Anti-SIRPα antibodies and their applications

Abstract

The present invention provides anti-SIRPα antibodies and antigen-binding fragments thereof that can effectively block the binding of SIRPα and CD47, thereby effectively promoting macrophage phagocytosis of tumor cells and effectively inhibiting tumor growth, and have good pharmaceutical prospects.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention is in the field of biology and relates to anti-SIRPα antibodies.

[0002] 〔background〕 SIRPα is a transmembrane protein expressed on the surface of myeloid cells such as macrophages, monocytes, dendritic cells, and granulocytes, and is a member of the immunoglobulin superfamily (IgSF). SIRPα is composed of an N-terminal extracellular region, a transmembrane region, and a C-terminal intracellular region. The extracellular region contains three IgSF domains (the N-terminus is an Ig-V domain), and the intracellular region contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The primary ligand of SIRPα is CD47, a group of transmembrane glycoproteins that are widely expressed in normal tissues and pathologies. Binding of CD47 to SIRPα on myeloid cells such as macrophages leads to phosphorylation of the ITIM in the SIRPα intracellular region, which in turn recruits and activates the phosphatases SHP-1 and SHP-2, thereby inhibiting macrophage phagocytosis via downstream signaling. Normal tissues express CD47 and release "don't eat me" signals as a self-defense mechanism by inducing CD47-SIRPα signaling.

[0003] CD47 has been found to be overexpressed on almost all tumor cells. Tumor cells escape from immune cell surveillance by sending a "don't eat me" signal through CD47 binding to SIRPα. Clinically, CD47 expression levels are closely associated with poor patient prognosis. In vitro and in vivo studies have also demonstrated that blocking CD47 / SIRPα can promote tumor cell phagocytosis and inhibit tumor growth in animal models. The above indicates that targeting CD47 / SIRPα can be used as a new approach to develop tumor immunotherapy. Considering that CD47 is widely expressed on normal cells or tissues, especially on red blood cells and platelets, targeting CD47 may cause hematological toxicity. Furthermore, in addition to SIRPα, CD47 can also interact with TSP-1 and integrins. CD47 is involved in various signaling pathways, and targeting CD47 may lead to many safety concerns. Therefore, the development of anti-SIRPα antibodies to block the CD47 / SIRPα signaling pathway may be a more effective strategy for cancer treatment.

[0004] 〔overview〕 An object of the present invention is to provide a novel anti-SIRPα antibody that can specifically bind to SIRPα and has great potential for tumor therapy.

[0005] In a first aspect, the present invention provides an anti-SIRPα antibody, or antigen-binding fragment thereof, that binds to SIRPα or a fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three CDRs, VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region comprising three CDRs, VL CDR1, VL CDR2 and VL CDR3; The VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, 13, 23 or 33, the VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, 14, 24 or 34, the VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, 15, 25 or 35, the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, 18, 28 or 38, the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, 19, 29 or 39, and the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, 20, 30 or 40.

[0006] In a preferred embodiment, the antibody comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3, the amino acid sequence of said VH CDR1 is set forth in SEQ ID NO:3; the amino acid sequence of the VH CDR2 is set forth in SEQ ID NO:4; the amino acid sequence of the VH CDR3 is set forth in SEQ ID NO:5; the amino acid sequence of said VL CDR1 is set forth in SEQ ID NO:8; the amino acid sequence of the VL CDR2 is set forth in SEQ ID NO:9; and The amino acid sequence of the VL CDR3 is set forth in SEQ ID NO:10.

[0007] In a preferred embodiment, the antibody comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3, the amino acid sequence of said VH CDR1 is set forth in SEQ ID NO: 13; the amino acid sequence of said VH CDR2 is set forth in SEQ ID NO: 14; the amino acid sequence of the VH CDR3 is set forth in SEQ ID NO: 15; the amino acid sequence of the VL CDR1 is set forth in SEQ ID NO: 18; the amino acid sequence of the VL CDR2 is set forth in SEQ ID NO: 19; and The amino acid sequence of the VL CDR3 is set forth in SEQ ID NO:20.

[0008] In a preferred embodiment, the antibody comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3, the amino acid sequence of said VH CDR1 is set forth in SEQ ID NO:23; the amino acid sequence of the VH CDR2 is set forth in SEQ ID NO:24; the amino acid sequence of the VH CDR3 is set forth in SEQ ID NO:25; the amino acid sequence of the VL CDR1 is set forth in SEQ ID NO:28; the amino acid sequence of the VL CDR2 is set forth in SEQ ID NO:29; and The amino acid sequence of the VL CDR3 is set forth in SEQ ID NO:30.

[0009] In a preferred embodiment, the antibody comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3, the amino acid sequence of said VH CDR1 is set forth in SEQ ID NO:33; the amino acid sequence of the VH CDR2 is set forth in SEQ ID NO:34; the amino acid sequence of the VH CDR3 is set forth in SEQ ID NO:35; the amino acid sequence of the VL CDR1 is set forth in SEQ ID NO:38; the amino acid sequence of the VL CDR2 is set forth in SEQ ID NO:39; and The amino acid sequence of the VL CDR3 is set forth in SEQ ID NO:40.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:1, 11, 21, or 31, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0011] In some embodiments, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 6, 16, 26 or 36, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0012] In certain preferred embodiments, the three CDRs in a heavy chain variable region and / or the three CDRs in a light chain variable region are defined according to the Kabat or Chothia numbering systems.

[0013] In some embodiments, the antibody or antigen-binding fragment disclosed herein may further comprise one or more of a heavy chain constant region, a light chain constant region, and an Fc region. In further preferred embodiments, the light chain constant region is a lambda chain or a kappa chain constant region. In some preferred embodiments, the antibody or antigen-binding fragment thereof is of IgG1, IgG2, IgG3, or IgG4 type.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody, or an antigen-binding fragment thereof.

[0015] In a second aspect, a nucleic acid molecule is provided that comprises nucleotides encoding an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the nucleic acid molecule encodes the heavy chain variable region and / or the light chain variable region of the antibody or antigen-binding fragment thereof.

[0016] In preferred embodiments, the nucleic acid molecule encodes a heavy chain variable region and the corresponding nucleotide sequence is set forth in, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, as set forth in SEQ ID NO: 2, 12, 22, or 32. In other preferred embodiments, the nucleic acid molecule encodes a light chain variable region and the corresponding nucleotide sequence is set forth in, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, as set forth in SEQ ID NO: 7, 17, 27, or 37.

[0017] In a third aspect, the present invention provides a biomaterial which is: (1) a vector, host cell, microorganism, etc., comprising a nucleic acid molecule disclosed herein; or (2) The expression product, suspension, supernatant, etc. of (1) above.

[0018] According to the amino acid sequence of the antibody, a person skilled in the art can easily select and prepare a vector, a host cell or a microorganism containing the coding sequence of the antibody, and know how to culture such a host cell or microorganism to obtain the corresponding expression product, suspension, supernatant, etc., and obtain the corresponding antibody, all of which are conventional techniques in the art.

[0019] In a fourth aspect, there is provided a composition comprising an antibody or antigen-binding fragment thereof disclosed herein, preferably said composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0020] In a fifth aspect, there is provided a method for preparing an antibody or antigen-binding fragment thereof disclosed herein, comprising culturing the host cell as described above to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment.

[0021] In a sixth aspect, there is provided a use of an antibody or antigen-binding fragment thereof, nucleic acid molecule, biomaterial, or composition disclosed herein in the preparation of a medicament for treating a tumor; preferably, the tumor is a CD47-positive tumor; more preferably, the tumor is a hematological or solid tumor, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, renal cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma, and other solid tumors.

[0022] In a seventh aspect, there is provided a use of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a biomaterial or a composition disclosed herein in the preparation of a formulation that blocks the binding of SIRPα to CD47.

[0023] In an eighth aspect, there is provided a use of an antibody or antigen-binding fragment thereof, nucleic acid molecule, biomaterial, or composition disclosed herein in combination with one or more additional cancer therapeutic agents in the preparation of a medicament for treating a tumor; preferably, the tumor is a CD47-positive tumor.

[0024] In some preferred embodiments, the additional cancer therapeutic agent includes, but is not limited to, a chemotherapeutic agent, a radiotherapeutic agent, and a biopolymer drug.More preferably, the biopolymer drug is a monoclonal antibody drug that targets tumor cell surface antigens, including anti-CD20 antibody (e.g., zuberitamab or rituximab), cetuximab, and trastuzumab.

[0025] In a ninth aspect, a method for treating a tumor is provided, wherein the tumor is a CD47 positive tumor; preferably, the tumor is a hematological or solid tumor, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, renal cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma and other solid tumors; said method comprising administering to an individual in need thereof an antibody or antigen-binding fragment thereof, nucleic acid molecule, biomaterial or composition disclosed herein.

[0026] In a tenth aspect, there is provided a method for blocking the binding of SIRPα to CD47 comprising using an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a biomaterial or a composition disclosed herein.

[0027] In an eleventh aspect, a method for treating a tumor with combination therapy is provided, wherein the tumor is a CD47-positive tumor; the method comprises administering to an individual in need an antibody or antigen-binding fragment thereof, nucleic acid molecule, biomaterial or composition disclosed herein, and one or more additional cancer therapeutic agents; preferably, the additional cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, and biopolymer drugs; more preferably, the biopolymer drugs are monoclonal antibody drugs that target tumor cell surface antigens, including anti-CD20 antibodies (e.g., suberitamab or rituximab), cetuximab, and trastuzumab.

[0028] The anti-SIRPα antibodies or antigen-binding fragments disclosed herein can effectively block the binding of SIRPα to CD47, thereby effectively promoting macrophage phagocytosis of tumor cells and effectively inhibiting tumor growth, and are blocking antibodies with good pharmaceutical prospects.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the blocking effect of mouse monoclonal antibodies on the binding of SIRPα to CD47.

[0030] FIG. 2 shows the binding of humanized monoclonal antibodies to SIRPα.

[0031] FIG. 3 shows the blocking effect of humanized monoclonal antibodies on the binding of SIRPα to CD47.

[0032] FIG. 4A shows the blocking effects of antibody #14 and KWAR23 on the binding of SIRPα (V1) to CD47.

[0033] FIG. 4B shows the blocking effect of antibodies #14, 18D5 and 1H9 on the binding of SIRPα (V1) to CD47.

[0034] FIG. 4C shows the blocking effects of antibody #14, KWAR23, 18D5 and 1H9 on the binding of SIRPα (V2) to CD47.

[0035] FIG. 4D shows the blocking effects of antibody #14, KWAR23, 18D5 and 1H9 on the binding of SIRPα (V8) to CD47.

[0036] FIG. 5 shows the binding kinetics of antibody #14 and KWAR23 to recombinant SIRPα protein.

[0037] FIG. 6A shows the promotion of anti-CD20 antibody-induced phagocytosis by antibody #14; FIG. 6B shows the promotion of anti-HER2 antibody-induced phagocytosis by antibody #14.

[0038] FIG. 7 shows the anti-tumor efficacy of antibody #14 in an MC38 tumor model.

[0039] FIG. 8 shows that antibody #14 and anti-CD20 antibody can synergistically inhibit tumor growth.

[0040] Detailed Description The present invention will be described with reference to the following specific examples. The reagents and equipment used in the following procedures are conventional in the art and commercially available unless otherwise specified; the methods used are all conventional in the art, and those skilled in the art can certainly carry out the methods and obtain the corresponding results according to the descriptions in the examples.

[0041] Definition: In the present invention, the term "antibody" refers to an immunoglobulin capable of specifically recognizing and binding to an antigen, and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies or antibody fragments, synthetic antibodies, recombinantly produced antibodies, human antibodies, non-human antibodies (e.g., murine antibodies), humanized antibodies, chimeric antibodies, and intracellular antibodies and antibody fragments. Antibody fragments include, for example, Fab fragments, Fab' fragments, F(ab') 2 The antibodies include, but are not limited to, fragments, Fv fragments, Fd fragments, Fd' fragments, single chain Fv (scFv), single chain Fab (scFab), or antigen-binding fragments of any of the above antibodies. The antibodies provided herein include members of any class (e.g., IgG, IgM, IgD, IgE, IgA, or IgY), any subclass (e.g., IgG1, IgG2, lgG3, IgG4, IgA1, or IgA2) or type (e.g., IgG2a or IgG2b) of immunoglobulins. In preferred embodiments, the antibodies disclosed herein are murine, chimeric, or humanized antibodies.

[0042] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more binding sites of the antibody) and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetic derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, as well as other fragments including modified fragments (e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). Fragments may include, for example, multiple chains linked together by disulfide bonds and / or peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), generates an antibody that immunospecifically binds to an antigen.

[0043] As used herein, a "conventional antibody" refers to an antibody that comprises two heavy chains (which may be designated H and H'), two light chains (which may be designated L and L') and two antigen-binding sites, where each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability (e.g., heavy chains include, but are not limited to, VH, VH-CH1 and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region thereof (e.g., light chains include, but are not limited to, VL and VL-CL chains). Each heavy chain (H or H') is paired with one light chain (L or L', respectively).

[0044] As used herein, a full length antibody is an antibody having two full length heavy chains (e.g., VH-CH1-CH2-CH3) and two full length light chains (VL-CL), as well as a hinge region, e.g., antibodies naturally produced by antibody-secreting B cells, and synthetically produced antibodies having the same domains.

[0045] As used herein, dsFv refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

[0046] As used herein, a Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin or a fragment of the same structure produced synthetically, for example by recombinant methods. The Fab fragment contains an L chain (containing VL and CL) and another chain containing the variable domain of the heavy chain (VH) and one constant domain of the heavy chain (CH1).

[0047] As used herein, an F(ab')2 fragment is an antibody fragment obtained by digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment of the same structure produced synthetically, for example by recombinant methods. An F(ab')2 fragment essentially contains two Fab fragments, in which each heavy chain portion contains several additional amino acids, including a cysteine ​​that forms a disulfide bond linking the two fragments.

[0048] As used herein, a Fab' fragment is a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain).

[0049] As used herein, an ScFv fragment refers to an antibody fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH) covalently linked in any order by a polypeptide linker.

[0050] The term "variable region" refers to the domain of an antibody heavy or light chain that recognizes and specifically binds to an antigen epitope, which contains an amino acid sequence that varies between different antibodies. Each light or heavy chain has a variable region domain, VL or VH, respectively. The variable domain provides antigen specificity and is therefore involved in antigen recognition. Each variable region contains CDRs and framework regions (FRs), and the CDRs are part of the antigen binding site domain.

[0051] CDR region or "complementarity determining region" refers to the region in the antibody variable region that is hypervariable in sequence, forms structurally defined loops, and / or contains antigen contact amino acid residues. CDR is mainly involved in binding of antibody to antigen epitope and determines the specificity of the antibody. In a given amino acid sequence of heavy chain variable region or light chain variable region, the specific amino acid sequence of CDR is determined using any one or combination of many well-known numbering schemes, including, for example, Kabat, Contact, AbM and Chothia. The CDR of the antibody of the present invention can be determined according to any scheme or combination in the art.

[0052] As used herein, "hypervariable region", "complementarity determining region" and "CDR" can be used interchangeably and refer to one of the multiple parts in each variable region that together form the antigen binding site of an antibody. Each variable region domain contains three CDRs, designated CDR1, CDR2 and CDR3. For example, the light chain variable region domain contains three CDRs, designated VL CDR1, VL CDR2 and VL CDR3; the heavy chain variable region domain contains three CDRs, designated H CDR1 (or VH CDR1), H CDR2 (or VH CDR2) and H CDR3 (or VH CDR3). The three CDRs in the variable region are not contiguous along the linear amino acid sequence, but are adjacent in the folded polypeptide. The CDRs are located in the parallel-stranded loops that connect the β-sheets of the variable domain. As described herein, CDRs are known to those of skill in the art and can be identified based on the Kabat or Chotnia numbering by those of skill in the art (e.g., Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, AIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

[0053] As used herein, framework regions (FR) are domains within the variable region domains of antibodies that are located in the β-sheets, and FR regions are more conserved in terms of amino acid sequence compared to the hypervariable regions.

[0054] As used herein, a "constant region" domain is a domain in an antibody's heavy or light chain that contains an amino acid sequence that is more conserved compared to the amino acid sequence of the variable region domain. In a conventional full-length antibody molecule, each light chain has a single light chain constant region (CL) domain, and each heavy chain contains one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3 and CH4. Antibody constant regions can perform effector functions, such as, but not limited to, eliminating antigens, pathogens, and toxins that the antibody specifically binds to by interacting with various cells, biomolecules, and tissues.

[0055] As used herein, a functional region of a VH domain is at least a portion of an intact VH domain that retains at least some of the binding specificity of the intact VH domain (e.g., by retaining one or more CDRs of the intact VH domain), which functional region of a VH domain binds to an antigen alone or in combination with another antibody domain (e.g., a VL domain) or region thereof. An exemplary functional region of a VH domain is a region comprising CDR1, CDR2 and / or CDR3 of the VH domain.

[0056] As used herein, a functional region of a VL domain is at least a portion of an intact VL domain that retains at least some of the binding specificity of the intact VL domain (e.g., by retaining one or more CDRs of the intact VL domain), and which functional region of a VL domain binds to an antigen alone or in combination with another antibody domain (e.g., a VH domain) or region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2 and / or CDR3 of the VL domain.

[0057] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by phosphodiester bonds.

[0058] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular materials or culture medium when prepared by recombinant technology, or may be substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include the isolated nucleic acid molecules that code for the antibody or antigen-binding fragments provided herein.

[0059] Sequence "identity" has a meaning well known in the art, and the percentage of sequence identity between two nucleic acid molecules, peptide molecules, or regions can be calculated using techniques disclosed in the art. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule. (e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0060] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide.The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, the method for determining the amount of polypeptide produced in a host cell.Such methods include, but are not limited to, ELISA, Coomassie blue staining after gel electrophoresis, quantification of polypeptide in cell lysate by Lowry protein assay and Bradford protein assay.

[0061] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. When a host cell divides, the nucleic acid in the vector replicates and is thus amplified. A host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0062] As used herein, a "vector" is a replicable nucleic acid that can express one or more heterologous proteins when the vector is transformed into a suitable host cell. Vectors include those into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, typically by restriction enzyme digestion and ligation. Vectors also include those that contain a nucleic acid encoding a polypeptide. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell to amplify the nucleic acid or to express / present the polypeptide encoded by the nucleic acid. Vectors are usually free, but can be designed to integrate a gene or a part thereof into a chromosome of a genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such delivery means are well known to those skilled in the art.

[0063] As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors may generally be derived from or contain elements of plasmid or viral DNA. Thus, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses, or other vectors that, when introduced into a suitable host cell, cause expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that can replicate in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or that integrate into the genome of a host cell.

[0064] As used herein, "treating" an individual with a disease or condition means that the individual's symptoms are partially or completely alleviated or remain unchanged after treatment. Thus, treatment includes prevention, treatment and / or cure. Prevention refers to the prevention of the underlying disease and / or the prevention of worsening of symptoms or disease progression. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof and any composition provided herein.

[0065] As used herein, "efficacy" means the effect resulting from a particular treatment that alters, typically improves or ameliorates, the symptoms of a disease or condition, or cures the disease or condition.

[0066] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, the amount is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition. Also, as used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition containing a compound that, when administered to a subject, can have a desired prophylactic effect, such as preventing or delaying the occurrence or recurrence of a disease or condition, or reducing the likelihood of the occurrence or recurrence of a disease or condition. A complete prophylactically effective dose is not necessarily achieved by administration of a single dose, but may be achieved after administration of a series of doses. Thus, a prophylactically effective amount can be administered through one or more doses.

[0067] As used herein, the term "individual" refers to a mammal, such as a human.

[0068] Anti-SIRPα Antibodies Disclosed herein The present invention provides an anti-SIRPα antibody with high affinity to human SIRPα protein. The antibody can effectively inhibit the binding of SIRPα to its ligand CD47, thereby blocking downstream signaling to CD47 / SIRPα, promoting phagocytosis of tumor cells by macrophages, and further achieving tumor cell elimination. When binding to SIRPα protein on the cell surface, the anti-SIRPα antibody described herein can cause endocytosis, resulting in reduced expression of SIRPα protein on the cell surface, thereby further reducing CD47 / SIRPα signaling.

[0069] The anti-SIRPα antibodies or antigen-binding fragments thereof disclosed herein include substitutions, insertions or deletions. The anti-SIRPα antibodies disclosed herein include modifications to the light chain variable region, the heavy chain variable region, the light chain or the heavy chain, and the modified amino acid sequence is different from the amino acid sequence from which the antibody is derived. For example, the amino acid sequence derived from the same given protein may be similar to the initial sequence, for example, may have a certain percentage of identity. For example, the amino acid sequence may have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to the initial sequence.

[0070] As used herein, "identity" refers to the percentage of identical bases (or amino acids) in the two compared sequences when the sequences of two peptides or two nucleic acid molecules are aligned. Such alignment and homology percentage or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al., eds., (2007), Current Protocols in Molecular Biology. Preferably, alignment is performed using default parameters. One alignment program is BLAST, using default parameters. In particular, the program is BLASTN or BLASTP.

[0071] In certain embodiments, amino acid modifications, which may be one or more, can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc variant. The Fc variant can comprise a human Fc region sequence that includes an amino acid modification at one or more amino acid positions.

[0072] Suitable "antibodies and antigen-binding fragments thereof" in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, xenogeneic, chimeric, humanized or deimmunized antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single chain antibodies, nanobodies, and epitope-binding fragments of any of the foregoing.

[0073] In some embodiments, the antibodies disclosed herein may be monospecific, bispecific, or multispecific. An anti-SIRPα antibody may be linked to another antibody or antibody fragment to generate a bispecific or multispecific antibody with secondary or higher binding specificities.

[0074] In certain embodiments, the antibody can be further modified to add functional moieties.Suitable moieties for antibody derivatization include, but are not limited to, PEG, dextran, proteins, lipids, therapeutic agents and toxins.Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, ligation to other proteins, etc.

[0075] Tumor Treatment and Use of Antibodies The anti-SIRPα antibodies and antigen-binding fragments thereof and pharmaceutical compositions comprising the same provided herein can be used to diagnose, prognose, treat or inhibit cancer. The present invention relates to a method for treating cancer in a subject by administering to a subject in need thereof an anti-SIRPα antibody or fragment thereof disclosed herein. The therapeutic compounds described herein include, but are not limited to, the antibodies disclosed herein (including variants and derivatives disclosed herein) and nucleic acids or polynucleotides encoding the antibodies disclosed herein (including variants and derivatives disclosed herein).

[0076] The present invention further provides combination therapy comprising the anti-SIRPα antibody disclosed herein in combination with at least one additional therapeutic agent, including, but not limited to, chemotherapeutic agents, radiotherapeutic agents, and biopolymer drugs. In one embodiment, the biopolymer drug is a monoclonal antibody drug that targets a tumor cell surface antigen, including anti-CD20 antibodies (e.g., zuberitamab or rituximab), cetuximab, and trastuzumab.

[0077] The antibodies (and any additional therapeutic agents) disclosed herein may be administered through any suitable means, including, but not limited to, intraperitoneal, intravenous, subcutaneous, intranasal and intramuscular injection. The antibodies and variants thereof or compositions may be administered by any convenient route, for example, by bolus injection or infusion, or by absorption through epithelial or mucocutaneous membranes.

[0078] Exemplary anti-SIRPα antibody sequences herein [Table 1] [Table 2] [Table 3] JPEG0007620807000004.jpg228169 JPEG0007620807000005.jpg225169 JPEG0007620807000006.jpg231169 JPEG0007620807000007.jpg231169 JPEG0007620807000008.jpg20169 [Table 4]

[0079] Example 1: Preparation of mouse anti-SIRPα monoclonal antibody This example describes the preparation of mouse anti-human SIRPα monoclonal antibodies using hybridoma technology. The extracellular domain of SIRPα protein (Uniprot: P78324) was expressed as an immunogen. A human IgG1 Fc tag was fused to the C-terminus of the amino acid sequence (Glu31-Arg370) of the extracellular domain of SIRPα protein. This gene was then cloned into vector V152 (obtained from Huabio) to generate vector V152-SIRPα ECD-Fc. This vector was transiently transfected into 293 cells (obtained from ATCC). After 5 days, the cell culture supernatant was collected and purified with Protein A (manufacturer: Solarbio, catalog number I8090). To prepare mouse anti-human SIRPα monoclonal antibodies, 4-week-old BAL B / c mice (Huabio) were immunized with 100 μg of SIRPα protein. On days 14 and 28 after the first immunization, the immunized mice were re-immunized with 50 μg of SIRPα protein. Serum titers of the immunized mice were measured by ELISA. 96-well plates were coated with SIRPα (1 μg / mL) diluted in PBS (manufacturer: ZSGB-BIO, catalog number ZLI-9062) overnight at 4° C. The plates were blocked with 1% BSA-PBS blocking solution for 1 h at 37° C. and washed with PBST. Serum dilutions from immunized mice were added to the plates and the plates were incubated for 1 h at 37° C. The plates were washed and HRP-labeled goat anti-mouse IgG (manufacturer: Abcam, catalog number Ab205719, 1:10000 dilution) was added. After 0.5 h incubation at 37° C., the plates were washed and TMB solution (manufacturer: InnoReagents, catalog number TMB-S-00) was added. After 5 min of incubation at room temperature in the dark, the reaction was stopped by adding 2N H2SO4. The absorbance at 450 nm was read using a microplate reader. Mice with sufficient titers of anti-SIRPα antibodies were immunized with 50 μg of SIRPα protein as a booster dose on day 42. Mice were sacrificed 3-5 days later.Spleen cells were collected and washed 2-3 times with IMDM basal medium (manufacturer: BasalMedia, catalog number L610KJ) by centrifugation, then mixed with mouse myeloma cells SP2 / 0 (obtained from Huabio) at a 1:1 ratio. PEG (manufacturer: Roche, catalog number 25771700) was added to the mixed cells, gently stirred, and the mixture was left at 37°C for 30 seconds. The fused cells were diluted in IMEM selection medium containing 15% fetal bovine serum (manufacturer: Tianhang Biotechnology, catalog number 11011-8611) and 1×HAT (manufacturer: Sigma, catalog number H262-1VL), and 200 μL / well was added to a 96-well cell culture plate and incubated at 37°C in a 5% CO2 incubator. After 10-14 days, anti-SIRPα antibodies in the supernatant of the hybridoma cells were detected by ELISA. Eleven different hybridoma clones, 10F11-5-6, 1B6-1-1, 27A11-1-8, 14B11-5-4, 31D4-4-5, 7C2-3-8, 30C1-6-4, 4A3-5-1, 6H1-8-6, 9A12-5 and 10F4-15, were identified and subjected to further analysis.

[0080] Example 2: Binding of mouse antibodies to SIRPα The binding ability of mouse antibody to SIRPα protein was detected by ELISA. The specific method is as described in Example 1. Mouse antibody was serially diluted 3-fold with 1 μg / mL PBS to prepare a total of seven concentration gradients. The results are shown in Table 5.

[0081] [Table 5]

[0082] The binding of mouse monoclonal antibodies to membrane SIRPα on human myeloid leukemia monocytes (THP-1 obtained from the Cell Bank of Chinese Academy of Sciences) that endogenously express SIRPα was analyzed by flow cytometry (FACS). Mouse monoclonal antibodies at various concentrations (three-fold serial dilutions from 1 μg / mL, a total of seven concentration gradients) were co-incubated with THP-1 cells at 4°C for 30 min. The cells were washed twice before adding FITC-labeled goat anti-mouse IgG (manufacturer: Jackson, catalog number 115-095-003, 1:1000 dilution), and then incubated in the dark at 4°C for 30 min. After washing twice, the cells were detected by a flow cytometer BD C6. The results are shown in Table 6.

[0083] [Table 6]

[0084] Example 3: Blocking effect of mouse anti-SIRPα antibodies on the binding of SIRPα to CD47 According to the results of Example 2, when combining the binding ability of mouse monoclonal antibodies against SIRPα and their binding ability to THP-1 cells, mAb#4, mAb#11, mAb#13 and mAb#14 are the molecules with the best activity. Therefore, mAb#4, mAb#11, mAb#13 and mAb#14 were selected to determine whether these mouse mAbs can block the binding of SIRPα to CD47. A 96-well plate was coated with CD47-Fc (manufacturer: Acrobiosystems, catalog number CD7-H5256) overnight at 4°C. The plate was washed and blocked with 1% BSA-PBST blocking solution. After 1 hour of incubation at 37°C, the plate was washed and different concentrations of antibodies (from 66.7nM to 3-fold dilutions, a total of 7 concentration gradients) and biotin-labeled SIRPα (manufacturer: Acrobiosystems, catalog number CDA-H82F2) were added to the plate. After 1 hour of incubation at 37°C, the plate was washed and HRP-labeled streptavidin (manufacturer: Abcam, catalog number: ab7403) was added. After 0.5 hour of incubation at 37°C, the plate was washed and TMB solution was added. After 0.5 hour of incubation in the dark at room temperature, the reaction was stopped by adding 2N H2SO4. The absorbance at 450 nm was read by a microplate reader. IgG (manufacturer: GenScript) was used as a control. The results demonstrated that all of these mouse antibodies could inhibit the binding of SIRPα to CD47 to various degrees, as shown in Figure 1.

[0085] Example 4: Humanization of mouse antibodies Based on the results of Examples 2 and 3, mAb#4, mAb#11, mAb#13 and mAb#14 were selected for humanization. The heavy and light chain variable regions of these antibodies were compared to available databases of human IgG gene sequences to identify optimal human germline Ig gene sequence matches. Specifically, the human germline IgG heavy chains selected for mAb#4, mAb#11, mAb#13 and mAb#14 were selected from IGHV1-69-2, IGHV2-69-2, IGHV3-69-2, IGHV4-69-2, IGHV5-69-2, IGHV6-69-2, IGHV7-69-2, IGHV8-69-2, IGHV9-69-2, IGHV10-69-2, IGHV11-69-2, IGHV12-69-2, IGHV13-69-2, IGHV14 ... * 01, IGHV2-26* 01, IGHV7-4-1 * 01 and IGHV4-31 * 02, and the human germline IgG L chains selected for mAb#4, mAb#11, mAb#13 and mAb#14 were IGKV4-1 * 01, IGKV1-9 * 01, IGKV4-1 * 01 and IGKV4-1 * The humanized antibodies were designated #4, #11, #13 and #14. The heavy chain CDRs of mAb#4, #11, #13 and #14 were grafted onto the framework region of a human germline IgG heavy chain, and the light chain CDRs of mAb#4, #11, #13 and #14 were grafted onto the framework region of a human germline IgG light chain. The new humanized molecules were named #4, #11, #13 and #14, respectively. The sequences of the heavy chain variable region, light chain variable region and CDR region of the constructed humanized antibodies are shown in Table 3. The heavy and light chain sequences of the #14 antibody are shown in Table 4. The heavy and light chain constant regions of the #4, #11 and #13 antibodies are identical to the heavy and light chain constant regions of the #14 antibody. The complete amino acid sequences of the heavy and light chains of the #4, #11 and #13 antibodies are known to those skilled in the art based on the information disclosed above.

[0086] The antibodies disclosed herein were expressed in 293 cells (obtained from ATCC) and purified. The coding sequences of the antibody heavy and light chains were cloned into the V152 vector (obtained from Huabio). The V152 vector carrying the coding sequences of the antibody heavy and light chains was transferred into 293 cells using the transfection reagent PEI (manufacturer: Polyscience, catalog number 23966-2). The plasmid DNA and transfection reagent were prepared in a biosafety cabinet. Plasmid DNA and PEI (mass ratio = 0.15:1.75) were mixed uniformly and left for 10 minutes. The mixture was gently added to the 293 cells and mixed gently. After culturing the cells at 37 °C and 5% CO2 for 5 days, the cell culture supernatant was centrifuged at 3000 rpm for 10 minutes. The supernatant was purified by Protein A (manufacturer: Solarbio, catalog number I8090) to obtain an antibody purity of more than 95%.

[0087] Example 5: Binding of humanized antibodies to SIRPα protein Binding of humanized antibodies to SIRPα protein was detected by ELISA. 96-well plates were coated with SIRPα (manufacturer: Acrobiosystems, catalog number SIA-H5225) (1 μg / mL) in PBS overnight at 4° C. Then, the plates were blocked with 1% BSA-PBS blocking solution for 1 h at 37° C. The plates were washed with PBST. Humanized antibodies were diluted to different concentrations (3-fold serial dilutions from 66.7 nM, total 11 concentration gradients) and added to the plates. After 1 h incubation at 37° C., the plates were washed and HRP-labeled goat anti-human IgG (manufacturer: Abcam, catalog number Ab98595, 1:10000 dilution) was added. After 0.5 h incubation at 37° C., the plates were washed and TMB solution was added. After 5 min incubation in the dark at room temperature, the reaction was stopped by adding 2N H2SO4. The absorbance at 450 nm was read using a microplate reader. The results are shown in Figure 2. EC 50 were 1.833 nM, 0.4642 nM, 0.9517 nM and 0.6831 nM, respectively, indicating that all of these humanized molecules had high binding activity to SIRPα.

[0088] Example 6: Blocking effect of humanized anti-SIRPα antibodies on the binding of SIRPα to CD47 The blocking effect of humanized anti-SIRPα antibodies, #4, #11, #13 and #14, on the binding of SIRPα to CD47 was tested as described in Example 3. The antibodies were serially diluted 3-fold from 66.7 nM to prepare a total of 11 concentration gradients. The results are shown in Figure 3. The IC of #4, #11, #13 and #14 50The values ​​were 6.03 nM, 0.2086 nM, 1.5 nM and 0.1315 nM, respectively, indicating that all of these humanized antibodies could effectively block the binding of SIRPα to CD47, and antibodies #11 and #14 had a stronger blocking effect than the other two humanized antibodies.

[0089] KWAR23, 18D5 and 1H9 were used as controls. KWAR23 was prepared according to the sequence disclosed in US2018037652 (SEQ ID NO: 1 and SEQ ID NO: 2), 18D5 was prepared according to the sequence disclosed in US20190127477 (SEQ ID NO: 24 and SEQ ID NO: 31), and 1H9 was prepared according to the sequence disclosed in US20190119396 (SEQ ID NO: 7 and SEQ ID NO: 8). Human SIRPα protein is highly polymorphic in the extracellular domain, and common genotypes include V1, V2 and V8. Therefore, the blocking effect of #14 antibody and the above control antibody on the binding of common SIRPα genotypes to CD47 was further compared (in other examples, SIRPα proteins without specific genotypes are all V1 genotypes).

[0090] The blocking effect of different antibodies on the binding of SIRPα (V1) to CD47 was tested as described in Example 3. Antibodies were serially diluted 2-fold from 83.4 nM to prepare a total of 9 concentration gradients. The results are shown in Figure 4A. The IC of antibody #14 50 is 3.404nM, and the IC 50 The IC value of antibody #14 was 13.54 nM, indicating that antibody #14 has a stronger blocking effect on the binding of SIRPα (V1) to CD47 than KWAR23. In FIG. 4B, the IC values ​​of antibody #14, 18D5, and 1H9 were 50 were 4.4 nM, 4.1 nM and 4.2 nM, respectively, indicating that the blocking activities of antibody #14, 18D5 and 1H9 against the binding of SIRPα (V1) to CD47 are similar.

[0091] The blocking effect of different antibodies on the binding of SIRPα(V2) to CD47 was determined by the method described in Example 3, where the biotin-labeled SIRPα used was SIRPα(V2) (manufacturer: Acrobiosystems, catalog number SI2-H82W8). The antibodies were serially diluted 3-fold from 200 nM to prepare a total of nine concentration gradients. The results are shown in Figure 4C. Antibodies #14, KWAR23 and 1H9 can block the binding of SIRPα(V2) to CD47, but 18D5 cannot block the binding of SIRPα(V2) to CD47. The blocking activity of antibody #14 is stronger than that of 1H9. The IC of antibody #14 50 The IC value for KWAR23 is 7.8 nM. 50 The value was 6.4 nM, indicating that antibody #14 and KWAR23 have similar blocking activity against SIRPα (V2).

[0092] The blocking effect of different antibodies on the binding of SIRPα(V8) to CD47 was determined by the method described in Example 3, where the biotin-labeled SIRPα used was SIRPα(V8) (manufacturer: Acrobiosystems, catalog number SI8-H82W6). The antibodies were serially diluted 3-fold from 200 nM to prepare a total of nine concentration gradients. The results are shown in Figure 4D. Antibodies #14, KWAR23 and 1H9 can block the binding of SIRPα(V8) to CD47, but 18D5 cannot block the binding of SIRPα(V8) to CD47. The blocking activity of antibody #14 is stronger than that of 1H9. The IC of antibody #14 50 The IC value for KWAR23 is 11.5 nM. 50 The value was 10.8 nM, indicating that antibody #14 and KWAR23 have similar blocking activity against SIRPα (V8).

[0093] According to the above results, antibody #14 can block the binding of common SIRPα genotypes (V1, V2 and V8) to CD47, and has stronger blocking activity against SIRPα (V1) than KWAR23, and stronger blocking activity against SIRPα (V2) and SIRPα (V8) than 18D5 and 1H9.

[0094] Example 7: Binding kinetics of antibodies to SIRPα Antibody-antigen binding kinetics was determined using an Octet Red96 system (manufacturer: ForteBio) equipped with an H1S1K biosensor (manufacturer: ForteBio). The biosensor was used directly to capture the antigen and immediately immersed in the analyte sample (antibody). The experiment consisted of five steps: 1. baseline (100 s), 2. loading (capture of antigen SIRPα) (500 s), 3. baseline (100 s), 4. association (binding to antibody) (500 s), 5. dissociation (antibody dissociation) (1000 s). After the test was completed, the sensor was regenerated by alternately immersing it in regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS) for 5 s, performing a total of 5 cycles to regenerate the sensor. The operating buffer in this experiment was PBS.

[0095] Sample processing: Antigen SIRPα protein (manufacturer: Acrobiosystems, catalog number SIA-H5225) was diluted to a working concentration of 2.5 μg / mL with working buffer, and analyte samples (#14 antibody and KWAR23) were serially diluted to five working concentrations: 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL and 0.0625 μg / mL. For data analysis, Octet Data Analysis (version 7.0 or latest) was used to calculate response signal values ​​(bound analyte sample signal subtracted by blank analyte sample signal) and the data was fitted using a 1:1 association model.

[0096] A fitting diagram of the binding kinetics is shown in Figure 5, and the association, dissociation and equilibrium constants are shown in Table 7. It can be seen that antibody #14 has very high affinity, comparable to the positive control antibody KWAR23.

[0097] [Table 7]

[0098] Example 8: Promoting effect of anti-SIRPα antibodies disclosed herein on phagocytosis of tumor cells by macrophages PBMC cells (manufacturer: Allcells, catalog number PB004F-C) were cultured in 1640 basal medium (manufacturer: Gibco, catalog number 22400-089) at 37°C and 5% CO2 for 2-3 hours. Non-adherent cells were removed by pipette, and induction medium (80ng / mL M-CSF) was added for induction. Fresh medium containing sufficient cytokines was replaced every 3 days. Cells were cultured for 7 days to obtain macrophages. Raji cells (obtained from Cell Bank of Chinese Academy of Sciences) were fluorescently labeled according to the instructions of CFSE reagent (manufacturer: Abcam, catalog number AB113853). The labeled Raji target cells and the above differentiated macrophages were mixed uniformly at a ratio of 3:1, and anti-CD20 antibody (Zuberitamab, CAS RN: 2251143-19-6, WHO Drug Information, Vol. 33, No. 4, 2019, 914-915; concentration: 0.05 μg / mL, 0.5 μg / mL; manufacturer: BioRay), a combination of anti-CD20 antibody (concentration: 0.05 μg / mL, 0.5 μg / mL) and #14 antibody (concentration: 10 μg / mL), or anti-CD20 antibody (concentration: 0.05 μg / mL, 0.5 μg / mL) and KWAR23 antibody (concentration: 10 μg / mL) were added. The cells were cultured at 37 °C and 5% CO2 for 4 hours. The cells were then washed with PBS, and anti-CD14-APC (manufacturer: BD Biosciences, catalog number 555399) was added. After incubation in the dark at 4°C for 30 minutes, the cells were washed and analyzed by flow cytometry. The phagocytosis rate (ADCP) was calculated according to the following formula: phagocytosis rate (%) = (APC + CFSE) positive cell ratio / APC positive cell ratio × 100%. Figure 6A shows that #14 antibody can enhance the phagocytosis of Raji cells induced by anti-CD20 antibody.

[0099] The promoting effect of the combination of #14 antibody and anti-Her2 antibody on macrophage phagocytosis of Her2-overexpressing SK-BR-3 cells was also investigated. According to the above method, the labeled SK-BR-3 cells (obtained from the Cell Bank of Chinese Academy of Sciences) and differentiated macrophages were mixed equally in a 2:1 ratio, and anti-Her2 antibody (concentration: 0.5 μg / mL; manufacturer: BioRay) or a combination of anti-Her2 antibody (concentration: 0.5 μg / mL) and #14 antibody (concentration: 10 μg / mL) was added. The cells were cultured at 37°C and 5% CO2 for 2 hours. The phagocytosis rate was analyzed and calculated by flow cytometry. Figure 6B shows that #14 antibody can enhance the phagocytosis of SK-BR-3 cells induced by anti-Her2 antibody.

[0100] Example 9: Anti-SIRPα antibodies disclosed herein induce endocytosis of SIRPα on the cell surface. THP-1 cells (obtained from the Cell Bank of Chinese Academy of Sciences) were co-incubated with 10 μg / mL antibody for 30 min at 4°C. The cells were washed twice and divided into two. One was incubated at 37°C for 4 h, and the other was further incubated at 4°C as a control. The cells were washed twice and FITC-labeled goat anti-human IgG (Manufacturer: Abcam, Cat. No. ab97224) was added. After incubation at 4°C for 30 min in the dark, the cells were washed and then analyzed by flow cytometry. The endocytosis rate was calculated from the decrease in cell surface fluorescence at 37°C vs. 4°C. The endocytosis rate was calculated by the following formula: endocytosis rate (%) = (fluorescence intensity at 4°C MFI-fluorescence intensity at 37°C MFI) / fluorescence intensity at 4°C MFI x 100%.

[0101] In Table 8, the endocytosis rate of antibody #14 was 52.9%, while the endocytosis rate of KWAR23 was 34.7%, indicating that antibody #14 is more effective in reducing the expression level of cell surface SIRPα.

[0102] [Table 8]

[0103] Example 10: Tumor growth inhibition induced by anti-SIRPα antibodies disclosed herein The anti-tumor efficacy of antibody #14 was evaluated in a hCD47-MC38 mouse colon cancer model. hCD47-MC38 cells (obtained from Shanghai Model Biology) were inoculated into hSIRPα / hCD47 double-humanized C57BL / 6 mice (obtained from Shanghai Model Biology). Tumor volumes of approximately 150 mm 3 At the stage where tumor growth rate reached 100%, the mice were intraperitoneally injected with antibody #14 (200 μg / mouse) twice a week, and the control group was administered the same amount of PBS. The results are shown in Figure 7. Compared with the control group, antibody #14 can continuously inhibit tumor growth.

[0104] The antitumor efficacy of the combination of antibody #14 with an anti-CD20 antibody (suberitamab, obtained from BioRay) in a lymphoma model was also evaluated. Raji-Luc cells (obtained from Biocytogen) were inoculated into B-NDG-hSIRPα mice (obtained from Biocytogen) via the tail vein. The mean tumor imaging signal intensity was approximately 1 × 10 6 When p / sec was reached, mice were administered the indicated doses of IgG control (obtained from Biocytogen, 200 μg / mouse), #14 antibody alone (200 μg / mouse), anti-CD20 antibody alone (2 μg / mouse), or #14 antibody (200 μg / mouse) + anti-CD20 antibody (2 μg / mouse). As shown in Figure 8, #14 antibody and anti-CD20 antibody have a synergistic anti-tumor effect. [Brief description of the drawings]

[0105] [Figure 1] FIG. 1 shows the blocking effect of mouse monoclonal antibodies on the binding of SIRPα to CD47. [Diagram 2] FIG. 2 shows the binding of humanized monoclonal antibodies to SIRPα. [Diagram 3] FIG. 3 shows the blocking effect of humanized monoclonal antibodies on the binding of SIRPα to CD47. [Figure 4A] FIG. 4A shows the blocking effects of antibody #14 and KWAR23 on the binding of SIRPα (V1) to CD47. [Figure 4B] FIG. 4B shows the blocking effect of antibodies #14, 18D5 and 1H9 on the binding of SIRPα (V1) to CD47. [Figure 4C] FIG. 4C shows the blocking effects of antibody #14, KWAR23, 18D5 and 1H9 on the binding of SIRPα (V2) to CD47. [Figure 4D] FIG. 4D shows the blocking effects of antibody #14, KWAR23, 18D5 and 1H9 on the binding of SIRPα (V8) to CD47. [Diagram 5] FIG. 5 shows the binding kinetics of antibody #14 and KWAR23 to recombinant SIRPα protein. [Figure 6A] FIG. 6A shows the promotion of anti-CD20 antibody-induced phagocytosis by antibody #14. [Figure 6B] FIG. 6B shows the promotion of anti-HER2 antibody-induced phagocytosis by antibody #14. [Figure 7] FIG. 7 shows the anti-tumor efficacy of antibody #14 in an MC38 tumor model. [Figure 8] FIG. 8 shows that antibody #14 and anti-CD20 antibody can synergistically inhibit tumor growth.

Claims

1. a heavy chain variable region comprising three CDRs, VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region comprising three CDRs, VL CDR1, VL CDR2 and VL CDR3; the amino acid sequence of the VH CDR1 is set forth in SEQ ID NO:33; the amino acid sequence of the VH CDR2 is set forth in SEQ ID NO:34; the amino acid sequence of the VH CDR3 is set forth in SEQ ID NO:35; the amino acid sequence of the VL CDR1 is set forth in SEQ ID NO:38; the amino acid sequence of the VL CDR2 is set forth in SEQ ID NO:39; and An anti-SIRPα antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the VL CDR3 is set forth in SEQ ID NO:

40.

2. 2. The anti-SIRPα antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:31 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:36 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

3. 3. The anti-SIRPα antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising one or more of a heavy chain constant region, a light chain constant region and an Fc region; preferably, the light chain constant region is a lambda or kappa chain constant region; more preferably, the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3 or IgG4 type; even more preferably, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

4. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; preferably, said nucleic acid molecule comprises nucleotide sequences encoding the heavy chain variable region and the light chain variable region, respectively, of said antibody or antigen-binding fragment thereof; more preferably, the nucleotide sequence encoding said heavy chain variable region is set forth in SEQ ID NO:32 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and the nucleotide sequence encoding said light chain variable region is set forth in SEQ ID NO:37 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

5. 1. A biomaterial, said biomaterial being: (1) A vector, a host cell or a microorganism comprising the nucleic acid molecule according to claim 4; or (2) The expression product, suspension or supernatant of (1) above, which comprises an antibody or an antibody-binding fragment thereof according to any one of claims 1 to 3.

6. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; preferably said composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

7. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising the steps of culturing a host cell according to claim 5 to express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

8. 13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5 or the composition according to claim 6 in the preparation of a medicament for treating a tumor; preferably, the tumor is a CD47-positive tumor; more preferably, the tumor is a hematological or solid tumor, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, renal cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma and other solid tumors.

9. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a biomaterial according to claim 5, or a composition according to claim 6 in the preparation of a formulation that blocks the binding of SIRPα to CD47.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, or the composition according to claim 6, in combination with one or more additional cancer therapeutic agents, in the preparation of a medicament for treating a tumor; preferably, the tumor is a CD47-positive tumor; preferably, the additional cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, and biopolymer drugs; more preferably, the biopolymer drug is a monoclonal antibody drug that targets a tumor cell surface antigen, such as an anti-CD20 antibody, cetuximab, or trastuzumab; more preferably, the anti-CD20 antibody is suberitamab or rituximab.

Images