has, or is susceptible to a VISTA-mediated disease, disorder or condition. Any additional therapeutic agent can be administered with other additional therapeutic agents (eg, anti-PSGL-1 antibody and anti-VISTA antibody) in any order or at any time point. In some embodiments, antibodies may be administered in combination with one or more therapeutic agents (e.g., non-antibody therapeutic agents currently administered to prevent, treat, inhibit, and/or alleviate a VISTA-mediated disease, disorder, or state). Non-limiting examples of therapeutic agents that can be administered in combination with an antibody include the following: a coinhibitory molecule antagonist, a co-stimulatory molecule agonist, a chemotherapeutic agent, radiation, analgesic agents, anesthetics, antibiotics or immunomodulatory agents, or any other agent listed in the US Pharmacopoeia and/or Doctor's Desk Reference.

An "isolated" antibody is substantially free of cellular material or other contaminating proteins from the cellular or tissue source and/or other contaminating components from which the antibody is derived, or substantially free of chemical precursors or other chemical reagents used during chemical synthesis. The expression "substantially free of cellular material" includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations containing less than about 30%, 20%, 10%, or 5% (by dry weight) of a heterologous protein (also referred to herein as a “contaminant protein "). In some embodiments, when the antibody is produced recombinantly, it is substantially free of culture medium, for example, the culture medium constitutes less than about 20%, 10%, or 5% of the volume of the protein preparation. In some embodiments, when an antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemical reagents, for example, it is separated from chemical precursors or other chemical reagents involved in protein synthesis. Accordingly, such antibody preparations contain less than about 30%, 20%, 10%, 5% (by dry weight) chemical precursors or compounds other than the antibody of interest. Contaminants may also include, but are not limited to, materials that would interfere with therapeutic applications of 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 the antibody, as determined by the Lowry method (Lowry et al., J. Bio. Chem., 193: 265-275, 1951), for example, to 99% by weight mass, (2) to an extent sufficient to obtain at least 15 residues at the N-terminus or within the amino acid sequence using a spinning beaker sequencer, or (3) to a homogeneous state as determined by SDS-PAGE (polyacrylamide gel electrophoresis in the presence of SDS) under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. An isolated antibody includes an antibody found in situ within recombinant cells after at least one component of the antibody's natural environment is no longer present. Typically, however, at least one purification step will be used in obtaining the isolated antibody. In a specific embodiment, the antibodies provided herein are isolated.

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

The term "light chain", when used in relation to an antibody, refers to a polypeptide chain of about 25 kDa in size, where the amino-terminal portion includes a variable region consisting of from about 100 to about 110 or more amino acids, and the carboxy-terminal portion includes a constant region . The approximate length of the light chain is 211-217 amino acids. Based on the amino acid sequence of the constant domains, there are two different types of chains called kappa (k) or lambda (A). The amino acid sequences of the light chains are well known in the art. The light chain may be a human immunoglobulin light chain.

As used herein, the terms “inhibit development,” “implement developmental inhibition,” and “inhibit development” refer to the beneficial effects obtained by a subject from a therapy (eg, as a result of the use of a prophylactic or therapeutic agent) that does not cure the disease. In some embodiments, a subject is administered one or more than one therapy (e.g., administered a prophylactic or therapeutic agent, such as an antibody provided herein) to "inhibit the progression" of a VISTA-mediated disease, disorder, or condition, including one or more one of its symptoms in order to prevent the progression or exacerbation of the disease, disorder or condition.

The term "monoclonal antibody" refers to an antibody derived from a population of homogeneous or substantially homogeneous antibodies, i.e. The antibodies that make up this population are largely identical to each other, with the exception of possible natural mutations that may be present in minute quantities. In other words, a monoclonal antibody is a homogeneous antibody that arises from the growth of a clone of a single cell (for example, a hybridoma, a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, etc. .d.) and is usually characterized by the presence of heavy chains of one and only one class and subclass and light chains of only one type. Such antibodies are highly specific and directed against a single antigen. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants or epitopes, each monoclonal antibody is directed against a single epitope of an antigen. In some embodiments, the term "monoclonal antibody" as used herein means an antibody produced by a single hybridoma or other cell, wherein the antibody binds only to a VISTA epitope, as determined, for example, using an ELISA or other antigen binding or competitive binding assay , known in the art. The term "monoclonal" is not limited to any particular method of producing an antibody. For example, the monoclonal antibodies provided herein can be produced by the hybridoma method as described in Kohler et al., Nature, 256:495 (1975), or can be produced using isolation methods from phage libraries. Other methods for generating clonal cell lines and the monoclonal antibodies they express 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 for producing other monoclonal antibodies are set forth herein in the Examples section.

The term "natural" when used in reference to biological materials such as nucleic acid molecules, polypeptides, host cells and the like, refers to those that occur naturally and are not derived from human activities.

The term "pharmaceutically acceptable" as used herein means "approved for use by a federal or state regulatory agency" or listed in the United States Pharmacopoeia, European Pharmacopoeia, or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans.

The term "polyclonal antibodies" as used herein refers to a population of antibodies formed as a result of an immune response to a protein that has many epitopes, and therefore includes many different antibodies directed to the same and different epitopes of a given protein. Methods for producing polyclonal antibodies are 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).

As used herein, the terms "polynucleotide", "nucleotide", "nucleic acid", "nucleic acid molecule" and other similar terms are used interchangeably and include DNA, RNA, mRNA and the like.

As used herein, the terms “prevent,” “warning,” and “preventing” refer to the complete or partial inhibition of the development, recurrence, onset, or spread of a VISTA-mediated disease, disorder, or condition and/or associated symptom as a result of administration of a therapeutic agent or combinations of therapeutic agents provided herein (eg, combinations of prophylactic or therapeutic agents, such as an antibody provided herein).

As used herein, the term “prophylactic agent” refers to any agent that can completely or partially inhibit the development, relapse, onset or spread of a VISTA-mediated disease, disorder or condition and/or associated symptom in a subject. In some embodiments, the term “prophylactic agent” refers to the anti-PSGL-1 antibody provided herein. In certain other embodiments, the term “prophylactic agent” refers to an agent other than the anti-PSGL-1 antibody provided herein. In some embodiments, a prophylactic agent is an agent that is known to be useful or has been used or is currently used to prevent or prevent the onset, development, progression and/or attenuation of a VISTA-mediated disease, disorder or condition and/or an associated symptom. the severity of the VISTA-mediated disease, disorder or condition and/or associated symptom. 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, P-selectin ligand, SELPLG, CLA and CD162) refers to a polypeptide ("polypeptide", "peptide" and "protein" are used interchangeably herein) , encoded by the SELPLG gene, for example, containing the amino acid sequence:

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

As will be apparent to those skilled in the art, the anti-PSGL-1 antibody provided herein can bind to a polypeptide, polypeptide fragment, antigen, and/or epitope of PSGL-1, as long as the epitope is part of a longer antigen, e.g. part of a longer fragment of a polypeptide, which in turn, for example, is part of a longer polypeptide. PSGL-1 can be in native or denatured form. The PSGL-1 polypeptides described herein can be isolated from various sources, for example, from different types of human tissue or other source, or obtained by recombinant or synthetic methods. A “native sequence PSGL-1 polypeptide” is a polypeptide having the same amino acid sequence as the corresponding naturally occurring PSGL-1 polypeptide. Such native sequence PSGL-1 polypeptides can be isolated from natural sources or can be produced by recombinant or synthetic methods. The term “native sequence PSGL-1 polypeptide” specifically covers naturally occurring truncated or secreted forms of a particular PSGL-1 polypeptide (e.g., extracellular domain sequence), naturally occurring variant forms (e.g., forms resulting from alternative splicing), and naturally occurring allelic variants of this polypeptide.

The nucleic acid sequence of the cDNA encoding the PSGL-1 polypeptide, for example, contains:

Orthologues of the human PSGL-1 polypeptide are also well known in the art. For example, orthologs of PSGL-1 can be found among organisms such as mouse (Mus musculus), rat (Rattus norvegicus), dog (Canis lupus familiaris), cow (Bos taurus), zebrafish (Danio rerio), horse (Equus caballus), chimpanzees (Pan troglodytes) and so on.

The terms "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 wholly or partly caused by or which results from the presence of PSGL -1. Such diseases, disorders or conditions include those caused by or otherwise associated with PSGL-1, including those caused by or associated with PSGL-1-expressing cells (eg, tumor cells, myeloid-derived suppressor cells (MDSCs), suppressive dendritic cells cells (having a suppressive effect on DC) and/or regulatory T cells (T _reg )). In some embodiments, PSGL-1 is aberrantly (eg, highly) expressed on the cell surface. In some embodiments, PSGL-1 may be aberrantly activated on a particular cell type. In other embodiments, normal, aberrant or excessive cell signaling is due to the binding of PSGL-1 to a PSGL-1 ligand (eg, VISTA), which can bind or otherwise interact with PSGL-1. In a preferred embodiment, PSGL-1-mediated disease is caused by the binding of PSGL-1 to a specific PSGL-1 ligand (eg, VISTA), but not to other ligands (eg, selectins).

The term "irradiation", when used in a therapeutic context, refers to a type of treatment that uses a beam of intense energy to kill target cells (eg, cancer cells). Radiation therapy uses x-rays, protons or other forms of energy delivered through an external beam. Radiation therapy also includes radiation therapy in which a radiation source is placed into the patient's body (such as brachytherapy), with a small container of radioactive material implanted directly into or near the tumor.

The term "relative expression level" refers to the quantification of the expression level of a protein in a given sample relative to another reference protein in the same sample and/or relative to another comparison sample. In the context of the methods described herein, the level of expression of PSGL-1 can be expressed in absolute numbers, for example, taking into account a standard curve, or can be expressed in relative levels of expression compared to one or more other proteins that are to be analyzed in a given sample ( eg VISTA, CD11b, CD33, CD4 or CD8).

The term "recombinant antibody" refers to an antibody that is produced, expressed, created or isolated using recombinant methods. Recombinant antibodies can be antibodies expressed using a recombinant expression vector with which a host cell has been transfected, antibodies isolated from combinatorial libraries of recombinant antibodies, antibodies isolated from an animal (for example, mouse or cow) that is transgenic and/or transchromosomal for immunoglobulin genes human (see, for example, Taylor L. D. et al. (1992) Nucl. Acids Res., 20: 6287-6295), or antibodies produced, expressed, created or isolated using any other method that involves splicing immunoglobulin gene sequences with other DNA sequences. Such recombinant antibodies may 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). However, in some embodiments, such recombinant antibodies are subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which, being derived from and related to VH and VL sequences from human germline antibodies, cannot naturally exist in the human germline antibody repertoire in vivo.

As used herein, the term “side effects” includes undesirable and unfavorable effects of a therapy (eg, a prophylactic or therapeutic agent). Undesired effects are not necessarily unfavorable. An adverse effect of a therapy (eg, a prophylactic or therapeutic agent) may be harmful or unpleasant, or may be associated with risk. Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, hypohydration, alopecia, shortness of breath, insomnia, dizziness, mucositis, nerve and muscle effects , fatigue, dry mouth and decreased appetite, rashes or swelling at the injection site, flu-like symptoms such as fever, chills and fatigue, digestive problems and allergic reactions. Additional undesirable effects experienced by patients are numerous and known in the art. Many of them are described in the Doctor's Desk Reference (67th ed., 2013).

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

As used herein, "substantially all" means 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 to treat, prevent or alleviate a disease, disorder or condition, including treating, preventing or alleviating one or more symptoms of a VISTA-mediated disease, disorder or condition and/or symptom associated with it. In some embodiments, the term “therapeutic agent” refers to the anti-PSGL-1 antibody provided herein. In some embodiments, the term “therapeutic agent” refers to an agent other than the anti-PSGL-1 antibody provided herein. In some embodiments, a therapeutic agent is an agent that is known to be useful or has been or is currently used for treating, preventing, or alleviating one or more symptoms of a VISTA-mediated disease, disorder, condition, or related symptom.

The effect of a combination of therapeutic agents (eg, the use of therapeutic agents) may be greater than the additive effects of any two or more monotherapies. For example, the synergistic effect of a combination of therapeutic agents allows the use of lower dosages of one or more agents and/or the use of less frequent administration of these agents to a subject with a VISTA-mediated disease, disorder or condition and/or associated symptom. The ability to use lower dosages when treating therapeutics and/or administering therapeutics less frequently reduces the toxicity associated with administering therapeutics to a subject without reducing the effectiveness of these therapies in preventing, treating, or alleviating one or more symptoms of a VISTA-mediated disease, disorder or condition and/or symptom associated with it. In addition, the synergistic effect may result in improved effectiveness of the therapies in preventing, treating, or alleviating one or more symptoms of a VISTA-mediated disease, disorder, or condition and/or a related symptom. Finally, the synergistic effect of a combination of therapeutic agents (eg, therapeutic agents) may prevent or reduce adverse or undesirable side effects associated with the use of any monotherapy.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent (e.g., an anti-PSGL antibody or any other therapeutic agent, including those described herein, including, for example, an anti-VISTA antibody) that is sufficient to reduce and/or reducing the severity and/or duration of a particular disease, disorder or condition and/or symptom associated therewith. A therapeutically effective amount of a therapeutic agent may be the amount necessary to reduce or attenuate the development or progression of a particular disease, disorder or condition, reduce or reduce the likelihood of relapse, development of onset of a particular disease, disorder or condition, and/or improve or enhance the prophylactic or therapeutic effect of another therapy (eg, therapy other than the anti-PSGL antibody, including that described herein).

As used herein, the term “therapy” refers to any protocol, method and/or agent that can be used to prevent, inhibit, treat and/or alleviate a VISTA-mediated disease, disorder or condition. In some embodiments, the terms "therapies" and "therapy" refer to biologic therapies, supportive therapies, and/or other therapies useful for treating, preventing, and/or ameliorating a VISTA-mediated disease, disorder, or condition known to one of ordinary skill in the art. such as a member of the medical staff.

As used herein, the terms “treat,” “treating,” and “treating” refer to reducing and/or attenuating the progression, severity, and/or duration of a VISTA-mediated disease, disorder, or condition as a result of one or more therapies (including (but not limited to, administration of one or more therapeutic agents, such as an anti-PSGL-1 antibody, including those described herein). In some embodiments, such terms refer to attenuating or inhibiting a cancer (eg, a hematologic malignancy). In some embodiments, such terms refer to reducing and/or attenuating the progression, severity and/or duration of a disease, disorder or condition that is susceptible to immune modulation, such as modulation resulting from increased T cell activation.

The term "tumor microenvironment" refers to the cellular environment in which the tumor resides. The tumor microenvironment may include surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix.

The term "variable domain" or "variable region" refers to a portion of the light or heavy chains of an antibody that is typically located at the amino-terminal portion of the light or heavy chain and is about 120-130 amino acids long in the heavy chain and about 100-110 amino acids long in the light chain. chain, is used to bind each specific antibody to its specific antigen and determines the specificity of each specific antibody for its specific antigen. Variable domains vary greatly in sequence among different antibodies. Variation in the sequence is concentrated in the CDRs, while the less variable parts in the variable domain are called framework regions (FRs). Each variable region contains three CDRs that are connected to four FRs. The light and heavy chain CDRs are primarily responsible for the interaction of the antibody with the antigen. Although FR is not directly involved in antigen binding, it determines the folding of molecules and thus the number of CDRs that are presented on the surface of the variable region for interaction with the antigen. In some embodiments, the variable region is a human immunoglobulin variable region.

The term “Kabat variable domain residue numbering” or “Kabat amino acid position numbering” and variants thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains in the antibody data summary of Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD. (1991). When using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or contain additional amino acids in accordance with the shortening of the FR or CDR of the variable domain or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (Kabat residue 52a) after residue 52 in H2 and inserted residues (eg, Kabat residues 82a, 82b and 82c, etc.) after residue 82 in the heavy chain FR. Kabat residue numbering can be determined for a given antibody by aligning regions of homology of the antibody sequence with the Kabat numbered “standard” sequence. The Kabat numbering system is typically used when referring to a residue in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (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 typically used when referring to a residue in the constant region of an immunoglobulin heavy chain (eg, the EU index given by Kabat et al., supra). The term "Kabat EU index" refers to the residue numbering of the EU antibody representing human IgG1. Unless otherwise indicated herein, references to residue numbers in the antibody variable domain indicate residue numbering according to the Kabat numbering system. Other numbering systems have been described, including, for example, the AbM database, Chothia, the contact method, and IMGT.

The term “variant,” when used with respect to PSGL-1, VISTA, or an anti-PSGL-1 antibody or an anti-VISTA antibody, refers to a peptide or polypeptide containing one or more (e.g., from about 1 to about 25, from about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) substitutions, deletions and/or insertions in the amino acid sequence compared to the native or unmodified sequence. For example, a PSGL-1 or VISTA variant may result from one or more (e.g., 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 approximately 5) changes in the amino acid sequence of native PSGL-1 or VISTA, respectively. Also by way of example, a variant anti-PSGL-1 antibody or anti-VISTA antibody may result from one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about about 10 or about 1 to about 5) changes in the 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 engineered. Polypeptide variants can be derived from the corresponding nucleic acid molecules encoding the variants. In some embodiments, the PSGL-1 variant, VISTA variant, or anti-PSGL-1 antibody or anti-VISTA antibody variant at least retains the functional activity of PSGL-1, VISTA, anti-PSGL-1 antibody, or anti-VISTA antibody, respectively. In some embodiments, the anti-PSGL-1 antibody variant binds to PSGL-1 and/or exhibits PSGL-1 antagonistic activity. In some embodiments, the anti-VISTA antibody variant binds to VISTA and/or exhibits VISTA antagonistic activity. In some embodiments, the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes the PSGL-1, VISTA, VH or VL regions or subregions of an anti-PSGL-1 antibody or an anti-VISTA antibody.

The term "vector" refers to a substance that is used to introduce a nucleic acid molecule into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which may include selection sequences or selectable markers suitable for stable integration into the host cell chromosome. In addition, such vectors may include one or more selectable marker genes and corresponding expression control sequences. Selectable marker genes that can be introduced provide, for example, resistance to antibiotics or toxins, auxotrophic complement deficiency, or the supply of essential nutrients not available in culture media. Expression control sequences may 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 expressed together (eg, for both heavy and light chains of an antibody), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. In the case of a single expression vector, the encoding nucleic acids may be operably linked to one common expression control sequence or may be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. 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 blotting, or mRNA amplification using polymerase chain reaction (PCR), or immunoblotting to analyze the expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will appreciate that the nucleic acid molecule is expressed in sufficient quantities to produce the desired product (e.g., the anti-PSGL-1 antibody provided herein), and it will also be understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

DETAILED DESCRIPTION

In the practical application of this invention, unless otherwise indicated, traditional methods of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, synthesis and modification of oligonucleotides, nucleic acid hybridization and methods of related fields that are within the competence of a specialist are used. in this field of technology. These methods are described in the references cited herein and are fully disclosed in the literature. See, for example, 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 & Sons (1987 and annual revisions); Current Protocols in Immunology, John Wiley & Sons (1987 and annual revisions); 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 that includes the extracellular matrix and non-tumor host cells, including mesenchymal cells, vascular endothelial cells and inflammatory or immune cells. The microenvironment plays a critical role in suppressing tumor-specific T cell responses. To ensure that the immune inflammatory response is not continuously activated after tumor antigens have stimulated the response, multiple checkpoints exist or are activated. These checkpoints are primarily the T cell receptor, which binds to ligands on cells in the tumor microenvironment to form immunological synapses, which then regulate T cell function.

VISTA is one such immune checkpoint. Expression of this protein is restricted to hematopoietic cells, and in many cancer models it has been detected only on tumor-infiltrating leukocytes and not on tumor cells. VISTA negatively regulates T cell immunity through direct effects on T cells involving various receptors/ligands because, unique among immune checkpoint proteins, it acts as both a ligand and a receptor (Le Mercier, supra).

We have now identified that PSGL-1 is a binding partner (eg, ligand or receptor) of the VISTA protein. PSGL-1 is a 120-kDa homodimeric transmembrane glycoprotein bearing O- and N-linked glycans whose best known role is in immune cell trafficking through binding to selectins. PSGL-1 is expressed in cells of the lymphoid, myeloid and dendritic lineages (Laszik et al., Blood, 88(8): 3010-21 (1996)). Naive T cells express a non-selectin-binding form of PSGL-1, which can interact with potentially other currently unknown binding partners (Veerman et al, Nat. Immunol., 8(5), 532-539 (2007)). Expression in tumor cells was also observed. Recently, PSGL-1 was demonstrated to stimulate T cell exhaustion, thereby promoting melanoma tumor growth through the induction of an unknown partner (Tinoco et al., Immunity, 44: 1190-03 (2016)).

We demonstrated direct binding between these two proteins and showed that PSGL-1 and VISTA act together to prevent T cell activation. Indeed, the physical interaction between VISTA and PSGL-1 underlies the functional interaction, as both genes are coexpressed in a number of tumors. None of the other putative VISTA receptors exhibit such co-localization, highlighting the specificity of this relationship. In addition, VISTA and PSGL-1 are expressed in the microenvironment of tumor cells. More specifically, in situ hybridization revealed that both genes are expressed in neighboring cells in the tumor microenvironment. In immune infiltrates, each PSGL-1-expressing cell is located close to a VISTA-expressing cell, indicating that PSGL-1 is a reliable surrogate for activated VISTA.

DIAGNOSIS OF VISTA-MEDIATED DISORDERS

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

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

a) contacting a biological sample from said subject with a reagent capable of binding to the PSGL-1 protein or PSGL-1 nucleic acid; And

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

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

The reagent capable of binding to the PSGL-1 protein or nucleic acid may be any reagent or any compound known to those skilled in the art that has the ability to specifically bind to PSGL-1. For example, one skilled in the art will readily recognize that a DNA or RNA probe that specifically hybridizes to PSGL-1 specifically binds to PSGL-1. Likewise, one skilled in the art will readily recognize that an anti-PSGL-1 antibody, such as the antibody described herein, specifically binds to PSGL-1.

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

a) contacting a biological sample from said subject with a reagent capable of binding to the PSGL-1 protein or PSGL-1 nucleic acid; And

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

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

As will be apparent to one skilled in the art, the level of PSGL-1 binding reagent can be quantified by any method known to those skilled in the art, as described in detail below. Preferred methods include the use of enzyme-linked immunosorbent assays such as ELISA or enzyme-linked immunospot assay (ELISPOT), immunofluorescence, immunohistochemistry (IHC), radioimmunoassay (RIA) or FACS.

The result of the quantitation in step b) of the method of the present invention is a direct reflection of the level of PSGL-1 expression in the sample, primarily in the immune infiltrates of the tumor microenvironment. Thus, the method of the present invention provides the ability to identify VISTA-mediated cancer by determining the expression level of PSGL-1, as described above. In a preferred embodiment, the expression level of PSGL-1 in said sample, primarily in immune infiltrates of the tumor microenvironment, is compared with a reference level.

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

a) determining the level of expression of PSGL-1 in a biological sample from the specified subject; And

b) comparing the expression level at stage a) with the reference level;

in this case, an increase in the analyzed level of PSGL-1 at stage a) compared to the reference level indicates the presence of a VISTA-mediated disease, disorder or condition.

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

a) determining the level of expression of PSGL-1 in a biological sample from the specified subject; And

b) comparing the expression level at stage a) with the reference level;

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

The level of PSGL-1 expression is preferably compared or measured relative to levels in a control cell or control sample, also referred to as a “reference level” or “reference expression level”. The terms "reference level", "reference expression level", "control level" and "control" are used interchangeably herein. “Reference level” means a single baseline measured in a comparable control cell that is typically not affected by disease or cancer. This control cell can be obtained from the same individual because, even in a cancer patient, the tissue that is the site of tumor development still contains healthy tissue that is not affected by the tumor. It may also come from another individual who is normal or who does not have the same disease as the individual from whom the affected or test sample was obtained. As used herein, the term “reference level” refers to a “control level” of PSGL-1 expression used to assess the test level of PSGL-1 expression in a cancer cell-containing sample from a patient. For example, if the level of PSGL-1 in a biological sample from a patient is higher than the PSGL-1 reference level, then such cells will be considered to have a high level of expression or show overexpression of PSGL-1. The reference level can be determined by a variety of methods. Therefore, expression levels may be determined by cells bearing PSGL-1, or, alternatively, the level of PSGL-1 expression is independent of the number of cells expressing PSGL-1. Thus, the reference level for each patient can be prescribed by the reference ratio for PSGL-1, and such reference ratio can be determined by any of the methods for determining reference levels set forth herein.

For example, control may be a predetermined value that may take various forms. This could be a single cut-off value such as the median or mean. The “reference level” may be expressed as a single number that is equally applicable to each patient individually, or the reference level may vary depending on specific subpopulations of patients. Thus, for example, older men may have a different reference level than younger men for the same cancer, and women may have a different reference level than men for the same cancer. Alternatively, a “reference level” can be determined by measuring the expression level of PSGL-1 in non-tumorigenic cancer cells from the same tissue as the tissue of the malignant tumor cells to be tested. Equally, a “reference level” may be a defined ratio between the levels of PSGL-1 in a patient's malignant tumor cells and PSGL-1 in non-tumor cells from the same patient. In addition, the "reference level" may be the level of PSGL-1 in cells cultured in vitro, which can be manipulated to mimic tumor cells or can be manipulated in any other manner that produces expression levels that accurately define the reference level. On the other hand, a "reference level" may be set in consideration of the comparison groups, such as groups not having elevated PSGL-1 levels and groups having elevated PSGL-1 levels. Another example of groups used for comparison would be groups having a particular disease, condition or symptoms, and groups without such disease. The predetermined value may be classified, for example, as follows: the test population is divided equally (or unequally) into groups such as a low-risk group, a medium-risk group, and a high-risk group.

A reference level can also be determined by comparing PSGL-1 levels in patient populations with the same type of cancer. This can be done, for example, by analyzing a histogram in which the entire cohort of patients is represented graphically, with the first axis representing the level of PSGL-1 and the second axis representing the number of patients in the cohort whose tumor cells express PSGL-1 at a given level . By identifying subsets of this cohort's populations that have the same or similar levels of PSGL-1, two or more distinct groups of patients can be identified. The determination of the reference level can then be made based on the level that best shows the difference for these individual groups. The reference level may also represent the levels of two or more markers, one of which is PSGL-1. Two or more markers may be represented, for example, by a ratio of values for the levels of each marker.

Likewise, it is apparent that a healthy population will have a different "normal" range compared to the range of a population known to have a condition associated with PSGL-1 expression. Accordingly, the selected predetermined value may take into account the category into which the individual falls. Routine experimentation may be sufficient for those of ordinary skill in the art to select appropriate ranges and categories. By "increased" or "higher" is meant "high relative to the selected control." Typically, the reference level will be determined for apparently healthy normal individuals of the appropriate age category.

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

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

In some embodiments, expression of a given protein indicates the presence of a particular cell type in the sample. For example, expression of PSGL-1, CD4 and/or CD8 cells in a sample may indicate the presence of T cells in the sample. Likewise, expression of VISTA, alone or in combination with CD11b or CD33 cells in a sample, may indicate the presence of VISTA-bearing tumor cells, regulatory T cells (eg, CD4 ⁺ Foxp3 ⁺ regulatory T cells), myeloid suppressor cells ( for example, CD11b ⁺ or CD11 b ^high and/or CD33 ⁺ myeloid suppressor cells) and/or suppressive dendritic cells (for example, CD11b ⁺ or CD11 b ^high dendritic cells). Preferably, expression of VISTA, CD11b, CD33, CD4 and CD8, primarily in immune infiltrates of the tumor microenvironment, indicates the presence of VISTA-mediated cancer in the subject.

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

a) determining the level of expression of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 in a biological sample from the specified subject; And

b) comparing the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 at stage a) with a reference level;

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

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

a) determining the level of expression of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 in a biological sample from the specified subject; And

b) comparing the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 at stage a) with a reference level;

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

A more accurate diagnosis of a VISTA-mediated disease, disorder, or condition may allow health care providers to implement preventive measures or aggressive treatment earlier, thereby preventing the development or further progression of the VISTA-mediated disease, disorder, or condition.

IDENTIFICATION OF PATIENTS SENSITIVE TO ANTI-VISTA THERAPEUTIC AGENTS

The above data indicate that PSGL-1 is a valid biomarker for the diagnosis of VISTA-mediated disorder such as VISTA-mediated cancer. Patients thus identified are susceptible to anti-VISTA therapeutic agents.

In another aspect, the invention relates to a method for identifying in vitro tumor patients who will be susceptible to treatment with an anti-VISTA therapeutic agent. Preferably, said patients express PSGL-1, primarily in immune infiltrates, and expression of PSGL-1 indicates that said patients will be susceptible to treatment with an anti-VISTA therapeutic agent.

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

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

b) comparing the expression level at stage a) with the reference level; And

c) diagnosing that the cancer is responsive to treatment with a VISTA blocking agent based on said comparison.

In another embodiment, said reference level is the expression level 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 receptive to the treatment. In a preferred embodiment, a similar level of PSGL-1 expression in the first biological sample compared to the level of PSGL-1 expression in the second biological sample indicates that the first patient will be responsive to treatment.

In another embodiment, step a) comprises determining the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 in said biological sample, preferably using immune infiltrates. Preferably, the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8, or relative expression levels thereof, in the first biological sample is compared with the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8, or the relative levels of expression thereof, in a second biological sample from a second patient having the same VISTA-mediated cancer as the first patient, the second patient being receptive to the treatment. In a preferred embodiment, similar or relative expression levels of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 in the first biological sample compared to the expression level of PSGL-1 and at least one of VISTA, CD11 , CD33, CD4 and CD8 or the relative levels of their expression in the second biological sample indicates that the first patient will be responsive to treatment.

MEASUREMENT OF PSGL-1 EXPRESSION

PSGL-1 expression can be measured by any means available to those skilled in the art. Thus, PSGL-1 expression can be assessed by measuring the level of PSGL-1 nucleic acid (eg, mRNA or corresponding cDNA for PSGL-1) or by measuring the level of PSGL-1 protein.

In this case, the method of the invention may include one or more intermediate steps between collecting a biological sample and measuring the level of PSGL-1 expression, said steps corresponding to isolating a sample of mRNA (or corresponding cDNA) or a sample of protein from said biological sample. Obtaining or isolating mRNA (and its reverse transcription into cDNA) or proteins from a cellular sample are simply routine techniques well known to those skilled in the art.

Once a sample of mRNA (or corresponding cDNA) or protein has been obtained, the level of PSGL-1 expression can be measured, either by mRNA (i.e., relative to the total amount of mRNA or cDNA present in the sample) or by protein (i.e., relative to the total amount protein present in the sample). In such a case, the method used for this purpose depends on the type of transformation (mRNA, cDNA or protein) and the type of sample available.

When marker expression is measured from mRNA (or corresponding cDNA), any method commonly used by those skilled in the art can be used. These technologies for analyzing gene expression levels, such as transcriptome analysis, involve the use of well-known methods such as PCR (polymerase chain reaction if DNA is used), RT-PCR (reverse transcription PCR if RNA is used) or quantitative RT-PCR, or nucleic acid arrays (including DNA arrays and oligonucleotide arrays) for greater throughput.

The term “nucleic acid arrays” as used herein refers to a variety of different nucleic acid probes attached to a support, which may be a microchip, a glass slide, or a microsphere-sized bead. The microchip can be made of polymers, plastics, resins, polysaccharides, silica or silica-based material, carbon material, metals, inorganic glass or nitrocellulose.

The probes may be nucleic acids such as cDNA ("cDNA array"), mRNA ("mRNA array") or oligonucleotides ("oligonucleotide array"), these oligonucleotides typically being suitably about 25-60 nucleotides in length.

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

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

In a preferred embodiment, the expression profile is determined using quantitative PCR. The quantitative PCR or real-time PCR method is a well-known and readily available technology to those skilled in the art and does not need to be described in detail.

In a specific embodiment, which should not be construed as limiting the scope of the present invention, expression profiling using quantitative PCR can be performed as follows. Briefly, real-time PCR reactions were performed using TaqMan® Universal PCR Master Mix (Applied Biosystems). Add 6 µl of cDNA to 9 µl of PCR mix containing 7.5 µl of TaqMan® Universal PCR Master Mix, 0.75 µl of 20x probe and primer mix and 0.75 µl of water. This reaction consists of one initial step, lasting 2 min at 50°C, then 10 min at 95°C, and 40 amplification cycles, including 15 s at 95°C and 1 min at 60°C. This reaction and data collection can be performed using an ABI PRISM 7900 sequence detection system (Applied Biosystems). The number of molecules transcribed from the template in the sample is determined by recording the amplification cycle in exponential phase (threshold cycle value or _Ct ) at the time when a fluorescence signal above background fluorescence can be detected. Thus, the initial number of molecules transcribed from the matrix is inversely proportional to C _t .

In another preferred embodiment, the expression profile is determined using a nucleic acid microarray.

The present invention also relates to a microchip intended for implementing the methods of the invention, containing no more than 500, preferably no more than 300, no more than 200, more preferably no more than 150, no more than 100, even more preferably no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 different probes, at least 1 of which specifically binds to the mRNA (or corresponding cDNA) for PSGL-1 or PSGL-1 protein. In a preferred embodiment, said microchip is a nucleic acid microchip containing no more than 500, preferably no more than 300, no more than 200, more preferably no more than 150, no more than 100, even more preferably no more than 75, no more than 50, no more than 40, no more 30, no more than 20, no more than 10 different probes (thus excluding, for example, pangenomic microarrays), at least 1 of which specifically binds to the mRNA (or corresponding cDNA) for PSGL-1. The microarray may also contain at least one probe that specifically hybridizes to a housekeeping gene in addition to a probe that specifically hydridizes to PSGL-1. For example, the housekeeping gene is the beta-2-microglobulin gene.

Alternatively, any existing or future technology suitable for determining gene expression based on the amount of mRNA in a sample can be used. For example, those skilled in the art can measure gene expression by hybridization with a labeled nucleic acid probe, such as Northern blotting (in the case of mRNA) or Southern blotting (in the case of cDNA), but also using methods such as serial analysis gene expression (SAGE) and its variants, such as longSAGE, superSAGE, deepSAGE and so on.

Additionally, tissue microchips (also known as TMAs) can be used as starting material. TMAs consist of paraffin blocks in which up to 1000 individual tissue “cylinders” (tissue cores) are assembled in an array, which allows for multiplex histological analysis. The tissue microarray technique uses a hollow needle as small as 0.6 mm in diameter to extract tissue “cylinders” from areas of interest in paraffin-embedded tissue, such as clinical biopsies or tumor samples. Then these tissue “cylinders” are built into a paraffin block recipient with a strictly ordered arrangement pattern. Sections from this block are prepared using a microtome, mounted on a microscope slide, and then analyzed by any standard histological method. From each microarray block, 100-500 sections can be prepared, which can be independently tested. Tests commonly used for tissue microarrays include immunohistochemistry and fluorescence in situ hybridization. For analysis at the mRNA level, tissue microarray technology can be combined with fluorescence in situ hybridization. An example of in situ hybridization using RNAscope 2.5 (Advanced Cell Diagnostics, Hayward, CA) is shown in the Examples section.

Finally, massively parallel sequencing (using RNA sequencing (RNA-Seq) or "shotgun whole transcriptome sequencing") can be used to determine the amount of mRNA in a sample. Many massively parallel sequencing methods are available for this purpose. Such methods are described, for example, in US 4882127; US 4849077; US 7556922; US 6723513; WO 03/066896; WO 2007/111924; US 2008/0020392; WO 2006/084132; US 2009/0186349; US 2009/0181860; US 2009/0181385; US 2006/0275782; EP-B1-1141399; Shendure & Ji, Nat Biotechnol., 26(10): 1135-45 (2008); Pihlak et al. War. Biotechnol., 26(6): 676-684 (2008); Fuller et al., Nature Biotechnol., 27(11): 1013-1023 (2009); Mardis, Genome Med., 1(4): 40 (2009); Metzker, Nature Rev. Genet, 11(1): 31-46 (2010).

If marker expression is assessed in relation to the protein, then antibodies specific to PSGL-1 can be used. Binding to anti-PSGL-1 antibody can be detected and/or quantified and/or assessed using various assays available to one skilled in the art, such as, for example, immunoprecipitation, immunohistochemistry (IHC), Western blotting, dot blotting , ELISA, ELISPOT, use of protein chips, antibody chips or tissue chips in combination with immunohistochemistry. Other techniques that may be used include fluorescence activated cell sorting (FACS), fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) techniques, microscopy or histochemistry techniques, especially including confocal microscopy and electron microscopy techniques. based on the use of one or more excitation wavelengths and a suitable optical method, as well as electrochemical methods (voltametry and amperometry methods), atomic force microscopy methods and radio frequency methods, such as multipole resonance spectroscopy, confocal and non-confocal, fluorescence signal detection, luminescence, chemiluminescence, absorption, reflection, transmission and birefringence or refractive index (e.g. surface plasmon resonance, ellipsometry, resonance mirror, etc.), flow cytometry, radioisotope or magnetic resonance imaging, analysis by polyacrylamide electrophoresis gel in the presence of SDS (SDS-PAGE); through a combination of high-performance liquid chromatography (HPLC) and mass spectrophotometry, through liquid chromatography/mass spectrophotometry/mass spectrometry (LC-MS/MS). All of these methods are well known to those skilled in the art and need not be described in detail herein.

Although any of the above methods are suitable for carrying out the methods of the present invention, particular mention may be made of FACS, ELISA, ELISPOT, Western blotting and IHC. Preferred methods include ELISPOT, FACS and IHC.

DETERMINATION OF PSGL-1 TUMOR STATUS

Determination of reagent binding to PSGL-1 (as described above) allows the PSGL-1 status of the tumor to be treated to be determined. PSGL-1 status can be determined by any method or method known or currently used by one skilled in the art, typically based on determining the level of PSGL-1 expression. Based on the PSGL-1 status of the tumor, it can then be predicted whether the patient will be receptive to an anti-VISTA therapeutic agent.

It has recently become apparent that immunological data (the type, density and location of immune cells in tumor samples) are a more reliable predictor of patient survival than the histopathological methods currently used to determine the stage of colorectal cancer.

In addition, increasing evidence from clinical trials supports the potential of immune-targeted therapies in certain types of cancer (Robert et al., Stagg et al.). This has led to the development of more standardized ways to characterize the tumor immune infiltrate in cancer, for example by “immune score”, with the goal of quantifying in situ immune infiltrate in addition to standardized clinical parameters to aid in prognosis and patient selection for immunotherapy in cancer. various types of cancer (see, for example, Galon et al., J. Pathol, 232(2): 199-209 (2014); Galon et al., J. Transl. Med., 14: 273 (2016)).

Thus, the method for detecting or diagnosing VISTA-mediated cancer described in this application includes determining a PSGL-1 score for the tumor.

According to this embodiment, the method includes the steps of:

a) contacting a biological sample from said subject with a reagent capable of binding to the PSGL-1 protein or PSGL-1 nucleic acid;

b) quantifying the binding of said reagent to said biological sample; And

c) assessing tumor cells by comparing the quantitative value of stage a) with an appropriate scale based on two parameters, the intensity of the staining and the percentage of positive cells.

In a preferred embodiment, step b) comprises quantifying the binding of said reagent to PSGL-1 in immune infiltrates of the tumor microenvironment in said biological sample.

According to this preferred embodiment the method comprises the steps of:

a) contacting a biological sample from said subject with a reagent capable of binding to the PSGL-1 protein or PSGL-1 nucleic acid;

b) quantifying the binding of said reagent to said biological sample; And

c) assessing the immune cells of the tumor by comparing the quantitative value of the level in stage a) with an appropriate scale based on two parameters representing the intensity of staining and the percentage of positive cells.

Tumor immune cells (or immune infiltrates) contain immune cells present in the tumor microenvironment, primarily immunosuppressive cells of the tumor microenvironment, like some macrophages, monocytes, and so on. In a preferred embodiment, immune infiltrates include lymphocytes (eg, T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells and macrophages. Accordingly, in this embodiment, step b) involves quantifying the binding of said reagent to PSGL-1 on lymphocytes (eg, T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells and macrophages present in the microenvironment tumors in the specified biological sample.

Any traditional hazard analysis method can be used to evaluate the predictive value of PSGL-1. Representative analysis methods include Cox regression analysis, which is a semiparametric method for modeling survival or time-to-event data in the presence of censored cases (Hosmer and Lemeshow, 1999; Cox, 1972). Unlike other survival analyses, such as life tables or the Kaplan-Meier procedure, Cox analysis allows prognostic variables (covariates) to be included in such models. Using a conventional assay such as the Cox assay, it is possible to test hypotheses regarding the correlation of PSGL-1 expression status in the primary tumor with either time to disease recurrence (disease-free survival time or time to disease metastasis) or time to death from disease recurrence. disease (overall survival time). Cox regression analysis is also known as Cox proportional hazards analysis. This method is the standard method for investigating the prognostic value of a tumor marker as a function of patient survival time. When used in a multivariate setting, the effects of several covariates are examined in parallel so that individual covariates that have independent predictive value, that is, the most useful markers, can be identified. The term negative or positive "PSGL-1 status" can also be referred to as [PSGL-1(-)] or [PSGL-1(+)].

"Evaluation" of a sample can be done during cancer diagnosis or monitoring. In its simplest form, the assessment may be categorized as negative or positive as judged by visual examination of specimens using immunohistochemistry. Evaluation in more quantitative terms involves making a judgment regarding two parameters, the intensity of staining and the proportion of stained (“positive”) cells that are present in the sample.

In one embodiment, to ensure standardization, samples can be assessed for PSGL-1 expression levels on different scales, most of which are based on the intensity of the reaction product and the percentage of positive cells (Payne et al., Predictive markers in breast cancer - the present, Histopathology, 2008, 52, 82-90).

In another embodiment, said evaluation includes the use of an appropriate scale based on the intensity of staining and the percentage of positive cells.

As a first example, similar to the rapid Allred scoring for estrogen receptor and progesterone receptor IHC testing, samples can be assessed for levels of PSGL-1 expression on a global scale combining scores from 0 to 8 for severity of reactivity and for proportions of stained cells (Harvey JM, Clarck GM, Osborne CK, Allred DC; J. Clin. Oncol., 1999; 17; 1474-1481). More specifically, the first criterion of severity of reactivity is scored on a scale from 0 to 3, with 0 representing “no reactivity” and 3 representing “high reactivity.” The second criterion for the proportion of reactivity is assessed on a scale from 0 to 5, with 0 corresponding to “no reactivity” and 5 corresponding to “67-100% reactivity proportion”. The Severity of Reactivity and Proportion of Reactivity scores are then summed to give a total score ranging from 0 to 8. A total score of 0–2 is considered negative, whereas a total score of 3–8 is considered positive.

According to this scale, the terms negative or positive "PSGL-1 status" of tumors used in the description of the present invention refer to levels of PSGL-1 expression that correspond to scores of 0-2 or 3-8 on the Allred scale, respectively.

Table 2 below illustrates guidelines for interpreting IHC results according to the Allred method.

According to the present invention, said corresponding scale may be a scale from 0 to 8, on which the absence of reactivity is assessed with a score of 0, and high reactivity, expressed by a reactivity fraction of 67-100%, is assessed with a score of 8.

In other words, a method is described for determining in vitro or ex vivo the status of a tumor from a subject, the method comprising the steps of (a) assessing the tumor from the subject according to the Allred score; and (b) determining that the tumor status is [PSGL-1(+)] with an Allred score of 3 to 8; or (c) determining that the tumor status is [PSGL-1 (-)] with an Allred score of 0 to 2.

In a specific aspect of the invention, the tumor is classified as [PSGL-1(+)] with an Allred score of 3.

In a specific aspect of the invention, the tumor is classified as [PSGL-1(+)] with an Allred score of 4.

In a specific aspect of the invention, the tumor is classified as [PSGL-1(+)] with an Allred score of 5.

In a specific aspect of the invention, the tumor is classified as [PSGL-1(+)] with an Allred score of 6.

In a specific aspect of the invention, the tumor is classified as [PSGL-1(+)] with an Allred score of 7.

In a specific aspect of the invention, the tumor has a [PSGL-1(+)] status with an Allred score of 8.

According to another specific aspect of the invention, the tumor has a [PSGL-1(+)] status with an Allred score of 3 to 8.

Another specific method described herein for determining in vitro or ex vivo PSGL-1 status of tumor cells in a subject is characterized in that it includes the steps of:

(a) assessing PSGL-1-bearing tumor cells as described above; And

(b) determining that the PSGL-1 status of the tumor cells corresponds to [PSGL-1(+)] with a score of 3 to 8; or

(c) determining that the PSGL-1 status of the tumor cells corresponds to [PSGL-1(-)] with a score of 0 to 2.

Another specific method described herein for determining in vitro or ex vivo PSGL-1 status of tumor immune cells in a subject is characterized in that it includes the steps of:

(a) assessing a PSGL-1-bearing tumor immune cell as described above; And

(b) determining that the PSGL-1 status of the tumor immune cells corresponds to [PSGL-1(+)] with a score of 3 to 8; or

(c) determining that the PSGL-1 status of the tumor immune cells corresponds to [PSGL-1(-)] with a score of 0 to 2.

In a preferred embodiment, tumor immune cells (ie, immune infiltrates) include lymphocytes (eg, T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells and macrophages. Accordingly, in this embodiment, step a) involves quantifying the binding of said reagent to PSGL-1 on lymphocytes (eg, T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells and macrophages present in the microenvironment tumors in the specified biological sample.

As a second example, similar to traditional assessment in the case of HER-2 receptor IHC testing, for example, samples can be assessed for PSGL-1 expression levels based on a somewhat simpler scoring method combining staining intensity (preferably membrane staining) and the proportion of cells that display staining on a combined scale of 0 to 3+.

According to this scale, called the simplified scale, 0 and 1+ refer to a negative staining result, while 2+ and 3+ refer to a positive staining result. However, scores ranging from 1+ to 3+ can be considered positive because each positive score may be associated with a significantly higher risk of disease recurrence and death compared with a score of 0 (negative), but increased staining intensity among samples with positive assessments may provide additional risk reduction.

Generally speaking, the terms negative or positive "PSGL-1 status" of tumors used in the description of the present invention refer to levels of PSGL-1 expression that correspond to scores of 0 to 1+ or 2+ to 3+ on this simplified scale. respectively. Only the membranous reactivity throughout the periphery of the invasive tumor, which often resembled a “chicken wire” appearance, should be considered. Current guidelines require further evaluation for specimens rated as borderline on PSGL-1 (score 2+ or 3+). The results of an IHC assay should be rejected and either repeated or tested using FISH or any other method if, as a non-limiting example, the controls are not as expected, artifacts affect a large portion of the sample, and the sample shows a high membrane-associated positive result for ducts mammary glands are normal (as internal controls), which suggests excessive antigen unmasking.

For clarity, Table 3 below summarizes these parameters.

The corresponding scale can be a scale from 0 to ³⁺ , where the absence of membrane reactivity of tumor cells or tumor immune cells is scored as 0, and high complete reactivity in more than 10% of tumor cells is scored as ³⁺ .

In more detail, as described above, the corresponding scale is a scale from 0 to 3, where the absence of membrane reactivity of tumor cells or tumor immune cells is scored as 0, faint membrane reactivity in more than 10% of tumor cells or tumor immune cells is scored as 1 +; mild to moderate complete membrane reactivity in more than 10% of tumor cells or tumor immune cells is scored 2+; and high complete reactivity in more than 10% of tumor cells or tumor immune cells is scored 3+.

In other words, a method is described for determining in vitro or ex vivo the status of a tumor from a subject, the method comprising the steps of (a) assessing the tumor from the subject according to a simplified scale as described above; and (b) determining that the tumor status is [PSGL-1(+)] with a score of 2+ or 3+; or (c) determining that the tumor status is [PSGL-1(-)] with a score of 0 or 1+.

In a specific aspect of the invention, the tumor has a [PSGL-1(+)] status with a score of 2+.

According to a specific aspect of the invention, the tumor has a [PSGL-1(+)] status with a score of 3+.

According to another specific aspect of the invention, the tumor has a [PSGL-1(+)] status with a score of 2+ or 3+.

In another embodiment, a method for determining in vitro or ex vivo the PSGL-1 status of tumor cells in a subject may comprise the steps of:

(a) assessing PSGL-1-bearing tumor cells from said subject according to the method described above; And

(b) determining that the PSGL-1 status of the tumor cells corresponds to [PSGL-1(+)] with a score of ²⁺ or ³⁺ ; or

(c) determining that the PSGL-1 status of the tumor cells corresponds to [PSGL-1(-)] with a score of 0 or ¹⁺ .

In another embodiment, a method of determining in vitro or ex vivo the PSGL-1 status of tumor immune cells in a subject may comprise the steps of:

(a) assessing PSGL-1-bearing tumor immune cells from said subject according to the method described above; And

(b) determining that the PSGL-1 status of the tumor immune cells corresponds to [PSGL-1(+)] with a score of ²⁺ or ³⁺ ; or

(c) determining that the PSGL-1 status of the tumor immune cells corresponds to [PSGL-1(-)] with a score of 0 or ¹⁺ .

In a preferred embodiment, tumor immune cells (ie, immune infiltrates) include lymphocytes (eg, T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells and macrophages. Accordingly, in this embodiment, step a) involves quantifying the binding of said reagent to PSGL-1 on lymphocytes (eg, T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells and macrophages present in the microenvironment tumors in the specified biological sample.

Typically, test or analysis results can be presented in any of a variety of formats. The presentation of results may be qualitative. For example, the test report may indicate only whether a particular polypeptide was detected or not detected, possibly also indicating detection limits. The display of results may be semi-quantitative. For example, various ranges may be defined, and these ranges may be assigned a score (eg, 0 to 3+ or 0 to 8 depending on the scale used) that provides some degree of quantitative information. This score may reflect various factors, such as the number of cells in which PSGL-1 is detected, signal intensity (which may indicate the expression level of PSGL-1 or PSGL-1-bearing cells), and so on. The results can be displayed quantitatively, for example as the percentage of cells in which PSGL-1 is detected, as protein concentration, and so on.

It will be apparent to one of ordinary skill in the art that the type of result obtained from testing will vary depending on the technical limitations of the test and the biological significance associated with the detection of the polypeptide. For example, for certain polypeptides, a purely qualitative result (eg, whether a given polypeptide is detected or not detected at a certain level of detection) provides important information. In other cases, a more quantitative result is needed (for example, the ratio of the expression level of the polypeptide in the test sample to the level in normal).

ANTIBODIES TO PSGL-1

Antibodies for use in the methods of the present invention are antibodies that bind to PSGL-1, including a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, or a PSGL-1 epitope. Anti-PSGL-1 antibodies include humanized anti-PSGL-1 antibodies. Also provided are antibodies (eg, humanized anti-PSGL-1 antibodies) that completely block the binding of the anti-PSGL-1 antibody provided herein to the PSGL-1 polypeptide.

The present invention also provides antibodies that bind to PSGL-1 and exhibit an agonizing or antagonizing effect on the interaction between PSGL-1 and VISTA. Preferably, the anti-PSGL-1 antibody inhibits or blocks the binding of PSGL-1 to VISTA, primarily to the extracellular domain of VISTA. In some embodiments, an anti-PSGL-1 antibody inhibits or blocks binding of a VISTA-expressing cell to a PSGL-1-expressing T cell, such as, for example, a myeloid cell, dendritic cell, macrophage, or T cell. In some embodiments, the anti-PSGL-1 antibody does not block or inhibit the binding of PSGL-1 to P-selectin, L-selectin, or E-selectin.

The anti-PSGL-1 antibodies (eg, humanized anti-PSGL-1 antibodies) provided herein may also be conjugated or recombinantly fused to a diagnostic agent, detection agent, or therapeutic agent (eg, an antibody-drug conjugate). For example, the detectable agent may be a detectable probe. In addition, compositions are provided, including pharmaceutical compositions, containing an anti-PSGL-1 antibody (eg, a humanized anti-PSGL-1 antibody).

Antibodies provided herein that bind to an antigen, such as PSGL-1, can be produced by any method known in the art for antibody synthesis, particularly using chemical synthesis or recombinant expression methods. For example, certain anti-PSGL-1 antibodies and methods for producing such antibodies have been described previously (see, for example, WO 2005/110475, WO 2003/013603; US Patent Application Publications Nos. 2009/0198044, 2005/0266003, 2009/0285812, 2013/0011391 and 2015/0183870; and US patent Nos. 7833530 and 8361472).

Polyclonal antibodies that bind to antigen can be produced using various techniques well known in the art. For example, a human antigen can be administered to a variety of animal hosts, including, but not limited to, rabbits, mice, rats, etc., to induce sera containing polyclonal antibodies specific for the human antigen. Various adjuvants can be used to enhance the immune response, depending on the host species, and these include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide gel, surfactants such as lysolecithin , Pluronic-type polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially beneficial human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be produced using a wide range of methods known in the art, including the use of hybridoma and recombinant technologies and phage display methods, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma methods, including methods known in the art, information about which is contained, for example, in Harlow et at, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, 563-681 (Elsevier, N.Y., 1981); these sources are incorporated by reference in their entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced using hybridoma technology. Other typical methods for producing monoclonal antibodies are discussed elsewhere in this specification, such as, for example, the use of KM mouse™ technology. Additional exemplary methods for producing monoclonal antibodies are provided herein in the Examples section. Alternatively, it is also possible to use an anti-PSGL-1 antibody, such as, for example, the antibodies described in WO 2003/013603, WO 2005/110475, WO 2009/140623, Dimitroff et al., Cancer Res., 65(13): 5750 -60 (2005), Veerman et al., Nature Immunol., 8(5): 532-9 (2007), Tinocco et al., Immunity, 44: 1190-1203 (2016).

Methods for producing and screening specific antibodies using hybridoma technology are well established and well known in the art. Briefly, mice can be immunized with PSGL-1 antigen, and once an immune response is detected, eg, PSGL-1 antigen-specific antibodies are detected in mouse serum, mouse spleens are harvested and splenocytes are isolated. Splenocyte fusion is then performed using well known methods with any suitable myeloma cells, for example cells of the SP20 cell line, which can be obtained from ATCC (American Type Culture Collection). Hybridomas are selected and cloned using the limiting dilution method.

Additionally, the repeat immunization multiple site (RIMMS) technique can be used to immunize an animal (Kilptrack et al., 1997, Hybridoma, 16:381-9, incorporated by reference in its entirety). The hybridoma clones are then analyzed by methods known in the art for cells that secrete antibodies that have the ability to bind to the polypeptide in question. Immunization of mice with positive hybridoma clones produces ascites fluid, which typically contains high levels of antibodies.

Accordingly, this invention also provides methods for producing antibodies by culturing a hybridoma cell secreting a modified antibody provided herein, wherein in some embodiments the hybridoma is obtained by fusing splenocytes isolated from a mouse immunized with PSGL-1, including a PSGL-1 polypeptide. 1, a PSGL-1 polypeptide fragment or PSGL-1 epitope, with myeloma cells and subsequently screening hybridomas resulting from this fusion for hybridoma clones that secrete an antibody having the ability to bind PSGL-1.

Antibodies to PSGL-1 having the ability to modulate (eg, enhance or inhibit) the interaction between PSGL-1 and VISTA can be identified by any method known to those skilled in the art. Example assays for detecting and measuring the interaction between PSGL-1 and VISTA are described in the experimental section. Either of these assays can be used to test whether anti-PSGL-1 antibody can modulate the interaction between PSGL-1 and VISTA.

Antibody fragments that recognize (eg, bind to) PSGL-1 can be prepared by any method known to those skilled in the art. For example, the Fab and F(ab') ₂ fragments proposed in this application can be obtained by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments). F(ab') ₂ fragments contain a variable region, a light chain constant region and a heavy chain CH1 domain. In addition, the antibodies proposed in this application can also be obtained using various phage display methods known in the art.

For example, antibodies can also be produced using various phage display methods. In phage display methods, the functional domains of antibodies are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from cDNA libraries of animal origin (eg, human or mouse diseased tissue cDNA libraries). The DNA encoding the VH and VL domains is recombined with the scFv linker using PCR and cloned into a phagemid vector. The vector is introduced using electroporation into E. coli and E. coli is infected with helper phage. The phage used in these methods is usually a filamentous bacteriophage, including fd and M13, and the nucleotide sequences of the VH and VL domains are usually fused recombinantly to either phage gene III or gene VIII. A phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified using the antigen, for example, using a labeled antigen or an antigen bound to or captured on a solid surface or bead. Examples of phage display methods that can be used to produce the antibodies provided herein include those described in Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50; Ames et al., 1995, J. Immunol. Methods, 184: 177-186; Kettleborough et al, 1994, Eur. J Immunol 24: 952–958; Persic et al., 1997, Gene, 187: 9-18; Burton et al., 1994, Advances in Immunology, 57: 191-280; PCT/GB91/01134; WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844; and US patents No. 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5 969108; all of which are incorporated herein by reference in their entirety.

As described in the above references, following selection using a phage, antibody-encoding portions of the phage DNA can be isolated and used to produce whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host , including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described below. Methods for producing Fab, Fab' and F(ab') ₂ fragments recombinantly can also be used using methods known in the art, such as those described in PCT Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques, 12(6): 864-869; Sawai et al., 1995, AJRI, 34: 26-34; and Better et al., 1988, Science, 240: 1041-1043 (these sources are incorporated by reference in their entirety).

To produce whole antibodies, PCR primers containing the VH or VL nucleotide sequences, a restriction site, and flanking sequences to protect the restriction site can be used to amplify VH or VL sequences in scFv clones. Using cloning techniques known to those skilled in the art, PCR-amplified VH domains can be cloned into vectors expressing a VH constant region, such as the human immunoglobulin gamma 4 constant region, and PCR-amplified VL domains can be cloned into vectors expressing a constant region. VL region, for example, human immunoglobulin kappa or lambda constant regions. The VH and VL domains can also be cloned into a single vector expressing the required constant regions. The heavy chain introducing vectors and the light chain introducing vectors are then co-transfected into cell lines to produce stably or transiently transfected cell lines that express full-length antibodies, eg IgG, using methods known to those skilled in the art.

For some applications, including in vivo use of antibodies in humans and in vitro detection assays, human or chimeric antibodies can be used. For therapeutic treatment of subjects such as humans, fully human antibodies are particularly desirable. Human antibodies can be produced by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. Also see US Patent Nos. 4,444,887 and 4,716,111 and Application Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741; all of which are incorporated herein by reference in their entirety.

In some embodiments, human antibodies are produced. Human antibodies and/or fully human antibodies can be obtained using any method known in the art. For example, using transgenic mice that are unable to express functionally active endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or using homologous recombination into mouse embryonic stem cells. Alternatively, in addition to the human immunoglobulin heavy and light chain genes, nucleotide sequences encoding the variable region, constant region and D-segment (diversity region) of human immunoglobulin can be introduced into mouse embryonic stem cells. The mouse immunoglobulin heavy and light chain genes can be rendered nonfunctional individually or simultaneously with the introduction of the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the J _H region prevents the formation of endogenous antibodies. Modified embryonic stem cells are grown and microinjected into blastocysts to produce chimeric mice. Chimeric mice are further bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual manner with a selected antigen, for example the whole polypeptide or part thereof. Monoclonal antibodies directed to a given antigen can be generated from immunized, transgenic mice using traditional hybridoma technology. Transgenic human immunoglobulins contained in transgenic mice undergo rearrangement during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced using this method. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol., 13: 65-93). For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, WO 98/24893, WO 96/34096 and WO 96/33735; and US Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated herein by reference in their entirety. Other methods are described in detail in this description in the Examples section. In addition, companies such as Abgenix, Inc. can be contacted to obtain human antibodies directed to a selected antigen using technology similar to that described above. (Freemont, CA) and Genpharm (San Jose, CA).

A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science, 229: 1202; Oi et al., 1986, BioTechniques, 4: 214; Gillies et al., 1989, J. Immunol. Methods, 125: 191-202 and US Patent Nos. 5807715, 4816567, 4816397 and 6331415, which are incorporated herein by reference in their entirety.

A humanized antibody is an antibody or variant thereof or fragment thereof that is capable of binding to a predetermined antigen and that contains a framework region having essentially the amino acid sequence of a human immunoglobulin and a CDR having essentially the amino acid sequence immunoglobulin of a non-human species. A humanized antibody contains substantially all of the variable domains, at least one and usually two variable domains (Fab, Fab', F(ab') ₂ , Fabc, Fv), in which all or substantially all of the CDR regions correspond to those of the immunoglobulin species non-human (eg, donor antibody), and all or substantially all of the framework regions are the same as the human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin. Typically, an antibody will contain both a light chain and at least a heavy chain variable domain. The antibody may also include a CH1 region, a hinge region, CH2, CH3 and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Typically, the constant domain is a complement fixation constant domain when it is desired that the humanized antibody exhibit cytotoxic activity, and this class is typically IgG1. When such cytotoxic activity is undesirable, the constant domain may be of the IgG2 class. Examples of VL and VH constant domains that may be used in some embodiments include, but are not limited to, C-kappa and C-gamma-1 (nG1m) described in Johnson et al. (1997), J. Infect. Dis., 176, 1215-1224, and those described in US Pat. No. 5,824,307. A humanized antibody may contain sequences from more than one class or isotype, and the selection of specific constant domains to optimize desired effector functions is within the skill of one of ordinary skill in the art. The framework and CDR regions of a humanized antibody do not necessarily correspond exactly to the original sequences; for example, the donor CDR or consensus framework may be subject to mutations with the substitution, insertion, or deletion of at least one residue such that a given CDR or framework residue at that site does not correspond to either consensus nor the administered antibody. Such mutations, however, will not be extensive. Typically, at least 75% of the residues of the humanized antibody will match the residues of the original FR and CDR sequences, more commonly 90% or greater than 95%. Humanized antibodies can be produced using various methods known in the art, including, but not limited to, CDR grafting (EP 239400; WO 91/09967; and US patent Nos. 5225539, 5530101 and 5585089), veining or alteration surfaces (EP 592106 and EP 519596; Padlan, 1991, Molecular Immunology, 28(4/5): 489-498; Studnicka et al., 1994, Protein Engineering, 7(6): 805-814; and Roguska et al. , 1994, Proc. Natl. Acad. Sci., 91: 969-973), chain shuffling (US Patent No. 5565332) and methods described, for example, in US Patent No. 6407213, US Patent No. 5766886, application WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5): 353-60 (2000), Morea et al., Methods, 20(3) : 267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10): 895 904 (1996), Couto et al., Cancer Res., 55 (23 Supp): 5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu JS, Gene, 150(2): 409-10 (1994) and Pedersen et al, J Mol. Biol., 235(3): 959-73 (1994). Also see US Patent Application Publication No. US 2005/0042664 A1, which is incorporated herein by reference in its entirety. Often, framework residues in framework regions will be replaced by the corresponding residue from the CDR of the donor antibody in order to alter (eg, improve) binding to the antigen. Such scaffold substitutions are identified by methods well known in the art, for example, by modeling the interaction of CDR residues and the scaffold to identify scaffold residues important for antigen binding, and by sequence comparison to identify unusual scaffold residues at specific positions. (See, for example, Queen et al., US Pat. No. 5,585,089; and Reichmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety).

Single domain antibodies, for example, antibodies that do not contain light chains, can be produced by methods well known in the art. See Riechmann et al., 1999, J. Immunol., 231: 25-38; Nuttall et al, 2000, Sip.Pharm. BiotechnoL, 1(3): 253-263; Muylderman, 2001, J. Biotechnol., 74(4): 277302; US Patent No. 6005079; and Application Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of which are incorporated herein by reference in their entirety.

In addition, antibodies that bind to PSGL-1 can in turn be used to produce anti-idiotypic antibodies that “mock” the antigen using methods well known to those skilled in the art. (See, for example, Greenspan & Bona, 1989, FASEB J., 7(5): 437-444; and Nissinoff, 1991, J. Immunol., 147(8): 2429-2438).

Antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, intraantibodies, anti-idiotypic antibodies (anti-Id) antibodies and functional fragments of any of the above. Non-limiting examples of functional fragments include single chain Fv (scFv) (including, for example, monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab) ₂ fragments, F(ab') ₂ Fv disulfide bonded fragments (sdFv), Fd fragments, Fv fragments, diabody, tribody, tetrabody and minibody.

In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules containing an antigen-binding site that binds to PSGL-1 (e.g., PSGL-1 polypeptide, PSGL-1 polypeptide fragment, PSGL epitope -1). The immunoglobulin molecules provided herein may be immunoglobulin molecules of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4 IgA1 and IgA2) or subclass.

Antibody variants and derivatives include functional antibody fragments that retain the ability to bind PSGL-1 (eg, PSGL-1 polypeptide, PSGL-1 polypeptide fragment, PSGL-1 epitope). Exemplary functional fragments include Fab fragments (an antibody fragment that contains an antigen binding domain and contains a light chain and a portion of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing one antigen-binding domain, which is a Fab, and an additional heavy chain portion connected through a hinge region); F(ab') ₂ (two Fab' molecules connected by interchain disulfide bonds at the hinge regions of the heavy chains; Fab' molecules can be directed to the same epitope or to different epitopes); bispecific Fab (Fab molecule having two antigen-binding domains, each of which can be directed to a different epitope); a single-chain Fab fragment containing a variable region, also known as sFv (an antigen-binding determining variable region consisting of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv or dsFv (a variable, antigen-binding-determining region consisting of one light and heavy chain of an antibody linked together by a disulfide bond); camel VH (the variable antigen-binding-determining region of a single heavy chain antibody in which some of the amino acids on the surface of the VH are the same as those found in the heavy chain of natural camel antibodies); bispecific sFv (sFv or dsFv molecule having two antigen-binding domains, each of which can be directed to a different epitope); diabody (a dimerized sFv formed when the VH domain of the first sFv binds to the VL domain of the second sFv, and the VL domain of the first sFv binds to the VH domain of the second sFv; these two antigen-binding sites of the diabody can be directed to the same epitope or different epitopes); and tribody (a trimerized sFv formed in a manner similar to that of a diabody, but in which three antigen-binding domains are combined into a single complex; the three antigen-binding domains may be directed to the same epitope or to different epitopes). Antibody derivatives also include one or more CDR sequences of an antibody combining site. CDR sequence data may be joined together using a backbone when there are two or more CDR sequences. In some embodiments, the antibody contains a single chain Fv ("scFv"). scFvs are antibody fragments containing the VH and VL domains of the antibody, with such domains present as a single polypeptide chain. Typically, the scFv polypeptide further contains a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for binding to the antigen. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The antibodies provided herein may be monospecific, bispecific, trispecific, or have a higher degree of specificity. Multispecific antibodies may have specificity for different epitopes of the PSGL-1 polypeptide or may have specificity for both the PSGL-1 polypeptide and a heterologous epitope, such as a heterologous polypeptide or solid support material. In some embodiments, the antibodies provided herein are monospecific for a particular epitope of the PSGL-1 polypeptide and do not bind to other epitopes.

In addition, the present invention provides fusion proteins comprising an antibody provided herein that binds PSGL-1 and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused is useful for targeting the antibody to cells having cell surface expressed PSGL-1.

In addition, the present invention provides panels of antibodies that bind to PSGL-1. In some embodiments, panels of antibodies have different association rate constants, different dissociation rate constants, different affinities for PSGL-1, and/or have different specificities for PSGL-1. In some embodiments, the panels comprise or consist of about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950 or about 1000 antibodies or more. Antibody panels can be used, for example, in 96-well or 384-well plates, for example, for assays such as ELISA.

USE OF PSGL-1 BINDING REAGENTS FOR DIAGNOSTICS

The anti-PSGL-1 antibodies provided herein can be used to analyze the levels of PSGL-1 in a biological sample using classical immunohistological methods as set forth herein or known to those skilled in the art (e.g., see Jalkanen et al., 1985, J. Cell Biol., 101: 976-985; and Jalkanen et al., 1987, J. Cell. Biol., 105: 3087-3096). Other antibody methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable labels for antibody assays are known in the art and include enzymatic labels such as glucose oxidase; radioactive isotopes such as iodine ( ¹²⁵ 1, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); luminescent tags such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin.

The present invention further provides a method for detecting and diagnosing a VISTA-mediated disease, disorder or condition in a human. In one embodiment, the diagnosis includes: a) administering (eg, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody that binds to PSGL-1; b) waiting for a period of time after administration to allow the labeled antibody to concentrate in the subject, preferably at sites of PSGL-1 expression (and to allow unbound labeled molecules to reach background levels); c) determining the background level; and d) detecting the labeled antibody in the subject, wherein detection of the labeled antibody above a background level indicates that the subject has a PSGL-1-mediated disease, disorder or condition. The background level can be determined by various methods, including comparing the number of detected labeled molecules with a standard value previously determined for a particular system.

It will be appreciated by those skilled in the art that the size of the subject and the imaging system used will determine the amount of imaging array required to obtain a diagnostic image. For a human subject, in the case of a radioactive isotope group, the amount of radioactive compound administered will typically range from about 5 to 20 millicuries for ⁹⁹ Tc. The labeled antibody will then preferentially accumulate at the site of cells that contain that particular protein. In vivo tumor imaging is described in SW Burchiel et at., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

Depending on several factors, including the type of label used and the route of administration, the amount of time after administration to allow the labeled antibody to concentrate in the subject preferentially at sites of antigen expression and for unbound labeled antibody to reach background levels ranges from 6 to 48 hours, or from 6 to 24 hours, or from 6 to 12 hours. In another embodiment, the period of time after administration is from 5 to 20 days or from 5 to 10 days.

In some embodiments, monitoring of the VISTA-mediated disease, disorder or condition is performed by repeating the method of diagnosing the VISTA-mediated disease, disorder or condition, for example, one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis and so on.

The presence of a labeled molecule in a subject can be detected using methods known in the art for in vivo scanning. These methods depend on the type of label being used. Those skilled in the art will be able to determine an appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods proposed herein include, but are not limited to, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging ( MRI) and echography.

In one embodiment, the molecule is labeled with a radioactive isotope and detected in a patient using a radiation-sensitive surgical instrument (Thurston et al., US Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence-sensitive scanning device. In another embodiment, the molecule is labeled with a positron-emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in a patient using magnetic resonance imaging (MRI).

ANTI-VISTA THERAPEUTIC AGENTS

In a first embodiment, the anti-VISTA therapeutic agent is an agent that inhibits the function of the VISTA checkpoint inhibitor. Inhibition of the inhibitory function of VISTA can occur at the DNA, RNA or protein level. In some embodiments, an inhibitory nucleic acid (eg, double-stranded RNA (dsRNA), small interfering RNA (siRNA), or small hairpin RNA (shRNA)) can be used to inhibit VISTA expression. In other embodiments, the VISTA inhibitory signal inhibitor is a polypeptide, such as a soluble ligand (e.g., PSGL-1-Fc), or an antibody or antigen-binding fragment thereof (also referred to herein as an “antibody molecule”) that contacts VISTA. Preferably, the anti-VISTA therapeutic agent is an antibody.

Antibodies that inhibit VISTA function are particularly useful in the treatment of cancer. The present inventors have previously described antibodies directed against VISTA that cause potent suppression of tumor growth (see applications WO 2014/197849 and WO 2016/094837, both of which are incorporated herein by reference). Other anti-VISTA antibodies having anti-cancer properties are also described in the art (see, for example, WO 2014/039983 A1, WO 2015/145360 A1, WO 2015/097536, WO 2017/137830, WO 2017/181139; all of them are hereby incorporated by reference in their entirety).

Such highly specific and/or VISTA-specific antibodies (referred to herein as “anti-VISTA antibodies”) may be polyclonal (“anti-VISTA PAb”) or monoclonal (“anti-VISTA MAb”), although for therapeutic applications and, in some cases, , diagnostic or other in vitro applications, monoclonal antibodies are preferred.

In specific embodiments, the antibody is a humanized antibody, a monoclonal antibody, a recombinant antibody, an antigen binding fragment, or any combination thereof. In specific embodiments, the antibody is a humanized monoclonal antibody as described in WO 2016/094837 (e.g., 5B, 46A, 97A, 128A, 146C, 208A, 215A, 26A, 164A, 230A, 76E1, 53A, 259A, 33A, 39A, 124A, 175A, 321D, 141A, 51A, 353A or 305A described therein (for example, in Tables 12-33 of WO 2016/094837), containing a V _H domain, a V _L domain, a V _H CDR1, a V _H CDR2, V _H CDR3, V _L CDR1, V _L CDR2 and/or V _L CDR3), or an antigen binding fragment thereof, that binds to a VISTA polypeptide (eg, cell surface expressed or soluble VISTA), a VISTA fragment or a VISTA epitope.

In other embodiments, the anti-VISTA antibodies used in the method of the invention are antibodies (1) that competitively block (e.g., in a dose-dependent manner) the binding of the anti-VISTA antibody described in WO 2016/094837 to a VISTA polypeptide (e.g., expressed on the cell surface or soluble VISTA), a VISTA fragment or a VISTA epitope, and/or (2) that binds to a VISTA epitope that binds to an anti-VISTA antibody (eg, humanized anti-VISTA antibodies) described in WO 2016/094837. In other embodiments, the antibody competitively blocks the binding (e.g., in a dose-dependent manner) of monoclonal antibody 5B, 46A, 97A, 128A, 146C, 208A, 215A, 26A, 164A, 230A, 76E1, 53A, 259A, 33A, 39A, 124A, 175A, 321D, 141A, 51A, 353A, or 305A described therein (eg, in Tables 12-33), or a humanized variant thereof with a VISTA polypeptide (eg, cell surface expressed or soluble VISTA), a VISTA fragment, or a VISTA epitope. In other embodiments, the antibody binds to a VISTA epitope that binds to (e.g., is recognized by) monoclonal antibody 5B, 46A, 97A, 128A, 146C, 208A, 215A, 26A, 164A, 230A, 76E1, 53A, 259A, 33A, 39A, 124A, 175A, 321D, 141A, 51A, 353A or 305A, described in WO 2016/094837 (for example, in Tables 12-33), or a humanized version thereof (for example, with humanized antibodies to VISTA).

More preferably, the anti-VISTA antibody from the method of this invention is the 26A antibody described in WO 2016/094837. In a first embodiment, the antibody contains a heavy chain containing 3 CDRs and a light chain containing 3 CDRs, with said CDRs shown in Table 4. In another embodiment, the anti-VISTA antibody contains a heavy chain containing 3 CDRs and a light chain containing 3 CDRs, with said CDRs shown in Table 5.

The anti-VISTA monoclonal antibodies of the invention include both intact molecules and antibody fragments (such as, for example, Fab and F(ab') ₂ fragments) that have the ability to specifically bind to VISTA. The Fab and F(ab') ₂ fragments lack the Fc fragment of the intact antibody, are cleared more quickly from the animal or plant bloodstream, and may have less nonspecific tissue binding than the intact antibody (Wahl et al., 1983, J. Nucl. Med ., 24: 316). Therefore, antibody fragments can be used for therapeutic purposes, among other applications.

The term "antibody fragment" refers to a portion of a full-length antibody, typically the target binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. The "Fv" fragment is the minimal antibody fragment that contains the complete target recognition and binding site. This region is a dimer composed of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association (as a VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact with each other to form a target binding site on the surface of the VH-VL dimer. Often six CDRs give an antibody specificity for binding to its target. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may have the ability to recognize and bind to the target, although with lower affinity than the entire binding site. "Single chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, such domains being present as a single polypeptide chain. Typically, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for binding to the target. "Single domain antibodies" consist of single VH or VL domains that exhibit sufficient affinity for VISTA. In a specific embodiment, the single domain antibody is a camel antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods, 231: 25-38).

The Fab fragment contains a light chain constant domain and a first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by several added residues at the carboxyl terminus of the CH1 heavy chain domain, including the presence of one or more cysteine residues from the antibody hinge region. F(ab') fragments are formed as a result of cleavage of the disulfide bond between the cysteine residues contained in the hinge region in the product F(ab') ₂ obtained after cleavage with pepsin. Additional chemical reactions of combining antibody fragments are known to those of ordinary skill in the art.

The anti-VISTA monoclonal antibodies of the invention may be chimeric antibodies. The term “chimeric” antibody as used herein refers to an antibody having variable sequences derived from immunoglobulins of non-human species, such as a rat or mouse antibody, and human immunoglobulin constant regions, typically selected from a human immunoglobulin matrix. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science, 229(4719): 1202-7; Oi et al., 1986, BioTechniques, 4: 214-221; Gillies et al., 1985, J. Immunol. Methods, 125: 191-202; US Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

The anti-VISTA monoclonal antibodies of the invention may be humanized. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other target-binding antibody subsequences) that contain minimal sequences originating from immunoglobulins of non-human species. In general, a humanized antibody will contain substantially all of the variable domains, at least one and typically two variable domains in which all or substantially all of the CDR regions correspond to those of a non-human species immunoglobulin, and all or substantially all of the FRs regions are the same as the human immunoglobulin consensus sequence and may be referred to as "CDR-grafted". The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence. Methods for humanizing antibodies, including methods for constructing humanized antibodies, are known in the art. See, for example, Lefranc et al., 2003, Dev. Comp. Immunol., 27: 55-77; Lefranc et al., 2009, Nucl. Acids Res., 37: D1006-1012; Lefranc, 2008, Mol. Biotechnol., 40: 101-111; Riechmann et al., 1988, Nature, 332: 323-7; US patent numbers: 5530101, 5585089, 5693761, 5693762 and 6180370 by Queen et al.; EP 239400; PCT publication of application WO 91/09967; US Patent No. 5225539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28: 489–498; Studnicka et al., 1994, Prot. Eng., 7: 805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci., 91: 969-973; and US Pat. No. 5,565,332, all of which are hereby incorporated by reference in their entirety.

POLYNUCLEOTIDES ENCODING ANTIBODY

In addition, the present invention provides polynucleotides containing a nucleotide sequence encoding an antibody provided herein that binds to PSGL-1 (eg, a PSGL-1 polypeptide, a fragment of a PSGL-1 polypeptide, a PSGL-1 epitope). In addition, the present invention provides polynucleotides that hybridize under high, intermediate, or low stringency hybridization conditions, for example, as defined above, with polynucleotides that encode an antibody or modified antibody provided herein.

In addition, the present invention provides polynucleotides containing a nucleotide sequence encoding an antibody provided herein that binds to VISTA (eg, a VISTA polypeptide, a fragment of a VISTA polypeptide, a VISTA epitope). In addition, the present invention provides polynucleotides that hybridize under high, intermediate, or low stringency hybridization conditions, for example, as defined above, with polynucleotides that encode an antibody or modified antibody provided herein.

In some embodiments, the nucleic acid molecules provided herein contain or consist of a nucleic acid sequence encoding the VH and/or VL amino acid sequence described herein, or any combination thereof (for example, such as a nucleotide sequence encoding an antibody proposed in this application, for example, a full-length antibody, a heavy and/or light chain antibody, or a single chain antibody proposed in this application).

RECOMBINANT ANTIBODY EXPRESSION

To express the antibodies of the present invention, for example, the anti-PSGL-1 antibody or the anti-VISTA antibody described herein, a variety of expression systems can be used. In one aspect, such expression systems are vehicles in which coding sequences of interest can be produced and subsequently purified, but are also cells that, after transient transfection with the appropriate nucleotide coding sequences, express in situ an antibody of the invention.

According to the invention, vectors are provided containing the polynucleotides described in this application. In one embodiment, the vector contains a polynucleotide encoding the heavy chain of an IgG antibody of the invention, i.e. an antibody that carries a mutation in the Fc domain. In another embodiment, said polynucleotide encodes the light chain of an IgG antibody of the invention. The invention also provides vectors containing polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments and probes thereof.

To express the heavy and/or light chain of an antibody described herein, such as an anti-PSGL-1 antibody or an anti-VISTA antibody, polynucleotides encoding said heavy and/or light chains are inserted into expression vectors such that the genes are functional associated with transcription and translation regulatory sequences.

The term "operably linked" sequences includes both expression control sequences that are adjacent to the gene of interest and expression control sequences that are trans-regulatory or act at a distance to regulate the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are associated. The expression control sequences include the corresponding transcription initiation, transcription termination, promoter and enhancer sequences; efficient signals that control RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, the Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance the secretion of proteins. The nature of such control sequences varies depending on the host organism; such control sequences in prokaryotes typically include a promoter, a ribosome binding site, and a transcription termination sequence; such control sequences in eukaryotes typically include promoters and transcription termination sequences. The term “control sequences” is intended to include, at a minimum, all components whose presence is important for expression and processing, and may also include additional components whose presence is preferred, such as leader sequences and fusion partner sequences.

The term "vector" as used herein is intended to mean a nucleic acid molecule having the ability to carry another nucleic acid that is attached to it. One type of vector is a “plasmid,” which refers to a circular loop of double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, whereby additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomously replicating in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal vectors for mammalian cells). Other vectors (eg, non-episome vectors for mammalian cells), once introduced into a host cell, can be integrated into the genome of the host cell and thereby replicate along with the host genome.

Some vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Typically, expression vectors used in recombinant DNA techniques are in the form of plasmids. In the present invention, "plasmid" and "vector" may be used interchangeably, since plasmid is the most commonly used form of vector. However, the present invention is intended to include such forms of expression vectors as bacterial plasmids, YAC (yeast artificial chromosomes), cosmids, retroviruses, EBV (Epstein-Barr virus)-derived episomes, and all other vectors known to skilled person, are convenient for ensuring the expression of heavy and/or light chains of antibodies according to the invention. One skilled in the art will appreciate that polynucleotides encoding the heavy and light chains can be cloned into different vectors or into the same vector. In a preferred embodiment, said polynucleotides are cloned into two vectors.

The polynucleotides of the invention and vectors containing these molecules can be used to transform a suitable host cell. The term “host cell” as used herein is intended to refer to a cell into which a recombinant expression vector has been introduced to express an antibody of the present invention (eg, an anti-PSGL-1 antibody or an anti-VISTA antibody). It should be understood that such terms are intended to refer not only to the particular cell being affected, but also to the progeny of such cell. Since some modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but will still be included within the scope of the term "host cell" as used herein.

Transformation can be accomplished by any method known for introducing polynucleotides into a host cell. Such methods are well known to one skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotide encapsulation in liposomes, biolistics, and microinjection of DNA directly into nuclei.

A host cell can be co-transfected with two or more expression vectors, including a vector expressing a protein of the invention. In particular, other expression vectors may encode enzymes involved in post-translational modifications such as glycosylation. For example, a host cell may be transfected with a first vector encoding an antibody described above (eg, an anti-PSGL-1 antibody or an anti-VISTA antibody) and a second vector encoding a glycosyltransferase polypeptide. Alternatively, the host cell may be transfected with a first vector encoding an antibody (eg, an anti-PSGL-1 antibody or an anti-VISTA antibody), a second vector encoding a glycosyltransferase described above, and a third vector encoding a different glycosyltransferase. Mammalian cells are commonly used for the expression of recombinant therapeutic immunoglobulins, especially for the expression of whole recombinant antibodies. For example, mammalian cells, such as HEK293 (human embryonic kidney line 293) or Chinese hamster ovary (CHO) cells, together with a vector containing an expression signal, such as a vector carrying the major immediate early gene promoter element from human cytomegalovirus, are an effective system. to express an antibody of the present invention, primarily an anti-PSGL-1 antibody or an anti-VISTA antibody (Foecking et al., 1986, Gene, 45:101; Cockett et al., 1990, Bio/Technology, 8:2).

In addition, a host cell can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a particular desired manner. Such modifications (eg, glycosylation) and processing of protein products may be important for the function of a given protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems are selected to ensure correct modification and processing of the expressed protein of interest. For this reason, it is possible to use eukaryotic host cells that have the cellular machinery to properly process the primary transcript, glycosylation of the gene product. Such host cells in the case of mammals include, but are not limited to, CHO, COS, HEK293, NS0, BHK (newborn Syrian hamster kidney), Y2/0, 3T3 or myeloma cells (all of these cell lines are available from government depository institutions such as as the National Collection of Cultures of Microorganisms, Paris, France, or in the American Collection of Type Cultures, Manassas, Virginia, USA).

For long-term production of recombinant proteins in high yield, stable expression is preferred. In one embodiment of the invention, cell lines can be constructed that stably express an antibody (eg, an anti-PSGL-1 antibody or an anti-VISTA antibody). Instead of using expression vectors that contain viral origins of replication, host cells are transfected with DNA controlled by appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other relevant sequences known to one skilled in the art, and containing a selectable marker. After introducing foreign DNA, the engineered cells can be left to grow for one to two days in an enriched medium, after which they are transferred to a selective medium. The selectable marker in the recombinant plasmid confers the stability necessary for selection, and its presence allows cells to stably integrate the plasmid into the chromosome and grow into a cell lineage. Other methods for creating stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, transfected DNA, under the control of appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites and other relevant sequences, is integrated into the genome of the host cell at a specific target site at which cleavage has previously been carried out (Moele et al., Proc . Natl. Acad. Sci. U.S.A., 104(9): 3055-3060; US 5792632; US 5830729; US 6238924; WO 2009/054985; WO 03/025183; WO 2004/067753, all of which are incorporated herein by reference ).

A number of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (tk) genes (Wigler et al., Cell, 11: 223, 1977), hypoxanthine guanine phosphoribosyltransferase (hgprt) genes (Szybalska et al., Proc. Natl. Acad. Sci. USA, 48: 202, 1992), glutamate synthase, with selection in the presence of methionine sulfoximide (Adv. Drug Del. Rev., 58: 671, 2006 and website or reference list from Lonza Group Ltd.), and adenine phosphoribosyltransferase (aprt) (Lowy et al., Cell, 22. 8-17, 1980), which can be used in tk-, hgprt- or aprt-bearing cells, respectively. In addition, antimetabolite resistance can be used as a basis for selection in the case of the following genes: dhfr (dihydrofolate reductase gene), which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 357, 1980 ); gpt (glutamate-pyruvate transaminase gene), which confers resistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA, 78: 2072, 1981); neo (neomycin phosphotransferase gene), which confers resistance to aminoglycoside G-418 (Wu et al., Biotherapy, 3: 87, 1991; and hygro (hygromycin resistance gene), which confers resistance to hygromycin (Santerre et al., Gene, 30: 147, 1984) Methods well known in the art of recombinant DNA technology can invariably be used to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons (1993). Antibody expression levels can be increased using vector amplification. When a marker in a vector system expressing an antibody is able to be amplified, increasing the level of inhibitor present in the culture will cause an increase in the number of copies of the marker gene. Because the amplified region is associated with the gene encoding a target antibody (eg, an anti-PSGL-1 antibody or an anti-VISTA antibody), the production of said antibody will also be enhanced (Crouse et al., Mol. Cell. Biol., 3: 257, 1983). There are alternative methods for expressing the gene of the invention, and these are known to those skilled in the art. For example, it is possible to construct a modified zinc finger protein gene that is capable of binding to expression regulatory elements located upstream of the gene of the invention; expression of said engineered zinc finger protein (ZFN) in a host cell of the invention results in increased protein production (see, for example, Reik et al., Biotechnol. Bioeng., 97(5), 1180-1189, 2006). Moreover, ZFN can stimulate DNA integration at a predetermined location in the genome, resulting in highly efficient site-specific gene addition (Moehle et al., Proc. Natl. Acad. Sci. USA, 104: 3055, 2007).

The antibody of the invention can be produced by growing a culture of transformed host cells under the culture conditions necessary for expression of the desired antibody. The resulting expressed antibody can then be purified from culture media or cell extracts. Soluble forms of the antibody can be recovered from the culture supernatant. They can then be purified by any method known in the art for purifying immunoglobulin molecules, for example, chromatography (e.g. ion exchange, affinity, in particular by affinity for Fc after purification on a protein A immobilized support, etc.), centrifugation , using differential solubility or any other standard method used for protein purification. Suitable cleaning methods will be apparent to those of ordinary skill in the art.

ANTIBODY CONJUGATES AND FUSION PROTEINS

In some embodiments, the antibodies provided herein are conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent or any other molecule. Conjugated or recombinantly fused antibodies may be useful, for example, for monitoring or predicting the onset, development, progression and/or severity of a VISTA-mediated disease, disorder or condition as part of a clinical trial procedure, such as determining the effectiveness of a particular therapy.

Such diagnosis and detection may be accomplished, for example, by reacting an antibody (eg, anti-PSGL-1 antibody) with detectable substances, including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin and aequorin; a chemiluminescent substance such as, but not limited to, an acridinium-based compound or HALOTAG; radioactive materials such as, but not limited to, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In and ¹¹¹ In), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn and ¹¹⁷ Sn; and positron-emitting metals using various types of positron emission tomography, and non-radioactive paramagnetic metal ions.

In addition, the present invention provides antibodies that are conjugated or recombinantly fused to a therapeutic moiety (or one or more therapeutic moieties), as well as uses thereof. The antibody may be conjugated or recombinantly fused to a therapeutic moiety, such as a cytotoxin, eg, a cytostatic or cell-destructive agent, a therapeutic agent, or a radioactive metal ion, eg, alpha radiation sources. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Therapeutic groups include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine); alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BCNU stands for bis-chloroethylnitrosourea) and lomustine (CCNU stands for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), cyclophosphamide, busulfan, dibromomannitol, streptozotocin , mitomycin C and cis-dichlorodiamineplatinum(II) (DDP) and cisplatin); anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (eg actinomycin D (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)); auristatin-based molecules (eg, auristatin PHE, auristatin F, monomethyl auristatin E, bryostatin 1 and solastatin 10; see Woyke et al., Antimicrob. Agents Chemother., 46: 3802-8 (2002), Woyke et al., Antimicrob Agents Chemother., 45: 3580-4 (2001), Mohammad et al., Anticancer Drugs, 12: 735-40 (2001), Wall et al., Biochem. Biophys. Res. Commun., 266: 76-80 (1999), Mohammad et al., Int. J. Oncol., 15: 367-72 (1999), all of which are incorporated herein by reference); hormones (eg, glucocorticoids, progestins, androgens and estrogens), inhibitors of DNA repair enzymes (eg, etoposide or topotecan), kinase inhibitors (eg, compound ST1571, imatinib mesylate (Kantarjian et al., Clin. Cancer Res., 8(7) ): 2167-76 (2002)); cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs and compounds disclosed in US patents No. 6245759, 6399633, 6383790, 6335156, 6271242, 6242196, 6218 410, 6218372, 6057300, 6034053, 5985877, 5958769, 5925376, 5922844, 5911995, 5872223, 5863904, 5840745, 5728868, 5648239, 5587459); farnesyltransferase inhibitors (for example, R1157 77, BMS-214662 and inhibitors described, for example, in US patents No.: 6458935, 6451812, 6440974, 6436960, 6432959, 6420387, 6414145, 6410541, 6410539, 6403581, 6399615, 6387905, 6372747, 6369034, 636218 8, 6342765, 6342487, 6300501, 6268363, 6265422, 6248756, 6239140, 6232338, 6228865, 6228856, 6225322, 6218406, 6211193, 6187786, 6169096, 6159984, 6143766, 6133303, 6127366, 6124465, 6124295, 6103723, 6093737, 6090948, 608087 0, 6077853, 6071935, 6066738, 6063930, 6054466, 6051582, 6051574 and 6040305); topoisomerase inhibitors (eg, camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022; TAN-1518A; TAN 1518 B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506 and rebeccamycin); bulgarein; substances that bind to the minor groove of DNA, such as Hoescht dye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine; coraline; beta-lapachone; VS-4-1; bisphosphonates (eg, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, pyridronate, pamidronate, zoledronate); HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors (eg, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statin, cerivastatin, lescol, Lipitor, rosuvastatin and atorvastatin); antisense oligonucleotides (for example, those described in US patent No. 6277832, 5998596, 5885834, 5734033 and 5618709); adenosine deaminase inhibitors (for example, fludarabine phosphate and 2-chlorodeoxyadenosine); ibritutomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) and their pharmaceutically acceptable salts, solvates, clathrates and prodrugs.

In addition, the antibody provided herein may be conjugated or recombinantly fused to a therapeutic or drug moiety that modifies a specific biological response. Therapeutic or drug groupings should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide having the desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, cholera toxin or diphtheria toxin; protein such as tumor necrosis factor (TNF), interferon-γ, interferon-α, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agent such as TNF-γ, TNF-γ, AIM (activation inducer molecule ) I (see international publication no. WO 97/33899), AIM II (see international publication no. WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6: 1567-1574) and VEGF (see International Publication No. WO 99/23105), an antiangiogenic agent, for example angiostatin, endostatin or a component of the coagulation cascade (eg tissue factor); or a biological response modifier such as, for example, a lymphokine (eg, interferon-gamma, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-5 (“IL-5”) , interleukin-6 ("IL-6"), interleukin-7 ("IL-7"), interleukin 9 ("IL-9"), interleukin-10 ("IL-10"), interleukin-12 ("IL -12"), interleukin-15 ("IL-15"), interleukin-23 ("IL-23"), granulocyte-monocyte colony-stimulating factor ("GM-CSF") and granulocyte-monocyte colony-stimulating factor ("G-CSF") ")) or growth factor (e.g., growth hormone ("GH")), or coagulation-inducing agent (e.g., calcium, vitamin K, tissue factors such as, but not limited to, Hageman factor (factor XII), high molecular weight kininogen (HMWK), prekallikrein (PK), coagulation-inducing protein factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid and fibrin monomer).

The present invention further provides antibodies that are recombinantly fused or chemically conjugated (via covalent or non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, e.g., a polypeptide consisting of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to form fusion proteins, as well as their use. In particular, the present invention provides fusion proteins comprising an antigen binding fragment of an antibody as provided herein (e.g., Fab fragment, Fd fragment, Fv fragment, F(ab) ₂ fragment, VH domain, VH CDR, VL domain or VL CDR), and a heterologous protein, polypeptide or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide to which the antibody is fused is useful for targeting the antibody to a specific cell type, for example, a cell that expresses PSGL-1 or VISTA. For example, an antibody that binds to a cell surface receptor expressed by a particular cell type (eg, an immune cell) can be fused or conjugated to a modified antibody provided herein.

In addition, the antibody provided herein may be conjugated to therapeutic moieties such as radioactive metal ions, such as alpha sources such as ²¹³ Bi, or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to limited to this, ¹³¹ In, ¹³¹ Lu, ¹³¹ Y, ¹³¹ Ho, ¹³¹ Sm, with polypeptides. In some embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are widely known in the art and are described in Denardo et al., 1998, Clin. Cancer Res., 4(10): 2483-90; Peterson et al, 1999, Bioconjug. Chem., 10(4): 553-7; and Zimmerman et al., 1999, Nucl. Med. Biol., 26(8): 943-50, each of which is incorporated by reference in its entirety.

In addition, to facilitate purification, the antibodies provided herein can be fused to marker sequences such as peptides. In specific embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc.), which is commercially available, among many others. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 821-824, for example, the presence of hexahistidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, a hemagglutinin (“HA”) tag, which corresponds to an epitope of the influenza virus hemagglutinin protein (Wilson et al., 1984, Cell, 37:767), and a “FLAG” tag. .

Methods for fusing or conjugating therapeutic moieties (including polypeptides) with antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd Ed.), Robinson et al, (eds.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press, 1985), Thorpe et al. 1982, Immunol. Rev., 62: 119-58; US Patent Nos. 5336603, 5622929, 5359046, 5349053, 5447851, 5723125, 5783181, 5908626, 5844095 and 5112946; EP 307434; EP 367166; EP 394827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631 and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331: 84-86, 1988; Zheng et al., J. Immunol., 154: 5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA, 89: 11337-11341, 1992, which are incorporated herein by reference in their entirety.

Fusion proteins can be produced, for example, using gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling techniques (collectively referred to as “DNA shuffling”). DNA shuffling can be used to alter the activity of the antibodies provided herein (eg, to produce antibodies with higher affinity and lower dissociation rate constants). In general, see US Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol., 8: 724-33; Harayama, 1998, Trends Biotechnol., 16(2): 76-82; Hansson et al., 1999, J. Mol. Biol., 287: 265-76; and Lorenzo and Blasco, 1998, Biotechniques, 24(2): 308-313 (each of these patents and each of these publications is hereby incorporated by reference in its entirety). Antibodies or encoded antibodies may be the result of random mutagenesis techniques using error-prone PCR, random nucleotide insertion, or other techniques prior to recombination. A polynucleotide encoding an antibody provided herein may be recombined with one or more components, motifs, sections, portions, domains, fragments, etc. one or more than one of the heterologous molecules.

The antibody provided herein may also be conjugated to a second antibody to form an antibody heteroconjugate, as described in US Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated or recombinantly fused to an antibody provided herein that binds PSGL-1 must be selected to achieve the desired preventive(s) or therapeutic(s) effect(s). In some embodiments, the antibody is a modified antibody. The attending physician or other health care professional should consider the following when deciding which therapeutic moiety or drug to conjugate or recombinantly fuse with an antibody provided herein: the nature of the disease, the severity of the disease, and the health status of the subject.

The antibodies provided herein (eg, anti-PSGL-1 or anti-VISTA) can also be attached to solid supports, which are particularly useful for performing immunoassays or purifying the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions, including therapeutic compositions containing one or more therapeutic agents provided herein (e.g., an anti-VISTA therapeutic agent such as an anti-VISTA antibody), can be prepared for storage by mixing the antibody having the desired purity, with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA) in the form of lyophilized compositions or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium ion; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONIC™ or polyethylene glycol (PEG).

In addition, anti-VISTA therapeutic agents proposed herein, primarily anti-VISTA antibodies, can, for example, be formulated into liposomes. Liposomes containing the molecule of interest are prepared by methods known in the art, such as those described in Epstein et al. (1985) Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA, 77: 4030; and US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended residence time in the bloodstream are described in US Pat. No. 5,013,556.

In particular, useful immunoliposomes with a lipid composition that includes phosphatidylcholine, cholesterol, and a derivative of phosphatidylethanolamine and PEG (PEG-PE) can be formed by reverse-phase evaporation. Liposomes are pressed through filters with pores of a certain size, obtaining liposomes of the desired diameter. Fab' fragments of the antibody provided herein can be conjugated to liposomes as described in Martin et al. (1982) J. Biol. Chem., 257: 286-288, via a disulfide exchange reaction. Such a liposome may contain a chemotherapeutic agent (such as doxorubicin) within it; see Gabizon et al., (1989) J. National Cancer Inst., 81(19): 1484.

Compositions, such as those described herein, may also contain more than one active compound that is needed for the particular indication being treated. In some embodiments, the compositions contain an anti-VISTA therapeutic agent provided herein (eg, an anti-VISTA antibody) and one or more active compounds with complementary activities that do not adversely affect each other. Accordingly, such molecules are presented in combination in amounts that are effective for the intended purpose. For example, an antibody provided herein may be combined with one or more other therapeutic agents. Such combination therapy may be administered to a patient intermittently or together or sequentially.

An anti-VISTA therapeutic agent provided herein (eg, an anti-VISTA antibody) may also be encapsulated in a microcapsule prepared, for example, by coacervation or interfacial polymerization techniques, for example, a hydroxymethylcellulose or gelatin microcapsule and a poly(methyl methacrylate) microcapsule, respectively; in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, PA.

Compositions intended for use by in vivo administration must be sterile. This is easily achieved by filtration, for example through sterilizing filtration membranes.

Extended-release formulations can also be made. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing a polypeptide, the matrices being in the form of extruded articles such as films or microcapsules. Examples of sustained release matrices include matrices of polyesters, hydrogels (eg, poly-2-hydroxyethyl methacrylate or polyvinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene vinyl acetate , degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid polymers allow release of molecules over 100 days, certain hydrogels release proteins over shorter periods of time. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to the aqueous environment at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational strategies for stabilization can be developed. For example, if the mechanism of aggregation is found to be the formation of intermolecular S--S bonds via thiodisulfide exchange, then stabilization can be achieved through modification of sulfhydryl residues, lyophilization from acid solutions, control of moisture content, use of appropriate excipients and development of specific compositions based on polymer matrices.

In some embodiments, the pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more anti-VISTA therapeutic agents provided herein (eg, anti-VISTA antibodies) and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in preventing, treating, or alleviating one or more symptoms of a VISTA-mediated disease, disorder, or condition.

Pharmaceutical carriers suitable for administering the compounds provided herein include any such carriers known to those skilled in the art as suitable for a particular mode of administration.

In addition, the anti-VISTA therapeutic agents provided herein, primarily anti-VISTA antibodies, may be formulated as a single pharmaceutically active ingredient or may be combined with other active ingredients (such as one or more other prophylactic or therapeutic agent).

These compositions may contain one or more antibodies proposed in this application. In some embodiments, the anti-VISTA therapeutic agents (e.g., anti-VISTA antibodies) provided herein are formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release compositions, or elixirs, for oral administration or sterile solutions or suspensions for parenteral administration, as well as the drug in the form of a transdermal patch and dry powder inhalers. In some embodiments, pharmaceutical compositions of the anti-VISTA therapeutic agents (e.g., anti-VISTA antibodies) described above are prepared herein using methods and techniques well known in the art (see, e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, ^4th ed, p.126).

In some embodiments of the compositions, one or more anti-VISTA therapeutic agents (eg, one anti-VISTA antibody) at effective concentrations are mixed with a suitable pharmaceutically acceptable carrier. In some embodiments, the content of the compounds in the compositions is effective to deliver an amount, upon administration, that treats, prevents or ameliorates a VISTA-mediated disease, disorder or condition or a symptom thereof.

In some embodiments, the compositions are formulated for administration as a single dose. To prepare the composition, a weight fraction of the compound at an effective concentration is dissolved, suspended, dispersed in, or otherwise mixed with a selected vehicle so as to provide relief, prevention of the condition being treated, or amelioration of one or more than one symptom.

In some embodiments, an anti-VISTA therapeutic agent provided herein (eg, an anti-VISTA antibody) is administered to a pharmaceutically acceptable carrier in an effective amount sufficient to provide a therapeutically beneficial effect in the absence of undesirable side effects in the patient being treated. The therapeutically effective concentration can be determined empirically by testing compounds in in vitro and in vivo systems using routine methods and then extrapolating these data to human dosages.

The content of the anti-VISTA therapeutic agent (eg, anti-VISTA antibody) in the pharmaceutical composition will depend, for example, on the physicochemical characteristics of the therapeutic agent, the dosage regimen and the amount administered, as well as other factors known to those skilled in the art.

In some embodiments, a therapeutically effective dosage achieves a serum concentration of the anti-VISTA therapeutic agent (eg, anti-VISTA antibody) from about 0.1 ng/mL to about 50-100 μg/mL. The pharmaceutical compositions, in another embodiment, provide a therapeutic agent dosage of from about 0.001 mg to about 2000 mg per kilogram of body weight per day. Pharmaceutical unit dosage forms can be prepared to achieve between about 0.01 mg; 0.1 mg or 1 mg to approximately 500 mg; 1000 mg or 2000 mg; and in some embodiments, from about 10 mg to about 500 mg of an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) and/or a combination of other possible essential ingredients per unit dosage form.

The anti-VISTA therapeutic agent (eg, anti-VISTA antibody) may be administered at one time or may be divided into several smaller doses to be administered at intervals. Obviously, the exact dosage and duration of treatment depend on the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro testing data. It should be noted that concentrations and dosages may also vary depending on the severity of the condition being treated. It should also be understood that for any particular subject, specific administration regimens may be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges stated herein are only representative and are not intended to to limit the scope or practical application of the claimed compositions.

Once the anti-VISTA therapeutic agent is mixed or added, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the chosen vehicle or diluent. The effective concentration is sufficient to reduce the symptoms of the disease, disorder or condition being treated and can be determined empirically.

In some embodiments, pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions and oral solutions or oil-in-water suspensions and emulsions containing suitable amounts compounds or pharmaceutically acceptable derivatives thereof. The anti-VISTA therapeutic agent (eg, an anti-VISTA antibody), in some embodiments, is formulated and administered in single-dose dosage forms or multi-dose dosage forms. The term “single-dose dosage forms” as used herein refers to physically discrete units suitable for human and animal subjects and individually packaged as is known in the art. Each dosage unit contains a predetermined amount of a therapeutic agent sufficient to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier, excipient or diluent. Examples of single-dose dosage forms include ampoules and syringes and individually packaged tablets or capsules. Single-dose dosage forms can be administered in fractions or multiples. A “multi-dose dosage form” is a plurality of identical single-dose dosage forms packaged in a single container, administered in a separate single-dose dosage form. Examples of multi-dose dosage forms include vials, tablet or capsule bottles, or pint or gallon bottles. Therefore, a multi-dose dosage form is a plurality of single-dose forms that are contained in a single package.

In some embodiments, one or more anti-VISTA therapeutic agents provided herein (eg, one anti-VISTA antibody) are contained in a liquid pharmaceutical composition. Liquid pharmaceutical compositions for administration can be prepared, for example, by dissolving, dispersing or otherwise mixing the active compound as defined above and possible pharmaceutical adjuvants in a carrier such as, for example, water, saline, aqueous dextrose, glycerin, glycols, ethanol and the like, thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic excipients such as wetting agents, emulsifying agents, solubilizing agents, pH maintaining agents and the like, for example, acetic acid salts, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate , triethanolamine, sodium acetate, triethanolamine oleate and other similar agents.

Current methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

Dosage forms or compositions may be prepared containing the therapeutic agent, specifically the antibody, in the range of 0.005% to 100%, with the remainder being composed of a non-toxic carrier. Methods for preparing such compositions are known to those skilled in the art.

Pharmaceutical dosage forms for oral administration are either in solid, gel or liquid form. Solid dosage forms include tablets, capsules, granules and bulk powders. Types of oral tablets include compressed tablets, chewable lozenges, and tablets that may be enteric-coated, sugar-coated, or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be offered in non-effervescent or effervescent form containing a combination of other ingredients known to those skilled in the art.

In some embodiments, the compositions are dosage forms in solid form. In some embodiments, the compositions are capsules or tablets. Tablets, pills, capsules, lozenges and the like may contain one or more of the following ingredients or compounds of a similar nature: a binder; lubricant; diluent; glidant; baking powder; dye; sweetener; flavoring; moisturizing agent; vomit-inducing coating and film coating. Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, gum acacia, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.

Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Dyes include, for example, any approved certified water-soluble dyes for use in foods, drugs and cosmetics (FD&C dyes), mixtures thereof; and water-insoluble FD and C dyes suspended in alumina hydrate. Sweeteners include sucrose, lactose, mannitol and artificial sweeteners such as saccharin, and any number of spray-dried flavors. Flavors include natural flavors extracted from plants, such as fruits, and mixtures of synthetic compounds that produce a pleasant taste sensation, such as, but not limited to, peppermint oil and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, grease, waxes, shellac, ammonia shellac, and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

The anti-VISTA therapeutic agents provided herein (eg, anti-VISTA antibodies) may be formulated to protect them from the acidic environment of the stomach. For example, the composition may be formulated with an enteric coating that maintains its integrity in the stomach and allows release of the active compound in the intestines. The composition may also be formulated in combination with an acid neutralizing agent or other such ingredient.

When the unit dosage form is a capsule, it may contain, in addition to a substance of the type described above, a liquid carrier such as a fixed liquid oil. In addition, unit dosage forms may contain various other materials that modify the physical form of the dosage unit, such as sugar coatings and other enteric agents. The compounds may also be administered as a component of an elixir, suspension, syrup, cachet, burst capsule, chewing gum, or the like. In addition to the active compound, syrup may contain sucrose as a sweetener and some preservatives, dyes and coloring agents and flavorings.

The therapeutic agent may also be mixed with other active substances that do not impair the desired effect, or with substances that complement the desired effect, such as antacids, histamine type 2 (H2) blockers, and diuretics. The active ingredient is an anti-VISTA therapeutic agent, primarily an antibody or a pharmaceutically acceptable derivative thereof described herein. The active ingredient may be included in higher concentrations, up to about 98% by weight.

In some embodiments, compositions in the form of tablets and capsules may be coated, as known to those skilled in the art, to modify or promote dissolution of the active ingredient. For example, they can be coated with traditional enteric coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.

In some embodiments, the compositions are liquid dosage forms. Liquid dosage forms for oral administration include aqueous solutions, emulsions, suspensions, solutions and/or suspensions obtained by reconstitution from non-effervescent granules, and effervescent preparations obtained by reconstitution from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil emulsions.

Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugar, such as sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small droplets in another liquid. Pharmaceutically acceptable carriers used in emulsions include non-aqueous liquids, emulsifying agents and preservatives. Pharmaceutically acceptable suspending agents and preservatives are used in suspensions. Pharmaceutically acceptable substances used in the non-effervescent granules to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in the effervescent granules to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Dyes and flavorings are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propyl paraben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, gum acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, veegum and gum acacia. Sweeteners include sucrose, syrups, glycerin and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Dyes include any approved certified water-soluble FD and C dyes and mixtures thereof. Flavors include natural flavors extracted from plants, such as fruits, and mixtures of synthetic compounds that produce a pleasant taste sensation.

In some embodiments, to prepare a solid dosage form, a solution or suspension, for example, in propylene carbonate, vegetable oils or triglycerides, is encapsulated in a gelatin capsule. Such solutions and their preparation and encapsulation are described in US Pat. Nos. 4,328,245, 4,409,239, and 4,410,545. To prepare a liquid dosage form, a solution, for example, in polyethylene glycol, can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, to easily measure for insertion.

Alternatively, liquid or semi-solid compositions for oral administration can be prepared by dissolving or dispersing the active compound or a salt thereof in vegetable oils, glycols, triglycerides, propylene glycol esters (eg, propylene carbonate) and other similar vehicles and encapsulating these solutions or suspensions in solid shells or soft gelatin capsules. Other useful compositions include those set forth in US Patent Nos. RE28819 and 4358603. Briefly, such compositions include, but are not limited to, compositions containing the compound provided herein, dialkylated mono- or polyalkylene glycol, including but not limited to these, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol dimethyl ether-350, polyethylene glycol dimethyl ether-550, polyethylene glycol dimethyl ether-750, where 350, 550 and 750 refer to the approximate average molecular weight of polyethylene glycol, and one or more antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its complexes ethers and dithiocarbamates.

Other compositions include, but are not limited to, aqueous alcoholic solutions containing a pharmaceutically acceptable acetal. Alcohols used in these compositions are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals (lower alkyl) aldehydes, such as acetaldehyde diethyl acetal.

Parenteral administration, in some embodiments, is characterized by injection, and administration either subcutaneously, intramuscularly, intratumorally, or intravenously is also covered herein. Injectable preparations may be prepared in conventional forms, either as solutions or suspensions in liquid, or as solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Injectable preparations, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic excipients such as wetting or emulsifying agents, pH maintaining agents, stabilizers, solubility enhancers and other similar agents such as, for example, sodium acetate, sorbitan monolaurate , triethanol aminooleate and cyclodextrins.

Implantation of a slow-release or sustained-release system to maintain a constant dosage level (see, for example, US Pat. No. 3,710,795) is also included herein. Briefly, the compound provided herein is dispersed in a solid internal matrix, for example, polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone new ones rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as acrylic and methacrylic acid ester hydrogels, collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolyzed polyvinyl acetate, which (matrix) is surrounded by an outer polymer membrane, e.g. polyethylene, polypropylene , ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymers, vinylidene chloride, ethylene and propylene, ion containing polyethylene terephthalate, butyl rubber , epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer and ethylene/vinyloxyethanol copolymer, which (membrane) is insoluble in body fluids. The therapeutic agent (eg, antibody) diffuses through the outer polymer membrane during the release step at a controlled rate. The amount of therapeutic agent contained in such parenteral compositions depends greatly on its specific nature, as well as the activity of the compound and the needs of the subject.

Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready for combination with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions, ready for injection, sterile dry insoluble products , ready to be combined with diluent immediately before use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

For intravenous administration, suitable carriers include saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral formulations include aqueous diluents, non-aqueous diluents, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, complexing or chelating agents and other pharmaceutically acceptable agents.

Examples of aqueous diluents include sodium chloride injection, Ringer's solution for injection, isotonic dextrose injection, sterile water for injection, dextrose-lactated Ringer's solution for injection. Non-aqueous parenteral diluents include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multi-dose containers, which include phenols or cresols, mercuric compounds, benzyl alcohol, chlorobutanol, l-hydroxybenzoic acid methyl and propyl esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate buffers. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). The metal ion complexing or chelating agent includes EDTA. Pharmaceutically acceptable carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water-miscible diluents; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

The concentration of the pharmaceutically active anti-VISTA therapeutic agent (eg, anti-VISTA antibody) is adjusted to provide an effective amount after injection to produce the desired pharmacological effect. The exact dosage will depend on the age, weight and health status of the patient or animal, as is known in the art.

Single-dose parenteral medications may be packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration may be sterile, as is known and practiced in the art.

By way of illustration, intravenous or intraarterial infusion of a sterile aqueous solution containing the active compound is considered an effective route of administration. Another embodiment is a sterile aqueous or oily solution or suspension in water or oil containing the active substance, administered by injection as needed to obtain the desired pharmacological effect.

Injectable drugs are intended for local and systemic administration. In some embodiments, the formulation at a therapeutically effective dosage is formulated such that the tissue(s) being treated contains the active compound at a concentration of at least about 0.1% (w/w) to about 90% (wt/wt) or higher, in certain embodiments, higher than 1% (wt/wt).

The therapeutic agent, such as an antibody, can be suspended into a micronized or other suitable form. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen vehicle or diluent. The effective concentration is sufficient to reduce the symptoms of a VISTA-mediated disease, disorder or condition and can be determined empirically.

In some embodiments, the pharmaceutical compositions are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. In addition, they can be re-diluted and based on them, compositions can be prepared in the form of solids or gels.

The lyophilized powder is prepared by dissolving a therapeutic agent, such as an antibody provided herein, or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain an excipient that improves stability or other pharmacological component of the powder or solution prepared by reconstitution from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as a citrate, sodium or potassium phosphate buffer, or other such buffer known to those skilled in the art, in some embodiments at approximately neutral pH. Subsequent sterile filtration of the solution, then lyophilization under standard conditions known to those skilled in the art, provides the desired composition. In some embodiments, the resulting solution will be portioned into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

By reconstituting this lyophilized powder with water for injection, a composition for parenteral administration is obtained. To effect reconstitution, the lyophilized powder is added to sterile water or any other suitable carrier. The exact amount depends on the selected compound. This amount can be determined empirically.

Mixtures for topical use are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, or the like and may be prepared in the form of creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, rinses, sprays, suppositories , dressings, dermal patches or any other composition suitable for topical administration.

The therapeutic agents proposed herein can be formulated into aerosols for topical use, for example, by inhalation (see, for example, US Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which disclose aerosols for delivering a steroid compound useful in the treatment of inflammatory diseases, in particular asthma). These respiratory compositions may be in the form of an aerosol or nebulizer solution, or as a micronized powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the composition will, in some embodiments, have a diameter of less than 50 microns, in some embodiments, less than 10 microns.

Based on these therapeutic agents, compositions can be prepared for topical or local use, for example, for topical application to the skin and mucous membranes, such as the eyes, in the form of gels, creams and lotions, and for application to the eye, or for intracisternal or intraspinal use . Topical administration is intended for transdermal delivery, as well as for ocular or mucosal administration, or for inhalation therapies. In addition, nasal solutions of the active compound, alone or in combination with other pharmaceutically acceptable excipients, can be administered.

Such solutions, particularly those intended for ophthalmic use, can be prepared as 0.01% to 10% isotonic solutions, pH approximately 5-7, using appropriate salts.

Other routes of administration, for example, using transdermal patches, including iontophoretic and electrophoretic agents, and rectal administration, are also included in this description.

Transdermal patches, including iontophoretic and electrophoretic agents, are well known to those skilled in the art. For example, such patches are described in US patents No. 6267983, 6261595, 6256533, 6167301, 6024975, 6010715, 5985317, 5983134, 5948433 and 5860957.

Pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic action. The term "rectal suppositories" as used herein means solid forms for administration to the rectum that melt or soften at body temperature, releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories include bases or fillers and melting point elevating agents. Examples of bases include cocoa butter (cocoa bean butter), glycerin-gelatin mixture, Carbowax (polyoxyethylene glycol) and corresponding mixtures of mono-, di- and triglycerides of fatty acids. Combinations of different bases can be used. Agents for increasing the melting point of suppositories include spermaceti and wax. Rectal suppositories can be prepared either by pressing or molding. The weight of the rectal suppository, in some embodiments, is approximately 2-3 grams.

Tablets and capsules for rectal administration can be prepared using the same pharmaceutically acceptable substance and by the same methods as in the case of compositions for oral administration.

Therapeutic agents (eg, antibodies) and other compositions provided herein may also be formulated to target a specific tissue, receptor, or other area of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are included herein for use in the compositions of the present invention. For non-limiting examples of targeting methods, see, for example, US Pat. Nos. 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975, 60 04534, 5985307, 5972366, 5900252, 5840674, 5759542 and 5709874.

In one embodiment, liposomal suspensions, including tissue-targeting liposomes such as tumor-targeting liposomes, can also be used as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art. For example, liposome compositions can be prepared as described in US Pat. No. 4,522,811. Briefly, liposomes such as multilayer vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (in a molar ratio of 7:3) on the inner surface of a flask. A solution of the compound provided herein in phosphate buffered saline (PBS) free of divalent cations is added and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, collected by centrifugation and then resuspended in PBS.

METHODS OF TREATMENT, PREVENTION AND/OR RELIEF

In another aspect, the present invention also provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in treating a VISTA-mediated disease, disorder or condition in a patient. The present invention provides an anti-VISTA therapy (e.g., the anti-VISTA antibody provided herein) for use in the prevention, treatment, and/or alleviation of one or more symptoms of a disease, disorder, or condition, such as a VISTA-mediated disease. disorder or condition, primarily VISTA-mediated cancer. Preferably, said VISTA-mediated disease, disorder or condition has been established or diagnosed in advance by one of the methods provided herein.

In one embodiment, the present invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of a VISTA-mediated disease, disorder or condition in a patient, wherein said VISTA-mediated disease, disorder or condition is established or diagnosed in advance one of the methods proposed in this application. In other words, the present invention thus provides an anti-VISTA therapeutic agent for treating a VISTA-mediated disease, disorder or condition in a patient, wherein the anti-VISTA therapeutic agent is administered to a patient diagnosed with a VISTA-mediated disease, disorder or condition with using the method described above.

Some embodiments of the present invention provide compositions comprising one or more antibodies (e.g., an anti-VISTA antibody) provided herein for use in inhibiting, preventing, or treating a VISTA-mediated disease, disorder, or condition, and/or for alleviating one or more than one symptom of a VISTA-mediated disease, disorder or condition. Exemplary VISTA-mediated diseases, disorders or conditions include cell proliferation disorder, cancer and graft-versus-host disease (GVHD) or symptoms thereof. Preferably, said VISTA-mediated disease, disorder or condition is cancer.

Thus, the present invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of VISTA-mediated cancer in a patient, said use including:

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

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

In a preferred embodiment, use of the present invention further includes the step of evaluating a tumor by comparing the level of PSGL-1 expression in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) on an appropriate scale based on two parameters, staining intensity and percentage cells giving a positive response.

In another embodiment, the present invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of VISTA-mediated cancer in a patient, said use comprising pre-determining the PSGL-1 status of said tumor as described above. In this embodiment, a tumor having a [PSGL-1(+)] status indicates the presence of a VISTA-mediated cancer and is thus susceptible to treatment with an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody).

According to another preferred embodiment, use of the present invention further includes comparing the expression level of PSGL-1 in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) with a reference level.

In this preferred embodiment, an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) is provided for use in the treatment of a VISTA-mediated cancer in a patient, said use including:

a) determining the level of expression of PSGL-1 in a biological sample from the specified subject, for example, using immune infiltrates of the tumor microenvironment in the specified biological sample;

b) comparison of the expression level at stage a) with the reference level; And

c) detection of VISTA-mediated cancer when the expression level in stage a) is above the reference level.

In another preferred embodiment, an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) is provided for use in the treatment of VISTA-mediated cancer in a patient, said use including:

a) determining the level of expression of PSGL-1 in a biological sample from the specified subject, for example, using immune infiltrates of the tumor microenvironment in the specified biological sample;

b) comparison of the expression level at stage a) with the reference level; And

c) diagnosing VISTA-mediated cancer when the expression level at stage a) is above the reference level.

Preferably, the method according to the invention includes both stages:

• assessing the tumor by comparing the level of PSGL-1 expression in a biological sample from the subject (eg, using immune infiltrates of the tumor microenvironment) on an appropriate scale based on two parameters, staining intensity and the percentage of positive cells; And

• comparing the expression level of PSGL-1 in a biological sample from the subject (using immune infiltrates of the tumor microenvironment) with a reference level.

Preferably, the above-mentioned use of an anti-VISTA therapeutic agent will further include determining the expression level of at least one of VISTA, CD11b, CD33, CD4 and CD8, as detailed above. In such cases, the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8, or relative expression levels thereof, above the reference level indicates the presence of VISTA-mediated cancer.

According to another embodiment, the invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of VISTA-mediated cancer in a patient, said use including:

a) contacting a biological sample from said subject with a reagent capable of specifically binding to a PSGL-1 encoding nucleic acid or PSGL-1 protein; And

b) quantifying the binding of said reagent to said biological sample, thereby determining the level of PSGL-1 expression in said sample; And

c) tailoring treatment with an anti-VISTA therapeutic agent based on the level in step a).

In a preferred embodiment, use of the present invention further includes the step of evaluating a tumor by comparing the level of PSGL-1 expression in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) on an appropriate scale based on two parameters, staining intensity and percentage cells giving a positive response.

In another embodiment, the present invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of VISTA-mediated cancer in a patient, said use comprising pre-determining the PSGL-1 status of said tumor as described above. In this embodiment, a tumor having a [PSGL-1(+)] status indicates the presence of a VISTA-mediated cancer and is thus susceptible to treatment with an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody).

According to another preferred embodiment, use of the present invention further includes comparing the expression level of PSGL-1 in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) with a reference level.

Said adaptation of treatment with an anti-VISTA therapeutic agent may consist of:

- reducing or suspending specified treatment with an anti-VISTA therapeutic agent if the patient is determined to be unresponsive to the anti-VISTA therapeutic agent, or

- continuation of specified treatment with an anti-VISTA therapeutic agent if the patient is determined to be susceptible to the anti-VISTA therapeutic agent.

A patient is considered responsive to a given treatment if there is a difference in PSGL-1 expression observed between the expression level in step a) and the reference level. For example, the difference in PSGL-1 expression observed between the expression level in step a) and the PSGL-1 expression level in a second biological sample from a patient obtained prior to treatment indicates whether said patient is responsive to said treatment. Preferably, a higher level of PSGL-1 expression in step a) compared to the level of PSGL-1 expression in the second biological sample from the patient obtained prior to treatment indicates that said patient is susceptible to said treatment.

In some embodiments, the above use includes determining the expression level of at least one of VISTA, CD11b, CD33, CD4 and CD8, in addition to PSGL-1, and comparing the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 or relative expression levels thereof in a first biological sample with the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 or relative expression levels thereof in a second biological sample from the patient obtained prior to treatment. In this case, the difference in the expression level or relative expression levels of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 in the first biological sample compared to the expression level or relative expression levels of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8 in the second biological sample indicates that the patient is responsive to treatment.

In some aspects of this method, treatment includes administration of an anti-VISTA antibody and/or an anti-PSGL-1 antibody described herein.

In some aspects, the method includes cases where the first biological sample contains immune infiltrates of the tumor microenvironment.

In one embodiment, the present invention provides methods for preventing or treating a disease, disorder, or condition set forth herein by administering to a subject a therapeutically effective amount of an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody), including an agent described herein , or compositions based on it. In some embodiments, a method of treating a disease, disorder, or condition includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an anti-VISTA antibody and a pharmaceutically acceptable carrier, excipient, and/or stabilizer. The method provided herein may also optionally employ at least one additional therapeutic agent, such as the agents described herein (eg, an anti-VISTA antibody), either as a single treatment or in a combination. Also disclosed herein are compositions, including pharmaceutical compositions, containing an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) provided herein for use in the treatment, prevention, and/or alleviation of one or more than one symptom. a disease, disorder or condition, such as a VISTA-mediated disease, disorder or condition. A typical VISTA-mediated disease, disorder or condition includes a disorder of cell proliferation (eg, cancer or tumor) or a symptom thereof.

Some embodiments of this application disclose compositions containing an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) for use in the prevention, treatment, and/or alleviation of one or more symptoms of a VISTA-mediated disease, disorder, or condition, such as a cell proliferation disorder . A disorder of cell proliferation includes cancer or tumor formation or a symptom thereof. In some embodiments, impaired cell proliferation is associated with increased expression and/or increased activity of VISTA. In some embodiments, impaired cell proliferation is associated with increased expression of VISTA on the surface of the cancer cell.

In another aspect, the present invention also provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in treating a PSGL-1-mediated disease disorder or condition in a patient. The present invention provides anti-VISTA therapy (e.g., the anti-VISTA antibody provided herein) for use in the prevention, treatment, and/or alleviation of one or more symptoms of a disease, disorder, or condition, such as PSGL-1-mediated disease, disorder or condition, primarily PSGL-1-mediated cancer. Preferably, said PSGL-1-mediated disease, disorder or condition is established or diagnosed in advance by one of the methods proposed herein.

In one embodiment, the present invention provides an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) for use in treating a PSGL-1-mediated disease, disorder, or condition in a patient, wherein said PSGL-1-mediated disease, disorder, or condition established or diagnosed in advance by one of the methods proposed in this application. In other words, the invention thus relates to an anti-VISTA therapeutic agent for treating a PSGL-1-mediated disease, disorder or condition in a patient, wherein the anti-VISTA therapeutic agent is administered to a patient diagnosed with a PSGL-1-mediated disease, disorder or condition using the method described above.

Some embodiments of the present invention provide compositions comprising one or more antibodies (e.g., an anti-VISTA antibody) provided herein for use in inhibiting, preventing, or treating a PSGL-1-mediated disease, disorder, or condition and/or to relieve one or more symptoms of a PSGL-1-mediated disease, disorder or condition. Exemplary PSGL-1-mediated diseases, disorders, or conditions include cell proliferation disorder, cancer, and graft-versus-host disease (GVHD) or symptoms thereof. Preferably, said PSGL-1-mediated disease, disorder or condition is cancer.

Thus, the present invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of PSGL-1-mediated cancer in a patient, said use including:

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

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

In a preferred embodiment, use of the present invention further includes the step of evaluating a tumor by comparing the level of PSGL-1 expression in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) on an appropriate scale based on two parameters, staining intensity and percentage cells giving a positive response.

In another embodiment, the present invention provides an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) for use in the treatment of a PSGL-1-mediated cancer in a patient, said use comprising first determining the PSGL-1 status of said tumor as described above . In this embodiment, a tumor having a [PSGL-1(+)] status indicates the presence of a PSGL-1-mediated cancer and is thus susceptible to treatment with an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody).

According to another preferred embodiment, use of the present invention further includes comparing the expression level of PSGL-1 in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) with a reference level.

In this preferred embodiment, an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) is for use in the treatment of PSGL-1-mediated cancer in a patient, said use including:

a) determining the level of expression of PSGL-1 in a biological sample from the specified subject, for example, using immune infiltrates of the tumor microenvironment in the specified biological sample;

b) comparison of the expression level at stage a) with the reference level; And

c) identification of PSGL-1-mediated cancer if the expression level at stage a) exceeds the reference level.

In another preferred embodiment, the anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) is for use in the treatment of PSGL-1-mediated cancer in a patient, wherein said use includes:

a) determining the level of expression of PSGL-1 in a biological sample from the specified subject, for example, using immune infiltrates of the tumor microenvironment in the specified biological sample;

b) comparison of the expression level at stage a) with the reference level; And

c) diagnosis of PSGL-1-mediated cancer if the expression level at stage a) exceeds the reference level.

Preferably, the method according to the invention includes both stages:

• assessing the tumor by comparing the level of PSGL-1 expression in a biological sample from the subject (eg, using immune infiltrates of the tumor microenvironment) on an appropriate scale based on two parameters, staining intensity and the percentage of positive cells; And

• comparison of the expression level of PSGL-1 in a biological sample from the subject (eg, using immune infiltrates of the tumor microenvironment) with a reference level.

Preferably, the above use of an anti-VISTA therapeutic agent will further include determining the expression level of at least one of VISTA, CD11b, CD33, CD4 and CD8, as detailed above. In such cases, the expression level of PSGL-1 and at least one of VISTA, CD11b, CD33, CD4 and CD8, or relative expression levels thereof, above the reference level indicates the presence of PSGL-1-mediated cancer.

According to another embodiment, the invention provides an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of PSGL-1-mediated cancer in a patient, said use including:

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

b) quantifying the binding of said reagent to said biological sample, thereby determining the level of PSGL-1 expression in said sample; And

c) tailoring treatment with an anti-VISTA therapeutic agent based on the level in step a).

According to a preferred embodiment, the use of the present invention further includes the step of assessing a tumor by comparing the level of PSGL-1 expression in a biological sample from a subject (for example, using immune infiltrates of the tumor microenvironment) on an appropriate scale based on two parameters, staining intensity and percentage proportion of positive cells.

In another embodiment, the present invention provides an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) for use in the treatment of a PSGL-1-mediated cancer in a patient, said use comprising first determining the PSGL-1 status of said tumor as described above . In this embodiment, a tumor having a [PSGL-1(+)] status indicates the presence of a PSGL-1-mediated cancer and is thus susceptible to treatment with an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody).

According to another preferred embodiment, use of the present invention further includes comparing the expression level of PSGL-1 in a biological sample from a subject (eg, using immune infiltrates of the tumor microenvironment) with a reference level.

Said adaptation of treatment with an anti-VISTA therapeutic agent may consist of:

- reducing or suspending specified treatment with an anti-VISTA therapeutic agent if the patient is determined to be unresponsive to the anti-VISTA therapeutic agent, or

- continuation of specified treatment with an anti-VISTA therapeutic agent if the patient is determined to be susceptible to the anti-VISTA therapeutic agent.

A patient is considered responsive to a given treatment if there is a difference in PSGL-1 expression observed between the expression level in step a) and the reference level. For example, the difference in PSGL-1 expression observed between the expression level in step a) and the PSGL-1 expression level in a second biological sample from a patient obtained prior to treatment indicates whether said patient is responsive to said treatment. Preferably, a higher level of PSGL-1 expression in step a) compared to the level of PSGL-1 expression in the second biological sample from the patient obtained prior to treatment indicates that said patient is susceptible to said treatment.

Examples of disorders of cell proliferation that may be treated, prevented, or ameliorated using the antibodies provided herein include, but are not limited to, hematologic malignancies (e.g., leukemia, lymphoma, or myeloma), bladder cancer, or sarcoma. breast, colon, connective tissue, rectum, stomach, esophagus, lung, larynx, kidney, oral cavity, ovarian or prostate, melanoma or glioma, or metastases of any of these cancers. Typical hematologic malignancies include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, localized myeloma or extramedullary myeloma.

In some embodiments, the hematologic malignancy is a lymphoma. In other embodiments, the hematologic malignancy is leukemia. In some embodiments, the hematologic malignancy is myeloma. In another embodiment, the hematologic malignancy is acute myeloid leukemia (AML). In another embodiment, the hematologic malignancy is acute lymphoblastic leukemia (ALL). In another embodiment, the hematologic malignancy is chronic myelogenous leukemia (CML). In another embodiment, the hematologic malignancy is chronic lymphocytic leukemia (CLL). In another embodiment, the hematologic malignancy is acute monocytic leukemia (AMoL). In another embodiment, the hematologic malignancy is Hodgkin's lymphoma. In another embodiment, the hematologic malignancy is non-Hodgkin's lymphoma. In another embodiment, the hematologic malignancy is multiple myeloma. In another embodiment, the hematologic malignancy is a plasmacytoma. In another embodiment, the hematologic malignancy is a localized myeloma. In another embodiment, the hematologic malignancy is an extra medullary myeloma.

In some embodiments, the hematologic malignancy is a myelodysplastic syndrome, acute leukemia, e.g., acute T-cell leukemia, acute myelogenous leukemia (AML), acute promyelocytic leukemia, acute myeloid leukemia, acute megakaryoblastic leukemia, acute lymphoblastic B-cell precursor leukemia, T-cell precursor acute lymphoblastic leukemia, Burkitt's leukemia (Burkitt's lymphoma) or acute biphenotypic leukemia; chronic leukemia, for example chronic myeloid lymphoma, chronic myelogenous leukemia (CML), chronic monocytic leukemia, small cell lymphocytic lymphoma or B-cell prolymphocytic leukemia; hairy cell lymphoma; T-cell prolymphocytic leukemia; or lymphoma, for example, histiocytic lymphoma, lymphoplasmacytic lymphoma (eg, Waldenström's macroglobulinemia), splenic marginal zone lymphoma, plasma cell tumor (eg, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulinopathy or heavy chain disease), extranodal B-cell marginal zone lymphoma zone (mucosal associated tissue lymphoma (MALT)), nodal marginal zone cell lymphoma (NMZL) B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, large cell B-cell lymphoma of the mediastinum (thymus), intravascular large cell B-cell lymphoma, primary lymphoma of the serous cavities, T-cell leukemia of large granular lymphocytes, aggressive NK-cell leukemia, T-cell leukemia/T-cell lymphoma of adults, extranodal NK/T-cell lymphoma of the nasal type, T-cell lymphoma of the enteropathy type, hepatolienal T-cell lymphoma, blast NK-cell lymphoma, mycosis fungoides (Sézary syndrome), primary cutaneous CD30-positive T-cell lymphoproliferative disorder (eg, primary cutaneous large cell anaplastic lymphoma or lymphomatoid papulosis), angioimmunoblastic T-cell lymphoma, lymphoma of cells with a peripheral T-cell immunophenotype, unspecified, anaplastic large cell lymphoma, Hodgkin's lymphoma or nodular-type Hodgkin's lymphoma with a large number of lymphocytes.

An anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) described herein can be administered to a human for therapeutic purposes. In addition, an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) can be administered to a non-human mammal expressing VISTA with which the antibody cross-reacts (eg, a primate, pig, rat or mouse), for veterinary purposes or as an animal models of the disease in humans. With regard to the latter, such animal models may be useful for assessing the therapeutic efficacy of the antibodies proposed in this application (eg, for testing dosages and time courses during administration).

In some embodiments, the anti-VISTA therapeutic agent is an antibody that can be used in a method of modulating T cell function mediated by the binding of VISTA to PSGL-1. Such methods may include contacting the T cell with an anti-VISTA antibody described herein. In some embodiments, the anti-PSGL-1 antibody does not block or inhibit the binding of PSGL-1 to P-selectin, L-selectin, or E-selectin. In some embodiments, a method of modulating T cell function comprises administering an effective amount of a composition comprising an anti-VISTA antibody provided herein to a subject. In some aspects, the T cell function being modulated is increased T cell activation. Such T cell activation may also involve increased T cell proliferation. Techniques for immune response modulation assays are well known to one of ordinary skill in the art, and such assays will readily be performed by one of ordinary skill in the art.

In some embodiments, an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) or a composition containing an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody), including an agent described herein, can be used either alone or in combination combinations with another compound or treatment. For example, in some embodiments, the other compound is an antagonist of a co-inhibitory molecule or an agonist of a co-stimulatory molecule. In such embodiments, the combination therapy results in renewed enhancement or de novo activation of the immune system through activated T cells, which is greater than the administration of either the compound or the other therapy alone. Such activation of the immune system will result in a highly effective physiological response in the treatment of a VISTA-mediated disease, disorder or condition, including in the context of cancer treatment (eg, treatment of a hematologic malignancy).

In some embodiments, the methods described herein may include administering a therapeutically effective amount of an anti-VISTA antibody in combination with a therapeutically effective amount of a coinhibitory molecule antagonist. In some embodiments, the coinhibitory molecule is selected from the group consisting of CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2, B7-NZ, B7-H4, butyrophilin, CD48, CD244, TIM-3 , CD200R, CD200, CD160, BTLA, HVEM, LAIR1, T1M1, galekgin 9, TIM3, CD48, 2 B4, CD155, CD112, CD113 and TIGIT. A coinhibitory molecule antagonist includes an antibody to the coinhibitory molecule. It is generally accepted that antagonists of other coinhibitory molecules are well known in the art, such as those described in Mercier et al., Frontiers in Immunology, 6: 418 (2015), Kyi et al., FEBS Letters, 588: 368-376 (2014) and Pardoll, Nature Reviews, 12: 252-264 (2012). According to this embodiment, the invention relates to the use of an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) for use in the treatment of a VISTA-mediated tumor as described above, said use further comprising administration of an antagonist of a coinhibitory molecule, wherein said coinhibitory molecule is selected from the group , consisting of CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2, B7-NZ, B7-H4, butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA , HVEM, LAIR1, T1M1, galectin 9, TIM3, CD48, 2 B4, CD155, CD112, CD113 and TIGIT.

In some embodiments, the methods described herein may include administering a therapeutically effective amount of an anti-VISTA antibody in combination with a therapeutically effective amount of a costimulatory molecule agonist. In some embodiments, the co-stimulatory molecule is selected from the group consisting of CD154, TNFRSF25, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L, CD70, HHLA2, ICOSL, cytokine, LIGHT , HVEM, CD30, CD30L, B7-H2, CD80, CD86, CD40L, TIM4, T1M1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2 and CD226. A costimulatory molecule agonist includes an agonistic antibody to a costimulatory molecule. It is generally accepted that agonists of other co-stimulatory molecules are well known in the art, such as those described in Mercier et al., Frontiers in Immunology, 6: 418 (2015), Kyi et al., FEBS Letters, 588: 368-376 (2014) and Sarese et al., J. Biomed. Biotechnol., 2012: 926321, 17, pages (2012). According to this embodiment, the invention relates to the use of an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of a VISTA-mediated tumor as described above, wherein said use further comprises administration of an agonist costimulatory molecule, wherein said costimulatory molecule is selected from the group , consisting of CD154, TNFRSF25, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L, CD70, HHLA2, ICOSL, cytokine, LIGHT, HVEM, CD30, CD30L, B7 -H2, CD80, CD86, CD40L, TIM4, T1M1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2 and CD226.

In some embodiments, the methods described herein may include administering a therapeutically effective amount of an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) in combination with a conventional form of therapy for treating cancer, such as administering a therapeutically effective amount of a chemotherapeutic agent described in this application, or the use of radiation therapy described in this application. According to this embodiment, the invention relates to the use of an anti-VISTA therapeutic agent (eg, an anti-VISTA antibody) for use in the treatment of a VISTA-mediated tumor, as described above, wherein said use further includes the administration of a conventional form of therapy for the treatment of cancer, such as the administration of therapeutically an effective amount of a chemotherapeutic agent described in this application, or the use of radiation therapy described in this application.

Various delivery systems are known and can be used to administer an anti-VISTA therapeutic agent (e.g., the anti-VISTA antibody described herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, the use of recombinant cells capable of expressing a given antibody, receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, and so on. Methods of administration of a therapeutic agent (e.g., the anti-VISTA antibody provided herein) or pharmaceutical composition include, but are not limited to, parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In some embodiments, the therapeutic agent (eg, the anti-VISTA antibody provided herein) or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, intratumorally, or subcutaneously. The therapeutic agents or compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, through the oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc. .) and can be administered together with other biologically active agents. Administration may be systemic or local. In addition, pulmonary administration can also be used, for example by using an inhaler or nebulizer and a composition with an aerosolizing agent. See, for example, US Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety.

In some embodiments, it may be desirable to locally administer a therapeutic agent or pharmaceutical composition provided herein to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion, by topical administration (eg by intranasal spray), by injection (primarily intratumoral injection) or by use of an implant, wherein the implant is porous , a non-porous or gel-like material, including membranes such as silastic membranes or fibers. In some embodiments, when administering an antibody provided herein, caution should be exercised when using materials that do not absorb the antibody.

In some embodiments, the therapeutic agent provided herein may be delivered in a vesicle, particularly in a liposome (see Langer, 1990, Science, 249: 1527-1533; Treat et al. in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; generally see ibid.).

In some embodiments, a therapeutic agent provided herein may be delivered in a controlled release or sustained release system. In some embodiments, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng., 14:20; Buchwald et al., 1980, Surgery, 88:507; Saudek et al., 1989, N. Engl. J. Med., 321: 574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a therapeutic agent (eg, an antibody provided herein) or a composition provided herein (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds .), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol Sci. Rev. Macromol. Chem., 23: 61; also see Levy et al., 1985, Science, 228: 190; During et al., 1989, Ann. Neurol., 25: 351; Howard et al. , 1989, J. Neurosurg., 71:105); US Patent No. 5679377; US Patent No. 5916597; US Patent No. 5912015; US Patent No. 5989463; US Patent No. 5128326; PCT publication No. WO 99/15154; and PCT publication No. WO 99/20253. Examples of polymers used in sustained release compositions include, but are not limited to, poly-2-hydroxyethyl methacrylate, polymethyl methacrylate, polyacrylic acid, ethylene vinyl acetate copolymer, polymethacrylic acid, polyglycolides (PLG), polyanhydrides, poly-N-vinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polyethylene glycol, polylactides (PLA), polylactide-co-glycolides (PLGA) and polyorthoesters. In some embodiments, the polymer used in the sustained release composition is inert, free of leachable impurities, shelf stable, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system may be placed in close proximity to the therapeutic target, such as the nasal passages or lungs, resulting in only a fraction of the systemic dose being required (see, e.g., Goodson, in Medical Applications of Controlled Release , supra, vol. 2, pp. 115-138 (1984)). Controlled release systems are discussed in a review by Langer (1990, Science, 249: 1527-1533). To prepare sustained release compositions containing one or more of the antibodies provided herein, any method known to one skilled in the art can be used. See, for example, US Pat. No. 4,526,938, PCT Publication WO 91/05548, PCT Publication WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel", Radiotherapy & Oncology, 39:179-189, Song et al., 1995, Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA Journal of Pharmaceutical Science & Technology, 50: 372-397, Cleek et at., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Pro. Int'l. Symp. Control. Rel. Bioact. Mater., 24: 853-854 and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery ", Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24: 759-760.

In some embodiments, the composition used in the method provided herein contains one, two or more antibodies provided herein (eg, an anti-VISTA antibody). In another embodiment, the composition used in the method proposed in this application contains one, two or more antibodies proposed in this application, and a therapeutic agent different from the antibody proposed in this application. In some embodiments, these agents are known to be useful or have been or are currently used for the prevention, treatment and/or alleviation of one or more symptoms of a VISTA-mediated disease, disorder or condition. In addition to therapeutic agents, the compositions provided herein may also contain a carrier.

The compositions provided herein include bulk dosage compositions useful in the manufacture of pharmaceutical compositions (eg, compositions suitable for administration to a subject or patient) that can be used to prepare unit dosage forms. In some embodiments, the composition provided herein is a pharmaceutical composition. Such compositions contain a prophylactically or therapeutically effective amount of one or more therapeutic agents (eg, an anti-VISTA therapeutic agent, such as the anti-VISTA antibody provided herein, or other therapeutic agent) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are formulated to be suitable for a particular route of administration to a subject.

In some embodiments, the composition is formulated in accordance with conventional techniques into a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine or lignocaine, to relieve pain at the injection site. Such compositions, however, can be administered by a route other than intravenous administration.

The ingredients of the compositions proposed herein may be supplied either individually or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent . If the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If such a composition is administered by injection, an ampoule of sterile water for injection or saline should be provided so that the ingredients can be mixed before administration.

In some embodiments, the antibody provided herein is packaged in a sealed container, such as an ampoule or sachet, indicating the amount of antibody. In one embodiment, the antibody is supplied as a dry, sterile lyophilized powder or water-free concentrate in a sealed container and can be reconstituted, for example, with water or saline to a concentration suitable for administration to a subject. In some embodiments, the antibody is supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dose of at least 0.1 mg; at least 0.5 mg; at least 1 mg; at least 2 mg; or at least 3 mg; for example, at least 5 mg; at least 10 mg; at least 15 mg; at least 25 mg; at least 30 mg; at least 35 mg; at least 45 mg; at least 50 mg; at least 60 mg; at least 75 mg; at least 80 mg; at least 85 mg; at least 90 mg; at least 95 mg; or at least 100 mg. The lyophilized antibody can be stored at a temperature of 2 to 8°C in its original container, and the antibody can be administered in no more than 12 hours, for example, in no more than 6 hours, in no more than 5 hours, in no more than 3 hours or within 1 hour after repeated dilution. In an alternative embodiment, the antibody is supplied in liquid form in a hermetically sealed container indicating the amount and concentration of the antibody. In some embodiments, the antibody in liquid form is supplied in a sealed container at a concentration of at least 0.1 mg/ml, at least 0.5 mg/ml, or at least 1 mg/ml, such as at least 5 mg/ml. ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 60 mg/ml, at least 70 mg/ml, at least 80 mg/ml, at least 90 mg/ml or at least 100 mg/ml.

The amount of an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) or composition provided herein that will be effective in preventing, treating, and/or alleviating one or more symptoms of a VISTA-mediated disease, disorder, or condition may be determined standard clinical methods.

Accordingly, to prevent, treat, and/or alleviate one or more than one symptom of a VISTA-mediated disease, disorder, or condition, an anti-VISTA therapeutic agent (e.g., an anti-VISTA antibody) or composition may be administered to a person at a dosage that results in a serum titer blood, ranging from about 0.1 μg/ml to about 450 μg/ml and in some embodiments at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.4 μg/ml , at least 0.5 µg/ml, at least 0.6 µg/ml, at least 0.8 µg/ml, at least 1 µg/ml, at least 1.5 µg/ml, according at least 2 µg/ml, at least 5 µg/ml, at least 10 µg/ml, at least 15 µg/ml, at least 20 µg/ml, at least 25 µg/ml, at least 30 µg/ml, at least 35 µg/ml, at least 40 µg/ml, at least 50 µg/ml, at least 75 µg/ml, at least 100 µg/ml, at least 125 µg /ml, at least 150 µg/ml, at least 200 µg/ml, at least 250 µg/ml, at least 300 µg/ml, at least 350 µg/ml, at least 400 µg/ml , at least 450 µg/ml. In addition, the results of in vitro assays can be used to facilitate the identification of optimal dosage ranges. The exact dosage used in the composition will also depend on the route of administration and the severity of the VISTA-mediated disease, disorder or condition, and its selection should be made in accordance with the judgment of the practitioner and the condition of each patient.

Effective dose values can be determined by extrapolation using dose-response curves obtained from in vitro test systems or animal models.

For the antibodies provided herein, the dosage administered to the patient in some embodiments may range from 0.1 mg/kg to 100 mg/kg of the patient's body weight. In some embodiments, the dosage administered to the patient is from about 1 mg/kg to about 75 mg/kg of the patient's body weight. In some embodiments, the dosage administered to the patient is from 1 mg/kg to 20 mg/kg of the patient's body weight, for example, from 1 mg/kg to 5 mg/kg of the patient's body weight. In general, human antibodies have a longer half-life in the human body than antibodies from other species due to the presence of an immune response to foreign polypeptides. For this reason, it is often possible to use lower dosages of human antibodies and less frequent administration. In addition, the dosage and frequency of administration of the antibodies proposed in this application can be reduced due to increased absorption and penetration of antibodies into tissues due to modifications, such as, for example, lipidation.

In one embodiment, the antibody provided herein is administered 5 times, 4 times, 3 times, 2 times, or 1 time at a dose of about 100 mg/kg or less, about 75 mg/kg or less, about 50 mg/kg, or less, about 25 mg/kg or less, about 10 mg/kg or less, about 5 mg/kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, or about 0.1 mg/kg kg or less to prevent, treat, or alleviate one or more symptoms of a VISTA-mediated disease, disorder, or condition. In some embodiments, the antibody provided herein is administered approximately 1-12 times, and these doses can be administered as needed, for example, once a week, once every two weeks, once a month, once every two months, once once every three months, etc., as determined by the doctor. In some embodiments, a lower dose (eg, 1-15 mg/kg) may be administered more frequently (eg, 3-6 times). In other embodiments, a higher dose (eg, 25-100 mg/kg) may be administered less frequently (eg, 1-3 times). However, as will be apparent to those skilled in the art, other dosage amounts and administration schedules are readily ascertainable and are included within the scope of the present invention.

In some embodiments, an antibody provided herein in a sustained release composition is administered to a subject, such as a human, at a dose of about 100 mg/kg, about 75 mg/kg or less, about 50 mg/kg or less, about 25 mg /kg or less, about 10 mg/kg or less, about 5 mg/kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, about 0.1 mg/kg or less for warning , treating and/or alleviating one or more symptoms of a VISTA-mediated disease. In another of certain embodiments, the antibody provided herein is administered to a subject, such as a human, not in a sustained release formulation, but rather as a bolus of about 100 mg/kg, about 75 mg/kg, or less, about 50 mg /kg or less, about 25 mg/kg or less, about 10 mg/kg or less, about 5 mg/kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, or about 0. 1 mg/kg or less, for the prevention, treatment and/or relief of one or more than one symptom of a VISTA-mediated disease, disorder or condition and over a period of time administered to the subject (eg, intranasally or intramuscularly) one, two, three or four times the antibody provided herein in a sustained release composition at a dose of about 100 mg/kg, about 75 mg/kg or less, about 50 mg/kg or less, about 25 mg/kg or less, about 10 mg /kg or less, about 5 mg/kg or less, about 1 mg/kg or less, about 0.5 mg/kg or less, or about 5 mg/kg or less. According to this embodiment, the "specified period of time" can be from 1 to 5 days, a week, two weeks or a month.

In some embodiments, a single dose of an antibody provided herein is administered to a patient to prevent, treat, and/or alleviate one or more symptoms of a VISTA-mediated disease, disorder, or condition, including one or more doses, e.g., two, three , four times, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five or twenty-six times, including once every two weeks (for example, approximately every 14 days) during the annual course of treatment, and this dose is selected from the group consisting of doses of about 0.1 mg/kg, about 0. 5 mg/kg, approximately 1 mg/kg, approximately 5 mg/kg, approximately 10 mg/kg, approximately 15 mg/kg, approximately 20 mg/kg, approximately 25 mg/kg, approximately 30 mg/kg, approximately 35 mg /kg, approximately 40 mg/kg, approximately 45 mg/kg, approximately 50 mg/kg, approximately 55 mg/kg, approximately 60 mg/kg, approximately 65 mg/kg, approximately 70 mg/kg, approximately 75 mg/kg , about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or combinations thereof (e.g., each once-monthly dose may or may not be the same ).

In some embodiments, a single dose of an antibody provided herein is administered to a patient to treat, prevent, and/or alleviate one or more symptoms of a VISTA-mediated disease, disorder, or condition, including one or more times, e.g., two, three times. , four times, five, six, seven, eight, nine, ten, eleven or twelve times, including at intervals of approximately once a month (for example, approximately 30 days) during the annual course of treatment, and this dose is selected from group consisting of doses of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg , approximately 25 mg/kg, approximately 30 mg/kg, approximately 35 mg/kg, approximately 40 mg/kg, approximately 45 mg/kg, approximately 50 mg/kg, approximately 55 mg/kg, approximately 60 mg/kg, approximately 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or combinations thereof ( for example, each monthly dose may or may not be the same).

In some embodiments, a single dose of an antibody provided herein is administered to a patient to treat, prevent, and/or alleviate one or more symptoms of a VISTA-mediated disease, disorder, or condition, including one or more times, e.g., two, three times. , four, five or six times, including at intervals of approximately once every two months (for example, approximately 60 days) during the annual course of treatment, this dose being selected from the group consisting of doses of approximately 0.1 mg/kg , approximately 0.5 mg/kg, approximately 1 mg/kg, approximately 5 mg/kg, approximately 10 mg/kg, approximately 15 mg/kg, approximately 20 mg/kg, approximately 25 mg/kg, approximately 30 mg/kg , approximately 35 mg/kg, approximately 40 mg/kg, approximately 45 mg/kg, approximately 50 mg/kg, approximately 55 mg/kg, approximately 60 mg/kg, approximately 65 mg/kg, approximately 70 mg/kg, approximately 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or combinations thereof (e.g., each bimonthly dose may be or may not be the same).

In some embodiments, a single dose of an antibody provided herein is administered to a patient to treat, prevent, and/or alleviate one or more symptoms of a VISTA-mediated disease, disorder, or condition, including one or more times, e.g., two, three times. or four times, with a frequency of approximately once every three months (for example, after approximately 120 days) during the annual course of treatment, and this dose is selected from the group consisting of doses of about 0.1 mg/kg, about 0.5 mg/kg kg, approximately 1 mg/kg, approximately 5 mg/kg, approximately 10 mg/kg, approximately 15 mg/kg, approximately 20 mg/kg, approximately 25 mg/kg, approximately 30 mg/kg, approximately 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or combinations thereof (eg, each trimonthly dose may or may not be the same).

In some embodiments, the route of administering a dose of an antibody provided herein to a patient is intranasal, intramuscular, intravenous, or a combination thereof, but other routes described herein are also acceptable. Each dose may be administered using the same route of administration or not. In some embodiments, an antibody provided herein can be administered using multiple routes of administration concurrently with or subsequent to other doses of the same or another antibody provided herein.

In some embodiments, anti-VISTA therapeutic agents (eg, anti-VISTA antibodies) provided herein are administered to a subject for prophylactic or therapeutic purposes. Anti-VISTA therapeutic agents (eg, anti-VISTA antibodies) can be administered to a subject for prophylactic or therapeutic purposes to prevent, attenuate or alleviate a VISTA-mediated disease, disorder or condition or symptoms thereof.

SETS

The present invention also provides a pharmaceutical package or pharmaceutical kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions provided herein, for example, one or more antibodies (for example, an anti-PSGL-1 antibody and /or to VISTA) proposed in this application. It is possible that such container(s) may be accompanied by a statement in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which statement reflects that agency's approval of the manufacture, use, or sale for administration. person. In some embodiments, the kit includes an insert leaflet. As used, the term “package insert” refers to instructions typically included in commercial packaging of therapeutic products that contain information regarding the indications, use, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products, as well as instructions for use.

In addition, the present invention provides kits that can be used in the methods described above. In one embodiment, the kit contains an antibody (e.g., anti-PSGL-1 and/or anti-VISTA) provided herein, such as an isolated antibody (e.g., anti-PSGL-1 and/or anti-VISTA), in one or more containers. In some embodiments, the kits provided herein contain substantially isolated PSGL-1 or VISTA as a control. In some embodiments, the kits provided herein further contain a control antibody that does not interact with PSGL-1 and/or VISTA. In some embodiments, the kits provided herein contain a means for detecting binding of the modified antibody to PSGL-1 and/or VISTA (e.g., the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound , or a second antibody that recognizes the first antibody can be conjugated to the detected substrate). In some embodiments, the kit may include recombinantly or chemically synthesized PSGL-1 and/or VISTA. The PSGL-1 and/or VISTA provided in such a kit may also be attached to a solid substrate. In some embodiments, the detection means of the kit described above includes a solid support to which PSGL-1 and/or VISTA is attached. Such a kit may also include an unattached reporter-labeled anti-human antibody. In some embodiments, antibody binding to PSGL-1 and/or VISTA can be detected by binding to a reporter-labeled antibody.

It will be appreciated that modifications which do not substantially affect the activity of the various embodiments of the present invention are also proposed herein. Accordingly, the following examples are intended to illustrate and not limit the present invention.

EXAMPLES

EXAMPLE I

Identification of the VISTA receptor

This example describes for the first time the identification of a VISTA receptor using the CAPTIREC™ technique, a TRICEPS™-based ligand-receptor capture system (Dualsystems Biotech AG).

The CAPTIREC™ technique with VISTA-Fc fusion protein as the ligand of interest and anti-CD28 antibody as control ligand was performed on naïve T cells isolated from primary human T cells. The nucleotide and amino acid sequences of the vista-fc fusion protein construct used in the following experiments are shown below.

Nucleotide sequence of the vista-fc fusion protein (the underlined sequence encodes VISTA; the bolded sequence encodes the Fc fragment of the human IgG1 antibody):

Amino acid sequence of the vista-fc fusion protein (underlined sequence refers to VISTA; bold sequence refers to the Fc portion of the human IgG1 antibody):

A general description of the CAPTIREC™ technique is presented in FIG. 1. Briefly, vista-fc fusion protein and anti-CD28 antibody were separately coupled to TRICEPS™. Naïve human T cells were isolated from commercially available primary human T cells. Surface glycoproteins of naïve T cells were selectively oxidized. Ligand binding and receptor attachment to the cell surface of oxidized naïve T cells were performed. The reacted T cells were then lysed and the final lysate was affinity purified to yield ligand-receptor protein complexes. Next, the purified proteins were digested by trypsin, thereby releasing the receptor peptides. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To perform statistical analyses, biochemical experiments were performed in triplicate.

Isolation of naive T cells was performed by negative selection of naive T cells from primary human T cells from healthy donors, which were purchased from ALLCELLS (Alameda, CA). To carry out negative selection of the desired cells, a naïve pan-T cell isolation kit from Miltenyi (No. 130-097-095) was used according to the manufacturer's protocol. Briefly, peripheral blood mononuclear cells (PBMCs) were resuspended in magnetically activated cell sorting (MACS) working buffer and incubated for 5 min with a mixture of biotin-conjugated monoclonal antibodies to HLA-DR, CD14, CD15, CD16, CD19, CD25, CD36, CD56, CD57, CD45RO, CD123, CD244, CD235a and anti-human T-cell receptor (TCR) antibodies, then incubated for 10 min with antibiotin magnetic beads conjugated with anti-CD61 and anti-biotin monoclonal antibodies. Negative selection of naïve T cells was then performed using an autoMACS® automated sorter (Miltenyi Biotech, San Diego, CA). The ligand capture reaction used 100×10 ⁶ naïve T cells on TRICEPS™.

The remaining steps of the CAPTIREC™ technique, which included binding of the vista-fc fusion protein or anti-CD28 antibody to TRICEPS™, selective oxidation of glycoproteins located on the surface of naive T cells, ligand binding and receptor binding to the cell surface of “oxidized” T cells, lysis of these T cells, affinity purification of the cell lysate and trypsin digestion were carried out according to modified procedures described in Frei et al., Nat. Protoc., 8(7): 1321-1336 (2013) and Frei et al., Nat. Biotechnol., 30(10): 997-1001 (2012).

Analysis of the resulting receptor peptides was performed by LC-MS/MS on a Thermo LTQ Orbitrap XL spectrometer equipped with an electrospray ion source. Samples were measured in information-dependent acquisition mode using a 120 min gradient and a 10 cm C18 packed column. Six individual samples from the CAPTIREC™ data set were analyzed using an analysis of variance (ANOVA) statistical model. According to this model, it is assumed that measurement error is described by a Gaussian distribution and that individual characteristics are considered to be a consequence of excess protein, and this excess is taken into account directly. This model allows each protein to be tested for individual excess in all pairwise comparisons between ligand and control samples and reports p-values. P-values are then adjusted for multiple comparisons to control the experiment-wide false positive rate (FDR).

Identified peptides were filtered according to FDR ≤ 1% and quantified using a label-free MS1-based approach. Non-linear proteomics software DYNAMICS Progenesis QI was used to perform MS1 quantification with a setup to account for all unique peptides. Identified proteins were filtered for membrane association, secretion, and glycosylation using information provided by the Uniprot database (Universal Protein Resource). Proteins identified by only one peptide were not taken into account in the analysis.

The processed CAPTIREC™ data were reported in the form of a scatter plot for the large protein-level data set, which shows fold change versus statistical significance. The adjusted p-value obtained for each protein is plotted against fold enrichment comparing the two experimental conditions. The range of receptor candidates is determined according to a greater than 4-fold increase in the enhancement factor and statistical significance (FDR-corrected p-value < 0.01).

Among the detected glycoproteins, 5 peptides from the control data set were used to identify CD28. This indicates the successful operation of CAPTIREC™. 6 peptides were used to identify PSGL-1, and in addition, 12 peptides from the vista-fc fusion protein data set were used to identify VISTA itself (see Table 6).

The identified binding partner, PSGL-1, was imaged using Protter (FIG. 2) indicating N-glycosylation sites (as squared residues) and experimentally detected peptides (as filled circles) (Omasits et al ., Bioinformatics: advance online pub., October, 2013). This mapping shows that all six detected peptides are localized in the intracellular domain of PSGL-1. Analysis of the extracellular domain of PSGL-1 shows that despite the size of this domain, there are several sites for trypsin cleavage of peptide bonds in the extracellular domain. PSGL-1 contains three N-glycosylation sites, and peptides with these sites may have been lost during the LC-MS/MS analysis described above. The remaining possible peptides are either too large, too small, or undergo processing during protein sorting, which is a reasonable explanation for the fact that only peptides mapped to the PSGL-1 intracellular domain were detected.

Based on the above analysis, PSGL-1 was found to be a heterophilic binding partner of VISTA. Since VISTA has previously been shown to be a broad-spectrum negative checkpoint regulator that is expressed on hematopoietic cells (Lines et al, Cancer Res., 74(7), 1924-1932 (2014)), the above results demonstrate that the interaction of VISTA with PSGL-1 appears to result in T cell suppression. Therefore, interfering (e.g., inhibiting or blocking) this interaction using agents that target PSGL-1 and/or VISTA, such as anti-PSGL-1 and/or anti-VISTA antibodies, may result in renewed upregulation or de novo activation immune system through activated T cells. This activation of the immune system will result in a highly effective physiological response in the treatment of a VISTA-mediated disease, disorder or condition, including in the context of cancer treatment (eg, treatment of a hematologic malignancy).

EXAMPLE II

Linking VISTA to PSGL-1

This example describes properties related to the binding of VISTA to PSGL-1.

A vista-fc fusion protein (eg, described in Example I) was immobilized on a solid surface and assayed for binding to the extracellular domain of PSGL-1. To carry out these experiments, two different constructs containing the extracellular domain of PSGL-1 were created. Both constructs were fused with the IgG kappa chain signal sequence and the Fc fragment. In addition, a propeptide sequence or tandem repeat element, which is important for proper function, was added (see, for example, Cummings R.D., Brazilian J. Med. Biol. Res., 32: 519-28 (1999)). Cells were co-transfected with constructs expressing the glycosyltransferases GCNT1 (glucosaminyl-(N-acetyl)-transferase 1) and FUT3 (fucosyltransferase 3) to ensure proper post-translational modification of the protein, which is known to be important for the high-affinity binding of PSGL-1 to P-selectin (Sako et al., Cell, 75(6): 1179-86 (1993); Yang et al., Thrombosis and Haemostasis, 81(1): 1-7 (1999); Carlow et al., Immunol. Rev. , 230(1): 75-96 (2009); Kumar et at., Blood, 88(10): 3872-9 (1996); Cummings R. D., Brazilian J. Med. Biol. Res., 32: 519-28 (1999)). The amino acid sequences of the PSGL-1 based constructs are shown below.

PSGL-1 based construct A - amino acid sequence (PSGL-1 fused to Fc and IgG kappa chain signal sequence and propeptide sequence). The IgG kappa chain signal sequence is shown in italics; the propeptide sequence is in bold; The Fc sequence is underlined:

PSGL-1 based construct B - amino acid sequence (PSGL-1 fused to Fc and to IgG kappa chain signal sequence and tandem repeat element). The IgG kappa chain signal sequence is shown in italics; the tandem repeat element sequence is in bold; The Fc sequence is underlined:

For these experiments, PSGL-1-based construct A (PSGL-1A) and PSGL-1-based construct B (PSGL-1B) were tested for binding to the immobilized vista-fc construct.

A sample of the immobilized vista-fc construct was equilibrated in HEPES (N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid) buffered saline (HBS) containing calcium ions (1.5 mM calcium chloride) and magnesium (1.0 mM magnesium chloride). The same buffer was used as a working buffer. Samples containing PSGL-1A or PSGL-1B, which also contained calcium and magnesium ions, were passed over a solid surface immobilized with the vista-fc construct at a rate of 60 μl/min, with a contact time of 120 seconds, and the interval required for dissociation after surface regeneration using a 3-second pulse of glycine solution (pH 1.5), was 300 seconds. The assay was performed for six different concentrations of PSGL-1A and PSGL-1B (0.3 μM, 0.60 μM, 1.20 μM, 2.4 μM, 4.8 μM, and 9.6 μM).

The experimental results were analyzed in two different ways: using (1) a two-state binding model; and (2) equilibrium binding assay (1:1 model). For PSGL-1A, the two-state binding model showed a binding affinity corresponding to a K _D of 32.1 μM, while the 1:1 model showed a K _D value of 3.01 μM (FIG. 3). For PSGL-1B, the two-state binding model showed a K _D value of 5.09 μM, while the 1:1 model showed a K _D value of 4.76 μM (FIG. 4).

The results of the above analyzes demonstrated the presence of a net response signal for both PSGL-1A and PSGL-1B constructs. The resulting sensorgrams show features associated with binding and dissociation. It should be noted that the affinity assessments were qualitative in nature. Quantification of binding may be possible when the surface-bound activity is higher than the activity in the present experiments (eg, >0.5% using the purified PSGL-1 construct). Additionally, in these experiments, the use of PSGL-1A injection resulted in sample carryover into subsequent runs. In these experiments, the estimated affinities between VISTA and PSGL-1 were relatively similar for PSGL-1A (about 3 μM) and for PSGL-1B (about 5 μM).

EXAMPLE III

Binding of VISTA to PSGL-1 on Cells

This example describes the binding of VISTA to PSGL-1 expressed by a promyelocytic leukemia cell line (HL-60; ATCC, CCL-240) using a bifunctional cross-linking approach.

In these experiments, PSGL-1 expression was assessed using a phycoerythrin (PE)-conjugated anti-PSGL-1 monoclonal antibody (Abeam; ab78188), termed KPL-1. Detection of PSGL-1 expression by HL-60 cells was performed by flow cytometry using standard methods (FIG. 5). The PSGL-1 protein copy number was estimated to be approximately 263,000±2800 per cell.

In addition, in these experiments, covalent coupling was carried out with a bifunctional linker (sulfo-SBED (sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido-benzamido)-hexanoamido)-ethyl-1,3 '-dithiopropionate), ThermoFisher Scientific; 33073) samples of the vista-fc fusion protein (described in Example I), as well as antibodies to CD28 (BioXcell, BE0248) and IgG1-Fc (R&D Systems; 110-HG-100) as negative controls using the manufacturer's recommended conditions. The resulting samples were incubated with HL-60 cells for 30 min at room temperature in the dark. Cross-linking was photoactivated using an ultraviolet (UV) light source for 20 min. Cells were then lysed and precipitated using Protein A Sepharose. Samples were analyzed by Western blotting using anti-PSGL-1 polyclonal antibody (R&D Systems; AF3345) or horseradish peroxidase (HRP)-conjugated streptavidin.

As shown in FIG. 6, VISTA-Fc interacts with PSGL-1 but not with the negative isotype control IgG-Fc or anti-CD28 antibody.

Additional experiments were carried out to confirm the specificity of this interaction. The above experiment was repeated except that anti-VISTA monoclonal antibody was added to the cells before incubating sulfo-SBED-tagged proteins with HL-60 cells. Analysis was performed using ImageQuant software.

As shown in FIG. 7, inoculation with an antibody to VISTA resulted in a weakening of the interaction between VISTA and PSGL-1.

These experiments indicate that PSGL-1 expressed on HL-60 cells is a binding partner for VISTA. These experiments also show that this interaction is specific and is attenuated by blocking antibodies to VISTA.

EXAMPLE IV

Binding of VISTA to PSGL-1 on PBMCs

This example describes the binding of VISTA to PSGL-1 expressed by peripheral blood mononuclear cells (PBMCs) using a cross-linking approach.

In these experiments, PSGL-1 expression was assessed using a PE-conjugated anti-PSGL-1 monoclonal antibody (Abeam; ab78188), termed KPL-1. Detection of PSGL-1 expression by PBMCs was performed by flow cytometry using standard methods (FIG. 8). The PSGL-1 protein copy number was estimated to be approximately 38,000 per cell.

In addition, PBMCs were either left untreated or incubated with a cross-linking agent (10 mM bis(sulfosuccinimidyl)suberate (BS ³ ); ThermoFisher Scientific; 21580) for 90 min on ice. After quenching the cross-linking reaction, where necessary, cell lysis was performed and the resulting lysates were pre-cleared using Herceptin and GammaBind™ Plus Sepharose (GE Healthcare; 17-0886-01). The resulting samples were immunoprecipitated with either anti-VISTA antibody or anti-PSGL-1 (KPL-1) antibody overnight. Immunoprecipitated samples were analyzed by Western blotting using anti-PSGL-1 polyclonal antibody (R&D Systems; AF3345).

As shown in FIG. 9, lane 4, after cross-linking, no PSGL-1-specific zones were detected after immunoprecipitation with anti-VISTA antibody. For example, this may be due to the blocking of specific epitopes during the formation of the VISTA-PSGL-1 complex and, as a result, the prevention of immunoprecipitation. Anti-PSGL-1 antibody caused precipitation of several higher molecular weight complexes (approximately 250-450 kDa) in BS3-treated PBMC (FIG. 9, last lane), which were also PSGL-1 positive.

In these experiments, both anti-VISTA and anti-PSGL-1 antibodies precipitated a protein of approximately 240 kDa from PBMCs not treated with either cross-linker. This complex was PSGL-1 positive, demonstrating that PSGL-1 interacts with VISTA (FIG. 9, lanes 3 and 5). Immunoprecipitation with an isotype control antibody did not produce such a zone (FIG. 9, lanes 1 and 2).

These experiments indicate that PSGL-1 expressed on PBMC is a binding partner for VISTA.

EXAMPLE V

PSGL-1 expression

This example describes the expression of PSGL-1 in various T cell subsets.

In these experiments, PSGL-1 expression was assessed using PE-conjugated anti-human CD162 antibody. The following T cell subsets were assessed: naïve and resting cells (eg, described phenotype: CD45RO ^- / CD45RA ⁺ / CCR7 ⁺ / CD62L ⁺ / CD27 ⁺ / CD28 ⁺ / CD127 ⁺ ), effector cells (eg, described phenotype: CD45RO ⁺ / CD57 ⁺ / CD279 ^- / CD95 ⁺ / CCR7 ^- / CD62L ^- ), exhausted effector cells (e.g., described phenotype: CD45RO ⁺ / CD57 ⁺ / CD279 + / CD95 ⁺ / CD45RA ^{- /} CCR7 ^- / CD62L ^- ) and circulating memory cells (e.g. described phenotype: central: CD45RO ⁺ / CD45RA ^- / CCR7 ⁺ / CD62L ⁺ or effector: CD45RO ⁺ / CD45RA ⁺ / CCR7 ^- / CD62L ⁺ ).

For these experiments, human PBMC samples were obtained from ALLCELLS (Emeryville, CA). Two panels of T cell markers were prepared as follows. A. Panel 1: T cell markers + specific markers for effector/exhausted effector cells

1. CD45RA-FITC (fluorescein isothiocyanate),

2. CD45RO-PerCP-eFluor710,

3. CD197 (CCR7) - (dye brilliant violet 510),

4. CD62L-APC-eFluor 780, APC means allophycocyanin,

5. CD57-(Pacific blue dye),

6. CD95 (Fas)-PE-Cy7 (cyanine 7),

7. CD279-APC,

8. CD162-PE.

b. Panel 2: T cell markers + naive/quiescent cell specific markers

1. CD45RA-FITC,

2. CD45RO-PerCP-eFluor710,

3. CD197 (CCR7) - (dye brilliant violet 510),

4. CD62L-APC-eFluor780,

5 CD27-(Pacific blue dye),

6. CD28-PE-Cy7,

7. CD127-APC,

8. CD162-PE.

In these experiments, approximately 10 ⁶ cells and Fc receptor (FcR) blocking reagent (Miltenyi Biotec, 130-092-247) were added to each panel, along with the appropriate isotype controls for each antibody and incubated for 30 min at 4°C. In the dark. Cells were then washed and mean fluorescence intensity (MFI) values were calculated for each sample using a MACSQuant analyzer. MFI values were quantified using anti-mouse IgG antibodies from the Quantum™ Simply Cellular® kit (Bangs Laboratories, 815B). For the viable population (negative 4,6-diamidino-2-phenylindole (DAPI) staining), fluorescence data for 105 events were collected and submitted for analysis using the FlowJo software package.

As shown in FIG. 10 and 11, PSGL-1 is present in subsets of naive/resting, effector, exhausted effector T cells, as well as in both subsets of circulating (central and effector) T cells. Also in FIG. 10 and 11 show that PSGL-1 expression was increased in effector T cell subtypes compared to naïve and exhausted T cells. The highest level of expression was found in the subset of effector cells, while the lowest level of expression was found in the subset of naïve/resting cells. Table 7 shows the copy number for expressed PSGL-1 in each subgroup.

These experiments show that PSGL-1 is differentially expressed among different T cell subsets in human PBMCs.

EXAMPLE VI

In silico analysis of VISTA and PSGL-1 expression

This example demonstrates that VISTA expression correlates with PSGL1 in several ways.

The Cancer Genome Atlas (TCGA) provides the opportunity to conduct; Comprehensive analysis of cancer genomic profiles using high-throughput technologies, including next-generation sequencing and microarray-based methods. The data deposited in the TCGA contains both nucleotide sequence and gene expression information. The cBioportal Cancer Genomics website (http://www.cbioportal.org/) thus provides the ability to view, analyze, and download large-scale cancer genomics datasets. It covers genomics and transcriptomics research from TCGA.

For each indicator, TCGA was queried on the cBioportal site to identify the mRNAs whose expression was most correlated with VISTA. This correlation analysis was performed using Spearman's test. The results of statistical analysis (p-value <0.05) are shown in Table 8; The mRNAs are ranked based on their correlation with VISTA.

Table 8 shows that PSGL1 expression is highly correlated with VISTA expression in several cancers. The highest correlation is observed in NSCLC cases. In comparison, other possible receptors (i.e., VSIG3 and VSIG8) showed only weak correlation.

EXAMPLE VII

Assessment of VISTA and PSGL1 mRNA expression using RNAscope® technology

This example shows the mRNA expression and co-localization pattern of VISTA and PSGL1 using tissue microarrays (TMAs) in squamous cell carcinoma (SCC) and adenocarcinoma (ADK) of the lung.

Materials and methods

Paraffin-embedded TMA blocks containing SCC and ADK lung samples (3 blocks in each case) were freshly sectioned and mounted on glass slides, followed by technical in situ hybridization (ISH) steps. Excised tissue samples were placed in freshly prepared 10% pH neutral buffered formalin (NBF) for 16-32 hours at room temperature (RT). The samples were then dehydrated, embedded in paraffin, and sectioned at 5 ± 1 μm thickness, which were then mounted on Superfrost® Plus slides. These slides were kept in an oven for 1 hour at 60°C.

Tissue sections 5 μm thick were deparaffinized in xylene, followed by a series of dehydration procedures in ethanol. Tissue sections were then incubated in citrate buffer (10 nmol/L, pH 6) maintained at boiling temperature (100°C to 103°C) using a hot plate for 15 minutes, washed in deionized water, and immediately treated with protease (10 µg/ml; Sigma-Aldrich, St. Louis, MO) at 40°C for 30 minutes in a HybEZ hybridization chamber (Advanced Cell Diagnostics, Hayward, CA).

The treated tissue sections were then hybridized with PSGL-1 or VISTA probes using the RNAscope® 2.5 assay kit (Advanced Cell Diagnostics, Hayward, CA) following the manufacturer's instructions.

Sections were stained with hematoxylin and eosin for quality control and microscopic evaluation of each nucleus. The study was carried out using a standard light microscope with 20-40x magnification. An Excel sheet was used to record the collected data.

Negative and positive control tests were performed using 2 specific probes. These controls were scored to confirm the absence of contamination (in the case of negative control probes) and the presence of accessible mRNA (in the case of positive control probes). The protocol was filled out manually.

Evaluation of labeled tissues was performed using a semi-quantitative method using a dual scoring system for each target.

Tissue distribution scores ranged from 0 to 3 and reflected the content of positively labeled cells within the population (in the immune infiltrate) and were considered as Immunoscore scores.

Immunoscore rating scale: ranging from 0 to 3 as follows:

• 0: absent,

• 1: low content,

• 2: moderate content.

• 3: high content.

To estimate the number of RNA spots inside cells, the ACD grading scale was used, i.e. grading scale recommended by the manufacturer of the RNAscope® 2.5 assay kit (Advanced Cell Diagnostics (ACD), Hayward, CA). Each spot represents a single RNA molecule because the RNAscope® 2.5 assay detects individual RNA molecules. In this system, the score is measured in a range from 0 to 4 depending on the number of spots and/or clusters (0: no spots; 4: numerous spots and clusters) in the cytoplasm, and can be considered as a scoring system applied to the internal contents of the cell.

results

TMA in case of lung SCC

For lung SCC, analysis was performed using three TMAs. Nuclei were assessed in triplicate for each patient.

The fact of mRNA labeling for both VISTA and PSGL-1 was observed mainly in the tumor microenvironment (in the infiltrates of immune cells). Positively labeled cells had myeloid morphology (associated with macrophages). However, occasionally some "positive" spots were noted in lymphocytes (both mRNA) and neutrophils (VISTA only).

mRNA for VISTA was predominant in tumor microenvironment infiltrates. This target was expressed to varying degrees in all cell nuclei in lung SCC. Small and numerous spots were observed in the cytoplasm. In some cases, corresponding VISTA spots were seen in endothelial cells. VISTA mRNA-positive tumor cells were observed in more than 80% of the nuclei.

Compared with VISTA, the expression level of PSGL-1 mRNA was lower in tumor microenvironment infiltrates. Larger spots were observed, but there were fewer of them in the cytoplasm than spots in the case of VISTA. Tumor cells expressed PSGL-1 mRNA in isolated cases (30% of nuclei); For the most part, the ACD score (i.e., the number of spots in the cytoplasm) was quite low.

Moderate to high levels of positive VISTA mRNA labeling were observed in nearly all nuclei, compared with 35% for PSGL-1 mRNA. None of the nuclei were negatively labeled for both targets, i.e. each of the nuclei was “positive” for either VISTA or PSGL-1 or both. These results are summarized in Table 10.

Finally:

• co-localization of VISTA and PSGL-1 mRNA was observed in the tumor microenvironment, one example of which is shown in FIG. 12;

• almost all PSG Li-positive cells were located near VISTA-positive cells;

• expression of both PSGL-1 and VISTA was observed in each nucleus of at least some cells;

• VISTA-positive cells could be observed in the absence of nearby PSGL-1-positive cells. Some VISTA-positive cells could be observed in the apparent absence of nearby PSGL-1-positive cells. However, no PSGL-1-positive cell was observed without a VISTA-positive cell located nearby, meaning that PSGL-1-positive cells were always located near VISTA-positive cells. This was partly due to the fact that VISTA-positive cells predominated in the immune infiltrate.

TMA in case of ADK lungs

For ADK lungs, analysis was performed using three TMAs. Nuclei were assessed in quadruplicate for each patient.

As in the case of lung SCC, VISTA and PSGL-1 mRNA labeling was observed in the tumor microenvironment (in immune cell infiltrates). Positively labeled cells had myeloid morphology (associated with macrophages). However, occasionally some "positive" spots were noted in lymphocytes (both targets) and neutrophils (VISTA only).

The expression pattern of both VISTA and PSGL-1 in ADK lungs was very similar to that observed in SCC lungs.

More than 50% of nuclei showed high levels of positive labeling for VISTA mRNA, while approximately 50% of nuclei showed positive labeling for PSGL1 mRNA. In the case of PSGL-1, a small number of nuclei were negatively labeled (7%). These results are summarized in Table 12.

In ADK lung, as in lung SCC, co-localization or correlated expression patterns were observed for VISTA and PSGL-1 mRNA.

Semi-quantitative analysis of VISTA and PSGL-1 mRNA expression patterns using dual RNAscope® revealed that these targets were often colocalized or expressed in closely spaced cells in the tumor microenvironment. It turned out that VISTA mRNA was expressed more frequently than PSGL1 mRNA. However, all nuclei in SCC lung and 83% of nuclei in ADK lung expressed both targets.

Using RNAscope®, it was revealed that PSGL-1 can be expressed in the same cells or near cells that express VISTA.

EXAMPLE VIII

Release of IL-2 from CD4 ⁺ T cells in the presence of PSGL-1-Fc +/- anti-VISTA antibodies or anti-PSGL1 antibodies (72 h)

This example describes PSGL-1-mediated inhibition of T cell activation.

Methods

Experiments were carried out in triplicate.

Isolation of CD ⁺ T cells from two healthy donors was performed by negative selection using kits from Milenyi.

Wells of 96-well plates were coated with anti-CD3 antibody (manufactured by eBiosciences, BioxCell, catalog number BE0001-2, clone OKTZ, lot 640417J1 (mIgG2a; mouse IgG2a)) at a concentration of 2.5 μg/ml in a volume of 100 μl for 4 hours at 37°C. The plates were then washed 2 times with PBS. Coating with c9G4 antibody and PSGL-1-Fc fusion protein was performed overnight at 4°C at 224 nM concentration in a volume of 100 μl in triplicate.

These plates were washed 4 times with PBS. 100,000 CD4 ⁺ T cells were added to each well in 200 μl of medium containing anti-CD28 antibody (2.5 μg/ml) with or without the addition of anti-VISTA 26A antibody (10 μg/ml), as described in WO 2014/ 197849 (more specifically, the antibody used was a humanized antibody having the following CDR sequences described in WO 2014/197849: CDRH1: SEQ ID NO 1297, CDRH2: SEQ ID NO 1559, CDRH3: SEQ ID NO 1394, CDRL1: SEQ ID NO 1432, CDRL2: SEQ ID NO 1477 and CDRL3: SEQ ID NO 1499), or control antibody c9G4 (described in WO 2015/162292 A).

After incubation for 72 h, supernatants were removed and centrifuged for 5 min at 1200 rpm. After centrifugation, supernatants were transferred to new 96-well plates and frozen at -80°C until analysis for IL-2.

The concentration of IL-2 in the supernatants was measured using a commercially available kit (Universal Human IL2 Detection System using BD™ Cytometric Bead Array (CBA Human IL2 Flex Set), Cat. No. 558270).

results

To test for the immunosuppressive properties of PSGL-1, the activation status of T cells was examined after stimulation in the presence or absence of this protein. To this end, a PSGL-1-Fc fusion protein consisting of the extracellular domain of PSGL-1 and the Fc region of human IgG was first constructed. CD4 ⁺ T cells were then activated with antibodies to CD3 and CD28 in the presence of PSGL-1-Fc or control IgG. The release of IL-2 was monitored as a marker of activation of these cells.

As shown in FIG. 13, incubation of T cells in the presence of PSGL-1 causes a 2-fold decrease in the release of IL-2, a marker of T cell activation, compared to control, i.e. irrelevant protein used for coating at the same concentration (c9G4). The results obtained were similar for two different donors. Thus, PSGL-1 inhibits T cell activation.

The addition of anti-VISTA antibodies partially reversed this inhibition (see FIG. 13). Indeed, in the presence of antibodies to VISTA, inhibition was weakened by more than 50%. Addition of a control antibody (c9G4) had no effect on the inhibition of IL-2 release by psgl1-fc, highlighting the specificity of this effect detected using anti-VISTA antibodies. This specific reversal by the addition of anti-VISTA antibodies demonstrates that PSGL-1-dependent inhibition of T cell activation is at least partially mediated by VISTA.

These results confirm that VISTA and PSGL1 interact at both physical and functional levels. Disruption of this interaction (in this case using anti-VISTA antibodies) increases IL-2 release and hence T cell activation.

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LIST OF SEQUENCES

<110> PIERRE FABRE MEDICAMENT

FERRE, Pierre

CRUZALEGUI, Francisco

LOUKILI, Noureddine

<120> RECEPTOR FOR VISTA

<130> B3764799PCTD38601

<150> PCT IB2018/000983

<151> 2018-07-20

<160> 40

<170> Patent In version 3.5

<210> 1

<211> 311

<212> PRT

<213> Homo sapiens

<400> 1

Met Gly Val Pro Thr Ala Leu Glu Ala Gly Ser Trp Arg Trp Gly Ser

1 5 10 15

Leu Leu Phe Ala Leu Phe Leu Ala Ala Ser Leu Gly Pro Val Ala Ala

20 25 30

Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln

35 40 45

Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His

50 55 60

Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val

65 70 75 80

Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp

85 90 95

Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp

100 105 110

Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn

115 120 125

Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr

130 135 140

Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg Val

145 150 155 160

His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser

165 170 175

Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Thr

180 185 190

Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu

195 200 205

Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn

210 215 220

Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln Gly Ile

225 230 235 240

Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala Gln Gly Ile Pro Glu

245 250 255

Ala Lys Val Arg His Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser

260 265 270

Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser Thr Pro Leu Ser Pro

275 280 285

Pro Gly Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp

290 295 300

Ser Pro Asn Phe Glu Val Ile

305 310

<210> 2

<211> 936

<212> DNA

<213>

<400> 2

atgggcgtcc ccacggccct ggaggccggc agctggcgct ggggatccct gctcttcgct 60

ctcttcctgg ctgcgtccct aggtccggtg gcagccttca aggtcgccac gccgtattcc 120

ctgtatgtct gtcccgaggg gcagaacgtc accctcacct gcaggctctt gggccctgtg 180

gacaaagggc acgatgtgac cttctacaag acgtggtacc gcagctcgag gggcgaggtg 240

cagacctgct cagagcgccg gcccatccgc aacctcacgt tccaggacct tcacctgcac 300

catggaggcc accaggctgc caacaccagc cacgacctgg ctcagcgcca cgggctggag 360

tcggcctccg accaccatgg caacttctcc atcaccatgc gcaacctgac cctgctggat 420

agcggcctct actgctgcct ggtggtggag atcaggcacc accactcgga gcacagggtc 480

catggtgcca tggagctgca ggtgcagaca ggcaaagatg caccatccaa ctgtgtggtg 540

tacccatcct cctcccagga tagtgaaaac atcacggctg cagccctggc tacgggtgcc 600

tgcatcgtag gaatcctctg cctccccctc atcctgctcc tggtctacaa gcaaaggcag 660

gcagcctcca accgccgtgc ccaggagctg gtgcggatgg acagcaacat tcaagggatt 720

gaaaaccccg gctttgaagc ctcaccacct gcccagggga tacccgaggc caaagtcagg 780

caccccctgt cctatgtggc ccagcggcag ccttctgagt ctgggcggca tctgctttcg 840

gagcccagca cccccctgtc tcctccaggc cccggagacg tcttcttccc atccctggac 900

cctgtccctg actctccaaa ctttgaggtc atctag 936

<210> 3

<211> 402

<212> PRT

<213> Homo sapiens

<400> 3

Met Pro Leu Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn

1 5 10 15

Ser Leu Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu

20 25 30

Gly Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr

35 40 45

Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg

50 55 60

Asn Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser

65 70 75 80

Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly

85 90 95

Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu

100 105 110

Ser Thr Asp Ser Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala

115 120 125

Thr Glu Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr

130 135 140

Arg Leu Thr Ala Thr Glu Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu

145 150 155 160

Ala Gln Thr Thr Pro Pro Ala Ala Thr Glu Ala Gln Thr Thr Gln Pro

165 170 175

Thr Gly Leu Glu Ala Gln Thr Thr Ala Pro Ala Ala Met Glu Ala Gln

180 185 190

Thr Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Pro Pro Ala Ala

195 200 205

Met Glu Ala Gln Thr Thr Gln Thr Thr Ala Met Glu Ala Gln Thr Thr

210 215 220

Ala Pro Glu Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Ala Thr Glu

225 230 235 240

Ala Gln Thr Thr Pro Leu Ala Ala Met Glu Ala Leu Ser Thr Glu Pro

245 250 255

Ser Ala Thr Glu Ala Leu Ser Met Glu Pro Thr Thr Lys Arg Gly Leu

260 265 270

Phe Ile Pro Phe Ser Val Ser Ser Val Thr His Lys Gly Ile Pro Met

275 280 285

Ala Ala Ser Asn Leu Ser Val Asn Tyr Pro Val Gly Ala Pro Asp His

290 295 300

Ile Ser Val Lys Gln Cys Leu Leu Ala Ile Leu Ile Leu Ala Leu Val

305 310 315 320

Ala Thr Ile Phe Phe Val Cys Thr Val Val Leu Ala Val Arg Leu Ser

325 330 335

Arg Lys Gly His Met Tyr Pro Val Arg Asn Tyr Ser Pro Thr Glu Met

340 345 350

Val Cys Ile Ser Ser Leu Leu Pro Asp Gly Gly Glu Gly Pro Ser Ala

355 360 365

Thr Ala Asn Gly Gly Leu Ser Lys Ala Lys Ser Pro Gly Leu Thr Pro

370 375 380

Glu Pro Arg Glu Asp Arg Glu Gly Asp Asp Leu Thr Leu His Ser Phe

385 390 395 400

Leu Pro

<210> 4

<211> 2226

<212> DNA

<213> Homo sapiens

<400> 4

atggggtgtg ggctgtcaca tggccctgcc taagtaacca cattctcgct tcctccttcc 60

acacacagcc attgggggtt gctcggatcc gggactgccg cagggggtgc cacagcagtg 120

cctggcagcg tgggctggga ccttgtcact aaagcagaga agccacttct tctgggccca 180

cgaggcagct gtcccatgct ctgctgagca cggtggtgcc atgcctctgc aactcctcct 240

gttgctgatc ctactgggcc ctggcaacag cttgcagctg tgggacacct gggcagatga 300

agccgagaaa gccttgggtc ccctgcttgc ccgggaccgg agacaggcca ccgaatatga 360

gtacctagat tatgatttcc tgccagaaac ggagcctcca gaaatgctga ggaacagcac 420

tgacaccact cctctgactg ggcctggaac ccctgagtct accactgtgg agcctgctgc 480

aaggcgttct actggcctgg atgcaggagg ggcagtcaca gagctgacca cggagctggc 540

caacatgggg aacctgtcca cggattcagc agctatggag atacagacca ctcaaccagc 600

agccacggag gcacagacca ctccactggc agccacagag gcacagacaa ctcgactgac 660

ggccacggag gcacagacca ctccactggc agccacagag gcacagacca ctccaccagc 720

agccacggaa gcacagacca ctcaacccac aggcctggag gcacagacca ctgcaccagc 780

agccatggag gcacagacca ctgcaccagc agccatggaa gcacagacca ctccaccagc 840

agccatggag gcacagacca ctcaaaccac agccatggag gcacagacca ctgcaccaga 900

agccacggag gcacagacca ctcaacccac agccacggag gcacagacca ctccactggc 960

agccatggag gccctgtcca cagaacccag tgccacagag gccctgtcca tggaacctac 1020

taccaaaaga ggtctgttca tacccttttc tgtgtcctct gttactcaca agggcattcc 1080

catggcagcc agcaatttgt ccgtcaacta cccagtgggg gccccagacc acatctctgt 1140

gaagcagtgc ctgctggcca tcctaatctt ggcgctggtg gccactatct tcttcgtgtg 1200

cactgtggtg ctggcggtcc gcctctcccg caagggccac atgtaccccg tgcgtaatta 1260

ctcccccacc gagatggtct gcatctcatc cctgttgcct gatgggggtg aggggccctc 1320

tgccacagcc aatggggggcc tgtccaaggc caagagcccg ggcctgacgc cagagcccag 1380

ggaggaccgt gagggggatg acctcaccct gcacagcttc ctcccttagc tcactctgcc 1440

atctgttttg gcaagacccc acctccacgg gctctcctgg gccacccctg agtgcccaga 1500

ccccattcca cagctctggg cttcctcgga gacccctggg gatggggatc ttcagggaag 1560

gaactctggc cacccaaaca ggacaagagc agcctggggc caagcagacg ggcaagtgga 1620

gccacctctt tcctccctcc gcggatgaag cccagccaca tttcagccga ggtccaaggc 1680

aggaggccat ttacttgaga cagatctct cctttttcct gtcccccatc ttctctgggt 1740

ccctctaaca tctcccatgg ctctccccgc ttctcctggt cactggagtc tcctccccat 1800

gtacccaagg aagatggagc tcccccatcc cacacgcact gcactgccat tgtcttttgg 1860

ttgccatggt caccaaacag gaagtggaca ttctaaggga ggagtactga agagtgacgg 1920

acttctgagg ctgtttcctg ctgctcctct gacttggggc agcttgggtc ttcttgggca 1980

cctctctggg aaaacccagg gtgaggttca gcctgtgagg gctgggatgg gtttcgtggg 2040

cccaagggca gacctttctt tgggactgtg tggaccaagg agcttccatc tagtgacaag 2100

tgacccccag ctatcgcctc ttgccttccc ctgtggccac tttccaggt ggactctgtc 2160

ttgttcactg cagtatccca actgcaggtc cagtgcaggc aataaatatg tgatggacaa 2220

acgata 2226

<210> 5

<211> 10

<212> PRT

<213> Mus musculus

<400> 5

Gly Phe Ser Phe Thr Gly Tyr Thr Met Asn

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Mus musculus

<400> 6

Gly Phe Ser Phe Thr Gly Tyr Thr

15

<210> 7

<211> 5

<212> PRT

<213> Mus musculus

<400> 7

Gly Tyr Thr Met Asn

15

<210> 8

<211> 7

<212> PRT

<213> Mus musculus

<400> 8

Gly Phe Ser Phe Thr Gly Tyr

15

<210> 9

<211> 6

<212> PRT

<213> Mus musculus

<400> 9

Thr Gly Tyr Thr Met Asn

15

<210> 10

<211> 17

<212> PRT

<213> Mus musculus

<400> 10

Leu Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 11

<211> 8

<212> PRT

<213> Mus musculus

<400> 11

Ile Ser Pro Tyr Asn Gly Gly Thr

15

<210> 12

<211> 4

<212> PRT

<213> Mus musculus

<400> 12

Pro Tyr Asn Gly

1

<210> 13

<211> 13

<212> PRT

<213> Mus musculus

<400> 13

Trp Ile Gly Leu Ile Ser Pro Tyr Asn Gly Gly Thr Ser

1 5 10

<210> 14

<211> 10

<212> PRT

<213> Mus musculus

<400> 14

Leu Ile Ser Pro Tyr Asn Gly Gly Thr Ser

1 5 10

<210> 15

<211> 9

<212> PRT

<213> Mus musculus

<400> 15

Arg Ala Tyr Gly Tyr Ala Met Asp Tyr

15

<210> 16

<211> 11

<212> PRT

<213> Mus musculus

<400> 16

Ala Arg Arg Ala Tyr Gly Tyr Ala Met Asp Tyr

1 5 10

<210> 17

<211> 7

<212> PRT

<213> Mus musculus

<400> 17

Ala Tyr Gly Tyr Ala Met Asp

15

<210> 18

<211> 10

<212> PRT

<213> Mus musculus

<400> 18

Ala Arg Arg Ala Tyr Gly Tyr Ala Met Asp

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Mus musculus

<400> 19

Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr

1 5 10

<210> 20

<211> 5

<212> PRT

<213> Mus musculus

<400> 20

Ser Ser Val Ser Tyr

15

<210> 21

<211> 6

<212> PRT

<213> Mus musculus

<400> 21

Ser Ser Ser Val Ser Tyr

15

<210> 22

<211> 6

<212> PRT

<213> Mus musculus

<400> 22

Ser Tyr Met Tyr Trp Tyr

15

<210> 23

<211> 7

<212> PRT

<213> Mus musculus

<400> 23

Asp Thr Ser Asn Leu Ala Ser

15

<210> 24

<211> 3

<212> PRT

<213> Mus musculus

<400> 24

Asp Thr Ser

1

<210> 25

<211> 10

<212> PRT

<213> Mus musculus

<400> 25

Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala

1 5 10

<210> 26

<211> 9

<212> PRT

<213> Mus musculus

<400> 26

Gln Gln Trp Ser Ser Tyr Pro Phe Thr

15

<210> 27

<211> 6

<212> PRT

<213> Mus musculus

<400> 27

Trp Ser Ser Tyr Pro Phe

15

<210> 28

<211> 8

<212> PRT

<213> Mus musculus

<400> 28

Gln Gln Trp Ser Ser Tyr Pro Phe

15

<210> 29

<211> 118

<212> PRT

<213> Mus musculus

<400> 29

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Val Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Tyr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 30

<211> 106

<212> PRT

<213> Mus musculus

<400> 30

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Leu Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 31

<211> 17

<212> PRT

<213> Mus musculus

<400> 31

Leu Ile Ser Pro Tyr Asp Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 32

<211> 8

<212> PRT

<213> Mus musculus

<400> 32

Ile Ser Pro Tyr Asp Gly Gly Thr

15

<210> 33

<211> 17

<212> PRT

<213> Mus musculus

<400> 33

Leu Ile Ser Pro Tyr Asp Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 34

<211> 13

<212> PRT

<213> Mus musculus

<400> 34

Trp Ile Gly Leu Ile Ser Pro Tyr Asp Gly Gly Thr Ser

1 5 10

<210> 35

<211> 10

<212> PRT

<213> Mus musculus

<400> 35

Leu Ile Ser Pro Tyr Asp Gly Gly Thr Ser

1 5 10

<210> 36

<211> 118

<212> PRT

<213> Mus musculus

<400> 36

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Val Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ser Pro Tyr Asp Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Tyr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 37

<211> 1260

<212> DNA

<213> Artificial

<220>

<223> VISTA-Fc fusion protein

<400> 37

atgggcgtgc ccacagccct ggaagctggc agctggaggt ggggaagcct gctgttcgcc 60

ctgtttctgg ccgcctccct gggacctgtg gccgccttta aggtcgccac cccttacagc 120

ctgtacgtgt gccccgaggg ccagaacgtg accctgacct gcagactgct gggccctgtg 180

gacaagggcc acgacgtgac cttctacaag acctggtaca ggagcagcag gggcgaggtc 240

cagacctgca gcgagaggag gcccatcagg aacctgacct tccaggacct gcacctgcac 300

cacggaggcc atcaggccgc caacacctcc cacgacctgg ctcagaggca cggactggag 360

agcgccagcg atcaccacgg caacttcagc atcaccatga ggaacctcac cctgctggac 420

agcggcctgt actgttgcct ggtggtggag atcaggcacc accacagcga gcacagagtg 480

cacggcgcca tggaactgca ggtgcagacc ggaaaggacg cccccagcaa ctgcgtggtg 540

taccccagca gctcccagga cagcgagaac atcaccgccg cgatctgt ggagtgccca 600

ccttgcccag caccacctgt ggcaggacct tcagtcttcc tcttcccccc aaaacccaag 660

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 720

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 780

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 840

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaaggcctc 900

ccatcctcca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 960

tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 1020

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1080

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1140

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1200

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1260

<210> 38

<211> 416

<212> PRT

<213> Artificial

<220>

<223> VISTA-Fc fusion protein

<400> 38

Met Gly Val Pro Thr Ala Leu Glu Ala Gly Ser Trp Arg Trp Gly Ser

1 5 10 15

Leu Leu Phe Ala Leu Phe Leu Ala Ala Ser Leu Gly Pro Val Ala Ala

20 25 30

Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln

35 40 45

Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His

50 55 60

Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val

65 70 75 80

Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp

85 90 95

Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp

100 105 110

Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn

115 120 125

Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr

130 135 140

Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg Val

145 150 155 160

His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser

165 170 175

Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Arg

180 185 190

Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

195 200 205

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

210 215 220

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

225 230 235 240

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

245 250 255

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

260 265 270

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

275 280 285

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

290 295 300

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

305 310 315 320

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

325 330 335

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

340 345 350

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

355 360 365

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

370 375 380

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

385 390 395 400

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

405 410 415

<210> 39

<211> 521

<212> PRT

<213> Artificial

<220>

<223> PSGL-1 Fc fusion with IgG kappa signal sequence and propeptide sequence

<400> 39

Met Pro Leu Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn

1 5 10 15

Ser Leu Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu

20 25 30

Gly Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr

35 40 45

Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg

50 55 60

Asn Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser

65 70 75 80

Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly

85 90 95

Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu

100 105 110

Ser Thr Asp Ser Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala

115 120 125

Thr Glu Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr

130 135 140

Arg Leu Thr Ala Thr Glu Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu

145 150 155 160

Ala Gln Thr Thr Pro Pro Ala Ala Thr Glu Ala Gln Thr Thr Gln Pro

165 170 175

Thr Gly Leu Glu Ala Gln Thr Thr Ala Pro Ala Ala Met Glu Ala Gln

180 185 190

Thr Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Pro Pro Ala Ala

195 200 205

Met Glu Ala Gln Thr Thr Gln Thr Thr Ala Met Glu Ala Gln Thr Thr

210 215 220

Ala Pro Glu Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Ala Thr Glu

225 230 235 240

Ala Gln Thr Thr Pro Leu Ala Ala Met Glu Ala Leu Ser Thr Glu Pro

245 250 255

Ser Ala Thr Glu Ala Leu Ser Met Glu Pro Thr Thr Lys Arg Gly Leu

260 265 270

Phe Ile Pro Phe Ser Val Ser Ser Val Thr His Lys Gly Ile Pro Met

275 280 285

Ala Ala Ser Asn Leu Ser Val Ala Arg Ser Val Glu Cys Pro Pro Cys

290 295 300

Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

305 310 315 320

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

325 330 335

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

340 345 350

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

355 360 365

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

370 375 380

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

385 390 395 400

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

405 410 415

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

420 425 430

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

435 440 445

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

450 455 460

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

465 470 475 480

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

485 490 495

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

500 505 510

Lys Ser Leu Ser Leu Ser Pro Gly Lys

515 520

<210> 40

<211> 523

<212> PRT

<213> Artificial

<220>

<223> PSGL-1 Fc fusion with IgG kappa chain signal sequence and tandem repeat element

<400> 40

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Gln Ala Thr Glu Tyr Glu Tyr Leu Asp Tyr Asp

20 25 30

Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg Asn Ser Thr Asp

35 40 45

Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser Thr Thr Val Glu

50 55 60

Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly Gly Ala Val Thr

65 70 75 80

Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu Ser Thr Asp Ser

85 90 95

Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala Thr Glu Ala Gln

100 105 110

Thr Thr Gln Pro Val Pro Thr Glu Ala Gln Thr Thr Pro Leu Ala Ala

115 120 125

Thr Glu Ala Gln Thr Thr Arg Leu Thr Ala Thr Glu Ala Gln Thr Thr

130 135 140

Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro Ala Ala Thr Glu

145 150 155 160

Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln Thr Thr Ala Pro

165 170 175

Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala Met Glu Ala Gln

180 185 190

Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr Thr Gln Thr Thr Ala

195 200 205

Met Glu Ala Gln Thr Thr Ala Pro Glu Ala Thr Glu Ala Gln Thr Thr

210 215 220

Gln Pro Thr Ala Thr Glu Ala Gln Thr Thr Pro Leu Ala Ala Met Glu

225 230 235 240

Ala Leu Ser Thr Glu Pro Ser Ala Thr Glu Ala Leu Ser Met Glu Pro

245 250 255

Thr Thr Lys Arg Gly Leu Phe Ile Pro Phe Ser Val Ser Ser Val Thr

260 265 270

His Lys Gly Ile Pro Met Ala Ala Ser Asn Leu Ser Val Asn Tyr Pro

275 280 285

Val Gly Ala Pro Asp His Ile Ser Val Ala Arg Ser Val Glu Cys Pro

290 295 300

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

305 310 315 320

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

325 330 335

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

340 345 350

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

355 360 365

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

370 375 380

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

385 390 395 400

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

405 410 415

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

420 425 430

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

435 440 445

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

450 455 460

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

465 470 475 480

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

485 490 495

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

500 505 510

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

515 520

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Claims (18)

1. An in vitro method for diagnosing a tumor caused by or otherwise associated with cells expressing V-domain immunoglobulin suppressor of T-cell activation (VISTA) in a subject, comprising the steps of: a) contacting a biological sample from said subject with a reagent capable of specifically binding to P-selectin glycoprotein ligand 1 (PSGL-1) nucleic acid or PSGL-1 protein; And b) quantifying the binding of said reagent to said biological sample, thereby determining the level of PSGL-1 expression in said sample. 2. The method according to claim 1, where the specified reagent is selected from a DNA probe, an RNA probe and an antibody to PSGL-1. 3. Method according to any one of paragraphs. 1 or 2, wherein binding to PSGL-1 in immune infiltrates of the tumor microenvironment is quantified. 4. Method according to any one of paragraphs. 1-3, further including grading the tumor stage by comparing the stage level (b) with a corresponding scale based on two parameters, staining intensity and percentage of positive cells. 5. Method according to any one of paragraphs. 1-4, further including the step of comparing the expression level in stage b) with a reference level, wherein an increase in the analyzed PSGL-1 level in stage b) compared to the reference level indicates the presence of a VISTA-mediated tumor. 6. The method of claim 5, wherein said reference level is the expression level of PSGL-1 in normal tissue samples. 7. Method according to any one of paragraphs. 5 or 6, wherein the VISTA-mediated tumor is selected from the group consisting of hematological malignancies, cancer or sarcoma of the bladder, breast, colon, connective tissue, rectum, stomach, esophagus, lung, larynx, kidney, oral cavity, ovarian or prostate cancer, melanoma or glioma, or metastasis of any of these cancers. 8. The method of claim 7, wherein the hematological malignancy is leukemia, lymphoma or myeloma. 9. The use of an anti-VISTA therapeutic agent for the preparation of a medicament for the treatment of cancer caused by or otherwise associated with VISTA-expressing cells in a patient, wherein said treatment includes the preliminary stage of diagnosing said cancer in said patient according to claims. 1-8, wherein said anti-VISTA therapeutic agent is selected from the group consisting of: a) antibodies to VISTA, wherein the antibody contains a heavy chain containing 3 complementarity determining regions (CDRs) with the sequences SEQ ID NO: 7, 10 and 15, defined by Kabat; and a light chain containing 3 CDRs with the sequences SEQ ID NO: 19, 23 and 26, determined by Kabat; And b) antibodies to VISTA, wherein the antibody contains a heavy chain containing 3 CDRs with the sequences SEQ ID NO: 17, 31 and 15, determined by Kabat; and a light chain containing 3 CDRs with the sequences SEQ ID NO: 19, 23 and 26, determined by Kabat. 10. Use according to claim 9, wherein said anti-VISTA antibody is a humanized antibody. 11. Application according to any one of paragraphs. 9 or 10, wherein said treatment further comprises the step of tailoring the treatment with an anti-VISTA therapeutic agent, wherein said tailoring the treatment is: - reducing or suspending specified treatment with an anti-VISTA therapeutic agent if the patient is determined to be refractory to the anti-VISTA therapeutic agent, or - continuation of specified treatment with an anti-VISTA therapeutic agent if the patient is determined to be susceptible to the anti-VISTA therapeutic agent.