PSGL-1 antagonists and uses thereof

Abstract

To provide methods of treating cancer.SOLUTION: Methods of identifying and using PSGL-1 antagonists are provided. Such methods include, but are not limited to, methods of treating cancer. PSGL-1 antagonists include, but are not limited to, antibodies that bind PSGL-1 and antibodies that bind VISTA, where the antibodies inhibit PSGL-1 binding to VISTA at acidic pH (e.g., pH 6.0), as well as PSGL-1 and VISTA extracellular domain polypeptides.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] Methods for identifying and using PSGL-1 antagonists are provided. The methods include, but are not limited to, methods of treating cancer. PSGL-1 antagonists include, but are not limited to, antibodies that bind to PSGL-1 and antibodies that bind to VISTA, and these antibodies inhibit the binding of PSGL-1 to VISTA at acidic pH (e.g., pH 6.0).

[0002] Sequence Listing This application is filed with a sequence listing in electronic form. The sequence listing is filed as a file entitled "2018-01-10_01134-0058-00PCT_Final_Seq_List_ST25.txt" created on January 10, 2018 and having a size of 49,152 bytes. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] V-domain immunoglobulin-containing T cell activation inhibitor (VISTA) is a cell surface-expressed protein that negatively regulates T cell activity (Wang et al., 2011, JEM 208(3) 577). VISTA is a single-pass type I transmembrane protein with a single extracellular IgV domain. In particular, the extracellular domain of VISTA is homologous to B7 family members such as PDL1, which also play a role in the regulation of immune responses (Wang et al., 2011, JEM 208(3) 577). The expression of VISTA is restricted to hematopoietic cells and is present in monocytes, T cells, and a subset of dendritic cells (Wang et al., 2011, JEM 208(3) 577 & Flies et al., 2011, JI 187:1537). Treatment with VISTA:Ig in vitro suppresses the proliferation and cytokine production by CD4+ T cells (Wang et al., 2011, JEM 208(3) 577). VISTA-specific monoclonal antibodies can induce an increase in T cell division in response to antigens presented by VISTA-expressing dendritic cells (Wang et al., 2011, JEM 208(3) 577). In vivo treatment of tumor-bearing animals with anti-VISTA monoclonal antibodies induced an immune-mediated antitumor response that inhibited tumor growth (Wang et al., 2011, JEM 208(3) 577). Collectively, these results highlight the importance of VISTA as a regulator of T cell-driven immune responses as observed during immune-mediated tumor rejection. The homophilic binding partner of VISTA is currently unknown.

[0004] Identification of the binding partner of VISTA would aid in the understanding of VISTA-mediated inhibition of T cell activation and provide many advantages in drug development, including the development of therapeutically effective and safe treatments, biomarkers for patient selection and companion diagnostics, targets for combination therapies, and new targets for the development of cancer immunotherapeutic agents. Summary of the Invention

[0005] In some embodiments, methods for identifying a PSGL-1 antagonist are provided. In some embodiments, the method comprises contacting a candidate molecule with a VISTA molecule (e.g., mature VISTA protein or a fragment thereof) and a PSGL-1 molecule (e.g., mature PSGL-1 protein or a fragment thereof), wherein the VISTA molecule comprises VISTA, the VISTA extracellular domain ("ECD"), or a VISTA ECD fusion molecule (e.g., excluding the signal sequence; i.e., mature VISTA or a fragment thereof), the PSGL-1 molecule comprises PSGL-1, the PSGL-1 ECD, or a PSGL-1 ECD fusion molecule (e.g., excluding the signal sequence; i.e., mature PSGL-1 or a fragment thereof), and the contacting occurs at an acidic pH, e.g., pH < 7.0, ≤ 6.8, ≤ 6.5, or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0, or 6.0 - 7.0, e.g., pH 6.0. In some embodiments, the method comprises forming a composition comprising the candidate molecule, the VISTA molecule, and the PSGL-1 molecule, wherein, e.g., at an acidic pH, e.g., pH < 7.0, ≤ 6.8, ≤ 6.5, or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0, or 6.0 - 7.0, e.g., pH 6.0, the VISTA molecule comprises VISTA, the VISTA ECD, or a VISTA ECD fusion molecule, and the PSGL-1 molecule comprises PSGL-1, the PSGL-1 ECD, or a PSGL-1 ECD fusion molecule. In some embodiments, the method further comprises detecting the binding of the VISTA molecule to the PSGL-1 molecule. In some embodiments, a decrease in the binding of the VISTA molecule to the PSGL-1 molecule in the presence of the candidate molecule as compared to the binding of the VISTA molecule to the PSGL-1 molecule in the absence of the candidate molecule indicates that the candidate molecule is a PSGL-1 antagonist. In some embodiments, the binding of the VISTA molecule to the PSGL-1 molecule decreases by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% in the presence of the candidate molecule. In some embodiments, the binding of the VISTA molecule to the PSGL-1 molecule is detected by a method selected from surface plasmon resonance, ELISA, amplified luminescent proximity homogeneous assay (ALPHA), and flow cytometry.

[0006] In any of the methods for identifying a PSGL-1 antagonist described herein, the PSGL-1 antagonist can be, for example, an antibody that binds to VISTA at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In any of the methods for identifying a PSGL-1 antagonist described herein, the PSGL-1 antagonist can be an antibody that binds to PSGL-1. In any of the methods for identifying a PSGL-1 antagonist described herein, the PSGL-1 antagonist can be a small molecule. In any of the methods for identifying a PSGL-1 antagonist described herein, the PSGL-1 antagonist can be a low molecular weight peptide.

[0007] In some embodiments, provided is a method for determining whether a VISTA antibody is a PSGL-1 antagonist. In some embodiments, the method comprises contacting the VISTA antibody with a VISTA molecule and a PSGL-1 molecule, wherein, for example, at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example pH 6.0, the VISTA molecule comprises VISTA, VISTA ECD or a VISTA ECD fusion molecule, and the PSGL-1 molecule comprises PSGL-1, PSGL-1 ECD or a PSGL-1 ECD fusion molecule. In some embodiments, the method comprises forming a composition comprising the VISTA antibody, the VISTA molecule and the PSGL-1 molecule, wherein, for example, at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example pH 6.0, the VISTA molecule comprises VISTA, VISTA ECD or a VISTA ECD fusion molecule, and the PSGL-1 molecule comprises PSGL-1, PSGL-1 ECD or a PSGL-1 ECD fusion molecule. In some embodiments, the method further comprises detecting the binding of the VISTA molecule to the PSGL-1 molecule. In some embodiments, a decrease in the binding of the VISTA molecule to the PSGL-1 molecule in the presence of the VISTA antibody as compared to the binding of the VISTA molecule to the PSGL-1 molecule in the absence of the VISTA antibody indicates that the VISTA antibody is a PSGL-1 antagonist. In some embodiments, the binding of the VISTA molecule to the PSGL-1 molecule decreases by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% in the presence of the VISTA antibody. In some embodiments, the binding of the VISTA molecule to the PSGL-1 molecule is detected by a method selected from surface plasmon resonance, ELISA, amplified luminescent proximity homogeneous assay and flow cytometry.

[0008] In any of the methods for identifying a PSGL-1 antagonist described herein, the VISTA molecule may be VISTA expressed on the surface of a cell, and / or the PSGL-1 molecule may be PSGL-1 expressed on the surface of a cell.

[0009] In some embodiments, methods are provided for inhibiting the binding of PSGL-1 to VISTA in a subject. In some embodiments, the method comprises administering to the subject at least one PSGL-1 antagonist. In some embodiments, methods are provided for inhibiting the binding of PSGL-1 on a cell to VISTA. In some embodiments, the method comprises contacting the cell with at least one PSGL-1 antagonist at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In some embodiments, the cell is in vitro.

[0010] In some embodiments, methods are provided for treating cancer. In some embodiments, the method comprises administering to a subject having cancer an effective amount of at least one PSGL-1 antagonist. In some embodiments, the method further comprises administering to the subject an effective amount of a therapeutic agent selected from chemotherapeutic agents, anti-angiogenic agents, growth inhibitors, immuno-oncology agents, and anti-tumor compositions. In any of the embodiments described herein, the PSGL-1 antagonist may block the binding of PSGL-1 to VISTA.

[0011] In any of the embodiments described herein, the method may comprise administering a PSGL-1 antagonist selected from a PSGL-1 antibody and a VISTA antibody, wherein the antibody inhibits the binding of PSGL-1 to VISTA at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In any of the embodiments described herein, the method may comprise administering a PSGL-1 antibody that inhibits the binding of PSGL-1 to VISTA at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In any of the embodiments described herein, the antibody may be selected from chimeric antibodies, humanized antibodies, and human antibodies. In any of the embodiments described herein, the antibody may be an antibody fragment. In some embodiments, the antibody fragment is selected from IgG (e.g., IgG1, IgG2 or IgG4), Fv, single-chain Fv (scFv), Fab, Fab’ and (Fab’)2.

[0012] In some embodiments, provided is the use of a PSGL-1 antagonist for the treatment of cancer in a subject. In any of the uses described herein, the PSGL-1 antagonist can be a PSGL-1 antibody or a VISTA antibody, wherein the antibody inhibits the binding of PSGL-1 to VISTA at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In some embodiments, the antibody is selected from chimeric antibodies, humanized antibodies, and human antibodies. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is selected from IgG (e.g., IgG1, IgG2 or IgG4), Fv, single-chain Fv (scFv), Fab, Fab’ and (Fab’)2.

[0013] Any embodiment described herein, or any combination thereof, is applicable to all of the methods of the invention described herein.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0015] The inventors have identified PSGL-1 as a binding partner of VISTA, where the binding preferentially occurs at acidic pH. Targeting the interaction between VISTA and PSGL-1 can enhance the immune response against cancer cells by inhibiting the immunosuppressive effect of VISTA. The target molecules include antibodies that bind to PSGL-1 and antibodies that bind to VISTA, and these antibodies block the binding of VISTA to PSGL-1. Exemplary target molecules are molecules that bind to VISTA or PSGL-1 at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example pH 6.0. Such target molecules are provided as therapeutic agents for treating cancer.

[0016] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0017] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described content.

[0018] Definitions Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars.

[0019] Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, among other places, in, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). In addition, exemplary techniques for chemical synthesis, chemical analysis, preparation of pharmaceuticals, formulation, delivery, and treatment of patients are also known in the art.

[0020] As used herein, unless otherwise specified, "or" means "and / or". In the context of multiple dependent claims, the use of "or" only cites more than one preceding independent or dependent claim in an alternative format. Unless otherwise indicated, the terms "comprise", "comprises", "comprising" have the same meaning as "comprise but not limited to". Similarly, the term "such as" has the same meaning as the term "such as, but not limited to". Also, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include a plurality of subunits, unless otherwise specified.

[0021] When used in accordance with the present disclosure, the following terms are understood to have the following meanings, unless otherwise specified.

[0022] The terms "nucleic acid molecule" and "polynucleotide" can be used interchangeably and refer to a polymer of nucleotides. Such a polymer of nucleotides can contain natural and / or non-natural nucleotides and includes, but is not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides including a nucleic acid molecule or polynucleotide.

[0023] The term "polypeptide" refers to a polymer of amino acid residues and is not limited to a minimum length. Such a polymer of amino acid residues may contain natural or non-natural amino acid residues and includes, but is not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. This term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, in the present invention, "polypeptide" refers to a protein (usually of a conservative nature) that includes modifications to the native sequence, such as deletions, additions, and substitutions, as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as due to mutations in the host producing the protein or errors resulting from PCR amplification. "Protein" includes polypeptides and complexes of two or more polypeptides (such as dimers and polymers). "Small peptide" refers to a peptide having 50 or fewer amino acids. In some embodiments, the small peptide has 40 or fewer or 35 or fewer or 30 or fewer or 25 or fewer amino acids. In some embodiments, the small peptide has 10 to 50 amino acids or 15 to 30 amino acids.

[0024] "Native sequence" polypeptides include polypeptides having the same amino acid sequence as polypeptides found in nature. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides can be isolated from nature or produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring cleavage or secreted forms of the polypeptide (e.g., extracellular domain sequences), naturally occurring variant forms of the polypeptide (e.g., alternative splicing forms), and naturally occurring allelic variants of the polypeptide.

[0025] The polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide, when aligned, introducing gaps if necessary to achieve maximal percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity. In some embodiments, the variant has at least about 95% amino acid sequence identity to the native sequence. In some embodiments, the variant has at least about 97% amino acid sequence identity to the native sequence.

[0026] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of sequence identity, after introducing gaps if necessary to obtain the maximum percent sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be obtained using a variety of methods within the skill in the art, such as generally available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to obtain the maximum alignment over the full length of the sequences being compared.

[0027] The terms "P-selectin glycoprotein ligand 1" and "PSGL-1" are used interchangeably to refer to native PSGL-1 unless otherwise specified. Thus, references to PSGL-1, unless otherwise specified as being otherwise (e.g., by discussing non-human or murine or cynomolgus monkey PSGL-1), have the same meaning as references to human PSGL-1 throughout the text. Non-human PSGL-1 can be from any vertebrate, including mammals such as primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats).

[0028] The term PSGL-1 includes not only full-length, unprocessed PSGL-1, but also any form of PSGL-1 that results from processing within a cell or any fragment thereof and that retains the ability to specifically bind to VISTA with an affinity (Kd) of, for example, ≤1 μM, ≤100 nM, or <≤10 nM. The term also encompasses naturally occurring variants of PSGL-1, such as splice variants or allelic variants. In some embodiments, PSGL-1 comprises the amino acid sequence of SEQ ID NO: 1 (human isoform 1 precursor, including the signal peptide) or SEQ ID NO: 2 (mature human isoform 1, without the signal peptide), or the amino acid sequence of SEQ ID NO: 14 (human isoform 2 precursor, including the signal peptide) or SEQ ID NO: 15 (mature human isoform 2, without the signal peptide).

[0029] The term "PSGL-1" includes full-length PSGL-1, PSGL-1 fragments, and PSGL-1 variants, with or without a signal peptide. As used herein, the term "full-length PSGL-1" refers not only to full-length, non-processed PSGL-1, but also to any form of PSGL-1 that results from processing within a cell or any fragment thereof and that retains the ability to specifically bind to VISTA with an affinity (Kd) of, for example, ≤1 μM, ≤100 nM, or <≤10 nM. In some embodiments, full-length PSGL-1 has the amino acid sequence of SEQ ID NO: 1 (isoform 1 precursor, including the signal peptide), or SEQ ID NO: 2 (mature isoform 1, without the signal peptide), or SEQ ID NO: 14 (isoform 2 precursor, including the signal peptide), or SEQ ID NO: 15 (mature isoform 2, without the signal peptide). As used herein, the term "PSGL-1 fragment" refers to a PSGL-1 in which one or more residues have been deleted from the N-terminus and / or C-terminus of full-length PSGL-1 and that retains the ability to bind to VISTA. The PSGL-1 fragment may or may not include the N-terminal signal peptide. As used herein, the term "PSGL-1 variant" refers to a PSGL-1 that includes additions, deletions, and substitutions of amino acids and that retains the ability to bind to VISTA. Such variants are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the parent PSGL-1. The % identity between two polypeptides can be measured by the similarity score determined by comparing the amino acid sequences of the two polypeptides using the Bestfit program with default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the segment of highest similarity between two sequences.

[0030] Unless otherwise specified, the terms "V-region Ig suppressor of T cell activation", "platelet receptor Gi24 isoform 1 precursor", "B7H5", and "VISTA" refer to native human VISTA herein. Accordingly, the expressions "VISTA" and "human VISTA" have the same meaning unless it is clear from the context (e.g., by specifically referring to non-human VISTA species) that they do not. When VISTA is non-human, it can be from any vertebrate, including mammals such as primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). This term includes not only full-length, unprocessed VISTA, but also any form of ISTA that results from processing within a cell or any of its fragments and retains the ability to specifically bind to PSGL-1 with an affinity (Kd) of, for example, ≤1 μM, ≤100 nM, or <≤10 nM. This term also encompasses naturally occurring variants of VISTA, such as splice variants or allelic variants. In some embodiments, VISTA comprises the amino acid sequence of SEQ ID NO: 5 (precursor, including the signal peptide) or SEQ ID NO: 6 (mature, without the signal peptide). Non-limiting and exemplary non-human VISTA is mouse VISTA having the amino acid sequence of SEQ ID NO: 7 (precursor, including the signal peptide) or SEQ ID NO: 8 (mature, without the signal peptide).

[0031] The term "antagonist" is used in the broadest sense and refers to any molecule that partially or completely inhibits or neutralizes the biological activity of a polypeptide such as PSGL-1 or VISTA, or that partially or completely inhibits the transcription or translation of the nucleic acid encoding the polypeptide. Exemplary antagonist molecules include, but are not limited to, antagonist antibodies, small molecule peptides, oligopeptides, organic molecules (including small molecules), aptamers, and antisense nucleic acids. In some embodiments, antagonist agents may be referred to as blocking agents (such as blocking antibodies).

[0032] The term "PSGL-1 antagonist" refers to a molecule that interacts with PSGL-1 or VISTA and inhibits PSGL-1 and / or VISTA-mediated signaling. Exemplary PSGL-1 antagonists include antibodies that bind to PSGL-1 and antibodies that bind to VISTA. In some embodiments, the PSGL-1 antagonist is an antibody against PSGL-1. In some embodiments, the PSGL-1 antagonist blocks the binding of PSGL-1 to VISTA.

[0033] A PSGL-1 antagonist is considered to "block the binding of PSGL-1 to VISTA" if it reduces the amount of detectable binding of PSGL-1 to VISTA by at least 50%. In some embodiments, the PSGL-1 antagonist reduces the amount of detectable binding of PSGL-1 to VISTA by at least 60%, at least 70%, at least 80% or at least 90%. In some such embodiments, the antagonist is said to block ligand binding by at least 50%, at least 60%, at least 70% etc.

[0034] The term "inhibit" or "inhibiting" refers to a decrease or elimination of any phenotypic characteristic, or a decrease or cessation of the incidence, degree or likelihood of that characteristic. In some embodiments, "decrease" or "inhibit" means the ability to cause a decrease of 20% or more. In another embodiment, "decrease" or "inhibit" means the ability to cause a decrease of 50% or more. In yet another embodiment, "decrease" or "inhibit" means the ability to cause a decrease of 75%, 85%, 90%, 95% or more.

[0035] The term "PSGL-1 antibody" or "antibody that binds to PSGL-1", as used herein, refers to an antibody that binds to PSGL-1 at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In some embodiments, the PSGL-1 antibody inhibits PSGL-1 and / or VISTA-mediated signaling. In some embodiments, the PSGL-1 antibody blocks the binding of PSGL-1 to VISTA at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In some embodiments, the PSGL-1 antibody refers to an antibody that can bind to PSGL-1 with sufficient affinity such that it is useful as a diagnostic and / or therapeutic agent in targeting PSGL-1. In some embodiments, the degree of binding of the PSGL-1 antibody to an irrelevant non-PSGL-1 protein is less than about 10% of the binding of the antibody to PSGL-1 as measured, for example, by radioimmunoassay (RIA). In some embodiments, the PSGL-1 antibody binds to an epitope of PSGL-1 that is conserved among PSGL-1s from different species. In some embodiments, the PSGL-1 antibody binds to the same epitope as a human or humanized PSGL-1 antibody that binds to PSGL-1.

[0036] The term "VISTA antibody" or "antibody that binds to VISTA", as used herein, refers to an antibody that binds to VISTA at an acidic pH, e.g., pH < 7.0, ≦ 6.8, ≦ 6.5 or ≦ 6.3, or at pH 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0. In some embodiments, the VISTA antibody inhibits PSGL-1 and / or VISTA-mediated signaling. In some embodiments, the VISTA antibody blocks the binding of PSGL-1 to VISTA at an acidic pH as defined above, e.g., pH < 7.0, ≦ 6.8, ≦ 6.5 or ≦ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, e.g., pH 6.0. Thus, in some embodiments, the VISTA antibody is a PSGL-1 antagonist. In some embodiments, the VISTA antibody refers to an antibody that can bind to VISTA with sufficient affinity such that it is useful as a diagnostic and / or therapeutic agent in targeting VISTA. In some embodiments, the degree of binding of the VISTA antibody to an irrelevant non-VISTA protein is less than about 10% of the binding of the antibody to VISTA as measured, e.g., by radioimmunoassay (RIA). In some embodiments, the VISTA antibody binds to an epitope of VISTA that is conserved among VISTAs from different species. In some embodiments, the VISTA antibody binds to the same epitope as a human or humanized VISTA antibody that binds to human VISTA.

[0037] As used herein, the term "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and various antibody structures including, but not limited to, antibody fragments, as long as they exhibit the desired antigen-binding activity. The term "antibody" as used herein further refers to a molecule comprising heavy chain complementarity determining regions (CDRs) 1, 2, and 3 and light chain CDRs 1, 2, and 3, which molecule has the ability to bind to an antigen. The term antibody includes, but is not limited to, fragments having the ability to bind to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.

[0038] In some embodiments, an antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, an antibody comprises at least one heavy chain comprising at least a portion of the heavy chain variable region and at least a portion of the heavy chain constant region, and at least one light chain comprising at least a portion of the light chain variable region and at least a portion of the light chain constant region. In some embodiments, an antibody comprises at least two heavy chains, each heavy chain comprising at least a portion of the heavy chain variable region and at least a portion of the heavy chain constant region, and at least two light chains, each light chain comprising at least a portion of the light chain variable region and at least a portion of the light chain constant region. As used herein, a single-chain Fv (scFv), or any other antibody comprising a single polypeptide chain that includes, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs), is considered to have heavy and light chains. In some such embodiments, the heavy chain is the region of the antibody that includes the three heavy chain CDRs, and the light chain is the region of the antibody that includes the three light chain CDRs.

[0039] As used herein, the term "heavy chain variable region" refers to the region that includes heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the heavy chain variable region also includes at least a portion of FR1 at the N-terminus of CDR1 and / or at least a portion of FR4 at the C-terminus of CDR3.

[0040] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant regions, C H 1, C H 2 and C H 3. Non-limiting and exemplary heavy chain constant regions include γ, δ, and α. Also, non-limiting and exemplary heavy chain constant regions include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. Further, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Some isotypes can be further classified into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0041] As used herein, the term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence and with or without a C-terminal lysine.

[0042] As used herein, the term "light chain variable region" refers to a region comprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the light chain variable region also comprises FR1 and / or FR4.

[0043] As used herein, the term "light chain constant region" refers to a region comprising the light chain constant region C L . Non-limiting and exemplary light chain constant regions include λ and λ.

[0044] As used herein, the term "light chain" refers to a polypeptide that includes at least a light chain variable region, regardless of the presence or absence of a leader sequence. In some embodiments, the light chain includes at least a portion of the light chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide that includes a light chain variable region and a light chain constant region, regardless of the presence or absence of a leader sequence.

[0045] The term "antibody that binds to the same epitope as a reference antibody," as determined by an antibody competition assay, refers to an antibody that blocks the binding of the reference antibody to its antigen in the competition assay by 50% or more. Conversely, the reference antibody blocks the binding of the antibody to its antigen in the competition assay by 50% or more. The term "competes" as used in the context of antibodies that compete for the same epitope means that the competition between the antibodies is determined by an assay in which the test antibody prevents or inhibits the specific binding of the reference antibody to a common antigen (e.g., PSGL-1 or VISTA). For example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA using 1-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32 (77-82), etc. Many types of competitive binding assays can be used. Typically, such assays involve the use of a solid surface or cells bound to purified antigen having either an unlabeled test antigen-binding protein or a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually, the test antibody is present in excess.Antibodies identified by a competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope that is close enough to cause steric hindrance to the epitope to which the reference antibody binds. In some embodiments, the competitive antibody, when present in excess, inhibits the specific binding of the reference antibody to the common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, the binding is inhibited by at least 80%, 85%, 90%, 95% or 97% or more.

[0046] The term "antigen" refers to a molecule or a part of a molecule that can be bound by a selective binding agent such as an antibody or an immunofunctional fragment thereof, and can be used in a mammal to produce an antibody capable of binding to the antigen. The antigen may have one or more epitopes capable of interacting with the antibody.

[0047] The term "epitope" refers to the part of a molecule that is bound by a selective binding agent such as an antibody or a fragment thereof. This term includes any determinant that can specifically bind to an antibody. Epitopes can be continuous or discontinuous (e.g., in a polypeptide, amino acid residues that are not contiguous with each other in the polypeptide sequence but are bound by an antigen-binding protein in the context of the molecule). In some embodiments, an epitope can be mimetic in the sense that it contains a three-dimensional structure similar to the epitope used to generate the antibody, but contains none or only some of the amino acid residues found in that epitope used to generate the antibody. Epitope determinants include chemically active surface groupings of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, etc., and have specific three-dimensional structural characteristics and / or specific charge characteristics. In some embodiments, an "epitope" is defined by the method used to determine it. For example, in some embodiments, an antibody and a reference antibody bind to the same epitope determined by hydrogen-deuterium exchange (HDX) if they bind to the same region of an antigen. In certain embodiments, an antibody and a reference antibody bind to the same epitope determined by X-ray crystallography if they bind to the same region of an antigen.

[0048] As used herein, the term "chimeric antibody" refers to an antibody that includes at least one variable region derived from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region derived from a second species (such as human, cynomolgus monkey, chicken, etc.). In some embodiments, a chimeric antibody includes at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody includes at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are derived from a first species and all of the constant regions of the chimeric antibody are derived from a second species.

[0049] As used herein, "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region (mouse, rat, cynomolgus monkey, chicken, etc.) is replaced with the corresponding amino acid of a human variable region. In some embodiments, the humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is Fab, scFv, (Fab')2, etc.

[0050] As used herein, "CDR-grafted antibody" refers to a humanized antibody in which one or more complementarity-determining regions (CDRs) of a first (non-human) species are grafted into the framework region (FR) of a second (human) species.

[0051] As used herein, "human antibody" refers to an antibody produced in a human, an antibody produced in a non-human animal containing a human immunoglobulin gene such as XenoMouse®, and an antibody selected using an in vitro method such as phage display, wherein the antibody repertoire is based on human immunoglobulin sequences.

[0052] The term "PSGL-1 extracellular domain" ("PSGL-1 ECD") includes full-length PSGL-1 ECD, PSGL-1 ECD fragments, and PSGL-1 ECD variants, and refers to a PSGL-1 polypeptide that lacks an intracellular domain and a transmembrane domain and may or may not contain a signal peptide. Unless otherwise specified, the PSGL-1 ECD polypeptide is a native human ECD. As used herein, the term "full-length PSGL-1 ECD" refers to a PSGL-1 ECD that extends to the last amino acid of the extracellular domain, which may or may not contain an N-terminal signal peptide, and refers to the native rice variant of the extracellular domain. Non-limiting and exemplary PSGL-1 ECDs include amino acids 1 to 24 of SEQ ID NO: 1 (including the signal sequence) or amino acids 23 to 241 of SEQ ID NO: 1 (excluding the signal sequence), amino acids 1 to 219 of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Another exemplary PSGL-1 ECD includes amino acids 1 to 24 of SEQ ID NO: 14 (including the signal sequence) or amino acids 23 to 241 of SEQ ID NO: 14 (excluding the signal sequence), or amino acids 1 to 219 of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. As used herein, the term "PSGL-1 ECD fragment" refers to a PSGL-1 ECD in which one or more residues have been deleted from the N-terminus and / or C-terminus of the full-length ECD and that retains the ability to bind to VISTA. The PSGL-1 ECD fragment may or may not contain an N-terminal signal peptide. As used herein, the term "PSGL-1 ECD variant" refers to a PSGL-1 ECD that includes amino acid additions, deletions, and substitutions and retains the ability to bind to VISTA. Such variants are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the parental PSGL-1 ECD. The % identity between two polypeptides can be measured by the similarity score determined by comparing the amino acid sequences of the two polypeptides using the Bestfit program with the default settings for determining similarity.Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the segment of highest similarity between two sequences.

[0053] The term "PSGL-1 ECD fusion molecule" refers to a molecule that includes a PSGL-1 ECD and one or more "fusion partners". In some embodiments, the PSGL-1 ECD and the fusion partner are covalently linked ( "fused"). If the fusion partner is also a polypeptide ( "fusion partner polypeptide"), the PSGL-1 ECD and the fusion partner polypeptide can be part of a continuous amino acid sequence, and the fusion partner polypeptide can be linked to either the N-terminus or the C-terminus of the PSGL-1 ECD. In such cases, the PSGL-1 ECD and the fusion partner polypeptide can be translated as a single polypeptide from a coding sequence that encodes both the PSGL-1 ECD and the fusion partner polypeptide ( "PSGL-1 ECD fusion protein"). In some embodiments, the PSGL-1 ECD and the fusion partner are covalently linked via other means, such as a chemical bond other than a peptide bond, for example. Many known methods for covalently linking a polypeptide to another molecule (e.g., a fusion partner) can be used. In other embodiments, the PSGL-1 ECD and the fusion partner can be fused via a "linker" composed of at least one amino acid or chemical moiety. SEQ ID NO: 19 provides a linker used in the PSGL-1 Fc fusion molecule of the examples herein. Non-limiting and exemplary PSGL-1 ECD fusion molecules include the fusion molecules described in T. Pouyani et al., Cell 83: 333-343 (1995). Non-limiting and exemplary PSGL-1 ECD fusion molecules include, for example, (a) amino acids 1 to 241 of SEQ ID NO: 1 (including the signal sequence), or amino acids 23 to 241 of SEQ ID NO: 1 (excluding the signal sequence), amino acids 1 to 219 of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or amino acids 1 to 241 of SEQ ID NO: 14 (including the signal sequence), or amino acids 23 to 241 of SEQ ID NO: 14 (excluding the signal sequence), or amino acids 1 to 219 of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, (b) an Fc (such as the Fc of SEQ ID NO: 11, 12 or 13, etc.) or an Fc region derived from human IgG1, or a binding to an Fc, etc., including a PSGL-1 ECD bound to an Fc.

[0054] In some embodiments, the PSGL-1 polypeptide and the fusion partner are not covalently linked. In some such embodiments, they can be linked, for example, using a binding pair. Exemplary binding pairs include, but are not limited to, biotin and avidin or streptavidin, and an antibody and its antigen, etc.

[0055] Exemplary fusion partners include, but are not limited to, the immunoglobulin Fc region, albumin, and polyethylene glycol. Non-limiting and exemplary amino acid sequences of the Fc region are shown in SEQ ID NOs: 11 to 13.

[0056] As described above, the PSGL-1 ECD is derived from the native human PSGL-1 sequence unless otherwise specified. However, in some embodiments, the PSGL-1 ECD amino acid sequence is derived from that of a non-human mammal. In such embodiments, the non-human PSGL-1 ECD amino acid sequence can be derived from mammals including, but not limited to, rodents (including mice, rats, hamsters), rabbits, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. A PSGL-1 ECD fusion molecule incorporating a nonhumPSGL-1 ECD is referred to as a "nonhumPSGL-1 ECD fusion molecule" or "non-human PSGL-1 ECD fusion molecule". Similar to the human PSGL-1 ECD fusion molecule, the non-human fusion molecule may include a fusion partner, an optional linker, and a non-human PSGL-1 ECD. Such a non-human fusion molecule may also include a signal peptide.

[0057] The term "PSGL-1 ECD fragment" refers to a PSGL-1 ECD in which one or more residues have been deleted from the N-terminus and / or C-terminus of the full-length human ECD and which retains the ability to bind to human VISTA. The term "non-human PSGL-1 ECD fragment" refers to a non-humPSGL-1 ECD in which one or more residues have been deleted from the N-terminus and / or C-terminus of the full-length ECD and which retains the ability to bind to VISTA of the non-human animal from which the sequence is derived. This is in contrast to a "PSGL-1 ECD variant", which refers to a PSGL-1 ECD that contains amino acid additions, deletions, and substitutions compared to the native human PSGL-1 ECD and retains the ability to bind to human VISTA.

[0058] The term "non-humPSGL-1 ECD variant" refers to a non-human PSGL-1 ECD that contains amino acid additions, deletions, and substitutions and retains the ability to bind to VISTA of the animal from which the sequence is derived. This is in contrast to a "PSGL-1 ECD variant", which refers to a human PSGL-1 ECD that contains amino acid additions, deletions, and substitutions and retains the ability to bind to human VISTA.

[0059] In any of the embodiments described herein, PSGL-1, including but not limited to full-length PSGL-1, PSGL-1 fragments, PSGL-1 variants, PSGL-1 ECDs, and PSGL-1 ECD fusion proteins, may further include tags. Non-limiting and exemplary tags include FITC, His6, biotin, and other labels and tags known in the art.

[0060] The term "VISTA extracellular domain" ("VISTA ECD") includes full-length VISTA ECD, VISTA ECD fragments, and VISTA ECD variants, and refers to VISTA polypeptides that lack intracellular and transmembrane domains and may or may not contain a signal peptide. The polypeptide is a native human ECD unless otherwise specified. As used herein, the term "full-length VISTA ECD" refers to a VISTA ECD that extends to the last amino acid of the extracellular domain, which may or may not include an N-terminal signal peptide, and refers to the native rice variant of the extracellular domain.

[0061] As used herein, the term "VISTA ECD fragment" refers to a VISTA ECD that has one or more residues deleted from the N-terminus and / or C-terminus of the full-length ECD and retains the ability to bind to PSGL-1. The VISTA ECD fragment may or may not include an N-terminal signal peptide. As used herein, the term "VISTA ECD variant" refers to a VISTA ECD that includes amino acid additions, deletions, and substitutions and retains the ability to bind to PSGL-1. Such variants are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the parental VISTA ECD.

[0062] The % identity of two polypeptides can be measured by a similarity score determined by comparing the amino acid sequences of the two polypeptides using the Bestfit program with default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the segment of highest similarity between two sequences. In some embodiments, the VISTA ECD containing a His tag has the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VISTA ECD without a His tag has the amino acid sequence of amino acids 1-202 of SEQ ID NO: 10 (corresponding to the sequence of SEQ ID NO: 10 with the last 6 His residues of that sequence subtracted).

[0063] The term "VISTA ECD fusion molecule" refers to a molecule comprising a VISTA ECD and one or more "fusion partners". In some embodiments, the VISTA ECD and the fusion partner are covalently linked ( "fused"). When the fusion partner is also a polypeptide ( "fusion partner polypeptide"), the VISTA ECD and the fusion partner polypeptide can be part of a continuous amino acid sequence, and the fusion partner polypeptide can be linked to either the N-terminus or the C-terminus of the VISTA ECD. In such cases, the VISTA ECD and the fusion partner polypeptide can be translated as a single polypeptide from a coding sequence encoding both the VISTA ECD and the fusion partner polypeptide ( "VISTA ECD fusion protein"). In some embodiments, the VISTA ECD and the fusion partner are covalently linked via other means, such as a chemical bond other than a peptide bond. Many known methods can be used to covalently attach a polypeptide to another molecule (e.g., a fusion partner). In other embodiments, the VISTA ECD and the fusion partner can be fused via a "linker" composed of at least one amino acid or chemical moiety. Non-limiting and exemplary VISTA ECD fusion molecules include the sequence of SEQ ID NO: 9. Another exemplary VISTA ECD fusion molecule includes the amino acid sequence 1-202 of SEQ ID NO: 10 with the Fc sequence of SEQ ID NO: 11, 12, or 13 added thereto.

[0064] In some embodiments, the VISTA polypeptide and the fusion partner are not covalently bound. In some such embodiments, they can be bound, for example, using a binding pair. Exemplary binding pairs include, but are not limited to, biotin and avidin or streptavidin, and an antibody and its antigen.

[0065] Exemplary fusion partners include, but are not limited to, an immunoglobulin Fc region, albumin, and polyethylene glycol. Non-limiting and exemplary amino acid sequences of the Fc region are shown in SEQ ID NOs: 11 to 13.

[0066] Here too, unless otherwise specified, the VISTA ECD amino acid sequence is derived from the human sequence. However, in some embodiments, the VISTA ECD amino acid sequence is derived from that of a non-human mammal. In such embodiments, the VISTA ECD amino acid sequence can be derived from mammals including, but not limited to, rodents (including mice, rats, hamsters), rabbits, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. A VISTA ECD fusion molecule incorporating a non-human VISTA ECD is referred to as a "non-human VISTA ECD fusion molecule". Similar to a human VISTA ECD fusion molecule, a non-human fusion molecule may include a fusion partner, an optional linker, and a VISTA ECD. Such a non-human fusion molecule may also include a signal peptide. In contrast, the term "VISTA ECD fragment" refers to a native VISTA ECD in which one or more residues have been deleted from the N-terminus and / or C-terminus of the full-length human ECD and which retains the ability to bind to human PSGL-1.

[0067] A "non-human VISTA ECD fragment" refers to a non-human VISTA ECD in which one or more residues have been deleted from the N-terminus and / or C-terminus of the full-length ECD and which retains the ability to bind to PSGL-1 of the non-human animal from which the sequence is derived. In contrast, a "VISTA ECD variant" refers to a VISTA ECD that includes amino acid additions, deletions, and substitutions compared to the native human VISTA ECD and which retains the ability to bind to human PSGL-1. A "non-human VISTA ECD variant" refers to a VISTA ECD that includes amino acid additions, deletions, and substitutions compared to its parental VISTA ECD and which retains the ability to bind to PSGL-1 of the animal from which the sequence is derived.

[0068] In any of the embodiments described herein, VISTA, including but not limited to full-length VISTA, VISTA fragments, VISTA variants, VISTA ECD, and VISTA ECD fusion proteins, may further include tags. Non-limiting and exemplary tags include FITC, His6, biotin, and other labels and tags known in the art.

[0069] The term "signal peptide" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that promotes the secretion of the polypeptide from mammalian cells. The signal peptide is cleaved when the polypeptide is transported out of mammalian cells, forming a mature protein. Signal peptides may be natural or synthetic and may be heterologous or homologous to the protein to which they are attached. Exemplary signal peptides include, but are not limited to, the signal peptides of PSGL-1 and VISTA. Exemplary signal peptides also include signal peptides derived from heterologous proteins. The term "signal sequence" refers to the polynucleotide sequence encoding the signal peptide.

[0070] The term "vector" is used to describe a polynucleotide that can be engineered to contain a polynucleotide cloned or capable of being propagated within a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as a promoter and / or enhancer, etc.) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (such as an antibiotic resistance gene and genes that can be used in colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest within a host cell.

[0071] "Host cell" refers to a cell that can be a recipient of a vector or an isolated polynucleotide, or a cell that is a recipient. A host cell is a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells; fungal cells such as yeast; plant cells; and insect cells. Non-limiting and exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells and their derivatives (such as 293-6E and DG44 cells, respectively).

[0072] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components that are normally found together in nature, or a molecule that is separated from at least some of the components that are normally produced together. For example, a polypeptide is "isolated" if it is separated from at least some of the components of the cell in which it is produced. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced the polypeptide is considered to "isolate" the polypeptide. Similarly, a polynucleotide is "isolated" if it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) that is normally found in nature, or if, in the case of an RNA polynucleotide, for example, it is separated from at least some of the components of the cell in which it is produced. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated" unless the polynucleotide is naturally found in that vector.

[0073] The terms "subject" and "patient" are used interchangeably herein and refer to a human. In some embodiments, methods of treating other mammals including, but not limited to, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets are also provided. In some cases, "subject" or "patient" refers to a subject or patient in need of treatment for a disease or disorder.

[0074] As used herein, the term "sample" or "patient sample" refers to a material obtained from or derived from a subject of interest that contains cell entities and / or other molecular entities that are characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological properties. For example, the phrases "disease sample" and variations thereof refer to a sample obtained from a subject in which it is expected or known to contain characterized cell and / or molecular entities. "Tissue or cell sample" means a collection of similar cells obtained from the tissue of a subject or patient. The source of the tissue or cell sample can be fresh, frozen, and / or preserved organ or tissue specimens or solid tissues from biopsies or aspirates; blood or any blood component; body fluids such as sputum, cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid; cells from any point during the gestational or developmental period of the subject. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from diseased tissue / organs. The tissue sample may contain compounds that do not naturally coexist with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0075] As used herein, "reference sample", "reference cell" or "reference tissue" refers to a sample, cell or tissue obtained from a source known or believed not to be affected by the disease or condition sought to be identified using the methods or compositions of the present invention. In one embodiment, the reference sample, reference cell or reference tissue is obtained from a healthy part of the same subject or patient whose disease or condition has been identified using the compositions or methods of the present invention. In one embodiment, the reference sample, reference cell or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient whose disease or condition has been identified using the compositions or methods of the present invention. In some embodiments, the reference sample, reference cell or reference tissue is obtained from a patient in advance before the onset of the disease or condition or at an early stage of the disease or condition.

[0076] A condition was "previously characterized as having [a characteristic]" when the characteristic of that condition was demonstrated in at least a subset of patients having that condition or in one or more animal models of that condition. In some embodiments, such a characteristic of a condition need not be determined in a patient being treated with one or more PSGL-1 antagonists of the invention. The presence of such a characteristic in a particular patient being treated using the methods and / or compositions need not be determined for the patient to be considered to have a condition that was previously characterized as having that characteristic.

[0077] A "disorder" or "disease" is any condition that would benefit from treatment with one or more PSGL-1 antagonists of the invention. This includes chronic and acute disorders or diseases, including pathological conditions that render a mammal susceptible to the disorder in question. Non-limiting examples of disorders treated herein include cancer.

[0078] The term "cancer" as used herein refers to a context of cells that exhibit abnormally high levels of proliferation and growth. Cancer can be benign (also called a benign tumor), pre-malignant or malignant. Cancer cells can be solid cancer cells (i.e., the formation of solid tumors) or leukemia cancer cells. The term "cancer growth" as used herein refers to the proliferation or growth by cells (singular or plural) including cancer that results in a corresponding increase in the size or extent of the cancer.

[0079] Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific and non-limiting examples of such cancers include squamous cell cancer, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, stomach cancer, melanoma, and various head and neck cancers.

[0080] "Chemotherapeutic agents" are chemical compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, thiotepa and cytoxan (registered trademark) cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, piposulfan; aziridines such as benzodopa, carbocone, meturedopa, uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bratasin and bratasinone); camptothecin (including synthetic analog topotecan); bryostatin, calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); erythrobins; pancratistatin; sarcodictyin; spongistatin; alkylating agents such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobucarcin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: Engl. (1994)); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein-engineered antibiotic chromophores), actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardifilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin (registered trademark), doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin and other antibiotics; methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine and other antimetabolites; androgens such as calusterone, drostanolone propionate, epithiostanol, mepithiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone;Elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oregon); razoxane; lysoxine; schizophyllan; spirogermanium; tenuazonic acid; triazicone; 2,2’,2”-trichloro-triethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids such as Taxol (registered trademark) (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, New Jersey), Abraxane (registered trademark) (American Pharmaceutical Partners, Schaumburg, Illinois) (cremophor-free), (albumin-engineered nanoparticle formulation of paclitaxel), and Taxotere (registered trademark) (docetaxel) (Rhone-Poulenc Rorer, Antony, France); chlorambucil; Gemzar (registered trademark) (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine (registered trademark) (vinorelbine); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; Zoladex; ibandronate; irinotecan (Camptosar, CPT-11) (j including the treatment regimen of 5-FU and leucovorin with irinotecan);Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including the oxaliplatin treatment regimen (FOLFOX)); inhibitors of PKC-alpha, Raf, H-Ras, VEGF-A, EGFR that reduce cell proliferation (e.g., erlotinib (Tarceva (registered trademark))), and pharmaceutically acceptable salts, acids or derivatives of any of the above are included.;

[0081] Additional non-limiting and exemplary chemotherapeutic agents include antihormonal agents that act to regulate or inhibit the hormonal action on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), for example tamoxifen (including Norvadex® (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® (toremifene); aromatase inhibitors that inhibit the enzyme aromatase that regulates estrogen production in the adrenal gland, such as 4(5)-imidazoles, aminoglutethimide, Megase® (megestrol acetate), Arimidex® (anastrozole), exemestane, formestane, fadrozole, Rivisor® (vorozole), Femara® (letrozole), and Aromasin® (exemestane); and antiandrogenic drugs such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes (such as PKC-alpha, Ralf, and H-Ras, etc.) in signal transduction pathways related to abherant cell proliferation; ribozymes such as VEGF expression inhibitors (e.g., Angiozyme® (ribozyme), etc.) and HER2 expression inhibitors; vaccines such as gene therapy vaccines, for example, Alovecti® vaccine, Leuvcti® vaccine, and Vaxid® vaccine; Proleukin® (rIL-2); Lartotecan® (topoisomerase 1 inhibitor); Abarelix® (rmRH); and pharmaceutically acceptable salts, acids, or derivatives of any of the above are included.

[0082] An "anti-angiogenic agent" or "angiogenesis inhibitor" refers to a low molecular weight substance, polynucleotide (including inhibitory RNAs (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or a conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability. Anti-angiogenic agents include agents that bind to block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents include antibodies or other antagonists against angiogenic agents, such as antibodies against VEGF-A (e.g., bevacizumab (Avastin®) or VEGF-A receptors (e.g., KDR receptor or Flt-1 receptor), antibodies against VEGF-C, anti-PDGFR inhibitors such as Gleevec® (imatinib mesylate); small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SU11248 (sunitinib malate), AMG706, or those described, for example, in WO 2004 / 113304). Also included in anti-angiogenic agents are natural angiogenesis inhibitors, such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D’Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 showing a list of anti-angiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2 showing a list of known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 showing a list of anti-angiogenic agents used in clinical trials).

[0083] As used herein, the "growth inhibitor" refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Thus, a growth inhibitor is one that significantly reduces the proportion of cells (VEGF-expressing cells) in the S phase. Examples of growth inhibitors include, but are not limited to, agents that block the progression of the cell cycle (at stages other than the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (doxorubicin, epirubicin, daunorubicin, etoposide, bleomycin, etc.). Also, agents that arrest cells in the G1 phase, such as DNA alkylating agents (tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, ara-C, etc.), also affect S phase arrest. Further information can be found in Murakami et al., "Cell cycle regulation, oncogenes, and antineoplastic drugs", edited by Mendelsohn and Israel, The Molecular Basis of Cancer, Chapter 1, (W.B. Saunders, Philadelphia, 1995), for example, p. 13. Taxanes (paclitaxel and docetaxel) are both anticancer agents derived from the yew tree. Docetaxel (Taxotere (registered trademark), Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analog of paclitaxel (Taxol (registered trademark), Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting the assembly of microtubules from tubulin dimers and preventing depolymerization, which results in the inhibition of cell mitosis.

[0084] The term "anti-tumor composition" refers to a composition useful for treating cancer, containing at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapeutic agents (also called immuno-oncology agents), apoptosis agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva®)), platelet-derived growth factor inhibitors (e.g., Gleevec® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab (Yervoy®)), PD-1 inhibitors (e.g., anti-PD1 antibody, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibody), VISTA inhibitors (e.g., anti-VISTA antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, VISTA or VEGF receptor(s), TRAIL / Apo2, as well as other bioactive agents and organic chemical agents, etc. Combinations of these are also included in the present invention.

[0085] "Treatment", as used herein, is not only aimed at, for example, slowing down (mitigating) a targeted pathological condition or disorder, but also at, for example, preventing recurrence of the pathological condition or disorder. "Treatment", as used herein, encompasses any administration or application of a therapeutic agent for a disease (also referred to herein as a "disorder" or "condition") in mammals including humans, and includes preventing a disease or the progression of a disease, arresting or retarding a disease or its progression, stopping its occurrence, removing the disease partially or completely, removing one or more symptoms of the disease partially or completely, or restoring or repairing a damaged, defective, or malfunctioning function, or stimulating an inefficient process. The term "treatment" also includes reducing the severity of any phenotypic characteristic and / or reducing the incidence, degree, or likelihood of that characteristic. Those in need of treatment include not only those who already have the disorder, but also those at risk of recurrence of the disorder, or those in whom the disorder should be prevented or slowed down.

[0086] The term "effective amount" or "therapeutically effective amount" refers to the amount of a drug that is effective for treating a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective at the required dosage and over the required period to achieve the desired therapeutic or prophylactic result. The therapeutically effective amount of the PSGL-1 antagonist of the present invention can vary depending on factors such as the medical condition, age, sex, and weight of the individual, and the ability of the antagonist to induce the desired response in the individual. A therapeutically effective amount includes an amount in which the therapeutically beneficial effects exceed the toxic or harmful effects of the PSGL-1 antagonist.

[0087] "Prophylactically effective amount" refers to an amount effective at the required dosage and over the required period to achieve the desired prophylactic result. Usually, but not necessarily, prophylactic dosages are less than therapeutically effective amounts because they are used in a subject prior to or at an early stage of a disease.

[0088] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier common in the art for use with a therapeutic agent that together with a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed. For example, if the therapeutic agent is administered orally, the carrier may be a gel capsule. When the therapeutic agent is administered subcutaneously, the carrier is preferably not irritating to the skin and does not cause a reaction at the injection site.

[0089] "Manufactured article" means any manufactured product (e.g., a package or container) or kit that includes at least one reagent, such as a medicament for the treatment of a disease or disorder, or a probe for specifically detecting a biomarker described herein. In some embodiments, the manufactured product or kit is promoted, distributed or sold as a unit for performing the methods described herein.

[0090] Therapeutic Compositions and Methods Methods of Treating a Disease PSGL-1 antagonists are provided for use in methods of treating humans and other mammals. Provided are methods of treating a disease comprising administering a PSGL-1 antagonist to a human or other mammal.

[0091] Methods of Treating Cancer In some embodiments, provided are methods of treating or preventing cancer comprising administering to a subject in need an effective amount of a PSGL-1 antagonist.

[0092] The inventors of the present invention have identified PSGL-1 as a binding partner of VISTA. VISTA is a receptor on the surface of various immune cells (T cells, dendritic cells, natural killer cells, monocytes, macrophages, etc.) and functions as an inhibitor of the active immune response. The expression of PSGL-1 and / or VISTA on the surface of cancer and / or immune cells (e.g., T cells and NK cells) may inhibit the immune response by binding PSGL-1 and / or VISTA. Inhibition of the VISTA-PSGL-1 interaction may promote the immune-mediated killing of cancer cells.

[0093] In some embodiments, provided is a method of treating cancer, comprising administering a PSGL-1 antagonist to a subject having cancer. In some embodiments, provided is the use of a PSGL-1 antagonist for treating cancer. Non-limiting and exemplary cancers treatable with a PSGL-1 antagonist are provided herein, including carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific and non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, and various head and neck cancers. In some embodiments, the lung cancer is non-small cell lung cancer or squamous cell carcinoma of the lung. In some embodiments, the leukemia is acute myeloid leukemia or chronic lymphocytic leukemia. In some embodiments, the breast cancer is invasive breast cancer. In some embodiments, the ovarian cancer is ovarian serous cystadenocarcinoma. In some embodiments, the kidney cancer is kidney renal clear cell carcinoma. In some embodiments, the colon cancer is colon adenocarcinoma. In some embodiments, the bladder cancer is bladder urothelial carcinoma.

[0094] In some embodiments, the PSGL-1 antagonist is selected from a PSGL-1 antibody and a VISTA antibody. In some embodiments, the PSGL-1 antagonist is a PSGL-1 antibody. The PSGL-1 antagonist for treating cancer may be a non-antibody protein (such as PSGL-1 or VISTA) or a portion thereof (e.g., ECD) that inhibits the interaction between PSGL-1 and VISTA, and may optionally further include a fusion partner in the form of a fusion molecule. Various exemplary PSGL-1 antagonists are described in more detail in the following sections.

[0095] Route of Administration and Carrier In various embodiments, the PSGL-1 antagonist can be administered subcutaneously or intravenously. In some embodiments, the PSGL-1 antagonist can be administered in vivo by various routes including, but not limited to, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, inhalation, intradermal, topical, transdermal, and intrathecal, or alternatively, for example, by implantation. The subject compositions can be formulated into solid, semi-solid, liquid or gaseous preparations including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. In some embodiments, the PSGL-1 antagonist is delivered using gene therapy. As a non-limiting example, a nucleic acid molecule encoding the PSGL-1 antagonist can be coated with gold microparticles and delivered intradermally by a particle gun device or "gene gun", as described in the literature (e.g., see Tang et al., Nature 356:152-154 (1992)).

[0096] In various embodiments, a composition comprising a PSGL-1 antagonist is provided in a formulation comprising a variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, including vehicles, adjuvants, and diluents. Additionally, various pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, isotonic agents, stabilizers, wetting agents, etc. are also available. Non-limiting and exemplary carriers include physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0097] In various embodiments, a composition comprising a PSGL-1 antagonist can be formulated for injection, including subcutaneous injection, by dissolving, suspending, or emulsifying it in a vegetable oil or other oil, synthetic fatty acid glyceride, ester of a higher fatty acid, or an aqueous or non-aqueous solvent such as propylene glycol, along with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives if desired. In various embodiments, the composition can be formulated for inhalation using an acceptable pressurized propellant such as dichlorodifluoromethane, propane, nitrogen, etc. In various embodiments, the composition can also be formulated into sustained-release microcapsules using a biodegradable or non-biodegradable polymer, etc. Non-limiting and exemplary biodegradable formulations include polylactic-co-glycolic acid polymers. Non-limiting and non-biodegradable formulations include polyglycerol fatty acid esters. Specific methods for making such formulations are described, for example, in EP No. 1125584.

[0098] Also provided is a pharmaceutical package comprising one or more containers each containing one or more doses of a PSGL-1 antagonist. In some embodiments, unit doses are provided, where a unit dose contains a predetermined amount of the composition comprising the PSGL-1 antagonist, regardless of the presence or absence of one or more additional agents. In some embodiments, such unit doses are provided in disposable pre-filled syringes for injection. In some embodiments, the composition contained in the unit dose may contain physiological saline, sucrose, etc.; a buffer such as phosphate, etc., and / or is formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder that can be reconstituted by the addition of a suitable liquid, such as sterile water. In some embodiments, the composition contains one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. In some embodiments, the composition of the present invention contains heparin and / or proteoglycan.

[0099] The pharmaceutical composition is administered in an amount effective for the treatment or prevention of a particular indication. The therapeutically effective amount typically depends on the weight, physical condition or health status of the subject being treated, the extent of the condition being treated, or the age of the subject being treated. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

[0100] In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 10 mg to about 1000 mg per dose. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the PSGL-1 antagonist can be administered in an amount in the range of about 20 mg to about 200 mg per dose.

[0101] The PSGL-1 antagonist composition can be administered to a subject as needed. In some embodiments, an effective dose of the PSGL-1 antagonist is administered to the subject one or more times. In various embodiments, the effective dose of the PSGL-1 antagonist is administered to the subject once a month, less than once a month, e.g., every two months, every three months, or every six months. In other embodiments, the effective dose of the PSGL-1 antagonist is administered to the subject more than once a month, e.g., every two weeks, weekly, twice a week, three times a week, daily, or multiple times a day. The effective dose of the PSGL-1 antagonist is administered to the subject at least once. In some embodiments, the effective dose of the PSGL-1 antagonist can be administered multiple times over a period of at least one month, at least six months, or at least one year, etc. In some embodiments, the PSGL-1 antagonist is administered to the subject as needed to alleviate one or more symptoms of the condition.

[0102] Combination therapy For the treatment of a disease, the PSGL-1 antagonist according to the present invention, including its functional fragments, can be administered to a subject in need thereof in combination with other biologically active substances or other treatment procedures. For example, the PSGL-1 antagonist can be administered alone or in combination with other treatment methods. The PSGL-1 antagonist can be provided before, substantially simultaneously with, or after other treatment methods such as radiation therapy.

[0103] For the treatment of cancer, the PSGL-1 antagonist may be administered in combination with one or more anti-cancer agents such as chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, or anti-tumor compositions. Non-limiting examples of chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, and anti-tumor compositions that can be used in combination with one or more PSGL-1 antagonists of the present invention are provided in the "Definitions" herein.

[0104] In certain embodiments, a PSGL-1 antagonist that specifically binds to PSGL-1 (a "PSGL-1 binding antagonist"), such as a PSGL-1 antagonist antibody, is administered to a subject having a disease in which stimulation of the immune system is beneficial, such as cancer or an infectious disease, together with an antagonist that specifically binds to VISTA (a "VISTA binding antagonist"), such as a VISTA antagonist antibody. These two antagonists can be administered simultaneously or sequentially, as described below for example for combinations of PSGL-1 antagonists and immuno-oncology agents. For example, for cancer or an infectious disease, one or more additional therapies, such as checkpoint modulators, may be added to the treatment with a PSGL-1 binding antagonist and a VISTA binding antagonist.

[0105] In certain embodiments, a PSGL-1 antagonist is administered to a subject, such as a subject having cancer, simultaneously or sequentially with another treatment. For example, a PSGL-1 antagonist can be administered with one or more of radiation therapy, surgery, or chemotherapy, such as targeted chemotherapy or immunotherapy. Immunotherapy, such as cancer immunotherapy, includes cancer vaccines and immuno-oncology agents. A PSGL-1 antagonist may be a protein, antibody, antibody fragment, or small molecule that binds to PSGL-1. A PSGL-1 antagonist may be an antibody or an antigen-binding fragment thereof that specifically binds to PSGL-1. A PSGL-1 antagonist may be a protein, antibody, antibody fragment, or small molecule that binds to VISTA. A PSGL-1 antagonist may be an antibody or an antigen-binding fragment thereof that specifically binds to VISTA.

[0106] In certain embodiments, a method of treating a subject having cancer comprises administering to the subject having cancer a PSGL-1 antagonist (e.g., a PSGL-1 antibody or a VISTA antibody) and one or more immuno-oncology agents. Immunotherapy, such as therapy using immuno-oncology agents, is effective to enhance, stimulate, and / or upregulate the immune response in a subject. In one aspect, the administration of the immuno-oncology agent and the PSGL-1 antagonist has a synergistic effect in the treatment of cancer, such as in the inhibition of tumor growth.

[0107] As used herein in the context of a combination of a PSGL-1 antagonist and another agent (e.g., an immuno-oncology agent), when the PSGL-1 antagonist is a PSGL-1 binding antagonist, the immuno-oncology agent is a VISTA binding antagonist, and when the PSGL-1 antagonist is a VISTA binding antagonist, the immuno-oncology agent is a PSGL-1 binding antagonist.

[0108] In one aspect, the PSGL-1 antagonist is administered sequentially before the administration of the immuno-oncology agent. In one aspect, the PSGL-1 antagonist is administered concomitantly with the immuno-oncology agent. In one aspect, the PSGL-1 antagonist is administered sequentially after the administration of the immuno-oncology agent. The administration of these two agents may be initiated, for example, at times separated by 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or more than one week, or the administration of the second agent may be initiated, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or more than one week after the first agent is administered.

[0109] In certain embodiments, the PSGL-1 antagonist and the immuno-oncology agent are administered concomitantly, for example, by co-injecting the patient simultaneously over 30 minutes or 60 minutes. The PSGL-1 antagonist may be co-formulated with the immuno-oncology agent.

[0110] Immune-oncology agents include, for example, small molecule drugs, antibodies or fragments thereof, or other biomolecules or small molecules. Examples of biological immune-oncology agents include, but are not limited to, antibodies, antibody fragments, vaccines, and cytokines. In one aspect, the antibody is a monoclonal antibody. In certain aspects, the monoclonal antibody is a humanized or human antibody.

[0111] In one aspect, the immune-oncology agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule (e.g., receptor or ligand) on immune cells such as T cells or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., receptor or ligand), both of which result in amplification of the antigen-specific T cell response. In certain aspects, the immune-oncology agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule (e.g., receptor or ligand) on cells involved in innate immunity such as NK cells or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., receptor or ligand), and the immune-oncology agent enhances innate immunity. Such immune-oncology agents are generally referred to as immune checkpoint regulators, such as immune checkpoint inhibitors or immune checkpoint stimulators.

[0112] In certain embodiments, the immuno-oncology agent targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, the immuno-oncology agent is an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, including B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5, and B7-H6, or a co-stimulatory or co-inhibitory receptor that specifically binds to a member of the B7 family. The immuno-oncology agent may be an agent that targets a member of the TNF family of membrane-bound ligands, or a co-stimulatory or co-inhibitory receptor (e.g., a TNF receptor family member) that specifically binds thereto. Exemplary TNF and TNFR family members that may be targeted by the immuno-oncology agent include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, and NGFR. The immuno-oncology agent that can be used in combination with a PSGL-1 antagonist agent to treat cancer may be an agent such as an antibody that targets an IgSF member such as the above-described B7 family member, B7 receptor family member, TNF family member, or TNFR family member.

[0113] In one aspect, the PSGL-1 antagonist is administered with one or more of (i) antagonists of proteins that inhibit T cell activation, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-4, and GL-1 (e.g., immune checkpoint inhibitors), and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3, and CD28H.

[0114] In one aspect, the immuno-oncology agent is an agent that inhibits cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines) (i.e., an antagonist of the cytokine), or an agonist (e.g., the cytokine itself) of cytokines that stimulate T cell activation and stimulate an immune response (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα).

[0115] For example, for the treatment of cancer and infectious diseases, other agents that can be combined with a PSGL-1 antagonist to stimulate the immune system include antagonists of inhibitory receptors of NK cells or agonists of activating receptors of NK cells. For example, an anti-PSGL-1 antagonist can be combined with an antagonist of KIR.

[0116] Additional other agents for combination therapy include, but are not limited to, agents that inhibit or deplete macrophages or monocytes, such as CSF-1R antagonist antibodies including RG7155 (WO 11 / 70024, WO 11 / 107553, WO 11 / 131407, WO 13 / 87699, WO 13 / 119716, WO 13 / 132044) or FPA008 (WO 11 / 140249; WO 13169264; (WO 14 / 036357)).

[0117] Immuno-oncology agents also include agents that inhibit TGF-β signaling.

[0118] Additional agents that can be combined with a PSGL-1 antagonist include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides and imiquimod, or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[0119] Additional other therapies that can be combined with a PSGL-1 antagonist include therapies that deplete or block Treg cells, such as agents that specifically bind to CD25.

[0120] Another therapy that can be combined with a PSGL-1 antagonist is a therapy that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthase.

[0121] Another class of agents that can be used includes agents that inhibit the formation of adenosine or inhibit the adenosine A2A receptor.

[0122] Other therapies that can be combined with a PSGL-1 antagonist for the treatment of cancer include therapies that reverse / prevent T cell anergy or exhaustion, and therapies that cause innate immune activation and / or inflammation at the tumor site.

[0123] The PSGL-1 antagonist may be combined with two or more immuno-oncology agents, for example, the following therapies: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccine, GM-CSF-secreting cell vaccine, CpG oligonucleotide, imiquimod); therapies that inhibit negative immune regulation by inhibiting, for example, the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Tregs or other immunosuppressive cells; therapies that stimulate positive immune regulation using agonists that stimulate, for example, the CD-137, OX-40 and / or GITR pathways and / or stimulate T cell effector functions; therapies that systemically increase the frequency of anti-tumor T cells; therapies that deplete or inhibit Tregs, such as Tregs in the tumor, for example, by using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect the function of suppressive myeloid cells in the tumor; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that cause innate immune activation and / or inflammation at the tumor site; and may be combined with combinatorial approaches that target multiple elements of the immune pathway, such as one or more of the administration of immunostimulatory cytokines or the blockade of immunosuppressive cytokines.

[0124] For example, a PSGL-1 antagonist can be used in combination with one or more agonist agents that bind to positive co-stimulatory receptors; one or more antagonists (blocking agents) that attenuate signal transduction through inhibitory receptors, such as antagonists that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking the PD-L1 / PD-1 / PD-L2 interaction); one or more agents that systemically increase the frequency of anti-tumor immune cells such as T cells and deplete or inhibit Tregs (e.g., by inhibiting CD25); one or more agents that inhibit metabolic enzymes such as IDO; one or more agents that reverse / prevent T cell anergy or exhaustion; and one or more agents that cause innate immune activation and / or inflammation at the tumor site.

[0125] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration of a PSGL-1 antagonist and an immuno-oncology agent to the subject, where the immuno-oncology agent is a CTLA-4 antagonist such as an antagonist CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

[0126] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration of a PSGL-1 antagonist and an immuno-oncology agent to the subject, where the immuno-oncology agent is a PD-1 antagonist such as an antagonist PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab) or MEDI-0680 (AMP-514; WO 2012 / 145493). Also, the immuno-oncology agent may include pidilizumab (CT-011), although there is some question as to its specificity for PD-1 binding. Another approach targeting the PD-1 receptor is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, also called AMP-224.

[0127] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration of a PSGL-1 antagonist and an immuno-oncology agent to the subject, where the immuno-oncology agent is a PD-L1 antagonist such as an antagonist PD-L1 antibody. Suitable PD-L1 antibodies include, for example, MPDL3280A (Rg7446; WO 2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO 2007 / 005874), MSB0010718C (WO 2013 / 79174) or rHigM12B7.

[0128] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration of a PSGL-1 antagonist and an immuno-oncology agent to the subject, where the immuno-oncology agent is a LAG-3 antagonist such as an antagonist LAG-3 antibody. Suitable LAG3 antibodies include, for example, BMS-986016 (WO 10 / 19570, WO 14 / 08218), or IMP-731 or IMP-321 (WO 08 / 132601, WO 09 / 44273).

[0129] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration of a PSGL-1 antagonist and an immuno-oncology agent to the subject, where the immuno-oncology agent is a CD137 (4-1BB) agonist such as an agonist CD137 antibody. Suitable CD137 antibodies include, for example, urelumab or PF-05082566 (WO 2012 / 32433).

[0130] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is a GITR agonist such as an agonist GITR antibody. Suitable GITR antibodies include, for example, TRX-518 (International Publication No. WO 06 / 105021, International Publication No. WO 09 / 009116), MK-4166 (International Publication No. WO 11 / 028683) or the GITR antibodies disclosed in International Publication No. WO 2015 / 031667.

[0131] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is an OX40 agonist such as an agonist OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; International Publication No. WO 06 / 029879).

[0132] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is a CD40 agonist such as an agonist CD40 antibody. In certain embodiments, the immuno-oncology agent is a CD40 antagonist such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7 / 4.

[0133] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is a CD27 agonist such as an agonist CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab.

[0134] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is MGA271 (against B7H3) (International Publication No. WO 2011 / 109400).

[0135] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is a KIR antagonist such as lirilumab.

[0136] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (International Publication No. WO 2006 / 122150, (International Publication No. WO 2007 / 075598, International Publication No. WO 2008 / 036653, International Publication No. WO 2008 / 036642), indoximod, NLG-919 (WO 2009 / 073620, International Publication No. WO 2009 / 1156652, International Publication No. WO 2011 / 56652, International Publication No. WO 2012 / 142237) or F001287.

[0137] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is a Toll-like receptor agonist, such as a TLR2 / 4 agonist (e.g., heat-killed Mycobacterium bovis (Bacillus Calmette-Guerin)); a TLR7 agonist (e.g., hiltonol or imiquimod); a TLR7 / 8 agonist (e.g., resiquimod); or a TLR9 agonist (e.g., CpG7909).

[0138] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administration to the subject of a PSGL-1 antagonist and an immuno-oncology agent, where the immuno-oncology agent is a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197 or IMC-TR1.

[0139] Exemplary PSGL-1 antagonists In some embodiments, the PSGL-1 antagonist is selected from a PSGL-1 antibody and a VISTA antibody. In some embodiments, the PSGL-1 antagonist is a PSGL-1 antibody. In some embodiments, the PSGL-1 antagonist is a VISTA antibody. The PSGL-1 antagonist for treating cancer may be a non-antibody protein (such as PSGL-1 or VISTA) or a portion thereof (e.g., ECD) that inhibits the interaction between PSGL-1 and VISTA, and may optionally further include a fusion partner in the form of a fusion molecule. Also, in other embodiments, this antagonist can be a small molecule or a low molecular weight peptide.

[0140] PSGL-1 antibody and VISTA antibody In some embodiments, antibodies are provided that block the binding of PSGL-1 to VISTA, for example at an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example at pH 6.0. In some embodiments, antibodies are provided that inhibit PSGL-1-mediated signaling. In some embodiments, the antibody is a PSGL-1 antibody. In some embodiments, the PSGL-1 antibody binds to the PSGL-1 extracellular domain (ECD). In some embodiments, the PSGL-1 antibody inhibits the binding of PSGL-1 to VISTA. In some embodiments, the PSGL-1 antibody inhibits VISTA-mediated signaling. In some embodiments, the PSGL-1 antibody inhibits PSGL-1-mediated signaling.

[0141] In some embodiments, the PSGL-1 antibody has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M - 10 -13 M, e.g., 10 -9 M - 10 -13 M) with respect to PSGL-1, e.g., humPSGL-1. In certain embodiments, the PSGL-1 antibody has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M - 10 -13 M, e.g., 10 -9 M - 10 -13 M) with respect to PSGL-1, e.g., humPSGL-1, at an acidic pH, e.g., pH < 7.0, ≤ 6.8, ≤ 6.5, or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0, or 6.0 - 7.0, e.g., at pH 6.0.

[0142] In some embodiments, a PSGL-1 antibody having the features provided herein inhibits the binding of VISTA to PSGL-1 by at least 25%, 50%, 75%, 80%, 90%, or 100%.

[0143] In some embodiments, the antibody binds to PSGL-1 from multiple species. For example, in some embodiments, the antibody binds to human PSGL-1 and also binds to PSGL-1 of at least one mammal selected from mouse, rat, dog, guinea pig, and cynomolgus monkey.

[0144] In some embodiments, the antibody is a VISTA antibody. In some embodiments, the VISTA antibody binds to the VISTA extracellular domain (ECD). In some embodiments, the VISTA antibody inhibits the binding of VISTA to PSGL-1. In some embodiments, the VISTA antibody inhibits VISTA-mediated signaling. In some embodiments, the VISTA antibody inhibits PSGL-1-mediated signaling. In some embodiments, the VISTA antibody has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M - 10 -13 M, e.g., 10 -9 M - 10 -13 M) with respect to VISTA, e.g., human ISTA. In some embodiments, the VISTA antibody has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M - 10 -13 M, e.g., 10 -9 M - 10 -13 M) with respect to VISTA, e.g., human VISTA, at an acidic pH, e.g., pH < 7.0, ≤ 6.8, ≤ 6.5, or ≤ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0, or 6.0 - 7.0, e.g., at pH 6.0.

[0145] In some embodiments, a VISTA antibody having the features provided herein inhibits the binding of VISTA to PSGL-1 by at least 25%, 50%, 75%, 80%, 90%, or 100%.

[0146] In some embodiments, the antibody binds to VISTA from multiple species. For example, in some embodiments, the antibody binds to human VISTA and also binds to VISTA of at least one mammal selected from mouse, rat, dog, guinea pig, and cynomolgus monkey.

[0147] In some embodiments, multispecific antibodies are provided. In some embodiments, bispecific antibodies are provided. Non-limiting and exemplary bispecific antibodies include antibodies comprising a first arm comprising a heavy chain / light chain combination that binds to a first antigen and a second arm comprising a heavy chain / light chain combination that binds to a second antigen. Further non-limiting and exemplary multispecific antibodies are bispecific variable domain antibodies. In some embodiments, the bispecific antibody comprises a first arm that inhibits the binding of PSGL-1 to VISTA and a second arm that stimulates T cells, for example, by binding CD3. In some embodiments, the first arm binds to PSGL-1.

[0148] Humanized antibody In some embodiments, the PSGL-1 or VISTA antibody is a humanized antibody. Humanized antibodies are useful as therapeutic molecules to reduce or eliminate human immune responses to non-human antibodies (such as human anti-mouse antibody (HAMA) responses), which can cause an immune response to the therapeutic antibody and lead to a reduction in therapeutic efficacy.

[0149] Antibodies can be humanized by any method. Non-limiting and exemplary methods of humanization include, for example, those described in U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-27 (1988); Verhoeyen et al., Science 239: 1534-36 (1988); and U.S. Patent Application Publication No. 2009 / 0136500.

[0150] As described above, a humanized antibody is an antibody in which at least one amino acid in the framework region of the non-human variable region is replaced with an amino acid from the corresponding position in the human framework region. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 15 or at least 20 amino acids in the framework region of the non-human variable region are replaced with amino acids from one or more corresponding positions in one or more human framework regions.

[0151] In some embodiments, some of the corresponding human amino acids used for substitution are from the framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more non-human amino acids are replaced with the corresponding amino acids from the human framework region of a first human antibody or can be encoded by a first human immunoglobulin gene, one or more non-human amino acids are replaced with the corresponding amino acids from the human framework region of a second human antibody or can be encoded by a second human immunoglobulin gene, one or more non-human amino acids are replaced with the corresponding amino acids from the human framework region of a third human antibody or can be encoded by a third human immunoglobulin gene, etc. Further, in some embodiments, not all of the corresponding human amino acids used for substitution in a single framework region, such as FR2, need to be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for substitution are from the same human antibody or are encoded by the same human immunoglobulin gene.

[0152] In some embodiments, the antibody is humanized by replacing the entire one or more framework regions with the corresponding human framework regions. In some embodiments, a human framework region having the highest level of homology to the non-human framework region being replaced is selected. In some embodiments, such humanized antibodies are CDR-grafted antibodies.

[0153] In some embodiments, following CDR grafting, one or more framework amino acids are reverted to the corresponding amino acids of the murine framework region. In some embodiments, such "back mutations" are made to retain one or more murine framework amino acids that appear to contribute to the structure of one or more CDRs and / or may be involved in antigen contact and / or appear to be involved in the overall structural integrity of the antibody. In some embodiments, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or 0 back mutations are made to the framework region of the antibody after CDR grafting.

[0154] In some embodiments, the humanized antibody also comprises a human heavy chain variable region and / or a human light chain variable region.

[0155] Chimeric antibody In some embodiments, the PSGL-1 antibody or VISTA antibody is a chimeric antibody. In some embodiments, the PSGL-1 antibody or VISTA antibody comprises at least one non-human variable region and at least one human constant region. In some such embodiments, all of the variable regions of the PSGL-1 antibody or VISTA antibody are non-human variable regions, and all of the constant regions of the PSGL-1 antibody or VISTA antibody are human constant regions. In some embodiments, one or more regions of the chimeric antibody are mouse variable regions. The human constant region of the chimeric antibody need not be of the same isotype as the non-human constant region (if any) that it replaces. Chimeric antibodies are discussed, for example, in U.S. Patent No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984).

[0156] Human antibody In some embodiments, the PSGL-1 antibody or VISTA antibody is a human antibody. Human antibodies can be made by any suitable method. Non-limiting and exemplary methods include the production of human antibodies in transgenic mice that contain the human immunoglobulin loci. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature 362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,458,06.

[0157] Non-limiting and exemplary methods also include making human antibodies using phage display libraries. See, for example, Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); and International Publication No. 99 / 10494.

[0158] Human antibody constant region In some embodiments, the humanized, chimeric or human antibodies described herein include one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, the antibodies described herein include a human IgG constant region, such as human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody or Fc fusion partner includes, for example, a C237S mutation in the IgG1 constant region. See, for example, SEQ ID NO: 17. In some embodiments, the antibodies described herein include a human IgG2 heavy chain constant region. In some such embodiments, the IgG2 constant region includes a P331S mutation as described in U.S. Patent No. 6,900,292. In some embodiments, the antibodies described herein include a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein include an S241P mutation in the human IgG4 constant region. See, for example, Angal et al. Mol. Immunol. 30(1): 105-108 (1993). In some embodiments, the antibodies described herein include a human IgG4 constant region and a human κ light chain.

[0159] By selecting the heavy chain constant region, it is possible to determine whether the antibody has effector functions in vivo. In some embodiments, such effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can lead to the death of cells to which the antibody is bound. Usually, antibodies containing human IgG1 or IgG3 heavy chains have effector functions.

[0160] In some embodiments, effector functions are not desirable. For example, in some embodiments, effector functions may not be desirable in the treatment of inflammatory conditions and / or autoimmune disorders. In some such embodiments, the human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, the IgG4 constant region contains the S241P mutation.

[0161] Exemplary properties of the antibody Exemplary properties of the PSGL-1 antibody In some embodiments, the PSGL-1 antibody binds to PSGL-1, for example, at an acidic pH, such as pH < 7.0, ≦6.8, ≦6.5 or ≦6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example at pH 6.0, and inhibits PSGL-1-mediated signal transduction. In some embodiments, the PSGL-1 antibody blocks the binding of PSGL-1 to VISTA. In some embodiments, the PSGL-1 antibody blocks the binding of PSGL-1 to VISTA by at least 25%, 50%, 75%, 80%, 90% or 100%. In some embodiments, the PSGL-1 antibody binds to PSGL-1 with a binding affinity (Kd) of less than 50 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In some embodiments, the degree of binding of the PSGL-1 antibody to an irrelevant non-PSGL-1 protein is less than about 10% of the binding of the antibody to PSGL-1, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the PSGL-1 antibody binds to an epitope of PSGL-1 that is conserved among PSGL-1s from different species. In some embodiments, the PSGL-1 antibody binds to the same epitope as a human or humanized PSGL-1 antibody that binds to humPSGL-1.

[0162] Exemplary properties of VISTA antibodies In some embodiments, the VISTA antibody binds to VISTA, for example, at an acidic pH, such as pH < 7.0, ≦ 6.8, ≦ 6.5 or ≦ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example, at pH 6.0, and inhibits PSGL-1-mediated signaling. In some embodiments, the VISTA antibody blocks the binding of PSGL-1 to VISTA. In some embodiments, the VISTA antibody blocks the binding of PSGL-1 to VISTA by at least 25%, 50%, 75%, 80%, 90% or 100%. In some embodiments, the VISTA antibody binds to VISTA with a binding affinity (Kd) of less than 50 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In some embodiments, the degree of binding of the VISTA antibody to an irrelevant non-VISTA protein is less than about 10% of the binding of the antibody to VISTA, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the VISTA antibody binds to an epitope of VISTA that is conserved among VISTAs from different species. In some embodiments, the VISTA antibody binds to the same epitope as a human or humanized VISTA antibody that binds to human VISTA.

[0163] Antibody conjugate In some embodiments, the PSGL-1 or VISTA antibody is conjugated to a label. As used herein, a label is a moiety that facilitates the detection of the antibody and / or the detection of the molecule to which the antibody binds. Non-limiting and exemplary labels include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin tags, metal-binding tags, and the like. One of ordinary skill in the art can select an appropriate label according to the intended use in the art.

[0164] In some embodiments, chemical methods are used in vitro to conjugate a label to an antibody. Non-limiting and exemplary methods of conjugation are known in the art and include, for example, commonly available services, methods, and / or reagents from Thermo Scientific Life Science Research Produces (formerly Pierce; Rockford, Illinois), Prozyme (Hayward, California), SACRI Antibody Services (Calgary, Canada), AbD Serotec (Raleigh, North Carolina), and the like. In some embodiments, where the label is a polypeptide, the label can be expressed from the same expression vector having at least one antibody chain to produce a polypeptide comprising the label fused to the antibody chain.

[0165] PSGL-1 and VISTA ECD, ECD fusion molecules, and small peptides In some embodiments, a PSGL-1 antagonist is a PSGL-1 polypeptide that inhibits the binding of PSGL-1 to VISTA, such as full-length PSGL-1 or a fragment of PSGL-1. In some embodiments, the PSGL-1 antagonist is the extracellular domain (ECD) of PSGL-1. In some embodiments, the PSGL-1 antagonist is the full-length PSGL-1 ECD. In some embodiments, the PSGL-1 ECD is a PSGL-1 ECD fragment that comprises, for example, at least 80%, at least 85%, at least 90%, or at least 95% of the full-length PSGL-1 ECD amino acid sequence from which it is derived. In some embodiments, the PSGL-1 ECD is a PSGL-1 ECD variant that comprises, for example, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the full-length PSGL-1 ECD from which it is derived. In other embodiments, the PSGL-1 ECD is from a non-human PSGL-1 ECD and can be either full-length, a fragment, or a variant.

[0166] In some embodiments, PSGL-1 or a PSGL-1 fragment is combined with at least one fusion partner. Thus, in some such embodiments, a PSGL-1 antagonist can include a full-length PSGL-1 ECD and at least one fusion partner to form a PSGL-1 ECD fusion molecule. In some embodiments, the PSGL-1 ECD portion of the fusion molecule is a PSGL-1 ECD fragment that includes, for example, at least 80%, at least 85%, at least 90% or at least 95% of the full-length PSGL-1 ECD amino acid sequence from which it is derived. In some embodiments, the PSGL-1 ECD portion of the fusion molecule is a PSGL-1 ECD variant that has, for example, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with the full-length PSGL-1 ECD from which it is derived. In other embodiments, the PSGL-1 ECD component is from a non-human PSGL-1 ECD and can be any of full-length, fragment or variant. In any of the above fusion molecule embodiments, the fusion partner can be an immunoglobulin Fc molecule (e.g., a human Fc molecule), or in some embodiments can include an Fc having a sequence selected from SEQ ID NOs: 11-13. In other embodiments, the fusion partner can be a different molecule such as albumin or polyethylene glycol (PEG). In some embodiments, one or more fusion partners can be bound to the PSGL-1 ECD. In some embodiments, the fusion partner(s) is bound to the C-terminus of the ECD, although other bindings are possible, such as on an amino acid side chain or at the N-terminus. The binding of the fusion partner to the PSGL-1 ECD can be direct (i.e., by a covalent bond) or indirect through a linker. The linker can play a role of linking the fusion partner to the ECD by either a covalent or non-covalent bond and can include, for example, at least one intervening amino acid or other chemical moiety.

[0167] In any of the above embodiments, the PSGL-1 polypeptide is either in a form that includes a signal sequence or is in its mature form (i.e., does not include a signal sequence). The signal sequence can be from the native PSGL-1 molecule or from a different protein, such as a protein selected to enhance the expression of the PSGL-1 polypeptide in cell culture.

[0168] In some embodiments, the PSGL-1 ECD can include the amino acid sequence 1-241 of SEQ ID NO: 1 or the amino acid sequence 1-241 of SEQ ID NO: 14 (human isoforms 1 and 2 including the signal sequence, respectively). In other embodiments, the PSGL-1 ECD can include the amino acid sequence 23-241 of SEQ ID NO: 1 or the amino acid sequence 23-241 of SEQ ID NO: 14 or the amino acid sequence 1-219 of SEQ ID NO: 2 or the amino acid sequence 1-219 of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 16 or SEQ ID NO: 17 (mature forms of human isoforms 1 and 2 without the signal sequence) or the amino acid sequence of SEQ ID NO: 18 (another exemplary ECD sequence). In some embodiments, the PSGL-1 ECD can consist of one of the above amino acid sequences. In any of the above cases, the PSGL-1 ECD can be part of a fusion molecule such that the above amino acid sequences can be directly or indirectly, via a linker such as Fc, albumin or PEG, bound to a fusion partner. For example, in some embodiments where the antagonist is a PSGL-1 ECD fusion molecule, the fusion molecule can include at least one of SEQ ID NOs: 11-13 (immunoglobulin Fc sequences) or Fc from human IgG1 in addition to one of the above sequences. The PSGL-1 ECD Fc fusion molecule can be formed by direct binding of the Fc amino acid sequence to the PSGL-1 ECD amino acid sequence or via a linker (either intervening amino acids or an amino acid sequence or another chemical moiety). For example, SEQ ID NO: 19 provides a linker used in the PSGL-1 Fc molecule used in the examples described below. Additional PSGL-1 ECD Fc fusion molecules are described in T. Pouyani et al., Cell 83: 333-343 (1995).

[0169] In some embodiments, the PSGL-1 antagonist is a VISTA polypeptide, such as a full-length VISTA or a fragment of VISTA, that inhibits the interaction between VISTA and PSGL-1. For example, in some embodiments, the PSGL-1 antagonist is a full-length VISTA polypeptide. In some embodiments, the PSGL-1 antagonist is the VISTA extracellular domain (ECD). In some embodiments, the PSGL-1 antagonist is the full-length human VISTA ECD. In some embodiments, the VISTA ECD can be a VISTA ECD fragment that comprises, for example, at least 80%, at least 85%, at least 90% or at least 95% of the full-length VISTA ECD amino acid sequence from which it is derived. In some embodiments, the VISTA ECD is a VISTA ECD variant that comprises, for example, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with the full-length VISTA ECD from which it is derived. In other embodiments, the VISTA ECD is from a non-human VISTA ECD and can be any of full-length, fragment or variant.

[0170] In some embodiments, VISTA or a VISTA fragment is combined with at least one fusion partner. Thus, in some such embodiments, a PSGL-1 antagonist can include a full-length VISTA ECD and at least one fusion partner to form a VISTA ECD fusion molecule. In some embodiments, the VISTA ECD portion of the fusion molecule is a VISTA ECD fragment that includes, for example, at least 80%, at least 85%, at least 90% or at least 95% of the full-length VISTA ECD amino acid sequence from which it is derived. In some embodiments, the VISTA ECD portion of the fusion molecule is a VISTA ECD variant that has, for example, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with the full-length VISTA ECD from which it is derived. In other embodiments, the VISTA ECD component is from a non-human VISTA ECD and can be either full-length, a fragment or a variant. In any of the above-described fusion molecule embodiments, the fusion partner can include an immunoglobulin Fc molecule (e.g., a human Fc molecule) having a sequence selected from SEQ ID NOs: 11-13 in some embodiments. In other embodiments, the fusion partner can be a different molecule such as albumin or polyethylene glycol (PEG). In some embodiments, one or more fusion partners can be bound to the VISTA ECD. In some embodiments, the fusion partner(s) is bound to the C-terminus of the ECD, although other bindings are possible, such as on an amino acid side chain or at the N-terminus. The binding of the fusion partner to the VISTA ECD can be direct (i.e., by a covalent bond) or indirect via a linker. The linker can play a role in linking the fusion partner to the ECD either by a covalent or non-covalent bond and can include, for example, at least one intervening amino acid or other chemical moiety.

[0171] In any of the above embodiments, the VISTA polypeptide is either in a form that includes a signal sequence or in a mature form (i.e., does not include a signal sequence). The signal sequence can be from the native VISTA molecule or from a different protein, such as a protein selected to enhance the expression of the VISTA polypeptide in cell culture.

[0172] In some embodiments, the VISTA ECD can include the amino acid sequence 1 - 202 of SEQ ID NO: 10. In some embodiments, the VISTA ECD may consist of the above amino acid sequence. In any of the above cases, the VISTA ECD may be part of a fusion molecule such that the above amino acid sequence can be directly or indirectly linked to a fusion partner via a linker such as Fc, albumin, or PEG. For example, in some embodiments where the antagonist is a VISTA ECD fusion molecule, the fusion molecule may include at least 1 of SEQ ID NOs: 11 - 13 (immunoglobulin Fc sequence) in addition to the amino acid sequence 1 - 202 of SEQ ID NO: 10, or the entire fusion molecule may include or consist of the amino acid sequence of SEQ ID NO: 9 (exemplary VISTA ECD Fc fusion protein). The VISTA ECD Fc fusion molecule can be formed by direct binding of the Fc amino acid sequence to the VISTA ECD amino acid sequence or via a linker (either intervening amino acids or an amino acid sequence or any other chemical moiety).

[0173] In some embodiments, the PSGL-1 antagonist may be a small molecule or a peptide, such as a small peptide. In some embodiments, the PSGL-1 antagonist may be a small peptide comprising the amino acid sequence of the PSGL-1 ECD fragment. In some embodiments, the PSGL-1 antagonist may be a small peptide comprising the amino acid sequence of the VISTA ECD fragment. In some embodiments, the PSGL-1 antagonist is a small peptide having, for example, from 3 to 20, such as from 3 to 15 or from 3 to 10 amino acids, which peptide may be linear or cyclic and comprises a sequence comprising a PSGL-1 fragment, a PSGL-1 ECD fragment, a VISTA fragment or a VISTA ECD fragment, or a variant of a PSGL-1 fragment, a PSGL-1 ECD fragment, a VISTA fragment or a VISTA ECD fragment. Such variants of the PSGL-1 or VISTA fragment may have, for example, at least 95%, at least 97%, at least 99% sequence identity to the native fragment sequence from which it is derived.

[0174] Signal peptide For some secreted proteins to be expressed and secreted in large amounts, a signal peptide from a heterologous protein may be desirable. Using a heterologous signal peptide can be advantageous in that the resulting mature polypeptide remains unchanged since the signal peptide is removed in the ER during the secretion process. In some cases, the addition of a heterologous signal peptide may be required to express and secrete some proteins.

[0175] Non-limiting and exemplary signal peptide sequences are described, for example, in the online Signal Peptide Database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al., BMC Bioinformatics, 6: 249 (2005); and WO 2006 / 081430.

[0176] Cotranslational modification and post-translational modification In some embodiments, the PSGL-1 or VISTA antibody or polypeptide such as PSGL-1 or VISTA ECD is differentially modified during or after translation, for example, by glycosylation, sialylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or conjugation to an antibody molecule or other cell ligand. Any of a plurality of chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease; NaBH4; acetylation; formylation; oxidation; reduction; and / or metabolic synthesis in the presence of tunicamycin.

[0177] Additional post-translational modifications included within the present invention include, for example, N-linked or O-linked sugar chains; N-terminal or C-terminal processing; attachment of chemical moieties to the amino acid backbone; chemical modification of N-linked or O-linked sugar chains; and addition or removal of N-terminal methionine residues as a result of cellular expression.

[0178] Nucleic acid molecule encoding a PSGL-1 antagonist There is provided a nucleic acid molecule that comprises a polynucleotide encoding one or more chains of an antibody described herein, such as a PSGL-1 or VISTA antibody. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy chain or the light chain of an antibody described herein. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0179] In some embodiments, the heavy and light chains are expressed as two separate polypeptides, from one nucleic acid molecule, or from two separate nucleic acid molecules. In some embodiments, such as when the antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0180] In some embodiments, the polynucleotide encoding the heavy or light chain of the antibody described herein includes a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain when translated. As described above, the leader sequence may be the leader sequence of the native heavy or light chain or another heterologous leader sequence.

[0181] Nucleic acids encoding other PSGL-1 antagonists are also provided, such as fragments or variants of PSGL-1 that include a PSGL-1 ECD molecule or a PSGL-1 ECD fusion molecule, and fragments or variants of VISTA that include a VISTA ECD molecule or a VISTA ECD fusion molecule. The nucleic acid molecule can be constructed using recombinant DNA techniques common in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0182] Expression and production of polypeptides Vector Vectors are provided that include a polynucleotide encoding the heavy chain and / or light chain of the antibody described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector includes a first polynucleotide sequence encoding the heavy chain and a second polynucleotide sequence encoding the light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is an scFv.

[0183] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into a host cell in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a molar ratio or mass ratio between 5:1 and 1:5 of the first vector to the second vector is transfected into the host cell. In some embodiments, a mass ratio between 1:1 and 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0184] In some embodiments, a vector optimized for the expression of a polypeptide in CHO or CHO-derived cells or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0185] In some embodiments, a vector is selected for the in vivo expression of a PSGL-1 antagonist in an animal including a human. In some such embodiments, the polypeptide or the expression of the polypeptide is under the control of a promoter (s) that functions tissue-specifically. For example, a liver-specific promoter is described, for example, in WO 2006 / 076288.

[0186] host cell In various embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in prokaryotic cells such as bacterial cells, or in eukaryotic cells such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include COS cells including COS 7 cells; 293 cells including 293-6E cells; CHO cells including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells, but are not limited thereto. In some embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on the heavy and / or light chains of a PSGL-1 or VISTA antibody. For example, in some embodiments, CHO cells produce polypeptides with a higher level of sialylation than the same polypeptides produced in 293 cells.

[0187] Introduction of the desired host cell(s) with one or more nucleic acids can be accomplished by any method including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting and exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acids can be transfected transiently or stably into the desired host cell according to any suitable method.

[0188] In some embodiments, one or more polypeptides can be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptide according to any suitable method.

[0189] Purification of the Polypeptide The antibodies described herein can be purified according to any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and / or epitope to which the antibody binds, and ligands that bind to the antibody constant region. For example, Protein A, Protein G, Protein A / G, or an antibody affinity column can be used to bind to the constant region and purify the antibody.

[0190] In some embodiments, hydrophobic interaction chromatography, such as a butyl or phenyl column, is also used to purify some polypeptides. Many methods for purifying polypeptides are known in the art.

[0191] Cell-Free Production of the Polypeptide In some embodiments, the antibodies described herein are produced in a cell-free system. Non-limiting and exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv.21: 695-713 (2003).

[0192] Methods for Identifying PSGL-1 Antagonists In some embodiments, methods for identifying a PSGL-1 antagonist are provided. In some embodiments, the method comprises contacting a candidate molecule (i.e., a molecule being tested for antagonist activity) with VISTA, VISTA ECD or a VISTA ECD fusion molecule (collectively referred to as "VISTA molecules") at, for example, an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In some embodiments, the method further comprises contacting the candidate molecule / VISTA molecule mixture with PSGL-1, PSGL-1 ECD or a PSGL-1 ECD fusion molecule (collectively referred to as "PSGL-1 molecules"). In some embodiments, the method comprises contacting the candidate molecule with the PSGL-1 molecule at, for example, an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0, and then contacting the candidate molecule / PSGL-1 molecule mixture with the VISTA molecule. In some embodiments, the method comprises contacting the candidate molecule with the VISTA molecule and the PSGL-1 molecule substantially simultaneously. In some embodiments, the method comprises forming a first composition comprising the VISTA molecule and the PSGL-1 molecule at, for example, an acidic pH, such as pH < 7.0, ≤ 6.8, ≤ 6.5 or ≤ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0, and then contacting the candidate molecule with the first composition. One of ordinary skill in the art will recognize that the order in which the components contact each other can vary depending on the assay design. In some embodiments, the contact of the VISTA molecule, the PSGL-1 molecule and the candidate molecule occurs at an acidic pH, or at a pH of less than 8.0, less than 7.0, less than 6.5 or less than 6.0, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. The pH of the composition can be pH 5.0 - pH 8.0, pH 5.5 - pH 7.0, pH 6.0 - pH 8 or pH 6.5 - pH 8.

[0193] In some embodiments, the VISTA molecule is full-length VISTA, such as VISTA expressed on the surface of a cell. In some embodiments, the VISTA molecule is a soluble VISTA, such as VISTA ECD or a VISTA ECD fusion molecule. In some embodiments, the PSGL-1 molecule is full-length PSGL-1, such as PSGL-1 expressed on the surface of a cell. In some embodiments, the PSGL-1 molecule is a soluble PSGL-1, such as PSGL-1 ECD or a PSGL-1 ECD fusion molecule.

[0194] In some embodiments, after contacting a candidate molecule with a VISTA molecule and / or a PSGL-1 molecule, one or more assays are performed to detect PSGL-1 molecules that bind to the VISTA molecule. Non-limiting and exemplary assays for detecting PSGL-1 molecules that bind to the VISTA molecule include ELISA assays, surface plasmon resonance assays (e.g., Biacore®), flow cytometry-based assays (e.g., where one or more components are bound to beads or expressed on the surface of cells), amplified luminescent proximity homogeneous assay (ALPHA), and the like. Methods for detecting protein-protein binding are known in the art, and those skilled in the art can select an appropriate assay method. Additionally, various reagents, such as antibodies (labeled or unlabeled), secondary antibodies (labeled or unlabeled), labeled assay components (including but not limited to labeled PSGL-1 molecules and / or labeled VISTA molecules), etc., can be used for detection as needed.

[0195] In some embodiments, a method of identifying a PSGL-1 antagonist comprises comparing the degree of VISTA molecule / PSGL-1 molecule binding in the presence and absence of a candidate molecule, for example, at an acidic pH, such as pH < 7.0, ≦ 6.8, ≦ 6.5 or ≦ 6.3, or pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, such as pH 6.0. In some embodiments, if the binding of the VISTA molecule / PSGL-1 molecule in the presence of the candidate molecule is decreased compared to the binding in the absence of the candidate molecule, the candidate molecule is a PSGL-1 antagonist. In some embodiments, the binding between the VISTA molecule and the PSGL-1 molecule is decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the presence of the candidate molecule. In some such embodiments, the candidate molecule is a PSGL-1 antagonist.

[0196] Exemplary types of candidate molecules include, but are not limited to, antibodies, peptides, small molecules, and aptamers. In some embodiments, the candidate molecule is an antibody known to bind to VISTA (i.e., a VISTA antibody). In some embodiments, the candidate molecule is an antibody known to bind to PSGL-1 (i.e., a PSGL-1 antibody).

[0197] In some embodiments, a method for determining whether a VISTA antibody is a PSGL-1 antagonist is provided. In such embodiments, the VISTA antibody is tested in the above assay as a candidate molecule. In some embodiments, for example, at an acidic pH, such as pH < 7.0, ≦ 6.8, ≦ 6.5 or ≦ 6.3, or at pH 5.5 - 6.5, 6.0 - 6.5, 6.5 - 7.0 or 6.0 - 7.0, for example at pH 6.0, a method for determining whether the VISTA antibody blocks the binding of PSGL-1 to VISTA is provided. In some embodiments, the method includes contacting the VISTA antibody with a VISTA molecule and a PSGL-1 molecule and detecting the binding of the VISTA molecule to the PSGL-1 molecule in the presence of the antibody, for example as described above and herein.

[0198] Manufactured article In some embodiments, provided are articles or kits that include materials useful for detecting a biomarker (e.g., PSGL-1 or VISTA) or treating, preventing, and / or diagnosing the above disorders. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from various materials such as glass or plastic. In some embodiments, the container can contain a composition that is effective for treating, preventing, and / or diagnosing a condition and can be used in combination with a single or other composition, and can have a sterile access port (e.g., the container can be a bag or vial of intravenous solution having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is to be used for treating an alternative condition. In some embodiments, the article may include (a) a first container containing a composition comprising a PSGL-1 antagonist of the present invention, and (b) a second container containing a composition comprising an additional cytotoxic agent. The article may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0199] In some embodiments, the molecules of the invention can be packaged alone or in combination with other therapeutic compounds as a kit. In one embodiment, the therapeutic compound is an anti-cancer agent. In another embodiment, the therapeutic compound is an immunosuppressive agent. The kit can include optional components that assist in the administration of unit doses to a patient, such as vials for reconstituting the powder form, syringes for injection, customized IV delivery systems, inhalers, etc. Further, the unit dose kit can include instructions for the preparation and administration of the composition. The kit can be manufactured as a single-use unit dose for one patient or as multiple uses (either at a fixed dose or where the efficacy of the individual compounds may vary as the treatment progresses) for a particular patient, or the kit can contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The components of the kit can be assembled in a carton, blister pack, bottle, tube, etc.

Example

[0200] The following examples are intended to merely illustrate the invention and should in no way be considered to limit the invention. The examples do not represent all or the only experiments in which the following experiments were conducted. Efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), but some experimental error and deviation need to be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0201] Example 1: VISTA ECD is histidine-rich and VISTA dextramer selectively binds to leukocytes at acidic pH The frequency of histidine residues within the extracellular domain of immunoglobulin receptor superfamily (IgSF) members was analyzed, and it was found that VISTA is highly histidine-rich compared to other receptors or ligands.

[0202] PBMC CD4+ T cells were isolated from blood using RosetteSep® (Stem Cell Technologies) and stimulated with anti-CD3 / CD28 Dynabeads® (ThermoFisher) at a 1:1 ratio and recombinant human IL-2 for 3 - 4 days. After stimulation, the CD4+ T cells were washed and then incubated with a fluorescent-conjugated human VISTA dextramer (streptavidin dextramer loaded with optimally molar ratio of monobiotinylated recombinant human VISTA molecules) at a pH ranging from 6.97 to 5.99. Binding was detected by flow cytometry.

[0203] The results, shown in Figure 1, demonstrate that VISTA selectively binds to T cells at acidic pH rather than neutral pH.

[0204] Example 2: Identification of PSGL-1 as a VISTA counter-receptor at acidic pH LRC-TriCEPS TM The LRC-TriCEPS technology was used to identify the VISTA receptor on T cells (Frei et al. (2013) Nat. Protoc. 8:1321; Frei and Jeon (2012) Nat. Biotechnol. 30:997 and Omasits et al. (2014) Bioinformatics 15:884). Human VISTA-Fc was conjugated to TriCEPS and incubated on primary human T cells at pH 6.0. Anti-CD3 was also conjugated to the TriCEPS reagent and served as a positive control. Several proteins were identified by screening, some of which appeared to bind only to the Fc portion of VISTA-Fc. Human PSGL-1 was identified as a protein that binds to human VISTA. The screening experiment was repeated once more under the same conditions, and PSGL-1 was identified again.

[0205] P-selectin glycoprotein ligand 1 (PSGL-1 or SELPL) is a ligand for P-, E-, and L-selectins, is highly glycosylated and tyrosine-sulfated, and is expressed by most leukocytes, including T cells. The involvement of PSGL-1 of selectins is essential for extravasation and intracellular trafficking of leukocytes.

[0206] Example 3: PSGL-1 expression correlates with VISTA dextramer binding PBMCs were isolated from blood by ficoll-paque gradient centrifugation, washed, and then incubated in Hank's balanced salt solution (HBSS) at pH 7.2 with a fluorescent-conjugated anti-PSGL-1 antibody or at pH 6.0 with a fluorescent-conjugated human VISTA dextramer (streptavidin dextramer loaded with an optimal molar ratio of monobiotinylated recombinant human VISTA molecules). Binding was detected by flow cytometry.

[0207] The results, shown in Figures 2A - B, show that PSGL-1 surface expression on PBMC lymphocytes correlates with VISTA dextramer binding at acidic pH.

[0208] Example 4: Soluble PSGL-1 and P-selectin block the binding of VISTA dextramer to activated CD4+ T cells The ability of soluble PSGL-1 to interfere with the binding of VISTA dextramer to T cells was evaluated by incubating activated T cells with a fluorescent-conjugated VISTA dextramer (streptavidin dextramer loaded with a sub-optimal molar ratio of monobiotinylated recombinant human VISTA molecule) in Hank's balanced salt solution at pH 6.0 in the presence of titrated concentrations of a control (i.e., non-VISTA or PSGL-1 specific) antibody (dots), recombinant human PSGL-1-Fc fusion protein (triangles) or recombinant human P-selectin (R&D Systems, diamonds). The recombinant human PSGL-1 Fc fusion molecule was obtained from R&D Systems, catalog number 3345-PS and contains the PSGL-1 ECD sequence shown in SEQ ID NO: 18, which is the amino acid positions 42-295 of the human PSGL-1 sequence of deposit number AA50061 linked at the C-terminus to an IEGRMD linker sequence (SEQ ID NO: 19), followed by amino acids P100-K330 of human IgG1. After incubation, the binding of the VISTA dextramer to T cells was measured by flow cytometry. IC50 values were calculated by non-linear regression using Prism software (GraphPad). The results are shown in Figure 3 and Table 1, which show that PSGL-1 and P-selectin inhibit the binding of VISTA to activated CD4+ T cells in a dose-dependent manner at acidic pH. Table 1 TIFF2025108413000002.tif39170

[0209] Example 5: PSGL-1-Fc binds to VISTA expressed on the cell surface at acidic pH The binding of humPSGL-1-Fc to 293T cells that ectopically express human VISTA was tested. 293T cells expressing human VISTA and GFP (dots and squares) and 293T cells that do not express either VISTA or GFP (diamonds and triangles) were cultured. These cells were washed and then incubated with the PSGL-1-Fc fusion protein (R&D Systems; catalog number 3345) in Hank's balanced salt solution at pH 7.2 (triangles and squares) or pH 6.0 (diamonds and dotted curve). After the primary incubation, the cells were washed and incubated with a fluorescent-conjugated anti-hIgG secondary detection antibody. After the secondary incubation, the cells were washed and binding was detected by flow cytometry. The results, shown in FIGS. 4A and B, indicate that humPSGL-1-Fc binds to 293T cells that express human VISTA (hVISTA) but not to 293T cells that do not express human VISTA. Furthermore, this result shows that the binding of PSGL-1-Fc is pH-dependent, as it binds at pH 6.0 (circles / dots in FIG. 4B and right curve in FIG. 4A) but not as much at pH 7.2 (squares in FIG. 4B and left curve in FIG. 4A).

[0210] Example 6: PBMC CD4 T cell PSGL-1 CRISPR Removes VISTA Dextramer Binding Human CD4 T cells were isolated from whole blood, seeded onto plates, and activated with OKT3 and CD28.2 coated for 2 days. Thereafter, the T cells were transfected with Cas9 ribonucleoprotein (RNP) loaded with guide RNA targeting either CD4, PSGL1, or a gRNA with no human sequence homology (non-targeting control). Transfection was performed in triplicate. After transfection, the cells were re-activated with CD3 / CD28 coated Dynabeads® for 4 days. Thereafter, the cells were stained with dextramers loaded with recombinant human avi-tagged VISTA to evaluate binding to VISTA. The percentage of maximum VISTA binding was determined by dividing the mean fluorescence intensity (MFI) of the VISTA dextramer of the knockout population by the mean ISTA dextramer MFI of the non-targeting control.

[0211] The results, shown in Figure 5, indicate that in T cells with PSGL-1 removed, VISTA binding to T cells decreased by approximately half. Furthermore, these results suggest that PSGL-1 is a VISTA counter-receptor on T cells.

[0212] [Sequence Listing] TIFF2025108413000003.tif248170TIFF2025108413000004.tif254170TIFF2025108413000005.tif255170TIFF2025108413000006.tif255170TIFF2025108413000007.tif255170TIFF2025108413000008.tif117170

Claims

1. A method of treating cancer, comprising administering to a subject having cancer an effective amount of at least one PSGL-1 antagonist.

2. The method of claim 1, further comprising administering to the subject an effective amount of a therapeutic agent selected from the group consisting of chemotherapeutic agents, anti-angiogenic agents, growth inhibitors, immuno-oncology agents, and anti-tumor compositions.

3. The method of claim 1 or 2, wherein the PSGL-1 antagonist is selected from a PSGL-1 antibody and a VISTA antibody.

4. The method of claim 3, wherein the PSGL-1 antagonist is an antibody that binds to PSGL-1 (e.g., to the PSGL-1 ECD).

5. The method of claim 3, wherein the PSGL-1 antagonist is an antibody that binds to VISTA (e.g., to the VISTA ECD).

6. The method according to any one of claims 3 to 5, wherein the antibody is selected from chimeric antibodies, humanized antibodies, and human antibodies.

7. The method according to any one of claims 3 to 6, wherein the antibody is an antibody fragment.

8. The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', and (Fab') ２ The method according to claim 7, wherein the antibody fragment is selected from the group consisting of Fv, single-chain Fv (scFv), Fab, Fab', and (Fab').

9. The method according to any one of claims 1 to 8, wherein the PSGL-1 antagonist blocks the binding of PSGL-1 to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (e.g., pH 6.0).

10. The method according to any one of claims 1 to 9, wherein the PSGL-1 antagonist is an antibody that binds to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (e.g., pH 6.0), or is an antibody that binds to PSGL-1 at a pH in the range of, for example, pH 5.5 to pH 6.5 (e.g., pH 6.0).

11. The antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less. Ｄ The method according to any one of claims 1 to 10, wherein the antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less.

12. The method according to any one of claims 1 to 11, wherein the antagonist is an immune-stimulatory molecule.

13. The method of claim 12, wherein the antagonist stimulates T cell activation.

14. Use of a PSGL-1 antagonist for treating cancer in a subject.

15. The use of claim 14, wherein the PSGL-1 antagonist is selected from a PSGL-1 antibody and a VISTA antibody.

16. The use of claim 15, wherein the PSGL-1 antagonist is an antibody that binds to PSGL-1 (e.g., to the PSGL-1 ECD).

17. The use of claim 15, wherein the PSGL-1 antagonist is an antibody that binds to VISTA (e.g., to the VISTA ECD).

18. The use according to any one of claims 15 to 17, wherein the antibody is selected from a chimeric antibody, a humanized antibody, and a human antibody.

19. The use according to any one of claims 15 to 18, wherein the antibody is an antibody fragment.

20. The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', and (Fab') ２ The use according to claim 19, wherein the antibody fragment is selected from the group consisting of Fv, single-chain Fv (scFv), Fab, Fab', and (Fab').

21. The use according to any one of claims 14 to 20, wherein the PSGL-1 antagonist blocks the binding of PSGL-1 to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0).

22. The use according to any one of claims 15 to 21, wherein the PSGL-1 antagonist is an antibody that binds to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0), or an antibody that binds to PSGL-1 at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0).

23. The antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less. Ｄ The use according to any one of claims 15 to 22, wherein the antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less.

24. The use according to any one of claims 14 to 23, wherein the antagonist is an immune stimulatory molecule.

25. The use according to claim 24, wherein the antagonist stimulates T cell activation.

26. A method for inhibiting the binding of PSGL-1 to VISTA in a subject, comprising administering to the subject at least one PSGL-1 antagonist.

27. The method according to claim 26, further comprising administering to the subject an effective amount of a therapeutic agent selected from a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitor, an immuno-oncology agent, and an anti-tumor composition.

28. The method according to claim 26 or 27, wherein the PSGL-1 antagonist is selected from a PSGL-1 antibody and a VISTA antibody.

29. The method according to claim 28, wherein the PSGL-1 antagonist is an antibody that binds to PSGL-1 (for example, to the PSGL-1 ECD).

30. The method according to claim 28, wherein the PSGL-1 antagonist is an antibody that binds to VISTA (for example, to the VISTA ECD).

31. The method according to any one of claims 28 to 30, wherein the antibody is selected from a chimeric antibody, a humanized antibody, and a human antibody.

32. The method according to any one of claims 28 to 31, wherein the antibody is an antibody fragment.

33. The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab’, and (Fab’) ２ The method according to claim 32, wherein the antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab’, and (Fab’).

34. The method according to any one of claims 26 to 33, wherein the PSGL-1 antagonist blocks the binding of PSGL-1 to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0).

35. The method according to any one of claims 26 to 34, wherein the PSGL-1 antagonist is an antibody that binds to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0), or an antibody that binds to PSGL-1 at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0).

36. The antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less. Ｄ The method according to any one of claims 26 to 35, wherein the antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less.

37. The method according to any one of claims 26 to 36, wherein the antagonist is an immune-stimulatory molecule.

38. The method according to claim 37, wherein the antagonist stimulates T cell activation.

39. A method for inhibiting the binding of PSGL-1 on a cell to VISTA, the method comprising contacting the cell with at least one PSGL-1 antagonist.

40. The method according to claim 27, wherein the cell is in vitro.

41. The method according to claim 39 or 40, wherein the PSGL-1 antagonist is selected from a PSGL-1 antibody and a VISTA antibody.

42. The method according to claim 41, wherein the PSGL-1 antagonist is an antibody that binds to PSGL-1 (for example, to the PSGL-1 ECD).

43. The method according to claim 41, wherein the PSGL-1 antagonist is an antibody that binds to VISTA (for example, to the VISTA ECD).

44. The method according to any one of claims 41 to 43, wherein the antibody is selected from a chimeric antibody, a humanized antibody, and a human antibody.

45. The method according to any one of claims 41 to 44, wherein the antibody is an antibody fragment.

46. The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', and (Fab') ２ The method according to claim 45, wherein the antibody fragment is selected from the group consisting of Fv, single-chain Fv (scFv), Fab, Fab', and (Fab').

47. The method according to any one of claims 39 to 46, wherein the PSGL-1 antagonist blocks the binding of PSGL-1 to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0).

48. The method according to any one of claims 39 to 47, wherein the PSGL-1 antagonist is an antibody that binds to VISTA at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0), or an antibody that binds to PSGL-1 at a pH in the range of, for example, pH 5.5 to pH 6.5 (for example, pH 6.0).

49. The antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less. Ｄ The method according to any one of claims 39 to 48, wherein the antagonist is an antibody that binds to VISTA or PSGL-1 at a K of 10 nM or less.

50. The method according to any one of claims 39 to 49, wherein the antagonist is an immune-stimulatory molecule.

51. The method according to claim 50, wherein the antagonist stimulates T cell activation.

52. A method for identifying a PSGL-1 antagonist, comprising: a) forming a composition comprising a candidate molecule, a VISTA molecule (e.g., a mature full-length VISTA protein or a fragment thereof), and a PSGL-1 molecule (e.g., a mature full-length PSGL-1 protein or a fragment thereof), wherein the VISTA molecule comprises VISTA, VISTA ECD, or a VISTA ECD fusion molecule, and the PSGL-1 molecule comprises PSGL-1, PSGL-1 ECD, or a PSGL-1 ECD fusion molecule; b) detecting the binding of the VISTA molecule to the PSGL-1 molecule; in a method comprising: the composition has a pH in the range of, for example, pH 5.5 to pH 6.5 (e.g., pH 6.0), and a decrease in the binding of the VISTA molecule to the PSGL-1 molecule in the presence of the candidate molecule as compared to the binding of the VISTA molecule to the PSGL-1 molecule in the absence of the candidate molecule indicates that the candidate molecule is a PSGL-1 antagonist; a method.

53. The method according to claim 52, wherein the binding of the VISTA molecule to the PSGL-1 molecule is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% in the presence of the candidate molecule.

54. The method according to claim 52 or 53, wherein the binding of the VISTA molecule to the PSGL-1 molecule is detected by a method selected from surface plasmon resonance, ELISA, amplified luminescence proximity homogeneous assay, and flow cytometry.

55. The method according to any one of claims 52 to 54, wherein the VISTA molecule is expressed on the surface of a cell.

56. The method according to any one of claims 52 to 55, wherein the PSGL-1 molecule is expressed on the surface of a cell.

57. The method according to any one of claims 52 to 56, wherein the PSGL-1 antagonist is an antibody that binds to VISTA.

58. The method according to any one of claims 52 to 56, wherein the PSGL-1 antagonist is an antibody that binds to PSGL-1.

59. The method according to any one of claims 52 to 56, wherein the PSGL-1 antagonist is a small molecule.

60. The method according to any one of claims 52 to 56, wherein the PSGL-1 antagonist is a low molecular weight peptide.

61. A method for determining whether a VISTA antibody is a PSGL-1 antagonist, comprising: a) forming a composition comprising a VISTA antibody, a VISTA molecule (e.g., a mature full-length VISTA protein or a fragment thereof), and a PSGL-1 molecule (e.g., a mature full-length PSGL-1 protein or a fragment thereof), wherein the VISTA molecule comprises VISTA, VISTA ECD, or a VISTA ECD fusion molecule, and the PSGL-1 molecule comprises PSGL-1, PSGL-1 ECD, or a PSGL-1 ECD fusion molecule; b) detecting the binding of the VISTA molecule to the PSGL-1 molecule; in a method comprising: the composition having a pH in the range of, for example, pH 5.5 to pH 6.5 (e.g., pH 6.0), and a decrease in the binding of the VISTA molecule to the PSGL-1 molecule in the presence of a VISTA or PSGL-1 antibody as compared to the binding of the VISTA molecule to the PSGL-1 molecule in the absence of the VISTA or PSGL-1 antibody, respectively, indicates that the VISTA or PSGL-1 antibody is a PSGL-1 antagonist; a method. **Claim 62** The method according to claim 61, wherein the binding of the VISTA molecule to the PSGL-1 molecule is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% in the presence of a candidate molecule. **Claim 63** The method according to claim 61 or 62, wherein the binding of the VISTA molecule to the PSGL-1 molecule is detected by a method selected from surface plasmon resonance, ELISA, amplified luminescence proximity homogeneous assay, and flow cytometry. **Claim 64** The method according to any one of claims 61 to 63, wherein the VISTA molecule is VISTA expressed on the surface of a cell. **Claim 65** The method according to any one of claims 61 to 64, wherein the PSGL-1 molecule is PSGL-1 expressed on the surface of a cell. **Claim 66** A PSGL-1 antagonist that at least partially inhibits the binding of VISTA to PSGL-1, for example, at a pH in the range of pH 5.5 to pH 6.5 (e.g., pH 6.0). **Claim 67** The PSGL-1 antagonist according to claim 66, which inhibits the binding of VISTA to PSGL-1 by at least 90%, for example, at a pH in the range of pH 5.5 to pH 6.5 (e.g., pH 6.0). **Claim 68** The PSGL-1 antagonist according to claim 66 or 67, which is an immune-stimulatory molecule. **Claim 69** The PSGL-1 antagonist according to claim 68, which inhibits T cell activation.

70. The PSGL-1 antagonist according to any one of claims 66 to 69, which is selected from a PSGL-1 antibody and a VISTA antibody.

71. The PSGL-1 antagonist according to claim 70, which is an antibody that binds to PSGL-1 (for example, to the PSGL-1 ECD).

72. The PSGL-1 antagonist according to claim 70, which is an antibody that binds to VISTA (for example, to the VISTA ECD).

73. The PSGL-1 antagonist according to any one of claims 70 to 72, wherein the antibody is selected from a chimeric antibody, a humanized antibody, and a human antibody.

74. The PSGL-1 antagonist according to any one of claims 70 to 73, wherein the antibody is an antibody fragment.

75. The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', and (Fab') ２ The PSGL-1 antagonist according to claim 74, which is selected from

76. A composition comprising the PSGL-1 antagonist according to any one of claims 66 to 75.

77. For example, a composition comprising (a) VISTA, or a PSGL-1 binding portion thereof, and (b) PSGL-1, or a VISTA binding portion thereof, for use in the method according to any one of claims 1 to 65.

78. For example, a complex comprising (a) VISTA, or a PSGL-1 binding portion thereof, and (b) PSGL-1, or a VISTA binding portion thereof, for use in the method according to any one of claims 1 to 65.

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