Multispecific binding constructs targeting PD-1 and VEGF and uses thereof

Abstract

The present disclosure relates to an anti-VEGF-anti-PD-1 multispecific antibody, a pharmaceutical composition of same, and uses thereof. The multispecific antibody may activate the immune system and block tumor angiogenesis simultaneously, thus providing increased antitumor properties.

Description

MULTISPECIFIC BINDING CONSTRUCTS TARGETING PD-1 AND VEGF AND USES THEREOF TECHNICAL FIELD

[0001] The present disclosure relates to multispecific antibodies that bind VEGF (VEGF- A) andPD-lor PD-L1, a pharmaceutical composition of the same, and uses thereof. In addition, the present invention relates to polynucleotides encoding such multispecific antibodies and vectors and host cells comprising such polynucleotides. The invention further relates to methods for selecting and producing such antibodies and to methods of using such antibodies in the treatment of diseases. The invention also relates to the therapeutic use of the VEGFxPD-1 bispecific antibodies in monotherapy and in combination therapy.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled CTX0095.PCTl_SEQ LISTING_FILE, was created on December 23, 2025 and is 20,600 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Cancer is one of the leading causes of death in both the United States and worldwide. While common treatments such as surgery, radiation, chemotherapy, hormone therapy, targeted therapies, and immunotherapy have decreased the rates of cancer-related deaths during the 20thcentury, there were 20 million new cancer cases diagnosed and 9.7 million cancer deaths worldwide as of 2022. Approximately 1 in 5 people will develop cancer in their lifetime, with 1 in 9 men and 1 in 12 women dying from the disease. See American Cancer Society, Global Cancer Facts & Figures 5th Edition, Atlanta: American Cancer Society; 2024.

[0004] An increasing body of evidence suggests that the immune system operates as a significant barrier to tumor formation and progression. The principle that naturally occurring T cells with anti-tumor potential or activity exist in a patient with cancer has rationalized the development of immunotherapeutic approaches in oncology. Immune cells, such as T cells, macrophages, and natural killer cells, can exhibit anti-tumor activity and effectively control the occurrence and growth of malignant tumors. Tumor-specific or -associated antigens can induce immune cells to recognize and eliminate malignancies (Chen & Mellman, (2013)Immunity 39(1 ): 1 -10). Despite the existence of tumor-specific immune responses, malignant tumors often evade or avoid immune attack through a variety of immunomodulatory mechanisms resulting in the failure to control tumor occurrence and progression (Motz & Coukos, (2013) Immunity 39(1) :61 -730). Indeed, an emerging hallmark of cancer is the exploitation of these immunomodulatory mechanisms and the disablement of anti-tumor immune responses, resulting in tumor evasion and escape from immunological killing (Hanahan and Weinberg (2011) Cell 144(5):646-674).

[0005] Traditionally, monoclonal antibodies (mAbs) blocking immune checkpoints have been used as monotherapy. Antibodies that block immune escape and that block angiogenesis have been used with success. Because the interaction of PD-L1 and PD-1 can inhibit the T-cell response, mAbs targeting programmed cell death protein 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) have been shown to reactivate suppressed T-cells to block the cancer-immune escape.

[0006] Moreover, mAbs or fusion proteins targeting vascular endothelial growth factor (VEGF) or VEGF receptor (VEGFR) have also been shown to inhibit cancer growth. Perhaps one of the most important cytokines in promoting cancer angiogenesis, VEGFs may promote mitosis of vascular endothelial cells to form new blood vessels. Of the known members of the human VEGF family, VEGF-A may be the most important regulator of blood vessel formation in health and diseases. Blocking the combination of VEGF and / or VEGFR1 may effectively inhibit angiogenesis and tumor growth. Bevacizumab (Avastin®, Genentech / Roche), an anticancer antibody drug that inhibits angiogenesis by targeting VEGF, was approved by the US Food and Drug Administration (FDA) in 2004 and has been largely successful as an anticancer therapeutic agent. Recent clinical model and preclinical animal model studies have indicated, however, that not all solid tumors respond to VEGF inhibitors. Further, several cases have been reported showing that some tumors treated with VEGF inhibitors in the initial stage show resistance after a certain time. In addition, some studies report that the administration of VEGF inhibitors may convert cancer cells into cancer cells that are more aggressive and easily metastasize. Such study reports have propelled research and development of novel anticancer targets that overcome bevacizumab resistance or that have efficacy superior to that of bevacizumab.

[0007] As previously mentioned, the occurrence and progression of malignant cancers have been attributed to a myriad of physiological processes, including cell proliferation,induction of angiogenesis, immune escape, resistance to apoptosis, tissue invasion and metastasis, the promotion of inflammatory responses of cancers, and abnormal energy metabolism (See e.g., Falcon BL, et al. (2011) Increased Vascular Delivery and Efficacy of Chemotherapy After Inhibition of Platelet-Derived Growth Factor-B. Am J Pathol 178(6):2920-30; and Sabina S, et al. (2011) Prospective Study on the Role of Glucose Metabolism in Breast Cancer Occurrence, hit J Cancers.). Consequently, the therapeutic efficacy of monotherapy is often ineffective. The use of bispecific antibodies capable of binding to multiple targets at once are therefore of great interest.SUMMARY

[0008] The present disclosure is based, in part, on novel multispecific and multivalent constructs targeting both PD-1 and VEGF, such as a bispecific and tetravalent constructs. As demonstrated herein, these multispecific constructs exhibit comparable or improved in vitro and / or in vivo potency as compared to combinations of individual antibodies, as well as compared to clinical checkpoint blockade agents. Further, the multispecific and multivalent constructs described herein may concurrently block both the PD-1 and VEGF pathways simultaneously, and thus may be more effective in inhibiting cancer growth than an anti-PD-1 or anti-VEGF mAh or VEGF fusion protein. Thus, the multispecific and multivalent constructs or bispecific antibodies described herein may bring about greater clinical benefits as well as lead to broader indications. The simultaneous targeting of VEGF- A and PD-1 in a single molecule may reduce the complexity of combination treatment regimens by concentrating PD-1 blockade at VEGF-rich, immunosuppressed tumor sites. Further, the simultaneous targeting of VEGF- A and PD-1 in a single molecule may also offer improved safety over VEGF tyrosine kinase inhibitor (TKI) containing combination regimens. The multispecific and multivalent constructs or bispecific antibodies described herein simultaneously inhibit tumor angiogenesis and PD- 1 -mediated immune suppression and eliminates tumors.

[0009] The present disclosure also provides multispecific (e.g., bispecific) antigen binding constructs that include at least two linked antigen binding units, wherein a first antigen binding unit specifically binds PD-1, and wherein a second antigen binding unit specifically binds VEGF. In some aspects, the multispecific antigen binding constructs may include an anti-VEGF-A IgGl with Fc-silencing LALA mutations fused to anti-PD-1 single chain variable fragments (scFvs) via a flexible linker (see Fig. 1).

[0010] Also disclosed herein are multispecific (e.g., bispecific) antigen binding constructs that effectively block VEGF-A / VEGFR2 and PD-1 / PD-L1 interactions. In some embodiments, the multispecific (e.g., bispecific) antigen binding constructs block VEGF-A / VEGFR2 and PD-1 / PD-L1 interactions in a dose-dependent manner. In some aspects, the multispecific antigen binding constructs may reduce engagement with Fey receptors, thus limiting off-target immune activation. Functionally, the multispecific antigen binding constructs may significantly enhanced IFN-y production in mixed lymphocyte reactions (MERs) and tumor cell killing by activated peripheral blood mononuclear cells (PBMCs).

[0011] Nucleic acids comprising nucleotide sequences encoding the polypeptides of the disclosure are provided herein, as are vectors comprising nucleic acids. Additionally, prokaryotic and eukaryotic host cells transformed with a vector comprising a nucleotide sequence encoding a disclosed polypeptide are provided herein, as well as eukaryotic (such as mammalian) host cells engineered to express the nucleotide sequences. Methods of producing polypeptides, by culturing host cells and recovering the polypeptides are also provided.

[0012] In some embodiments, the first antigen binding unit is a polypeptide, small molecule, or an aptamer. Optionally, the first antigen binding unit is an antibody or antigen binding fragment thereof. The antibody or antigen binding portion thereof of the first antigen binding unit is optionally a monoclonal antibody or antigen binding portion thereof. The antibody or antigen binding portion thereof of the first antigen binding unit can be a humanized antibody, a fully human antibody, or an antigen binding portion of either. The antibody or antigen binding portion thereof of the first antigen binding unit is optionally an scFv or Fab antibody fragment.

[0013] In some embodiments, the second antigen binding unit is a polypeptide, small molecule, or an aptamer. Optionally, the second antigen binding unit is an antibody or antigen binding fragment thereof. The second antigen binding unit is optionally a monoclonal antibody or antigen binding portion thereof. The second antigen binding unit can be a humanized antibody or a fully human antibody. The second antigen binding unit is optionally a scFv or Fab antibody fragment.

[0014] In some embodiments, the multispecific antigen-binding construct is capable of binding human PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding murine PD-1. In some embodiments, the multispccific antigen-bindingconstruct is capable of binding cynomolgus monkey PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding human, murine and cynomolgus monkey PD-1 with similar affinity. In some aspects, the multispecific antigen-binding construct may bind to both human and cynomolgus monkey VEGF-A and PD-1 with high affinity.

[0015] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein may be capable of reducing PD-1 levels on a cell. In some embodiments, the multispecific antigen-binding construct is capable of inducing PD-1 degradation. In some embodiments, the multispecific antigen-binding construct is capable of reducing PD-1 expression. In some embodiments, the multispecific antigen-binding construct is capable of reducing PD-1 cell surface expression. In some embodiments, the multispecific antigenbinding construct is capable of reducing PD-1 cell surface expression by inducing shedding of PD-1 from the cell surface. In some embodiments, the multispecific antigen-binding construct is capable of inducing shedding of PD-1 from an immune cell. In some embodiments, the immune cell (e.g., T cell) is a tumor infiltrating lymphocyte (TIL). In some embodiments, engagement of a multispecific antigen-binding molecule as described herein to PD-1 expressed by an immune cell in the tumor microenvironment results in the downregulation of PD- 1 by the immune cell.

[0016] In some embodiments, the disclosure provides for a multispecific antigenbinding construct comprising at least two antigen-binding units, wherein a first antigen binding unit binds PD-1 expressed by an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, a first antigen binding unit binds PD-1 expressed by a tumor cell. In some embodiments, the tumor cell is selected from the group consisting of a hematological cancer, a lymphoma, a myeloma, a leukemia, a neurological cancer, melanoma, breast cancer, a prostate cancer, a colorectal cancer, lung cancer (including e.g., non-small cell lung cancer), head and neck cancer, a gastrointestinal cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, a genitourinary cancer, a bone cancer, renal cancer (including e.g., advanced renal cell carcinoma), cervical cancer (e.g., endometrial carcinoma), and a vascular cancer. In some embodiments, the binding of one antigen binding unit to its target does not block the binding of the other antigen binding unitto its target. In some embodiments, the first antigen binding unit and second antigen binding unit bind to their respective targets and both antigen binding units remain bound concurrently. In some embodiments, the first antigen binding unit is an antagonist of PD-1.

[0017] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein may be capable of inducing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% greater interferon-gamma levels (e.g., as measured in a Staphylococcus aureus Enterotoxin A (“SEA”) assay) as compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab). In some embodiments, the multispecific antigen-binding construct induces at least 5%. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 200%, 300%, 400%, or 500% more killing of tumor cells (e.g., leukemia cells, lymphoma cells, melanoma, or breast cancer cells), as compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab). In some embodiments, the multispecific antigen-binding construct is capable of extending the survival of a subject suffering from a cancer (e.g., leukemia, lymphoma, melanoma, and / or breast cancer) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% longer than a subject administered a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab). In some embodiments, the multispecific antigen-binding construct is capable of inducing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% more shedding of PD-1 from an immune cell as compared to an untreated immune cell or as compared to an immune cell treated with reference antigen-binding construct (e.g., pembrolizumab or atezolizumab).

[0018] In some embodiments, the multispecific antigen-binding construct is capable of reducing PD-1 levels at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% (e.g., by causing PD-1 shedding from the cell surface and / or inducing PD-1 degradation and / or reducing PD-1 expression) as compared to an untreated immune cell or to an immune cell treated with a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab).

[0019] In some embodiments of any of the aspects described herein, the construct is a bispecific antibody. A bispecific binding construct may comprise two polypeptides linked together via disulfide bonds, with each polypeptide comprising, for example, a first scFv region and a second scFv region at its respective N- and C-termini. The two scFv regions may specifically bind different antigens. Between the first and second scFv regions of each polypeptide are sequences comprising the hinge, CH2, and CH3 domains of immunoglobulins.

[0020] Alternatively, a bispecific binding construct can comprise an IgG that has, for example, a scFv region linked at the C-temiinal end of each CH3 domain. In this case, the variable heavy and light chains in the Fab regions of the IgG may specifically bind one antigen, and the C-terminal scFv regions comprise different variable heavy and light chains that specifically bind a second antigen.

[0021] As noted above, a bispecific binding construct may bind to two different antigens via two domains, one at the N-terminal end and the other at the C-terminal end. In some embodiments, the bispecific binding construct binds the first domain of an immunomodulatory protein, for example, to activate an immune receptor, and the second domain of a tumor antigen. In such cases, the bispecific binding protein provides for targetspecific activation of the immune response in a tumor microenvironment. Such conditional activation of an immunomodulatory protein in the tumor microenvironment may avoid systemic toxicity, potentially allowing for higher dosing and a wider application of immunological anticancer agents.

[0022] In some embodiments, the bispecific antibody is an antagonist of PD-1 and VEGF / VEGF-A. In some embodiments, the construct comprises a common light chain. In some embodiments, one or both antigen binding units is an aptamer. In some embodiments, one or both antigen binding units is a protein other than an antibody. In some embodiments, the construct comprises at least two bispecific antibodies. In some embodiments, one of the at least two bispecific antibodies may be monovalent for PD-1. In some embodiments, at least one of the antigen binding units is a bivalent antibody specific for PD- 1. In some embodiments, the bispecific antibody binds two different epitopes on PD-1. In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises at least two monospecific antibodies. In some embodiments, at least one of the monospecificantibodies is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a bivalent anti-PD-1 antibody.

[0023] In some embodiments, the construct is a fusion construct in which a polypeptide comprising the variable heavy chain of the anti-PD- 1 antibody is fused to a polypeptide comprising the variable heavy chain of the anti-VEGF antibody. In some embodiments, the polypeptide comprising the variable heavy chain of the anti-PD-1 antibody is fused to the polypeptide comprising the variable heavy chain of the anti-VEGF antibody by means of a linker. In some embodiments, the fusion construct comprises a common light chain.

[0024] In some embodiments of the multispecific antigen-binding constructs provided herein, the first antigen binding unit binds to PD-1 and comprises a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 7 (GFTFSSYA); (ii) a CDRH2 comprising SEQ ID NO: 8 (ISNSGTYTY); and (iii) a CDRH3 comprising SEQ ID NO: 9 (ARGLDFIVGYTGNDY); and (b) a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 6 (QQSYSTPLT).

[0025] In some embodiments, the heavy chain variable region of the first antigen binding unit comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy chain variable region of the first antigen binding unit comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. In some embodiments, the heavy chain variable region of the first antigen binding unit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2. In some embodiments, the light chain variable region of the first antigen binding unit comprises an amino acid sequence of SEQ ID NO: 3. In some embodiments, the light chain variable region of the first antigen binding unit comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the light chain variable region of the first antigen binding unit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. Also provided herein, in some aspects and embodiments, is a multispecific antigen-binding construct comprising four units capable of antigen-binding, wherein two antigen-binding units bind PD-1 and two antigen-binding units bind VEGF, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85%, identical to the amino acid sequence of SEQ ID NO: 2 or10, and a light chain amino acid sequence that is at least 85%, identical to the amino acid sequence of S EQ ID NO: 3 or 11.

[0026] In some embodiments of the multispecific antigen-binding constructs provided herein, the second antigen binding unit binds to VEGF and comprises a heavy chain variable region comprising a CDRH1 comprising SEQ ID NO: 15, a CDRH2 comprising SEQ ID NO: 16, and a CDRH3 comprising SEQ ID NO: 17; and a light chain variable region comprising a CDRL1 comprising SEQ ID NO: 12, a CDRL2 comprising SEQ ID NO: 13, and a CDRL3 comprising SEQ ID NO: 14.

[0027] In some embodiments, the heavy chain variable region of the second antigen binding unit comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable region of the second antigen binding unit comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the heavy chain variable region of the second antigen binding unit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 10. In some embodiments, the light chain variable region of the second antigen binding unit comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the light chain variable region of the second antigen binding unit comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the light chain variable region of the second antigen binding unit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.

[0028] In some embodiments, the first and the second antigen binding units each comprise a common light chain variable region. In some embodiments, the common light chain comprises a variable region comprising SEQ ID NO: 3 or SEQ ID NO: 11. In some embodiments, the common light chain comprises a variable region at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 11. In some embodiments, the common light chain comprises a variable region at least 85% identical to SEQ ID NO: 3 or SEQ ID NO: 11. In some embodiments, the N-terminal variable heavy chain of the multispecific antigen-binding construct binds to PD-1 in the presence of the common light chain, and the C-terminal variable heavy chain of the antigen-binding construct binds to VEGF in the presence of the common light chain. In some embodiments, the N-terminal variable heavy chain of the fusion construct binds to VEGF in the presence of the common light chain, and the G-terminal variable heavy chain of the fusion construct binds to PD-1 in the presence of the common light chain.

[0029] In some embodiments, the multispecific antigen-binding construct may be a form in which the protein that binds specifically to VEGF and the IgG (immunoglobulin G)-type antibody that binds specifically to PD-1 are connected to each other by a linker. The linker may be a peptidyl linker or a non-peptide linker, preferably the peptide linker has an amino acid sequence represented by SEQ ID NO: 18.

[0030] Tumor angiogenesis, the formation of new blood vessels in solid tumors, plays an important role in tumor cell survival, growth, and metastasis. A major driving force of tumor angiogenesis is the signaling pathway involving VEGF and VEGFRs. Several angiogenesis inhibitors, including antibodies and small molecule compounds targeting the VEGF / VEGFR signaling pathway, have been approved by the FDA, and used for the treatment of many different types of cancers. Besides cancer treatment, VEGF / VEGFR inhibitors, including antibody fragments, aptamers, and VEGF-Traps were also approved and used for the treatment of ocular diseases caused by pathological angiogenesis. VEGF / VEGFR blockade can inhibit VEGF-driven tumor angiogenesis, and the regression of tumor vessels is dependent on the VEGF signaling pathway. However, VEGF inhibitors alone are not capable of destroying all tumor blood vessels. In addition, preclinical studies indicate that VEGF inhibitors alone resulted in an increasingly aggressive and invasive pattern of tumors. Some cancer patients are eventually refractory to anti-VEGF therapy; hence, next-generation angiogenesis inhibitors are being sought to augment the effects of VEGF inhibitors. Provided herein is a bispecific antibody that simultaneously targets both PD-1 and VEGF, by linking each C-terminal of an anti-VEGF antibody (bevacizumab-similar) with a at least one PD-1 antigen binding unit.

[0001] When the interaction between VEGF and a VEGF receptor (VEGF-R) is inhibited by the VEGF-specific antigen binding unit of the present disclosure, VEGF signaling is inhibited. It is known that when VEGF binds to VEGF-R on tumor cells, it triggers signaling pathways that promote tumor survival, proliferation, invasion, and metastasis while also stimulating angiogenesis (new blood vessel growth) in the surrounding microenvironment. This combined effect enhances tumor growth and spread. Disruption of VEGF / VEGF receptor signaling in stromal / endothelial cells of cancer tissue, thus inhibits angiogenesis and reduces the number of blood vessels and weakens vascular function in tumors, thereby inhibiting cancer proliferation and metastasis.

[0032] Thus, the dual-targeting protein or multispecific antigen-binding construct of the present disclosure, which in some aspects, is specific for PD-1 and VEGF, shows the ability to inhibit angiogenesis in cancer tissue by a different mechanism, and thus can be used as a therapeutic agent having better anticancer activity.

[0033] The present disclosure may include multispecific antigen-binding constructs comprising an antigen binding unit or an antibody that binds specifically to VEGF. The VEGF-specific binding antibody may include all antibodies that bind specifically to a VEGF antigen. Specifically, the antibody may be bevacizumab, a therapeutic antibody that targets VEGF, but is not limited thereto. Such antibodies that bind specifically to VEGF may include full-length antibodies or antibody fragments, and may be IgG antibodies, but are not limited thereto. VEGF is a ligand playing an important role in angiogenesis, and when VEGF is inhibited, no angiogenesis will occur, and thus cancer can be treated. Bevacizumab, approved by the FDA, is a therapeutic antibody that can be stably used. In some embodiments, the VEGF-specific binding antibody may include a heavy chain variable region, wherein the heavy chain variable region may comprise one or more mutations. For example, the VEGF-specific binding antibody may comprise a double mutation in the hinge-region at L234A / L235A.

[0034] In some embodiments, the multispecific antigen-binding construct is capable of binding human VEGF. The present disclosure provides a multispecific antigen-binding constructs that may bind specifically to human-derived VEGF to effectively inhibit the VEGF / VEGFR signaling pathways and may also minimize the risk of immunogenicity. In some aspects, the multispecific antigen-binding construct may be a bispecific antibody that may bind specifically to human VEGF. In some aspects, the bispecific antibody may be a fusion protein and include a first antigen binding unit targeting PD-1 connected to the C-terminal region of a protein similar to IgG -type bevacizumab. Thus, in some aspects the bispecific antibody described herein may effectively inhibit PD-1 as well as the interaction between VEGF and VEGF receptor, thereby exhibiting excellent anticancer effects. In the present disclosure, the multispecific antigen-binding construct may be a form wherein an immunoglobulin G (IgG)-type antibody that binds specifically to VEGF and a full-length antibody, Fab', F(ab’h, Fab, Fv, rlgG or scFv- type protein that binds specifically to PD-1 are connected to each other by a linker.

[0035] The multispecific antigen-binding constructs of the present disclosure, which may comprise a first antigen binding unit that binds specifically to PD- 1 and a second antigen binding unit that binds specifically to VEGF, show a strong affinity for human-derived VEGF, and effectively inhibits an angiogenic process in which vascular endothelial cells expressing VEGF receptor are activated by VEGF that is overexpressed in cancer cells. Thus, the multispecific antigen-binding constructs of the present disclosure may exhibit a stronger therapeutic effect in the treatment of diseases such as cancer. The multispecific antigenbinding construct may be a bispecific antibody. The bispecific antibody may include a VEGF-specific antigen binding unit and PD- 1 antigen binding unit that may maintain their specific binding, and particularly, can simultaneously inhibit two targets (antigens), for example VEGF and PD-1. Thus, the bispecific antibody may be more effective than a protein or antibody that binds to and inhibits a single target. Further, the bispecific antibody as described herein may simultaneously inhibit two signals. The VEGF-specific binding unit may be in the form of a full-length antibody and / or antibody fragments.

[0036] In some embodiments, the multispecific antigen-binding construct or bispecific antibody may comprise an antigen binding unit that specifically binds to VEGF and includes: (a) a heavy chain variable region comprising heavy chain CDRH1 having an amino acid sequence represented by SEQ ID NO: 15, heavy chain CDRH2 having an amino acid sequence represented by SEQ ID NO: 16, and heavy chain CDRH3 having an amino acid sequence represented by SEQ ID NO: 17, and (b) a light chain variable region comprising light chain CDRL1 having an amino acid sequence represented by SEQ ID NO: 12, light chain CDRL2 having an amino acid sequence represented by SEQ ID NO: 13, and light chain CDRL3 having an amino acid sequence represented by SEQ ID NO: 14.

[0037] In some embodiments, the antigen-binding unit that specifically binds to VEGF may include a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 10 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 11. In some embodiments, the heavy chain variable region of the VEGF antigen binding unit comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the heavy chain variable region of the VEGF antigen binding unit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 10. In some embodiments, the light chain variable region of the VEGF antigen binding unit comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:11. In some embodiments, the light chain variable region of the VEGF antigen binding unit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11. Also provided herein, in some aspects and embodiments, is a multispecific antigen-binding construct comprising four units of antigen-binding, wherein two units of antigen-binding bind PD-1 and two units of antigen-binding bind VEGF, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2 or 10, and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3 or 11.

[0038] The present disclosure also provides for a bispecific antigen binding construct. In some embodiments the construct may comprise at least two amis that bind PD- 1 and at least two arms that bind VEGF. In some embodiments, the construct may comprise a bivalent antibody specific for PD- 1 , and a bivalent antibody specific for VEGF, wherein the bivalent antibody specific for PD- 1 and the bivalent antibody specific for VEGF arc linked. In some embodiments, the construct may comprise a bivalent antibody specific for PD-1, and a fusion protein specific for VEGF, wherein the bivalent antibody specific for PD-1 and the fusion protein specific for VEGF are linked.

[0039] In some embodiments, the first antigen binding unit or second antigen binding unit, or both, comprise a heavy chain comprising one or more immunoglobulin Fc modifications. In some embodiments, the immunoglobulin Fc domain of the heavy chain comprises one or more amino acid mutations that promote heterodimerization of the first and second arms. In some embodiments, the mutation is present in a CH3 domain of the heavy chain. In some embodiments, the multispecific antigen-binding construct is produced in a quadroma cell. In some embodiments, the construct comprises one or more immunoglobulin constant region modifications. In some embodiments, the immunoglobulin constant region comprises one or more amino acid mutations that promote hctcrodimcrization of antibodies. In some embodiments, one or more mutations is present in the light chain constant region of one arm and one or more mutations is present in the heavy chain constant region of another arm. In some embodiments, the bispecific antibody is of a format selected from the group consisting of a bispecific IgG, bispecific antibody fragment, bispecific fusion protein, appended IgG, and bispecific antibody conjugate. In some embodiments, the Fc region has reduced effector function. In some embodiments, the Fc region enhances half-life of the construct. In some embodiments, the Fc region may be an IgGl. In some aspects, the Fcregion may be an IgGl that includes two mutations. For example, the Fc region may include the mutations L234A and L235A.

[0040] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein are aglycosylated. In some embodiments, the multispecific antigen-binding construct is capable of binding human PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding murine PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding cynomolgus monkey PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding human, murine and cynomolgus monkey PD-1 with similar affinity.

[0041] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein may be capable of reducing PD- 1 levels on a cell. In some embodiments, the multispecific antigen-binding construct may be capable of inducing PD- 1 degradation. In some embodiments, the multispecific antigen-binding construct may be capable of reducing PD-1 expression. In some embodiments, the multispecific antigen-binding construct may be capable of reducing PD- 1 cell surface expression. In some embodiments, the multispecific antigen-binding construct may be capable of reducing PD-1 cell surface expression by inducing shedding of PD-1 from the cell surface. The VEGF-specific binding unit of the multispecific antigen-binding construct according to the present disclosure may bind specifically to VEGF that is overexpressed in tumor cells, and thus may concentrate the multispecific antigen-binding construct of the present disclosure on tumor cells expressing VEGF. The multispecific antigen-binding construct may also exhibit anticancer activity by binding to VEGF. In addition, the VEGF-binding antigen binding unit of the multispecific antigen-binding construct may inhibit the interaction between human VEGF and VEGF receptor and furthermore, the VEGF-binding antigen binding unit of the multispecific antigen-binding construct may inhibit the interaction between VEGF and VEGFR-2 but is not limited thereto.

[0042] In some embodiments, the multi specific antigen-binding construct may be capable of binding both PD-1 and VEGF and reducing PD-1 levels on a cell. In some embodiments, the multispecific antigen-binding construct may be capable of binding both PD-1 and VEGF and inducing PD-1 degradation. In some embodiments, the multispecific antigen-binding construct may be capable of binding both PD- 1 and VEGF and reducing PD-1 expression. In some embodiments, the multispecific antigen-binding construct may becapable of inducing shedding of PD-1 from an immune cell. In some embodiments, the multispecific antigen-binding construct may be capable of binding both PD- 1 and VEGF and inducing PD- 1 shedding from an immune cell.

[0043] The disclosure also provides compositions that include a multispecific (i.e., bispecific) antigen binding construct disclosed herein and a pharmaceutically acceptable earner. In some embodiments, the composition may include more than one bispecific antigen binding construct disclosed herein.

[0044] Further, the bispecific antigen binding construct provided herein may bind specifically to two antigens, such as an immunomodulatory protein and a tumor antigen, and activate immune cells within a tumor microenvironment to achieve antitumor activity.Accordingly, the present disclosure provides methods of treating subjects, such as human subjects, diagnosed with a solid tumor. The method generally involves administering to the subject an amount of a bispecific antigen binding construct described herein effective to provide therapeutic benefit. The subject may be diagnosed with any one of a number of solid tumors that may be newly diagnosed, relapsed, or relapsed and refractory.

[0045] The bispecific antigen binding constructs may be administered as single therapeutic agents (monotherapy) or adjunctive to or with other therapeutic agents typically, but not necessarily, those used for the treatment of a solid tumor. Therapeutic agents typically will be used at their approved dose, route of administration, and frequency of administration, but may be used at lower dosages.

[0046] The bispecific antigen binding constructs described herein may be administered via a variety of routes or modes of administration, including but not limited to, intravenous infusion and / or injection, intratumoral injection, and subcutaneous injection. The amount administered will depend upon the route of administration, the dosing schedule, the stage of cancer being treated, and other parameters such as the age and weight of the patient, as is well known in the art.

[0047] The disclosure further provides methods for treating cancer in a subject, including the step of administering to the subject having cancer an effective amount of a multispecific antigen binding construct described herein or a composition including a bispecific antigen binding construct described herein. The subject may be a human or other mammal. The cancer is optionally selected from the group consisting of a hematologicalcancer, neurological cancer, breast cancer, prostate cancer, skin cancer, lung cancer, bladder cancer, kidney cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, and vascular cancer. The methods for treating cancer may further include the step of administering a second anticancer therapy to the subject. In certain aspects, the anticancer therapy is chemotherapy, immunotherapy, hormone therapy, cytokine therapy, radiotherapy, cryotherapy, or surgical therapy. The multispecific antigen binding construct or composition is administered, for example, subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

[0048] The disclosure also provides methods of enhancing an immune response in a subject in need thereof, comprising administering to the subject (e.g., a human or other mammal) a therapeutically effective amount of a multispecific antigen binding construct described herein or a composition that includes the disclosed bispecific antigen binding construct. The enhanced immune response may include one or more enhanced T cell function, enhanced NK cell function, or enhanced macrophage function. The multispecific antigen binding construct or composition may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.BRIEF DESCRIPTION OF DRAWINGS

[0049] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0050] FIG. 1 shows the structure of a dual-targeting bispccific molecule capable of binding to both PD-1 and VEGF and amino acid sequence for an Anti-PD-1 x Anti-VEGF-A bispecific (referred to as CTX- 10726). The bispecific antibody combines an anti- VEGF- A binding region which may be, for example, bevacizumab and a Fc region that may be IgGl with mutations at L234A and / or L235A, and a second region that includes Anti-PD-1. The anti-VEGF and anti-PD-1 regions of the bispecific antibody are connected via a linker;

[0051] FIG. 2 CTX- 10726 binds with human and cynomolgus macaque targets with high affinity. Figs. 2A-2C present sensorgrams showing binding of CTX- 10726 to soluble human and cynomolgus macaque PD- 1 and human VEGF- A as assessed by biolayer interferometry (BLI). His-tagged antigens were captured onto biosensors and then exposed to CTX- 10726. Cynomolgus monkey and human VEGF- A are identical. Fig. 2D shows CTX-10726 binding simultaneously to human VEGF-A and PD-1. Plates were coated with soluble VEGF-A, incubated with serially diluted CTX- 10726, followed by PD-1 -biotin and Streptavidin-HRP detection (ELISA);

[0052] FIG. 3 shows CTX-10726 and Pembrolizumab bind to PD-1 in the presence or absence of VEGF-A. Fig. 3A shows in the presence of VEGF-A, the affinity of CTX-10726 binding to PD-1 was substantially improved compared to the binding in the absence of VEGF-A; While adding VEGF-A to Pembrolizumab had no meaningful impact on its binding to PD-1 as demonstrated in Fig. 3B;

[0053] FIG. 4 shows CTX-10726 binds to PD-1 over-expressed Jurkat cells in the presence or absence of VEGF-A. Fig. 4 demonstrates that preincubating CTX-10726 and VEGF-A in an 1:2 molar ratio enhanced CTX-10726 binding to PD-1 expressing Jurkat cells as indicated by the increased overall signal;

[0054] FIG. 5 showsPD-1 signaling blockade by CTX-10726 in the presence of VEGF-A. Fig. 5 shows in a signaling assay that adding VEGF-A to the CTX-10726 (closed square) enhanced its PD- 1 blockade as compared to its potency in the absence of VEGF-A (open square);

[0055] FIG. 6 shows CTX-10726 blocks PD-1 in a mixed lymphocyte reaction (MLR) with and without the presence of VEGF-A. Fig. 6 shows interferon gamma production in one-way MLR assays with Keytruda (1 nM), CTX-10726 (0.2, 1, 5, and 1025 nM), and CTX-10726 mixed with VEGF (1:2 molar ratio). IFN-y levels were measured after a five-day incubation;

[0056] FIG. 7 CTX-10726 binds human and cynomolgus macaque cell surface PD-1 with comparable affinity. The graph shown in Fig. 7A presents Staphylococcus aureus Enterotoxin B (SEB)-activated human PBMCs that were exposed to increasing concentrations of CTX-10726, ivonescimab, or human IgGl (isotype control), followed by fluorochrome-conjugated anti-human F(ab)2 secondary antibodies, FACS acquisition and analysis. Fig. 7B shows human PBMCs treated as in 7A, incubated with increasing concentrations of CTX-10726, pembrolizumab, or human IgGl in the presence of VEGF-A at 1:2 molar ratio. Figs. 7C-D show CTX-10726 cross-reactivity to PD-1 from human and cynomolgus monkey;

[0057] FIG. 8 CTX- 10726 has potent immunomodulatory activity in vitro. Fig. 8A shows anti-PD-1 activity of CTX- 10726 evaluated using the Jurkat PD-1 / PD-L1 reporter assay (Promega). Fig. 8B shows PBMCs from three donors paired (1:1) in two-way MLR assays with 35 nM PD-l-blocking antibodies. IFN-y levels were measured after a three-day incubation. Fig. 8C shows TCR-activated PBMCs from three donors co-cultured with HCC827 target cells (1:1) in the presence of 50 nM anti-PD-1 antibodies for two days (TTR, Tumor / T cell Reaction assay):

[0058] FIG. 9 CTX- 10726 blocks VEGF-A / VEGFR2 activity in vitro. Fig. 9 shows VEGF-A / VEGFR2 (KDR, Kinase insert domain receptor) blockade by CTX- 10726 evaluated using a qualified KDR / NFAT-RE HEK293 cell-based reporter assay:

[0059] FIG. 10 CTX-10726 demonstrates potent anti-tumor efficacy against HCC827 xenografts. Fig. 10A shows PD-L1 expression in HCC827 human NSCLC cell line evaluated by FACS. Fig. 10B shows VEGF-A levels in HCC827 cell culture supernatant measured by ELISA and plotted against time. Fig. 10C shows SEB-activated human PBMCs that were coinjected subcutaneously with HCC827 tumor cells into the flanks of female SCID / Beige mice (n=8 / group) at an effector-to-target ratio of 1:100. CTX-10726, an isotype control antibody, an anti- VEGF-A monoclonal antibody, or ivonescimab were administered intraperitoneally on the day of implantation and then weekly for a total of four doses (dotted lines);

[0060] FIG. 11 CTX-10726 shows curative anti-tumor activity against MC38hPD-Ll tumors implanted in hPD-l / hPD-Ll transgenic double knock-in mice. Fig. 11A presents a graph in which each mouse (n=5 / group) was inoculated subcutaneously with MC38 hPD-Ll tumor cells. Fig. 1 IB presents a graph in which each mouse (n=10 / group), was inoculated subcutaneously with MC38 hPD-Ll tumor cells. Fig. 11C shows individual tumor responses to hlgGL CTX-10726, and pembrolizumab over time;

[0061] FIG. 12 CTX-10726 demonstrates superior efficacy against MC38 hPD-Ll / h VEGF-A tumors, driven mainly by PD-1 / PD-L1 check-point inhibition. Fig. 12A shows the combined anti- VEGF-A and anti-PD-1 activity of CTX-10726 assessed in the hPD-1 / hPD-Ll / h VEGF-A triple-knock-in mice (Biocytogen, n=8 / group), where both targets of the drug are human. Fig. 12B shows the tumor volume of individual mice treated with bevacizumab, CTX-10726, ivonescimab, or PBS (control) overtime; and

[0062] FIG. 13 CTX- 10726 pharmacokinetics profile in non-human primates (NHPs) after a single dose demonstrates a dose-proportional increase in serum concentration. Fig. 13 shows CTX- 10726 serum concentration-time profiles in cynomolgus monkeys following a single intravenous infusion of CTX-10726 at a dose of 1 mpk, 10 mpk, or 50 mpk.DETAILED DESCRIPTION

[0063] The following description recites various aspects and embodiments of the disclosed compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide nonlimiting examples that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included. It is also to be understood that as used herein, the singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise.Bispecific Antigen Binding Constructs

[0064] The immune system has the capability of recognizing and eliminating tumor cells; however, tumor cells can use multiple strategies to evade the immune system. Blockade of immune checkpoints is one of the approaches to activating or reactivating therapeutic antitumor immunity. Another approach to inhibit cancer formation or progression is by inhibiting angiogenesis facilitated by the expression of VEGF. The present disclosure provides bispecific antigen binding constructs that block both the PD-1 / PD-L1 and VEGF pathways, thereby enhancing anti-tumor efficacy. In some aspects, the multispccific or bispecific antigen binding constructs may simultaneously target VEGF (VEGF- A) and PD-1 in a single molecule, thus reducing the complexity of combination treatment regimens by concentrating PD-1 blockade at VEGF-rich, immunosuppressed tumor sites. Further, the multispecific or bispecific antigen binding constructs may also provide improved safety over VEGF TKI-containing combination regimens.

[0065] In some embodiments, the bispecific antigen binding constructs may exhibit high-affinity binding to both human and cynomolgus monkey VEGF- A and PD- 1. The bispecific antigen binding constructs (e.g., CTX-10726) may also effectively block VEGF-A / VEGFR2 and PD-1 / PD-L1 interactions in a dose-dependent manner. In some aspects, the bispecific antigen binding constructs may reduce engagement with Fey receptors, limitingoff-target immune activation. Further, the bispecific antigen binding constructs may enhance IFN-y production in mixed lymphocyte reactions (MLRs) and tumor cell killing by activated PBMCs. In still additional aspects, the bispecific antigen binding constructs presented herein may outperform benchmark biosimilar anti-VEGF or PD-1 antibodies in controlling tumor growth across multiple xenograft and syngeneic models, in vivo.

[0066] The present disclosure provides bispecific antigen binding constructs that include at least two linked antigen binding units that recognize specific target antigens. Thus, the terms bispecific antigen binding construct and multispecific antigen binding construct are used interchangeably herein to refer to bispecific, tri-specific, or multispecific antigen binding constructs.

[0067] A bispecific antigen binding construct can be a single multifunctional polypeptide, small molecule, or aptamer, or it can be a multimeric complex of two or more molecules that are covalently or non-covalently associated with one another. Bispecific antigen binding constructs include antibodies (or antigen binding fragments thereof) that may be linked to or co-expressed with another functional molecule, e.g., another peptide, protein, and / or aptamer. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a bispecific antigen binding construct with a second binding specificity. In certain embodiments, an antibody or antigen binding fragment thereof is functionally linked to one or more antibody or antigen binding fragment thereof having a different binding specificity to produce a bispecific antigen binding construct. Each antibody or antigen binding portion thereof of the construct may have one or more antigen binding specificities.

[0068] As used herein, an antigen binding unit refers to a domain, region, or the like, of the bispecific antigen binding construct that forms an area of the construct that binds to an antigen. A first antigen binding unit forms a separate binding area of the bispecific antigen binding construct from a second antigen binding unit of the construct, each unit forming a separate region of antigen binding. Generally, one unit (first unit) is distinct from the other unit (second unit) in its antigen binding. For example, one antigen binding unit of the antibody may be monovalent for and bind to PD-1 while the other antigen binding unit of the bispecific antigen binding construct may be monovalent for and bind to VEGF.

[0069] Alternatively, or in addition, the bispecific antigen binding construct provided herein may be tetravalent. For example, the first antigen binding unit may be bivalent for PD-1, each binding the same epitope on PD- 1, while the other antigen binding unit may be bivalent for VEGF, each binding the same epitope on VEGF. In some embodiments, the bispecific antigen binding construct may be tetravalent, wherein one antigen binding unit is bivalent for PD-1, each binding two different epitopes on PD-1. In some embodiments, the bispecific antigen binding construct is tetravalent, wherein one antigen binding unit is bivalent for VEGF, each binding two different epitopes of VEGF. In some embodiments, the bispecific antigen binding construct may be tetravalent, wherein one antigen binding unit is bivalent for PD-1, each binding two different but overlapping epitopes on PD-1. In some embodiments, the bispecific antigen binding construct may be tetravalent, wherein one antigen binding unit is bivalent for VEGF, each binding two different but overlapping epitopes of VEGF.

[0070] The term valency, when used to describe an antigen binding construct or protein, refers to the number of recognition (binding) sites in the antigen binding construct or protein. Each recognition site specifically recognizes, and is therefore capable of binding, one epitope (binding site) on an antigen. When an antigen binding protein comprises more than one recognition site (e.g., when an antigen binding protein is an IgG, which has two recognition sites in its variable regions), each recognition site can specifically recognize the same epitope on the same antigen, or different epitopes, whether on the same or different antigens.

[0071] In some embodiments, the bispecific antigen binding constructs comprise a first antigen binding unit that specifically binds PD-1, and a second antigen binding unit that specifically binds VEGF. With regard to the binding of an antigen binding unit to a target molecule, the terms specific binding, specifically binds to, specific for, selectively binds, selective for, and the like as related to a particular target antigen or molecule (e.g., a polypeptide target) or an epitope on a particular target antigen or molecule mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a target molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case,specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0072] In some embodiments, the binding of one antigen binding unit (e.g., the first antigen binding unit) of the bispecific antigen-binding construct to its target does not block or sterically hinder the binding of the other antigen binding unit (e.g., the second antigen binding unit) to its target. For example, upon the binding of a first antigen binding unit to PD-1, the second antigen binding unit is free to bind a VEGF antigen. Thus, in some embodiments, the first antigen binding unit and second antigen binding unit bind to their respective targets concurrently.

[0073] As described herein, the constructs of the present disclosure may be capable of binding to an immune cell that expresses PD-1. The type of immune cell depends on the context of the disease to be treated, and the particular type of immune cell can be determined by one of skill in the art depending on the disorder under consideration. In some embodiments, the immune cell is a T cell, including but not limited to a CD8+ T cell and CD4+ T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a macrophage.

[0074] In some embodiments, the VEGF is expressed by a tumor cell (a solid or nonsolid tumor cell). As used herein, tumor cell is sometimes used interchangeably with cancer cell but also encompasses non-malignant (non-cancerous) cells exhibiting increased proliferation as compared to a normal cell.

[0075] By way of example, the tumor cell is selected from the group consisting of a hematological cancer, a lymphoma, a myeloma, a leukemia, a neurological cancer, melanoma, breast cancer, a prostate cancer, a colorectal cancer, lung cancer, head and neck cancer, a gastrointestinal cancer, liver cancer, pancreatic cancer, a genitourinary cancer, a bone cancer, renal cancer, and a vascular cancer. In some embodiments, the tumor cell is selected from the group consisting of Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt’s lymphoma and marginal zone B cell lymphoma, Polycythemia vera Lymphoma,Hodgkin's disease, non- Hodgkin’s disease, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pincaloma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cavity cancer (for example lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system.

[0076] As described herein, the disclosed bispecific antigen binding constructs include bispccific, trispccific, tctraspccific, or multispccific antibodies or antigen binding fragments thereof.

[0077] Antigen binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv; and (viii) a single domain antibody (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Nall. Acad. Sei. USA 85:5879-5883). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0078] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are disclosed in WO 94 / 04678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.

[0079] In some embodiments, an antigen binding unit can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are, generally, obtained through combinatorial chemistry -based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al. (1999) J. Biol. Chem. 274:24066-24073. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol 23( 10):514-522.

[0080] One of skill in the art would appreciate that the scaffold portion of the nonantibody scaffold protein can include, e.g., all or part of the Z domain of .S'. aureus protein A, human transferrin, human tenth fibronectin type III domain, Kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins, such as those described in, e.g., W02015 / 104406), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.

[0081] In some embodiments, the bispecific antigen binding construct comprises a bispecific antibody, having specificity for at least two antigens but optionally having more than two binding sites. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first antigen (e.g., VEGF) and a second immunoglobulin variable domain sequence that has binding specificity for a second antigen (e.g., PD-1). In some embodiments, a bispecific antibody molecule comprises a sc Fv or fragment thereof having binding specificity for a first antigen and a scFv or fragment thereof having binding specificity for a second antigen. (See, e.g., Kontermann and Brinkmann (2015) Drug Discovery Today 20(7):838-47.)

[0082] Various bispecific antibody formats are known in the art, including, for example, a bispecific IgG, a bispecific antibody fragment, a bispecific fusion protein, an appended IgG, and a bispecific antibody conjugate, described herein. Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl / lgG2, dual acting Fab (DAF)-lgG, and Mab2bispecific formats (see, e.g., Klein et al. (2012) mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). (See also Spiess et al. (2015) Mol Immunol 67:95-106.) Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al. (2013) J Am Chem Soc 135(1 ):340-6). In some embodiments, the bispecific antigen binding construct disclosed herein comprises a common light chain.

[0083] In certain embodiments, the antibody or antigen binding fragment thereof comprises known antibodies or the CDRs of known PD-1 antibodies.

[0084] In some aspects, the antibody or antigen binding fragment thereof may comprise a first antigen binding unit that specifically binds PD-1 and comprises a heavy chain variable region comprising a CDRH1 comprising SEQ ID NO: 7, a CDRH2 comprising SEQ ID NO: 8, and a CDRH3 comprising SEQ ID NO: 9; and a light chain variable region comprising a CDRL1 comprising SEQ ID NO: 4, a CDRL2 comprising SEQ ID NO: 5, and a CDRL3 comprising SEQ ID NO: 6. In some aspects, the antibody or antigen binding fragment thereof of the first antigen binding unit comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to amino acids of SEQ ID NO: 2 and a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some aspects, the bispecific antigen binding construct comprises at least one heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. In certain aspects, the bispecific antigen binding construct comprises at least one heavy chain comprising an amino acid sequence of SEQ ID NO: 2. In some aspects, the bispecific antigen binding construct comprises at least one light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. Optionally, the bispecific antigen binding construct comprises at least one light chain comprising an amino acid sequence of SEQ ID NO: 3. Identity or similarity with respect to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0085] In certain embodiments, the antibody or antigen binding fragment thereof may comprise a second antigen binding unit that specifically binds VEGF and comprises a heavy chain variable region comprising a CDRH1 comprising SEQ ID NO: 15, a CDRH2 comprising SEQ ID NO: 16, and a CDRH3 comprising SEQ ID NO: 17; and a light chain variable region comprising a CDRL1 comprising SEQ ID NO: 12, a CDRL2 comprising SEQ ID NO: 13, and a CDRL3 comprising SEQ ID NO: 14. In some aspects, the antibody or antigen binding fragment thereof of the second antigen binding unit comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to amino acids of SEQ ID NO: 10 and a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 11. In some aspects, the bispecific antigen binding construct comprises at leastone heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In certain aspects, the bispecific antigen binding construct comprises at least one heavy chain comprising an amino acid sequence of SEQ ID NO: 10. In some aspects, the bispecific antigen binding construct comprises at least one light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 11. Optionally, the bispecific antigen binding construct comprises at least one light chain comprising an amino acid sequence of SEQ ID NO: 11. Identity or similarity with respect to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In some aspects, the antigen binding construct comprises at least one copy of a heavy chain that includes both CDRH sequences for PD-1 and CDRH sequences for VEGF.

[0086] The present disclosure also encompasses antibodies or fragments thereof that bind to the same epitope of PD- 1 or VEGF as the antibodies disclosed herein. Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.

[0087] Methods for generating bispecific antibody molecules are known in the art, including but not limited to, for example, the “knob in a hole” approach described in, e.g., U.S. Pat. No. 5,731,168; the electrostatic steering Fc pairing as described in, e.g., WO 09 / 089004, WO 06 / 106905 and WO 2010 / 129304; Strand Exchange Engineered Domains (SEED) heterodimer formation as described in, e.g., WO 07 / 110205; Fab arm exchange as described in, e.g., WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867: double antibody conjugate, e.g., by antibody cross-linking to generate a bi-specific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl reactive group as described in, e.g., U.S. Pat. No. 4,433,059; bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab' fragments cross-linked through sulfhydryl reactive groups, as described in, e.g., U.S. Pat. No.5,273,743; biosynthetic binding proteins, e.g., pair of scFvs cross-linked through C-terminal tails preferably through disulfide or amine-reactive chemical cross-linking, as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies, e.g., Fab fragments with differentbinding specificities dimerized through leucine zippers (e.g., c-fos and c-jun) that have replaced the constant domain, as described in, e.g., U.S. Pat. No. 5,582,996; bispecific and oligospecific mono- and oligo-valent receptors, e.g., VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CHI region of one antibody and the VH region of the other antibody typically with associated light chains, as described in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-anribody conjugates, e.g., crosslinking of antibodies or Fab fragments through a double stranded piece of DNA, as described in, e.g., U.S. Pat. No. 5,635,602; bispecific fusion proteins, e.g., an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a full constant region, as described in, e.g., U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., dimer of polypeptides having first domain with binding region of Ig heavy chain variable region, and second domain with binding region of Ig light chain variable region, generally termed diabodies (higher order structures are also encompassed creating for bispccific, trispccific, or tctraspccific molecules, as described in, e.g., U.S. Pat. No.5,837,242; minibody constructs with linked VL and VH chains further connected with peptide spacers to an antibody hinge region and CH3 region, which can be dimerized to form bispecific / multivalent molecules, as described in, e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all in either orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String of VH domains (or VL domains in family members) connected by peptide linkages with cross-linkable groups at the C-terminus further associated with VL domains to form a series of FVs (or scFvs), as described in, e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptides with both a VII and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV or diabody type format, as described in, e.g., U.S. Pat. No. 5,869,620. Additional exemplary multispecific and bispecific molecules and methods of making the same are found, for example, in U.S. Pat. Nos. 5,910,573; 5,932,448; 5,959,083; 5,989,830; 6,005,079; 6,239,259; 6,294,353; 6,333,396; 6,476,198; 6,511,663; 6,670,453; 6,743,896; 6,809,185; 6,833,441; 7,129,330; 7,183,076; 7,521,056; 7,527,787; 7,534,866; 7,612,181; US Patent Publication Nos. 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[0088] In some embodiments, the bispecific antigen binding construct of the present disclosure selectively binds PD-1 and VEGF. The antigen binding units can be generated by standard techniques, as disclosed herein. In some embodiments, any known antibodies against PD- 1 or VEGF can be used to generate a bispecific antibody according to the present disclosure, or the bispecific antigen binding constructs can be generated using known and / or available antibodies in the art.

[0089] The first antigen binding unit or second antigen binding unit, or both, can comprise a heavy chain comprising one or more immunoglobulin Fc modifications. For example, the VEGF-specific antigen binding unit may comprise an Fc region that is IgGl with mutations at E234A and / or E235A.

[0090] In some embodiments, the immunoglobulin Fc domain of the heavy chain comprises one or more amino acid mutations that, e.g., promote heterodimerization of the first and second antigen binding unit, promote serum half-life, and / or modify effector function. In some embodiments, the mutation is present in a CH3 domain of the heavy chain. (See, e.g., Xu et al. (2015) mAbs 7(1): 231-42.).

[0091] While traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, a variety of engineered Fc domains have been designed as asymmetric interaction pairs (Spicss ct al. (2015) Molecular Immunology 67(2A): 95-106) to promoteheterodimerization, e.g., of a first antigen binding unit and a second antigen binding unit. Various methods are known in the art that increase desired pairing of Fc-containing polypeptide chains in a single cell line to produce a preferred asymmetric fusion protein at acceptable yields (see, for example, Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015) Molecular Immunology 67(2PartA): 95-106). Methods to obtain desired pairing of Fc-containing polypeptides include, but are not limited to, charge-based pairing (electrostatic steering), “knobs-into-holes” steric pairing, SEED body pairing, and leucine zipper-based pairing. (See, for example, Ridgway et al. (1996) Protein Eng 9:617-621 ; Merchant et al. (1998) Nat Biotech 16:677-681; Davis et al. (2010) Protein Eng Des Sei 23:195-202;Gunasekaran et al. (2010) J Biol Chem 285:19637-19646; Wranik et al. (2012) J Biol Chem 287:43331-43339; US Patent No. 5932448; and PCT Publication Nos. WO 1993 / 011162; WO 2009 / 089004, and WO 2011 / 034605.

[0092] For example, one means by which interaction between specific polypeptides may be promoted is by engineering protuberance-into-cavity (knob-into-holes) complementary regions such as described in U.S. Patent Nos. 7,183,076 and 5,731,168; and PCT Publication No. WO 2016 / 164089. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., a first interaction pair) with larger side chains (e.g., tyrosine or tryptophan). Complementary “cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide (e.g., a second interaction pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Where a suitably positioned and dimensioned protuberance or cavity exists at the interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface.

[0093] At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged and lysine, arginine, and histidine are positively charged. These charged residues can be used to promote heterodimer formation and at the same time hinder homodimer formation. Attractive interactions take place between opposite charges and repulsive interactions occur between like charges. In part, protein complexes disclosed herein make use of the attractive interactions for promoting heteromultimer formation (e.g., heterodimer formation), and optionally repulsive interactions for hindering homodimer formation (e.g., homodimer formation) by carrying out site directed mutagenesis of charged interface residues.

[0094] For example, the IgGl CH3 domain interface comprises four unique charge residue pairs involved in domain-domain interactions: Asp356-Lys439', Glu357-Lys370', Lys392-Asp399', and Asp399-Lys409' [residue numbering in the second chain is indicated by (')]. It should be noted that the numbering scheme used here to designate residues in the IgGl CH3 domain conforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction is represented twice in the structure (e.g., Asp-399-Lys409' and Lys409-Asp399'). In the wild-type sequence, K409-D399' favors both heterodimer and homodimer formation. A single mutation switching the charge polarity (e.g., K409E; positive to negative charge) in the first chain leads to unfavorable interactions for the formation of the first chain homodimer. The unfavorable interactions arise due to the repulsive interactions occurring between the same charges (negative-negative; K409E-D399' and D399-K409E’). A similar mutation switching the charge polarity (D399K'; negative to positive) in the second chain leads to unfavorable interactions (K409'-D399K' and D399K-K409') for the second chain homodimcr formation. But, at the same time, these two mutations (K409E and D399K') lead to favorable interactions (K409E-D399K' and D399-K409') for the heterodimer formation. The electrostatic steering effect on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360. (See, e.g., PCT Publication No. WO 2016 / 164089.).

[0095] Thus, in some embodiments, the bispecific antigen binding constructs described herein may comprise a constant domain of an immunoglobulin, including, for example, the Fc portion of an immunoglobulin. For example, the bispecific antigen binding construct may include a first antigen binding unit that may comprise an amino acid sequence that is derived from an Fc domain of an IgG (IgGl, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM immunoglobulin. Alternatively, the bispccific antigen binding construct may include a second antigen binding unit may comprise an amino acid sequence that is derived from an Fc domain of an IgG (IgGl, lgG2, lgG3, or IgG4), IgA (IgAl or lgA2), IgE, or IgM. Such immunoglobulin domains may comprise one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimer formation. In some embodiments, a first antigen binding unit and a second antigen binding unit comprise Fc domains derived from the same immunoglobulin class and subtype. In some embodiments, a first and second antigen binding unit comprise Fc domains derived from differentimmunoglobulin classes or subtypes. Similarly, a first and / or a second antigen binding unit (e.g., an asymmetric pair or an unguided interaction pair) comprise a modified constant domain of an immunoglobulin, including, for example, one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimer formation. Methods of generating Fc modifications having the desired heterodimer formation are known in the art.

[0096] In some embodiments, the Fc domain can be modified to enhance serum halflife of the bispecific antigen binding construct disclosed herein. Fc domain comprising one or more mutations which enhance or diminish antibody binding to the Fc receptor, e.g., at acidic pH as compared to neutral pH, are known in the art. For example, the constructs disclosed herein may comprise a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the construct when administered to an animal. Methods of modifying the Fc domain for desired characteristics, such as enhanced serum half-life are known in the art.

[0097] Antibodies or fragments thereof can be further selected for binding to more than one species. For example, antibodies or fragments that bind both mouse and human can be selected by screening with both mouse and human target cells.

[0098] The regions that comprise the bispecific antigen binding construct of the disclosure comprise VH and VL regions that together specifically bind an antigen. The VH and VF regions and CDRs incorporated into bispecific antigen binding constructs may be derived from multiple sources, including pre-existing antibodies, newly generated antibodies, VH and VF chain libraries, CDR libraries or synthetic sequences. The VH and VF chains within, for example, a scFv are linked by way of a polypeptide linker that does not significantly interfere with the antigen binding properties of the VH and VF regions. The scFv regions may have a VH-VF or a VF-VH orientation (in N^C direction).

[0099] In some embodiments, a bispecific antigen binding construct exhibit conditional activation, by which they provide activation of an immunomodulatory protein in the presence of a cell-surface tumor antigen, and reduced, minimal or no activation of the immunomodulatory protein in the absence of the cell-surface tumor antigen.

[0100] In some embodiments, a bispecific antigen binding construct as described herein, comprises a linker linking the single chain variable fragment VH chain with the VL chain. The linker linking the single chain variable fragment VH chain with the VL chain may be selected to increase expression, solubility, stability (for example, as measured by lower aggregation levels, lower rate of aggregation, higher melting temperature, and / or longer plasma half-life), and / or titer of a bispecific antigen binding construct of the present disclosure.Methods for Producing the Bispecific Antigen Binding Constructs[00101 J Provided herein are methods for producing any of the bi specific antigen binding constructs described herein. The methods for producing the disclosed constructs include methods for preparing an antibody and antigen binding fragments thereof. Such methods are well-known in the art and include, e.g., immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. To generate an antibody that binds to PD- 1 or VEGF, for example, a skilled artisan immunizes a suitable subject (e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate) with full-length PD-1 or VEGF polypeptide or a variant or fragment thereof. Antigenic fragments of a polypeptide (PD-1 or VEGF) can be selected to generate antibodies based on known structural features of the polypeptide. For example, regions within PD-1 or VEGF, based on receptor / ligand interface information available in the art, can be used to design a suitable antigenic fragment to generate antibodies having desirable properties. Resulting antibodies or antigen binding constructs can then be screened for desired binding properties (e.g., binding affinities for PD-1 and VEGF and capacity to bridge cells on which PD-1 and VEGF are expressed).

[0102] A suitable subject (e.g., a non-human mammal) can be immunized with the appropriate antigen along with subsequent booster immunizations a number of times sufficient to elicit the production of an antibody by the mammal. The immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant. Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum or Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund’s adjuvant; viral adjuvants; and chemical adjuvants, e.g., aluminum hydroxide, andiodoacetate and cholesteryl hemisuccinate. Other adjuvants that can be used in the methods for inducing an immune response include, e.g., cholera toxin and parapoxvirus proteins. (See also Bieg et al. (1999) Autoimmunity 31(1): 15-24. See also, e.g., Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) J Med Chem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Gupta et al. (1995) Vaccine 13(14): 1263-1276.)

[0103] In some embodiments, the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen. For example, a suitable mammal such as a laboratory mouse is immunized with a polypeptide (e.g., PD-1 or VEGF) or antigenic fragment as described above. Antibody-producing cells (e.g., B cells of the spleen) of the immunized mammal can be isolated two to four days after at least one booster immunization of the immunogen and then grown briefly in culture before fusion with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter, such as, e.g., vaccinia vims or polyethylene glycol. The hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells of Balb / c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag 14. After the fusion, the cells are expanded in suitable culture medium, which is supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The obtained hybridoma cells are then screened for secretion of the desired antibodies, e.g., an antibody that binds to the desired antigen.

[0104] In some embodiments, the methods described herein involve, or be used in conjunction with, e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques. (See, e.g., Etz et al. (2001) J Bacterio! 183:6924-6935;Cornells (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-444;Grabherr et al. (2001) Comb Chem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel et al. (1999) J Immunol Methods 231:119-135; and Hanes et al. (2000) Nat Biotechnol 18:1287-1292).

[0105] Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on thesurface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-bond stabilized Fv antibody fragments, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage used in these methods are typically filamentous phage such as fd and Ml 3. The antigen binding domains are expressed as a recombinantly-fused protein to any of the phage coat proteins pill, pVIII, or pIX. (See, e.g., Shi et al. (2010) JMB 397:385-396.) Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184:177-186; Kettleborough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57:191-280; and PCT publication nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, and WO 95 / 20401. Suitable methods are also described in, e.g., U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727: 5,733,743 and 5.969,108.

[0106] In some embodiments, the phage display antibody libraries can be generated using mRNA collected from B cells from the immunized mammals. For example, a splenic cell sample comprising B cells can be isolated from mice immunized with a PD-1 or VEGF polypeptide as described above. mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. (See, e.g., Green and Sambrook (2012) Molecular Cloning- A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001; Harlow and Lane (1988) Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y; Lo (2004) Antibody Engineering:Methods and Protocols, Springer Science & Business Media; and Borrebaeck (1995) Antibody Engineering, Oxford University Press. The cDNA coding for the variable regions of the heavy chain and light chain polypeptides of immunoglobulins arc used to construct the phage display library. Methods for generating such a library are described in, e.g., Merz et al. (1995) J Neurosci Methods 62(l-2):213-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.

[0107] In some embodiments, a combination of selection and screening can be employed to identify an antibody of interest from, e.g., a population of hybridoma-derived antibodies or a phage display antibody library. Suitable methods are known in the art and aredescribed in, e.g., Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995) J Immunol Methods 182(l):41-50.; Ames et al. (1995) J Immunol Methods 184(2): 177-86.; Kettleborough et al. (1994) Eur J Immunol 24(4):952-8; and Persic et al. (1997) Gene 187(1 ): 9- 18. For example, a plurality of phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pill, pVIII, or pIX of M13 phage) and a different antigen-combining region are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coll). Expression of the bacteriophage in bacteria can, in some embodiments, require use of a helper phage. In some embodiments, no helper phage is required (see, e.g., Chasteen et al., (2006) Nucleic Acids Res 34(21):el45). Phage produced from the bacteria are recovered and then contacted to, e.g., a target antigen bound to a solid support (immobilized). Phage may also be contacted to antigen in solution, and the complex is subsequently bound to a solid support.

[0108] A subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular antigen (e.g., PD-1 or VEGF) using any immunological or biochemical based method known in the art. For example, specific binding of an antibody to PD-1 or VEGF may be determined, for example, using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above. Immunoassays that can be used to analyze immunospecific binding and cross-reactivity of the antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. It is understood that the above methods can also be used to determine if an antibody to PD-1 or VEGF docs not bind to human PD-1 or VEGF proteins.

[0109] In embodiments where the selected CDR amino acid sequences are short sequences (e.g., fewer than 10-15 amino acids in length), nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. (2007) Nucleic Acids Symposium Series 51 (1 ): 129-130 and U.S. Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced with the chemically synthesized nucleic acids using standardmolecular biology techniques. The 5’ and 3’ ends of the chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody.Alternatively, fragments of chemically synthesized nucleic acids, together capable of encoding an antibody, can be joined together using DNA assembly techniques known in the art (e.g. Gibson Assembly).

[0110] Any antibody of choice can be further modified to generate an antigen-binding fragment, as described herein, and / or manipulated using known techniques in the art to generate the bispecific antigen binding constructs as described herein. For example, crosslinking methods can be used to generate a bispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl reactive group as described in, e.g., U.S. Pat. No. 4,433,059; bispecific antibody determinants can be generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab' fragments can be crosslinked through sulfhdryl reactive groups, as described in, e.g., U.S. Pat. No. 5,273,743. Methods of generating bispecific constructs include, e.g., methods of generating bispecific constructs having common light chains.Expression and Purification of Bispecific Antigen Binding Constructs

[0111] The bispecific antigen binding constructs disclosed herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding the bispecific antigen binding construct (as a single multifunctional polypeptide, or as separate molecules of a multimeric complex - e.g., one antigen binding unit separately from the other antigen binding unit) can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, such that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0112] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drag resistance genes such as E. coll gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1 :327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0113] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPC precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0114] Appropriate host cells for the expression of antibodies or antigen binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines.

[0115] In some embodiments, an antibody or fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine (2002) Curr Opin Biotechnol 13(6) :625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2): 155- 159; and Pollock et al. (1999) J Immunol Metter 231(1-2): 147-157.

[0116] The antibodies and fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encodingthe antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell and are easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley & Sons: and Green and Sambrook (2012) Molecular Cloning-A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells vary depending on a number of factors, and may be easily optimized as needed. An antibody (or fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0117] Following expression, the antibodies and fragments thereof can be isolated. An antibody or fragment thereof can be isolated or purified in a variety of ways known in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) Protein Purification, 3rdedition, Springer-Verlag, New York City, New York. The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof is necessary.

[0118] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).Modification of the Bispecific Antigen Binding Constructs

[0119] The bispecific antigen binding constructs can be modified as a single multifunctional polypeptide or as separate molecules of a multimeric complex - e.g., one antigen binding unit separately from the other antigen binding unit. The modifications can be covalent or non-covalent modifications. Such modifications can be introduced into the antibodies or antigen binding fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments.

[0120] In some embodiments, the antibodies or antigen binding fragments thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag, polyhistidine, hemagglutinin, glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radioactive labels include, e.g.,32P,33P,14C,125I,1311,35S, and3H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Dy Light™ 488, phycoerythrin (PF), propidium iodide (PI), PerCP, PE- Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTP A) or tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOT A). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0121] Two proteins (e.g., an antibody and a heterologous moiety) can be crosslinked using any of a number of known chemical cross linkers. Examples of such cross linkers are those that link two amino acid residues via a linkage that includes a “hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected(by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

[0122] In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g.,125I in meta-[123I]iodophenyl-N-hydroxysuccinimide ([12H]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) or chelate (e.g., to DOTA or DTP A), which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules / chelates containing them to the antibodies or antigen binding fragments described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and / or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

[0123] Methods for conjugating a fluorescent label (sometimes referred to as a fluorophorc) to a protein (e.g., an antibody) arc known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein or fragment thereof with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0124] In some embodiments, the antibodies or fragments can be modified, e.g., with a moiety that improves the stabilization and / or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, the antibody or fragment can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic et al. (2010) IntJ Pharm 387( 1-2): 110- 119). The stabilization moiety can improve the stability, or retention of, the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

[0125] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some embodiments, an antibody or antigen-binding fragment thereof described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the antibody or fragment has reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10( 10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.Pharmaceutical Compositions and Formulations

[0126] Compositions comprising a bispecific antigen binding construct of the present disclosure and a pharmaceutically acceptable carrier arc also provided. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used in a subject having cancer or other condition that would benefit from the bispecific antigen binding constructs described herein.

[0127] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for s.c. and / or I.V. administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants(such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Allen (2012) Remington -The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Phamraceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the bispecific antigen-binding construct.

[0128] In certain embodiments, the primary vehicle or carrier in a phamraceutical composition can be either aqueous or non-aqueous in nature. Eor example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments,pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In certain embodiments, a composition comprising the bispecific antigen binding constructs disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising the bispecific antigen binding construct disclosed herein can be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0129] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0130] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[0131] In certain embodiments when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogcn-frcc, parenterally acceptable aqueous solution comprising a bispecific antigen binding construct, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which a bispecific antigen binding construct is formulated as a sterile, isotonic solution, and properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0132] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, a bispecific antigen binding construct can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising a bispecific antigen binding construct can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0133] In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, a bispecific antigen binding construct that is administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of a bispecific antigen binding construct. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0134] In certain embodiments, a pharmaceutical composition can involve an effective quantity of a bispecific antigen binding construct in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0135] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving a bispecific antigen binding construct in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT / US 93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained- release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and European Patent No. EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1993) Biopolymers 22:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al. (1981) J Biomed Mater Res. 15: 167-277; and Langer (1982) Chem Tech 12:98-105), ethylene vinyl acetate (Langer et al.) or poly-D(-)-3-hydroxybutyric acid (European Patent No, EP 133,988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Nos. EP 036,676; EP 088,046; and EP 143,949.

[0136] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0137] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0138] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0139] In certain embodiments, the effective amount of a pharmaceutical composition comprising a bispccific antigen binding construct to be employed therapeutically depend, forexample, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a bispecific antigen binding construct is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0140] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the bispecific antigen binding construct in the formulation used. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0141] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of the combination therapy may be administered by different routes.

[0142] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0143] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising a bispecific antigen-binding construct in an ex vivo manner. In such instances, cells, tissues (including, e.g., blood) and / or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising a bispecific antigenbinding construct after which the cells, tissues and / or organs are subsequently implanted back into the patient.

[0144] In certain embodiments, a bispecific antigen-binding construct can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient’s immune system or by other detrimental factors from the surrounding tissues.Methods of Use of the Bispecific or Multispecific Antigen Binding Constructs

[0145] As described herein, the present disclosure provides a method of treating a proliferative disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antigen binding construct of the present disclosure. In some embodiments, the present disclosure provides a method of enhancing an immune response (e.g., enhanced T cell function; enhanced T cell-mediated response; increased IFNy secretion and / or production from T cells; enhanced NK cell function; enhanced macrophage function) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antigen binding construct or composition comprising the construct of the present disclosure. As exemplified herein, the enhancement of the immune response is greater upon administration of the bispecific antigen binding construct as compared to an agent that has a single target. In some embodiments, the enhancement of the immune response is greater by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), or more as compared to an agent that binds either PD-1 alone or VEGF alone.

[0146] In some aspects, the bispecific and / or multispecific antigen-binding construct according to any embodiment described herein may be a method for preventing and / or treating a tumor, wherein preferably, the tumor is selected from colon cancer, rectal cancer, lung cancer such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer and leukemia.

[0147] In some embodiments, the bispecific and / or multispecific antigen-binding construct according to any embodiment described herein may be used in preparing a medicament for preventing and / or treating a tumor, wherein preferably, the tumor is selected from colon cancer, rectal cancer, lung cancer such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer and leukemia.

[0148] Another aspect of the present disclosure relates to a method for preventing and / or treating a tumor, comprising administering to a subject in need an effective amount of the and / or multispecific antigen-binding construct according to any embodiment wherein the tumor is selected from colon cancer, rectal cancer, lung cancer such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer and leukemia.

[0149] In some aspects, the use of a bispccific and / or multispccific antigen-binding as described herein, may be non-therapeutic and / or non-diagnostic.

[0150] The compositions described herein are useful in, inter alia, methods for treating or preventing a variety of cancers in a subject. The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), oral, intracranial injection, or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion.

[0011] As used herein, the term subject means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats and sheep. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have a disease or condition that can be treatedwith a bispecific antigen-binding construct provided herein. In some aspects, the disease or condition is a cancer. In some embodiments, the subject is a human with a cancer that can be treated with a bispecific antigen-binding construct provided herein. In some embodiments, the subject is a human that is suspected of having cancer that can be treated with a bispecific antigen-binding construct provided herein.

[0152] Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Treating or treatment includes delaying or preventing metastasis.

[0153] As used herein, administer or administration refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a bispecific antigen binding construct provided herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0154] Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sikalastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; and European Patent Nos. EP488401 and EP 430539. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanicalsystems. In some embodiments, a bispecific antigen-binding construct of the present disclosure is therapeutically delivered to a subject by way of local administration.

[0155] As used herein, the term enhanced T cell function or activation of T cells refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell-based effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co- stimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof. In some embodiments, enhanced T cell function also encompasses enhanced survival and / or enhanced proliferation of the T cell. Methods for measuring such activities are routine and known in the art.

[0156] As used herein, the term T cell-mediated response refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+cells) and helper T cells (e.g., CD4+cells). T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0157] As used herein, the term therapeutically effective amount or effective amount refers to an amount of a bispecific antigen binding construct that, when administered to a subject, is effective to treat a disease or disorder. A suitable dose of an antibody or fragment thereof described herein, which dose is capable of treating or preventing cancer in a subject, can depend on a variety of factors including the particular construct used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a whole bispecific antigen binding construct may be required to treat a subject with cancer as compared to the dose of a fragment of the bispecific antigen-binding construct (e.g., Fab’ antibody fragment) required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the cancer. For example, a subject having metastatic melanoma may require administration of a different dosage of bispecific antigen binding construct than a subject with glioblastoma. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, thegeneral health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0158] A pharmaceutical composition can include a therapeutically effective amount of a bispecific antigen binding construct described herein. Such effective amounts can be readily determined by one of ordinary skill in the art as described above. Considerations include the effect of the administered bispecific antigen binding construct, or the combinatorial effect of the bispecific antigen binding construct with one or more additional active agents, if more than one agent is used in or with the pharmaceutical composition.

[0159] Suitable human doses of any of the bispecific antigen binding constructs described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part l):523-531 ; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0160] Toxicity and therapeutic efficacy of such bispccific antigen binding constructs can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. A bispecific antigen binding construct that exhibits a high therapeutic index is preferred. While constructs that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

[0161] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such bispecific antigen binding construct thereof lies generally within a range of circulating concentrations of thebispecific antigen binding construct that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For bispecific antigen binding constructs described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50 (i.e., the concentration of the construct - e.g., antibody - which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. l evels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration (e.g., to the eye or a joint) is desired, cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

[0162] In some embodiments, a bispecific antigen binding construct described herein can be administered to a subject as a monotherapy. Alternatively, the bispecific antigen binding construct can be administered in conjunction with other therapies for cancer. For example, the composition can be administered to a subject at the same time, prior to, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplant therapy, precision medicine, genome editing therapy, or other pharmacotherapy.

[0163] Chemotherapeutic agents suitable for co-administration with compositions of the present disclosure include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Further agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlordiamine platinum (II)(DDP), procarbazine, altretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracycline (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. dactinomcin (formerly actinomycin), bleomycin, mithramycin,and anthramycin (AMC)), and anti-mitotic agents (e.g. vincristine and vinblastine) and temozolomide. In some embodiments, the bispecific antigen binding construct and the one or more additional active agents are administered at the same time. Optionally, the bispecific antigen binding construct is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the bispecific antigen binding construct is administered second in time. Optionally, the bispecific antigen binding construct and the one or more additional agents are administered simultaneously in the same or different routes. For example, a composition comprising the antigen binding construct optionally contains one or more additional agents.

[0164] A bispecific antigen binding described herein can replace or augment a previously or currently administered therapy. For example, upon treating with a bispecific antigen binding construct, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy is maintained until the level of the bispecific antigen binding construct reaches a level sufficient to provide a therapeutic effect.

[0165] Monitoring a subject (e.g., a human patient) for an improvement in a cancer, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in tumor growth or size. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a cancer described herein.

[0166] In some embodiments, a therapeutically effective amount of a bispecific antigen binding construct, or a composition comprising the construct, described herein is administered to a subject to modulate an immune response in a subject in need thereof. Insome aspects, the enhanced immune response includes one or more of enhanced T cell function, enhanced NK cell function, or enhanced macrophage function. In certain aspects, the bispecific antigen binding construct enhances the subject’s immune response by agonizing VEGF function by bridging an immune cell that expresses VEGF with a second cell (e.g., another immune cells, or a tumor cell) that expresses PD-1.

[0167] Disclosed are materials, compositions, and ingredients that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0168] Publications cited herein and the material for which they are cited are hereby specifically incoiporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods.Definitions

[0169] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent adifference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0170] As used herein, the following definitions are provided to facilitate the understanding of the present disclosure.

[0171] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0172] In the specification and claims, the term “about” is used to modify, for example, the quantity of an ingredient in a composition, concentration, volume, process temperature, process time, yield, flow rate, pressure, and like values, and ranges thereof, employed in describing the embodiments of the disclosure. The term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and like proximate considerations. The term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture. Where modified by the term “about” the claims appended hereto include equivalents to these quantities. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0173] With regard to the binding of an antigen-binding protein / region / arm to a target molecule, the terms “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0174] As used herein, the term "antagonist" refers to any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein (e.g., PD1 and / or VEGF / VEGF-A). Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. In some embodiments, inhibition in the presence of the antagonist is observed in a dosedependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured with a negative control under comparable conditions.

[0175] As used herein, the term "antibody" refers to a protein molecule which comprises an immunoglobulin molecule immunologically reactive with a particular antigen, and which serves as a receptor that specifically recognizes an antigen. The term may include all polyclonal antibodies, monoclonal antibodies, full-length antibodies, and antibody fragments. In addition, the term may include forms produced by the genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterogeneous antibodies (e.g., bispecific antibodies). A full-length antibody has two full-length light chains and two full-length heavy chains, in which each of the light chains is linked to the heavy chain by a disulfide bond. The full-length antibody may comprise IgA, IgD, IgE, IgM and IgG, and subtypes of IgG include IgGl, IgG2, IgG3 and IgG4. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody. In addition, the term antibody may include bivalent molecules,diabodies, tribodies, and tetrabodies. Specifically, the antibodies that bind specifically to VEGF may be IgG type.

[0176] In the present disclosure, the multispecific antigen binding construct or bispecific antibody may be a form wherein an immunoglobulin G (IgG)-type antibody that binds specifically to VEGF and a full-length antibody, Fab', F(ab'h, Fab, Fv, rlgG or scFv-type protein that binds specifically to PD-1 are connected to each other by a linker.

[0177] As used herein, the phrase "antibody that binds specifically to VEGF" or "VEGF- specific binding antibody" includes all antibodies that bind specifically to the antigen VEGF expressed in tumor cells. Specifically, the antibody may be bevacizumab, a therapeutic antibody that targets VEGF, but is not limited thereto. Such antibodies that bind specifically to VEGF may include full-length antibodies or antibody fragments as described above, and may be IgG antibodies, but are not limited thereto. VEGF is a ligand playing an important role in angiogenesis, and when VEGF is inhibited, no angiogenesis will occur, and thus cancer can be treated. Bevacizumab approved by the US FDA is a therapeutic antibody that can be stably used.

[0178] As used herein, "cancer antigen" refers to (i) tumor- specific antigens, (ii) tumor- associated antigens, (iii) cells that express tumor- specific antigens, (iv) cells that express tumor- associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor- specific membrane antigens, (viii) tumor- associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.

[0179] As used herein, the term "cancer-specific immune response" refers to the immune response induced by the presence of tumors, cancer cells, or cancer antigens. In certain embodiments, the response includes the proliferation of cancer antigen specific lymphocytes. In certain embodiments, the response includes expression and upregulation of antibodies and T-cell receptors and the formation and release of lymphokines, chemokines, and cytokines. Both innate and acquired immune systems interact to initiate antigenic responses against the tumors, cancer cells, or cancer antigens. In certain embodiments, the cancer-specific immune response is a T cell response.

[0180] The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The multispecific antigen binding constructs or bispecific antibodies described herein can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0181] As used herein, the terms “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; the terms “consisting essentially of’ and “consists essentially of’ likewise have the meaning ascribed in U.S. Patent law, and the terms are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0182] As used herein, the term “disease” refers to any condition or disorder that damages, interferes with, or dysregulates the normal function of a cell, tissue, or organ. In a disease such as cancer (e.g., lung cancer), the normal function of a cell, tissue, or organ can be altered to enable immune evasion and / or escape of cancer cells or tumors.

[0183] As used herein, the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient’s own immune system.

[0184] As used herein, the term “human antibody” includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. Human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (Sec, e.g., Eonbcrg ct al., (1994) Nature368(6474): 856-859); Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93, and Harding & Lonberg, (1995) Ann. N.Y. Acad. Sci. 764:536-546). However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e. humanized antibodies).

[0185] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. The term “induce” as used with respect to inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms.

[0186] As used herein, the terms “inhibits,” “blocks” or “reduces” (e.g., when referring to inhibition / blocking of the PD- 1 and / or VEGF / VEGF-A signaling pathway) are used interchangeably and encompass both partial and complete inhibition / blocking as well as direct and allosteric inhibition / blocking. As used herein, “inhibition,” “blocking” or “reducing” are also intended to include any measurable decrease in biological function and / or activity of a target (e.g., PD-1 or VEGF). For example, when an antibody or an antigenbinding fragment thereof (e.g., an anti-PD-1 or an anti-VEGF antibody) is in contact with the target as compared to the target not in contact with an antibody or antigen-binding fragment. In some embodiments, an antibody or antigen-binding fragment thereof, that targets PD-1 or VEGF, inhibits or reduces PD- 1 or VEGF function and / or activity in a given system by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0187] As used herein, the term “inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the growth of a cell, e.g., the inhibition of growth of a cell by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, 70%, about 80%, about 90%, about 99%, or 100%.

[0188] As used herein, a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the caseof non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-VEGF antibody and / or PD-1 antagonist).

[0189] As used herein, the term "in vivo" refers to processes that occur in a living organism.

[0190] As used herein, the terms “immune therapy,” “immunotherapy,” and “immunologic therapy” refer to the treatment of disease by activating or suppressing the immune system. Activation immunotherapies amplify immune responses, and suppression immunotherapies reduce or suppress immune response.

[0191] As used herein, the term “monoclonal antibody” refers to an antibody which displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0192] As used herein, the term "PD-1 antagonist" refers to any chemical compound or biological molecule that inhibits the PD- 1 signaling pathway or that otherwise inhibits PD-1 function in a cell (e.g., an immune cell). In some embodiments, a PD-1 antagonist blocks binding of PD-L1 to PD-1 and / or PD-L2 to PD-1. In some embodiments, the PD-1 antagonist specifically binds PD-1. In some embodiments, the PD-1 antagonist is an isolated monoclonal antibody that specifically binds human PD-1, or an antigen-binding fragment thereof.Exemplary PD-1 antagonists comprising anti-PD-1 antibodies, or antigen-binding fragments thereof, are described herein. In some embodiments, the PD-1 antagonist is an isolated monoclonal antibody that specifically binds human PD-L1, or an antigen-binding fragment thereof. Exemplary PD-1 antagonists comprising anti-PD-Ll antibodies, or antigen-binding fragments thereof, are described herein.

[0193] As used herein, the term “preventing” when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

[0194] As used herein, the term “prognoses,” “prognosis,” “prognosed,” “prognosticate,” “prognosticated,” or “prognosing” relates to providing a forecast or prediction of the likely outcome of a cancer after treatment with an anti-VEGF antibody and / or an anti-PD-1 antibody. Furthermore, the difference between “prognosis” and “treatment response” is a key concept in oncology. While patients can have a good prognosis, they still can have no treatment response and experience only side effects. Thus, it is essential to predict the prognosis or the overall survival of a patient and separately predict the treatment outcome and treatment response.

[0195] As used herein, the term “Programmed Cell Death Protein 1” or “PD-1" refers to the Programmed Cell Death Protein 1 polypeptide, an immune-inhibitory receptor belonging to the CD28 family and is encoded by the PDCD1 gene in humans. Alternative names or synonyms for PD-1 include: PDCD1, PD1, CD279, and SLEB2. PD-1 is expressed predominantly on previously activated T cells, B cells, and myeloid cells in vivo, and binds to two ligands, PD-L1 and PD-L2. The term "PD-1 " as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. AAC51773.

[0196] As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

[0197] As used herein, the term “stratifying” or “stratification” refers to sorting patients into those who may or may not benefit from cancer therapy.

[0198] As used herein, the term “tumor” or “tumor cells” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Within the context of the present disclosure, the treatment of malignant tumors, i.e. cancers, is preferred. Thus, uses and methods described herein may be used to treat tumors, including both solid and non-solid tumors.

[0199] As used herein, the term “treat” or “treatment” means to reduce, stabilize, or inhibit progression of a symptom, such as tumor size, number of metastases or othersymptoms which are caused by / associated with the presence and / or progression of a tumor. A non- limiting exemplary list of cancerous diseases and tumors which can be treated with cancer therapy, is provided herein. These tumors described herein may be metastatic or non-metastatic.

[0200] As used herein, the term “tumor microenvironment” (alternatively “cancer microenvironment”; abbreviated TME) refers to the cellular environment or milieu in which the tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME. The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0201] As used herein, the term “tumor sample” (also referred to as “tissue sample”) is preferably derived from a subject and may be obtained via biopsy such as needle biopsy, surgical biopsy, bone marrow biopsy etc. A tumor sample may therefore include a tumor, parts of a tumor, tumor cells derived from a tumor (including tumor cell lines which may be derived from a tumor and which are grown in cell culture), but also tumor cell lines as such, and cells and / or tissue which are / is derived from a subject and which are / is suspected of being tumorigenic or even cancerous or which are / is suspected of comprising tumorigenic or cancerous cells. A tumor tissue sample also encompasses pieces or slices of tissue that have been removed from the tumor and / or the surrounding tissue, including surgical tumor resection or the collection of a tissue sample by biopsy. A tissue sample may be obtained for the purpose of in vitro evaluation. In some embodiments, the tumor sample may result from the tumor resected from the patient. In some embodiments, the tumor sample may result from a biopsy performed in the primary tumor of the patient or performed in metastatic sample distant from the primary tumor of the patient. Generally, the tumor tissue sample may be fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mold and later hardened to produce a block which is readily cut. Thin slices of material can be then prepared using a microtome, placed on a glass slide and submitted e.g. to immunohistochemistry. The tumor tissue sample can be used in microarrays, called as tissue microarrays (TMAs). TMA consists of paraffin blocks in which up to 1000 separate tissuecores are assembled in array fashion to allow multiplex histological analysis. This technology allows rapid visualization of molecular targets in tissue specimens at a time, either at the DNA, RNA or protein level.

[0202] As used herein, the term “T cell” refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (a.k.a. TH cells or CD4+T cells) and subtypes, including THI, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (i.e., Tc cells, CD8+T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Tregcells or suppressor T cells) and subtypes, including CD4+FOXP3+Treg cells, CD4+FOXP3_Tregcells, Tri cells, Th3 cells, and Trcg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (y5 T cells), including Vy9 / V52 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.

[0203] As used herein, the terms “T cell activation” or “activation of T cells” refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cellbased effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co-stimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0204] As used herein, the term “T cell-mediated response” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+cells) and helper T cells (e.g., CD4+cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

[0205] As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., an anti-CD137 antibody or an antigen-binding fragment thereof or PD-1 antagonist) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a cancer).

[0206] The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0207] As used herein, the term "vascular endothelial growth factor” or “VEGF" refers to a kind of growth factor that enhances the growth activity of vascular endothelial cells and is secreted by various kinds of cells, including macrophages, smooth muscle cells and tumor cells. VEGF plays an important role in fetal angiogenesis, and also functions to induce angiogenesis in order to supply oxygen to tumor tissue in which rapid growth and metabolism occur. Pathways in which VEGF protein and its receptor are involved have been studied as target signaling pathways of anticancer agents in adults.

[0208] In addition, the VEGF-binding unit of the multispecific antigen binding constructs provided herein means inhibiting the interaction between human VEGF and VEGF receptor. Specifically, it means that the antigen binding unit specific for VEGF binds to VEGF to inhibit the interaction between VEGF and VEGFR-2 but is not limited thereto. Further, the VEGF receptor may be any protein that binds to mammalian VEGF. Specifically, it may be a protein that binds to human VEGF.

[0209] When the interaction between VEGF and VEGF receptor is inhibited by the VEGF- specific antigen binding unit of the present disclosure, VEGF / VEGF signaling by the binding of VEGF to VEGF receptor will be inhibited. It is known that when VEGF and VEGF receptor in tumors bind to each other, VEGF / VEGF receptor signaling in stromal / cndothclial cells of cancer tissue is activated to strongly inhibit angiogenesis toreduce the number of blood vessels and weaken a vascular function in tumors, thereby inhibiting cancer proliferation and metastasis. Thus, the multispecific antigen binding constructs of the present disclosure, which is specific for PD-1 and VEGF, shows the ability to inhibit angiogenesis in cancer tissue, and thus can be used as a therapeutic agent having better anticancer activity.

[0210] In some embodiments the expression of at least one immune marker is measured using mass spectrometry by time of flight (CyTOF) or Next-generation sequencing (NGS), or quantitative polymerase chain reaction (qPCR) or any other method known in the art to measure expression levels of an immune marker. In various embodiments at least one immune marker is CD56.

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0212] To clarify the use of and to hereby provide notice to the public, the phrases "at least one of , , ... and <N>" or "at least one of , , ... <N>, or combinations thereof" or ", , ... and / or <N>" are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B, ... and N. In other words, the phrases mean any combination of one or more of the elements A, B, ... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed. Unless otherwise indicated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or "one or more."

[0213] While various embodiments have been described, it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible. Accordingly, the embodiments described herein are examples, not the only possible embodiments and implementations.

[0214] Bispecific Antibody CTX-10726

[0215] Immunotherapies targeting programmed cell death protein- 1 (PD- 1 ) and its ligand (PD-L1) are standard treatments in an ever-growing list of indications. However, limited patient response rates have driven the development of next-generation therapies such as those that pair PD-1 / PD-L1 inhibitors with anti-angiogenic agents (anti-VEGF-A antibodies or tyrosine kinase inhibitors, TKIs). These types of combinations are established first-line treatments in, for example, non-small cell lung cancer, cervical cancer, advanced renal cell carcinoma, hepatocellular carcinoma, and endometrial carcinoma. The simultaneous targeting of VEGF-A and PD-1 in a single molecule could conceivably reduce the complexity of combination treatment regimens by potentially concentrating PD- 1 blockade at VEGF-rich, immunosuppressed tumor sites. It may also offer improved safety over VEGF TKI-containing combination regimens. As described herein, CTX-10726 is a novel, bispecific, tetravalent antibody that concurrently targets and simultaneously binds both VEGF-A and PD-1. CTX-10726 is a potent bispccific antibody that simultaneously inhibits tumor angiogenesis and PD-1 -mediated immune suppression and eliminates tumors in preclinical mouse models.Examples

[0216] Example 1 CTX-10726 Binding

[0217] CTX-10726 showed high-affinity binding to both human and cynomolgus monkey VEGF-A and PD-1. CTX-10726 effectively blocked VEGF-A / VEGFR2 and PD-1 / PD-L1 interactions in a dose-dependent manner and showed reduced engagement with Fey receptors, limiting off-target immune activation. Functionally, it significantly enhanced IFN-y production in mixed lymphocyte reactions (MLRs) and tumor cell killing by activated PBMCs. In vivo, CTX-10726 outperformed benchmark biosimilar anti- VEGF or PD-1 antibodies in controlling tumor growth across multiple xenograft and syngeneic models.

[0218] As shown in Fig. 1, CTX-10726 is a bispccific, tctravalcnt antibody consisting of an anti-VEGF-A IgGl-LALA (bevacizumab biosimilar with engineered Fc-silencing mutations) with an anti-PD-1 single chain variable fragment (scFv) fused to each C-terminus of the Fc through a (G4S)3 linker. Table 1 presents a summary of CTX-10726 binding parameters (nM).TABLE 1. Summary of CTX-10726 Binding Parameters (nM)

[0219] As shown via sensorgrams in Figs. 2A-C, binding of CTX-10726 to soluble human and cynomolgus macaque PD-1 and human VEGF-A was assessed by biolayer interferometry (BLI). His-tagged antigens were captured onto biosensors and then exposed to CTX-10726. Cynomolgus monkey and human VEGF-A are identical. Fig. 2D shows that CTX-10726 binds simultaneously to human VEGF-A and PD-1. Plates were coated with soluble VEGF-A, incubated with serially diluted CTX-10726, followed by PD-l-biotin and Streptavidin-HRP detection (ELISA).

[0220] As shown in Fig. 3, both CTX-10726 and Pembrolizumab bind to PD-1 and do so in the presence or absence of VEGF-A. Fig. 3 A shows that in the presence of VEGF-A, the affinity of CTX-10726 binding to PD-1 was substantially improved compared to the binding in the absence of VEGF-A. Comparison was made to pembrolizumab by adding VEGF-A, as demonstrated in Fig. 3B, and the addition of VEGF-A to pembrolizumab had no meaningful impact on its binding to PD- 1.

[0221] As shown in Fig. 4, CTX-10726 also binds to PD-1 over-expressed Jurkat cells in the presence or absence of VEGF-A. CTX-10726 is compared to CTX-10726 and VEGF-A preincubated at a molar ratio of 1:2. The 1:2 molar ratio enhanced CTX- 10726’ s binding to PD-1 expressing Jurkat cells as indicated by the increased overall signal.

[0222] Binding of CTX-10726 to PD-1 (human, mouse, and cynomolgus macaque) and FcyRs was assessed by biolayer interferometry (BLI) using purified proteins and by flow cytometry (FACS) in primary and target-expressing cell lines.

[0223] Example 2 CTX-10726 Blockade

[0224] As shown in Fig. 5, CTX-10726’s PD-1 blockade is also enhanced as shown through signaling assays in the presence of VEGF-A. Fig. 5 shows that adding VEGF-A to the CTX-10726 (closed square) enhanced its PD-1 blockade as compared to its potency in the absence of VEGF-A (open square).

[0225] Fig. 9 illustrates that CTX-10726 blocks VEGF-A / VEGFR2 activity in vitro. As shown in Fig. 9, VEGF-A / VEGFR2 (KDR, Kinase insert domain receptor) blockade by CTX-10726 was evaluated using a qualified KDR / NFAT-RE HEK293 cell-based reporter assay. Binding of VEGF-A to KDR expressed on these cells is detected as luminescence after the samples are incubated with luciferase substrate. Increasing concentrations of CTX-10726 blocked VEGF-A / KDR interaction in a dose-dependent manner, with an IC50 of 2.09 nM (comparable to the values for bevacizumab provided with the Promega kit).

[0226] Example 3 Dose Dependent Concentration

[0227] As seen in Fig. 7A, Staphylococcus aureus Enterotoxin B (SEB)-activated human PBMCs were exposed to increasing concentrations of CTX-10726, ivonescimab, or human IgGl (isotype control), followed by fluorochrome-conjugated anti-human F(ab)2 secondary antibodies, FACS acquisition and analysis. Shown in Fig 7B are human PBMCs treated as in 7 A, and were incubated with increasing concentrations of CTX-10726, pembrolizumab, or human IgGl in the presence of VEGF-A at 1 :2 molar ratio. Figs 7C-7D show CTX-10726 cross-reactivity to PD-1 from human and cynomolgus monkey. CHO cells were transfected with either human or cynomolgus monkey PD- 1 were incubated with titration series of CTX-10726 or control antibodies followed by detection with fluorescently labeled secondary antibodies using a flow cytometer.

[0228] Example 4 Immunomodulatory Activity

[0229] As shown in Fig. 6, CTX-10726 also blocks PD-1 in a mixed lymphocyte reactions (MLR) with and without the presence of VEGF-A. Fig. 6 shows interferon gamma production in one-way MLR assays with Keytruda (1 nM), CTX-10726 (0.2, 1, 5, and 1025 nM), and CTX-10726 mixed with VEGF (1:2 molar ratio). IFN-y levels were measured after a five-day incubation.

[0230] Fig. 8 demonstrates that CTX-10726 has potent immunomodulatory activity in vitro. As shown in Fig. 8A, anti-PD-1 activity of CTX-10726 was evaluated using the Jurkat PD-1 / PD-L1 reporter assay (Promega). In this system, PD-1 -expressing Jurkat NFAT-Luciferase reporter cells are activated by CHO cells expressing both PD-L1 and a TCR activator. CTX-10726 blocked the suppressive PD-1 / PD-L1 interaction and restored TCR activation in a dose-dependent manner. Fig. 8B shows PBMCs from three donors paired (1:1) in two-way MLR assays with 35 nM PD-l-blocking antibodies and IFN-y levels were measured after a three-day incubation. CTX-10726 treatment more than doubled IFN-y production by activated PBMCs. Data represent the average of three different donorcombinations. In Fig. 8C, TCR-activated PBMCs from three donors were co-cultured with HCC827 target cells ( 1 : 1 ) in the presence of 50 nM anti-PD- 1 antibodies for two days (TTR, Tumor / T cell Reaction assay). In this experimental setup, CTX- 10726 enhanced T cell activation driven by alloreactive immune responses resulting from the HLA mismatch between tumor and immune cells. As in B, CTX-10726 nearly doubled IFN-y secretion. Data represent the average of biological triplicates.

[0231] Immunomodulatory functions in vitro were measured by interferon-gamma (IFN-y) release in a two-way mixed lymphocyte reaction (MLR) and in vitro cytotoxicity assays with activated PBMCs.

[0232] Example 5 CTX-10726 Anti-tumor Efficacy

[0233] As seen in Fig. 10, CTX-10726 demonstrates potent anti-tumor efficacy against HCC827 xenografts. Shown in Fig. 10A, PD-L1 expression on HCC827 human NSCLC cell line was evaluated by FACS. PD-L1 is highly expressed at steady state (red histogram) and its expression is increased by IFN-y (blue histogram). Fig. 10B shows VEGF-A levels in HCC827 cell culture supernatant measured by ELISA and plotted against time. Shown in Fig. 10C, SEB-activated human PBMCs were co-injected subcutaneously (s.c.) with HCC827 tumor cells into the flanks of female SCID / Beige mice (n=8 / group) at an effector-to-target ratio of 1:100. CTX-10726, an isotype control antibody, an anti-VEGF-A monoclonal antibody, or ivonescimab were administered intraperitoneally (i.p.) on the day of implantation and then weekly for a total of four doses (dotted lines). The dual blockade of PD-1 and VEGF-A by CTX-10726 produced superior anti-tumor activity compared to anti-VEGF-A treatment or ivonescimab (****, P < 0.0001, Two-way ANOVA), demonstrating enhanced in vivo potency in this model.

[0234] As shown in Fig. 11, CTX-10726 shows curative anti-tumor activity against MC38hPD-Ll tumors implanted in hPD-l / hPD-Ll transgenic double knock-in mice. CTX-10726 checkpoint inhibitory activity was evaluated against MC38hPD-Ll tumors implanted subcutaneously in hPD-l / hPD-Ll double-knock-in mice. In fig. 11A each mouse (n=5 / group) was inoculated subcutaneously with MC38 hPD-Ll tumor cells. Treatment with hlgGl isotype control, ivonescimab, or CTX-10726 was initiated when tumors reached an average of approximately 230 mm3. In fig. 1 IB each mouse (n=10 / group), was inoculated subcutaneously with MC38 hPD-Ll tumor cells. Treatment with hlgGl, pembrolizumab, or CTX-10726 was initiated when tumors reached an average of about 100 mm3. Fig. 11C shows individual tumor responses to hlgGl, CTX-10726, and pembrolizumab over time. Inthis model, CTX-10726 potently blocked tumor growth, similarly to ivonescimab (Fig. 11A) or pembrolizumab (Figs. 1 IB, 11C). Treatment regimens are shown by dotted lines.

[0235] Seen in Fig. 12, CTX-10726 demonstrates superior efficacy against MC38 hPD-Ll / hVEGF-A tumors, driven mainly by PD-1 / PD-L1 check-point inhibition. Fig. 12A shows the combined anti-VEGF-A and anti-PD-1 activity of CTX-10726 assessed in the hPD-l / hPD-Ll / hVEGF-A triple-knock-in mice (Biocytogen, n=8 / group), where both targets of the drug are human. Each mouse was inoculated subcutaneously with MC38 hPD-Ll / hVEGF-A tumor cells. Treatment with PBS, bevacizumab, ivonescimab, or CTX-10726 started when tumors reached an average of approximately 150 mm3 and mice received three treatments (dotted vertical lines). CTX-10726 treatment led to significant reduction of tumor size compared to PBS control or bevacizumab-treated animals (****, P < 0.0001, Two-way ANOVA. Fig. 12B shows the tumor volume of individual mice over time. As of day 39, after tumor cell inoculation, 2 out of 8 mice treated with CTX-10726 reached complete responses.

[0236] In vivo anti-tumor efficacy was tested in PBMC-humanized xenograft models or in double hPD-l / hPD-Ll transgenic mice implanted with tumor cells and treated withCTX-10726 or control antibodies. Models were selected to assess the relative contribution of the VEGF and PD-1 targeting arms.

[0237] Example 6 CTX-10726 Pharmacokinetics

[0238] Fig. 13 illustrates that the CTX-10726 pharmacokinetics profile in non-human primates (NHPs) after a single dose demonstrates a dose-proportional increase in serum concentration. Fig. 13 shows CTX-10726 serum concentration-time profiles in cynomolgus monkeys following a single intravenous infusion. Male cynomolgus monkeys (n=3 / group) received a single dose of CTX-10726 at 1, 10, or 50 mg / kg. Blood samples were collected from each monkey pre-dose, immediately post end of infusion (EOI), 5 minutes post end of infusion, 1, 2, 4-, 24-, 96- and 168-hours post EOI. Samples were analyzed for CTX-10726 concentration using a qualified ELISA method with a lower limit of quantitation (LLOQ) of 100 ng / mL and upper limit of quantification (ULOQ) of 3200 ng / mL. Mean serum concentration-time profiles from all the groups are shown. Table 2 below shows experimental groups and mean CTX-10726 pharmacokinetic parameters in male cynomolgus monkeys after a single i.v. infusion of CTX-10726.TABLE 2. Experimental Groups and Mean CTX-10726 Pharmacokinetic Parameters in Male Cynomolgus Monkeys After a Single I.V. InfusionGroup | Dose | Dose | C max | Tmax~ | Tl / 2 | AUCp-Tlast | AUClNF obs | Cl obs ~|TABLE 3: SEQUENCE LISTING

Claims

1. CL IMS1. A multispecific antigen binding construct comprising at least two linked antigen binding units, wherein a first antigen binding unit specifically binds PD-1 and wherein a second antigen binding unit specifically binds VEGF.

2. The multispecific antigen binding construct of claim 1, wherein the first antigen binding unit is an antibody or antigen binding fragment thereof.

3. The multispecific antigen binding constmct of claim 2, wherein the antibody or antigen binding portion thereof of the first antigen binding unit is a monoclonal antibody or antigen binding portion thereof.

4. The multispecific antigen binding construct of claim 2, wherein the antibody or antigen binding portion thereof of the first antigen binding unit is a humanized antibody, a fully human antibody, or an antigen binding portion of either.

5. The multispecific antigen binding construct of claim 2-4, wherein the antibody or antigen binding portion thereof of the first antigen binding unit is a scFv or Fab antibody fragment.

6. The multispecific antigen binding construct of claim 1 , wherein the first antigen binding unit is a polypeptide, small molecule, or an aptamer.

7. The multispecific antigen binding construct of any one of claims 1-6, wherein the second antigen binding unit is an antibody or antigen binding fragment thereof.

8. The multispecific antigen binding constmct of claim 7, wherein the antibody or antigen binding fragment thereof of the second antigen binding unit is a monoclonal antibody or antigen binding portion thereof.

9. The multispccific antigen binding constmct of claim 7, wherein the antibody or antigen binding fragment thereof of the second antigen binding unit is a humanized antibody or a fully human antibody.

10. The multispccific antigen binding constmct of claim 7-9 wherein the antibody or antigen binding fragment thereof of the second antigen binding unit is a scFv or Fab antibody fragment.

11. A composition comprising the multispecific antigen binding construct of any one of claims 1-10 and a pharmaceutically acceptable carrier.

12. A method for treating a cancer in a subject, comprising administering to the subject having cancer an effective amount of the multispecific antigen binding construct of any one of claims 1-10 or the composition of claim 11.

13. The method of claim 12, wherein the subject is a human.

14. The method of claim 12, wherein the multispecific antigen binding construct enhances the subject’s immune response by agonizing PD-1 function.

15. The method of claim 13 or 14, wherein the cancer is selected from the group consisting of a hematological cancer, neurological cancer, breast cancer, prostate cancer, skin cancer, lung cancer, bladder cancer, kidney cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, and vascular cancer.

16. The method of any one of claims 12-15, further comprising administering a second anticancer therapy to the subject.

17. The method of any one of claims 12-16, wherein the anticancer therapy is chemotherapy, immunotherapy, hormone therapy, cytokine therapy, radiotherapy, cryotherapy, or surgical therapy.

18. A method of enhancing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multispecific antigen binding construct of any one of claims 1-10 or the composition of claim 11.

19. The method of claim 18, wherein the enhanced immune response comprises any one or more of enhanced T cell function, enhanced NK cell function, or enhanced macrophage function.

20. The method of any one of claims 15-19, wherein the multispecific antigen binding construct is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

21. The multispecific antigen binding construct of any one of claims 1-20, wherein the first and second antigen binding units are each an antibody or an antigen-binding fragment thereof.

22. The multispecific antigen binding construct of any one of claims 1-20, wherein the first antigen binding unit comprises a single-chain variable fragment (scFv) or a Fab fragment that specifically binds PD-1.

23. The multispecific antigen binding construct of any one of claims 1-20, wherein the second antigen binding unit comprises an IgG or an antigen-binding fragment thereof that specifically binds VEGF or VEGF-A.

24. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct is an appended IgG comprising an anti- VEGF- A IgGl Fc region and an anti-PD-1 scFv fused to each C-terminus of the Fc region.

25. The multispecific antigen binding construct of any one of claims 1-20, wherein the Fc region comprises L234A and L235A substitutions (LALA) that reduce Fey receptor engagement.

26. The multi specific antigen binding construct of any one of claims 1-20, wherein each anti-PD-1 scFv is fused to the Fc C-terminus through a peptide linker comprising SEQ ID NO: 18.

27. The multispecific antigen binding construct of any one of claims 1-20, wherein the linker comprises a (G4S)3 sequence (GGGGSGGGGSGGGGS, SEQ ID NO: 18) or a (G4S)4 sequence (GGGGSGGGGSGGGGSGGGGS. SEQ ID NO: 19).

28. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct is tetravalent and comprises two antigen-binding units specific for PD- 1 and two antigen-binding units specific for VEGF.

29. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-PD-1 unit comprises a heavy chain variable region having CDRH1, CDRH2, and CDRH3 comprising SEQ ID NOs: 7, 8, and 9, respectively, and a light chain variable region having CDRL1, CDRL2, and CDRL3 comprising SEQ ID NOs: 4, 5, and 6, respectively.

30. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-PD-1 heavy chain variable region is at least 90% identical to SEQ ID NO: 2 and the light chain variable region is at least 90% identical to SEQ ID NO: 3.

31. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-VEGF antigen binding unit comprises a heavy chain variable region having CDRH1, CDRH2, and CDRH3 comprising SEQ ID NOs: 15, 16, and 17, respectively, and a light chain variable region having CDRL1, CDRL2, and CDRL3 comprising SEQ ID NOs: 12, 13, and 14, respectively.

32. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-VEGF heavy chain variable region is at least 90% identical to SEQ ID NO: 10 and the light chain variable region is at least 90% identical to SEQ ID NO: 11.

33. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct comprises a common light chain variable region for both antigen-binding units.

34. The multispecific antigen binding construct of any one of claims 1-20, wherein the Fc region has reduced effector function and / or enhances serum half-life.

35. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct binds human PD- 1 and human VEGF- A with dissociation constants (Kd) of less than 1 nM for each target.

36. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct simultaneously binds PD- 1 and VEGF- A.

37. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct is aglycosylated.

38. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct blocks VEGF-A / VEGFR2 interaction.

39. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct reduces Fey receptor engagement.

40. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct is produced using an Fc pairing selected from knobs-into-holes, electrostatic steering, SEED pairing, or leucine zipper pairing.

41. The multispecific antigen binding construct of any one of claims 1-20, wherein the dissociation constant for VEGF-Ais <0.5 nM and the dissociation constant for PD-1 is <0.2 nM.

42. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-PD-1 scFv is configured in a VH-linker-VL orientation and the linker comprises SEQ ID NO: 18.

43. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-PD-1 scFv is configured in a VL-linker-VH orientation and the linker comprises SEQ ID NO: 18.

44. The multispecific antigen binding construct of any one of claims 1-20, wherein the linker comprises three repeats of Gly4-Ser (G4S) and has a length of 15 amino acids.

45. The multispecific antigen binding construct of any one of claims 1-20, wherein the linker comprises four repeats of Gly4-Scr (G4S) and has a length of 20 amino acids.

46. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-PD-1 unit consists of CDRH1, CDRH2, and CDRH3 comprising SEQ ID NOs: 7, 8, and 9.

47. The multispecific antigen binding construct of any one of claims 1-20, wherein the anti-VEGF unit consists of CDRH1, CDRH2, and CDRH3 comprising SEQ ID NOs: 15, 16, and 17.

48. The multispecific antigen binding construct of any one of claims 1-20, wherein the common light chain comprises a variable region at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 11.

49. The multispecific antigen binding construct of any one of claims 1-20, wherein the Fc region further comprises CH3 domain mutations promoting heterodimerization.

50. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct induces at least a 50% increase in interferon-gamma secretion in a mixed lymphocyte reaction relative to pembrolizumab.

51. The multispecific antigen binding construct of any one of claims 1-20, wherein the construct enhances tumor cell killing by activated peripheral blood mononuclear cells.

52. An isolated polynucleotide encoding a heavy chain variable region of an anti-PD-1 antigen-binding unit that is at least 85% identical to SEQ ID NO: 2 and / or a light chain variable region that is at least 85% identical to SEQ ID NO: 3.

53. An isolated polynucleotide encoding a heavy chain variable region of an anti-VEGF antigen-binding unit that is at least 85% identical to SEQ ID NO: 10 and / or a light chain variable region that is at least 85% identical to SEQ ID NO: 11.

54. An expression vector comprising a polynucleotide encoding a heavy and / or light chain variable region as recited herein, operably linked to a promoter suitable for expression in a mammalian cell.

55. The expression vector of the preceding claim, wherein the promoter is selected from CMV, EFla, or SV40, and the vector further comprises a selectable marker.

56. A host cell comprising an expression vector comprising a polynucleotide encoding a heavy and / or light chain variable region as recited herein, wherein the host cell is selected from CHO cells, HEK293 cells, NS0 cells, or PER.C6 cells.

57. The host cell of the preceding claim, wherein the host cell expresses both heavy and light chains of the multispecific antigen binding construct and assembles the construct into an IgG-scFv fusion format.

58. A method of producing a multispecific antigen binding construct, comprising culturing a host cell comprising an expression vector comprising a polynucleotide encoding a heavy and / or light chain variable region as recited herein under conditions suitable for expression of the construct and recovering the construct from the culture medium.

59. The method of the preceding claim, further comprising purifying the construct using Protein A affinity chromatography.

60. A kit comprising the pharmaceutical composition of claim 11 and instructions for treating a subject having a cancer.

61. The pharmaceutical composition of claim 11 provided as a lyophilized formulation or a ready -to-use solution for intravenous infusion.

62. The multi specific antigen binding constmct of any one of claims 1-20, wherein the Fc region comprises knobs-into-holes mutations.

63. The multispecific antigen binding construct of any one of claims 1-20, wherein the Fc region comprises electrostatic steering mutations.

64. The multispecific antigen binding construct of any one of claims 1-20, wherein the Fc region comprises SEED pairing mutations.

65. The multispecific antigen binding constmct of any one of claims 1-20, wherein the Fc region comprises leucine zipper pairing sequences.

66. The multi specific antigen binding constmct of any one of claims 1-20, wherein the constmct reduces PD-1 cell surface expression by at least 75% compared to untreated immune cells.

67. The multi specific antigen binding constmct of any one of claims 1-20, wherein the constmct blocks VEGF-A / VEGFR2 interaction with an IC50 of 2 nM or less.

68. The multispecific antigen binding constmct of any one of claims 1-20, wherein the constmct binds VEGF-A with a dissociation constant of 0.5 nM or less and PD-1 with a dissociation constant of 0.2 nM or less.

69. A pharmaceutical composition comprising the multispecific antigen binding constmct of any one of claims 1-20 formulated as a lyophilized powder for reconstitution.

70. A kit comprising the pharmaceutical composition of the preceding claim and instmctions for intravenous administration to a subject with a solid tumor.

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