Anti-SIRPα antibodies or antigen-binding fragments thereof and applications

Abstract

Anti-SIRPα antibody or antigen-binding fragment thereof and application. Mice are immunized to obtain anti-human SIRPα monoclonal antibodies, and further obtain chimeric and humanized antibodies. The provided anti-SIRPα antibody or antigen-binding fragment thereof can bind to human SIRPα protein, block the CD47-SIRPα signal pathway, and have immunomodulatory effects such as enhancing phagocytosis. Pharmaceutical compositions and nucleic acid molecules, as well as the use of the anti-SIRPα antibody or antigen-binding fragment thereof in the preparation of drugs for inhibiting or treating diseases, are further provided.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to the biomedical technical field, specifically to anti-SIRPα antibodies or antigen-binding fragments thereof and applications.

[0002] [Background technology] According to AACR estimates, the number of patients newly diagnosed with malignant tumors in the United States in 2018 will reach 1.8 million, and the number of deaths due to cancer will exceed 600,000, ranking second only to heart disease. According to data released by the China National Cancer Center, a total of 3.8 million new cases of cancer were diagnosed in China in 2014, and the number of deaths due to cancer in the same year reached 2.3 million. Uncontrolled cell proliferation is a key factor leading to the formation of malignant tumors, and in the long course of evolution, tumor cells have acquired a series of pathways to escape the host's immune surveillance of tumor cells in order to accelerate their own proliferation and protect themselves from attacks by the body's immune system. According to the cancer immunoediting theory proposed by Schreiber, the relationship between tumor cells and the host immune system can be divided into three different stages: elimination, equilibrium, and escape. Under the "elimination" state, new tumor cells have strong antigenicity and are easily identified and eliminated by the immune system, while tumor cells that survive elimination enter into an "equilibrium" state with the immune system, indicating a survival state with the host's tumor. However, after a certain amount of genetic mutations in tumor cells subjected to the selective pressure of the host immune system accumulate, the equilibrium is broken and they enter a final "evasion" state. At this stage, tumor cells can be identified as normal cells because they generate a series of malignant phenotypes, halt tumor suppressor response mechanisms, and induce normal immune responses. The cell apoptosis signal pathway in tumor cells is also altered, and the apoptosis mechanism of tumor cells induced by immune cells is ineffective. In addition, tumor cells grow rapidly and unrestrained, so the resulting tissue structure creates a microenvironment that inhibits immune cells. In the tumor microenvironment, tumor cells release molecules with immunosuppressive functions, such as VEGF, TGF-β, and IL-10, which inhibit the activation and differentiation of myeloid dendritic cells, thereby inhibiting the adaptive immune system. At the same time, they can induce regulatory T cells (Tregs) that express CTLA-4 in peripheral blood and lymph nodes, which can inhibit other immune cells and cause the immune system to develop immune tolerance to tumors.

[0003] The phagocytosis of tumor associated macrophages (TAMs) in the tumor microenvironment is inhibited because almost all tumor cells highly express CD47 protein on their surface, which can bind to signal regulatory protein α (SIRPα) on the surface of myeloid cells to send a "don't eat me" or "self" signal to the body, thereby inhibiting phagocytosis. CD47, called integrin-associated protein (IAP), is a widely expressed transmembrane glycoprotein that belongs to the immunoglobulin (Ig) superfamily. The molecular weight of CD47 is 50 kD, and its structure contains multiple glycosylated N-terminal IgV variable domains, five highly hydrophobic transmembrane domains, and a short C-terminal cytoplasmic tail, and four alternative splicing forms of the C-terminal cytoplasmic tail determine the expression of CD47 in different tissues. Its counterpart, SIRPα, is called SHPS-1, BIT or CD172a protein, and is a transmembrane protein that is expressed primarily in myeloid cells, including macrophages, myeloid dendritic cells, granulocytes, mast cells and their precursor cells. SIRPα consists of three extracellular Ig-like domains and four cytoplasmic tyrosine residues that are presumed to be phosphorylation sites. When phosphorylated, SIRPα activates downstream signaling pathways by binding to and activating the SH2 domain of SHP-1 / 2 proteins. Since the expression of SHP-1 and SHP-2 proteins is tissue specific, SIRPα is a docking protein that recruits and activates downstream protein phosphatases in response to extracellular stimuli. Oldenborg first reported that mature red blood cells (RBCs) are protected from erythrocyte elimination by binding of CD47 to splenic macrophage SIRPα.Researchers then discovered that RBCs can also bind to monocyte SIRPα and inhibit Fcγ receptor-dependent phagocytosis, which is achieved by dephosphorylation of myosin-IIA, a key molecule in phagocytosis. Clinically, the phenomenon of high CD47 expression has been found in various solid tumors and hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma (NHL), breast cancer, bladder cancer, ovarian cancer, colon cancer, etc., the essence of which is that tumor cells can avoid cytotoxicity by macrophages through the above mechanisms. CD47 also affects other biological processes by binding to other receptors or signaling through its intracellular cytoplasmic region. The interaction of CD47 with thrombospondin-1 (TSP-1) or vascular endothelial growth factor receptor 2 (VEGFR-2) inhibits angiogenesis, thereby limiting tumor growth.

[0004] Many studies have demonstrated that CD47 antibodies or SIRPα-Fc recombinant protein can play a role in different mouse PDX models, but also have good efficacy when used in combination with traditional chemotherapy drugs such as cytarabine, doxorubicin, paclitaxel, cisplatin, or rituximab, alemtuzumab, cetuximab, and trastuzumab. Sockolosky et al. were the first to report that CD47 nanobody and PD-L1 can be used in combination to show good antitumor efficacy in a syngeneic model of melanoma B16F10 cells. Recently, Hu5F9-G4 (malignant solid tumors: NCT02216409, hematological malignancies: NCT02678338, NCT03248479, colon cancer: NCT02953782, B-cell non-Hodgkin lymphoma: NCT02953509), CC-90002 (AML and myelodysplastic syndromes: NCT02641002, advanced solid tumors and hematological malignancies: NCT02367196), SRF231 (advanced solid tumors and Several antibodies acting on the CD47-SIRPα pathway have entered clinical phase I, including TTI-662 (solid tumors: NCT02890368, hematological malignancies and solid tumors: NCT02663518, myeloma and lymphoma: NCT03530683), and ALX148 (advanced solid tumors and lymphomas: NCT03013218).

[0005] Due to the biological function of CD47 itself, it has been found that CD47 therapeutic antibodies and SIRPα-Fc recombinant proteins may have the risk of causing hematologic toxicity or anemia, which has been reported in CD47 gene knockout NOD mice and mouse models treated with CD47 antibodies. Furthermore, it has been reported that endothelial cell CD47 promotes the transendothelial migration of T cells by interacting with SIRPγ via cell adhesion, although SIRPγ is mainly expressed in T cells, not myeloid cells.

[0006] Therefore, SIRPα antibodies are used as a preferred choice to block the CD47-SIRPα signaling pathway. The Weissman research group at Stanford University demonstrated that the combination of humanized SIRPα antibody KWAR23 and rituximab can effectively inhibit the growth of Burkitt lymphoma in SRG mice (Rag2- / -Il2rγ- / -) with human SIRPα gene knock-in, but KWAR23 alone has no obvious effect.

[0007] [Summary of the Invention] [Problem to be solved by the invention] The object of the present invention is to provide an anti-SIRPα antibody or antigen-binding fragment thereof and application thereof, which can bind to human SIRPα protein and block the CD47-SIRPα signal pathway.

[0008] [Means for solving the problem] In order to achieve the above object, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3, the amino acid sequences of which are shown in SEQ ID NOs: 3, 4 and 5, respectively, and the light chain variable region comprising VHCDR1, VHCDR2 and VHCDR3, the amino acid sequences of which are shown in SEQ ID NOs: 3, 4 and 5, respectively. (1) SEQ ID NO:37, 38, 9; (2) SEQ ID NO: 39, 38, 9; (3) SEQ ID NO:7, 40, 9; (4) SEQ ID NO:7, 8, 41; (5) SEQ ID NO:7, 8, 42; (6) SEQ ID NO:7, 8, 43; (7) SEQ ID NO: 37, 38, 41; (8) SEQ ID NO: 44, 38, 41; (9) Contains VLCDR1, VLCDR2 and VLCDR3 as shown in any one of SEQ ID NOs:7, 8 and 9.

[0009] The sequences of VLCDRs can be seen in the following table.

[0010] [Table 1] TIFF0007663265000002.tif172170

[0011] Optionally, the variable region further comprises murine or human derived FR regions. Optionally, the sequences of the FR regions are of murine origin, the sequence of the heavy chain variable region is as shown in SEQ ID NO:2 or has at least 85% sequence identity thereto, and the sequence of the light chain variable region is as shown in SEQ ID NO:6 or has at least 85% sequence identity thereto.

[0012] Optionally, the human-derived FR region comprises a heavy chain FR region sequence, which is derived from the combined sequence of human germline heavy chains IGHV1-18 and IGHJ2*01, comprising the FR1, FR2, FR3 regions of human germline heavy chain IGHV1-18 and the FR4 region of IGHJ2*01.

[0013] Optionally, the human-derived FR region comprises a light chain FR region sequence, which is derived from the combined sequence of human germline light chains IGKV4-1 and IGKJ2*01, comprising the FR1, FR2, FR3 regions of human germline light chain IGKV4-1 and the FR4 region of IGKJ2*01.

[0014] Optionally, the FR region sequence of the heavy chain variable region is derived from human germline, and the sequence of the heavy chain variable region is as set forth in SEQ ID NO:17 or has at least 85% sequence identity thereto.

[0015] Optionally, the FR region sequence of the light chain variable region is derived from human germline, and the sequence of the light chain variable region is selected from any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, or has at least 85% sequence identity thereto.

[0016] Optionally, the anti-SIRPα antibody or antigen-binding fragment thereof further comprises a heavy chain constant region selected from human-derived IgG1, IgG2, IgG3, or IgG4, or a variant thereof, and a light chain constant region selected from human-derived κ chain, λ chain, or a variant thereof.

[0017] Optionally, the heavy chain constant region comprises an Fc fragment or a variant thereof. Optionally, the Fc fragment variant is derived from IgG1 and contains the mutation sites L234A, L235A, K338A according to EU numbering.

[0018] Optionally, the heavy chain sequence of the anti-SIRPα antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:26 or comprises at least 85% sequence identity thereto.

[0019] Optionally, the anti-SIRPα antibody or antigen-binding fragment thereof is a monoclonal antibody, a bispecific antibody, or a multispecific antibody, or the antibody or antigen-binding fragment thereof is used in the preparation of an antibody-drug conjugate.

[0020] Optionally, the structural form of the anti-SIRPα antibody or antigen-binding fragment thereof is Fab, F(ab')2, Fv, or ScFv. The present invention further provides a pharmaceutical composition comprising the above-described anti-SIRPα antibody or antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0021] The present invention further provides a nucleic acid molecule encoding the above-described anti-SIRPα antibody or antigen-binding fragment thereof. The present invention further provides a carrier comprising the above-mentioned nucleic acid molecule.

[0022] The present invention further provides a host cell transformed with the above carrier. The invention further provides the use of the above-described anti-SIRPα antibody, or antigen-binding fragment thereof, in the preparation of a medicament for inhibiting or treating a disease, symptom or condition.

[0023] Optionally, the medicament is prepared by combining the anti-SIRPα antibody or antigen-binding fragment thereof with one or more other cancer therapeutic agents.

[0024] Optionally, the disease, condition or condition comprises cancer, a solid tumor, a chronic infection, an inflammatory disease, multiple sclerosis, an autoimmune disease, a nervous system disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, graft dysfunction or arthritis.

[0025] Optionally, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, digestive cancer, skin cancer, The cancer is selected from carcinoma, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T-cell or B-cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, hepatocellular carcinoma, or adenocarcinoma.

[0026] The present invention further provides the use of the above-mentioned anti-SIRPα antibody or antigen-binding fragment thereof in the preparation of a formulation for blocking the binding of SIRPα to CD47, said formulation comprising a detection agent.

[0027] [Effects of the invention] Compared with the prior art, the present invention has the following beneficial effects: (1) The anti-SIRPα antibody or antigen-binding fragment thereof provided by the present invention can bind to human SIRPα protein and block the CD47-SIRPα signal pathway, and is expected to be used for treating tumors or preparing tumor antibody pharmaceuticals.

[0028] (2) The anti-SIRPα antibody or antigen-binding fragment thereof provided by the present invention can bind to all subtypes of human SIRPα protein, which is beneficial for clinical development. [Brief description of the drawings]

[0029] [Figure 1] 1 shows the detection results of binding ELISA. [Diagram 2] 1 shows the detection results of binding ELISA. [Diagram 3] 1 shows the detection results of binding ELISA. [Figure 4] 1 shows the detection results of binding ELISA. [Diagram 5] 1 shows the detection results of binding ELISA. [Figure 6] 1 shows the detection results of binding ELISA. [Figure 7] These are the detection results of Blocking-ELISA. [Figure 8] This is the result of FACS detection of SIRPα antibody binding to human renal clear cell adenocarcinoma cells 786-O cells, which naturally express human SIRPa. [Figure 9] 1 shows the ADCP results of an in vitro functional experiment of anti-SIRPα antibody. [Figure 10] 1 shows the ADCP results of an in vitro functional experiment of anti-SIRPα antibody. [Figure 11]1 shows the ADCP results of an in vitro functional experiment of anti-SIRPα antibody. [Figure 12] Graph showing tumor growth curves and D18 image signal intensity results of tumor image signal value response in each group. [Figure 13] These are survival curves for each group. [Figure 14] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 15] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 16] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 17] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 18] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 19] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 20] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 21] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 22] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Diagram 23] 1 is a graph showing the results of ELISA detection of the binding of antibody CHO71 of the present invention and control antibodies 18D5 and KWAR23 to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 24] 1 is a comparison of the amino acid sequences of known human SIRP alpha binding domain alleles, with the amino acid sequences of V1-V10 being as set forth in SEQ ID NO:51-SEQ ID NO:60. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and examples. For the experimental methods for which no specific conditions are specified in this study, we usually follow the conventional conditions or the conditions provided by the raw material or product manufacturers. Reagents without specific sources are conventional reagents purchased on the market.

[0031] term: "Antibody" (Ab) refers to an immunoglobulin molecule (immunoglobulin (Ig)) that contains at least one antigen-binding site and is capable of specifically binding to an antigen.

[0032] An "antigen" is a substance that induces an immune response in the body and can bind specifically to an antibody. The binding between an antibody and an antigen is mediated by interactions that include hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds formed between the two. The area on the surface of an antigen that binds to an antibody is an "antigenic determinant" or "epitope," and typically there are multiple determinants on each antigen.

[0033] The term "antibody" referred to in the present invention is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) that contain at least two different antigen-binding domains. Antibodies further include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies of other origins. Antibodies of the present invention can be derived from any animal, including, but not limited to, immunoglobulin molecules of humans, non-human primates, mice, rats, cows, horses, chickens, camels, Llamas, Alpacas, Guanacos, Vicunas, and the like. Antibodies can include additional alterations such as unnatural amino acids, mutations of Fc effector functions, and mutations of glycosylation sites. Antibodies further include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as these antibodies exhibit the desired biological activity.

[0034] The basic structure of an antibody is a Y-shaped monomer consisting of two identical heavy chains (H) and two identical light chains (L) linked by disulfide bonds. Each chain is composed of 2 to 5 domains (called functional regions) of about 110 amino acids that have similar sequences but different functions. The amino acid sequences of the light and heavy chains near the N-terminus of an antibody molecule vary greatly, and the resulting domains are called variable regions (V regions), while the relatively constant region of amino acid sequence near the C-terminus is called constant region (C regions).

[0035] The V regions of the heavy and light chains are called VH and VL, respectively. VH and VL each have three regions with highly variable amino acid composition and sequence, called hypervariable regions (HVRs), which form a spatial structure complementary to the antigenic epitope and are called complementarity determining regions (CDRs). The three CDRs of VH are represented by VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL are represented by VLCDR1, VLCDR2, and VLCDR3, respectively. A total of six CDRs of VH and VL together form an antigen-binding site. The diversity of amino acids in the CDR regions is the molecular basis for the specific binding of antibodies to a large number of different antigens. The composition and sequence of amino acids other than the CDRs of the V regions are relatively less variable, which is called the skeleton region or framework region (FR). VH and VL each have four skeleton regions represented by FR1, FR2, FR3, and FR4. Each VH and VL is composed of three CDRs and four FRs, and the sequences from the amino terminus to the carboxyl terminus are FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0036] According to the amino acid sequence of the antibody heavy chain constant region, human immunoglobulins can be classified into five classes: IgM, IgG, IgA, IgD, and IgE. It can be classified into various subclasses (isotypes), for example, human IgG can be classified into IgG1, IgG2, IgG3, and IgG4, and IgA can be classified into IgA1 and IgA2. It is not yet known that IgM, IgD, and IgE have subclasses. According to the amino acid sequence of the light chain, the light chain can be classified into κ chain and λ chain. The antibody of the present invention can be of any class (e.g., IgM, IgG, IgA, IgD, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).

[0037] The heavy and light chain constant regions are called CH and CL, respectively. The heavy chain constant regions of IgG, IgA, and IgD have three domains, CH1, CH2, and CH3, while the heavy chain constant regions of IgM and IgE have four domains, CH1, CH2, CH3, and CH4.

[0038] Between CH1 and CH2 is the hinge region, which is rich in proline and therefore easily stretched and bent, allowing the distance between the two Y-shaped arms to be changed, making it easier for both arms to bind to the antigen epitope simultaneously.

[0039] "Antigen binding fragment" refers to Fab fragment, F(ab')2 fragment, Fv fragment, ScFv fragment, etc., having antigen binding activity. "Fab fragment" (fragment of antigen binding, Fab) refers to an antibody fragment composed of VL, VH, CL and CH1 domains that binds to a single antigenic epitope (monovalent). Those skilled in the art will understand that papain hydrolyzes IgG to form two identical Fab segments and one Fc segment, and pepsin hydrolyzes IgG to form one F(ab')2 segment and several polypeptide fragments (pFc'). When the disulfide bond between the F(ab')2 heavy chains is cleaved, two Fab' fragments are formed, the latter of which is further enzymatically degraded to form Fv fragments. Fv fragments contain the heavy and light chain variable regions of an antibody, but do not contain the constant region. A single chain variable fragment scFv (single chain antibody fragment) or single chain antibody is composed of the heavy and light chain variable regions of an antibody linked by a linker.

[0040] The terms "Fc", "Fc segment" or "Fc fragment" refer to a fragment crystallizable that does not have antigen-binding activity and is the site where an antibody interacts with an effector molecule or cell surface Fc receptor (FcR). The Fc fragment includes the polypeptides of the constant region of an antibody, excluding the heavy chain constant region CH1. The Fc fragment binds to cells that have the corresponding Fc receptor on their surface, producing various biological effects. In the ADCC effect (antibody-dependent cell-mediated cytotoxicity), the Fab segment of an antibody binds to an antigen epitope on a virus-infected cell or tumor cell, and the Fc segment binds to an FcR on the surface of a killer cell (NK cell, macrophage, etc.), mediating the direct killing of the target cell by the killer cell. ADCP is. It is antibody-dependent cellular phagocytosis, and the mechanism of ADCP is that target cells acted upon by antibodies activate the FcγR mechanism on the macrophage surface, inducing phagocytosis, internalizing the target cells, and acidifying and degrading the phagosome. In some specific cases, it may be beneficial to abolish the Fc function of antibodies. These scenarios include the use of antibodies (1) as receptor agonists to induce cell signaling, (2) as receptor antagonists to block the binding of receptors to ligands and inhibit signaling, or (3) as drug carriers to deliver drugs to target cells expressing the corresponding antigen. If the function of Fc is maintained, antibody drugs may mistakenly damage cells expressing the corresponding receptor, and drugs coupled with antibodies may mistakenly damage important immune cells.

[0041] The Fc variants or combinations of mutations are not limited to the following groups (based on EU numbering):

[0042] [Table 2] TIFF0007663265000004.tif76170

[0043] The CDR amino acid residues of the antibodies or antigen-binding fragments according to the invention follow the known Kabat numbering convention in number and position. Currently, mouse-derived antibodies are the main source of antibody drugs. Mouse antibodies are generally humanized because of their immunogenicity. In the following examples, mouse antibodies, chimeric antibodies, and humanized antibodies are provided. A "chimeric antibody" is an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response induced by mouse antibodies. The constant region of the human antibody can be selected from the heavy chain constant region of human-derived IgG1, IgG2, IgG3, IgG4 or a mutant thereof, as well as the light chain constant region of human-derived κ chain, λ chain or a mutant thereof. A "humanized antibody" refers to an antibody in which the CDR sequence of a mouse antibody is grafted onto the variable region framework of a human antibody, and can overcome the strong reaction caused by chimeric antibodies because it retains a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. To avoid a decrease in activity due to a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal back mutation or reverse mutation to maintain activity.

[0044] Theoretically, improving antibody affinity is helpful to improve antibody specificity and efficacy, which helps to reduce drug dosage and reduce toxicity and side effects. Actual research work has demonstrated that affinity improvement and antibody titer improvement are not always linear, especially in the treatment of solid tumors, but in many cases, this linear relationship is clear. The humanized antibody of the present invention also includes humanized antibodies after further affinity maturation of CDRs by phage display. The theoretical basis of in vitro antibody affinity maturation is to mimic the process of antibody affinity in vivo. High affinity antibodies can be screened by constructing random mutation libraries and mimicking the hypermutation of B cells in vivo.

[0045] The medicament provided by the present invention may contain a "therapeutically effective amount" of the antibody or antigen-binding fragment. The term "therapeutically effective amount" refers to the amount of a therapeutic agent to effectively prevent or alleviate a specific disease, which may vary depending on various factors such as the disease state, age, and weight of the patient, and the ability of the drug to produce the desired effect for each patient.

[0046] "Sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides, and is the degree to which two polynucleotides or two polypeptides have the same bases or amino acids. "Having at least 85% sequence identity" according to the present invention refers to reaching at least 85%, 90%, 95%, 97%, or 99% identity.

[0047] Antibody-Drug Conjugates (ADCs) refer to binding proteins linked to one or more chemical agents, which can optionally be therapeutic or cytotoxic agents. Antibody-drug conjugates can be obtained by linking a cytotoxic small molecule (cytotoxin) and an antibody via a permanent or labile chemical linker. ADCs can selectively and persistently deliver cytotoxic agents to tumors.

[0048] The gene encoding SIRPα is a polymorphic gene, and 10 SIRPα variants are known in the human population. Katsuto Takenaka and colleagues sequenced the SIRP alpha domain encoding IgV in 37 unrelated normal Caucasians, Africans, Chinese, and Japanese individuals from the Human HapMap Genome Project and found 10 different SIRP alpha IgV-encoding alleles (Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells, NATURE IMMUNOLOGY VOLUME 8 NUMBER 12 DECEMBER 2007). The 10 SIRPα variants are SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes, respectively. SIRPalpha is highly polymorphic, but a comparison of the amino acid sequences of known human SIRPalpha alleles by ChiaChi M.Ho et al. indicates that there are only two unique sequences at the binding interface between SIRPalpha and CD47, the alleles V1(a2d1) and V2(a1d1), respectively ("Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein(SIRPalpha)Antagonists That Enhance Antibody-dependent Cellular Phagocytosis,JOURNAL OF BIOLOGICAL CHEMISTRY,VOLUME 290 ·NUMBER 20·MAY 15,2015).

[0049] As shown in Figure 24, amino acid sequence comparison of known human SIRP alpha binding domain alleles revealed only two mutations in the CD47 contact interface, a1d1 and a2d1. The first line of text in Figure 24 is the amino acid sequence of the most prominent human SIRP alpha allele V1 (a2d1), and the second line of text in Figure 1 is the amino acid sequence of the most prominent human SIRP allele V2 (a1d1). Black boxes indicate residues that interact with CD47, and shading indicates residues that differ from the V1 sequence. Sanger sequencing of 2535 individual SIRPα sequences and 510 samples by Janet Sim et al. identifies two SIRPα variants v1 and v2, representing three allelic groups: homozygous v1 / v1, homozygous v2 / v2, and heterozygous v1 / v2. The distribution and frequency of SIRPα v1 and v2 allelic groups are determined in various populations and unrelated subpopulations. Here, the distribution of v1 / v2 heterozygosity in the five supergroups of Europe (EUR), America (AMR), East Asia (EAS), Africa (AFR) and South Asia (SAS) is similar, with a distribution range of 42.0% to 47.2%. The number of v2 / v2 in East Asian populations is significantly higher than the number of v1 / v1, with an occurrence frequency of 42.3% and 13.3%, respectively, while in African, European, American, and South Asian populations, the number of v1 / v1 is higher than the number of v2 / v2, with an occurrence frequency range of 30.3-49.1% and 8.9-24.2%, respectively (for references, see MABS, 2019, VOL. 11, NO. 6, 1036¨C1052, https: / / doi.org / 10.1080 / 19420862.2019.1624123). Aduro Biotech also investigated that the frequency of v2 / v2 homozygosity in East Asian population is 41.3%, and the frequency of v1 / v1 homozygosity is 34.6%, which also proves that 41.3% of people in East Asian population are V2 / V2 homozygosity (for references, see Voets et al. Journal for ImmunoTherapy of Cancer (2019) 7:340).

[0050] Based on the results of SIRPα polymorphism analysis, it is important that anti-SIRPα antibodies can bind to both the SIRPα v1 gene and the SIRPa v2 gene to facilitate clinical development.

[0051] Example 1: Obtaining anti-SIRPα mouse antibody (1) Mouse immunization: Anti-human SIRPα monoclonal antibodies are produced by immunizing mice. Six-week-old female Balb / c white mice are used for the experiment. Breeding environment: SPF grade. After purchasing the mice, they are bred for one week in a laboratory environment with a 12 / 12-hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. Balb / c mice are immunized with recombinant protein QP009(SIRPα) with Freund's complete adjuvant (CFA) 50μg / mouse for two weeks, and then reciprocally immunized with QP009(SIRPα) and Freund's incomplete adjuvant (IFA) or QP009(SIRPα) and aluminum salt Alum+CpG ODN 1826 at 25μg / mouse once a week.

[0052] QP009 (SIRPα) has the amino acid sequence as shown below (SEQ ID NO:1).

[0053] [Table 3] The sequence is referenced from UNIPROT accession number P78324(31-149) (SIRPA-Tyrosine-protein phosphatase non-receptor type substrate 1 precursor-Homo sapiens(Human)-SIRPA gene & protein(uniprot.org)).

[0054] (2) Cell fusion: Mice with high antibody titers in the serum are selected for splenocyte fusion. 72 hours prior to fusion, the selected mice are immunized by intraperitoneal injection. Using an optimized PEG-mediated fusion step, splenic lymphocytes are fused with myeloma Sp2 / 0 cells to obtain hybridoma cells. The fused hybridoma cells are resuspended in HAT complete medium (IMDM medium containing 20% ​​FBS, 1x HAT and 1x OPI) and plated in 96-well cell culture plates (1x10 5 Distribute 100 µl / well of 100 cells / well (150 µl / well) and incubate at 37 °C, 5% CO2. On the 5th day after fusion, add IMDM medium (2x HAT and 1x OPI) with 20% FBS and incubate at 50 µl / well, 37 °C, 5% CO2. On the 7th to 8th day after fusion, depending on the cell growth density, completely replace the medium with HT complete medium (IMDM medium containing 20% ​​FBS, 1x HT and 1x OPI) and incubate at 250 µl / well, 37 °C, 5% CO2.

[0055] (3) Screening of hybridoma cells: According to the cell proliferation density, ELISA detection is performed on the 10th to 14th day after fusion to screen the anti-SIRPα antibody in the hybridoma supernatant. The supernatant of the hybridoma fusion well is taken, and the 96-well plate is subjected to primary screening by ELISA, and it is detected that the anti-SIRPα antibody in the supernatant can block the binding of SIRPα / CD47, that is, it is the positive well of the primary screening. The supernatant of the positive well of the primary screening is taken, and the clones that can bind to QP009 (SIRPα), bind to SIRPα, and block the binding of SIRPα / CD47, and are all positive, that is, it is detected that it is the positive clone well of the anti-SIRPα antibody. The positive clones are expanded and transferred to a 24 / 6-well plate in a timely manner, and it is detected that the binding of the cell culture supernatant to SIRPα and the binding that can block SIRPα / CD47 are all positive clone wells, that is, it is the clone well that is positive for the anti-SIRPα antibody, by ELISA. The positive clones were subjected to limiting dilution 2-3 times to obtain single cell clones, and the positive single cell line was cryopreserved to obtain the single cell clone 71C10.

[0056] (4) Antibody sequences obtained by sequencing hybridoma monoclonal antibodies: Take the hybridoma positive monoclonal cell line 71C10, extract the mRNA, reverse transcribe the mRNA into cDNA, use the cDNA as a template for PCR amplification, select the PCR positive clones and sequence them, analyze the sequences to obtain the sequences of the light and heavy chain variable regions of the monoclonal antibody.

[0057] The heavy chain variable region sequence of 71C10 is SEQ ID NO:2, specifically, as follows:

[0058] [Table 4]

[0059] The light chain variable region sequence of 71C10 is SEQ ID NO:6, specifically:

[0060] [Table 5]

[0061] Example 2: SPR detection of the affinity of anti-SIRPα chimeric antibodies (1) The mouse variable region sequence of the monoclonal cell line 71C10 is fused with the human constant region gene to obtain a chimeric antibody molecule. The antibody light chain uses the kappa light chain constant region CL. At the same time, different antigen sequences are set up to be used for performance testing of the antibody molecule. For the molecular cloning design of the antigen and chimeric antibody, see Table 1 and Table 2.

[0062] [Table 6] TIFF0007663265000009.tif114170

[0063] pQD is the name of the carrier with signal peptide and constant region gene (CH1-FC / CL) fragment, where pQDH is used for linking and expressing the heavy chain variable region with signal peptide and constant region gene (CH1-FC) fragment, and pQDK is used for linking and expressing the light chain variable region with signal peptide and constant region gene (CL) fragment. "H" represents the heavy chain, and "L" represents the light chain. "(IgG4)" represents that the heavy chain uses the constant region of human IgG4. If "(IgG4)" is not marked, the constant region of human-derived IgG1 is used by default. 180122VH represents the heavy chain variable region derived from monoclonal cell line 71C10, and 180122VL represents the light chain variable region derived from monoclonal cell line 71C10.

[0064] Exemplarily, "pQDH-KWAR23-H" represents that the control sequence KWAR23 is fused to the pQDH carrier, where pQDH has a signal peptide and a constant region gene (CH1-FC) fragment, and uses the constant region of human-derived IgG1. "pQDH-180122VH" represents that the heavy chain variable region sequence 180122VH is fused to the pQDH carrier, and uses the constant region of human-derived IgG1. The sequences shown in the above SEQ ID NOs are specifically as follows:

[0065] [Table 7]

[0066] [Table 8]

[0067] [Table 9]

[0068] [Table 10]

[0069] [Table 11]

[0070] [Table 12]

[0071] [Table 13] TIFF0007663265000017.tif91169

[0072] (2) Expression and purification of antigen and chimeric antibody The culture density of 293E cells was (0.2–3) × 10 6 The cells were maintained at a density of (0.5–0.8) × 10 / ml and cultured in a maintenance medium (GIBCO Freestyle 293 expression medium). On the day before transfection, the transfected cells were centrifuged to replace the medium, and the cell density was adjusted to (0.5–0.8) × 10 6 On the day of transfection, the 293E cell density was adjusted to (1–1.5) × 10 6 / ml. Prepare the plasmid and transfection reagent PEI, the amount of plasmid to be transfected is 100μg / 100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI evenly, leave for 15 minutes, and then add the mixture of plasmid and PEI slowly to the 293E cells, and culture them in a shaker at 8% CO2, 120 rpm, and 37℃. On the 5th day after transfection, centrifuge in a horizontal centrifuge at 4700 rpm for 20 minutes to collect the cell supernatant.

[0073] Protein A affinity chromatography purification: use at least 3 CV of equilibration solution through the column, the actual volume is 20 ml, ensure that the pH and conductivity of the solution exiting the final device match the equilibration solution, the flow rate is 1 ml / min, pass the supernatant of the medium after centrifugation through the column, load 40 ml of sample, the flow rate is 0.33 ml / min, use at least 3 CV of equilibration solution through the column, the actual volume is 20 ml, ensure that the pH and conductivity of the solution exiting the final device match the equilibration solution, the flow rate is 0.33 ml / min, pass the eluent through the column, start collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU, stop collecting when UV280 falls to 15 mAU, the flow rate is 1 ml / min. After the sample collection is completed, adjust the PAC-EP to neutral with pH adjustment solution.

[0074] (3) Affinity detection by surface plasmon resonance (SPR) The affinity of SIRPα chimeric antibody QP163164 to human SIRPα V1 (protein number QP094) and human SIRPα V2 (protein number QP096) is measured by Biacore T200 (GE). Table 3 and Table 4 show the detection results of QP163164 and QP026027. The results show that SIRPα chimeric antibody QP163164 has an affinity KD value of 5.27E-10M for binding to human SIRPα V1 and an affinity KD value of 6.78E-10M for binding to human SIRPα V2. The binding affinity of human SIRPα V1 and human SIRPα V2 is significantly better than that of control antibody KWAR23 (QP026027).

[0075] [Table 14]

[0076] The affinity between the chimeric antibodies and cynomolgus SIRPα as measured by biacore is as follows:

[0077] [Table 15]

[0078] Example 3: Humanization of anti-SIRPα hybridoma monoclonal antibody By comparing with the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy and light chain variable region germline genes with high homology to QP163164 are selected as templates, and the CDRs of the mouse antibody are respectively grafted into the corresponding human templates to form the variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Select several important amino acid residues for the combination of back mutations, where the amino acid residues are determined and annotated according to the Kabat numbering system. In the following examples, the heavy chain FR region sequence is derived from the combination sequence of human germline heavy chain IGHV1-18 and IGHJ2*01, which includes the FR1, FR2, FR3 regions of human germline heavy chain IGHV1-18 and the FR4 region of IGHJ2*01. The light chain FR region sequence is derived from the combined sequence of human germline light chains IGKV4-1 and IGKJ2*01, and includes the FR1, FR2, FR3 regions of human germline light chains IGKV4-1 and the FR4 region of IGKJ2*01.

[0079] (1) Molecular cloning of humanized anti-SIRPα antibodies Primer PCR was designed to construct each humanized antibody VH / VK gene fragment, and homologous recombination was performed with the expression carrier pQD (containing the signal peptide and constant region gene (CH1-FC / CL) fragment) to construct the full-length antibody expression carrier VH-CH1-FC-pQD / VK-CL-pQD.

[0080] Using the online software DNAWorks (v3.2.2) (http: / / helixweb.nih.gov / dnaworks / ), design multiple primers to synthesize VH / VK containing the gene fragments required for recombination 5'-30bp signal peptide + VH / VK + 30bp CH1 / CL-3'. According to the instruction manual of Primer STAR GXL DNA polymerase of TaKaRa company, use the multiple primers designed above to obtain VH / VK containing the gene fragments required for recombination by dividing into two-step PCR amplification. Construct and digest the expression carrier pQD, and use several special restriction enzymes such as BsmBI identification sequence and different characteristics of restriction site to design and construct the expression carrier pQD. Digest the carrier with BsmBI, cut and collect the gel and stockpile. Construction of heavy chain expression carrier pQD-VH-CH1-FC and light chain expression carrier pQD-VL-CL: Mix the heavy chain variable region VH gene fragment with the carrier pQD (having signal peptide and heavy chain constant region (CH1-FC) fragment) digested with BsmBI in a ratio of 3:1, mix the light chain variable region VL gene fragment with the carrier pQD (having signal peptide and light chain constant region (CL) fragment) digested with BsmBI in a ratio of 3:1, transfer the mixture to DH5a competent cells respectively, ice bath at 0 ° C for 30 minutes, heat shock at 42 ° C for 90 seconds, add 5 times the amount of LB medium, incubate at 37 ° C for 45 minutes, spread on LB-Amp plate, culture at 37 ° C overnight, select single clones, and send to sequence to obtain each target clone.

[0081] The following table shows the specific information of the humanized design of QP163164. The protein expression number is QP256253. In this table, the antibody light chain uses the kappa light chain constant region CL, and the antibody heavy chain uses the human IgG4 constant region (the specific sequence of the constant region may refer to Example 2). The sequences of the light chain and heavy chain variable region of the humanized design are not limited to the sequences shown in the following table.

[0082] [Table 16]

[0083] [Table 17]

[0084] The heavy chain variable region of QP256253 is encoded by a plasmid numbered QD256. The specific sequence of the heavy chain variable region sequence SEQ ID NO:17 is as follows:

[0085] [Table 18]

[0086] (2) Expression of humanized anti-SIRPα antibody protein The culture density of 293E cells was (0.2–3) × 10 6 The cells were maintained at a density of (0.5–0.8) × 10 / ml and cultured in a maintenance medium (GIBCO Freestyle 293 expression medium). On the day before transfection, the transfected cells were centrifuged to replace the medium, and the cell density was adjusted to (0.5–0.8) × 10 6 On the day of transfection, the 293E cell density was adjusted to (1–1.5) × 10 6 / ml. Prepare the plasmid and transfection reagent PEI, the amount of plasmid to be transfected is 100μg / 100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI evenly, leave for 15 minutes, and then add the mixture of plasmid and PEI slowly to the 293E cells, and culture them in a shaker at 8% CO2, 120 rpm, and 37℃. On the 5th day after transfection, centrifuge in a horizontal centrifuge at 4700 rpm for 20 minutes to collect the cell supernatant.

[0087] (3) Purification of humanized anti-SIRPα antibody protein Protein A affinity chromatography purification: use at least 3 CV of equilibration solution through the column, the actual volume is 20 ml, ensure that the pH and conductivity of the solution exiting the final device match the equilibration solution, the flow rate is 1 ml / min, pass the supernatant of the medium after centrifugation through the column, load 40 ml of sample, the flow rate is 0.33 ml / min, use at least 3 CV of equilibration solution through the column, the actual volume is 20 ml, ensure that the pH and conductivity of the solution exiting the final device match the equilibration solution, the flow rate is 0.33 ml / min, pass the eluent through the column, start collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU, stop collecting when UV280 falls to 15 mAU, the flow rate is 1 ml / min. After the sample collection is completed, adjust the PAC-EP to neutral with pH adjustment solution.

[0088] (4) Identification of humanized SIRPα antibody activity (binding-ELISA) Binding-ELISA experimental method: Coat with QP094 (SIRPαV1-flag-his), QP096 (SIRPαV2-Flag-his), QP100 (cynoSIRPα-flag-his) 0.5μg / ml, 50μl / well, overnight at 4℃. Wash 3 times with PBS, incubate with 3%BSA / PBS 200μl / well at room temperature for 2 hours, wash 3 times with PBST, add antibodies at different concentrations, incubate at room temperature for 1 hour, wash 3 times with PBST, wash 3 times with PBS, incubate with secondary antibody HRP-anti Fab, dilute 1:2500, incubate at room temperature for 1 hour, wash 3 times with PBST, wash 3 times with PBS, develop with TMB, stop with 2M H2SO4, read at 450nm.

[0089] (5) SPR Identification of Humanized SIRPα Antibody Affinity The affinity between the humanized antibody and human SIRPαV1, human SIRPαV2 and cynomolgus monkey SIRPα measured by biacore is as shown in Table 6 below, and the results show that the anti-SIRPα humanized antibody QP256253 has an affinity KD value of 3.36E-10M for binding to human SIRPαV1 and an affinity KD value of 3.19E-10M for binding to human SIRPαV2.

[0090] [Table 19]

[0091] Example 4: Affinity maturation of anti-SIRPα antibody QP163164 (1) Construction of humanized phagemid carrier Humanized QP256253 was synthesized using the wild-type sequence (i.e., the original or disclosed sequence, and the mutant sequence obtained by affinity maturation screening), respectively in scFv mode (VH-3 × GGGGS (SEQ ID NO:64) -VL) on a phagemid carrier. VH, (GGGGS (SEQ ID NO:64) ) 3 linker, the VL is spliced ​​and ligated into the phagemid carrier using the NcoI and NotI restriction sites.

[0092] (2) Construction of a phage display library Using the constructed wild-type scFv as a template, codon-based primers are used, in the primer synthesis process, the codons in the mutation region have 50% wild-type codons and 50% NNK (the reverse primer is MNN), and mutations are introduced into all CDR regions to construct a mutation library. The PCR fragments are digested with NcoI and NotI, ligated into a phagemid carrier, and finally electrotransformed into E. coli TG1. Each codon-based primer creates an independent library.

[0093] (3) Library Panning After the library is rescued and packaged to generate phage particles for panning, biotinylated QP098 (cynoSIRPα (ECD)) antigen and streptavidin magnetic beads are used for liquid phase panning, and each round of selection reduces the antigen concentration compared to the previous round. After three rounds of panning, 250 clones are selected for phage ELISA to detect binding activity, and positive clones are sequenced. After comparing and analyzing the sequenced clones, redundant sequences are removed, and then non-redundant sequences are converted to full-length IG (heavy chain constant region is selected CH1-CH2-CH3 of hIgG4, and light chain constant region is selected kappa light chain CL) for expression in mammalian cells. Full-length IG protein is obtained after affinity purification. The specific sequences are as shown in the following table. In this table, the antibody light chain uses kappa light chain constant region CL, and the antibody heavy chain uses human IgG4 constant region (the specific sequence of the constant region may refer to Example 2).

[0094] [Table 20] TIFF0007663265000025.tif105170

[0095] [Table 21]

[0096] [Table 22]

[0097] [Table 23]

[0098] [Table 24]

[0099] [Table 25]

[0100] [Table 26]

[0101] [Table 27]

[0102] [Table 28]

[0103] The bold and underlined parts above are VLCDR1, VLCDR2, and VLCDR3 of each antibody molecule, respectively, and a specific comparison with the wild-type sequence QP256253 is as follows.

[0104] [Table 29] TIFF0007663265000035.tif187169

[0105] (4) ELISA detection Binding-ELISA experimental method: QP094 (SIRPαV1-flag-his), QP096 (SIRPαV2-Flag-his), QP098 (cynoSIRPα-flag-his), QP100 (cynoSIRPα-flag-his) 0.5μg / ml, 50μl / well, coated overnight at 4℃. Washed 3 times with PBS, incubated with 3%BSA / PBS 200μl / well at room temperature for 2 hours, washed 3 times with PBST, added antibodies at different concentrations, incubated at room temperature for 1 hour, washed 3 times with PBST, washed 3 times with PBS, incubated with secondary antibody HRP-anti Fab, diluted 1:2500, incubated at room temperature for 1 hour, washed 3 times with PBST, washed 3 times with PBS, developed with TMB, stopped with 2M H2SO4, read at 450nm. The EC50 values ​​are as shown in the table below. The table below also shows the detection results of humanized antibody QP256253, chimeric antibody QP163245, and control antibody QP026249. The results are as shown in Figures 1 to 6.

[0106] [Table 30] TIFF0007663265000037.tif128169

[0107] Blocking-ELISA experimental method: coated with QP001.22μg / ml overnight at 4℃, washed with PBS 3 times, blocked with 5% milk 250μl / well, 1:1 mixture of biotin-QP002 0.05μg / ml+Abs 50μg / ml, HRP-streptavidin (1:5000), incubated at 25℃ for 1 hour. The results are shown in Figure 7.

[0108] (5) Affinity detection by surface plasmon resonance (SPR) Some results of the affinity of anti-SIRPα antibodies to human SIRPαV1, human SIRPαV2 and cynomolgus monkey SIRPα measured by biacore are shown in Table 10. From Table 10, it can be seen that anti-SIRPα antibodies QP2561589, QP2561586, QP2561581, QP256279, QP2561770 can all bind to human SIRPαV1 and human SIRPαV2. At the same time, QP2561589, QP2561586, QP256279, QP2561770, QP256253 all bind to different cynomolgus monkey and rhesus monkey SIRPα proteins.

[0109] [Table 31] TIFF0007663265000039.tif23169

[0110] From the above table it can be seen that the affinity of the affinity matured antibody QP2561589, QP2561586, QP256279 proteins for human SIRPα V1 and SIRPα V2 is more than 50-fold higher than that of the control antibody KWAR23 (QP026249).

[0111] Example 5: FACS detection of anti-SIRPα antibodies binding to human renal clear cell adenocarcinoma 786-O cells that naturally express human SIRPα Experimental phase: Harvest 786-O cells 2E5 / well, wash once with PBS, centrifuge at 300g for 3 min, discard supernatant. Blocking: Resuspend in 2% FBS, seed 2E5 / well, 200μl / well into 96-well U-bottom plate, ice bath for 1 h. Centrifuge at 300g for 3 min, discard supernatant. Antibody incubation: Incubate antibody 10μg / ml, dilute 1:3, 100μl / well, ice bath for 1 h. Centrifuge, discard supernatant. Add pre-chilled 200μl / well PBS, centrifuge at 300g for 5 min, discard supernatant, repeat twice. Secondary antibody: PE-anti-human FC (1:200) 50μl / well, ice bath for 30 min. Centrifuge, discard supernatant. Add 200μl / well pre-chilled PBS, centrifuge at 300g for 5 min, discard the supernatant, and repeat 3 times. FACS read mean fluorescence value. The results are shown in Figure 8, SIRPα antibodies QP163245, QP256253, QP256279, QP2561586, QP2561589 all bind to human renal clear cell adenocarcinoma 786-O cells that naturally express human SIRPα, and their binding affinity is better than that of the control antibody QP026249 (KWAR23).

[0112] Example 6: In vitro functional experiment of anti-SIRPα antibody ADCP (1) Anti-SIRPα antibodies were generated in various IgG subtypes, and the molecular cloning design was as follows:

[0113] [Table 32] TIFF0007663265000041.tif227170TIFF0007663265000042.tif99170

[0114] Here, the specific sequence of the QP32700279 target heavy chain (SEQ ID NO:26) is as follows:

[0115] [Table 33]

[0116] The sequence of the light chain variable region of QP32700279 is as shown in SEQ ID NO:18. (2) Anti-SIRPα antibody in vitro functional experiment ADCP Preparation of macrophages: Resuscitating PBMCs and using the EasySep kit TM Isolate monocytes using Human Monocyte Isolation Kit (Stemcell-19359), add human recombinant M-CSF (final concentration is 50ng / mL), mix thoroughly, culture the cells at 37℃ for 6 days to induce macrophages, collect cells, count and stockpile. Label Raji cells with CFSE. Raji cells are diluted at 2×10 6 Resuspend cells / ml and then add 50 μl / well (1 × 10 5 Antibody dilution: Rituximab was diluted to 80μg / ml with complete medium, and the nine gradients were diluted 3-fold. Antibody mixing: In the Combination group, the two diluted antibodies were mixed 1:1, and in the Rituximab group, they were mixed with the same amount of medium. 50μl / well was added to the 96-well plate with the cells placed in advance, and the cells were cultured at 37℃ for 2 hours. FACS detection: Phagocytosis was measured by gating live CFSE+ / CD14+ cells.

[0117] The affinity matured molecule and the control antibody were used in ADCP experiments in synergy with rituximab, and the experimental results showed that the combination of SIRPα antibody and rituximab had a smaller EC50 than rituximab alone, and the ADCP synergistic effect was significantly enhanced. The results are shown in Figures 9, 10, and 11.

[0118] Example 7: Evaluating the inhibitory effect of anti-SIRPα antibodies on Raji-Luc tumor growth in the B-NDG-hSIRPα mouse model QP32700279 To investigate the killing effect of anti-SIRPα antibody on tumors, B-NDG-hSIRPα was used to inoculate Raji-Luc tumor models intravenously to evaluate the inhibitory effects of SIRPα antibody and rituximab on tumor growth. Raji-Luc cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Raji-Luc cells resuspended in PBS were cultured at 5 × 10 5 The cells were inoculated into the tail vein of B-NDG-hSIPRa mice at a concentration of 0.2 mL per cell and a volume of 0.2 mL per mouse. On days 0 and 3 after inoculation, tumor image signal values ​​were measured using a small animal imager, and the average image signal intensity was approximately 1 × 10 6 After reaching P / S, the appropriate animals were selected and grouped according to the tumor image signal value and animal weight, and then equally distributed into four experimental groups, each with 8 animals. Dosing began on the day of grouping, and the specific dosing schedule is shown in the table below.

[0119] [Table 34]

[0120] The grouping and administration day was counted as D0, and at the D18 time point, the tumor growth curves and D18 image signal intensity data of the tumor image signal value response of each group were as shown in FIG. 12 and the table below.

[0121] [Table 35]

[0122] The tumor growth curve results showed that rituximab, QP32700279 and the combination of QP32700279 and rituximab could significantly inhibit the growth of Raji-Luc tumors, with tumor inhibition rates (TGI) of 58.6%, 46.4% and 84.5%, respectively, indicating that the combination group showed stronger antitumor activity than the single-administration group.

[0123] Due to the characteristics of the model, the mice began to show abnormalities and paralysis towards the end of the test, at which point they were euthanized and their survival curves were recorded. When all the mice in the G1 group had died (D25), the survival curves for each group were as shown in Figure 13.

[0124] Survival analysis was performed using the Kaplan-Meier method, and the log rank test was used for comparison between groups, with p<0.05 being considered significant. Compared with the control group, QP32700279 and the combined treatment group (QP32700279+rituximab) can all significantly extend the survival time of Raji-Luc tumor-bearing mice (p=0.0445*, p<0.001**), but the rituximab group cannot effectively extend the survival time of tumor-bearing mice (p=0.23). The results suggest that the combined treatment of QP32700279 and QP32700279+rituximab can effectively inhibit the tumor growth of Raji-Luc tumor-bearing mice, thereby improving the survival of the mice.

[0125] Example 8: ELISA detection of anti-SIRPα antibodies that bind to all subtypes of human SIRPα According to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 sequences reported in known literature ("Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein (SIRP alpha) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis, JOURNAL OF BIOLOGICAL CHEMISTRY, VOLUME 290 · NUMBER 20 · MAY 15, 2015), the above SIRPα C-terminus was fused to the Fc of mouse IgG2a subtype (mouse IgG2a) through gene synthesis to construct eukaryotic expression carrier pQD, and then protein A purification and the 5th day transient supernatant were obtained by 293E transient transfection, and the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 fusion Fc (mouse IgG2a) protein was obtained and an ELISA was performed to detect binding of SIRPα antibodies to all subtypes of SIRPα. The sequences are as follows:

[0126] [Table 36]

[0127] [Table 37]

[0128] [Table 38]

[0129] [Table 39]

[0130] [Table 40]

[0131] [Table 41]

[0132] [Table 42]

[0133] [Table 43]

[0134] [Table 44]

[0135] [Table 45]

[0136] [Table 46]

[0137] SIRPα antibodies detected: The SIRPα antibody QP256279 was stably expressed in CHOS cells, and the CHOS stable expression protein number is CHO71.

[0138] According to the sequence provided by patent WO2017178653, molecular cloning constructs and expresses the purified OSE company anti-SIRPα antibody 18D5 as an experimental control. At the same time, as mentioned above, QP026249 is the anti-SIRPα antibody KWAR23 from 47 company (Forty Seven), hereby referred to as KWAR23.

[0139] ELISA detection of binding of SIRPα antibodies to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 Experimental steps: Coat SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10, 1μg / ml, 60μl / well overnight at 4℃, wash twice with PBST, block with 5% non-fat milk (SANGON BIOTECH), 200μl / well, incubate 1h at room temperature, wash twice with PBST, incubate antibody 10μg / ml, 5x dilution, 10 gradient, 60μ / well, incubate 1h at room temperature, wash 5 times with PBST, secondary antibody incubation: anti-hFab1:10000, 60μl / well, incubate 1h at room temperature, wash 5 times with PBST, development: equilibrate TMB 1h before room temperature, 100μl / well, develop 10min, stop with 2M H2SO4 50ul / well, read at 450nm by microplate reader.

[0140] As shown in Figures 14 to 23, the experimental results show that the SIRPα antibody CHO71 of the present invention binds to all subtypes of SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10. The SIRPa antibody 18D5 from OSE does not bind to SIRPa V2 / V3 / V7 / V8 / V10.

[0141] Although the contents of the present invention have been described in detail through the above preferred embodiments, it should be understood that the present invention is not limited to the above description. Those skilled in the art will be aware of various modifications and changes to the present invention upon reading the above contents. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. 1. An anti-SIRPα antibody or antigen-binding fragment thereof, and a light chain variable region, the heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3, the amino acid sequences of which are as set forth in SEQ ID NOs: 3, 4 and 5, respectively; the light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3, the amino acid sequences of which are as set forth in SEQ ID NOs: 3, 4 and 5, respectively. (1) SEQ ID NO: 37, 38 and 9; (2) SEQ ID NO: 39, 38 and 9; (3) SEQ ID NO: 7, 40 and 9; (4) SEQ ID NO: 7, 8 and 41; (5) SEQ ID NO: 7, 8 and 42; (6) SEQ ID NO: 7, 8 and 43; (7) SEQ ID NO: 37, 38 and 41; (8) SEQ ID NO: 44, 38 and 41; (9) The anti-SIRPα antibody or antigen-binding fragment thereof, characterized in that it is as shown in any one of the sequences of SEQ ID NO: 7, 8, and 9.

2. The variable region further comprises a FR region. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 1.

3. The sequences of the FR regions are of mouse origin, the sequence of the heavy chain variable region is as shown in SEQ ID NO: 2, and the sequence of the light chain variable region is as shown in SEQ ID NO: 6; Alternatively, the sequence of the FR region is derived from a human, and the human-derived FR region comprises a heavy chain FR region sequence and a light chain FR region sequence, the heavy chain FR region sequence being derived from a combined sequence of human germline heavy chain IGHV1-18 and IGHJ2*01, comprising the FR1, FR2, and FR3 regions of human germline heavy chain IGHV1-18 and the FR4 region of IGHJ2*01, and the light chain FR region sequence being derived from a combined sequence of human germline light chain IGKV4-1 and IGKJ2*01, comprising the FR1, FR2, and FR3 regions of human germline light chain IGKV4-1 and the FR4 region of IGKJ2*01. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 2.

4. The heavy chain variable region sequence is as shown in SEQ ID NO: 17, and the light chain variable region sequence is selected from any one of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, and 25. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 2.

5. The anti-SIRPα antibody or antigen-binding fragment thereof is characterized in that it further comprises a heavy chain constant region selected from human-derived IgG1, IgG2, IgG3, or IgG4, and a light chain constant region selected from human-derived κ chain and λ chain. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 1.

6. The heavy chain constant region comprises an Fc fragment derived from IgG1 or a variant thereof, and the mutation sites of the variant based on EU numbering are L234A, L235A, and K338A. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 5.

7. The heavy chain sequence of the anti-SIRPα antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:

26. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 1.

8. The antibody is characterized in that it is a monoclonal antibody, a bispecific antibody, or a multispecific antibody; Alternatively, the structural form is characterized as being Fab, F(ab')2, Fv, or ScFv. The anti-SIRPα antibody or antigen-binding fragment thereof described in claim 1.

9. 1. An antibody-drug conjugate comprising: An antibody-drug conjugate comprising the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. 1. A pharmaceutical composition comprising: The pharmaceutical composition, comprising an anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and one or more pharma- ceutically acceptable carriers, diluents or excipients.

11. 1. A nucleic acid molecule comprising: A nucleic acid molecule encoding the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

12. A vector comprising: A vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising: A host cell characterized in that it is obtained by transformation with a vector according to claim 12.

14. 10. Use of an anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a medicament for inhibiting or treating a disease, condition or condition, comprising: The disease, condition or condition includes cancer, solid tumors, chronic infections, inflammatory diseases, multiple sclerosis, autoimmune diseases, nervous system diseases, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, graft dysfunction or arthritis, and the cancer includes anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer. the cancer being selected from the group consisting of cancers of the spine, brain, cervical, uterine, endometrial, colon, colorectal, rectal, esophageal, digestive, skin, prostate, pituitary, vaginal, thyroid, laryngeal, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T-cell or B-cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma.

15. The drug is used in combination with one or more other drugs.

15. The use according to claim 14.

16. The other drug includes rituximab.

16. The use according to claim 15.

17. Use of an anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a formulation, comprising: The above use, wherein the formulation comprises a detection agent.

Images