Bispecific antibody combining PD-L1 and VEGF and application thereof

Abstract

The invention provides a bispecific antibody capable of binding to PD-L1 and VEGF (vascular endothelial growth factor). The bispecific antibody comprises a first binding domain capable of binding to PD-L1 and a second binding domain capable of binding to VEGF. The bispecific antibody provided by the invention is combined with PD-L1, so that the immunosuppression of tumor cells on T cells can be relieved, and the anti-tumor function of an immune system can be recovered; meanwhile, by combining the bispecific antibody with VEGF, the growth of the tumor can be slowed down, the blood supply of the tumor can be reduced, and even the tumor blood vessel can be normalized, so that the immune environment is improved, and the attack ability of immune cells on the tumor is enhanced.

Description

A bispecific antibody that binds PD-L1 and VEGF and its uses Technical Field

[0001] This invention relates to the fields of biomedicine and tumor immunology, and more particularly to a bispecific antibody or a fragment thereof that binds PD-L1 and VEGF and its uses. Background Technology

[0002] According to the latest data from the National Cancer Center based on tumor registry and follow-up monitoring, cancer has become one of the leading causes of death in China. Non-small cell lung cancer, liver cancer, stomach cancer, colorectal cancer, and breast cancer are the most common types of cancer in China and also the leading causes of cancer-related deaths; in particular, non-small cell lung cancer ranks among the top in both incidence and mortality among all cancers. Furthermore, although bone tumors account for a relatively small proportion of all cancers, they are one of the leading malignant tumors in children and adolescents, especially in the 10-20 age group. Bone tumors are diverse, including malignant types such as osteosarcoma, chondrosarcoma, and giant cell tumor of bone, as well as benign types such as bone cysts and fibrous dysplasia of bone. Although good treatment outcomes have been achieved for some types of bone tumors (such as localized osteosarcoma), tumor recurrence and distant metastasis remain challenges, especially in high-grade malignant bone tumors, where current treatments often fail to achieve satisfactory results.

[0003] For solid tumors, current treatment options typically include chemotherapy, radiotherapy, surgery, and targeted therapy. However, these treatments often come with varying degrees of side effects, despite their effectiveness. Particularly for patients with relapsed or refractory solid tumors, their response to existing treatments may decrease over time, with many exhibiting intolerance or resistance. Furthermore, the complexity of the tumor microenvironment, such as tumor-induced immunosuppression, further complicates treatment.

[0004] With a deeper understanding of tumor biology, particularly the increasing knowledge of the interactions between tumors and the host immune system, developing novel therapeutic strategies that target specific tumor biomarkers has become crucial. In the treatment of solid tumors, there is an urgent need for new therapies to overcome patient resistance to existing treatments, reduce treatment-related toxicities, and ultimately improve patient survival and quality of life.

[0005] PD-L1 (Programmed Death-ligand 1), also known as CD274 (cluster of differentiation 274) or B7-H1 (B7 homolog 1), is a protein encoded by the human CD274 gene. PD-L1 is a 40kDa type I transmembrane protein and an important immune checkpoint, capable of transmitting immunosuppressive signals by interacting with PD-1 (Programmed Death-1) on T cells. Some tumor cells express high levels of PD-L1, achieving immune escape through the PD-1 / PD-L1 signaling pathway. Vascular endothelial growth factor (VEGF), originally called vascular permeability factor (VPF), is a subfamily of growth factors that stimulates angiogenesis and is an important signaling protein involved in angiogenesis (the formation of the embryonic circulatory system) and the growth of blood vessels from existing blood vessels. High expression of VEGF in tumors promotes tumor angiogenesis, thereby promoting tumor growth and metastasis. Therapeutic strategies targeting molecular targets such as PD-L1 and VEGF have shown great potential for treating a variety of solid tumors due to their role in regulating key processes such as tumor immune escape and angiogenesis.

[0006] Therefore, the development of bispecific antibodies targeting PD-L1 and VEGF is of great significance for the treatment of solid tumors. These drugs, by simultaneously acting on tumor immune escape mechanisms and angiogenesis pathways, hold the promise of providing more effective and selective treatment options for patients with solid tumors, especially those who do not respond well to traditional treatments. Summary of the Invention

[0007] To address the aforementioned technical problems, the object of this invention is to provide a bispecific antibody or a fragment thereof capable of binding to PD-L1 and VGFR, and to provide uses based on this bispecific antibody or its fragment. The term "fraction" in the antibody molecule of this invention encompasses various functional fragments of the bispecific antibody, such as its antigen-binding portion, like Fab, F(ab')2, or scFv fragments.

[0008] The technical solution of the present invention is as follows.

[0009] On one hand, the present invention provides a bispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody or an antigen-binding fragment thereof comprises:

[0010] (a) A first binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) capable of binding PD-L1, wherein the VH comprises the heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, as shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and the VL comprises the light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, as shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively; and

[0011] (b) A second binding domain containing a polypeptide capable of binding VEGF.

[0012] In the context of this invention, the term “PD-L1” is Programmed death-ligand 1, also known as CD274 (cluster of differentiation 274) or B7-H1 (B7 homolog 1); unless otherwise stated, “PD-L1” covers any form or structural region of PD-L1.

[0013] In the context of this invention, the term "VEGF" stands for vascular endothelial growth factor; unless otherwise stated, "VEGF" encompasses any form or structural region of VEGF.

[0014] In the context of this invention, the term "antigen-binding fragment" encompasses various functional fragments of the bispecific antibody, but retains the binding ability of the bispecific antibody to the target proteins (i.e., PD-L1 and VEGF) and the corresponding biological activity. It is known in the art that the binding ability of a bispecific antibody to the target protein and the corresponding biological activity can be achieved from fragments of the intact antibody, which can be obtained using conventional techniques known to those skilled in the art and can be screened for functionality in the same manner as for the intact antibody. For example, antigen-binding fragments of the bispecific antibody can be generated by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact antibody.

[0015] Furthermore, in the bispecific antibody provided by the present invention, the VH may contain the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.3, and the VL may contain the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.7.

[0016] In the context of this invention, "at least 90% identity" means any percentage of identity between 90% and 100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. Further optionally, "at least 90% identity" means that there is at most 10% difference in amino acid residues between the two amino acid sequences, which can be due to amino acid deletions, additions, or substitutions.

[0017] Furthermore, in the bispecific antibody provided by the present invention, the first binding domain may further comprise a constant region, such as a heavy chain constant region (CH) and a light chain constant region (CL). The constant region may be a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region (CL). Preferably, the first binding domain comprises a heavy chain constant region of human IgG, particularly human IgG1 or IgG4, and / or a light chain constant region. According to a specific embodiment of the present invention, the first binding domain comprises a heavy chain constant region, which comprises the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 4; and / or, the first binding domain comprises a light chain constant region, which comprises the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 8.

[0018] Furthermore, in the bispecific antibody provided by this invention, the first binding domain can be an IgG antibody, which comprises a heavy chain (HC) and / or a light chain (LC). For example, the heavy chain (HC) in the first binding domain contains "VH-CH" from the amino terminus to the carboxyl terminus, and the light chain (LC) contains "VL-CL" from the amino terminus to the carboxyl terminus, and the heavy chain and the light chain are linked by disulfide bonds.

[0019] In the bispecific antibody provided by the present invention, the second binding domain may be a VEGF-binding polypeptide, wherein the amino terminus of the polypeptide is fused to the carboxyl terminus of HC in the first binding domain via a linker.

[0020] In the context of this invention, the term "linker" refers to a short peptide formed from multiple amino acids. It is known in the art that flexible or rigid short peptides can be used when fusing two polypeptides to form a single polypeptide, thereby separating the two polypeptides without affecting their various functions or activities. The linker in the bispecific antibody provided by this invention can use the following amino acid sequences: (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, (SGGS)n, or SGGGGS, where n is independently an integer from 1 to 10. According to a specific embodiment of the invention, in the bispecific antibody provided by this invention, the linker is (GGGGS)n, where n is an integer from 1 to 10, preferably 2 to 5, more preferably 3 to 4. According to a specific embodiment of the invention, the linker comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 5.

[0021] Furthermore, the VEGF-binding polypeptide can be VEGFR or any of its domains.

[0022] In the context of this invention, the term "VEGFR" stands for vascular endothelial growth factor receptor; unless otherwise stated, "VEGFR" encompasses any form or structural region of VEGF and covers any subtype. It is known in the art that three VEGF receptors (VEGFRs) exist in mammals: VEGFR-1, VEGFR-2, and VEGFR-3. These receptors all belong to the transmembrane tyrosine kinase receptor family and comprise three main regions: an extracellular domain, a transmembrane domain, and an intracellular carboxyl terminus. The term "VEGFR" encompasses these receptor types and their domains.

[0023] Preferably, the VEGF-binding polypeptide may contain the extracellular domain of VEGFR-1, such as the second domain of the extracellular region of VEGFR-1. According to a specific embodiment of the present invention, the VEGF-binding polypeptide may contain the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 6.

[0024] Furthermore, the bispecific antibody provided by the present invention comprises an Fc domain, and the Fc domain comprises an amino acid mutation selected from the following: E233P, L234A, L235A, G236Del, P329A or any combination thereof, wherein the amino acid residues are numbered according to the EU index described in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

[0025] According to a specific embodiment of the present invention, the present invention provides a bispecific antibody comprising a heavy chain and a light chain, particularly preferably comprising two identical heavy chains and two identical light chains, wherein the heavy chain comprises an amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.1, and the light chain comprises an amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.2.

[0026] In a second aspect, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody or an antigen-binding fragment thereof provided in the first aspect of the present invention.

[0027] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in another aspect, this invention also provides a vector containing the nucleic acid molecules provided by this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector, etc., for purposes such as preserving or expressing the bispecific antibodies.

[0028] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells for purposes such as preservation or antibody expression. Therefore, in a third aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, fungal, insect, or animal cells.

[0029] The bispecific antibody or its antigen-binding fragment provided by this invention can be obtained using any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the bispecific antibody can be obtained first from the nucleic acid molecule provided by this invention, or the heavy chain and / or light chain of the bispecific antibody can be obtained, and then assembled with optional other domains of the bispecific antibody to form an antibody; or, the host cells can be cultured while allowing the host cells provided by this invention to express the heavy chain variable region and / or light chain variable region of the bispecific antibody or the heavy chain and / or light chain of the bispecific antibody to assemble the antibody. Optionally, the method further includes the step of recovering the generated antibody.

[0030] The bispecific antibody or its antigen-binding fragment, nucleic acid molecule, vector, and / or host cell provided by this invention can be included in compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed. Therefore, in a fourth aspect, this invention also provides a composition comprising the bispecific antibody or its antigen-binding fragment, nucleic acid molecule, vector, and / or host cell described herein. Further, the composition provided by this invention can be a pharmaceutical composition, optionally further comprising pharmaceutically acceptable excipients.

[0031] Fifthly, the present invention also provides the use of the bispecific antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell and / or pharmaceutical composition in the preparation of a medicament for the prevention or treatment of a disease, including tumors or cancers, such as tumors or cancers that highly express PD-L1 and / or VEGF, or tumors or cancers associated with positive expression of PD-L1 and / or VEGF.

[0032] Preferably, the disease is a solid tumor. Alternatively, the disease may include, for example, non-small cell lung cancer, small cell lung cancer, malignant pleural mesothelioma, thymic carcinoma, thymic epithelial tumor, breast cancer, esophageal cancer, esophageal adenocarcinoma, gastric adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, colonic adenocarcinoma, rectal adenocarcinoma, hepatocellular carcinoma, pancreatic cancer, pancreatic duct adenocarcinoma, biliary tract cancer, head and neck squamous cell carcinoma, salivary gland carcinoma, adenoid cystic carcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer (recurrent / advanced), cervical cancer, fallopian tube cancer, primary peritoneal cancer, tumors of the sexual reproductive organs, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the skin, soft tissue sarcoma, recurrent glioblastoma, gastric cancer, prostate cancer, and thyroid cancer. Cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, malignant hematological diseases, nasopharyngeal carcinoma, laryngeal cancer, uterine tumor, osteosarcoma, bone cancer, anal cancer, testicular cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, oral squamous cell carcinoma, urothelial carcinoma, advanced malignant solid tumors.

[0033] Accordingly, in a sixth aspect, the present invention also provides a method for preventing or treating a disease, the method comprising administering to a subject in need a bispecific antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, and / or a pharmaceutical composition thereof, the disease including tumors or cancers, such as tumors or cancers that highly express PD-L1 and / or VEGF, or tumors or cancers associated with positive expression of PD-L1 and / or VEGF.

[0034] Preferably, the disease is a solid tumor. Alternatively, the disease may include, for example, non-small cell lung cancer, small cell lung cancer, malignant pleural mesothelioma, thymic carcinoma, thymic epithelial tumor, breast cancer, esophageal cancer, esophageal adenocarcinoma, gastric adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, colonic adenocarcinoma, rectal adenocarcinoma, hepatocellular carcinoma, pancreatic cancer, pancreatic duct adenocarcinoma, biliary tract cancer, head and neck squamous cell carcinoma, salivary gland carcinoma, adenoid cystic carcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer (recurrent / advanced), cervical cancer, fallopian tube cancer, primary peritoneal cancer, tumors of the sexual reproductive organs, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the skin, soft tissue sarcoma, recurrent glioblastoma, gastric cancer, prostate cancer, and thyroid cancer. Cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, malignant hematological diseases, nasopharyngeal carcinoma, laryngeal cancer, uterine tumor, osteosarcoma, bone cancer, anal cancer, testicular cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, oral squamous cell carcinoma, urothelial carcinoma, advanced malignant solid tumors.

[0035] Preferably, the subject is a mammal, more preferably a primate or rodent; more preferably, the subject is a human.

[0036] In a seventh aspect, the present invention also provides the use of the bispecific antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell and / or pharmaceutical composition in the preparation of reagents for diagnosing diseases, including tumors or cancers, such as tumors or cancers that highly express PD-L1 and / or VEGF, or tumors or cancers associated with positive expression of PD-L1 and / or VEGF.

[0037] Preferably, the disease is a solid tumor. Alternatively, the disease may include, for example, non-small cell lung cancer, small cell lung cancer, malignant pleural mesothelioma, thymic carcinoma, thymic epithelial tumor, breast cancer, esophageal cancer, esophageal adenocarcinoma, gastric adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, colonic adenocarcinoma, rectal adenocarcinoma, hepatocellular carcinoma, pancreatic cancer, pancreatic duct adenocarcinoma, biliary tract cancer, head and neck squamous cell carcinoma, salivary gland carcinoma, adenoid cystic carcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer (recurrent / advanced), cervical cancer, fallopian tube cancer, primary peritoneal cancer, tumors of the sexual reproductive organs, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the skin, soft tissue sarcoma, recurrent glioblastoma, gastric cancer, prostate cancer, and thyroid cancer. Cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, malignant hematological diseases, nasopharyngeal carcinoma, laryngeal cancer, uterine tumor, osteosarcoma, bone cancer, anal cancer, testicular cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, oral squamous cell carcinoma, urothelial carcinoma, advanced malignant solid tumors.

[0038] More preferably, the disease is a tumor, preferably rectal cancer, osteosarcoma, non-small cell lung cancer, melanoma, bladder cancer, or a combination thereof.

[0039] Eighthly, the present invention also provides a method for diagnosing a disease, the method comprising contacting a bispecific antibody or antigen-binding fragment thereof, a nucleic acid molecule, a carrier, a host cell and / or a pharmaceutical composition of the present invention with a sample from a subject, the disease including tumors or cancers, such as tumors or cancers that highly express PD-L1 and / or VEGF, or tumors or cancers associated with positive expression of PD-L1 and / or VEGF.

[0040] Preferably, the disease is a solid tumor. Alternatively, the disease may include, for example, non-small cell lung cancer, small cell lung cancer, malignant pleural mesothelioma, thymic carcinoma, thymic epithelial tumor, breast cancer, esophageal cancer, esophageal adenocarcinoma, gastric adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, colonic adenocarcinoma, rectal adenocarcinoma, hepatocellular carcinoma, pancreatic cancer, pancreatic duct adenocarcinoma, biliary tract cancer, head and neck squamous cell carcinoma, salivary gland carcinoma, adenoid cystic carcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer (recurrent / advanced), cervical cancer, fallopian tube cancer, primary peritoneal cancer, tumors of the sexual reproductive organs, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the skin, soft tissue sarcoma, recurrent glioblastoma, gastric cancer, prostate cancer, and thyroid cancer. Cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, malignant hematological diseases, nasopharyngeal carcinoma, laryngeal cancer, uterine tumor, osteosarcoma, bone cancer, anal cancer, testicular cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, oral squamous cell carcinoma, urothelial carcinoma, advanced malignant solid tumors.

[0041] Preferably, the subject is a mammal, more preferably a primate or rodent; more preferably, the subject is a human.

[0042] In a ninth aspect, the present invention also provides a kit comprising the bispecific antibody or its antigen-binding fragment of the present invention, a nucleic acid molecule, a vector, a host cell, and / or a pharmaceutical composition. The kit can be used for the aforementioned prevention or treatment, or for the aforementioned diagnosis. Depending on the intended application, the kit may also contain other reagents. For example, the kit is for detecting PD-L1 and / or VEGF expression (including overexpression) in any biological sample using an ELISA.

[0043] Compared with existing technologies, this invention provides a novel bispecific antibody capable of binding to PD-L1 and VEGF with high affinity. PD-L1, as an immunosuppressive molecule, inhibits T cell activity, allowing tumor cells to evade the immune system's attack; while VEGF is an important factor promoting angiogenesis, crucial for tumor growth and spread. The bispecific antibody provided by this invention, by binding to PD-L1, can relieve the immunosuppression of T cells by tumor cells, restoring the anti-tumor function of the immune system; simultaneously, by binding to VEGF, it can slow tumor growth, reduce its blood supply, and even normalize tumor angiogenesis, thereby improving the immune environment and enhancing the ability of immune cells to attack tumors. Therefore, the bispecific antibody provided by this invention exerts its anti-tumor effect by simultaneously blocking two key targets, PD-L1 and VEGF, and can be developed into a highly effective anti-tumor drug. Attached Figure Description

[0044] Figure 1A and Figure 1B are schematic diagrams of the structures of two bispecific antibodies that bind to PD-L1 and VEGF, respectively.

[0045] Figure 2 shows the detection results of the blocking activity of different antibodies on the VEGF signaling pathway in Example 1 of the present invention.

[0046] Figures 3A to 3J show the non-specific binding detection results of KH003 and B7 family proteins in Example 4 of this invention.

[0047] Figures 4A to 4C are graphs showing the detection results of protein binding activity of 5KH003 and VEGF165-HIS, PDL1-His, and Rat PDL1-His in Example 5 of the present invention.

[0048] Figures 5A to 5B are graphs showing the binding activity detection results of KH003 in Example 6 of the present invention with PDL1 molecules from different species.

[0049] Figures 6A to 6E are graphs showing the binding activity detection results of KH003 and cell surface PDL1 in Example 7 of the present invention.

[0050] Figure 7 is a graph showing the detection results of the binding activity of KH003 in Example 8 of the present invention in blocking PDL1 and PD1.

[0051] Figure 8 is a graph showing the results of detecting the functional blocking activity of KH003 on the PDL1 reporter cell line in Example 9 of the present invention.

[0052] Figure 9 is a graph showing the results of detecting the functional blocking activity of 10KH003 on VEGF reporter cell lines in Example 10 of the present invention.

[0053] Figure 10 is a graph showing the detection results of the blocking activity of KH003 in Example 11 of the present invention against VEGF and VEGFR binding.

[0054] Figures 11A to 11E show the results of the binding activity detection of KH003 and different Fc receptors in Example 12 of the present invention.

[0055] Figures 12A to 12B are curves showing the results of detecting the killing activity of 293T-PDL1 overexpressing cells by mediated CDC in Example 13KH003 of the present invention.

[0056] Figures 13A to 13C show the results of detecting the non-specific stimulatory activity of KH003 on the release of PBMC secreted cytokines IL-2, IFN-γ, and TNF-α in Example 14 of this invention.

[0057] Figure 14 is a curve showing the inhibitory activity of 16KH003 in Example 1 of the present invention on the growth of osteosarcoma HOS subcutaneous tumor.

[0058] Figure 15 is a curve showing the inhibitory activity of KH003 in Example 17 of the present invention on the growth of subcutaneous osteosarcoma SJSA1 tumor.

[0059] Figure 16 is a curve showing the inhibitory activity of 18KH003 in Example 1 of the present invention on the subcutaneous tumor growth of non-small cell lung cancer HCC827.

[0060] Figure 17 is a curve showing the inhibitory activity of Example 19KH003 of the present invention on the growth of subcutaneous tumors of lung cancer NCI-H292.

[0061] Figures 18A to 18C are the PK result detection curves of Example 22KH003 of the present invention in Balb / c mice or SCID mice. Detailed Implementation

[0062] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions. Unless otherwise specified, all experimental methods described in the embodiments are conventional methods. Unless otherwise specified, the medicinal materials, reagents, and other materials used in the following embodiments are commercially available products.

[0063] The control antibodies used in the examples are shown in Table 1.

[0064] Table 1. Control Antibodies

[0065]

[0066]

[0067] *: The amino acid sequence of the linker is GGGGSGGGGSGGGGS (SEQ ID NO.5);

[0068] **: VEGFR1-D2 protein is the second extracellular domain of VEGFR1. Its amino acid sequence is shown in VEGFR1(P17948-1,Ser129-Thr228) and below.

[0069] Example 1 Construction of the structure of a bispecific antibody combining PD-L1 and VEGF

[0070] In this embodiment, two different forms of fusion antibodies were constructed by fusing VEGFR1 domain 2 to the heavy chain or light chain of the PD-L1 antibody (KA-1396), respectively. The PD-L1 antibody (KA-1396) is a humanized monoclonal antibody against PD-L1 (the variable region sequences of the heavy and light chains are shown in patent publication CN116789833B). The amino acid sequences of the heavy and light chains are as follows:

[0071] Table 2. Structural domains of the PD-L1 antibody (KA-1396)

[0072]

[0073]

[0074] The amino acid sequence of the second extracellular domain of VEGFR1 (SEQ ID NO.6) was fused to the C-terminus of the heavy chain and the C-terminus of the light chain of the PD-L1 antibody via a linker (SEQ ID NO.5) to obtain bispecific antibodies with different structures. The structural schematic diagrams are shown in Figure 1A (named KA-9976) and Figure 1B (named KA-9977).

[0075] Then, the VEGF signaling pathway blocking ability of both genes was tested in the reporter gene cell line 293T-NFAT-Luc2-KDR (Kyinno, Cat#: KC-1840). The experimental procedures are as follows:

[0076] 293T-NFAT-Luc2-KDR cells were suspended in complete culture medium and added to 96-well plates at a concentration of 20,000 cells per well (80 μL). Solutions of two antibody structures and a control antibody were prepared using complete culture medium, starting at a concentration of 20 μg / mL and diluted 3.16-fold to obtain 10 concentrations. Each antibody solution was added to the 96-well plate at a concentration of 10 μL / well, with three replicates for each concentration. A VEGF165-His protein solution (Acrobio, Cat#: VE5-H5248; hereinafter the same) was prepared using complete culture medium and added to the 96-well plate at a concentration of 10 ng / mL (10 μL / well). Additionally, a solvent control group and a blank cell control group were included in the 96-well plate.

[0077] The 96-well plates were incubated at 37°C and 5% CO2 for 6 hours. Then, 100 μL of Bright-Lite Luciferase Assay System solution was added to each well, and cells were lysed by shaking for 15 minutes. The values ​​were then read using a microplate reader (BMG LABTECH). The raw data were analyzed using GraphPad Prism 7.0 software, and a dose-response curve was fitted using log(inhibitor) vs. response – Variable slope (four parameters) to calculate the IC50. The results are shown in Figure 2.

[0078] The results showed that the VEGF-binding VEGFR domain fused to the heavy chain of the PD-L1 antibody (Figure 1A) was able to more effectively block VEGF-induced reporter cell activation compared to the fusion to the light chain (Figure 1B), suggesting that heavy chain fusion is beneficial for maintaining and enhancing the inhibitory activity of the VEGF signaling pathway.

[0079] Example 2 KH003 expression and purification

[0080] According to Example 1, a bispecific antibody was constructed by fusing an anti-PD-L1 antibody with a VEGF-binding polypeptide. This bispecific antibody is a homodimer, abbreviated as "KH003", which is a humanized IgG1 with the following heavy and light chain amino acid sequences.

[0081] Heavy chains (HC)

[0082] QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVATITSDGRYS

[0083] YYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRENWFTYWGQGTTVTVS

[0084] SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

[0085] SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPA

[0086] AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0087] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVY

[0088] TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

[0089] LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA GGGGSGGGGSGGGGS SDT

[0090] GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT(SEQ ID NO.1)

[0091] > Light chain (LC)

[0092] DIQITQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGSSPKPWIYLTSNLASGVPSR

[0093] FSGSGSGTDFTLTISSLQPEDFATYYCQQWSSGPTFGQGTRLEIKRTVAAPSVFIFPPSDE

[0094] QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.2)

[0095] The structural diagram of KH003 is shown in Figure 1A; the amino acid sequences of the domains contained in the heavy and light chains are shown in Table 3.

[0096] Table 3. Structural domains of KH003 (CDR uses Kabat encoding)

[0097]

[0098]

[0099] KH003 contains 36 cysteine ​​residues, including 14 intrachain disulfide bonds and 4 interchain disulfide bonds. Each heavy chain contains a glycosylation site at Asn295, Asn496, and Asn528, respectively. After deglycosylation and formation of pyroglutamic acid at the N-terminus of the heavy chain, the average theoretical molecular weight of KH003 is 168160.5 Da (Pyroglutamic Acid Q N-TERM).

[0100] The heavy and light chain coding sequences of the bispecific antibody KH003 were cloned into the eukaryotic expression vector pcDNA3.4, respectively. Subsequently, the expression vectors encoding the heavy and light chains were transfected into CHO-S cells to express the bispecific antibody (using the ExpiCHO expression system, vendor: Thermo Fisher). The specific steps are as follows: First, at 3 × 10⁻⁶… 6 EXPI-CHOS cells were seeded at a density of cells / mL in 125 mL shake flasks and cultured at 110 rpm and 8% CO2. The next day, two pre-prepared expression vectors were mixed with transfection reagents at a specific ratio and then injected at 6 × 10⁻⁶ rpm. 6 The transfection complex was added to the cells at a cell density of [number] cells / mL. After 24 hours, nutrients and DNA inhibitors were added. Cells were cultured for 10-12 days post-transfection, and the supernatant was collected when cell viability dropped to 80%. The antibody expressed in the supernatant was purified using the MabSelect Sure protein A affinity chromatography system (GE Healthcare). The concentration of the purified antibody was determined by Nanodrop, and the purity was assessed by SDS-PAGE electrophoresis.

[0101] Example 3KH003's affinity for target proteins PDL1 and VEGF

[0102] KH003 is a bispecific antibody that targets PDL1 and VEGF molecules. It can bind to both PDL1 protein and vascular endothelial growth factor (VEGF) simultaneously, thus blocking both the PDL1 / PD1 signaling pathway and the VEGF / VEGFR signaling pathway.

[0103] This experiment used Fortebio technology to detect the affinity of KH003 antibody for hPDL1-His protein (Kyinno, Cat#: KP-1002; hereinafter the same) and VEGF165-His protein (Acrobio, Cat#: VE5-H5248; hereinafter the same). The experimental method is as follows:

[0104] Prepare the experimental buffer: 1×PBS + 0.02% Tween 20 + 1 mg / mL BSA;

[0105] Sample preparation: The working concentration of antibody is 5 μg / mL, 250 μL per well; the highest working concentration of antigen is 200 nM, and it is diluted 2× in sequence to obtain 7 concentrations, 250 μL per well.

[0106] Pretreatment of detection probes: The probes should be soaked in the prepared buffer solution for 10 minutes before the experiment.

[0107] Transfer the sample to a black 96-well plate and perform analysis.

[0108] Based on the experimental results, five curves with good fit were selected for data analysis at antigen concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. The results showed that the affinities of KH003 with PDL1 and VEGF were 0.17 nM and 0.21 nM, respectively.

[0109] The results are shown in Table 4 below:

[0110] Table 4. Results of binding affinity assays between KH003 antibody and hPDL1-His and VEGF165-His proteins.

[0111]

[0112] Example 4 Nonspecific binding of KH003 to B7 family proteins

[0113] PD-L1 belongs to the B7 family of proteins. This experiment used enzyme-linked immunosorbent assay (ELISA) to detect the non-specific binding of KH003 antibody to other B7 family proteins, including: ICOSL-His (Kyinno, Cat#: KP-1108), B7-H3-mFc (Kyinno, Cat#: KP-1112), B7H4-mFc (Kyinno, Cat#: KP-1279), Human B7-H5 / VISTA-His (Acro, Cat#: 1760-20CAF1-15J), Human B7-H6 / NCR3LG1-His (Acro, Cat#: 2058b-8B7F1-ZS), Human PD-L2 / B7-DC Protein-His (Acro, Cat#: 1040-2143F1-17C), and Human B7-1 / CD80. Nonspecific binding of Protein, Mouse IgG2a Fc (Acro, Cat#: 2514-205DF1-11U), and Human B7-2 / CD86 Protein-His (Acro, Cat#: 743-208VF1-13R).

[0114] The experimental method is as follows:

[0115] Antigens were coated in 96-well plates with hPDL1-His, ICOSL-His, B7-H3-mFc, B7H4-mFc, HumanB7-H5 / VISTA-His, Human B7-H6 / NCR3LG1-His, Human PD-L2 / B7-DC Protein-His, HumanB7-1 / CD80 Protein, Mouse IgG2a Fc, Human B7-2 / CD86 Protein-His, and 2% BSA protein at 100 ng / 50 μL / well, and incubated overnight at 4°C.

[0116] Washing: Wash 3 times with 1×PBST the next day.

[0117] Blocking: Add 200 μL of blocking solution (1×PBS containing 2% BSA) to each well and incubate at 37°C for 2 h.

[0118] Wash plate: Wash 3 times with 1×PBST.

[0119] Add the test sample and incubate: serially dilute KH003 antibody and human IgG1 isotype κ control antibody (starting concentration 20 μg / mL, 10-fold dilution, 3 concentrations) with 1×PBS containing 0.1% BSA, 50 μL / well, incubate at 37℃ for 1 h.

[0120] Wash plate: Wash 5 times with 1×PBST.

[0121] Secondary antibody incubation: Add 50 μL of Anti-Human IgG (Fc specific)-Peroxidase antibody produced in goat (1:10000 dilution) to each sample well and 2nd well, and add 50 μL of PBS to the blank well. Incubate at 37°C for 40 min.

[0122] Wash plate: Wash 5 times with 1×PBST.

[0123] Color development: Add 100 μL of color development solution (a 1:1 mixture of solution A and solution B, freshly prepared and used immediately) to each well and incubate at room temperature in the dark for 5 min-15 min.

[0124] Termination: Observe the color development and add 50 μL of stop solution to each well. Reading: Measure the OD value of each well at a single wavelength of 450 nm.

[0125] As shown in Figures 3A to 3J, the results show that KH003 can be detected to have a significant binding with hPDL1-His, while no significant binding with other proteins in the B7 family can be detected.

[0126] Example 5 KH003 binding activity with target proteins VEGF, PDL1, and rat PDL1

[0127] In this embodiment, the binding activity of KH003 with human PDL1, rat PDL1 (Rat PD-L1 Protein, His Tag, Sino biological, Cat#: 80450-R08H) and human VEGF molecules was detected by ELISA.

[0128] The experimental method is as follows:

[0129] Coating antigens: PDL1-His, Human VEGF165-His, Rat PD-L1-His protein 50ng / 50μL / well, incubated overnight at 4°C.

[0130] Blocking: Equilibrate the coated plate to room temperature, wash three times with PBS, add 200 μL of blocking buffer (1×PBS containing 2% BSA) to each well, incubate at 37°C for 2 h, wash five times with PBST, and pat dry.

[0131] Add the test sample: Prepare KH003 antibody and control antibody (final concentration: 20 μg / mL, 3.16-fold dilution, 10 concentrations) with PBS containing 0.1% BSA; add 50 μL of antibody to each well, incubate at 37℃ for 1 hour, and wash five times with PBST.

[0132] Add secondary antibody: Add 100 μL of Anti-Human IgG (Fc specific)-Peroxidase antibody produced in goat (1:10000 dilution) to each sample well and 2nd well. Add 100 μL of PBS to the blank wells and incubate at 37°C for 40 min. Wash 5 times with PBST.

[0133] Color development: Add 100 μL of color development solution (a 1:1 mixture of solution A and solution B, freshly prepared and used) to each well, and develop at 37℃ for 15-30 minutes.

[0134] Termination: Add 50 μL of termination solution per well.

[0135] Reading: The OD value of each well was measured at a single wavelength of 450 nm.

[0136] The results showed that KH003 could bind to human PDL1-His and had a similar affinity to Atezolizumab; KH003 could bind to Human VEGF165-His protein, and its binding ability was stronger than that of Bevacizumab; KH003 did not bind to rat PD-L1 protein. The results are shown in Figures 4A to 4C and Table 5.

[0137] Table 5. Detection results of KH003 binding to VEGF165-His, PDL1-His, and Rat PDL1-His proteins

[0138]

[0139] Example 6 Binding activity of KH003 with PDL1 molecules from different species

[0140] 293T-PDL1 and 293T-cyno-PDL1 cells can express PDL1 molecules derived from humans and cynomolgus monkeys on their cell surfaces, respectively. This experiment used 293T-PDL1 and 293T-cyno-PDL1 cells and employed flow cytometry to detect the binding activity of KH003 cells to PDL1 molecules from different species on the cell surface. The experimental methods are as follows:

[0141] Resuscitate 293T-PDL1 (Kyinno, Cat#: KC-0205; hereinafter the same) and 293T-cyno-PDL1 (Kyinno, Cat#: KC-1001) cells. After two passages, collect cells, centrifuge at 1500 rpm for 5 minutes, and remove the supernatant. After cell counting, resuspend cells in FACS buffer (PBS containing 2% FBS) and adjust the cell density to 2.0 × 10⁶ cells / year.5 Cells / mL, add 50 μL of cell suspension to each well of a U-bottom 96-well plate.

[0142] Antibody preparation: Start with a concentration of 40 μg / mL, then serially dilute 3.16 times to obtain 10 concentrations, and add 50 μL to each well of the prepared cells.

[0143] Mix cells thoroughly and incubate at 4°C for 1 hour. Wash three times with 400 μL of FACS buffer. Add secondary antibody Goat anti-human IgG-PE at a 1:500 ratio and incubate at 4°C for 1 hour. Wash three times with 400 μL of FACS buffer. Resuspend cells in 200 μL of FACS buffer for the final wash. Perform flow cytometry analysis, collecting 2000 cells per well. Analyze EC50.

[0144] The results showed that the human IgG1M Isotype in the control group did not bind to 293T-PDL1 or 293T-cyno-PDL1, while KH003 could bind to PDL1 molecules from both human and cynomolgus monkeys, with similar binding affinities. The results are shown in Figures 5A to 5B and Table 6.

[0145] Table 6. Detection results of KH003 binding to PDL1 molecules from different species

[0146]

[0147]

[0148] Example 7 Binding activity of KH003 and cell surface PDL1

[0149] This experiment used 293T-PDL1 cells overexpressing PDL1, NCI-H292 lung cancer cells (Kyinno, Cat#: KC-0578), HCC827 cells (non-small cell lung cancer cells; Kyinno, Cat#: KC-0347), osteosarcoma cells 143B (Kyinno, Cat#: KC-0287) and HOS (Kyinno, Cat#: KC-0734) to detect the binding activity of KH003 to PDL1 molecules on the surface of overexpressing cells and primary tumor cells using flow cytometry.

[0150] Resuscitate the cells to be tested, passage them twice, collect the cells, centrifuge at 1500 rpm for 5 minutes, and remove the supernatant. After cell counting, resuspend the cells in FACS buffer (1×PBS containing 2% FBS) and adjust the cell density to 2.0 × 10⁶ cells / day. 5Add 90 μL of cell suspension to each well of a U-bottom 96-well plate at cells / mL.

[0151] Antibody preparation: Starting at a concentration of 1 mg / mL, perform 10 serial dilutions (3.16-fold) to obtain 10 concentrations, adding 10 μL to each well of the plated cells. Mix the cells thoroughly and incubate at 4°C for 1 h. Wash three times with 400 μL of FACS buffer. Add the fluorescently labeled secondary antibody APC anti-human IgG Fc Antibody (Biolegend, Cat#: 410712) at a 500-fold dilution and incubate at 4°C for 1 h. Wash the cells three times with 400 μL of FACS buffer. Resuspend the cells in 200 μL of FACS buffer. Detect APC signal by flow cytometry and analyze the EC50 of binding to KH003 and PDL1 molecules on the surface of overexpressing cells and primary tumor cells.

[0152] The results showed that no binding of IgG1 Isotype to 293T-PDL1, NCI-H292, HCC827, 143B, or HOS was detected in the control group. KH003, however, was detected to bind to 293-PDL1 cells overexpressing PDL1, NCI-H292 and HCC827 lung cancer cells, and 143B and HOS osteosarcoma cells, indicating that KH003 specifically binds to tumor cells expressing PDL1. The binding curves of KH003 with various primary cells are shown in Figures 6A to 6E, and the EC50 values ​​are shown in Table 7.

[0153] Table 7. Detection results of the binding of KH003 and cell surface PDL1

[0154]

[0155] Example 8 KH003's blocking activity against PDL1 and PD1 binding

[0156] Human PD1 molecules are overexpressed on the surface of 293T-PD1 cells. The extracellular domain of PDL1 protein can bind to the surface of 293T-PD1 cells (Kyinno, Cat#: KC-0204) via PD1. This experiment used 293T-PD1 cells and employed flow cytometry to detect the effect of KH003 on blocking the binding of PDL1 and PD1. The experimental methods are as follows:

[0157] Resuscitate 293T-PD1 cells. After passage twice, collect cells, centrifuge at 1500 rpm for 5 minutes, and remove the supernatant. After cell counting, resuspend in FACS buffer (1×PBS containing 2% FBS), adjust the cell density to 2.0×10⁵ cells / mL, and add 80 μL of cell suspension to each well of a U-bottom 96-well plate.

[0158] Antibody preparation: Starting concentration 1 mg / mL, serially diluted 3.16 times to obtain 11 concentrations, add 10 μL to each well of the plated cells. Mix the cells thoroughly and incubate at 4°C for 1 h.

[0159] Preparation of blocking protein: PDL1-mFC (Kyinno, Cat#: KP-1008) was prepared as a 10× protein stock solution (10 μg / mL), and 10 μL was added to each well of the seeded cells. The cells were thoroughly mixed and incubated at 4°C for 1 h. The cells were washed three times with 400 μL of FACS buffer. Secondary antibody APC Goat anti-mouse IgG (410712, Biolegend) was added at a 1:500 ratio, and the cells were incubated at 4°C for 1 h. The cells were washed three times with 400 μL of FACS buffer. The final wash was with 200 μL of FACS buffer. EC50 was detected and analyzed by flow cytometry.

[0160] The results showed that the control group Human IgG1M Isotype could not block the binding of PDL1 to PD1 on the surface of 293T-PD1 cells, while KH003 and the control antibody Durvalumab could block the binding of PDL1 molecules to PD1 on the surface of 293T-PD1 cells, and the blocking effect of KH003 was slightly stronger than that of the control antibody Durvalumab. The results are shown in Figure 7 and Table 8.

[0161] Table 8. Results of KH003's blocking activity against PDL1 and PD1 binding

[0162]

[0163] Example 9 KH003's inhibitory activity against PDL1 reporter cell lines

[0164] Jurkat NFAT-Luc2-PD1 reporter cells (Kyinno, Cat#: KC-1503) overexpress PD1 molecules, and CD3 signaling activation stimulates luciferase expression. 293T OS8-PDL1 cells (Kyinno, Cat#: KC-1148) overexpress PDL1 and express the CD3 antibody protein OS8 on their cell surface. Co-expression of Jurkat NFAT-Luc2-PD1 reporter cells and 293T OS8-PDL1 cells activates CD3 signaling through OS8 binding to CD3 molecules on the Jurkat surface, while PDL1 and PD1 binding activate Jurkat's PD1 signaling. This experiment used Jurkat NFAT-Luc2-PD1 reporter cells and 293T OS8-PDL1 cells, adding different PDL1 antibodies, and measured luciferase activity to compare the degree of CD3 signaling activation, thereby assessing the blocking effect of KH003 and other PDL1 control antibodies on the PD1-PD-L1 signaling pathway. The experimental method is as follows:

[0165] Harvest cells in the logarithmic growth phase and count them using a platelet counter. Ensure cell viability is above 90%. Add 45 μL of 293T-OS8-PDL1 cell suspension to 96-well plates, resulting in 20,000 cells / well. Prepare antibody solutions at a concentration of 20 μg / mL, with 9 concentrations and 5-fold dilutions; add 10 μL of antibody solution to each well of the cell-seeded 96-well plate, with 3 replicates for each antibody solution concentration. Incubate the cells in the 96-well plates at 37°C and 5% CO2 for 30 minutes. Add 45 μL of Jurkat-NFAT-Luc2-PD-1 cell suspension to 96-well plates, resulting in 20,000 cells / well. Cells in 96-well plates with the drug added were cultured at 37°C and 5% CO2 for 6 hours. Luciferase activity in the cells was detected using the Bright-Lite Luciferase Assay System (Novozymes, Cat#: DD1204).

[0166] The results showed that the test antibody KH003 blocked the PD1-PD-L1 signaling pathway and activated CD3 signaling in T cells. Its activity in blocking the PD1-PD-L1 signaling pathway was similar to that of the control antibody Atezolizumab. The results are shown in Figure 8 and Table 9.

[0167] Table 9. Results of KH003's inhibitory effect on PDL1 reporter cell lines

[0168] Antibody EC 50(μg / mL)TopH14A7M0.0756.805Atezolizumab0.1126.878H48B10-6V1-hIgG1M0.1476.201KH0030.1006.483Human IgG1M IsotypeN / AN / A surface

[0169] Example 10 KH003's inhibitory activity against VEGF reporter cell line function

[0170] 293T-NFAT-Luc2-KDR cells (Kyinno, Cat#: KC-1840) express human VEGFR molecules on their surface. When VEGF binds to VEGFR on the cell surface, it activates downstream VEGFR signaling pathways and the expression of the reporter gene luciferase. Therefore, the activation of VEGFR signaling can be measured by detecting the expression of the reporter gene luciferase. In this experiment, the blocking effect of KH003 on the binding of VEGF and its ligands was detected by co-incubating 293T-NFAT-Luc2-KDR reporter cells with an antibody. The experimental method is as follows:

[0171] Harvest cells in the logarithmic growth phase and count them using a platelet counter. Ensure cell viability is above 90%. Adjust cell concentration; add 80 μL of 293T-OS8-PDL1 cell suspension to a 96-well plate, resulting in 20,000 cells / well. Incubate the cells in the 96-well plate overnight at 37°C and 5% CO2. Prepare a 10×VEGF165 working solution at a concentration of 100 ng / mL. Prepare a 200 μg / mL 10× antibody gradient solution, with an initial detection concentration of 20 μg / mL, for a total of 9 concentration points, serially diluted 3.16-fold. Add 10 μL of the serially diluted antibody solution to each well of the cell-seeded 96-well plate, with 3 replicates for each concentration. Incubate the cells in the drug-treated 96-well plate at 37°C and 5% CO2 for another 30 min. Add 10 μL of VEGF165 solution to the corresponding well of a 96-well plate seeded with cells, and continue culturing at 37°C and 5% CO2 for 6 hours. Detect Luciferase activity in reporter cells using the Bright-Lite Luciferase Assay System (Novozymes, Cat#: DD1204).

[0172] The results showed that the addition of VEGF protein (VEGF165) to reporter cells 293T-NFAT-Luc2-KDR significantly increased luciferase expression. However, the addition of the VEGF antagonists KH003 or Bevacizumab significantly inhibited luciferase expression, indicating that KH003 or Bevacizumab blocked the VEGF-VEGFR signaling pathway. Furthermore, KH003 exhibited significantly stronger blocking activity than the control antibody Bevacizumab. Additionally, H48B10-6V1-hIgG1M failed to block the VEGF-VEGFR signaling pathway in this experiment, suggesting that KH003 blocks VEGF signaling through the C-terminal-linked extracellular region of VEGFR. The results are shown in Figure 9 and Table 10.

[0173] Table 10. Results of KH003's inhibitory effect on VEGF reporter cell lines

[0174] Antibody IC 50 (μg / mL)BottomBevacizumab0.3251.043H48B10-6V1-hIgG1MN / AN / AKH0030.0151.151Human IgG1M IsotypeN / AN / A surface

[0175] Example 11 KH003's blocking activity against VEGF and VEGFR binding

[0176] 293T-NFAT-Luc2-KDR cells express human VEGFR molecules on their surface, and VEGF can bind to VEGFR on the cell surface. This experiment used 293T-NFAT-Luc2-KDR cells and employed flow cytometry to detect whether KH003 could block the binding of VEGF to VEGFR on the cell surface. The experimental method is as follows:

[0177] After counting 293T-NFAT-Luc2-KDR cells, the cells were resuspended in FACS buffer (PBS containing 2% FBS) and the cell density was adjusted to 2.0 × 10⁻⁶ cells / mL. 5 Cells / mL: Add 80 μL of cell suspension to each well of a U-bottom 96-well plate. Prepare antibody solution (1 mg / mL), serially dilute 11 times (3.16-fold), and add 10 μL to each well of the plated cells. Mix the cells thoroughly and incubate at 4°C for 1 h.

[0178] Prepare the blocking protein VEGF165-HIS: Prepare a 10-fold detection concentration (1 μg / mL), adding 10 μL to each well of the seeded cells. Mix the cells thoroughly and incubate at 4°C for 1 h. Wash three times with 400 μL of FACS buffer. Add the secondary antibody Alexa at a 1:500 ratio. Add 647 anti-His tag and incubate at 4°C for 1 hour. Wash three times with 400 μL FACS buffer. Resuspend in 200 μL FACS buffer for the final wash. Analyze by flow cytometry and determine the IC50.

[0179] The results showed that neither the IgG1 Isotype nor the KH003 anti-PDL1 parent antibody H48B10-6V1-hIgG1M had any blocking effect on the binding of VEGF and VEGFR. KH003, Bevacizumab, and IgG1×VEGFR could all block the binding of VEGF and VEGFR, and the blocking effect of KH003 was significantly stronger than that of Bevacizumab. The results are shown in Figure 10 and Table 11.

[0180] Table 11. Results of KH003's blocking activity against VEGF and VEGFR binding

[0181] Antibody IC 50 (μg / mL)KH0031.140Bevacizumab3.230IgG1×VEGFR4.857H48B10-6V1-hIgG1MN / AHuman IgG1M IsotypeN / A surface

[0182] Example 12 Binding activity of KH003 and different Fc receptors

[0183] The Fc terminus of KH003 contains mutations (E233P / L234A / L235A / G236Del / P329A), which prevent it from binding to the Fc receptor (FcR), thus eliminating antibody-dependent cell killing (ADCC), antibody-dependent phagocytosis (ADCP), and antibody-dependent complement killing (CDC) activities. In this study, the binding of KH003 antibodies to different Fc receptors was measured using enzyme-linked immunosorbent assay (ELISA).

[0184] The proteins used include: CD16a-F158-His (Kyinno, Cat#: KP-1218), CD16b-NA1-mFc (Kyinno, Cat#: KP-1222), CD1-H5223 Fc gamma RIIA / CD32a-His Tag (Acro, Cat#: 1823-2182F1-168), CDB-H5228 Fc gamma RIIB / C / CD32b / c-His Tag (Acro, Cat#: 478-203UF1-17V), and FCA-H52H1 Fc gamma RI / CD64-His Tag (Acro, Cat#: RC163P1-20BUF1-131).

[0185] The experimental method is as follows:

[0186] Coating antigens: CD16a-F158-His, CD16b-NA1-mFc, Fc gamma RIIA / CD32a, Fc gamma RIIB / C / CD32b / c protein 50 ng / 50 μL / well, Fc gamma RI / CD64 protein 200 ng / 50 μL / well, incubated overnight at 4°C.

[0187] Washing: Wash 3 times with 1×PBST the next day.

[0188] Blocking: Add 200 μL of blocking solution (1×PBS containing 2% BSA) to each well and incubate at 37°C for 2 h.

[0189] Wash plate: Wash 3 times with 1×PBST.

[0190] Add the test sample and incubate: serially dilute KH003 antibody, Human IgG1 Isotypeκ, and Human IgG4 Isotype with 1×PBS containing 0.1% BSA (starting concentration 100 μg / mL, 10-fold dilution, 6 concentrations, plus one well with a concentration of 0), 50 μL / well, and incubate at 37°C for 1 h.

[0191] Wash plate: Wash 5 times with 1×PBST.

[0192] Secondary antibody incubation: Add 50 μL of Anti-Human IgG (Fc specific)-Peroxidase antibody produced in goat (1:10000 dilution) to each sample well and 2nd well. Add 50 μL of PBS to each blank well. Incubate at 37°C for 40 min. Wash plate: Wash 5 times with 1×PBST.

[0193] Color development: Add 100 μL of color development solution (a 1:1 mixture of solution A and solution B, freshly prepared and used immediately) to each well and incubate at room temperature in the dark for 5 min-15 min.

[0194] Termination: Observe the color development and add 50 μL of stop solution to each well.

[0195] Reading: The OD value of each well was measured at a single wavelength of 450 nm.

[0196] The results showed that, in this experiment, the wild-type IgG1 Isotype and IgG4 Isotype in the control group could be detected binding to FcRI, FcRII, and FcRIII under high concentration conditions; however, no binding to the Fc receptor was detected in KH003. These results demonstrate that the Fc-introduced mutation in KH003 eliminated the binding to the Fc receptor. The results are shown in Figures 11A to 11E and Table 12.

[0197] Table 12. Results of KH003 binding activity assay with different Fc receptors

[0198]

[0199]

[0200] Example 13 KH003-mediated CDC

[0201] This experiment used 293T-PDL1 cells and 293T cells to detect the KH003-mediated CDC effect using FACS. The experimental method is as follows:

[0202] Cells were digested with EDTA cell digestion solution, and digestion was terminated with DMEM medium. An appropriate amount of the suspension was centrifuged at 400g for 5 minutes, and the supernatant was removed. The cells were resuspended in DMEM medium, cell viability was measured, and the cell density was adjusted to ~5.0 × 10⁶ cells / year. 5 / mL, add 80μL of cell suspension to each well of a U-bottom 96-well plate, and set up 3 replicates.

[0203] Prepare a 10× antibody stock solution: 200 μg / mL, serially diluted 3.16-fold to nine concentrations, plus a 0 concentration. Add 10 μL to each well of the plated cells and incubate for 30 minutes. Add 10 μL of Human AB Serum to each well, mix well, and incubate for 3 hours. Transfer the cells to ice and centrifuge at low temperature to remove the supernatant. Resuspend the cells in 100 μL of FACS buffer (PBS containing 2% FBS), add 2 μL of 7-AAD to each well, incubate at room temperature for 10 minutes, and then add another 100 μL of FACS buffer to each well. Analyze by flow cytometry, collecting 5000 cells per well.

[0204] The results showed that both KH003 and MPDL3280A-hIgG1 could mediate CDC, but due to the introduction of mutations (E233P / L234A / L235A / G236Del / P329A) at the Fc terminus of KH003, the CDC effect was significantly weakened, with almost no killing effect on 293T cells. Meanwhile, IgG1 Isotypeκ had no CDC activity. The cell-killing results of CDC mediated by KH003 and the control antibody Atezolizumab (MPDL3280A-hIgG1, without Fc mutation) are shown in Figures 12A to 12B and Table 13.

[0205] Table 13. Results of KH003-mediated CDC-mediated cell killing activity.

[0206]

[0207] Example 14 Non-specific stimulatory activity of KH003 on PBMC-secreted cytokines

[0208] This experiment involved co-culturing PBMCs from different individuals with KH003, and using ELISA to detect the non-specific stimulant effect of KH003 on PBMC cytokine secretion, thus evaluating the potential immunotoxicity of KH003. The experimental methods are as follows:

[0209] Isolate and count PBMC cells; based on the counting results, take a certain volume of PBMC cell suspension and prepare 30 mL of cell suspension at 50,000 cells / 180 μL using RPMI-1640 + 10% FBS + 1% Pen / Strep solution medium; seed PBMCs at 180 μL / well into the corresponding wells of a clear 96-well U-shaped cell culture plate; prepare a 10× antibody solution with a concentration of 100 μg / mL, dilute 10-fold, and create 6 concentration gradients; add the antibody at 20 μL / well to the corresponding wells of the seeded 96-well U-shaped cell culture plate; centrifuge the 96-well U-shaped cell culture plate at 1500 rpm for 5 min to allow cells to aggregate in the center of the wells; continue culturing the treated cell culture plate at 37℃, 5% CO2, and 95% humidity for 72 h; detect the cytokine concentration in the supernatant using ELISA, referring to the instructions of the commercially available kit for specific experimental procedures.

[0210] As shown in Figures 13A to 13C (Donor A, Donor B, Donor C), the results indicate that when the concentration of the test antibody KH003 is 10 μg / mL or below, the concentrations of cytokines secreted by PBMCs are all low and there is no significant dose-dependent relationship with the KH003 antibody concentration. Therefore, KH003 has no non-specific stimulatory effect on PBMC-secreted cytokines. These results demonstrate that KH003 has very low non-specific immunotoxicity.

[0211] Example 15 Effects of KH003 on in vitro cytokine release from human peripheral blood mononuclear cells

[0212] Using human peripheral blood mononuclear cytokines (PBMCs) as the experimental system, Anti hCD28 mAb and Anti hCD3 mAb as positive controls, and sodium chloride injection as the solvent control, the levels of cytokines IL-2, IL-4, IL-6, IL-10, TNF, and IFN-γ in the supernatant were measured by flow cytometry using the CBA method with solid-phase and liquid-phase methods. The effect of KH003 on the release of human peripheral blood mononuclear cytokines was studied in vitro.

[0213] The results showed that, under the experimental conditions, data from the solid-phase method (0.094–188 μg / well) and the liquid-phase method (0.47–940 μg / mL) confirmed that KH003 had no significant effect on the release of IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2.

[0214] Example 16 KH003's inhibitory activity on the growth of osteosarcoma HOS subcutaneous tumors

[0215] This experiment used an immunodeficient mouse model inoculated with a mixture of HOS osteosarcoma and human PBMCs to verify the efficacy of KH003 in inhibiting HOS tumor growth.

[0216] The experimental method is as follows:

[0217] Female B-NDG hIL15 mice (except for the IgG-C group) were subcutaneously inoculated with mixed cells (1×10⁻⁶). 7 HOS + 1.2 × 10 6 10 PBMCs), IgG-C group mice were subcutaneously injected with 1×10 7Three days after inoculation with HOS tumor cells, mice were randomly divided into groups (7 mice per group, 8 mice in the IgG-C group) and intravenously injected with antibodies (antibodies diluted in PBS containing 0.5% FBS). The dosage and cycle of administration were as follows: Group 1: Durvalumab (8.5 mg / kg); Group 2: H48B10-6V1-hIgG1M (8.5 mg / kg); Groups 3, 4, and 5: KH003 (10, 5, or 2 mg / kg); Group 6: IgG1×VEGFR (10 mg / kg); Group 7: IgG (8.5 mg / kg); Group 8 (IgG-C group): IgG (8.5 mg / kg) (IgG: Human IgG1 Isotypeκ); once a week for six weeks. After group administration, tumor size and body weight were measured three times a week, and mouse survival was recorded. At the endpoint (day 46), peripheral blood was collected and tumor detection was performed to assess the proportions of hCD45+CD3+, hCD45+CD3+CD4+ T cells, and hCD45+CD3+CD4- T cells.

[0218] The results showed that: 1) Compared with the control group (hIgG treatment group), treatment with KH003 antibody at 2 mg / kg inhibited the growth of HOS tumors, and the inhibition of HOS tumor growth by KH003 was dose-dependent, with higher doses of KH003 showing stronger tumor-inhibiting effects. 2) The tumor-inhibiting effect of KH003 was significantly stronger than that of anti-PDL1 antibody Durvalumab or H48B10-6V1-hIgG1M alone, or the use of IgG1×VEGFR alone to block VEGF signaling. 3) Compared with the control group (hIgG treatment group) and the groups treated with anti-PDL1 antibody Durvalumab or H48B10-6V1-hIgG1M, there were no significant differences in the proportions of 7-AAD-hCD45+CD3+ cells, 7-AAD-hCD45+CD3+CD4- cells, and 7-AAD-hCD45+CD3+CD4+ cells in the peripheral blood and tumor tissues of mice in the KH003 treatment group. 4) Compared with the control group (hIgG treatment group) and the Durvalumab or H48B10-6V1-hIgG1M administration groups, the low-dose KH003 administration groups (5, 2 mg / kg) had no significant effect on mouse body weight, indicating that KH003 did not produce significant toxicity in mice at the experimental dose of 5 mg / kg; furthermore, this experiment shows that the immunotoxicity of KH003 is less than that of the clinically approved PDL1 antibody Durvalumab. The mouse tumor growth curve is shown in Figure 14.

[0219] Example 17 KH003's inhibitory activity on the growth of osteosarcoma SJSA1 subcutaneous tumors

[0220] This experiment used an immunodeficient mouse model inoculated with a mixture of SJSA1 osteosarcoma and human PBMCs to verify the efficacy of KH003 in inhibiting SJSA1 tumor growth.

[0221] The experimental method is as follows:

[0222] hIL15 mice (except the IgG-C group) were subcutaneously inoculated with mixed cells (1×10⁻⁶). 7 SJSA1+1.2×10 6 PBMC); IgG-C group mice were subcutaneously injected with 1×10 7 SJSA1 tumor cells (osteosarcoma cells) were inoculated. On day 3 after cell inoculation, mice were randomly divided into groups (n=8 per group) and intravenously injected with antibodies (antibodies diluted in PBS containing 0.5% FBS). The dosage and cycle of administration were as follows: Group 1: Durvalumab 8.5 mg / kg; Group 2: H48B10-6V1-hIgG1M (8.5 mg / kg); Groups 3, 4, and 5: KH003 (10, 5, or 2 mg / kg); Group 6: IgG1×VEGFR (10 mg / kg); Group 7: IgG (8.5 mg / kg); Group 8 (IgG-C group): IgG (8.5 mg / kg) (IgG: Human IgG1 Isotypeκ). Administration was once a week for six weeks. After group administration, tumor size and mouse weight were measured three times a week, and mouse survival was recorded. On day 31, peripheral blood was collected, and at the endpoint (day 49), tumor samples were collected to detect the proportions of hCD45+CD3+ cells, hCD45+CD3+CD4+ T cells, and hCD45+CD3+CD4- T cells.

[0223] The results showed that: 1) Compared with the control group (hIgG1 treatment group), treatment with KH003 antibody at 2 mg / kg completely inhibited the growth of SJSA1 tumors. 2) KH003 showed significantly stronger tumor-inhibiting effects than the use of anti-PDL1 antibody Durvalumab or KH003 anti-PDL1 precursor antibody H48B10-6V1-hIgG1M alone, or the blocking of VEGF signaling (IgG1×VEGFR) alone. 3) Compared with the control group (hIgG1 treatment group), there were no significant differences in the proportions of 7-AAD-hCD45+CD3+ cells, 7-AAD-hCD45+CD3+CD4- cells, and 7-AAD-hCD45+CD3+CD4+ cells in the peripheral blood of mice in the KH003 treatment group, indicating that KH003 does not cause T cell depletion. 4) Compared with the control group (hIgG1 treatment group), the low-dose KH003 administration groups (5 or 2 mg / kg) had no significant effect on mouse body weight, indicating that KH003 did not produce significant toxicity in mice at the experimental dose of 5 mg / kg. The mouse tumor growth curve is shown in Figure 15.

[0224] Example 18 KH003's inhibitory activity on the growth of subcutaneous tumors of non-small cell lung cancer HCC827

[0225] This experiment used a humanized mouse model of HCC827 non-small cell lung cancer cell xenograft tumors to verify the efficacy of KH003 in inhibiting the growth of HCC827 tumors. The experimental methods are as follows:

[0226] Female B-NDG hIL15 mice were subcutaneously injected with 1×10 7 HCC827 tumor cells were collected. On day 8, 8 mice were randomly selected as the IgG1-C group, and the remaining mice were each injected intravenously with 1×10⁻⁶ HCC827 tumor cells. 7 Activated PBMCs were collected. On day 14 after tumor cell inoculation, mice were randomly divided into groups (n=8 per group) and intravenously injected with antibodies (antibodies diluted in PBS containing 0.5% FBS). The dosage and cycle of administration were as follows: Group 1: Durvalumab (10 mg / kg); Group 2: H48B10-6V1-hIgG1M (10 mg / kg); Groups 3 and 4: KH003 (10 or 2 mg / kg); Group 5: IgG1×VEGFR (10 mg / kg); Group 6: IgG1 (10 mg / kg); Group 7 (IgG1-C group): IgG1 (10 mg / kg) (IgG1: Human IgG1 Isotypeκ); once a week for four consecutive weeks. After group administration, tumor size and mouse weight were measured three times a week, and mouse survival was recorded. On day 21, peripheral blood was collected to detect the proportions of hCD45+CD3+, hCD45+CD3+CD4+ T, and hCD45+CD3+CD4- T cells.

[0227] The results showed that in this experiment, immunodeficient mice injected with human PBMCs (human peripheral blood mononuclear cells) were used. The proportion of human T cells in the peripheral blood of each group of mice was 5-10%. HCC827 tumor cells were sensitive to human T cells. As T cells grew in the mice, the HCC827 tumors shrank. However, when mice were injected with PDL1 antibody, the rate of tumor shrinkage significantly increased. A KH003 injection dose of 2 mg / kg significantly accelerated the shrinkage of HCC827 tumors. Furthermore, KH003's tumor-inhibiting effect was significantly stronger than that of using the anti-PDL1 antibody Durvalumab or the KH003 anti-PDL1 precursor antibody H48B10-6V1-hIgG1M alone, or simply blocking VEGF signaling. The mouse tumor growth curve is shown in Figure 16.

[0228] Example 19 KH003's inhibitory activity against the growth of subcutaneous tumors of lung cancer NCI-H292

[0229] This experiment used an immunodeficient mouse model inoculated with a mixture of NCI-H292 lung cancer tumors and human PBMCs to verify the efficacy of KH003 in inhibiting the growth of NCI-H292 tumors.

[0230] The experimental method is as follows:

[0231] Female B-NDG hIL15 mice (except for the IgG1-C group) were subcutaneously injected with 1×10 7 NCI-H292 and 1.2×10 6 PBMC mixed cells / mouse (8:1 with matrix gel) (ABW, Cat#: 082706); IgG1-C group mice were subcutaneously inoculated with 1×10 7 Three days after inoculation with NCI-H292 tumor cells, mice were randomly divided into groups (n=8 per group) and intravenously injected with antibodies (antibodies diluted in PBS containing 0.5% FBS). The dosage and cycle of administration were as follows: Group 1: H48B10-6V1-hIgG1M (8.5 mg / kg); Groups 2 and 3: KH003 (10 or 2 mg / kg); Group 4: IgG1×VEGFR (10 mg / kg); Group 5: IgG (8.5 mg / kg); Group 6 (IgG1-C group): IgG (8.5 mg / kg) (IgG: Human IgG1 Isotypeκ); once a week for five consecutive weeks. After group administration, tumor size and mouse weight were measured three times a week, and mouse survival was recorded. At the endpoint (day 46), peripheral blood was collected to detect the proportions of hCD45+CD3+ cells, hCD45+CD3+CD4+ T cells, and hCD45+CD3+CD4- T cells.

[0232] The results showed that: 1) Compared with the control group (hIgG1 treatment group), treatment with KH003 antibody at 2 mg / kg completely inhibited the growth of NCI-H292 subcutaneous tumors. Furthermore, the inhibition of NCI-H292 tumor growth by KH003 was dose-dependent, with a dose of 10 mg / kg showing stronger tumor-inhibiting effects than an injection dose of 2 mg / kg. 2) The tumor-inhibiting effect of KH003 was significantly stronger than that of using KH003 anti-PDL1 parent antibody H48B10-6V1-hIgG1M alone, or using IgG1×VEGFR alone to block VEGF signaling. 3) Compared with the H48B10-6V1-hIgG1M and IgG1×VEGFR treatment groups, the KH003 10mg / kg treatment group showed higher proportions of 7-AAD-hCD45+CD3+ cells, 7-AAD-hCD45+CD3+CD4- cells, and 7-AAD-hCD45+CD3+CD4+ cells in the peripheral blood of mice, indicating that KH003 does not cause T cell exhaustion. 4) Compared with the control group (hIgG1 treatment group), KH003 antibody treatment had no significant effect on mouse body weight, indicating that at a dose of 10mg / kg, KH003 did not produce significant immunotoxicity in mice. The mouse tumor growth curve is shown in Figure 17.

[0233] Example 20 Effects of KH003 on the central nervous system function of SD rats

[0234] The effects of a single intravenous infusion of KH003 on the central nervous system function of SD rats were assessed using a functional combination observation (FOB) method. The experimental methods are as follows:

[0235] Forty rats (20 per sex) were divided into four groups (5 rats per sex per group) according to their body weight. All rats were administered the drug intravenously (excipient control, KH003: 10, 30, 100 mg / kg). The technicians observing all animals were blinded, and the administration volume was 10 mL / kg. Functional combination tests were performed on all animals one day before administration (D-1), and at 10 min, 4 h, 24 h, 72 h, and D8 after administration.

[0236] The results showed that during the experiment, no abnormal behaviors related to drug administration were observed in any of the treatment groups during cage observation, hand-grabbing observation, open environment observation, and irritant response observation. No changes were observed in the number of animals standing, fecal masses, forelimb gripping strength, or body temperature related to the test substance. Under the experimental conditions, a single intravenous infusion of 10, 30, and 100 mg / kg of KH003 into SD rats had no effect on the central nervous system function of the rats.

[0237] Example 21 Effects of KH003 on the cardiovascular and respiratory systems of cynomolgus monkeys

[0238] In a toxicity study involving repeated administration of KH003 to cynomolgus monkeys for 4 weeks and a recovery period of 6 weeks, safety pharmacology parameters (respiration and electrocardiogram) were investigated.

[0239] The results showed that repeated intravenous infusions of KH003 at doses of 6, 20, and 60 mg / kg, twice a week for four consecutive weeks, did not cause any neurological abnormalities or significant effects on the respiratory and cardiovascular systems in the cynomolgus monkeys.

[0240] Example 22 KH003 in the PK of Balb / c mice or SCID mice

[0241] This experiment investigated the pharmacokinetics of KH003 in mice. The experimental methods are as follows:

[0242] Eighteen healthy female mice (BALB / c or SCID mice) were randomly divided into 6 groups of 3 mice each, based on body weight. Groups 1, 2, 3, 4, and 5 were administered the drugs intravenously: Group 1 (BALB / c mice): PBS; Groups 2 (BALB / c mice) and 3 (SCID mice): KH003 10 mg / kg; Group 4 (SCID mice): KH003 3 mg / kg; Group 5 (SCID mice): KH003 1 mg / kg; and Group 6 (SCID mice) was administered KH003 10 mg / kg subcutaneously. Each administration was a single dose, and blood samples were collected via the orbital rim or vein (anticoagulated). Blood samples were collected at 10 min, 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days, and 14 days, and analyzed using ELISA.

[0243] As shown in Figures 18A to 18C and Table 14, the results are as follows:

[0244] 1) KH003 is rapidly cleared in Balb / c mice (t 1 / 2 =1.88 days, CL = 0.233 mL / Day), while the clearance rate was slow in immunodeficient mice (SCID) (t = 1.88 days, CL = 0.233 mL / Day). 1 / 2 =3-4.1 days, CL = 0.11-0.136 mL / Day); 2) After KH003 injection, the volume of distribution in mice, Vd = 0.6-0.67 mL, which is consistent with the volume of mouse plasma, indicating that the antibody is distributed in the blood; 3) After subcutaneous injection of KH003 in SCID mice, the peak plasma concentration was reached after 24 hours, with a bioavailability F = 0.798; 4) After subcutaneous injection of KH003 in SCID mice, it has a longer half-life and a slower clearance rate (t). 1 / 2 =5.25 days, CL = 0.085 mL / Day).

[0245] Table 14. Pharmacokinetic parameters of a single dose of KH003 in BALB / c or SCID mice

[0246]

[0247] Note: NA: Not applicable.

[0248] Example 23 Pharmacokinetics of KH003 after a single intravenous infusion in Sprague-Dawley rats

[0249] This experiment evaluated the pharmacokinetic characteristics of a single intravenous infusion of KH003 in SD rats. The experimental methods are as follows:

[0250] Eighteen SD rats, half male and half female, were randomly divided into three groups (n=3 per sex per group). They were administered KH003 at doses of 6, 20, and 60 mg / kg via tail vein infusion, respectively. The infusion volume was 10 mL / kg, the infusion rate was 2 mL / kg / min, and the infusion time was 5 min. Serum samples were collected from all animals before administration, immediately after administration (±1 min), and at 2 h, 8 h, 24 h (D2), 48 h (D3), 72 h (D4), 96 h (D5), 120 h (D6), 168 h (D8), 240 h (D11), 336 h (D15), and 408 h (D18) for pharmacokinetic analysis. The concentration of KH003 in the serum of SD rats was detected using ELISA.

[0251] The results showed that: 1) AUC in male animals at doses of 6, 20, and 60 mg / kg was: last and AUC INF The CL in male animals was significantly higher than that in female animals, while the CL in male animals was significantly lower than that in female animals; in the 6 mg / kg dose group, the t in male animals was significantly higher than that in female animals. 1 / 2 and C max The MRT in male animals was significantly higher than that in female animals, while the Vz in male animals was significantly lower than that in female animals; the MRT in male animals in the 20 mg / kg dose group was significantly higher than that in female animals. last The concentration of the drug was significantly higher in males than in females, while there were no significant sex differences in the main parameters among the other dosage groups (p>0.05). 2) Blood drug concentration increased with increasing dosage, but the blood drug concentration in males was generally higher than that in females. max and AUC last The effect increases with increasing dosage. 3) The half-life of KH003 in SD rats ranges from 54.45 h to 90.18 h, the mean residence time ranges from 37.38 h to 66.84 h, the clearance rate ranges from 2.44 mL / h / kg to 6.93 mL / h / kg, and the apparent volume of distribution ranges from 255.55 mL / kg to 745.06 mL / kg. Pharmacokinetic parameters after administration to SD rats are detailed in Table 15.

[0252] Table 15. Pharmacokinetic parameters (Mean) of KH003 administered via a single intravenous infusion to SD rats.

[0253]

[0254] Example 24 Pharmacokinetics of KH003 administered via a single intravenous infusion in cynomolgus monkeys

[0255] This experiment evaluated the pharmacokinetic characteristics of a single intravenous infusion of KH003 in cynomolgus monkeys. The experimental method is as follows:

[0256] Eighteen cynomolgus macaques, half male and half female, were randomly divided into three groups of three (3 animals / sex / group). They were administered KH003 intravenously at doses of 2, 6, and 20 mg / kg, respectively, at a volume of 4 mL / kg, an infusion rate of 8 mL / kg / h, and a duration of 30 min. Serum samples were collected from all animals before administration, immediately after administration, and at 2 h, 8 h, 24 h (D2), 48 h (D3), 72 h (D4), 96 h (D5), 120 h (D6), 168 h (D8), 240 h (D11), 336 h (D15), and 408 h (D18) for pharmacokinetic analysis. The concentration of KH003 in the macaque serum was determined using ELISA.

[0257] The results showed that: 1) AUC in male animals at the 2 mg / kg dose group last and AUC INF The concentration of the drug in male animals was significantly higher than that in female animals, while the concentration in female animals was significantly lower than that in female animals. There were no significant sex differences in the main parameters among the other dosage groups (p>0.05). 2) Blood drug concentration increased with increasing dosage, but the blood drug concentration in male animals was slightly higher than that in female animals. max and AUC last The pharmacokinetics of KH003 in cynomolgus monkeys increased substantially proportionally with increasing dosage, exhibiting linear pharmacokinetic characteristics. 3) The half-life of the drug in cynomolgus monkeys ranged from 21.35 to 59.55 h, the mean residence time from 13.31 to 28.33 h, the clearance rate from 3.96 to 7.80 mL / h / kg, and the apparent volume of distribution from 148.60 to 599.16 mL / kg. Detailed pharmacokinetic parameters after administration to cynomolgus monkeys are shown in Table 16.

[0258] Table 16. Pharmacokinetic parameters (Mean) of a single intravenous infusion of KH003 into cynomolgus monkeys.

[0259]

[0260]

[0261] discuss

[0262] KH003 is a bispecific antibody developed by the applicant that targets PDL1 and VEGF molecules. Based on the various activities or functions of KH003 detected in the above examples, it can be concluded that:

[0263] Pharmacology

[0264] In vitro efficacy: (1) KH003 can bind to PDL1-His and VEGF165-His proteins. Its affinity for PDL1-His is similar to that of Atezolizumab, and its affinity for VEGF165-His protein is slightly stronger than that of Bevacizumab. (2) In 293-PDL1 cells overexpressing PDL1, as well as lung cancer cells NCI-H292 and HCC827, osteosarcoma cells 143B and HOS, KH003 can specifically bind to PDL1 on the cell surface. (3) KH003 can bind to PDL1 molecules derived from humans and cynomolgus monkeys, respectively, with similar binding affinity. (4) In 293-PD1 cells overexpressing PD1, KH003 can block the binding of PDL1 molecules to PD1 on the surface of 293T-PD1 cells, and the blocking effect is slightly stronger than that of the control antibody Durvalumab; (5) KH003 can enhance T cell function by blocking the PD1-PDL1 signaling pathway, and its blocking activity of the PD1-PDL1 signaling pathway is similar to that of the control antibody Atezolizumab; (6) KH003 blocks VEGF signaling through the extracellular region of VEGFR linked to the C-terminus of the antibody. In 293T-NFAT-Luc2-KDR cells expressing human VEGFR molecules, KH003 can block the binding of VEGF to VEGFR, and the blocking effect of KH003 is significantly stronger than that of Bevacizumab; (7) KH003 has removed the binding to the Fc receptor due to the introduction of Fc mutation. (8) Both KH003 and Atezolizumab can mediate CDC, but the CDC effect of KH003 is significantly weakened due to Fc mutation; it has almost no killing effect on 293T cells; (9) KH003 has no non-specific stimulatory effect on PBMC secreted cytokines and has no significant effect on cytokine secretion and release. KH003 has very low non-specific immunotoxicity.

[0265] In vivo efficacy: (1) In the HOS and SJSA1 osteosarcoma efficacy model of immunodeficient mice, KH003 was administered intravenously at doses of 2, 5, and 10 mg / kg once a week for six weeks. Compared with the control group (hIgG treatment group), treatment with KH003 antibody at 2 mg / kg could inhibit tumor growth, and the inhibition of tumor growth by KH003 was dose-dependent. The higher the dose of KH003, the stronger the tumor inhibition effect. The tumor inhibition effect of KH003 was significantly stronger than that of anti-PDL1 antibody Durvalumab or KH003 anti-PDL1 parent antibody H48B10-6V1-hIgG1M alone, or blocking VEGF signal (IgG1×VEGFR) alone. The low and medium doses of KH003 (5, 2 mg / kg) did not have a significant effect on the body weight of mice, indicating that KH003 did not produce significant toxicity to mice at the experimental dose of 5 mg / kg. (2) In the HCC827 and NCI-H292 lung cancer tumor efficacy models in immunodeficient mice, KH003 was administered intravenously at doses of 2 and 10 mg / kg once a week. In the HCC827 lung tumor model, continuous administration for four weeks, at a dose of 2 mg / kg, significantly accelerated the shrinkage rate of HCC827 tumors. Furthermore, the tumor-inhibiting effect of KH003 was significantly stronger than that of using the anti-PDL1 antibody Durvalumab or the KH003 anti-PDL1 parent antibody H48B10-6V1-hIgG1M alone, or blocking VEGF signaling alone. In an NCI-H292 lung cancer tumor model, after continuous administration for five weeks, compared with the control group (hIgG treatment group), KH003 antibody treatment at 2 mg / kg completely inhibited the growth of NCI-H292 subcutaneous tumors. Furthermore, the inhibition of NCI-H292 tumor growth by KH003 was dose-dependent, with 10 mg / kg showing stronger tumor-inhibiting effects than the injected dose of 2 mg / kg. The tumor-inhibiting effect of KH003 was significantly stronger than that of KH003 anti-PDL1 parent antibody H48B10-6V1-hIgG1M alone, or the blocking of VEGF signaling (IgG1×VEGFR) alone. In addition, KH003 antibody treatment had no significant effect on mouse body weight, and KH003 at a dose of 10 mg / kg did not produce significant immunotoxicity in mice.

[0266] Safety pharmacology: KH003 has no effect on the central nervous system, cardiovascular system and respiratory system.

[0267] Pharmacokinetics

[0268] PK assays in mice (Balb / c mice or SCID mice) showed that after a single dose of KH003, the volume of distribution (Vd) in mice was 0.6-0.67 mL, which is consistent with the volume of mouse plasma, indicating that the antibody is distributed in the blood. Following intravenous injection of KH003, the clearance rate in Balb / c mice was rapid (t...). 1 / 2=1.88 days, CL = 0.233 mL / Day), while the clearance rate was slow in immunodeficient mice (SCID) (t = 1.88 days, CL = 0.233 mL / Day). 1 / 2 =3-4.1 days, CL = 0.11-0.136 mL / Day).

[0269] Pharmacokinetic studies in SD rats showed that after a single dose of KH003, the blood concentration increased with increasing dose, and the blood concentration in males was generally higher than that in females. max and AUC last The half-life of KH003 in SD rats ranged from 54.45 h to 90.18 h, the mean residence time ranged from 37.38 h to 66.84 h, the clearance rate ranged from 2.44 mL / h / kg to 6.93 mL / h / kg, and the apparent volume of distribution ranged from 255.55 mL / kg to 745.06 mL / kg.

[0270] Pharmacokinetic studies in cynomolgus monkeys showed that after a single dose of KH003, the blood concentration increased with increasing dose, but the blood concentration in males was slightly higher than that in females. max and AUC last The pharmacokinetic profile of KH003 in cynomolgus monkeys is basically proportional to the increase of dosage. The half-life of the drug in cynomolgus monkeys ranges from 21.35 to 59.55 h, the mean residence time ranges from 13.31 to 28.33 h, the clearance rate ranges from 3.96 to 7.80 mL / h / kg, and the apparent volume of distribution ranges from 148.60 to 599.16 mL / kg.

[0271] In summary, KH003 has a clear efficacy and can be used as a broad-spectrum anti-tumor drug, especially in osteosarcoma and lung cancer, where it is expected to have very good anti-tumor effects.

[0272] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. A bispecific antibody or an antigen-binding fragment thereof, said bispecific antibody or antigen-binding fragment comprising: (a) a first binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) capable of binding PD-L1, wherein the VH comprises heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, respectively, and the VL comprises light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively; and (b) a second binding domain comprising a polypeptide capable of binding VEGF.

2. The bispecific antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH may contain the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.3, and the VL may contain the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.

7.

3. The bispecific antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The first binding domain may further include constant regions, such as a heavy chain constant region (CH) and a light chain constant region (CL); preferably, the constant region is a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region (CL); preferably, the first binding domain includes a heavy chain constant region and / or a light chain constant region of human IgG, especially human IgG1 or IgG4; more preferably, the first binding domain includes a heavy chain (HC) and a light chain (LC).

4. The bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The second binding domain is a VEGF-binding polypeptide, the amino terminus of which is fused to the carboxyl terminus of HC in the first binding domain via a linker; preferably, the amino acid sequence of the linker is (GGGGS)n, where n is an integer from 1 to 10, preferably 2 to 5, more preferably 3 to 4; preferably, the VEGF-binding polypeptide is VEGFR or any domain thereof, more preferably containing an extracellular region of VEGFR-1, such as the second domain of the extracellular region; particularly preferably, the VEGF-binding polypeptide contains the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.

6.

5. The bispecific antibody or its antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The bispecific antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.1, and the light chain comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.2; preferably, the bispecific antibody or its antigen-binding fragment comprises two identical heavy chains and two identical light chains.

6. A nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7, or transformed or transfected by the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A composition comprising the bispecific antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, the nucleic acid molecule as described in claim 6, the vector as described in claim 7, or the host cell as described in claim 8.

10. The use of the bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, or the composition of claim 9 in the preparation of a medicament for the prevention or treatment of a disease, said disease including tumors or cancers, such as tumors or cancers that highly express PD-L1 and / or VEGF, or tumors or cancers associated with positive expression of PD-L1 and / or VEGF; preferably, said disease is a solid tumor; more preferably, said disease is non-small cell lung cancer, small cell lung cancer, malignant pleural mesothelioma, thymic carcinoma, thymic epithelial tumor, breast cancer, esophageal cancer, esophageal adenocarcinoma, gastric adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, colonic adenocarcinoma, rectal adenocarcinoma, hepatocellular carcinoma, pancreatic cancer, pancreatic duct adenocarcinoma, biliary tract cancer, head and neck squamous cell carcinoma, salivary gland carcinoma, adenoid cystic carcinoma, renal cell carcinoma, clear cell renal cell carcinoma. Ovarian cancer, endometrial cancer (recurrent / advanced), cervical cancer, fallopian tube cancer, primary peritoneal cancer, tumors of the sexual reproductive organs, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the skin, soft tissue sarcoma, recurrent glioblastoma, gastric cancer, prostate cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, hematologic malignancies, nasopharyngeal carcinoma, laryngeal cancer, uterine fibroids, osteosarcoma, bone cancer, anal cancer, testicular cancer, vaginal cancer. Vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small bowel cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, oral squamous cell carcinoma, urothelial carcinoma, advanced malignant solid tumors.

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