Bispecific Anti-vista and Anti-CD28 binding proteins and uses thereof

Abstract

Provided herein are bispecific antigen-binding proteins binding to V-domain immunoglobulin suppressor of T-cell activation (VISTA) and CD28, as well as related compositions, nucleic acid molecules, vectors and host cells. Also provided herein are medical uses of such bispecific proteins.

Description

BISPECIFIC ANTI- VISTA AND ANTI-CD28 BINDING PROTEINS ANDUSES THEREOFRELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 631,577, filed on April 9, 2024. The contents of the aforementioned patent application are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SEBI_028_001WO_SeqList_ST26.xml; Size: 47,367; and Date of Creation: March 27, 2025) are herein incorporated by reference in their entirety.TECHNICAL FIELD

[0003] The disclosure is related to bispecific antigen-binding proteins and therapeutic uses of such proteins.BACKGROUND

[0004] Immunotherapy is being explored for treatment of many cancers. One of the serious adverse effects associated with some immunotherapies is cytokine release syndrome, a systemic inflammatory response that can progress to multiple organ failure and death. There is a need for bispecific therapeutic molecules that activate an immune response specifically in the tumor microenvironment while reducing the risk of systemic T-cell activation and cytokine release syndrome.SUMMARY

[0005] Provided herein is a bispecific binding protein comprising a first binding domain that binds to V-domain immunoglobulin suppressor of T-cell activation (VISTA), a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises five polypeptide chains, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the third polypeptide chain comprises the amino acid sequence of SEQID NO: 6, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34; or (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34.

[0006] Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35; or (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35.

[0007] Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA and a second binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39.

[0008] Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the aminoacid sequence of SEQ ID NO: 42; or (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43.

[0009] Provided herein is a pharmaceutical composition comprising a bispecific binding protein disclosed herein, and a pharmaceutically acceptable carrier.

[0010] Provided herein are one or more nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and a first nucleic acid sequence encoding a fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34; or (b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and a first nucleic acid sequence encoding a fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34.

[0011] Provided herein are one or more nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35; or (b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and afirst nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35.

[0012] Provided herein are one or more nucleic acid molecules comprising a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39.

[0013] Provided herein are one or more nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; or (b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43.

[0014] Provided herein is an expression vector comprising the one or more nucleic acid molecules disclosed herein. Provided herein is a recombinant host cell comprising the one or more nucleic acid molecules disclosed herein. Provided herein is a recombinant host cell comprising an expression vector disclosed herein.

[0015] Provided herein is a method of producing a bispecific protein, the method comprising: culturing a recombinant host cell disclosed herein under conditions whereby the one or more nucleic acid molecules are expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0016] Provided herein is a method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a bispecific protein or a pharmaceutical composition disclosed herein. In some embodiments, the cancer is head and neck cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer,esophageal cancer, prostate cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, thyroid cancer, lung cancer, melanoma, or sarcoma. In some embodiments, the cancer is a hematologic cancer.

[0017] In some embodiments, a method for treating a cancer further comprises administering to the subject an inhibitor of PD-1 or an inhibitor of PD-L1. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody or antigen-binding portion thereof. In some embodiments, the inhibitor of PD-L1 is an anti-PD-Ll antibody or antigen-binding portion thereof.

[0018] In some embodiments, a method for treating a cancer further comprises administering to the subject a T-cell engager. In some embodiments, the T-cell engager is a bispecific T-cell engager.

[0019] Provided herein is a bispecific protein or a pharmaceutical composition disclosed herein for use as a medicament.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A - FIG. IB show diagrams of co-stimulation approaches. FIG. 1A shows the conventional CD28xTAA (tumor-associated antigen) co-stimulation approach. FIG. IB shows the CD28x VISTA co-stimulation approach.FIG. 2A - FIG. 21 show results from experiments measuring protein binding of exemplary CD28x VISTA bispecific geometries. FIG. 2A shows diagrams of bispecific geometries / formats BS1 to BS5. Results from ELISA experiments are shown for binding to VISTA (FIG. 2B), binding to CD28 (FIG. 2C), and simultaneous binding to VISTA and CD28 (FIG. 2D - FIG. 2F). Binding was measured at pH 6.0 (FIG. 2E) and pH 7.4 (FIGs. 2B, 2C, 2D, and 2F). Results from flow cytometry experiments are shown for binding to native VISTA on an overexpressing CHO cell line (FIG. 2G, 375,000 copies / cell) or the Kasumi-3 myeloid cell line (FIG. 2H, 70,000 copies / cell), or binding to native CD28 on Jurkat T-cells (FIG. 21) analyzed by flow cytometry. Conventional anti-VISTA (55873 (“anti -VIS TA”, non-pH selective parental clone) and 67375 (“anti-VISTApH'sens”, pH-selective optimized version of 55873)) and anti-CD28 antibodies (CD28 mono) were used as controls. Mean Fluorescent Intensity (MFI) was plotted as a function of mAb concentration.

[0021] FIG. 3A - FIG. 3C shows results from experiments testing CD28xVISTA bispecific induction of IL-2-luciferase reporter expression in cis and in trans. Resultsfrom monovalent CD28 binding are shown in FIG. 3A. Anti-CD28 antibody TGN1412 was used as a positive control. Anti-VISTA antibody 55873 was used as a negative control. Results are shown for bispecific induction in cis (FIG. 3B) and in trans (FIG. 3C). “TCR” = T-cell receptor.

[0022] FIG. 4 shows results from experiments testing CD28x VISTA BS3 bispecific effects on cytokine release and shows results from an in vitro cytokine release assay. In each graph, results from the following proteins are shown from left to right: CD28x67375 BS3 (pH selective), CD28x55873 BS3 (non-pH selective), or TGN1412 (anti-CD28 positive control). Each point represents the results from one donor.

[0023] FIG. 5A - FIG. 5B show results from experiments testing CD28xVISTA bispecific effects on human T-cell mediated killing of LNCaP prostate cancer cells.

[0024] FIG. 6A - FIG. 6F show results from experiments testing CD28xVISTA bispecific effects on tumor growth inhibition (TGI) of a MC38 cell population overexpressing human VISTA in a humanized CD28 mouse model in combination with anti-murine PD-1 (anti-mPD-1). Day 17 was used for TGI analysis due to low N in groups 1-3 at final time point. Tumor growth inhibition (TGI) and statistical significance of endpoint MC38 tumor volumes evaluated using the Mann-Whitney two- sided unpaired t test with exact P values (*0.01 < P < 0.05; ** 0.001 < P < 0.01; *** 0.0001 < P < 0.001).

[0025] FIG. 7A - FIG. 7H shows a cytokine release profile of CD28x67375 BS2 in an ex vivo system that mimics human blood circulation. FIG. 7A is a schematic of the closed-loop system that mimics human blood circulation. FIG. 7B - FIG. 7F are measured levels of IFN-y, IL-2, IL-8, IL-6, and TNFa, respectively, after incubation for 4h with the antibodies and concentrations indicated in freshly collected, circulating blood from 6 healthy human volunteers. Activated T-cells (FIG. 7G) and NK cells (FIG. 7H) gated as CD69+cells and presented as the percentage of total cells of the respective cell type as measured by flow cytometry. Appropriate positive controls with known effects on the test parameters were included, i.e., alemtuzumab (anti-CD52), and anti-CD28 (ANC28.1). Phosphate buffered saline (PBS), cetuximab (anti-epidermal growth factor receptor [EGFR] antibody) and formulation buffer for the 3 tested bsAbs were used as negative controls. BS2: CD28xVISTApH'sens; BS2M: CD28MxVISTApH'sens; R-5678: CD28xPSMA. Lower level of quantification indicated by dotted lines; mean values in red line. A single n= 1 experiment was performed with n= 6 independent donor samples. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 comparison toformulation buffer (B through H) by Paired Student’s t-test with Holm-Sidak correction.

[0026] FIG. 8A - FIG. 8B show expression of VISTA on Kasumi-3 and LNCaP cells. Distribution of hVISTA+ Kasumi-3 human myeloid cells as measured by flow cytometry with a fluorescently labeled anti-h VISTA mAb (green, right) versus isotype control staining (gray, left) is shown in FIG. 8A. Distribution of hVISTA+ LNCaP human prostate cancer cells (green, right) versus isotype control staining (gray, left) is shown in FIG. 8B.

[0027] FIG 9. is a PK profile of CD28xVISTApH-sens BS2 in huCD28 KI mice. Human CD28 knock-in (KI) mice (n=4 per group) were given a single intravenous injection of 5 mg / kg CD28xVISTApH'sensBS2 (circle) or comparators CD28MxVISTApH'sensBS2 (square) or R-5678 (triangle). Serum samples were collected over a 28-day observation period and levels of bsAb were determined by ELISA. Data are presented as mean values + / - SD. Calculated PK parameters are shown in the inset table. KD values collected by Octet BLI (BS2) or Surface Plasmon Resonance (“SPR”)(R-5678).

[0028] FIG. 10A - FIG. 10B is a series of graphs illustrating the results of in vitro CRS assessment in HUVEC:PBMC co-culture assays. Levels of cytokines GM-CSF, IL-6, IFN-y, IL-8, IL-2, IL- 10, IL-4, and TNFa were measured 2 days after incubation with CD28MxVISTApH'sensBS2 (column 1, all graphs), CD28xVISTApH-sensBS2 (column 2, all graphs), TGN1412 (column 3, all graphs) or R-5678 (column 4, all graphs) at concentrations of 1, 10, 30, and 100 pg / ml. Quantification was done with 6 replicates for each concentration of each mAb. Each dot represents the results from one human donor (4 donors tested).

[0029] FIG. HA - FIG. 11G is a series of graphs illustrating the results of a full blood cell characterization from ex vivo human whole blood ID. Flow assay. Granulocyte counts (FIG. 11 A) and, activation (CD1 lb+)(FIG. 11B) were not significantly affected by any of the test items at all three concentrations. Monocyte counts (FIG. 11C) and, activation (CD83+)(FIG. HD) were not significantly affected by any of the test items at all three concentrations. Indicated treatments had no effect on blood cell viability including platelet (PLT, FIG. HE), white blood cell (WBC, FIG. HF); and red blood cell (RBC, FIG. HG) counts. A single n= 1 experiment was performed with n= 6 independent donor samples. Symbols represent individual donor samples, mean values are indicated by the solid line. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001comparison to formulation buffer (FIG. 11B - FIG. 11G) by Paired Student’s t-test with Holm-Sidak correction. BS2: CD28xVISTApH-sens; BS2M: CD28MxVISTApH-sens; R-5678: CD28xPSMA.

[0030] FIG. 12 is a graph illustrating the body weight of animals in Example 10. Initial body weight was tested by One-way ANOVA with Multiple Comparisons (GraphPad Prism 10). There was a trend toward group 6 having lower initial body weight compared to other groups, though the only comparison reaching statistical significance was between groups 4 and 6 (P= 0.0322).

[0031] FIG. 13A - FIG. 13B is a series of spider plots illustrating the individual tumor growth trajectories in groups 1-4 (FIG. 13A) and groups 5-6 (FIG. 13B) from Example 10. Day 18 was the cutoff for statistical significance and is indicated with a dashed vertical line.

[0032] FIG. 14A - FIG. 14B is a series of graphs illustrating the mean tumor volume vs time for animals in groups 1-4 (FIG. 14A) and groups 5-6 (FIG. 14B) from Example 10. Day 18 was the cutoff for statistical significance. Tabulations of the data can be found in Table 13.DETAILED DESCRIPTION

[0033] Provided herein are bispecific anti-VISTA and anti-CD28 binding proteins and medical uses thereof. VISTA is a B7-family member immune checkpoint that promotes T-cell and myeloid quiescence and is a potential target for cancer treatment in humans. As VISTA is highly expressed on myeloid cells, including those in the blood, antibodies binding VISTA at physiological pH 7.4 can result in rapid elimination from circulation through target-mediated drug disposition (TMDD), making efficacious drug occupancy levels difficult to reach and potentially narrowing the therapeutic window. Further, the interaction of VISTA with its receptor P-selectin glycoprotein ligand-1 (PSGL-1) was demonstrated to be significantly enhanced by the acidic tumor microenvironment (TME) (Johnston et al. Nature, 2019; 574:565-570). CD28 is a homodimeric glycoprotein expressed on T lineage cells. CD28 transduces a positive signal that promotes proliferation of T-cell receptor (TCR)-stimulated T-cells.

[0034] Provided herein are pH-selective CD28x VISTA bispecific binding proteins that act within the acidic TME. pH-selective VISTA binding domains may be derived from from clone 67375 and may be indicated by “VISTApH'sens”. These bispecific bindingproteins are designed for selective “cv.s-activation” or tripartite “ / ra / z.s-activation” of CD28 in the TME, aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and cytokine release syndrome (CRS) risk. Cis- activation relies on engagement of VISTA on tumor cells, in the synapse formed between a T-cell and a tumor cell. The / ra / z.s-activation mechanism relies on engagement of VISTA on myeloid cells, where this immune checkpoint acts to suppress T-cell activation in the low pH environment (~pH 6) found in many tumors. CD28x VISTA bispecific binding proteins provided herein are useful for tumor-targeted CD28 agonism and T-cell co-stimulation.

[0035] The CD28x VISTA co-stimulation approach (FIG. IB) takes advantage of the abundant tumor infiltration by VISTA+myeloid cells. pH-selective VISTA binding of a bispecific binding protein ensures CD28 clustering on T-cells in the low pH tumor microenvironment with minimal risk of systemic CRS. The approach described here bypasses the requirement for specific tumor associated antigen (TAA) of a conventional CD28xTAA co-stimulation approach shown in FIG. 1A.BISPECIFIC BINDING PROTEINS

[0036] Provided herein are bispecific binding proteins that bind VISTA (e.g., human VISTA) and CD28 (e.g., human CD28). Bispecific proteins provided herein comprise at least one binding domain that binds to VISTA and at least one binding domain that binds to CD28.

[0037] In some embodiments, a bispecific protein comprises a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28. In some embodiments, a bispecific protein comprises a first binding domain that binds to VISTA and a second binding domain that binds to CD28.

[0038] Bispecific proteins provided herein comprise domains and regions of antibody molecules. The term “antibody” broadly refers to an immunoglobulin (Ig) molecule, generally, comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential target binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0039] In a full-length antibody, each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH domain) and a heavy chain constant region. Theheavy chain constant region comprises three domains, CHI, CH2 and CH3. IgG, IgA, and IgD constant regions comprise a flexible hinge region between the CHI domain and the CH2 domain. Each light chain comprises a light chain variable domain (abbreviated herein as VL domain) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL domains 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 domain and VL domain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0040] The term “Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof. The numbering of the residues in the Fc region is according to the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. An Fc region can be present in dimer or monomeric form. The Fc region binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. In some embodiments, a bispecific protein provided herein comprises an Fc region.

[0041] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY) and class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2) or subclass. IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen combining domains, each composed of a VH) and a VL. Generally, IgA antibodies are composed of two monomers, each monomer composed of two heavy chains and two light chains (as for IgG, IgD, and IgE antibodies); in this way the IgA molecule has four antigen binding domains, each again composed of a VH and a VL. Certain IgA antibodies are monomeric in that they are composed of two heavy chains and two light chains. Secreted IgM antibodies are generally composed of five monomers, each monomer composed of two heavy chains and two light chains (as for IgG and IgE antibodies). Thus, the IgM molecule has ten antigen binding domains, each again composed of a VH and a VL. A cell surface form of IgM has a two heavy chain / two light chain structure similar to IgG, IgD and IgE antibodies.

[0042] As used herein, the terms “immunological binding” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule (e.g., antibody or antigen-binding portion thereof), or a protein comprising an immunoglobulin-derived binding domain(s) and an antigen for which the immunoglobulin or protein is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigenbinding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koir) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See, Malmqvist, Nature 361 : 186-187 (1993)). The ratio of Koir / Kon enables the cancellation of all parameters not related to affinity and is equal to the dissociation constant Kd. (See, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody or antigen-binding portion provided herein is said to specifically bind PD-L1 or CD3 when the equilibrium binding constant (Kd) is <10 |1M, preferably < 10 nM, more preferably < 10 nM, and most preferably < 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. One method for determining the Kd of an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system.

[0043] Functionally, the binding affinity of a protein provided herein may be within the range of 10'5M to 10'12M. For example, the binding affinity of a protein provided herein is from 10'6M to 10'12M, from 10'7M to 10'12M, from 10'8M to 10'12M, from 10'9M to IO’12M, from 10’5M to 10’11M, from IO’6M to 10’11M, from IO’7M to 10’11M, from 10'8M to 10'11M, from 10'9M to 10'11M, from 10'10M to 10'11M, from 10'5M to IO’10M, from IO’6M to 10-10M, from IO’7M to IO’10M, from IO’8M to IO’10M, from 10'9M to 10'10M, from 10'5M to 10'9M, from 10'6M to 10'9M, from 10'7M to 10'9M, from 10'8M to 10'9M, from 10'5M to 10'8M, from 10'6M to 10'8M, from 10"7M to 10'8M, from 10'5M to 10'7M, from 10'6M to 10'7M or from 10'5M to 10'6M.

[0044] Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, optionally, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28. In some embodiments, the first binding domain that binds to VISTA comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the first VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 3; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 4; and a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 5; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 8; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 9; and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first binding domain that binds to VISTA comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 14; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and the VL domain amino acid sequence comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 8; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 18; and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 19.

[0045] In some embodiments, the first binding domain that binds to VISTA comprises a VH domain and a VL domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 2, and the VL domain comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first binding domain that binds to VISTA comprises a VH domain and a VL domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 12, and the VL domain comprises the amino acid sequence of SEQ ID NO: 17.

[0046] In some embodiments, the first binding domain that binds to VISTA and the second binding domain that binds to VISTA comprise identical amino acid sequences.Table 1. Amino acid sequences of anti- VISTA antibody 67375 (anti-VISTApH sens)CDR sequences are underlined in heavy chain, light chain, and variable domain sequences. Variable domain sequences are in bold font in heavy chain and light chain sequences.Table 2. Amino acid sequences of anti- VISTA antibody 55873 (anti-VISTA)CDR sequences are underlined in heavy chain, light chain, and variable domain sequences. Variable domain sequences are in bold font in heavy chain and light chain sequences.Table 3. Amino acid sequences of anti-CD28 antibody TGN1412CDR sequences are underlined in heavy chain, light chain, and variable domain sequences. Variable domain sequences are in bold font in heavy chain and light chain sequences.

[0047] In some embodiments, a bispecific binding protein has one of the formats shown in FIG. 2A

[0048] In some embodiments, a bispecific binding protein comprises a single chain Fv (scFv), wherein the two domains of the Fv fragment, VL and VH, are joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules. In some embodiments, a binding domain that binds to CD28 is an scFv. In some embodiments, a binding domain that binds to VISTA is an scFv. In some embodiments, the linker joining the VH and VL domains in an scFv is a glycine-serine linker. In some embodiments, the glycine-serine linker comprises or consists of the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 44. Exemplary bispecific binding protein formats that comprise an scFv CD28 binding domain are the BS3, BS4, and BS5 formats.

[0049] In some embodiments, a bispecific binding protein comprises two identical VH domains that bind to VISTA, wherein the identical VH domains are in the same polypeptide chain. In some embodiments, the two identical VH domains are joined by a linker. In some embodiments, the linker joining the VH domains is a glycine-serine linker. In some embodiments, the glycine-serine linker comprises or consists of the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 44. Exemplary bispecific binding protein formats that comprise identical anti-VISTA VH domains in the same polypeptide chain are BS2 and BS3.

[0050] In some embodiments, a bispecific protein comprises five polypeptide chains. In some embodiments, a bispecific protein comprises five polypeptide chains and comprises a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28.

[0051] In some embodiments, a bispecific binding protein comprises four polypeptide chains. In some embodiments, a bispecific protein comprises four polypeptide chains and comprises a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28. In some embodiments, a bispecific protein comprises four polypeptide chains and comprises a first binding domain that binds to VISTA and a second binding domain that binds to CD28.

[0052] In some embodiments, a bispecific binding protein has the BS2 format (FIG. 2A). In some embodiments a bispecific binding protein is CD28x67375 BS2 (pH selective) or CD28x55873 BS2 (non-pH selective). Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises five polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34. Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises five polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQID NO: 40, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34.

[0053] In some embodiments, a bispecific binding protein has the BS3 format (FIG. 2A). In some embodiments a bispecific binding protein is CD28x67375 BS3 (pH selective) or CD28x55873 BS3 (non-pH selective). Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35. Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35.

[0054] In some embodiments, a bispecific binding protein has the BS1 format (FIG. 2A). In some embodiments a bispecific binding protein is CD28x55873 BS1 (non-pH selective). Provided herein is a bispecific binding protein that comprises a first binding domain that binds to VISTA and a second binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39.

[0055] In some embodiments, a bispecific binding protein has the BS4 format (FIG. 2A). In some embodiments a bispecific binding protein is CD28x55873 BS4 (non-pHselective). Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42.

[0056] In some embodiments, a bispecific binding protein has the BS5 format (FIG. 2A). In some embodiments a bispecific binding protein is CD28x55873 BS5 (non-pH selective). Provided herein is a bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43.Table 4. Glycine-serine linker sequencesTable 5. Amino acid sequences of CD28x67375 BS2 bispecificCDR sequences are underlined and variable domain sequences are in bold font in heavy chain and light chain sequences.Table 6. Amino acid sequences of CD28x67375 BS3 bispecificCDR sequences are underlined and variable domain sequences are in bold font in heavy chain, light chain, and heavy / light chain sequences.Table 7. Amino acid sequences of CD28x55873 BS1 bispecificCDR sequences are underlined and variable domain sequences are in bold font in heavy chain and light chain sequences.Table 8. Amino acid sequences of CD28x55873 BS2 bispecificCDR sequences are underlined and variable domain sequences are in bold font in heavy chain and light chain sequences.Table 9. Amino acid sequences of CD28x55873 BS3 bispecificCDR sequences are underlined and variable domain sequences are in bold font in heavy chain, light chain, and heavy / light chain sequences.Table 10. Amino acid sequences of CD28x55873 BS4 bispecificCDR sequences are underlined and variable domain sequences are in bold font.Table 11. Amino acid sequences of CD28x55873 BS5 bispecificCDR sequences are underlined and variable domain sequences are in bold font.

[0057] In some embodiments, a bispecific binding protein provided herein comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region comprises one or more mutations to reduce or prevent FcyR binding, antibody-dependent cell-mediated cytotoxicity activity, and / or complement-dependent cytotoxicity activity. In some embodiments, the immunoglobulin constant region is a human IgG4 constant region comprising the amino acid substitution S228P, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A, or a human IgGl constant region comprising the amino acid substitutions L234A, L235A, G237A and P331 S, wherein numbering is according to the EU index as in Kabat. In some embodiments, a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to the EU index as in Kabat (Ward et al.. 1995 Therap. Immunol. 2:77-94).

[0058] In some embodiments of the bispecific proteins provided herein, the protein may comprise one polypeptide chain that comprises a first immunoglobulin constant region and another polypeptide chain that comprises a second immunoglobulin constant region, and wherein the first immunoglobulin constant region and the second immunoglobulin constant region comprise knob-in-hole mutations.

[0059] In some embodiments, a bispecific protein provided herein may comprise any heterodimerization mutations or heterodimerization technology. In some embodiments, a protein provided herein may not comprise any heterodimerization mutations or heterodimerization technology. In such embodiments, purification techniques may be used to isolate the protein.

[0060] In some embodiments, a bispecific protein provided herein may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. The bispecific protein may be mutated to altersuch post-translational modifications, for example by adding, removing or replacing one or more amino acid residues to form or remove a glycosylation site.

[0061] In some embodiments, the bispecific protein provided herein may be modified for example by amino acid substitution to remove potential proteolytic sites in the antibody or portion.

[0062] In some embodiments, a bispecific binding protein provided herein may be isolated.

[0063] Further provided herein is an immunoconjugate comprising a bispecific protein, linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic peptide, a therapeutic nucleic acid, an anti -angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0064] Examples of suitable therapeutic agents include, but are not limited to, immunomodulatory agents, cytotoxins, radioisotopes, chemotherapeutic agents, anti- angiogenic agents, antiproliferative agents, pro-apoptotic agents, and cytostatic and cytolytic enzymes (for example, RNAses). Further therapeutic agents include a therapeutic nucleic acid, such as a gene encoding an immunomodulatory agent, an anti- angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent. These drug descriptors are not mutually exclusive, and thus a therapeutic agent may be described using one or more of the above terms.

[0065] Examples of suitable therapeutic agents for use in immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansines, CC-1065 and the duocarmycins, the calicheamicins and other enediynes, and the auristatins. Other examples include the anti-folates, vinca alkaloids, and the anthracyclines. Plant toxins, other bioactive proteins, enzymes (z.e., ADEPT), radioisotopes, photosensitizers may also be used in immunoconjugates. In addition, conjugates can be made using secondary carriers as the cytotoxic agent, such as liposomes or polymers, Suitable cytotoxins include an agent that inhibits or prevents the function of cells and / or results in destruction of cells. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and disrupt DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins.

[0066] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin,nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin, tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, flurouracils, etoposide, taxol, taxol analogs, platins such as cis-platin and carbo-platin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterlins, esperamicins and maytansinoids.PHARMACEUTICAL COMPOSITIONS

[0067] The anti-VISTA and anti-CD28 bispecific binding proteins provided herein (also referred to herein as “active compounds”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an anti-VISTA and anti-CD28 bispecific binding protein (or an immunoconjugate comprising said protein), and a pharmaceutically acceptable carrier, diluent or excipient. Such materials should be non-toxic and should not interfere with the efficacy of the bispecific binding protein. The precise nature of the carrier or other material will depend on the route of administration, which may be by injection, bolus, infusion, or any other suitable route, as discussed below.

[0068] Provided herein is a pharmaceutical composition comprising an anti-VISTA and anti-CD28 bispecific binding protein comprising the amino acid sequences of any one of the proteins in Table 5 - Table 11, and a pharmaceutically acceptable carrier.

[0069] As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.” As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, whichis incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutically acceptable carrier, diluent or excipient may be a compound or a combination of compounds that does not provoke secondary reactions and that allows, for example, facilitation of the administration of the bispecific binding protein, an increase in its lifespan and / or in its efficacy in the body or an increase in its solubility in solution.

[0070] A pharmaceutical composition disclosed herein may be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (z.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0071] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0072] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0073] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0074] For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0075] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0076] The pharmaceutical agents can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0077] In some embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.

[0078] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0079] In some embodiments, the bispecific binding protein may be provided in a lyophilized form for reconstitution prior to administration. For example, lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0080] The pharmaceutical compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.NUCLEIC ACID MOLECULES, VECTORS, HOST CELLSAND METHODS OF PRODUCING BISPECIFIC BINDING PROTEINS

[0081] Provided herein is a nucleic acid molecule (or a set of nucleic acid molecules) encoding an amino acid sequence of an anti-VISTA and anti-CD28 bispecific binding proteins disclosed herein. In some embodiments, a single nucleic acid molecule encodes all the polypeptide chains forming the bispecific binding protein. In some embodiments, a set of nucleic acid molecules is provided, wherein each polypeptide chain of the bispecific binding protein is encoded by a separate nucleic acid molecule.

[0082] Provided herein are one or more nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and a first nucleic acid sequence encoding a fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34; or (b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and a first nucleic acid sequence encoding a fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34.

[0083] Provided herein are one or more nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, a first nucleic acid sequence encoding a secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35; or (b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35.

[0084] Provided herein are one or more nucleic acid molecules comprising a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39.

[0085] Provided herein are one or more nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; or (b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43.

[0086] Also provided herein is an expression vector comprising a nucleic acid molecule described herein. In some embodiments, a single expression vector expresses all the polypeptide chains forming the bispecific binding protein. In some embodiments, a set of expression vectors is provided, wherein each polypeptide chain of the bispecificbinding protein is expressed by a separate vector. In certain vectors, a nucleic acid molecule is operatively linked to one or more regulatory sequences suitable for expression of the nucleic acid segment in a host cell. In some cases, an expression vector comprises sequences that mediate replication and comprises one or more selectable markers. As used herein, “vector” means a construct that is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0087] Provided herein is a recombinant host cell comprising an expression vector or a nucleic acid molecule disclosed herein. Provided herein is a recombinant host cell comprising a set of expression vectors or a set of nucleic acid molecules disclosed herein. A “host cell” includes an individual cell, a cell line or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. An expression vector can be transfected into a host cell by standard techniques. Non-limiting examples include electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like.

[0088] Bispecific binding proteins provided herein can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies, computational technologies or combinations of such technologies or other technologies readily known in the art.

[0089] Further provided herein is a method for producing a bispecific binding protein, the method comprising: culturing a recombinant host cell comprising an expression vector described herein under conditions whereby the nucleic acid segment is expressed, thereby producing the bispecific binding protein. The bispecific binding protein may then be isolated from the host cell or culture. Bispecific binding proteins can be produced by any of a variety of methods known to those skilled in the art. In certain embodiments, bispecific binding proteins can be produced recombinantly. For example, nucleic acid sequences encoding one or more of the amino acid sequences inTable 5 - Table 11 may be introduced into a bacterial cell (e.g., E. coli, B. subtilis) or a eukaryotic cell (e.g., a yeast such as S. cerevisiae, or a mammalian cell such as a CHO cell line, various Cos cell lines, a HeLa cell, a HEK293 cell, various myeloma cell lines, or a transformed B-cell or hybridoma), or into an in vitro translation system, and the translated polypeptide may be isolated. In some embodiments, light chain polypeptides and heavy chain polypeptides (or combined heavy / light chain polypeptides) are produced in a cell with a signal sequence that is removed upon production of a mature bispecific binding protein.

[0090] Those skilled in the art will be able to determine whether a bispecific binding protein comprising a given amino acid sequence binds to VISTA and CD28 proteins without undue experimentation using standard methodologies, for example, Western blots, ELISA, and the like.USES OF BISPECIFIC BINDING PROTEINS

[0091] Provided herein are methods and uses of the anti-VISTA and anti-CD28 bispecific binding proteins, immunoconjugates and pharmaceutical compositions described herein for providing a therapeutic benefit to a subject with cancer.

[0092] In some embodiments, an anti-VISTA and anti-CD28 bispecific binding protein as described herein does not induce CRS when administered to a subject. In some embodiments, an anti-VISTA and anti-CD28 bispecific binding protein as described herein induces minimal CRS when administered to a subject.

[0093] An anti-VISTA and anti-CD28 bispecific binding protein as described herein may be used in a method of treatment of the human or animal body. In some embodiments, an anti-VISTA and anti-CD28 bispecific binding protein as described herein may be used in prophylactic or preventative treatment (e.g., treatment before the onset of a condition in a subject to reduce the risk of the condition occurring in the subject; delay its onset; or reduce its severity after onset). The method of treatment may comprise administering the anti-VISTA and anti-CD28 bispecific binding protein to a subject in need thereof.

[0094] Provided herein is a method for inducing an immune response in a subject, the method comprising administering to the subject an anti-VISTA and anti-CD28 bispecific binding protein, an immunoconjugate, or a pharmaceutical composition disclosed herein. Provided herein is a method for inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effectiveamount of an anti-VISTA and anti-CD28 bispecific binding protein, an immunoconjugate, or a pharmaceutical composition disclosed herein. In some embodiments, the immune response is an immune response against cancer cells. In some embodiments, the immune response is a cytotoxic response. In some embodiments, the immune response is antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or antibody-dependent cellular phagocytosis. In some embodiments, the immune response is an antibody response.

[0095] Provided herein is a method for treating or preventing a cancer in a subject, the method comprising administering to the subject an anti-VISTA and anti-CD28 bispecific binding protein, an immunoconjugate or a pharmaceutical composition disclosed herein. Provided herein is a method for for treating or preventing a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-VISTA and anti-CD28 bispecific binding protein, an immunoconjugate or a pharmaceutical composition disclosed herein.

[0096] Provided herein is a method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a bispecific protein or a pharmaceutical composition disclosed herein.

[0097] In some embodiments, the cancer is head and neck cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, thyroid cancer, lung cancer, melanoma, or sarcoma. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is a myeloid malignancy. In some embodiments, the cancer is acute myeloid leukemia (AML).

[0098] As used herein, the term “effective amount” or “therapeutically effective amount” refers to the amount of a pharmaceutical agent, e.g., an anti-VISTA and anti- CD28 bispecific binding protein, which is sufficient to reduce or ameliorate the severity and / or duration of a disease, e.g., cancer, or one or more symptoms thereof, prevent the advancement of a disease, cause regression of a disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disease, detect a disease, or enhance or improve the prophylactic or therapeutic effect(s) of another related therapy (e.g., prophylactic or therapeutic agent) for a VISTA-mediated disease.

[0099] The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammalbeing treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and / or progression of a disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann J. A. et aL, 1991, hit. J. Cancer T. 659-664; Bagshawe K.D. et aL, 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of an antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g., whole antibody, fragment) and the nature of any detectable label or other molecule attached to the antibody.

[0100] A typical bispecific binding protein dose will be in the range 100 pg to 1 g for systemic applications, and 1 pg to 1 mg for intradermal injection. An initial higher loading dose, followed by one or more lower doses, may be administered. This is a dose for a single treatment of an adult subject, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for a subject may be dependent on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration and the nature of the condition being treated.

[0101] Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g., about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Treatment may be given before, and / or after surgery, and / or may be administered orapplied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.

[0102] In some embodiments, a subject may be treated with a bispecific binding protein, an immunoconjugate or a pharmaceutical composition described herein and an additional therapeutic agent or therapy that is used to treat a VISTA-mediated disease or disorder (e.g., cancer) or a symptom or complication of a VISTA-mediated disease or disorder. The bispecific binding protein and the additional therapeutic agent or therapy may be administered simultaneously or sequentially.

[0103] In some embodiments, a method for treating a cancer further comprises administering to the subject an inhibitor of PD-1 or an inhibitor of PD-L1. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody or antigen-binding portion thereof. In some embodiments, the inhibitor of PD-L1 is an anti-PD-Ll antibody or antigen-binding portion thereof.

[0104] In some embodiments, a subject does not respond or has developed a resistance to cancer treatment with an inhibitor of PD-1 or PD-L1. In some embodiments, a method provided herein further comprises administering to the subject an inhibitor of PD-1 or PD-L1. In some embodiments, administering a bispecific binding protein provided herein along with an inhibitor of PD-1 or PD-L1 produces a synergistic anticancer effect.

[0105] In some embodiments, an inhibitor of PD-1 or PD-L1 is an antibody or an antigen-binding portion thereof that disrupts the interaction between the human PD-1 receptor and its ligand, human PD-L1. Antibodies known in the art which bind to PD- 1 and disrupt the interaction between the PD-1 and its ligand, PD-L1, and stimulate an anti-tumor immune response, are suitable for use in the methods disclosed herein. In some embodiments, the antibody or antigen-binding portion thereof binds specifically to or targets PD-1. For example, antibodies that target PD-1 include, e.g., nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475, Merck), dostarlimab, tislelizumab and cemiplimab. Other suitable antibodies for use in the methods disclosed herein are anti-PD-1 antibodies disclosed in US 8,008,449, herein incorporated by reference in its entirety. In certain embodiments, the antibody or antigen-binding portion thereof binds specifically to or targets PD-L1 and inhibits its interaction with PD-1. Antibodies known in the art which bind to PD-L1 and disrupt the interaction between the PD-1 and PD-L1, and stimulates an anti -turn or immune response, are suitable for use in the methods disclosed herein. For example, antibodiesthat target PD-L1 include BMS-936559 (also known as MDX 1105, Bristol-Myers Squibb), atezolizumab (Genentech), durvalumab (AstraZeneca) and avelumab (MSB0010718C). Other suitable antibodies that target PD-L1 are disclosed in US 7,943,743, herein incorporated by reference in its entirety. Any antibody that binds specifically to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response, is suitable for use in the methods disclosed herein.

[0106] In some embodiments, a method for treating a cancer further comprises administering to the subject a T-cell engager. In some embodiments, the T-cell engager is a bispecific T-cell engager. In some embodiments, a bispecific T-cell engager binds to a TAA and CD3. In some embodiments, a bispecific T-cell engager binds to PSMA and CD3. In some embodiments, a bispecific T-cell engager binds to HER2 and CD3. In some embodiments, a bispecific T-cell engager binds to EpCAM and CD3. In some embodiments, a T-cell engager is a checkpoint-inhibitory T-cell engager, a simultaneous multiple interaction T-cell engager, a trispecific killer engager, or a BiTE- expressing chimeric antigen receptor (CAR) T-cell.

[0107] In some embodiments, the therapeutic effect of a bispecific protein may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of a bispecific protein may persist in a subject for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.

[0108] In some embodiments, a subject is a human, a non-human primate, a cynomolgus monkey, a pig, a horse, a cow, a dog, a cat, a guinea pig, a mouse or a rat. In some embodiments, a subject is an adult human. In some embodiments, a subject is a pediatric human.

[0109] Further provided herein is a bispecific protein, an immunoconjugate, or a pharmaceutical composition described herein, for use in the treatment of a disease or a disorder (e.g., cancer).

[0110] Provided herein is a bispecific protein, an immunoconjugate, or a pharmaceutical composition disclosed herein for use as a medicament.DEFINITIONS[OHl] Unless otherwise noted, the terms used herein have definitions as ordinarily used in the art. Some terms are defined below, and additional definitions can be found within the rest of the detailed description.

[0112] The term “a” or “an” refers to one or more of that entity, i.e., can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0113] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0114] As used herein, the term “HCDR” refers to a heavy chain complementarity determining region. As used herein, the term “LCDR” refers to a light chain complementarity determining region.

[0115] The term “immunoconjugate” refers to a bispecific protein that binds to VISTA and CD28 and that is conjugated to cytotoxic, cytostatic and / or therapeutic agents.

[0116] The term “isolated molecule” (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be “isolated” from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known in the art for purification.

[0117] The term “potency” is a measurement of biological activity and may be designated as IC50, EC50, or effective concentration of an anti-VISTA bispecific binding protein or antibody to inhibit 50% of activity measured in a VISTA activity assay as described herein.

[0118] The term “inhibit” or “neutralize” as used herein with respect to bioactivity of a bispecific binding protein or antibody disclosed herein means the ability of the antibody to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, eliminate, stop, reduce or reverse for example progression or severity of that which is being inhibited including, but not limited to, a biological activity or binding interaction of the bispecific protein or antibody molecule to VISTA.

[0119] As used herein, the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) mean that the severity of the subject's condition is reduced, at least partially improved or stabilized and / or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder.

[0120] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term’s definition in the application, the definition that appears in this application controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0121] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of thealternatives. As used herein, the terms “include” and “comprise” are used synonymously.

[0122] The disclosure will be further clarified by the following examples, which are intended to be purely exemplary of the disclosure and in no way limiting.EXAMPLESExample 1. Generation of bispecific proteinsCD28xVISTA bispecific proteins

[0123] CD28xVISTA bispecific proteins (bsAbs) were based on a single CD28- binding Fab arm or disulfide-stabilized scFv derived from mAb TGN1412 (including a mutation changing a Cys sequence liability in VH CDR2 to Ser) and VISTA-binding Fab arms from anti-VISTA mAb 55873 (“anti -VIS TA”, non-pH selective parental clone) or 67375 (“VISTApH'sens”, pH-selective lead optimized version of 55873).

[0124] These mAbs were obtained through selection of yeast-based platform libraries alternating between positive enrichment rounds at pH 6.0 and negative selection rounds at pH 7.4. pH-dependent agonism was achieved by incorporating VISTA binding Fab domains from clone 67375 with highly pH-selective target binding: KD= 0.8 nM at pH 6.0, and >400-fold lower binding at pH 7.4 (KD= 353 nM; (see e.g., Thisted T et al. VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response. Nat Commun 2024;15(l):2917). Metabolism, gene and protein expression and survival of primary human T-cells is thought to be affected by even short term exposure to low pH in vitro. Therefore, a parallel set of “surrogate” bispecific antibodies were generated by incorporating VISTA Fab arms from parental antibody clone 55873 (to ensure binding to the same epitope) which bind VISTA at pH 7.4 and pH 6.0 with similar binding affinities (KD,PH7.4= 1 nM vs. KD,PH6.O= 0.6 nM, respectively; data not shown). These surrogate bsAbs allowed cell-based in vitro assays to be conducted at neutral pH while all in vivo experiments and CRS assessment studies were conducted using the pH- selective, VISTApH'sens, described above.

[0125] To ensure proper heavy and light chain pairing and purification of correctly formed homodimer, CrossMab (CH1-CL) technology was utilized (see, e.g., Schaefer W et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci U S A 2011;108(27): 11187-92.) combined with Knob-side heavy chain (HC) mutations: T366W; disulfide stabilizingmutation: S354C; and Hole-side HC mutations: T366S / L368A / Y407V, disulfide stabilizing mutation: Y349C, and mutations: H435R / Y436F minimizing Protein A binding. The CD28-binding scFv domain derived from TGN1412 was engineered for enhanced stability by introducing a disulfide bond between VH44 and VL100 (Kabat numbering). CD28xVISTA bsAbs all contain the L234S / L235T / G236R mutations silencing FcyR interactions.

[0126] CD28xVISTA bispecific proteins (bsAbs) were expressed by transient cotransfection of the relevant heavy, light, and mixed chain expression constructs into CHO cells. Synthetic genes carrying an N-terminal IL-2 signal peptide (GeneArt, Thermo Fisher Scientific) were cloned into the expression vector pcDNA™3.4 TOPO® (Thermo Fisher Scientific). Heavy and light chain sequences as indicated in Table 5 - Table 11 were co-expressed using the ExpiCHO™ Expression System (Thermo Fisher Scientific) and antibody purification performed on a Protein A affinity column (HiTrap™ MabSelect SuRe™ pcc resin; Cytiva 17549112)) with 0.1M Glycine, pH 3.2 elution.

[0127] After neutralization and pooling of main peak fractions, material was buffer exchanged into PBS (Lonza 17-517Q) using a pPulse Tangential Flow System. An IEX polishing step was conducted as needed, and protein concentration of purified material was determined from the absorbance at 280 nm, where the molar extinction coefficient at 280 nm was calculated based on the amino acid sequence. Endotoxin levels were measured using an Endosafe® nexgen-PTS™ instrument (Charles River).CD28xPSMA bispecific proteins

[0128] The CD28xPSMA comparator R-5678 was generated based on the variable regions of REGN5678 (employing a common LC for the CD28 and PSMA binding arm; construct bsl6429D as described in US Patent No. 11,548,974) fused to IgGl HC and Kappa LC with the HC pairing and Fc-null mutations mentioned above, and a common LC for the CD28 and PSMA binding arm.Other antibodies

[0129] TGN1412 (anti-CD28) was generated according to the methods defined in US patent application No. 10 / 399,056, the contents of which are herein incorporated by reference in their entirety.

[0130] In vivo studies adhered to FDA guidance, maintaining endotoxin levels below 5EU / kg / h. For ex vivo CRS studies, endotoxin concentrations were kept below 0.01EU / ml in blood, following Immuneed’s specifications. QC included analytical SEC with a typical monomeric purity of >95%. Specifically, for CD28xVISTApH'sensBS2, an SEC-HPLC purity of >99% monomer consisting exclusively of correctly paired heterodimer as analyzed by intact LC / MS was achieved after Protein A capture and a single IEX polishing step.Example 2. Cell Culture Methods

[0131] The source of the cell lines as follows: MC38 (Kerafast ENH204-FP), Jurkat (Clone E6-1; ATCC TIB- 152), LNCaP (Clone FCG; ATCC CRL-1740) HEK-293 (ATCC CRL-1573), CHO-K1 (ATCC CCL-61), Kasumi-3 (ATCC CRL-2725). The Jurkat-IL-2-luciferase reporter cells were sourced from Promega (CD28 Bioassay, Core Kit; Promega JA6701). The CHO-K1 / VISTA stable cell line was purchased from GenScript (GenScript M00533)

[0132] HEK-293 cells were engineered by using the plasmid pcDNA3.1(+) (Thermo) for expression of membrane bound scFv derived from anti-CD3 clone OKT3 fused to the CD8 a-chain hinge and transmembrane domain or a fusion of this construct to the human VISTA gene through a furin cleavage site and T2A sequence allowing for coexpression of OKT3 scFv and VISTA from the same plasmid (as described in Yang S, et al. Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Ther 2008;15(21): 1411-23.)

[0133] The expression constructs were introduced into HEK-293 cells by Nucleofection (Lonza), and subsequent selection was performed in Neomycin- containing media. MC38 cells overexpressing human VISTA were generated by transducing MC38 cells with lentivirus encoding a hVISTA-IRES-Puromycin gene fusion construct from an EFla promoter (custom gene expression lentivirus; VectorBuilder). All other genes were codon optimized and custom synthesized by GeneArt (Thermo Fisher Scientific).Example 3. Target binding of exemplary CD28x VISTA antibodiesELISA Methods

[0134] Recombinant protein binding to various monospecific antibodies and bispecific proteins (bsAbs) was tested by ELISA experiments. A CD28 binding arm was combined with either a pH-selective VISTA binding arm or a “surrogate” arm with similar binding affinity at pH 6.0 and 7.4 for in vitro assays. As a comparator, a CD28binding arm with Fc silencing and chain-pairing mutations was combined with a pH- selective VISTA binding arm (“CD28MxVISTApH'sens”). These were all in a human IgGl backbone containing mutations silencing FcyR interactions (see, e.g., Wilkinson I, etal. Fc-engineered antibodies with immune effector functions completely abolished. PLoS One 2021;16(12):e0260954) to avoid clustering by interaction with Fc receptors on various cell surfaces. Without wishing to be bound by theory, it is thought that these mutations minimize VISTA-independent agonism and associated CRS risks such that CD28 agonism is dependent on the VISTA Fab arms engaging with their target on VISTA+ cells.

[0135] 96-well flat bottom plates (Coming) were coated with recombinant human VISTA-His (Sensei Biotherapeutics) or CD28-His (Aero Biosystems), 5 pg / ml in PBS pH 7.4 overnight at 4°C. The plates were blocked with PBS 7.4 or PBS pH 6.0 containing 2% NFDM for 2h at RT. The CD28xVISTA bsAbs (bispecific geometries / formats shown in FIG. 2A) as well as anti-VISTA and anti-CD28 monospecific control monoclonal antibodies (mAbs) were three-fold serially diluted in PBS pH 7.4 or PBS pH 6.0 containing 1% NFDM starting at 300 nM before being added to the coated plates. Plates were incubated at room temperature (RT) for 2h. After washing 5x with PBS 7.4 or PBS pH 6.0 containing 0.05% Tween 20 (PBS-T), the antigen-antibody complexes were detected with a 1 :60,000 dilution of goat-anti- human IgG-HRP (Ih at RT).

[0136] To display target binding, including simultaneous binding to both targets, antigen-antibody complexes were incubated with 100 ng / ml of biotinylated VISTA-His (Aero Biosystems) in PBS pH 7.4 or PBS pH 6.0 containing 1% NFDM for Ih at RT followed by incubation with 1 :400 dilution of Streptavidin-HRP (Pierce) in PBS pH 7.4 or PBS pH 6.0 containing 1% NFDM (FIG. 2B - FIG. 2F). Plates were washed 5x with PBS-T pH7.4 or PBS-T pH 6.0, and remaining HRP activity detected with TMB substrate (SeraCare). Optical density was measured at 450 nm.ELISA Analysis

[0137] VISTA binding was tested in both monovalent and bivalent formats. Both HC- LC and LC-HC orientations for the CD28-binding scFv were tested at different positions within the molecule; data for the most potent scFv configuration for each geometry is shown. Data was analyzed by GraphPad Prizm version 10 using non-linear curve fitting (log[agonist] vs. response; variable slope (four parameters)).Flow Cytometry Methods

[0138] Binding of serial diluted CF647-labelled antibodies (Mix-n-Stain™ CF® Dye Antibody Labeling Kit, Biotium Cat. #92238) to native proteins on Jurkat T-cells (CD28), Kasumi-3 (VISTA) or a CHO cell line overexpressing VISTA were analyzed by flow cytometry (FIG. 2G-FIG. 21). Briefly, 2.5xl05cells were added to diluted antibodies in FACS buffer (MACSQuant® buffer, Miltenyi Biotec, Cat# 130-092-747) containing FcR Blocking Reagent (Miltenyi Biotec Cat. No. 130-059-901) and incubated for 20 minutes at 4°C. Cells were washed twice with FACS buffer and resuspended in 150 pL of FACS buffer containing I pM SYTOX™.Flow Cytometry Analysis

[0139] Blue dead cell stain (Thermo, Cat# S34857) for live / dead cell discrimination. Samples were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). Raw data were extracted using FlowJo and plotted using GraphPad Prism version 10. The number of surface-expressed VISTA molecules / cell for Kasumi-3 and CHO-K1 / VISTA was determined using the Quantum™ Simply Cellular® (QSC) microsphere kit (Bangs Laboratories, Inc. 816) according to the manufacturer’s instructions. Mean Fluorescent Intensity (MFI) was plotted as a function of mAb concentration (nM).Results

[0140] BS1 monovalent VISTA binding (FIG. 2A) was improved through incorporation of bivalent VISTA binding, which led to improved VISTA binding efficiency through avidity (FIG. 2B). All CD28xVISTA bsAb formats show dual engagement of recombinant target proteins with BS2 and BS3 formats containing duplicate VISTA binding Fab domains in a stacked arrangement connected by a flexible linker showing the most efficient simultaneous target binding in ELISA assays (FIG. 2D) The mono-specific bivalent parental mAbs (from which the Fab or single-chain variable fragment (scFv) domains were derived) used as controls displayed minimal simultaneous binding in this assay (anti-VISTA and anti-CD28, FIG. 2D).

[0141] BS2 and BS3 formats with the pH-selective VISTA binding arm showed pH- dependent simultaneous binding to both targets, with no detectable interaction at pH 7.4 (FIG. 2E, FIG. 2F). In parallel, the control constructs with non-selective 55873Fab arms appeared to display efficient binding at both pH 6.0 and 7.4 (FIG. 2E, FIG. 2F). The effect of avidity was further demonstrated by BS1 and BS2 bsAb binding to native VISTA on VISTA-positive cell lines with two different target densities (FIG. 2G - FIG. 2H), while binding to native CD28 was similar for the two formats (FIG. 21).

[0142] Without wishing to be bound by theory, CD28x VISTA bispecific formats BS2 and BS3 appear to be equally potent in simultaneous target binding.Example 4. Induction of IL-2-luciferase reporter expression by CD28x VISTA

[0143] Upregulation of IL-2 expression is one of the key signatures of CD28 costimulation. To assess CD28xVISTA bsAb-mediated CD28 signaling, Jurkat-IL-2- luciferase reporter cell line was utilized.Luciferase Methods

[0144] CD28xVISTA bispecifics were tested for induction of luciferase expression from Jurkat-IL-2-luciferase reporter cells (CD28 Bioassay Core Kit; Promega) in the presence of HEK293 cells expressing membrane bound OKT3-scFv (anti-CD3) or OKT3-scFv+VISTA and CHO-K1 cells or CHO-K1 overexpressing human VISTA. The relevant HEK293 and CHO-K1 cells were seeded at 20,000 cells each line / well and co-cultured in F12K medium with 10% FBS at 37°C, 5% CO2 overnight. After removing culture supernatant, cells were treated with CD28x55873 (CD28xVISTA BS1) BS1 in 40 pl / well media (RPML1640 with 10% FBS) at 0, 0.0003, 0.001, 0.003, 0.01, 0.04, 0.11, 0.33, 1, and 3 pg / ml for 1 hour at room temperature. A 40 pl / well suspension of TCR / CD3 Effector Cells (IL-2) was added at 80,000 cells / well, and cultured at 37°C, 5% CO2 for 5 hours. To determine the luminescence intensity, 80 pl / well Bio-Gio™ Reagent was added to the plates and these were immediately read in a Molecular Devices Spectramax iD5 plate reader. The net relative luminescent unit (RLU) for each Ab-treated group was obtained after deducting signal of non- Ab -treated group.Results

[0145] The addition of anti-CD28 (TGN1412) to this cell line resulted in dosedependent expression of luciferase (FIG. 3A, circle). In contrast, neither the prototype CD28x VISTA BS1 bsAb (FIG. 3A, triangle) nor the monospecific parental anti- VISTA IgGl control mAb 55873 (FIG. 3A, square) induced luciferase expression ateven the highest 10 pg / mL concentration, indicating monovalent CD28 engagement does not enable CD28 clustering and signaling. In other words, the CD28x VISTA bsAb with monovalent CD28 binding did not display superagonism in this experimental setting.

[0146] In contrast, when the IL-2 reporter cell line was co-cultured with HEK293 cells co-expressing membrane-anchored anti-CD3 scFv (OKT3-scFv) and VISTA, a robust dose-response effect on luciferase expression was seen with BS1 in this c / .s-activation setting (FIG. 3B, solid circle). Luciferase expression was somewhat higher than seen with the TGN1412 control (FIG. 3B, solid triangle). A parallel control experiment with the Jurkat reporter cell line co-cultured with HEK293 cells expressing only OKT3-scFv (without VISTA) appeared to lead to only minimal luciferase expression by BS1 (FIG. 3B, open triangle), highlighting that monovalent CD28 engagement by BS1 without simultaneous binding to VISTA does not lead to CD28 signaling. In both settings, the monospecific parental anti-VISTA IgGl control mAb 55873 appeared to only lead to background luciferase expression levels (FIG. 3B, solid circle, open square).

[0147] To investigate whether CD28x VISTA bsAb BS 1 could potentially induce trans- activation (i.e. Signal 2 provided by interaction between T-cell and a cell different from the Signal 1 -inducing cell line), the Jurkat reporter line was co-cultured with the HEK293 cells expressing only OKT3-scFv (no VISTA) as well as CHO-K1 cells engineered to overexpress VISTA. While luciferase expression from the Jurkat cells was not induced by BS1 when co-cultured with the 293 OKT3 cell line and CHO cells not expressing VISTA (FIG. 3C, circle, top line), inclusion of CHO cells expressing VISTA enabled a robust induction of luciferase expression by addition of CD28x VISTA BS1 (FIG. 3C, square), albeit not as strong as seen when OKT3 and VISTA was co-expressed on the same HEK293 cells, i.e. cis activation (FIG. 3C, circle, bottom line).

[0148] Without wishing to be bound by theory, the prototype CD28x VISTA BS1 bsAb induced VISTA-dependent IL-2-luciferase reporter expression in cis as well as in trans.

[0149] Example 5. Assessment of CD28 VISTA BS3 on cytokine release

[0150] Cytokine release from human peripheral blood mononuclear cells (PBMCs), co- cultured with human umbilical vein endothelial cells (HUVECs) and treated with CD28x67375 (CD28xVISTApH’sens) BS3, CD28x55873 (CD28x VISTA) BS3, or TGN1412 as a positive control, was examined in an in vitro cytokine release assay.Co-culture assay methods

[0151] HUVEC:PBMC co-culture assays were conducted essentially as described in Findlay et al. Cytokine 55, 141-151 (2011) using soluble antibody. HUVECs, allogenic to PBMCs (Lonza), were expanded in Full EBM-2 Medium containing all BulletKit supplements (Lonza). Cells were seeded into clear flat-bottom TC-treated 96-well plates at a density of 30,000 cells / well in 100 pl medium. After culturing for 24h, the medium was replaced with human PBMC’s in full RPMI 1640 medium (Gibco) containing 2% AB serum (Sigma) and lx non-essential amino acids (NEAA) (200 pl / well of 500,000 cells / ml). Antibodies were diluted and titrated to reach final assay concentrations of 0.33, 3.33, 10 and 33.3 pg / ml in full RPMI 1640, added to the plates (100 pl / well), and the co-culture incubated at 48h prior to cytokine analysis on a BioRad Bio-Plex 200 instrument using the Bio-Plex Pro Human Cytokine 8-Plex Kit (BioRad) following the kit manual, with freshly prepared, reconstituted standards and positive controls diluted in Full RPMI- 1640.Results

[0152] A favorable safety profile in HUVEC:PBMC co-culture due to pH-selective VISTA engagement was observed (FIG. 4).Example 6. Assessment of CD28 VISTA BS2 on cytokine release

[0153] Cytokine release from human peripheral blood mononuclear cells (PBMCs), cocultured with human umbilical vein endothelial cells (HUVECs) and treated with CD28x67375 (CD28xVISTApH-sens) BS2, CD28MxVISTApH-sensBS2 (both with pH selective VISTA binding), R-5678 or TGN1412 as a positive control, was examined in an in vitro cytokine release assay.Co-culture Methods

[0154] HUVECPBMC co-culture assays were conducted as described in Example 5, except antibodies were diluted and titrated to reach final assay concentrations of 1, 10, 30, and 100 pg / ml in full RPMI 1640.Results

[0155] TGN1412 resulted in the highest level of induction of all 8 cytokines tested, while CD28xVISTApH'sensBS2 showed only minimal propensity for cytokine release (FIG. 10A, top left). CD28MxVISTApH'sensBS2 appeared to induce a slightly increasedresponse compared to CD28xVISTApH'sensBS2, particularly for IFN-y, IL-2 and IL-6. The comparator R-5678 with Fab arms from Regeneron’s clinical CD28xPSMA IgG4 candidate Nezastomig placed in the IgGl Fey null backbone used in CD28xVISTApH'sensBS2 did appear to display cytokine release in multiple donors at higher concentrations, particularly of IFN-y, IL-4, IL-6 and IL-8. Without wishing to be bound by theory, this could be a result of low level PSMA expression on HUVECs.Example 7. Cytokine release profiles of CD28xVISTApH sensBS2 in an ex vivo system that mimics human blood circulation

[0156] It has been shown that a circulating whole blood loop assay (such as ID.Flow®) is a powerful human-based extra-corporal assay to predict CRS, as it is superior to a standard plate assay in presenting distinctively lowered background of cytokine release, which translates to increased sensitivity of the assay.Ex vivo human whole blood ID.Flow Assay Methods

[0157] Ex vivo cytokine release in human whole blood from 6 heathy donors by CD28x67375 (CD28xVISTApH’sens) BS2, and R-5678 was tested by Immuneed, AB using their ID.Flow® circulating blood platform (Fletcher et al., Int Immunopharmacol. 2018; 54: 1-11), ex vivo system that mimics human blood circulation was generated (FIG. 7A). Anti-CD28 (ANC.28.1; 1 pg / ml), Alemtuzumab (3 pg / ml) or Cetuximab (250 pg / ml) were used as controls.

[0158] Fresh whole blood was taken from healthy volunteers and a low amount of soluble heparin (allowing for analysis of drug-related effects on complement or coagulation cascade systems) was added. Blood was immediately transferred to the ID.Flow system, followed by administration of the test items, and set to circulate at 37°C to prevent clotting. Blood was extracted at baseline and at 4 hours and automatically counted using a Sysmex XN-L350 Hematology Analyzer. Cytokines in blood samples processed to plasma by centrifugation were measured using the MultiArray platform from Meso Scale Discovery (MSD).

[0159] Proportions of T-cells (CD3+) and NK cells (CD56+CD3 ) expressing the activation marker CD69 was determined by flow cytometry using CD3- BV510 (BioLegend 300448), CD56-APC (Biolegend; 362504), CD69-PE / Cy7 (BioLegend 310912) and viability dye staining (LIVE / DEAD violet Viability Dye; Invitrogen L34964). Flow cytometry analysis was performed on a Cytoflex instrument (Beckman Coulter), and data analyzed using FlowJo vl0.9.0.Results

[0160] Ex vivo cytokine release in fresh human whole blood from 6 healthy donors by CD28xVISTApH'sensBS2 was compared to CD28MxVISTApH’sensBS2 and R-5678 at concentrations of 1, 10 and 100 pg / ml (mirroring realistic clinical plasma levels) and the controls anti-CD28 (ANC.28.1; 1 pg / ml), Alemtuzumab (3 pg / ml) or Cetuximab (250 pg / ml). These controls appeared to exhibit the expected effects on cytokine release (FIG. 7B-FIG.7F) while none of the tested bispecific Abs induced cytokine release appeared significantly different from PBS or formulation buffer up to and including the highest tested concentration of 100 pg / mL (an estimated peak serum concentration reached by a 5 mg / kg IV dose of the pH-selective anti-VISTA IgGl mAb SNS-101 in humans (see Sen S etal. Initial results from a first-in-human phase 1 study of SNS-101 (pH-selective anti-VISTA antibody) alone or in combination with cemiplimab in patients with advanced solid tumors. Journal of Clinical Oncology 2024;42(16_suppl):2600.)

[0161] In addition, it did not appear that there was a significant effect on T-cell (FIG. 7G) or NK cell activation (FIG. 7H) was observed. No effect on blood cell viability including platelet (PLT), white blood cell (WBC), and red blood cell (RBC) was found (FIG. 9), and in agreement with the lack of cytokine release, granulocyte and monocyte counts and activation, as well as T and NK cell counts were not significantly affected by any of the test items at all three concentrations (FIG. 11A - FIG. 11F).

[0162] Without wishing to be bound by theory, a favorable safety profile of CD28xVISTApH'sensBS2 in two different in vitro assays for CRS risk assessment was observed.Example 8. Effects of CD28xVISTA BS2 on T-cell mediated killing of LNCaP prostate cancer cells by CD3xPSMA TCE and primary human T-cells

[0163] To characterize the co- stimulatory activity of a CD28xVISTA bsAb in a physiologically more relevant setting, the effect of CD28xVISTA BS2 on primary human T-cell-mediated killing of LNCaP prostate cancer cells was tested in vitro. Methods

[0164] Human T-cell mediated killing of LNCaP prostate cancer cells was analyzed on the xCELLigence real-time cell analysis platform (Agilent) by co-culturing LNCaP cells with PBMCs and VISTA+Kasumi-3 cells in the presence of a CD3xPSMA bispecific T-cell engager (BPS Bioscience; Cat. # 101242-2) alone or in combinationwith CD28x55873 (CD28xVISTA) BS2. LNCaP cells were seeded at 10,000 cells / well into an E-plate (Agilent) and cultured in RPMI-1640 medium with 10% FBS for 3 days at 37°C, 5% CO2. After removing culture supernatant, medium with CD3xPSMA bsAb was added for a final concentration of 0, 0.001, 0.004, 0.01, 0.04, 0.11, 0.33, or 1 pg / ml. Human PBMCs (10,000 cells / well), Kazumi-3 cells (10,000 cells / well) and 1412x55873 (CD28x VISTA) BS2 (0.1 pg / ml or medium in controls) was added to a final volume of 200 pl / well. Lysis buffer was added into control wells. The cells were cultured at 37°C, 5% CO2 and cell viability dynamically monitored on the xCelligence instrument for 6 days. Only growth of the adherent LNCaP cells caused a change in the impedance signal measured by the instrument.

[0165] Cytokines in culture supernatant samples retrieved from the plate after 1 day were analyzed with a Bio-Plex Pro Human Cytokine 8-plex Kit (Bio-Rad). T-cell activation and proliferation were measured in samples retrieved after 6 days using flow cytometry on a Miltenyi MACSQuant Analyzer 10 instrument with CD4-VioBlue, CD8-VioGreen, CD25-PE, CD3-APC, Fc blocking reagent in MACS wash buffer with Propidium Iodide solution added immediately before sample analysis for live / dead cell discrimination (all flow reagents from Miltenyi Biotec).Results

[0166] Co-cultures of LNCaP cells with human PBMCs and the VISTA-positive Kasumi-3 myeloid cell line in the presence of a CD3xPSMA TCE alone or in combination with CD28x VISTA BS2 were analyzed on the xCELLigence real-time cell analysis platform (FIG. 5A, left). The use of a TCE allowed for fine-tuning of TCR stimulation (“Signal 1”) to explore the co-stimulatory effect over a broad range of sub- maximal TCR stimulation levels. LNCaP cells do not express VISTA (FIG. 8A, FIG. 8B), so a potential effect on cell killing would not be mediated by a cis co-stimulatory effect (Signal 1 and Signal 2 both occurring simultaneously in the synapse between a T-cell and LNCaP cell). Only growth of the adherent LNCaP cells caused a change in the measured impedance signal; no effect from non-adherent PBMC’s or Kasumi-3 cells was detected.

[0167] LNCaPs cultured with Kasumi-3 and T-cells with no added test antibodies reach confluence in approximately 5 days (120 hr; FIG. 5A, bottom, square). Inclusion of CD3xPSMA at ~4, 10 and 40 ng / mL did not appear to substantially affect growth of the adherent LNCaP cells compared to co-cultures in the absence of the TCE (FIG. 5A,bottom). However, those same concentrations appeared lead to a dose-dependent decrease of LNCaP viability in the presence of 100 ng / mL CD28x VISTA BS2, with the highest 40 ng / mL TCE concentration leading to the killing of LNCaP cells as indicated by a signal at day 5 comparable to wells with the addition of lysis buffer (FIG. 5A, bottom). Without wishing to be bound by theory, the potentiation of LNCaP killing by the CD3xPSMA TCE was consistent across 3 different donors (data not shown).

[0168] Culture supernatant samples retrieved from the plate after 1 day of bsAb treatment were analyzed for cytokine induction, and T-cell activation (upregulation of CD25 expression on T-cells, a commonly used activation marker responsive to CD28 co-stimulation) and proliferation (CD3+T-cell count) were measured in samples retrieved after 6 days using flow cytometry. Enhanced killing in the presence of the CD28xVISTA bispecific antibody appeared to be accompanied by increased IL-2 and TNF-a cytokine release (FIG. 5B, top), T-cell activation (FIG. 5B, bottom left) and proliferation (FIG. 5B, bottom right). Without wishing to be bound by theory, no effect of the presence of CD28xVISTA BS2 on any of these readouts was observed in the absence of CD3xPSMA, demonstrating that a TCR signal is required to enable the CD28 co-stimulatory effect. No superagonistic properties of CD28x VISTA BS2 were seen at the 0.1 pg / mL concentration used in this experiment.Example 9. In vivo studies - Effects of CD28xVISTA on MC38-h VISTA tumor growth in hCD28 knock-in mice

[0169] Tumor growth inhibition (TGI) of a MC38 cell population overexpressing human VISTA was tested in a humanized CD28 mouse model (C57BL / 6N- Ct / 2Stml l(CD28)Geno, genOway) in combination with anti-murine PD-1 (anti-mPD-1) (see FIG. 6A - FIG. 6F) Tumor Growth Methods

[0170] MC38-h VISTA cells were pre-conditioned by a passage through hCD28-KI mice followed by tumor harvesting and cell line establishment. Expression of hVISTA on preconditioned cells was analyzed by flow cytometry using fluorescently labelled anti-human VISTA mAb h26A (Sensei Biotherapeutics) prior to implantation. These MC38-hVISTA cells (lxl06 / animal) were implanted subcutaneously into female hCD28 KI mice. Once the tumor volumes reached -80-100 mm3(-day 7) mice were randomized into 4 groups of 10 animals per group. Animals were administered isotype controls (rat IgG2a isotype control (clone 2A3, BioXCell BE0089), human IgGlisotype control (BioXCell BP0297), anti-mPD-1 (InVivoMAb rat anti -mouse PD-1 (clone RMP1-14, BioXCell BE0146), CD28xVISTApH’sensBS2, or anti-mPD-1 + CD28xVISTApH'sensBS2 intraperitoneally twice / week for 3 weeks. Tumor growth inhibition was calculated as previously described as described in Tsukihara H, et al. Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS- 102, together with bevacizumab, cetuximab, or panitumumab on human colorectal cancer xenografts. Oncol Rep 2015;33(5):2135-42. Significance was evaluated using Mann-Whitney unpaired t test with P < 0.05 considered to be statistically significant.Results

[0171] Tumor-specific T-cells were activated by their TCRs engaging peptide / MHC complexes on the tumor cells, i.e. a natural “Signal 1” which could potentially be enhanced by CD28 co-stimulation in cis between T-cells and hVISTA+tumor cells (FIG. 6A)

[0172] MC38 cells overexpressing human VISTA were used for implantation into female human CD28 knock-in (KI) mice. Expression of hVISTA on the preconditioned cells prior to implantation was analyzed by flow cytometry using a fluorescently labelled anti-human VISTA mAb. As indicated by the overlap of staining with an isotype control mAb, a substantial population of MC38 cells were not expressing hVISTA (FIG. 6B).

[0173] Animals were administered intraperitoneally twice / week with isotype controls, a suboptimal dose of 1 mg / kg anti-mPD-1, CD28xVISTApH'sensBS2 at 5 mg / kg, or anti- mPD-1 (1 mg / kg) + CD28xVISTApH'sensBS2 (5 mg / kg) combined (FIG. 6C, top). A modest effect on tumor growth of anti-mPD-1 or CD28xVISTApH'sensBS2 monotherapy of 27.4%, and 28.0%, respectively, was seen compared to the isotype control arm (FIG. 6E). However, combining CD28xVISTApH'sensBS2 with the low dose anti -PD-1 resulted in a tumor growth inhibition of 73.4% on day 17, a significant reduction both compared to the isotype control group (P= 0.0005) and either of the monotherapy arms (P= 0.0018; FIG. 6D, FIG. 6E). This effect appeared to translate to a significant increase in probability of survival between combination arm and control group (P= 0.0299; FIG. 6F).

[0174] Without wishing to be bound by theory, the tumor growth inhibition and enhanced survival were observed despite the highly heterogeneous tumor cellpopulation (of the MC38 cells inoculated, only 47% were hVISTA+), suggesting efficient tumor growth control of even MC38 cells not expressing hVISTA. Without wishing to be bound by theory, as the anti-VISTA Fab arms of CD28xVISTApH'sensBS2 are not cross-reactive to murine VISTA, the effect on tumor growth can be uniquely ascribed to the CD28 agonistic effect, while potential VISTA checkpoint inhibition does not play a role.

[0175] Without wishing to be bound by theory, CD28xVISTApH'sensBS2 enhanced natural “Signal 1” by CD28 co-stimulation in cis, resulting in significant TGI in combination with anti-mPDl.Example 10. In vivo studies - Effects of CD28xVISTA and CD28M1xVISTA on MC38-hVISTA tumor growth in hCD28 knock-in miceTGI of a MC38 cell population overexpressing human VISTA was tested in a humanized CD28 mouse model (C57BL / 6N-Ct / 2Stml l(CD28)Geno, genOway) in combination with anti-murine PD-1 (anti-mPD-1) (see FIG. 12 - FIG. 14). The effect of CD28xVISTApH'sensBS2 was compared to CD28M1xVISTApH-sens, a bispecific antibody including a unique CD28-binding Fab and the 67375 pH sensitive VISTA arm, in the presence and the absence of anti-mPD-1.Tumor Growth and Rechallenge Methods

[0176] MC38-h VISTA cells were pre-conditioned by a passage through hCD28-KI mice followed by tumor harvesting and cell line establishment. Expression of hVISTA on preconditioned cells was analyzed by flow cytometry using fluorescently labeled anti-human VISTA mAb h26A (Sensei Biotherapeutics) prior to implantation. These MC38-hVISTA cells (lxl06 / animal) were implanted subcutaneously into female hCD28 KI mice (n = 70). Once the tumor volumes reached -80-100 mm3(-day 7) mice were randomized into 4 groups of 10 animals per group for groups 1-4 and 2 groups of 15 animals per group for groups 5 and 6. Animals were administered isotype controls (rat IgG2a isotype control (clone 2A3, BioXCell BE0089), human IgGl isotype control (BioXCell BP0297), anti-mPD-1 (InVivoMAb rat anti-mouse PD-1 (clone RMP1-14, BioXCell BE0146), anti-mPD-1 (clone LALA-PG, BioXCell CP153), CD28xVISTApH'sensBS2, and / or as a comparator CD28M1xVISTApH'sens(a bispecific antibody including a unique CD28-binding Fab and the 67375 pH sensitive VISTA arm). The dosage schedule was administered according to the following schedule:Table 12: Dosing Schedule:

[0177] Tumor growth inhibition was calculated as described in Example 10. Significance was evaluated using Mann-Whitney unpaired nonparametric T-test with P<0.05 considered to be statistically significant. GraphPad Prism 9 and Prism 10 were used for analysis.Measurements and samples

[0178] Tumor volumes were taken 3 times per week and body weights of were taken 2 times per week.

[0179] Blood (-100-200 pL) was sampled by maxillary facial vein or submandibular bleed prior to tumor inoculation, at day 0 (treatment initiation) and at day 10 posttreatment initiation. Additionally, Blood (-100-200 pL) from 5 tumor bearing animals from Groups 1-4 was sampled prior to initiation of treatment (day 0) by maxillary facial vein or submandibular bleed.

[0180] Blood samples were collected and processed as follows: blood was collected into a microfuge tube. The blood collected was kept at room temperature (RT) undisturbed for 30 min. Blood was centrifuged at 2000xg for 20 minutes at 4°C. Serum samples was immediately stored in individually labeled 1.5 mL microcentrifuge tubesat approximately -80°C. Blood samples were stored at -80°C until further analysis was performed.Termination

[0181] Tumors (n=3-4 per group) were harvested at an endpoint, placed in MACS® Tissue Storage Solution (Miltenyi 130-100-008), and stored at 4-8°C until further analysis was performed.

[0182] At the end of the study, a Terminal bleed was performed (collect plasma) and the spleen and tumor(s) were harvested.

[0183] Terminal Blood was collected by cardiac puncture and 0.4-0.5 mL of blood was placed in microtainer K2EDTA tubes. The blood collected was kept on ice until centrifugation, which happened within 10 minutes of collection at 4°C for 10 minutes at an RCF of 1200 with refrigeration. The plasma samples were immediately stored in individually labeled 1.5 mL microcentrifuge tubes at approximately -80°C.

[0184] Spleen and tumor were excised and placed in 10% Neutral Buffered Formalin. After 24 hours, tissue was transferred to 70% EtOH.Animal care

[0185] Mice were sacrificed if their body weight loss was >20% and / or the tumors were ulcerated.

[0186] Animal housing was maintained at 21-24°C and 50±20% relative humidity in rooms with at least ten room air changes per hour. Cage racks were ventilated with micro-filtered tops and sterile bedding (n= 5 / cage).

[0187] The photoperiod was diurnal and Animals were housed in microisolators in a 12: 12 light-dark cycle. There was standard maintenance of the rodent chow diet (Rodent Maintenance Chow: Harlan Teklad Product: 2018 ad libitum). Food and tap water were provided ad libitum.Results

[0188] Initial body weight was tested by One-way ANOVA with Multiple Comparisons (GraphPad Prism 10). There was a trend toward group 6 having lower initial body weight compared to other groups, though the only comparison reaching statistical significance was between groups 4 and 6 (P= 0.0322)(FIG. 12). Withoutwishing to be bound by theory, Antibody treatment was well tolerated, as no significant body weight changes were observed.

[0189] Low dose anti-PD-1 monotherapy appeared to have had a modest effect on tumor growth in this setting. The combination of anti-PD-1 and CD28xVISTApH'sensBS2 treatments demonstrated significant anti-tumor effects (60% TGI, P=0.0115). The combination of anti-PD-1 and CD28M1xVISTApH'senswas not significantly different from isotype control. These results are illustrated in FIG. 13A and FIG. 13B, FIG. 14A and FIG. 14B, and Table 13 below.Table 13: Tabulated Data at Day 18TGI is relative to isotype control; + values indicated decreased tumor volume; - values indicate excess tumor growth; Day 18 chosen for least effect on data by dropouts (N). Statistics using Mann Whitney unpaired non-parametric T-test in GraphPad Prism 10.RTV = (tumor volume on measured day 21) / (tumor volume on day 0).TGI (%) = [1 - (RTV of the treated group) / (RTV of the control group)] x 100 (%).

[0190] Without wishing to be bound by theory, this suggests that the anti-CD28 Fab in CD28xVISTApH'sensdoes not engage CD28 in a productive way that facilitates T-cell stimulation and activation or promotes anti-tumor activity.

[0191] In this model system, both signal 1 and signal 2 are understood to be presented via interactions between tumor cells (over)-expressing hVISTA and native APCs andT-cells. In practice, tumor-associated myeloid cells are expected to be the primary source of VISTA. Without wishing to be bound by theory, in this setting, an anti- VISTAxCD28 bispecific antibody would provide signal 2 in trans, by bridging a myeloid-T-cell interaction, while signal 1 is provided by a simultaneous tumor cell-T- cell interaction.Example 11. In vivo studies - Pharmacokinetic profile of CD28 VISTA BS2 bsAb in huCD28 KI mice

[0192] CD28 expressed on T-cells in blood and lymph nodes could provide a significant antigen sink. The effect of CD28 binding affinity on the PK properties of a CD28-targeting bsAb was interrogated herein.PK Study Methods

[0193] The PK-study was performed in a mixed population of female and male hCD28 KI mice (C57BL / 6N-Ct / 2Stml l(CD28)Geno; GenOway); n=4 per timepoint subgroup. The 3 CD28 targeting bsAbs CD28xVISTApH-sensBS2, CD28MxVISTApH-sensBS2 and R- 5678 were dosed by bolus IV injection at 5 mg / kg. Blood was collected at 14 timepoints: pre-bleed; 5 min, 1, 4, 8, 12, 24, 48, 72, 120, 168, 336, and 672 hr, immediately put on ice and processed to serum which was frozen at -80°C until analysis. mAb serum levels were measured by ELISA using mouse anti-human IgG Fc (Abeam ab99757) immobilized in high bind microplates (Coming 2592) followed by detection using a peroxidase-conjugated mouse anti-human IgG F(ab)2 fragmentspecific reagent (Jackson Laboratories 209-035-097; 40,000-fold diluted in Blocking Buffer PBS + 2% BSA) and development using the HRP substrate TMB (Life Technologies 34028). The concentrations in the samples were determined using nonlinear regression with interpolation of unknown values from the prepared standard curve of the identical mAb using GraphPad Prizm 10 (GraphPad Software). Calculation of PK parameters using non-compartmental analysis was performed with Phoenix WinNonlin (version 8.3, Certara Corp.).Results

[0194] The pharmacokinetics of 3 highly similar bsAbs in huCD28 KI mice was compared, each having a different CD28 binding domain with binding affinities spanning two orders of magnitude (KD values of 1-2 nM for CD28xVISTApH'sensBS2 (SPR to 272 nM for R-5678 (SPR; 37°C (16)). CD28MxVISTApH-sensBS2 has anidentical architecture to CD28xVISTApH'sensBS2 except for a different anti-CD28 Fab arm with approx. 60-fold lower affinity compared to the CD28 binding arm of CD28xVISTApH'sensBS2 (data not shown). R-5678 is a CD28xPSMA bsAb using the same Fc region as CD28xVISTApH'sensBS2 . The PSMA arm of R-5678 is not cross- reactive to murine PSMA.

[0195] When dosed IV at 5 mg / kg in human CD28 knock-in mice, which systemically expresses human CD28, all 3 antibodies showed relatively slow clearances with differences that did not correlate with CD28 binding affinity (FIG. 9), indicating that other intrinsic factors besides CD28 affinity are dominant in terms of resulting PK. The calculated clearance rate of CD28xVISTApH'sensBS2 falls in the middle of the range found for 16 human non-mouse cross-reactive IgGl mAbs, and is similar to the parental monospecific, bivalent pH-selective anti-VISTA IgGl mAb with the same anti-VISTA Fab arms dosed at the same level and route in a wildtype (wt) C57BL6 / J mouse. The pH-selective VISTA Fab arms employed in CD28xVISTApH'sensBS2 in the context of a regular bivalent IgGl mAb format showed favorable PK and did not appear to provide TMDD in hVISTA KI mice, cynomolgus monkeys or human trial subjects (see, e.g., Thisted Tet al. VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response. Nat Commun 2024; 15(1):2917 ). However, as VISTA binding is not mouse cross-reactive, any negative effect on PK in the bispecific context would not be expected to be revealed in this hCD28 KI mouse model.

[0196] Without wishing to be bound by theory, the data suggests a favorable PK profile of CD28xVISTApH'sensBS2 in huCD28 KI mice, and that the low KD of the CD28 binding domain in a monovalent format did not result in unacceptable rapid clearance.

Claims

CLAIMSWhat is claimed is:

1. A bispecific binding protein comprising a first binding domain that binds to V- domain immunoglobulin suppressor of T-cell activation (VISTA), a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises five polypeptide chains, wherein:(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34; or(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:33, and the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO:34.

2. A bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein:(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35; or(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, the second polypeptide chain comprises the amino acid sequence of SEQ IDNO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35.

3. A bispecific binding protein comprising a first binding domain that binds to VISTA and a second binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:36, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:37, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39.

4. A bispecific binding protein comprising a first binding domain that binds to VISTA, a second binding domain that binds to VISTA, and a third binding domain that binds to CD28, wherein the bispecific binding protein comprises four polypeptide chains, wherein:(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; or(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43.

5. A pharmaceutical composition comprising the bispecific binding protein of any one of claims 1-4, and a pharmaceutically acceptable carrier.

6. One or more nucleic acid molecules comprising:(a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and a first nucleic acid sequence encoding a fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34; or(b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, and a first nucleic acid sequence encoding a fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34.

7. One or more nucleic acid molecules comprising:(a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35; or(b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35.

8. One or more nucleic acid molecules comprising a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:36, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39.

9. One or more nucleic acid molecules comprising:(a) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; or(b) a first nucleic acid sequence encoding a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, a first nucleic acid sequence encoding a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, a first nucleic acid sequence encoding a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16, and a first nucleic acid sequence encoding a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43.

10. An expression vector comprising the one or more nucleic acid molecules of any one of claims 6-9.

11. A recombinant host cell comprising the one or more nucleic acid molecules of any one of claims 6-9.

12. A recombinant host cell comprising the expression vector of claim 10.

13. A method of producing a bispecific protein, the method comprising: culturing the recombinant host cell of claim 11 or 12 under conditions whereby the one or more nucleic acid molecules are expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

14. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject the bispecific protein of any one of claims 1-4 or the pharmaceutical composition of claim 5.

15. The method of claim 14, wherein the cancer is head and neck cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, thyroid cancer, lung cancer, melanoma, or sarcoma.

16. The method of claim 14, wherein the cancer is a hematologic cancer.

17. The method of any one of claims 14-16, further comprising administering to the subject an inhibitor of PD-1 or an inhibitor of PD-L1.

18. The method of claim 17, wherein the inhibitor of PD-1 is an anti-PD-1 antibody or antigen-binding portion thereof.

19. The method of claim 17, wherein the inhibitor of PD-L1 is an anti-PD-Ll antibody or antigen-binding portion thereof.

20. The method of any one of claims 14-16, further comprising administering to the subject a T-cell engager.

21. The method of claim 20, wherein the T-cell engager is a bispecific T-cell engager.

22. The bispecific protein of any one of claims 1-4 or the pharmaceutical composition of claim 5 for use as a medicament.

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