Bispecific antigen binding proteins targeting VEGF pathway and PD-1, compositions and uses thereof

Abstract

Bispecific antigen binding proteins targeting the VEGF pathway and PD-1, compositions and uses thereof are provided. Nucleic acid molecules, expression vectors, and host cells encoding the bispecific antigen binding proteins are also provided. Compositions and uses comprising the bispecific antigen binding proteins are also provided, as well as the use of the bispecific antigen binding proteins for the treatment of diseases, in particular cancer.

Description

Bispecific antigen binding proteins targeting VEGF pathway and PD-1, compositions and uses thereof

[0001] This application claims priority to the Chinese patent application filed on March 10, 2023, with application number 202310230191.X and invention name “Bispecific antigen binding proteins targeting VEGF pathway and PD-1, compositions and uses thereof”, the contents of which should be understood as incorporated into this application by reference. Technical Field

[0002] The present application generally relates to the field of medicine, and in particular to a bispecific antibody that simultaneously targets the VEGF pathway and PD-1, a preparation method thereof, a pharmaceutical composition and uses thereof. Background Art

[0003] According to the World Health Organization, cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. Efforts have been made to diagnose cancer early and provide appropriate treatment, and the overall death rate and the number of people suffering from cancer are decreasing.

[0004] Cancer is actually a group of diseases involving abnormal cell growth and division. Cancer cells are remarkably intelligent and have developed several mechanisms to evade host immune surveillance. For example, they express high levels of the membrane proteins PD-L1 and PD-L2, which bind to PD-1 on the surface of T cells and induce T-cell apoptosis. Cancer cells also secrete angiogenic factors, such as vascular epithelial growth factor (VEGF), to promote the growth of new blood vessels into tumors. With these newly formed blood vessels, cancer cells grow larger and faster, thus possessing a greater ability to travel.

[0005] Current cancer treatment options include surgery, radiotherapy, chemotherapy, hormone therapy, targeted therapy, and immunotherapy. Among these, antibody therapy has developed rapidly and achieved considerable success in recent years. Generally speaking, antibodies can help the immune system kill cancer cells, block the development of tumor blood vessels, or directly kill cancer cells.

[0006] PD-1, also known as CD279, is a cell surface receptor. It has two ligands, PD-L1 and PD-L2. PD-L1 is upregulated on macrophages and dendritic cells in response to LPS and GM-CSF treatment, and on T cells and B cells during T cell receptor (TCR) and B cell receptor (BCR) signaling. PD-L1 mRNA is detectable in the heart, lung, thymus, spleen, and kidney of mice. PD-L1 is also expressed on tumor cells. PD-L2 has a more restricted expression profile.

[0007] When bound to PD-L1 or PD-L2, PD-1 downregulates the immune system and promotes self-tolerance by suppressing T cell inflammatory activity. PD-1's inhibitory effect on the immune system can prevent autoimmune diseases. On the other hand, increased local PD-L1 expression in cancer cells may prevent the immune system from killing these cells. Several anti-PD-1 antibodies, such as (BMS) and (Merck), has been shown to be useful in clinical cancer treatment alone or in combination with other anti-tumor drugs.

[0008] Vascular endothelial growth factor (VEGF) is a signaling protein produced by cells that is involved in vasculogenesis and angiogenesis. VEGF can be divided into VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF. Overexpression of VEGF may lead to disease. For example, cancers that express VEGF (such as VEGF-A and VEGF-D) can grow and metastasize with an adequate blood supply. Excessive release of VEGF-A has been found to play a role in rheumatoid arthritis, diabetic retinopathy, wet age-related macular degeneration, and glomerular hypertrophy.

[0009] is a humanized anti-VEGF antibody that has been approved for the treatment of various cancers, including colorectal, lung, and breast cancer.

[0010] VEGF receptor is the natural receptor of VEGF, and there are three main subtypes: VEGFR-1, VEGFR-2 and VEGFR-3, all of which have seven immunoglobulin-like domains, a single transmembrane spanning region and an intracellular part.

[0011] Ramucirumab is a human IgG1 monoclonal antibody that blocks vascular endothelial growth factor receptor 2 (VEGFR-2 / VEGFR2 / KDR), thereby inhibiting angiogenesis. It has been approved by the FDA for the treatment of gastric cancer, lung cancer, and liver cancer.

[0012] Antibodies targeting a single tumor-associated antigen have been found to have limited therapeutic efficacy. For example, anti-VEGF antibodies It has been shown to inhibit the growth of cancer cells to a certain extent, but cannot eliminate cancer cells.

[0013] Therefore, in clinical trials and clinical treatments, antibodies are usually administered in combination with another antibody or another therapeutic agent so that target cells, such as cancer cells, are attacked by the immune system in a variety of ways. Alternatively, single-molecule drugs with two or more target binding specificities, such as bispecific or multispecific antibodies / proteins, can be developed to achieve the treatment of more than one molecule of the target. Currently, the technologies that can be used to prepare bispecific or multispecific antibodies include Triomab, CrossMab, DVD-Ig, BiTE, DART, and TandAb. The bispecific antigen-binding proteins (such as bispecific antibodies) of the same target will exhibit different characteristics due to different structures and antibody combinations.

[0014] Summary of the Invention

[0015] Currently, there are no bispecific antigen binding protein-related drugs targeting the VEGF pathway and PD-1 on the market, so there is still a certain demand for drugs with the same target.

[0016] The bispecific antibody of the present application can specifically target the VEGF pathway and PD-1, specifically relieve the immune suppression of the body by PD-1, and at the same time inhibit tumor-induced angiogenesis by inhibiting the binding of VEGF to VEGFR, and has good application prospects.

[0017] The present application provides a bispecific antigen-binding protein comprising (a) a first antigen-binding portion that specifically binds to PD-1; and (b) a second antigen-binding portion that specifically targets the VEGF pathway; and the second antigen-binding portion comprises VEGFR1 domain 2 or comprises an antibody domain or antigen-binding domain that targets VEGFR2.

[0018] In some embodiments, the first antigen binding portion comprises: a heavy chain complementary determining region and a light chain complementary determining region, the heavy chain complementary determining region comprising, from N-terminus to C-terminus: HCDR1, the HCDR1 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1; HCDR2, the HCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2; and HCDR3, the HCDR3 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3; the light chain complementary determining region comprising, from N-terminus to C-terminus: LCDR1, the LCDR1 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: NO:4; a LCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:5; and a LCDR3 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:6;

[0019] The second antigen-binding portion comprises a heavy chain complementary determining region and a light chain complementary determining region, wherein the heavy chain complementary determining region comprises, from N-terminus to C-terminus: HCDR1, wherein the HCDR1 has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; HCDR2, wherein the HCDR2 has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8; and HCDR3, wherein the HCDR3 has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9; and the light chain complementary determining region comprises, from N-terminus to C-terminus: LCDR1, wherein the LCDR1 has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: NO:10; a LCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:11; and a LCDR3 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:12; and the second antigen binding portion targets VEGFR2; or

[0020] The second antigen binding portion comprises VEGFR1 domain 2 (abbreviated as R1D2).

[0021] In some embodiments, the second antigen binding portion comprises a VEGFR1 domain 2 having the sequence set forth in SEQ ID NO:20.

[0022] In some embodiments, the N-terminus of the second antigen binding moiety is connected to the C-terminus of the heavy chain of the first antigen binding moiety via a first linker to form a recombinant heavy chain (HC). Preferably, the first linker is flexible. Preferably, the first linker is an IgG4 hinge region or (G4S) n , where n is an integer from 1 to 5.

[0023] In some embodiments, the bispecific antigen-binding protein comprises a recombinant heavy chain (HC) and a light chain (LC), wherein the recombinant heavy chain (HC) comprises an anti-PD1 antibody heavy chain, an optional first linker, and ramucirumab ScFv; and the light chain (LC) comprises an anti-PD1 antibody light chain.

[0024] In some embodiments, the bispecific antigen-binding protein comprises a recombinant heavy chain (HC) and a light chain (LC), wherein the recombinant heavy chain (HC) comprises, from N-terminus to C-terminus, an anti-PD1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional first linker, and a ramucirumab ScFv, wherein the ramucirumab ScFv comprises a ramucirumab heavy chain variable region VH, a second linker, and a ramucirumab light chain variable region VL; the light chain (LC) comprises, from N-terminus to C-terminus, an anti-PD1 antibody light chain variable region VL and an anti-PD1 antibody light chain constant region CL. Preferably, the second linker is flexible. Preferably, the second linker may be the same as or different from the first linker.

[0025] In some embodiments, the bispecific antigen-binding protein comprises a recombinant heavy chain (HC) and a light chain (LC), wherein the recombinant heavy chain (HC) comprises, from N-terminus to C-terminus, an anti-PD1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional first linker, a heavy chain variable region VH of ramucirumab ScFv, a second linker, and a ramucirumab light chain variable region VL; the light chain (LC) comprises, from N-terminus to C-terminus, an anti-PD1 antibody light chain variable region VL and an anti-PD1 antibody light chain constant region CL. Preferably, the second linker is flexible. Preferably, the second linker may be the same as or different from the first linker.

[0026] In some embodiments, the anti-PD1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16; the IgG1 heavy chain constant region has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17; the first linker is an IgG4 hinge region; (G4S) n , wherein n is an integer from 1 to 5; the heavy chain variable region VH of the ramucirumab ScFv has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18; and the second linker is an IgG4 hinge region; (G4S) n , wherein n is an integer from 1 to 5; preferably, the second linker is (G4S) n, wherein n=3; the light chain variable region VL of the ramucirumab ScFv has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19; the light chain variable region VL of the anti-PD1 antibody has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21; the light chain constant region CL of the anti-PD1 antibody has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0027] In some embodiments, the recombinant heavy chain (HC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 13. Preferably, the recombinant heavy chain (HC) has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0028] In some embodiments, the light chain (LC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 14. Preferably, the light chain (LC) has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0029] In some embodiments, the second antigen binding portion is ramucirumab ScFv, preferably, the ramucirumab ScFv has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0030] In some embodiments, the bispecific antigen-binding protein consists of two amino acid sequences listed in SEQ ID NOs: 13 and 14.

[0031] In some embodiments, the bispecific antigen-binding protein comprises a recombinant heavy chain (HC) and a recombinant light chain (LC), wherein the recombinant heavy chain (HC) comprises, from N-terminus to C-terminus, an anti-PD1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional linker, and a VEGFR1 domain 2 (R1D2); and the light chain (LC) comprises, from N-terminus to C-terminus, an anti-PD1 light chain antibody variable region VL and a light chain constant region CL.

[0032] In some embodiments, the anti-PD1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16; the IgG1 heavy chain constant region has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17; the first linker is an IgG4 hinge region; (G4S) n , wherein n is an integer from 1 to 5; the VEGFR1 domain 2 (R1D2) has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20; the anti-PD1 light chain antibody variable region VL has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21; the light chain constant region CL has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0033] In some embodiments, the recombinant heavy chain (HC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 15. Preferably, the recombinant heavy chain (HC) has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15.

[0034] In some embodiments, the light chain (LC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 14. Preferably, the light chain (LC) has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0035] In some embodiments, the bispecific antigen binding protein consists of two amino acid sequences listed in SEQ ID NOs: 15 and 14.

[0036] In some embodiments, the second antigen binding moiety is VEGFR1 domain 2 (R1D2).

[0037] The present application also provides a nucleic acid sequence encoding the aforementioned bispecific antigen-binding protein.

[0038] In some embodiments, the IgG1 heavy chain constant region sequence contained in the HC has the following mutations compared to the wild-type IgG1 heavy chain constant region sequence: L234A, L235A, D265A, P329A.

[0039] The present application also provides a plasmid or vector comprising a nucleic acid sequence encoding the above-mentioned bispecific antigen-binding protein.

[0040] The present application also provides a host cell comprising and expressing the above nucleic acid sequence, plasmid or vector.

[0041] The present application also provides a composition comprising the above-mentioned bispecific antigen-binding protein.

[0042] The present application also relates to the use of the above-mentioned bispecific antigen-binding protein or composition in the preparation of a drug for treating cancer.

[0043] The present application also provides a method for treating cancer, comprising administering the above-mentioned bispecific antigen-binding protein or composition to a subject in need thereof.

[0044] In some embodiments, preferred cancers whose growth can be inhibited using the bispecific antigen-binding proteins of the present application or compositions comprising the same include cancers that are generally responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include lung cancer, lymphoma, mesothelioma, melanoma, and renal cell carcinoma. In addition, the present application also includes the use of the bispecific antigen-binding proteins of the present application or compositions comprising the same to inhibit the growth of refractory or recurrent malignancies.

[0045] Examples of other cancers that can be treated using the bispecific antigen binding proteins of the present application or compositions comprising the same include bone cancer; pancreatic cancer; skin cancer; cancer of the head or neck; malignant melanoma of the skin or eye; uterine cancer; ovarian cancer; rectal cancer; cancer of the anal region; stomach cancer; testicular cancer; fallopian tube cancer; endometrial cancer; cervical cancer; vaginal cancer; vulvar cancer; Hodgkin's disease; non-Hodgkin's lymphoma; esophageal cancer; small intestine cancer; cancer of the endocrine system; thyroid cancer; parathyroid cancer; adrenal cancer; soft tissue sarcoma; urethral cancer; penile cancer; chronic or acute leukemia, including acute myeloid leukemia, Chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; solid tumors in children; lymphocytic lymphoma; bladder cancer; kidney or ureteral cancer; renal pelvis cancer; central nervous system (CNS) tumors; primary CNS lymphoma; tumor angiogenesis; spinal cord axis tumors; brain stem gliomas; pituitary adenomas; Kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; T-cell lymphoma; environmentally induced cancers, including asbestos-induced cancers, and combinations of the cancers. The present application can also be used to treat metastatic cancers, particularly metastatic cancers expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144).

[0046] Other features and advantages of the application will become apparent from the following detailed description and examples, or will be understood by implementing the application, and the following detailed description and examples should not be construed as restrictive. Other advantages of the application can be realized and obtained by the scheme described in the specification and the accompanying drawings. The contents of all references, Genbank entries, patents and disclosed patent applications cited in this application are clearly incorporated herein by reference.

[0047] Summary of the Figures

[0048] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0049] FIG1 shows the structure of the bispecific antigen-binding protein of the present application, wherein R1D2 represents the second domain of VEGFR1.

[0050] Figure 2 shows that the 1B12-R antibody can relieve the inhibitory effect of PD-1 / PD-L1 binding on NFAT signaling.

[0051] FIG3 shows that the 1B12-R antibody can inhibit the binding of VEGF-A to VEGFR2.

[0052] Figure 4 shows that the 1B12-T antibody can relieve the inhibitory effect of PD-1 / PD-L1 binding on NFAT signaling.

[0053] FIG5 shows that the 1B12-T antibody can inhibit the binding of VEGF-A to VEGFR2.

[0054] FIG6 shows that the 1B12-T antibody can inhibit VEGF-A-induced HUVEC cell proliferation.

[0055] FIG7 shows the in vivo anti-tumor effects of the bispecific antigen binding proteins 1B12-R and 1B12-T.

[0056] Details

[0057] In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0058] As used herein, the term "and / or" should be considered as a specific disclosure of each of two or more specified features or components. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0059] It will be understood that wherever aspects are described using the language "comprising" or "including," similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0060] The term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies at an acceptable level in the art. In some embodiments, such variation can be as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values.

[0061] The terms "antigen-binding protein" and "antibody" are used interchangeably and refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors.

[0062] The term "bispecific antigen binding protein" refers to a protein molecule that has binding specificities for two different epitopes, wherein the first antigen binding moiety and the second antigen binding moiety can be linked together by, for example, a linker.

[0063] As used herein, an antibody that "specifically binds to human PD-1" is intended to refer to an antibody that binds to human PD-1 protein (and possibly PD-1 proteins from one or more non-human species), but does not substantially bind to non-PD-1 proteins.

[0064] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide sequences comprising deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0065] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0066] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0067] The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell but may include mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the original transformed cell are included herein.

[0068] IC 50 The half maximal inhibitory concentration (IC) is the half-maximal inhibitory concentration of the antagonist being measured. It indicates the half-maximal inhibitory concentration of a drug or substance (inhibitor) in inhibiting a biological process (or a substance involved in the process, such as an enzyme, cell receptor, or microorganism). 50 Can measure antibody sensitivity, IC 50 The lower the value, the higher the sensitivity of the antibody.

[0069] EC 50 (concentration for 50% of maximal effect) refers to the concentration that can cause 50% of the maximum effect.

[0070] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0071] A "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary according to a variety of factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to stimulate the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are outweighed by the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%.

[0072] "Percent (%) sequence identity" or "percent (%) identical" relative to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the residues in the reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps, if necessary, to obtain maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent sequence identity can be achieved in a variety of ways in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. When referring to percent sequence identity in this application, unless otherwise expressly stated, these percentages are calculated relative to the full length of the longer sequence. Calculations relative to the full length of the longer sequence are applicable to both nucleic acid sequences and polypeptide sequences.

[0073] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional components that are unacceptably toxic to a subject to which the preparation would be administered.

[0074] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, etc., as known in the art. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Each carrier should be pharmaceutically and physiologically acceptable in terms of compatibility with the other ingredients and harmless to the subject. Unless any conventional media or agents are incompatible with the active ingredient, their use in pharmaceutical compositions is contemplated.

[0075] It is understood that the term "treat," ... Example

[0076] Example 1: Construction, expression and purification of the bispecific antibodies 1B12-R and 1B12-T of the present application

[0077] This application constructed two bispecific antibodies with the structures shown in Figure 1, named "1B12-R" and "1B12-T" respectively. The anti-PD-1 antibody parts of these two bispecific antibodies are derived from fully human anti-PD-1 antibodies, derived from the PD1 antibody 21F12-1B12 in patent WO2019219064A.

[0078] The heavy chain constant region of the bispecific antibody is the heavy chain constant region of IgG1 with effector function eliminated, which carries L234A, L235A, D265A, and P329A, and its amino acid sequence is

[0079] The recombinant heavy chain (HC) of the bispecific antibody 1B12-R comprises, from N-terminus to C-terminus, an anti-PD-1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, a linker, and ramucirumab ScFv. The amino acid sequence of the recombinant heavy chain (HC) is SEQ ID NO: 13. The light chain (LC) of the bispecific antibody 1B12-R comprises, from N-terminus to C-terminus, an anti-PD-1 light chain antibody variable region VL and a light chain constant region CL. The amino acid sequence of the light chain (LC) is SEQ ID NO: 14.

[0080] The recombinant heavy chain (HC) of the bispecific antibody 1B12-T comprises, from N-terminus to C-terminus, an anti-PD1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, a linker, and a VEGFR1 domain 2 (R1D2). The amino acid sequence of the recombinant heavy chain (HC) is SEQ ID NO: 15. The light chain (LC) of the bispecific antibody 1B12-T comprises, from N-terminus to C-terminus, an anti-PD1 light chain antibody variable region VL and a light chain constant region CL. The amino acid sequence of the light chain (LC) is SEQ ID NO: 14.

[0081] Nucleotides encoding the recombinant heavy and light chains of the bispecific antibodies 1B12-T and 1B12-R were generated by gene synthesis (GenScript) and cloned into the expression vector pcDNA3.1 (Invitrogen).

[0082] The expression vectors encoding the heavy and light chains were separately transfected into CHO-S cells to express the bispecific antibodies (ExpiCHO Expression System, Thermo Fisher Scientific). After transfection, the cells were cultured for 10-12 days. When cell viability dropped to 60% to 70%, the supernatant was collected and the antibodies expressed in the supernatant were purified using the MabSelect Sure Protein A affinity chromatography system (GE Healthcare) to obtain 1B12-R and 1B12-T. The purified antibodies were concentrated and sterile filtered. Purity was determined by SDS-PAGE and size exclusion, and the purity was >90% for both, allowing for use in subsequent experiments.

[0083] Table 1. Sequence composition of bispecific antibodies

[0084] Example 2: Fortebio detects the affinity of 1B12-T binding to human PD-1

[0085] In this experiment, the binding affinity of 1B12-T to human PD-1 (ACROBiosystems, Cat# PD1-H5221) protein was detected using ForteBio Octet RED96 according to the manufacturer's instructions.

[0086] Briefly, an AHC sensor (ForteBio, Cat#18-5060) was placed in running buffer (1X PBS Hyclone, Cat#SH30256.01, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate as follows: a) baseline equilibration with running buffer for 100 seconds; b) 1B12-T diluted in running buffer was added to a final concentration of 5 μg / mL and allowed to solidify for 200 seconds; c) baseline equilibration with running buffer for 300 seconds; d) human PD-1 protein at 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, or 6.25 nM diluted in running buffer was added to each well and allowed to bind for 200 seconds and dissociate for 600 seconds. Experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software. Table 1 summarizes the binding affinity of 1B12-T to human PD-1 protein.

[0087] Table 2: Binding affinity of 1B12-T to human PD1 protein

[0088] Example 3: Fortebio detects the affinity of 1B12-T antibody binding to human VEGF-A

[0089] In this experiment, the binding affinity of 1B12-T to human VEGF-A (SinoBiological, Cat# 11066-HNAH) protein was detected using ForteBio Octet RED96 according to the manufacturer's instructions.

[0090] Briefly, an AHC sensor (ForteBio, Cat#18-5060) was placed in running buffer (1X PBS Hyclone, Cat#SH30256.01, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate as follows: a) baseline equilibration with running buffer for 100 seconds; b) addition of bispecific antibody diluted in running buffer to a final concentration of 5 μg / mL and immobilization for 200 seconds; c) baseline equilibration with running buffer for 300 seconds; d) addition of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, or 1.5625 nM of human VEGF-A protein diluted in running buffer to each well; binding for 200 seconds and dissociation for 600 seconds. Experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software. Table 3 summarizes the binding affinity of 1B12-T to human VEGF-A protein.

[0091] Table 3: Binding affinity of 1B12-T to human VEGF-A protein

[0092] Example 4: 1B12-R Antibody Relieves the Inhibitory Effect of PD-1 / PD-L1

[0093] Construction of Jurkat-PD-1 / NFAT cell line: Gene Pul Ser X Cell TM (BIO-RAD) NFAT-luc2P reporter gene element (Promega, Cat#E8481) was transfected into Jurkat cells, and then NFAT-luc2P polyclonal cell lines were obtained by screening with hygromycin B (Invitrogen, Cat#10687010). After limiting dilution of the polyclonal cells, monoclonal Jurkat / NFAT-luc cells with stable and high expression of NFAT-luc2P were obtained. TM (BIO-RAD) A eukaryotic expression vector containing the human PD1 gene was transfected into Jurkat / NFAT-luc monoclonal cells. The Jurkat-PD-1 / NFAT polyclonal cell line was then screened with puromycin (Invitrogen Cat#A1113802). Limiting dilution of the polyclonal cells yielded the Jurkat-PD-1 / NFAT monoclonal cell line, which stably and highly expressed human PD1.

[0094] Construction of CHO-K1 / OKT3 / PD-L1 cell line: using Lipofectamine TM2000 (Invitrogen, Cat#11668019) transfection reagent was used according to the manufacturer's instructions to encode CD3 antibody (OKT3) (sequence see amino acid sequence shown in SEQ ID NO: 18 of Chinese Patent Publication CN114616251A) and human PD-L1 (sequence see SEQ ID NO: 20 of Chinese Patent Publication CN114616251A) A eukaryotic expression vector encoding the amino acid sequence of NP_054862.1 (shown in NO:17) was transfected into CHO-K1 cells. 48 hours after transfection, cells were screened with 4 μg / mL puromycin (Invitrogen, Cat#A1113802) and 600 μg / mL neomycin (Invitrogen, Cat#10131027) to obtain a stable polyclonal CHO-K1 cell line that highly expresses human PD-L1 and OKT3. Limiting dilution of the polyclonal cells yielded monoclonal CHO-K1 / OKT3 / PD-L1 cells that stably and highly express human PD-L1 and OKT3.

[0095] The Jurkat-PD-1 / NFAT cell suspension was collected by centrifugation, the supernatant was discarded, and the cell pellet was resuspended in assay medium (90% RPMI 1640 medium + 10% fetal bovine serum). The cell pellet was counted using a Countstar cell counter, and the cell suspension was diluted to 1×10 6 / mL. CHO-K1 / OKT3 / PD-L1 cells were collected by digestion and centrifugation, the supernatant was discarded, the cell pellet was resuspended in assay medium, and counted using a Countstar cell counter. The cell suspension was diluted to 5×10 5 / mL. Prepare 5× the final detection concentration of the test antibody. The final detection concentrations of the test antibody are 0.0002nM, 0.0061nM, 0.0024nM, 0.0098nM, 0.0391nM, 0.1563nM, 0.625nM, 2.5nM, 10nM and 40nM. Mix the Jurkat-PD-1 / NFAT and CHO-K1 / OKT3 / PD-L1 cell suspensions in a volume ratio of 1:1, inoculate the mixture into a 384-well detection white plate, and add 50μL / well. Add 10μL / well of gradient antibody dilution. Oscillate the entire plate at 450rpm at room temperature with a microplate fast shaker to mix for 5-10 minutes, and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 6 hours. Thaw One-Glo at room temperature TM Buffer, then added to One-Glo TM Substrate, fully dissolved at room temperature and ready for use. Remove the test plate from the incubator and equilibrate at room temperature for 10-15 minutes. Add 30 μL One-Glo to each well. TMLuciferase Assay Reagent (Promega, catalog number: E6130) was mixed and lysed at room temperature for 5-10 minutes. Luminescence signals were measured using the fluorescence detection module of a multifunctional microplate reader (Tecan F200Pro) with a 384-well white plate selected. Data were entered into Graphpad Prism software, with the x-axis representing the log value of the molar concentration. The "Dose-response-StimμLation→Log(agonist) vs. response-Variable slope (four parameters)" method was used for fitting. EC values ​​were recorded. 50 value.

[0096] The experimental results are shown in Figure 2. 1B12-R antibody can relieve the inhibitory effect of PD-1 / PD-L1 binding on NFAT signaling, showing a dose-dependent trend in the reporter gene system. 50 The value is 0.257nM.

[0097] Example 5: 1B12-R Antibody Inhibits Binding of VEGF-A to VEGFR2

[0098] Construction of 293T / NFAT / VEGFR2 cell line: using Lipofectamine TM The eukaryotic expression vector encoding the human VEGFR2 gene and the NFAT-luc2P reporter gene element (Promega, Cat#E8481) were transfected into 293T cells using Promega 2000 transfection reagent (Invitrogen, Cat#11668019) according to the manufacturer's instructions. 48 hours after transfection, the cells were screened with 0.3 μg / mL puromycin (Invitrogen, Cat#A1113802) and 85 μg / mL hygromycin B (Invitrogen, Cat#10687010) to obtain a 293T polyclonal stable cell line that highly expresses human VEGFR2 and NFAT-luc2P. By limiting dilution of the polyclonal cells, the monoclonal Jurkat / NFAT / VEGFR2 cells that stably and highly express NFAT-luc2P and human VEGFR2 were obtained.

[0099] 293T / NFAT / VEGFR2 cells were collected by digestion and centrifugation, the supernatant was discarded, the cell pellet was resuspended in assay medium (90% DMEM + 10% fetal bovine serum), and the cell suspension was counted using a Countstar cell counter. The cell suspension was diluted to 5×10 5 / mL. Prepare the test antibody at 5× the final detection concentration using assay medium containing 15ng / mL VEGF-A. The final detection concentrations of the test antibody are 0.002nM, 0.0039nM, 0.0078nM, 0.0156nM, 0.0313nM, 0.0625nM, 0.125nM, 0.25nM, 0.5nM and 1nM. Inoculate the 293T / NFAT / VEGFR2 cell suspension into a 384-well plate, 40μL per well. Add the gradient antibody dilution solution at 10μL / well. Oscillate the entire plate at 450rpm on a microplate shaker at room temperature for 5-10min and incubate in an incubator at 37°C, 5% CO2 and saturated humidity for 6 hours. Thaw One-Glo TM Buffer, then added to One-Glo TM Substrate, fully dissolved at room temperature and ready for use. Remove the test plate from the incubator and equilibrate at room temperature for 10-15 minutes. Add 30 μL One-Glo to each well. TM Mix the Luciferase Assay Reagent and lyse the cells at room temperature for 5-10 minutes. Use the fluorescence detection module of a multifunctional microplate reader, select a 384-well white plate, and then measure the luminescence signal. Enter the data into Graphpad Prism software, with the x-axis data as the log value of the molar concentration. Select the "Dose-response-Stimulation→Log (inhibitor) vs. response-Variable slope (four parameters)" method for fitting. Record the EC 50 value.

[0100] The experimental results are shown in Figure 3. 1B12-R antibody can inhibit the binding of VEGF-A to VEGFR2 in a dose-dependent manner. 50 The value is 0.5105nM.

[0101] Example 6: 1B12-T Antibody Relieves the Inhibitory Effect of PD-1 / PD-L1

[0102] Jurkat-PD-1 / NFAT cell lines and CHO-K1 / OKT3 / PD-L1 cell lines were constructed according to the method of Example 4 to detect the effect of 1B12-T antibody on the inhibition of PD-1 / PD-L1. The final concentrations of the antibodies to be tested were 0.0012nM, 0.0049nM, 0.0195nM, 0.0781nM, 0.3125nM, 1.25nM, 5nM and 20nM. The microplates were incubated and the luminescence signal readings were detected according to the method described in Example 4. The EC 50 value.

[0103] The experimental results are shown in Figure 4. 1B12-T antibody can relieve the inhibitory effect of PD-1 / PD-L1 binding on NFAT signaling, showing a dose-dependent trend in the reporter gene system. 50 The value is 0.1645nM.

[0104] Example 7: 1B12-T Antibody Inhibits Binding of VEGF-A to VEGFR2

[0105] The 293T / NFAT / VEGFR2 cell line was constructed using the method described in Example 5 to test the inhibitory effect of the 1B12-T antibody on the binding of VEGF-A to VEGFR2. The final concentrations of the antibody to be tested were 0.0410 nM, 0.1024 nM, 0.256 nM, 0.64 nM, 1.6 nM, 4 nM, 10 nM, and 25 nM. The microplate was incubated and the luminescence signal readings were measured according to the method described in Example 5. The IC was recorded. 50 The experimental results are shown in Figure 5. 1B12-T antibody can inhibit the binding of VEGF-A to VEGFR2, showing a dose-dependent trend in the reporter gene system. 50 The value is 0.7267nM.

[0106] Example 8: 1B12-T Antibody Inhibits VEGF-A-Induced Proliferation of Human Umbilical Vein Endothelial Cells (HUVEC)

[0107] HUVEC were collected by digestion and centrifugation (manufacturer: ATCC, catalog number: CRL-1730 TM ) cells, discard the supernatant, and resuspend the cell pellet in assay medium (90% Medium-199 1X Earle's Salts (Manufacturer: Gibco Catalog No. 11150-059) + 10% fetal bovine serum (Manufacturer Gibco Catalog No. 10099-141) + 10 mM HEPES (Manufacturer Gibco Catalog No. 15630080) + 100 μg / mL penicillin 100 μg / mL streptomycin (Manufacturer Gibco Catalog No. 10378016)). Count the cells using a Countstar cell counter, and dilute the cell suspension to 5 × 10 with assay medium. 4 / mL. 200ng / mL VEGF-A (Manufacturer: GenScript, Catalog No.: Z03073) working solution was prepared in assay medium. An experimental control containing only VEGF-A but no antibody was set up. Test antibodies were prepared in assay medium at 4X the final assay concentration. The final assay concentrations of the test antibodies were 0.1371nM, 0.4115nM, 1.2346nM, 3.7037nM, 11.1111nM, 33.3333nM, 100nM, and 300nM. 100μL of HUVEC cell suspension, 50μL of VEGF-A working solution, and 50μL of antibody were seeded into a 96-well plate. The 96-well plate was incubated in a 37°C, 5% CO2, saturated humidity incubator for 96 hours. Before testing, the culture supernatant was carefully aspirated and 50μL of fresh assay medium was added to each well. Add 50 μl of Cell Titer Glo to detect cell viability, read the plate on an F200 microplate reader, and record the data. Enter the data into Graphpad Prism software, with the x-axis data as the log value of the molar concentration, and select the "Dose-response-Stimulation→Log (inhibitor) vs. response-Variable slope (four parameters)" method for fitting. Record IC 50 value.

[0108] The experimental results are shown in Figure 6. 1B12-T antibody can inhibit VEGF-A-induced HUVEC cell proliferation in a dose-dependent manner. 50 It is 4.370nM.

[0109] Example 9: In vivo anti-tumor effects of bispecific antibodies 1B12-R and 1B12-T

[0110] Humanized mice expressing human PD-1 / KDR, C57BL / 6JSmoc-Cd274em1 (hCD274)Kdrtm2 (hKDR)Smoc, were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0111] The hPDL1-B16F10 murine melanoma cell line was provided by Shanghai Model Organisms Science Co., Ltd. hPDL1-B16F10 cells were maintained in DMEM complete medium supplemented with 10% fetal bovine serum. 1×10 6 hPDL1-B16F10 cells, and when the average tumor volume reached approximately 70 mm 3 (LxW 2 / 2) (day 0) and were randomly divided into 4 groups (N = 6 per group). On days 0, 3, 7, and 10, mice were intraperitoneally administered 63.72 nM / kg of 1B12-R (13 mg / kg), 1B12-T (11 mg / kg), 21F12-1F6 (9 mg / kg), and PBS, respectively. 21F12-1F6 is a highly homologous antibody to the anti-PD-1 antibody 1B12, and its sequence is available in International Patent Application Publication WO2019219064A. Tumor volume was monitored twice weekly by caliper measurement during the experiment.

[0112] Treatment with 1B12-R and 1B12-T resulted in significant tumor growth inhibition compared with the PBS group, and the 1B12-R and 1B12-T groups showed higher TGI rates compared with the anti-PD-1 antibody 21F12-1F6 group (63.4% and 74% vs. 30.6%), as shown in Table 4 below.

[0113] The status of mice in each group was observed daily and no abnormalities were found; the mice were weighed twice a week, and no significant weight loss occurred in the mice in each group. At the end of the experiment, the weight of mice in each group did not change significantly compared with the weight at the beginning of the treatment, and the drug had no obvious toxic side effects on the mice.

[0114] Table 4. hPDL1-B16F10 tumor growth inhibition

[0115] a Tumor volume data are expressed as mean ± SEM;

[0116] b TGI=(1-tumor volume change of the administration group / tumor volume change of the control group)×100%

[0117] c Compared with the PBS group, two-way ANOVA was performed, followed by Tukey's multiple comparison test.

[0118] This application describes multiple embodiments, but this description is exemplary rather than restrictive, and it will be apparent to those skilled in the art that more embodiments and implementations may be included within the scope of the embodiments described herein. Although many possible feature combinations are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

Claims

1. A bispecific antigen-binding protein comprising (a) a first antigen-binding portion that specifically binds to PD-1; and (b) a second antigen-binding portion that specifically targets the VEGF pathway; and the second antigen-binding portion comprises VEGFR1 domain 2 or comprises an antibody domain or antigen-binding domain that targets VEGFR2.

2. The bispecific antigen-binding protein according to claim 1, wherein The first antigen binding portion comprises: a heavy chain complementary determining region and a light chain complementary determining region, wherein the heavy chain complementary determining region comprises, from N-terminus to C-terminus, HCDR1, wherein HCDR1 has an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO: 1; HCDR2, wherein HCDR2 has an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO: 2; and HCDR3, wherein HCDR3 has an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO: 3; the light chain complementary determining region comprises, from N-terminus to C-terminus, LCDR1, wherein LCDR1 has an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO: 4; LCDR2, wherein LCDR2 has an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO: 5; and LCDR3, wherein LCDR3 has an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO: 6; The second antigen binding portion comprises: a heavy chain complementary determining region and a light chain complementary determining region, the heavy chain complementary determining region comprises, from N-terminus to C-terminus: HCDR1, the HCDR1 having an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO:7; HCDR2, the HCDR2 having an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO:8; and HCDR3, the HCDR3 having an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO:9; the light chain complementary determining region comprises, from N-terminus to C-terminus: LCDR1, the LCDR1 having an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO:10; LCDR2, the LCDR2 having an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO:11; and LCDR3, the LCDR3 having an amino acid sequence at least 75% identical to the amino acid sequence listed in SEQ ID NO:12; and the second antigen binding portion targets VEGFR2; or The second antigen binding portion comprises domain 2 of VEGFR1.

3. The bispecific antigen-binding protein according to claim 2, wherein the N-terminus of the second antigen-binding moiety is connected to the C-terminus of the heavy chain of the first antigen-binding moiety via a first linker to form a recombinant heavy chain; preferably, the first linker is flexible; preferably, the first linker is an IgG4 hinge region or (G4S) n , where n is an integer from 1 to 5.

4. The bispecific antigen-binding protein according to claim 2, wherein the bispecific antigen-binding protein comprises a recombinant heavy chain and a light chain, wherein the recombinant heavy chain comprises, from N-terminus to C-terminus, an anti-PD1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional first linker, and ramucirumab ScFv, and the ramucirumab scFv comprises ramucirumab heavy chain variable region VH, a second linker, and ramucirumab light chain variable region VL; and the light chain comprises, from N-terminus to C-terminus, an anti-PD1 antibody light chain variable region VL and an anti-PD1 antibody light chain constant region CL.

5. The bispecific antigen binding protein according to claim 4, wherein The anti-PD1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO: 16; The IgG1 heavy chain constant region has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 17; The first linker is an IgG4 hinge region; (G4S) n , where n is an integer from 1 to 5; The heavy chain variable region VH of the ramucirumab ScFv has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO: 18; The second linker is an IgG4 hinge region; (G4S) n , wherein n is an integer of 1-5; preferably, the second linker is (G4S) n , where n = 3; The light chain variable region VL of the ramucirumab ScFv has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO: 19; The anti-PD1 antibody light chain variable region VL has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO: 21; and The anti-PD1 antibody light chain constant region CL has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO:

22.

6. The bispecific antigen-binding protein of claim 5, wherein the recombinant heavy chain has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO:

13.

7. The bispecific antigen-binding protein of claim 6, wherein the light chain has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO:

14.

8. The bispecific antigen-binding protein according to claim 7, wherein the second antigen-binding moiety is ramucirumab ScFv, preferably, the ramucirumab ScFv has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO:

25.

9. The bispecific antigen binding protein according to any one of claims 4 to 8, wherein The bispecific antigen-binding protein consists of two amino acid sequences listed in SEQ ID NOs: 13 and 14.

10. The bispecific antigen-binding protein according to claim 2, wherein the bispecific antigen-binding protein comprises a recombinant heavy chain and a light chain, wherein the recombinant heavy chain comprises, from N-terminus to C-terminus, an anti-PD1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional first linker, and a VEGFR1 domain 2 (R1D2); and the light chain comprises, from N-terminus to C-terminus, an anti-PD1 light chain antibody variable region VL and a light chain constant region CL.

11. The bispecific antigen binding protein according to claim 10, wherein The anti-PD1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO: 16; The IgG1 heavy chain constant region has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 17; The first linker is an IgG4 hinge region or (G4S) n , where n is an integer from 1 to 5; The VEGFR1 domain 2 (R1D2) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 20; The anti-PD1 antibody light chain variable region VL has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO: 21; and The anti-PD1 antibody light chain constant region CL has an amino acid sequence that is at least 75% identical to the amino acid sequence listed in SEQ ID NO:

22.

12. The bispecific antigen-binding protein of claim 11, wherein the recombinant heavy chain has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO:

15.

13. The bispecific antigen-binding protein of claim 12, wherein the light chain has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO:

14.

14. The bispecific antigen binding protein according to any one of claims 10 to 13, wherein The bispecific antigen-binding protein consists of two amino acid sequences listed in SEQ ID NOs: 15 and 14.

15. The bispecific antigen binding protein according to any one of claims 3 to 14, wherein The IgG1 heavy chain constant region sequence contained in the HC has the following mutations compared to the wild-type IgG1 heavy chain constant region sequence: L234A, L235A, D265A, P329A.

16. A nucleic acid sequence encoding a bispecific antigen binding protein according to any one of claims 1 to 15.

17. A plasmid or vector comprising the nucleic acid sequence according to claim 16.

18. A host cell comprising and expressing the nucleic acid sequence according to claim 16 or the plasmid or vector according to claim 17.

19. A composition comprising the bispecific antigen binding protein according to any one of claims 1-15.

20. Use of the bispecific antigen binding protein according to any one of claims 1 to 15 or the composition according to claim 19 in the preparation of a medicament for treating cancer.

21. The use according to claim 20, wherein the cancer is selected from the group consisting of: bone cancer; pancreatic cancer; skin cancer; cancer of the head or neck; malignant melanoma of the skin or the eye; uterine cancer; ovarian cancer; rectal cancer; cancer of the anal region; stomach cancer; testicular cancer; fallopian tube cancer; endometrial cancer; cervical cancer; vaginal cancer; vulvar cancer; Hodgkin's disease; non-Hodgkin's lymphoma; esophageal cancer; small intestine cancer; endocrine system cancer; thyroid cancer; parathyroid cancer; adrenal cancer; soft tissue sarcoma; urethral cancer; penile cancer; chronic or Acute leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; solid tumors in children; lymphocytic lymphomas; bladder cancer; cancer of the kidney or ureter; cancer of the renal pelvis; central nervous system (CNS) tumors; primary CNS lymphomas; tumor angiogenesis; spinal axis tumors; brainstem gliomas; pituitary adenomas; Kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; T-cell lymphomas; environmentally induced cancers, including asbestos-induced cancers.

22. A method for treating cancer, comprising administering the bispecific antigen binding protein according to any one of claims 1-15 or the composition according to claim 19 to a subject in need thereof.

23. The method of claim 22, wherein the cancer is selected from the group consisting of: bone cancer; pancreatic cancer; skin cancer; cancer of the head or neck; malignant melanoma of the skin or the eye; uterine cancer; ovarian cancer; rectal cancer; cancer of the anal region; stomach cancer; testicular cancer; fallopian tube cancer; endometrial cancer; cervical cancer; vaginal cancer; vulvar cancer; Hodgkin's disease; non-Hodgkin's lymphoma; esophageal cancer; small intestine cancer; cancer of the endocrine system; thyroid cancer; parathyroid cancer; adrenal cancer; soft tissue sarcoma; urethral cancer; penile cancer; chronic or Acute leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; solid tumors in children; lymphocytic lymphomas; bladder cancer; cancer of the kidney or ureter; cancer of the renal pelvis; central nervous system (CNS) tumors; primary CNS lymphomas; tumor angiogenesis; spinal axis tumors; brainstem gliomas; pituitary adenomas; Kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; T-cell lymphomas; environmentally induced cancers, including asbestos-induced cancers.

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