CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 63/348,118 filed on June 2, 2022, U.S. Provisional Application No. 63/415,107 filed on October 11, 2022 and U.S. Provisional Application No. 63/419,511 filed on October 26, 2022, the contents of all of which are incorporated by reference in their entirety.

SEQUENCE LISTING INFORMATION

This application includes as part of its disclosure a biological sequence listing in the file named "1143260ol05813.xml", created on May 30, 2023, having a size of 782,382 bytes, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a negative checkpoint regulator of immune cells. VISTA has been recognized as a potential mediator of resistance to anti-PD-1 and anti-CTLA-4 immunotherapies in cancer patients. Targeting the VISTA signaling pathway using anti-VISTA antibodies has been suggested as a promising approach for overcoming resistance to current immune checkpoint therapies. However, methods for identifying subjects who will clinically respond to a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor are needed and for reversing resistance to existing checkpoint inhibitor therapies.

OBJECTS OF THE INVENTION

Toward that end it is an object of the present invention to provide methods for identifying subjects who will likely clinically respond to a VISTA antagonist or inhibitor, e.g., a e.g., therapeutic anti-VISTA antibody or anti-VISTA antibody fragment or small molecule VISTA inhibitors such as CA-170, based on the expression of specific biomarkers on immune cells and tumor cells of the subject, e.g., a subject with a solid tumor, e.g., a tumor associated with a non- hematological or hematological cancer.

Also it is an object of the present invention to provide methods for identifying subjects who will or have overcome resistance to current immune checkpoint therapies, e.g., PD-1 and CTLA4 antagonist therapeutics, by the administration of a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor, based on the expression of specific biomarkers on tumor and immune cells of the subject, e.g., a subject with a solid tumor, e.g., a tumor associated with a non-hematological or hematological cancer.

Further it is an object of the present invention to provide methods for evaluating the treatment status and/or developing dosing regimens in subjects who have received a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor based on the expression of specific biomarkers on immune cells and tumor cells of the subject, e.g., a subject with a solid tumor, e.g., a tumor associated with a non- hematological or hematological cancer.

More specifically it is an object of the invention to detect the biomarker status of a tumor in a subject with cancer, wherein the method comprises:

(i) obtaining one or more tumor biopsy samples from a subject with a cancerous solid tumor,

(ii) producing tissue sections from said one or more biopsy samples suitable for use in immunohistochemistry (I HC) analysis;

(iii) analyzing by use of IHC the expression and/or level of expression of specific biomarkers by immune cells and tumor cells in the tissue sections and/or the relative numbers of immune cells and tumor cells which express such biomarkers in the one or more tissue sections derived from said one or more tumor biopsy samples from the subject, and

(iv) based on the biomarker expression results in (iii) (1) determining whether to treat the subject with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor; (2) determining a dosing regimen for treatment of the subject with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor; (3) determining whether treatment of the subject with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor has been clinically effective; and/or (4) determining whether treatment of the subject with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor has reversed and/or alleviated resistance to treatment with another checkpoint antagonist.

In some embodiments the at least one biopsy sample is derived from a malignant cancer, e.g., a non- hematological cancer or hematological cancer or solid tumor obtained from a subject.

In some embodiments the at least one biopsy sample is derived from a non- hematological cancer or hematological cancer associated tumor obtained from a subject which is a carcinoma, lymphoma, blastoma, sarcoma, or leukemia.

In some embodiments the at least one biopsy sample is derived from a malignant non- hematological cancer or hematological cancer or solid cancer obtained from a subject which cancer is optionally selected from squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon or colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, liver or hepatic carcinoma, head and neck cancers, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, T and B- cell lymphomas (including low grade/fol licular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL), NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); leukemias such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL); Hairy cell leukemia, Acute Myeloid Leukemia (AML), Chronic myeloid or myeloblastic leukemia (CML), also known as chronic myelogenous leukemia, Hairy cell leukemia, chronic leukemia; post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), Meigs' syndrome; mesothelioma, multiple myeloma, cancer of the peritoneum, brains cancers such as glioblastoma, vulval cancer, thyroid cancer, by way of example. In exemplary embodiments the at least one biopsy sample is derived from a subject with a NSCLC tumor. In some exemplary embodiments the detected biomarkers include VISTA and at least one other immune cell biomarker, optionally one or more of CD8, CD68, CDllb, CD19, CD4, and CD56 and/or further include PD-L1, PD-1, or PD-L2.

In some embodiments the IHC methods detect the expression of the biomarkers on tissue sections derived from biopsies comprised on different portions of the tumor or different tumors.

In some embodiments the IHC results are used alone or in association with other clinical parameters to decide whether to treat the subject with an antagonist anti-VISTA antibody or antibody fragment.

In some embodiments the IHC results are used alone or in association with other clinical parameters to decide a particular dosing regimen for treating the subject with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor.

In some embodiments the IHC results are used alone or in association with other clinical parameters to decide whether to start or resume treatment with another checkpoint antagonist, optionally a PD-1, PD-L1, PD-L2, CTLA-4 or TIM-3 antagonist, further optionally wherein the other checkpoint antagonist, optionally a PD-1, PD-L1, PD-L2, CTLA-4 or TIM-3 antagonist comprises a small molecule, antibody or fusion protein.

In some embodiments the relative numbers of tumor and/or immune cells in the one or more specimens which express VISTA and at least one other immune cell biomarker, optionally one or more of CDS, CD68, CDllb, CD19, CD4, and CD56 in the sample is determined and used to design a treatment protocol.

In some embodiments the therapeutic anti-VISTA antibody comprises a human or humanized anti-human VISTA antibody or anti-human VISTA antibody.

In some embodiments the therapeutic anti-VISTA antibody comprises CI-8993 or an antibody comprising the same CDRs as CI-8993.

In some embodiments the therapeutic anti-VISTA antibody comprises the same CDRs and/or variable regions as any of the antagonist anti-human VISTA antibodies comprising the sequences in Figure 11A-11JJ.

In some embodiments the subject from which samples are analyzed for biomarker expression has been previously treated with another checkpoint inhibitor antagonist, optionally a small molecule, fusion protein or antibody which blocks or inhibits the activity of a checkpoint inhibitor, optionally PD-1, PD-L1, PD-L2, or CTLA4 or TIM-3.

In some embodiments the invention provides a method for determining whether a subject is clinically responding to treatment with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor comprising detecting the levels of expression of VISTA and at least one other immune cell biomarker, optionally one or more of CDS, CD68, CDllb, CD19, CD4, and CD56 on immune cells in the tumor microenvironment of a subject and/or by detecting the levels of expression of VISTA and/or at least one other checkpoint inhibitor on tumor cells of the same subject, preferably PD-L1 or PD-1, during and/or after treatment with a therapeutic anti-VISTA antibody or anti-VISTA antibody fragment and based on the results of said detection assessing whether the subject is clinically responding to treatment with a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor, optionally wherein the subject is further being treated with another checkpoint inhibitor antagonist, optionally a small molecule, fusion protein or antibody which blocks or inhibits the activity of a checkpoint inhibitor, optionally a PD-1, PD- Ll, PD-L2, CTLA-4 or TIM-3 antagonist.

Also more specifically it is an object of the present invention to provide methods for identifying subjects who will or have overcome resistance to an immune checkpoint therapy, e.g., PD-1 and CTLA4 antagonist therapeutics, by the administration of a VISTA antagonist or inhibitor, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor, by determining the expression of specific biomarkers by immune and tumor cells comprised in one or more tissue samples derived from a solid tumor biopsy specimen of the subject, optionally a human subject with cancer, by the use of immunohistochemistry (IHC), wherein such biomarkers comprise VISTA and other immune cell biomarkers or checkpoint inhibitors, optionally one or more of CDS, CD68, CDllb, CD19, CD4, and CD56, and based on the expression results determining whether the subject will or has overcome resistance to an immune checkpoint therapy, e.g., PD-1 and CTLA4 antagonist therapy.

Also more specifically it is an object of the invention to evaluate the treatment status and/or develop a VISTA antagonist or inhibitor dosing regimen, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor dosing regimen in a subject who will or has received an antagonist anti-VISTA antibody or anti-VISTA antibody fragment based on the expression of specific biomarkers on immune cells and tumor cells of the subject, e.g., a subject with a solid tumor, e.g., a tumor associated with a non- hematological or hematological cancer, by determining the expression of specific biomarkers by immune and tumor cells comprised in one or more tissue samples derived from one or more solid tumor biopsy specimen of the subject, optionally a human subject with cancer, by the use of immunohistochemistry (IHC), wherein such biomarkers comprise VISTA and other immune cell biomarkers or checkpoint inhibitors, optionally one or more of CD8, CD68, CDllb, CD19, CD4, and CD56, and based on the expression results determining the treatment status and/or developing a VISTA antagonist, e.g., a VISTA antibody or CA-170 dosing regimen.

In exemplary embodiments the anti-VISTA antibody used in the subject methods will comprise CI-8993 or an antibody comprising the same CDRs as CI-8993 (said antibody comprising the VH and VL CDR sequences of SEQ ID NO: 25-27 and 28-30 respectively, the VH and VL sequences of SEQ ID NO: 37 and SEQ ID NO 44 respectively, and the heavy chain and light chain in SEQ ID NO: 61 and SEQ ID NO 56 respectively, disclosed in US Patent No. 10,273,301 and PCT/IB2014/002868 both of which are incorporated by reference in their entireties).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 summarizes the expression profile of specific biomarkers (VISTA, CD8 CD68, CDllb, CD4, CD56) on immune cells and checkpoint inhibitors (VISTA and PD-L1 or both) on tumor cells of 10 subjects with non-small cell lung cancer.

Figure 2 contains an immunohistogram of a non-small cell lung cancer biopsy sample of a specific patient (NV-65) doubly stained with anti-VISTA and anti-CD68 antibodies. In the immunohistogram VISTA is detected with DAB label (brown color) and CD68 detected using Permanent Red (red color). VISTA expression on cancer cells at tumor invasion front is shown.

Figure 3 contains an immunohistogram of a non-small cell lung cancer biopsy sample from the same patient (NV-57) doubly stained with anti-VISTA and anti-CD8 antibodies. In the immunohistogram VISTA is detected with DAB label (brown color) and CDS detected using Permanent Red (red color).

Figure 4 contains an immunohistogram of a non-small cell lung cancer biopsy sample from the same patient (NV-57) doubly stained with anti-VISTA and anti-CD68 antibodies. In the immunohistogram VISTA is detected with DAB label (brown color) and CD68 detected using Permanent Red (red color).

Figure 5 contains the immunohistogram of 2 non-small cell lung cancer biopsy samples from 2 patients (NV-61 and NV-66) doubly stained with anti-VISTA and anti-CD68 antibodies. In the immunohistogram VISTA is again detected with DAB label (brown color) and CD68 detected using Permanent Red (red color). The results indicate that VISTA is not expressed by CD68 ⁺ cells in these NSCLC patients.

Figure 6 contains the immunohistogram of a non-small cell lung cancer biopsy sample obtained from a NSCLC patient (NV-62) doubly stained with anti-VISTA and anti-CD4 antibodies. In the immunohistogram VISTA is again detected with DAB label (brown color) and CD4 detected using Permanent Red (red color). The results indicate that VISTA is not expressed by CD4+ cells in this NSCLC patient.

Figure 7 contains immunohistogram of serial section biopsy samples from a NSCLC patient (NV-67) doubly stained with anti-VISTA and anti-CD19 antibodies or anti-VISTA and anti- CDllb antibodies. In the immunohistogram VISTA is again detected with DAB label (brown color) and CD19 and CDllb are detected using Permanent Red (red color). The results show VISTA expression by B cells (CD19 ⁺ ) but not in myeloid cells (CDllb ⁺ ) in this NSCLC patient. Figure 8 contains representative immunohistograms of serial section biopsy samples from a NSCLC patient (NV-64) doubly stained with anti-VISTA and anti-CD8 antibodies, anti-VISTA and anti-CDllb antibodies, anti-VISTA and anti-CD19 antibodies, or anti-VISTA and anti-PD- L1 antibodies. In the immunohistograms VISTA is again detected with DAB label (brown color) and CDS, CD19, CDllb and PD-L1 are each detected using Permanent Red (red color). The results show that the cancer cells of this NSCLC patient express PD-L1 but do not express VISTA and that some immune cells were VISTA positive; more particularly it shows that VISTA was expressed by CD19+ B cells of this patient but was not expressed by CD8+or CDllb+ myeloid cells. Figure 9 contain 2 representative immunohistograms of biopsy samples from 2 different NSCLC patients (NV-61 and NV-65) doubly stained with anti-VISTA and anti-CDllb antibodies. VISTA-positive myeloid cells (CDllb+) can be seen therein.

Figure 10 contain 2 representative immunohistograms of biopsy samples from 2 different NSCLC patients (NV-63 and NV-60) doubly stained with anti-VISTA and anti-PD-Ll antibodies. In both tumors, VISTA expression was not detectable in PD-Ll-positive cancer cells as can be seen therein.

Figure 11A-11JJ contains the CDR and variable sequences of exemplary anti-VISTA antibodies.

Figure 12 shows the average cytokine concentration for CI-8993 pre and post infusion at 0.15 mg/kg (n=7), 0.3 mg/kg (n=5) and 0.6 mg/kg (n=4) dose. (* P<0.05).

Figure 13 shows the average values of immunophenotypes at baseline and 24 hours after CI- 8993 infusion at 0.15 (n=7), 0.3 (n=5) and 0.6 (n=4) mg/kg dose.

Figure 14A shows the arithmetic mean of the concentration-time profiles of CI-8993 following iv administration at 0.15, 0.3, and 0.6 mg/kg to patients with solid tumors in cycle 1.

Figure 14B shows Cmax levels versus CI-8993 dose. (* P<0.05)

DETAILED DESCRIPTION OF THE INVENTION

Prior to describing the invention in detail the following definitions of terms used herein are provided.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the invention or testing of the present invention, suitable methods and materials are described herein. The materials, methods and examples are illustrative only, and are not intended to be limiting. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. "Aids in the diagnosis" or "aids in the detection" of a disease herein means that the expression level of a particular marker polypeptide detected alone or in association with one or more other markers correlates to a particular disease or treatment. Herein, "Aids in the diagnosis" or "aids in the detection" generally will mean determining the expression level of a particular marker polypeptide detected alone or in association with one or more other markers by tumor and immune cells in order to assess whether a subject will be amenable to a VISTA antagonist or inhibitor treatment, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor treatment and/or whether the subject has responded to a VISTA antagonist or inhibitor treatment, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor treatment and/or to facilitate the design of a VISTA antagonist or inhibitor treatment, optionally an antagonist anti-VISTA antibody or antibody fragment or small molecule VISTA inhibitor treatment in a subject, alone or in association with other treatments, e.g., other checkpoint inhibitors, e.g., PD-1 or CTLA-4 antagonists.

[0240] "Amino acid," as used herein refers broadly to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, .gamma. -carboxyglutamate, and O-phosphoserine.) Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group), and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.) Analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. "Antagonist" herein refers to a molecule, generally an antibody or fusion proteins which blocks or reduces the effects of a specific molecule on immunity. Generally in the present application this will refer to anti-human VISTA antagonist antibodies and antibody fragments and small molecule inhibitors such as CA-170 which block or reduce the effects of human VISTA on immunity, particularly VISTA's suppressive effects on T cell immunity (CD4 ⁺ and/or CD8 ⁺ T cell immunity), the expression of proinflammatory cytokines and VISTA's effects of the expression of specific chemokines and chemoattractants.

"Antibody", as used herein, refers broadly to an "antigen-binding portion" of an antibody (also used interchangeably with "antibody portion," "antigen-binding fragment," "antibody fragment"), as well as whole antibody molecules. The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., VISTA or specific portions thereof)). The term "antibody" as referred to herein includes whole polyclonal and monoclonal antibodies and any antigenbinding fragment (i.e., "antigen-binding portion") or single chains thereof as well as bispecific and multispecific antibodies, e.g., those that bind to multiple antigens or multiple antigen epitopes. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of at least one heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, Cm and Cm- Each light chain is comprised of at least one light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL-The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FRS, CDRS, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. More generally, the term "antibody" is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be "antibodies."

The antigen-binding function of an antibody can be effected using fragments of a full-length antibody. Non-limiting examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (a) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (b) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (c) a Fd fragment consisting of the VH and CHI domains; (d) a Fv fragment consisting of the Viand VH, domains of a single arm of an antibody; (e) a dAb fragment (Ward, et al. (1989) Nature 341:544-546), which consists of a VH domain; and (f) an isolated complementarily determining region (CDR). Furthermore, although the two domains of the Fv fragment, the Viand VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). See e.g., Bird, et al. (1988) Science 242: 423-426; Huston, et al. (1988) Proc Natl. Acad. Sci. USA 85: 5879-5883; and Osbourn, et al. (1998) Nat. Biotechnol. 16: 778. Single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any the Viand VH sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG molecules or other isotypes. VH and VL can also be used in the generation of Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which the Viand VH domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. See e.g. Holliger, et al. (1993) Proc Natl. Acad. Sci. USA 90: 6444-6448; Poljak, et al. (1994) Structure 2:1121-1123. Still further, an antibody or antigen-binding portion thereof (antigen-binding fragment, antibody fragment, antibody portion) may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, et al. (1995) Hum. Antibodies Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules. Kipriyanov, et al. (1994) Mol. Immunol. 31: 1047- 1058. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein. Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, e.g., humanized, chimeric, bispecific or multispecific antibodies. "Antibody recognizing an antigen" and "an antibody specific for an antigen" is used interchangeably herein with the term "an antibody which binds specifically to an antigen" and refers to an immunoglobulin or fragment thereof that specifically binds an antigen. "Antigen," as used herein, refers broadly to a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen may have one epitope, or have more than one epitope. The specific reaction referred to herein indicates that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

"Antigen presenting cell," as used herein, refers broadly to professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). [0249] "Antisense nucleic acid molecule," as used herein, refers broadly to a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule) complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, antisense nucleic acid molecules can hydrogen bond to sense nucleic acid molecules. "CA-170" is a small molecule inhibitor developed by Curis which is the first-in-class VISTA protein-protein interaction inhibitor. The structure of CA-170, which is disclosed in Sasikumar et a I., "PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy", Communications Biology, 4, Article number: 699 (2021), is set forth below.

"Cancer," as used herein, refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor (e.g., unregulated cell growth.) The term "cancer" or "cancerous" as used herein should be understood to encompass any neoplastic disease (whether invasive, non- invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor, non-limiting examples of which are described herein. This includes any physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer are exemplified in the working examples. Further cancers include but are not limited to In some embodiments the at least one biopsy sample is derived from a non- hematological cancer or hematological cancer associated tumor obtained from a subject which is a carcinoma, lymphoma, blastoma, sarcoma, or leukemia.

In some embodiments the cancer is a malignant non-hematological cancer or hematological cancer or solid cancer obtained from a subject which cancer is optionally selected from squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon or colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, liver or hepatic carcinoma, head and neck cancers, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, T and B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL), Hodgkin's lymphoma (H L), intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia); leukemias such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, Acute Myeloid Leukemia (AML), Chronic myeloid or myeloblastic leukemia (CML), also known as chronic myelogenous leukemia, Hairy cell leukemia, chronic leukemia; and post-transplant lymphoproliferative disorder (PTLD), Meigs' syndrome; mesothelioma, multiple myeloma, cancer of the peritoneum, brains cancers such as glioblastoma, vulval cancer, thyroid cancer, et seq. In exemplary embodiments the cancer comprises a NSCLC tumor.

The cancerous conditions amenable for treatment of the invention include cancers that express or do not express VISTA and further include non-metastatic or non-invasive as well as invasive or metastatic cancers wherein VISTA expression by immune, stromal or diseased cells suppress antitumor responses and anti-invasive immune responses. The method of the present invention is particularly suitable for the treatment of vascularized tumors. Cancers according to the invention include cancers that express or do not express VISTA and further include non-metastatic or non-invasive as well as invasive or metastatic cancers wherein VISTA expression by immune, stromal or diseased cells suppress antitumor responses and anti-invasive immune responses, and those characterized by vascularized tumors.

"Chimeric antibody," as used herein, refers broadly to an antibody molecule in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigenbinding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, the variable region or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

"Coding region," as used herein, refers broadly to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term "noncoding region" refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

"Conservatively modified variants," as used herein, applies to both amino acid and nucleic acid sequences, and with respect to particular nucleic acid sequences, refers broadly to

14 conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. "Silent variations" are one species of conservatively modified nucleic acid variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) may be modified to yield a functionally identical molecule.

"Complementarity determining region," "hypervariable region," or "CDR," as used herein, refers broadly to one or more of the hyper-variable or complementarily determining regions (CDRs) found in the variable regions of light or heavy chains of an antibody. See Kabat, et al. (1987) Sequences of Proteins of Immunological Interest National Institutes of Health, Bethesda, Md. These expressions include the hypervariable regions as defined by Kabat, et al. (1983) Sequences of Proteins of Immunological Interest, U.S. Dept, of Health and Human Services or the hypervariable loops in 3-dimensional structures of antibodies. Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917. The CDRs in each chain are held in close proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site. Within the CDRs there are select amino acids that have been described as the selectivity determining regions (SDRs) which represent the critical contact residues used by the CDR in the antibody-antigen interaction. (Kashmiri, Methods 36: 25-34 (2005)).

"Control amount," as used herein, refers broadly to a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker may be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition. A control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

"Diagnostic," as used herein, refers broadly to identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives". The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

"Diagnosing," or "aiding in the diagnosis" as used herein refers broadly to classifying a disease or a symptom, and/or determining the likelihood that an individual has a disease condition (e.g., based on absence or presence of VISTA or other biomarker expression, and/or increased or decreased expression by immune, stromal and/or putative diseased cells); determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the foregoing. Diagnosis of a disease according to the present invention may, in some embodiments, be affected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject.

"Effective amount," as used herein, refers broadly to the amount of a compound, typically a VISTA antagonist or inhibitor, e.g., an antibody or antibody fragment or small molecule that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The effective amount may be an amount effective for prophylaxis, and/or an amount effective for prevention. The effective amount may be an amount effective to reduce, an amount effective to prevent the incidence of signs/symptoms, to reduce the severity of the incidence of signs/symptoms, to eliminate the incidence of signs/symptoms, to slow the development of the incidence of signs/symptoms, to prevent the development of the incidence of signs/symptoms, and/or effect prophylaxis of the incidence of signs/symptoms. The "effective amount" may vary depending on the disease and its severity and the age, weight, medical history, susceptibility, and pre-existing conditions, of the patient to be treated. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of this invention.

"Expression vector/' as used herein, refers broadly to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.

"Family," as used herein, refers broadly to the polypeptide and nucleic acid molecules of the invention is intended to mean two or more polypeptide or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Family members can be naturally or non-natu rally occurring and can be from either the same or different species. For example, a family can contain a first polypeptide of human origin, as well as other, distinct polypeptides of human origin or alternatively, can contain homologues of non-human origin (e.g., monkey polypeptides.) Members of a family may also have common functional characteristics. "Fc receptor" (FcRs) as used herein, refers broadly to cell surface receptors for the Fc portion of immunoglobulin molecules (Igs). Fc receptors are found on many cells which participate in immune responses. Among the human FcRs that have been identified so far are those which recognize IgG (designated FcyR), IgE (FceRI), IgA (FcaR), and polymerized IgM/A (FcepP). FcRs are found in the following cell types: FceRI (mast cells), FceRI I (many leukocytes), FcaR (neutrophils), and FcpR (glandular epithelium, hepatocytes). (Hogg, Immunol. Today 9: 185-86 (1988)). The widely studied FcyRs are central in cellular immune defenses, and are responsible for stimulating the release of mediators of inflammation and hydrolytic enzymes involved in the pathogenesis of autoimmune disease. (Unkeless, Anna. Rev. Immunol. 6:251-87 (1988)). The FcyRs provide a crucial link between effector cells and the lymphocytes that secrete Ig, since the macrophage/monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell FcyRs confer an element of specific recognition mediated by IgG. Human leukocytes have at least three different types of FcyRs for IgG: hFcyRI(CD64) (found on monocytes/macrophages), hFcyRIIA or h FcyRII B, (CD32 or CD32A) (found on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line) and FcyRIIIA (CD16A) or FcyRIIIB (CD16B) (found on NK cells, neutrophils, eosinophils, and macrophages).

"Framework region" or "FR," as used herein refers broadly to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody. See Kabat, et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.

"Heterologous," as used herein, refers broadly to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid (e.g., a promoter from one source and a coding region from another source.) Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

"High affinity," as used herein, refers broadly to an antibody or fusion protein having a KD of at least 10‘ ⁵ M, more preferably 10‘ ⁷ M, even more preferably at least 10‘ ⁸ M and even more preferably at least 10‘ ⁹ M, 10 ¹⁰ M, 10 ¹¹ M, or 10- ¹² M for a target antigen or receptor. "High affinity" for an IgG antibody or fusion protein herein refers to an antibody having a KD of 10 ⁵ M or less, more preferably 10 ⁷ M or less, preferably 10 ⁸ M or less, more preferably 10‘ ⁹ M or less and even more preferably 10 ¹⁰ M, 10 ¹¹ M, or 10 ¹² M or less for a target antigen or receptor. With particular respect to antibodies, "high affinity" binding can vary for different antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of 10‘ ⁷ M or less, more preferably 10‘ ⁸ M or less.

"Homology," as used herein, refers broadly to a degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between a polypeptide sequence and a reference polypeptide sequence. Homology may be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison, for example using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. The term "sequence identity" may be used interchangeably with "homology."

"Host cell," as used herein, refers broadly to refer to a cell into which a nucleic acid molecule of the invention, such as a recombinant expression vector of the invention, has been introduced. Host cells may be prokaryotic cells (e.g., E. coli), or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa, HEK-293, e.g., cultured cells, explants, and cells in vivo. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"Human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germ line immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. This includes fully human monoclonal antibodies and conjugates and variants thereof, e.g., which are bound to effector agents such as therapeutics or diagnostic agents.

"Humanized antibody," as used herein, refers broadly to include antibodies made by a non- human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non- human antibody amino acid sequence to incorporate amino acids found in human germ line immunoglobulin sequences. The humanized antibodies of the invention may include amino acid residues not encoded by human germ line immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term "humanized antibody", as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. "Hybridization," as used herein, refers broadly to the physical interaction of complementary (including partially complementary) polynucleotide strands by the formation of hydrogen bonds between complementary nucleotides when the strands are arranged antiparallel to each other.

"Immune cell," as used herein, refers broadly to cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include but are not limited to lymphocytes, such as B cells and T cells, e.g., CDS T cells, CD4 T cells, Tregs et al., natural killer cells; dendritic cells, and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes, inter alia.

"Immunoassay," as used herein, refers broadly to an assay that uses an antibody to specifically bind an antigen. The immunoassay may be characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. "Immune response," as used herein, refers broadly to T cell-mediated and/or B cell- mediated immune responses that are influenced by modulation of T cell costimulation. Exemplary immune responses include B cell responses (e.g., antibody production) T cell responses (e.g., cytokine production, and cellular cytotoxicity) and activation of cytokine responsive cells, e.g., macrophages. As used herein, the term "downmodulation" with reference to the immune response includes a diminution in any one or more immune responses, while the term "upmodulation" with reference to the immune response includes an increase in any one or more immune responses. It will be understood that upmodulation of one type of immune response may lead to a corresponding downmodulation in another type of immune response. For example, upmodulation of the production of certain cytokines (e.g., IL-10) can lead to downmodulation of cellular immune responses.

"Immunologic", "immunological" or "immune" response herein refer to the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. Without wishing to be limited by a single hypothesis, a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class II or Class I MHC molecules to activate antigen-specific CD4 ⁺ T helper cells and/or CD8 ⁺ cytotoxic T cells, respectively. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells,

20 astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 ⁺ T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

"Immunogenic agent" or "immunogen" is a moiety capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

"Immunohistochemistry" or "IHC" is a common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing an antibody-antigen interaction is generally accomplished using Chromogenic immunohistochemistry (CIH), wherein an antibody is conjugated to an enzyme, such as peroxidase (the combination being termed immunoperoxidase), that can catalyze a color-producing reaction or Immunofluorescence, where the antibody is tagged to a fluorophore, such as fluorescein or rhodamine. Tissues used in IHC methods may be sliced or used whole, dependent upon the purpose of the experiment or the tissue itself. "IHC reporters" or "IHC Labels" refer to a moiety that permits visualization of bound antibody during IHC. IHC reporter molecules vary based on the nature of the detection method, the most popular being chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label generally reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, Permanent Red, Violet, among many others. Fluorescent reporters used for IHC detection include by way of example FITC, TRITC and AMCA, Alexa Fluors and Dylight Fluors, inter alia. "Isolated," as used herein, refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment and includes "recombinant" polypeptides. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated antibody"). For example, "isolated" or "purified," as used herein, refers broadly to a protein, DNA, antibody, RNA, or biologically active portion thereof, that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological substance is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. As used herein the term "isolated" refers to a compound of interest (for example a polynucleotide or a polypeptide) that is in an environment different from that in which the compound naturally occurs e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature. "Isolated" includes compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.

"Isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds VISTA) is substantially free of antibodies that specifically bind antigens other than VISTA). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

"Isotype" herein refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

"K-assoc" or "Ka", as used herein, refers broadly to the association rate of a particular antibody-antigen interaction, whereas the term "Kdiss" or "Kd," as used herein, refers to the dissociation rate of a particular antibody-antigen interaction.

The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art such as plasmon resonance (BIAcore®), ELISA and KINEXA. A preferred method for determining the KD of an antibody is by using surface Plasmon resonance, preferably using a biosensor system such as a BIAcore® system or by ELISA. Typically these methods are effected at 25° or 37°C. Antibodies for therapeutic usage generally will possess a KD when determined by surface Plasmon resonance of 50 nM or less or more typically 1 nM or less at 250 or 37°C.

"Label" or a "detectable moiety" as used herein, refers broadly to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

"Low stringency," "medium stringency," "high stringency," or "very high stringency conditions," as used herein, refers broadly to conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel, et al., Short Protocols in Molecular Biology (5th Ed.) John Wiley & Sons, NY (2002). Exemplary specific hybridization conditions include but are not limited to: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C., followed by two washes in 0.2XSSC, 0.1% SDS at least at 50°C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridization conditions in 6XSSC at about 45°C., followed by one or more washes in 0.2 X SSC, 0.1% SDS at 60°C.; (3) high stringency hybridization conditions in 6 X SSC at about 45° C. followed by one or more washes in 0.2 X SSC, 0.1% SDS at 65°C.; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C., followed by one or more washes at 0.2XSSC, and 1% SDS at 65°C.

"Mammal," as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the you ng. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington D.C. "Multispecific antibody" refers to an antibody with 2 or more antigen binding regions. This includes bispecific antibodies. These antigen binding regions may bind to different antigens or to different epitopes of the same antigen.

"Naturally-occurring nucleic acid molecule," as used herein, refers broadly refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

"Nucleic acid" or "nucleic acid sequence," as used herein, refers broadly to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

"Operatively linked", as used herein, refers broadly to when two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame. "Paratope," as used herein, refers broadly to the part of an antibody which recognizes an antigen (e.g., the antigen-binding site of an antibody.) Paratopes may be a small region (e.g., 15-22 amino acids) of the antibody's Fv region and may contain parts of the antibody's heavy and light chains. See Goldsby, et al. Antigens (Chapter s) Immunology (5th Ed.) New York: W. H. Freeman and Company, pages 57-75.

"Patient," or "subject" or "recipient", "individual", or "treated individual" are used interchangeably herein, and refers broadly to any animal that is in need of treatment either to alleviate a disease state or to prevent the occurrence or reoccurrence of a disease state. Also, "Patient" as used herein, refers broadly to any animal that has risk factors, a history of disease, susceptibility, symptoms, and signs, was previously diagnosed, is at risk for, or is a member of a patient population for a disease. The patient may be a clinical patient such as a human or a veterinary patient such as a companion, domesticated, livestock, exotic, or zoo animal.

"Polypeptide," "peptide" and "protein," are used interchangeably and refer broadly to a polymer of amino acid residues s of any length, regardless of modification (e.g., phosphorylation or glycosylation). The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide" and "protein" expressly include glycoproteins, as well as nonglycoproteins.

"Recombinant" as used herein, refers broadly with reference to a product, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germ line immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

25 "Specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," or "specifically interacts or binds," as used herein, refers broadly to a protein or peptide (or other epitope), refers, in some embodiments, to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. For example, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than about 10 to 100 times background.

"Specifically hybridizable" and "complementary" as used herein, refer broadly to a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. The binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art. (See, e.g., Turner, et al. CSH Symp. Quant. Biol. LI 1: 123-33 (1987); Frier, et al. PNAS 83: 9373-77 1986); Turner, et al. J. Am. Chem. Soc. 109:3783-85 (1987)). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., about at least 5, 6, 7, 8, 9, 10 out of 10 being about at least 50%, 60%, 70%, 80%, 90%, and 100% complementary, inclusive).

"Perfectly complementary" or 100% complementarity refers broadly all of the contiguous residues of a nucleic acid sequence hydrogen bonding with the same number of contiguous residues in a second nucleic acid sequence.

"Substantial complementarity" refers to polynucleotide strands exhibiting about at least 90% complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be noncomplementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically may differ by at least 5 nucleotides. "Soluble VISTA protein(s)/molecule(s)" herein also include VISTA molecules with the transmembrane domain removed to render the protein soluble, or fragments and derivatives thereof; fragments, portions or derivatives thereof, and soluble VISTA mutant molecules. The soluble VISTA molecules used in the methods according to at least some embodiments of the invention may or may not include a signal (leader) peptide sequence. "Subject" or "patient" or "individual" in the context of therapy or diagnosis herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc., i.e., anyone suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably mammalian. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, and adult) can be treated according to the present invention. The present invention may also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cattle, goats, sheep, and horses for veterinary purposes, and for drug screening and drug development purposes. "Subjects" is used interchangeably with "individuals" and "patients."

"T cell," as used herein, refers broadly to all T cell types such as CD4+ T cells and CD8+ T cells and Tregs. The term T cell also includes both T helper 1 type T cells and T helper 2 type T cells.

"Therapy," "therapeutic," "treating," or "treatment", as used herein, refers broadly to treating a disease, arresting, or reducing the development of the disease or its clinical symptoms, and/or relieving the disease, causing regression of the disease or its clinical symptoms. Therapy encompasses prophylaxis, treatment, remedy, reduction, alleviation, and/or providing relief from a disease, signs, and/or symptoms of a disease. Therapy encompasses an alleviation of signs and/or symptoms in patients with ongoing disease signs and/or symptoms (e.g., inflammation, pain). Therapy also encompasses "prophylaxis". The term "reduced", for purpose of therapy, refers broadly to the clinical significant reduction in signs and/or symptoms. Therapy includes treating relapses or recurrent signs and/or symptoms (e.g., inflammation, pain). Therapy encompasses but is not limited to precluding the appearance of signs and/or symptoms anytime as well as reducing existing signs and/or symptoms and eliminating existing signs and/or symptoms. Therapy includes treating chronic disease ("maintenance") and acute disease. For example, treatment includes treating or preventing relapses or the recurrence of signs and/or symptoms (e.g., inflammation, pain).

"Treg cell" (sometimes also referred to as suppressor! cells or inducible Treg cells or iTregs) as used herein refers to a subpopulation of T cells which modulate the immune system and maintain tolerance to self-antigens and can abrogate autoimmune diseases. Foxp3 ⁺ CD4 ⁺ CD25 ⁺ regulatory ! cells (!regs) are critical in maintaining peripheral tolerance under normal conditions.

"Variable region" or "VR," as used herein, refers broadly to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (V at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

"Vector," as used herein, refers broadly to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. !he techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook, et al. Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring Harbor Laboratory Press (2001). Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.

[0329] Having defined certain terms and phrases used in the present application, the invention is further described below.

As noted above the present invention in part provides methods for identifying subjects who will likely clinically respond to a VISTA antagonist, optionally therapeutic anti-VISTA antibodies or antibody fragments or small molecule VISTA inhibitors such as CA-170 based on the expression of specific biomarkers by immune cells and tumor cells of the subject, and wherein the detection of such expression is preferably effected by use of immunochemistry (IHC) wherein the expression and level of expression of specific biomarkers on specific types of immune and tumor cells comprised in one or more tumor biopsy samples obtained from a subject is effected, and optionally such expression is compared to normal tissue samples from the same or different subject.

Also, the present invention provides methods for assessing whether subjects treated with VISTA antagonist, optionally therapeutic anti-VISTA antibodies or antibody fragments or small molecule VISTA inhibitors such as CA-170 have clinically responded based on the expression of specific biomarkers on immune and tumor cells, again wherein such expression is preferably detected by use of IHC wherein the expression and level of expression of specific biomarkers on specific immune and tumor cells comprised in one or more tumor biopsy samples obtained from a subject is detected by use of IHC before, during and/or after treatment with therapeutic anti-VISTA antibodies or antibody fragments and optionally such expression is compared to normal tissue samples from the same or different subject.

In particular we disclose herein the design, development, and analytical algorithms for comprehensive VISTA-centric tumor immunophenotyping to determine the biomarker expression profile of subjects likely to clinically respond to the administration of VISTA antagonist, optionally therapeutic anti-VISTA antibodies or antibody fragments or small molecule VISTA inhibitors such as CA-170, said antibodies including but not limited to an anti-human VISTA therapeutic antibody referred to as CI-8993, which antibody is currently under clinical development (Phase 1 trial) by Curis for use in treating solid tumors such as non-small cell lung cancer.

In the present IHC methods specific biomarkers are detected on one or more tissue samples derived from a patient sample, typically a tumor sample. Most typically, the tissue samples will be obtained from one or more biopsies which are isolated from patient tissue or tissues suspected or known to be cancerous. The tissue samples or sections analyzed by use of IHC will preferably be derived from different sections of the tumor so that the biomarker expression results are representative of the biomarker status of the cancerous and immune cells comprised in a tumor potentially to be treated with a VISTA antagonist such as an antagonist anti-VISTA antibody or antibody fragment, preferably CI-8993 or a small molecule inhibitor such asCA-170.

As noted previously, IHC is a common application of immunostaining and involves the process of selectively identifying antigens (proteins) in cells of one or more tissue sections by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing an antibody-antigen interaction in IHC can be accomplished in a number of ways, e.g., using Chromogenic immunohistochemistry (CIH), wherein an antibody is conjugated to an enzyme, such as peroxidase (the combination being termed immunoperoxidase), that can catalyze a color-producing reaction or Immunofluorescence, where the antibody is tagged to a fluorophore, such as fluorescein or rhodamine. In the working example CIH is used however, the invention further contemplates the use of Immunofluorescence to detect the biomarkers.

IHC Reporters

Different reporters may be used in the subject IHC detection methods. The typical reporter system used in IHC are chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label generally reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. Various chromogens are used in IHC detection.

While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel, producing a deep purple/black staining. Fluorescent reporters used for IHC detection include by way of example FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.

Examples of chromogens frequently used in IHC and the color they emit is further set forth below:

Exemplary HRP Chromogens:

Aminoethyl carbazole (AEC) (Red Color)

3,3'5,5'-tetramethylbenzidine (TMB) (Blue Color)

Emerald (Green color)

3,3'-diaminobenzidine (DAB) (1st generation) (Brown Color)

Cardassian DAB (2nd generation) (Black Brown Color)

Betazoid DAB (3rd generation) (Brown Color)

Romulin AEC (Brick Red)

Bajoran Purple: (Purple)

Vina Green: (Green Color)

Deep Space Black™ (Black Color)

Exemplary AP Chromogens:

Fast Red (Red Color)

Permanent Red (Red Color)

Mixture of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (NBT / BCIP) (Intense blue/purple Color)

Warp Red™ (Fuchsin red)

Vulcan Fast Red (VFR) (Fuchsin Red)

Ferangi Blue Royal Blue

In the exemplary IHC methods DAB and Permanent Red chromogens are used to detect the biomarkers. Sample Preparation

Tissues used in IHC methods may be sliced or used whole, dependent upon the purpose of the experiment or the tissue itself. Before sectioning, the tissue sample may be embedded in a medium, like paraffin wax or cryomedia. Sections can be sliced on a variety of instruments, most commonly a microtome, cryostat, or vibratome. Specimens are typically sliced at a range of 3 pm-5 pm. The slices are then mounted on slides, dehydrated using alcohol washes of increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using a detergent like xylene before being imaged under a microscope. Depending on the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen-retrieval is often necessary, and involves pre-treating the sections with heat or protease. These steps may make the difference between the target antigens staining or not staining.

Depending on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining. To reduce background staining in IHC methods the samples may be incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. Commercial blocking buffers with proprietary formulations are available for greater efficiency. Methods to eliminate background staining include dilution of the primary or secondary antibodies, changing the time or temperature of incubation, and using a different detection system or different primary antibody. Commonly used buffers used in IHC in Citrate buffer, Tris-EDTA buffer and EDTA buffer and Tris buffer. In the exemplary IHC methods citrate is used as the buffer.

Tumor Biopsy Samples Used to Derive Tissue Sections Used In Present IHC Methods The tissue sections used in the subject IHC detection samples are derived from biopsy samples, generally cancer biopsy samples obtained from human patients. The biopsy may be obtained from different hematological or non-hematological tumor samples including any of the following cancers: carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Particular examples of cancers where biopsy samples may be obtained include malignant non- hematological cancer or hematological cancer obtained from a subject which cancer is optionally selected from squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon or colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, liver or hepatic carcinoma, head and neck cancers, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, T and B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL), Hodgkin's lymphoma (HL), intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS- related lymphoma, and Waldenstrom's Macroglobulinemia); leukemias such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, Acute Myeloid Leukemia (AML), Chronic myeloid or myeloblastic leukemia (CML), also known as chronic myelogenous leukemia, Hairy cell leukemia, chronic leukemia; post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), Meigs' syndrome; mesothelioma, multiple myeloma, cancer of the peritoneum, brains cancers such as glioblastoma, vulval cancer, and thyroid cancer, by way of example. In exemplary embodiments the tissue samples analyzed using IHC are derived from tumor biopsies obtained from NSCLC patients.

Biomarkers Detected and Antibodies Used in In Subject IHC Methods The biomarkers detected in the present IHC methods include VISTA and potentially other checkpoint inhibitors as well as specific antigens expressed by particular immune cells, e.g., T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, eosinophils, inter alia.

In the exemplary embodiments the detected biomarkers include the following:

VISTA (Checkpoint inhibitor primarily expressed by hematopoietic cells, and particularly immune cells including T cells, myeloid cells, particularly microglia and neutrophils followed by monocytes, macrophages, and dendritic cells, and within the T lymphocyte compartment, VISTA is most highly expressed on naive CD4+ and Foxp3+ regulatory T cells. Also VISTA is expressed by some cancers including melanoma, pancreatic cancers, prostate cancer, renal cell carcinoma, non-small cell cancer, Acute myeloid leukemia, Colorectal cancer, Ovarian and endometrial cancers, Glioma and fibrosarcoma inter alia; PD-L1 (Checkpoint inhibitor expressed on macrophages, activated macrophages, monocytes and by some cancers including renal cell carcinoma, gastric cancer, Thymoma and thymic carcinoma , Adrenocortical carcinoma, Head and neck squamous cancer, Malignant brain tumors, glioma, lung cancer, esophageal cancer, colorectal cancer, pancreatic cancer, Malignant pleural mesothelioma, Merkel cell carcinoma., melanoma, , cervical cancer, bladder cancer, non-small-cell lung cancer, .Differentiated thyroid carcinoma, sarcoma, Oropharyngeal squamous cell carcinoma, intrahepatic cholangiocarcinoma; Multiple myeloma. Leukemia, HCC, renal cell carcinoma, urothelial cancer of the bladder, ovarian cancer, breast cancer, Wilms' tumor, Nasopharyngeal carcinoma) inter alia;

CDS (cytotoxic T cell marker);

CD4 (helper T cell marker);

CD68 (Monocyte or Macrophage Marker);

CD56 or NCAM1 (NK cell Marker; also tumor marker of the pulmonary neuroendocrine cell system, Tumors that reportedly may be CD56 positive include Myeloma, Myeloid Leukemia, Neuroendocrine tumors, Wilm’s Tumor, Adult Neuroblastoma, NK/T cell Lymphomas, Pancreatic Acinar-cell Carcinoma, Pheochromocytoma, and Small-cell Lung Carcinoma, some mesodermally-derived tumors (Rhabdomyosarcoma) inter alia;

CDllb (Myeloid Cell Marker) and

CD19 (B cell marker).

The subject IHC methods will detect the presence of the afore-mentioned biomarkers and potentially other biomarkers using antibodies which specifically recognize these antigens. Antibodies which specifically bind to these antigens which may be used in IHC detection methods are commercially available. Exemplary antibodies and commercial source thereof are disclosed in the working examples.

Tissue Sections

Tissue sections used in the subject IHC detection samples may be sliced or used whole, and typically will be sliced. As afore-mentioned the samples or sections used for IHC analysis will preferably be derived from different portions or regions of the tumor so that an accurate representation of the biomarker status of a particular tumor is obtained. Also, as previously noted the biomarker status of the analyzed tissue sections may be compared to that of normal tissues which are evaluated using the same IHC protocol, e.g., as exemplified in the working example. Cells Analyzed for Biomarker Expression In Present IHC Methods

As noted above, the tissue sections used in the subject IHC detection methods are derived from biopsy samples, generally cancer biopsy samples obtained from human patients. Biomarker expression by cells in the tumor sample section will be analyzed for biomarker expression using antibodies which specifically bind to the biomarkers. The cells which will be analyzed for expression of one or more of the above-identified biomarkers include cancer cells, and immune cells such as cytotoxic T cells, helperT cells, monocytes, NK cells, myeloid cells, B cells, among others.

In the exemplary IHC detection methods the samples analyzed for biomarker expression are derived from biopsies of non-small cell lung cancer tumors. The analyzed cells in the samples include non-small cell lung cancer and immune cells such as T cells, B cells, cytotoxic T cells, helper T cells, monocytes, macrophages, NK cells, myeloid cells, B cells, among others.

EXAMPLES

The invention is further described in the following examples.

EXAMPLE 1: Development of VISTA-centric tumor immunophenotyping as a novel approach for identification of potential biomarkers for anti-VISTA therapy

VISTA is a negative checkpoint regulator of immune cells and has been recognized as a potential mediator of resistance to anti-PD-1 and anti-CTLA-4 immunotherapies in cancer patients. Targeting the VISTA signaling pathway has been suggested as a promising approach for overcoming resistance to current immune checkpoint therapies. Herein, we report the design, development, and analytical algorithm for comprehensive VISTA-centric tumor immunophenotyping to identify potential tumor biomarkers for novel anti-VISTA therapeutic antibody CI-8993, currently under clinical development in a Phase 1 clinical trial in solid tumor patients.

METHODS:

Serial formalin-fixed paraffin embedded (FFPE) tumor tissue sections from 10 cases of non- small cell lung carcinoma (NSCLC) were double-immunostained with VISTA combined with CDS (cytotoxic T cell marker), CD4 (T helper cell marker), CDllb (myeloid cell marker), CD68 (monocyte/macrophage marker), CD56 (NK cell marker), CD19 (B cell marker) and Programmed Death-Ligand 1 (PD-L1). The specifics of the double staining IHC protocol are set forth below. Double IHC Staining protocol

1. Deparaffinize tissue sections in xylene 3x5 min.

2. Wash in 100% ethanol 2x5 min, then 95% ethanol 5 min.

3. Wash in tap water for 5 min.

4. Incubate for 10 min in Dual Endogenous Peroxidase Block (DakoEnvision, K4065).

5. Wash in tap water for 5 min

6. Place slides in container and cover with Citrate Buffer (cat. C9999-1000ML, Sigma), heat in microwave (slides should be covered by buffer during procedure, refill container if needed) for 10 min.

Allow slides to cool down in the buffer at RT.

7. Wash in IX Wash Buffer (Envision Flex Wash Buffer, Agilent, K800721-2), 3x5min.

8. Incubate with VISTA Ab, cat 64953, Cell Signaling (dilutions 1:100) in humidified chamber for lhr at RT.

9. Wash in Wash Buffer 3x5 min.

10. Apply Labeled Polymer-HRP solution (DAKO EnVision+ System-HRP) to the section.

Incubate 30 min at RT.

11. Wash in PBS 3x5 min.

12. Prepare Substrate-Chromogen solution by mixing 1 ml of Substrate Buffer and 20 pl of Liquid DAB+Chromogen. Apply Substrate-Chromogen solution on tissue to develop the staining for 5 min.

13. Wash slides in tap water for 5 min to stop reaction.

14. Antigen retrieval as described in #6. Wash in Wash Buffer 3x5 min.

15. Incubate with antibody for specific cell marker (the list of Abs is shown on slide #3)in humidified chamber for lhr at RT or overnight at +4C.

16. Wash in Wash Buffer 3x5 min.

17. Incubate with Rabbit/Mouse Link (Vial 1), cat K5355, DAKO for 30 min.

18. Wash in Wash Buffer 3x5 min.

19. Incubate with AP Enzyme (ENHANCER) (Vial 2), cat K5355, DAKO for 30 min.

20. Wash in Wash Buffer 3x5 min.

21. Prepare Substrate Working Solution: 1 ml (Vial 4) Permanent Red Substrate Buffer + 10 pl (Vial 3) Permanent Red Chromogen. Apply Substrate Working Solution on tissue to develop the staining for 5 min. 22. Wash slides in tap water for 5 min to stop reaction. Counterstain with fresh filtered Mayer's hematoxylin for 5 seconds. Wash slides in tap water 10 min.

23. Air-dry slides.

24. Place in xylene for 5 seconds, mount and cover with a glass coverslip.

The IHC staining kits used for staining were the DAKO EnVisionDual Link System-HRP (DAB+), cat. K4065 which elicits a brown color (used to detect VISTA) and the DAKO EnVisionG 12 System/AP, Rabbit/Mouse which elicits a Permanent Red color (used to detect immune cell markers).

Antibodies

The antibodies used in the IHC studies are in Table 1 below:

TABLE 1 RESULTS

As summarized in Figure 1, the expression of specific antigens on biopsy samples from 10 NSCLC patients was assayed by use of IHC in order to identify potential biomarkers and combinations of biomarkers that potentially may be used to assess whether a cancer patient is amenable to anti-VISTA therapy and/or whether the patient is responding to anti-VISTA therapy. IHC results from specific patients are further shown in Figures 2-10.

This immunohistochemical analysis revealed the presence of CD8 ⁺ cells (9/10 cases), CD4 ⁺ cells (3/10 cases), CDllb ⁺ cells (10/10 cases), CD68 ⁺ cells (10/10 cases), CD56 ⁺ cells (4/10 cases) and CD19 ⁺ cells (8/10 cases) in NSCLC lung tumors. Using double IHC staining, it was observed that VISTA was expressed in CD8 ⁺ cells (5/9 tumors), CDllb ⁺ cells (5/10 tumors) and CD19 ⁺ cells (5/8 tumors), whereas VISTA was hardly detectable in CD4 ⁺ , CD68 ⁺ or CD56 ⁺ cells.

The expression of PD-L1 was detected in cancer cells in 6/10 tumors, whereas VISTA-pos cancer cells were revealed in 1/10 tumors. Based on these observations an algorithm for evaluating VISTA-centric tumor immunophenotyping was developed which demonstrated that every tumor has a unique cell-type-specific pattern of VISTA expression which could serve as a potential biomarker and facilitate treatment design.

ALGORITHM AND APPLICATIONS

The biomarker algorithm for VISTA-centric immunophenotyping of the tumor will evaluate the specific pattern of VISTA expression in different immune cells and cancer cells in tumor tissue. The exact intratumoral VISTA expression pattern associated with a positive clinical response or resistance to anti-VISTA therapy will be determined in clinical studies. The subject methods (IHC assay) and such biomarker algorithm may be used for 1) biomarker discovery to identify a specific VISTA expression pattern in tumor as a potential predictive biomarker for anti-VISTA therapy (retrospective study with clinical response data) and 2) selection of cancer patients for anti-VISTA therapy (prospective study) as soon as the intratumoral expression pattern of VISTA in specific immune cells and/or cancer cells is identified as a predictive biomarker.

For example, if VISTA is expressed only in CDS, CDllb and CD19 cells (e.g. NSCLC cases 61 and 65, Figure 1) and if a clinical response is observed in those cases (retrospective study), this intratumoral VISTA expression pattern could potentially be determined as a predictive biomarker for efficacious anti-VISTA therapy and this expression pattern would be used as a selection biomarker for cancer patients (prospective study).

CONCLUSIONS:

Our results demonstrate that comprehensive VISTA-centric immunophenotyping enables spatially resolved and cell-type-specific characterization of VISTA expression on immune and tumor cells comprised in a tumor sample of a subject with a solid tumor. The results of such VISTA-centric immunophenotyping potentially can be used as a bioanalytical approach for identifying solid tumor patients who likely will clinically respond to treatment with a therapeutic anti-VISTA antibody or anti-VISTA antibody fragment.

Moreover, the results of such VISTA-centric immunophenotyping potentially can be used for identifying patients who after treatment with a therapeutic anti-VISTA antibody or anti- VISTA antibody fragment have responded to treatment and/or are no longer resistant to or are less resistant to treatment with current immune checkpoint therapies such as PD-1 antagonists (e.g., anti-PD-1 or anti-PD-Ll antagonist antibodies such as Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) developed by Merck; Nivolumab (Opdivo) developed by Bristol-Myers Squibb; Cemiplimab (Libtayo) developed by Regeneron Pharmaceuticals; Dostarlimab (Jemperli) - developed by GlaxoSmithKline, small molecule PD-1 antagonists such as CA-170 developed by Curis, which is an orally bioavailable antagonist of the VISTA/PD1H and PD-L1 immune checkpoint pathways [CA-170 is currently in clinical development by Aurigene and Curis and recently completed Phase 1 clinical trials]; CA-327, a small molecule, orally bioavailable antagonist of Tim-3 and PD-L1 checkpoint pathways which like CA-170 is in clinical development; and/or CTLA-4 antagonists (e.g., antagonist anti-CTLA-4 antibodies such as ipilimumab and tremelimumab and CTLA-4 fusion proteins).

Example 2: Pharmacokinetic and Pharmacodynamic data from a Phase 1 Study of CI-8993 Anti-VISTA Antibody in Patients with Advanced Solid Tumors

BACKGROUND

V-set immunoglobulin domain suppressor of T cell activation (VISTA) is a novel immunoregulatory protein that is broadly expressed on cells of the myeloid and lymphoid lineages, and is frequently implicated as a poor prognostic indicator in multiple cancers. VISTA is the only oncology checkpoint on quiescent T cells affecting earliest phase of response to tumor antigen.

Negative immune checkpoint regulation by VISTA has been described as a mechanism of resistance in melanoma and prostate patients treated with anti- CTLA 4 and anti-PD-l/PD-Ll inhibitors.

Preclinical ly, CI-8993 shows anti-tumor efficacy and synergy with other checkpoint inhibitors (CPIs) through TME modulation via increased peripheral tumor-specific T cells numbers, monocyte- and T cell activation.

CI-8993 Study Design

Patients with solid tumor malignancy (non-lymphoma) that is metastatic or unresectable and considered relapsed and/or refractory to prior therapy, were included for this study. Patients were enrolled into cohorts structured to receive step-dosing regimen before administering a single full dose of CI-8993 (see Study Design and Table 2). The full doses ranged from 0.15 mg/kg to 0.6mg/kg over three different cohorts.

** Initial step dose administered within 1 week prior to full dose.

Table 2: CI-8993-101 Step and Full Dosing Scheme Methods

Immune related PD, cytokine quantification and immune phenotyping was performed on peripheral blood from patients receiving a single full dose of CI-8993, divided in 3 cohorts (0.15, 0.3 and 0.6 mg/kg).

Plasma concentration of CI-8993 (PK) was quantified on samples from patients at time points following step-dose, and a single full dose administration of CI-8993.

Abbreviations

Pharmacokinetic (PK), Pharmacodynamic (PD), Dose Limiting Toxicities (DLT), Recommended Phase 2 Dose (RP2D), Maximum Concentration (Cmax), Intravenous (IV), once every 2 weeks (Q2W), Non-Small Cell Lung Cancer (NSCLC)

TABLE 3

CI-8993 Patient Baseline Characteristics

RESULTS

Cytokine Profile

Figure 12 shows the average cytokine concentration at CI-8993 pre and post infusion at 0.15 mg/kg (n=7), 0.3 mg/kg (n=5) and 0.6 mg/kg (n=4) dose. (* P<0.05). These results indicate a rapid, but transient, increase of inflammatory mediators including plasma cytokines (ILS, IL18) and chemokines (IP10, MCP1) (* P<0.05).

As further shown soluble markers such as TNFa and MIPip present differences between cohorts at 4 hours after treatment (* P<0.05). Still further shown it can be seen that the patients from CI-8993 cohort 3 (0.6 mg/kg) exhibit lower cytokine levels when compared to cohort 1 and 2 (0.15 and 0.3 mg/Kg).

Whole Blood Immunophenotyping

Figure 13 shows the average values of immunophenotypes at baseline and 24 hours after CI- 8993 infusion at 0.15 (n=7), 0.3 (n=5) and 0.6 ( n=4) mg/kg dose.

Particularly, activated T cells, suppression of CD16 on NK cells, and increased HLA-DR on monocytes, are observed between the three cohorts. Also, significant changes in neutrophils and activated monocytes populations are observed after 24 hours of initial treatment (* P<0.05).

CI-8993 Plasma Concentration

The experiments in Figure 14A & B assessed the concentration of CI-8993 in plasma postadministration. Figure 14A shows the arithmetic mean of the concentration-time profiles of CI-8993 following iv administration at 0.15, 0.3, and 0.6 mg/kg to patients with solid tumors in cycle 1. B. Cmax levels versus CI-8993 dose. (* P<0.05)

The results reveal greater-than-proportional exposure increases with higher and multiple doses of CI-8993. The increased half-life at higher doses suggests the ability to saturate the "VISTA sink" consistent with preclinical PK-distribution study predictions (5). No anti-drug antibodies were detected in any of the cohorts.

Conclusions

The clinical results with CI-8993-101 IV administration to cancer patients showed that these patients were safely managed without DLTs at 0.15, 0.3 and 0.6mg/kg dose levels. The clinical results further showed evidence of a rapid, transient and systemic inflammatory response indicated by an increase of the inflammatory soluble mediators.

The observed increase on activated immune cells, and decrease on suppressor cells suggests an early immune response with different anti-tumoral mechanisms.

The observed lower cytokine levels in patients from cohort 3 (0.6 mg/kg) may be associated with the higher step dosing regimen used in this cohort.

The observed saturation kinetics suggest favorable drug bioavailability at higher dose levels.

Further evaluation of the peripheral immune system and PK properties, are ongoing.

42 References

1. EITanbouly MA, Noelle RJ et al. VISTA is a checkpoint regulator for naive T cell quiescence and peripheral tolerance. Science. 2020; 367(5475)

2. Gao J, Ward JF, Pettaway CA, et al. VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer. Nat Med 2017; 23(5):551-55.

3. Kakavand H, Jackett LA, et al. Negative immune checkpoint regulation by VISTA: a mechanism of acquired resistance to anti-PD-1 therapy in metastatic melanoma patients. Mod Pathol. 2017;30(12):1666-76.

4. Johnson M , Lines JL, et al. Phase 1 Study of CI-8993 anti-VISTA antibody in patients with advanced solid tumor malignancies. SITC 2020 Abstract #392

5. Wichmann CW, Burvenich IJG, et al. Preclinical evaluation of anti-VISTA antibody CI-8993 in a syngeneic huVISTA-KI model. SITC 2021 Abstract #324

ADDENDUM TO APPLICATION

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