Anti-SIRP alpha antibodies and uses thereof

Abstract

The anti-SIRP alpha antibody and the antigen binding fragment thereof provided by the invention can effectively block the binding of SIRP alpha and CD47 so as to effectively promote the phagocytosis of macrophages to tumor cells and effectively inhibit the growth of tumors, and have a good patent medicine prospect.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202180012639.X, application date December 9, 2021, and invention name “Anti-SIRPα antibodies and their uses”. Technical Field

[0002] The invention belongs to the field of biology and relates to an anti-SIRPα antibody. Background Art

[0003] SIRPα is a transmembrane protein expressed on the surface of myeloid cells such as macrophages, monocytes, dendritic cells, and granulocytes. It is a member of the immunoglobulin superfamily (IgSF). SIRPα consists 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 the Ig-V domain), and the intracellular region contains a tyrosine immunoreceptor inhibitory motif (ITIM). The main ligand of SIRPα is CD47, which is a type of transmembrane glycoprotein widely expressed in normal and diseased tissues. The binding of CD47 to SIRPα on myeloid cells such as macrophages causes the ITIM in the intracellular region of SIRPα to be phosphorylated, thereby recruiting and activating the phosphatases SHP-1 and SHP-2, thereby inhibiting the phagocytic function of macrophages and the like through downstream signal transduction. Normal tissues release the "don't eat me" signal as a self-protection mechanism by expressing CD47 and CD47-SIRPα signaling.

[0004] Studies have found that CD47 is overexpressed on almost all tumor cells. Tumor cells use the combination of CD47 and SIRPα to release "don't eat me" signals to escape the monitoring of immune cells. In addition, the expression level of CD47 is closely related to the poor prognosis of patients in clinical practice. In vitro and in vivo studies have also found that blocking CD47 / SIRPα can promote tumor cell phagocytosis and inhibit tumor growth in animal models. The above shows that targeting CD47 / SIRPα can be used as a new approach for the development of tumor immunotherapy drugs. Considering the widespread expression of CD47 in normal cell tissues, especially on red blood cells and platelets, targeting CD47 may bring certain blood toxicity. In addition, in addition to SIRPα, proteins that interact with CD47 also include TSP-1, integrin, etc. The signaling pathways involved in CD47 are more complex, and targeting CD47 has more risks. Therefore, developing anti-SIRPα antibodies to block the CD47 / SIRPα signaling pathway may be a more effective strategy for tumor drug development. Summary of the invention

[0005] The purpose of the present invention is to provide a new anti-SIRPα antibody, which can specifically bind to SIPRα and has great potential for tumor treatment.

[0006] In a first aspect, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof that binds to SIRPα or a fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs, namely VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises three CDRs, namely VL CDR1, VL CDR2 and VLCDR3; wherein,

[0007] VH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, 13, 23 or 33, VH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, 14, 24 or 34, VH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, 15, 25 or 35, VL CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, 18, 28 or 38, VL CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, 19, 29 or 39, and VLCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, 20, 30 or 40.

[0008] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0009] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:3;

[0010] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:4;

[0011] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:5;

[0012] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:8;

[0013] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:9;

[0014] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:10.

[0015] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0016] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:13;

[0017] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:14;

[0018] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:15;

[0019] The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 18;

[0020] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 19;

[0021] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:20.

[0022] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0023] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:23;

[0024] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:24;

[0025] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:25;

[0026] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:28;

[0027] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:29;

[0028] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:30.

[0029] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0030] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:33;

[0031] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:34;

[0032] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:35;

[0033] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:38;

[0034] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:39;

[0035] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:40.

[0036] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown 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 with the above sequence.

[0037] In some embodiments, the amino acid sequence of the light chain variable region is as shown 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 with the above sequence.

[0038] In certain preferred embodiments, the three CDRs contained in the heavy chain variable region, and / or the three CDRs contained in the light chain variable region, are defined by the Kabat or Chothia numbering systems.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention may further comprise one or more of a heavy chain constant region, a light chain constant region, and an Fc region. In a further preferred embodiment, the light chain constant region is a λ chain or a κ chain constant region. In some preferred embodiments, the antibody or antigen-binding fragment thereof is of IgG1, IgG2, IgG3, or IgG4 type.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or antigen-binding fragment thereof.

[0041] In a second aspect, a nucleic acid molecule is provided, comprising nucleotides encoding an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the nucleic acid molecule encodes the heavy chain variable region and / or light chain variable region of the antibody or antigen-binding fragment thereof.

[0042] In a preferred embodiment, the nucleic acid molecule encodes a heavy chain variable region, and its nucleotide sequence is as shown in SEQ ID NO: 2, 12, 22 or 32, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence. In other preferred embodiments, the nucleic acid molecule encodes a light chain variable region, and its nucleotide sequence is as shown in SEQ ID NO: 7, 17, 27 or 37, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence.

[0043] In a third aspect, the present invention provides a biomaterial comprising:

[0044] (1) a vector, host cell or microorganism comprising the nucleic acid molecule of the present invention; or

[0045] (2) The expression product, suspension, supernatant, etc. of (1) above.

[0046] A person skilled in the art can easily select and prepare a vector, host cell or microorganism containing the coding sequence of the antibody according to the amino acid sequence of the antibody, and can know how to culture such a host cell or microorganism to obtain the corresponding expression product, suspension, supernatant, etc., to obtain the corresponding antibody. These are all conventional technical means in the art.

[0047] In a fourth aspect, a composition is provided, comprising the antibody or antigen-binding fragment thereof of the present invention; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0048] In a fifth aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising: culturing the above-mentioned host cell to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment.

[0049] In the sixth aspect, there is provided the use of the antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule of the present invention, the biomaterial of the present invention, or the composition of the present invention in the preparation of a medicament for treating a tumor; preferably, the tumor is a tumor that is positive for CD47 expression; further preferably, the tumor is various blood tumors and solid tumors, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma and other solid tumors.

[0050] In a seventh aspect, provided is the use of the antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule of the present invention, the biomaterial of the present invention, or the composition of the present invention in the preparation of a preparation for blocking the binding of SIRPα and CD47.

[0051] In an eighth aspect, provided is the use of the antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule of the present invention, the biomaterial of the present invention, or the composition of the present invention in combination with one or more other cancer therapeutic agents for the preparation of a medicament for treating tumors; preferably, the tumor is a CD47-positive tumor.

[0052] In some preferred embodiments, the other cancer therapeutic agents include but are not limited to chemotherapeutic agents, radiotherapeutic agents and biomacromolecule drugs. Further preferably, the biomacromolecule drug is a monoclonal antibody drug targeting tumor cell surface antigens, including anti-CD20 antibodies (such as zabetozumab or rituximab), cetuximab or trastuzumab.

[0053] In the ninth aspect, a method for treating a tumor is provided, wherein the tumor is a CD47-positive tumor, preferably, the tumor is various blood tumors and solid tumors, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma and other solid tumors, the method comprising administering the antibody or antigen-binding fragment thereof of the present invention or the nucleic acid molecule of the present invention or the biomaterial of the present invention or the composition of the present invention to an individual in need.

[0054] In a tenth aspect, a method for blocking the binding between SIRPα and CD47 is provided, comprising using the antibody or antigen-binding fragment thereof of the present invention, the nucleic acid molecule of the present invention, the biomaterial of the present invention, or the composition of the present invention.

[0055] In the eleventh aspect, a method for treating a tumor with a combined drug is provided, wherein the tumor is a CD47-positive tumor, and the method comprises administering to an individual in need thereof an antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule of the present invention, a biomaterial of the present invention, or a composition of the present invention and one or more other cancer therapeutic agents; preferably, the other cancer therapeutic agents include but are not limited to chemotherapeutic agents, radiotherapeutic agents, and biomacromolecule drugs; further preferably, the biomacromolecule drug is a monoclonal antibody drug targeting tumor cell surface antigens, including anti-CD20 antibodies (such as zabetozumab or rituximab), cetuximab, or trastuzumab.

[0056] The anti-SIRPα antibody and the antigen-binding fragment thereof of the present invention are blocking antibodies, which can effectively block the binding of SIRPα and CD47, thereby effectively promoting the phagocytosis of tumor cells by macrophages, effectively inhibiting the growth of tumors, and have good drug development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1The blocking effect of mouse monoclonal antibody on SIRPα binding to CD47 is shown.

[0058] Figure 2 Binding of humanized monoclonal antibodies to SIRPα is shown.

[0059] Figure 3 The blocking effect of humanized monoclonal antibody on the binding of SIRPα to CD47 is shown.

[0060] Figure 4A The blocking effect of #14 antibody and KWAR23 on the binding of SIRPα(V1) and CD47 is shown.

[0061] Figure 4B The blocking effect of antibody #14, 18D5 and 1H9 on the binding of SIRPα(V1) and CD47 is shown.

[0062] Figure 4C The blocking effect of antibody #14 and KWAR23, 18D5, and 1H9 on the binding of SIRPα(V2) and CD47 is shown.

[0063] Figure 4D The blocking effect of antibody #14 and KWAR23, 18D5, and 1H9 on the binding of SIRPα (V8) and CD47 is shown.

[0064] Figure 5 The binding kinetics of #14 antibody, KWAR23 and recombinant SIRPα protein are shown.

[0065] Fig. 6A The promoting effect of antibody #14 on phagocytosis induced by anti-CD20 antibody is shown; Figure 6B The promoting effect of antibody #14 on phagocytosis induced by anti-HER2 antibody is shown.

[0066] Figure 7 The anti-tumor efficacy of antibody #14 in the MC38 tumor model was shown.

[0067] Figure 8 It was shown that #14 antibody and anti-CD20 antibody can synergistically inhibit tumor growth. DETAILED DESCRIPTION

[0068] The present invention will be described below in conjunction with specific examples. Unless otherwise specified, the reagents and instruments used in the following methods are all commonly used reagents and instruments in the art and can be obtained commercially; the methods used are all conventional methods in the art, and those skilled in the art can undoubtedly perform the methods and obtain corresponding results based on the contents described in the examples.

[0069] definition:

[0070] In the present invention, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and covers 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 (such as mouse antibodies), humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as but not limited to Fab fragments, Fab' fragments, F(ab') 2 The antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). In preferred embodiments, the antibodies of the present invention are murine antibodies, chimeric antibodies, or humanized antibodies.

[0071] 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 the full length, but at least comprises 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 antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as 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, diabodies, Fd and Fd' fragments and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). The fragments can include multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and 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), results in an antibody that immunospecifically binds to an antigen.

[0072] As used herein, "conventional antibodies" refer to antibodies comprising two heavy chains (which may be designated as H and H') and two light chains (which may be designated as L and L') and two antigen binding sites, wherein 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 chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region (e.g., light chains include but are not limited to VL chains and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively).

[0073] 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) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies produced synthetically with the same domains.

[0074] As used herein, dsFv refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

[0075] As used herein, Fab fragments are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments with the same structure, for example, produced by recombinant synthesis. Fab fragments contain a light chain (comprising VL and CL) and another chain comprising a variable domain (VH) of a heavy chain and a constant region domain (CH1) of a heavy chain.

[0076] As used herein, a F(ab')2 fragment is an antibody fragment obtained by digesting an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, for example, by a recombinant method. A F(ab')2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, including cysteine ​​that forms a disulfide bond linking the two fragments.

[0077] As used herein, a Fab' fragment is a fragment comprising half (one heavy chain and one light chain) of a F(ab')2 fragment.

[0078] As used herein, ScFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker.

[0079] The term "variable region" refers to the domain of an antibody heavy chain or light chain that recognizes and specifically binds to an antigenic epitope, and is comprised of an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region domain, VL and VH, respectively. The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), and CDRs are parts of the antigen binding site domain.

[0080] CDR region or "complementarity determining region" refers to the region in the variable region of an antibody that is highly variable in sequence and forms a loop that is structurally determined and / or contains antigen contact amino acid residues. CDR is primarily responsible for the binding of the antibody to the antigen epitope and determines the specificity of the antibody. In a given heavy chain or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of many well-known numbering rules, including, for example, Kabat, Contact, AbM and Chothia. The CDR of the antibody of the present invention can be determined according to any rule or a combination of these techniques in the art.

[0081] As used herein, "hypervariable region", "complementarity determining region" and "CDR" are used interchangeably and refer to one of the multiple parts within each variable region that together form the antigen binding site of an antibody. Each variable region domain contains three CDRs, named CDR1, CDR2 and CDR3. For example, the light chain variable region domain contains three CDRs, named VL CDR1, VLCDR2 and VL CDR3; the heavy chain variable region domain contains three CDRs, named H CDR1 (or VH CDR1), H CDR2 (or VHCDR2) and H CDR3 (or VH CDR3). The three CDRs in the variable region are discontinuous along the linear amino acid sequence, but are close in the folded polypeptide. The CDRs are located in the loops connecting the parallel chains of the beta folds of the variable domain. As described herein, those of skill in the art know and can identify CDRs based on Kabat or Chotnia numbering (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Tests, 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).

[0082] As used herein, framework regions (FR) are domains within the antibody variable region domains that are located within the beta folds; in terms of amino acid sequence, FR regions are relatively more conserved than hypervariable regions.

[0083] As used herein, a "constant region" domain is a domain in an antibody heavy chain or light chain that contains an amino acid sequence that is relatively more conservative than 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). The antibody constant region can serve effector functions, such as but not limited to clearing antigens, pathogens, and toxins that the antibody specifically binds, such as by interacting with various cells, biomolecules, and tissues.

[0084] As used herein, a functional region of a VH domain is at least a portion of a complete VH domain that retains at least part of the binding specificity of the complete VH domain (e.g., by retaining one or more CDRs of the complete VH domain), such that the functional region of the VH domain binds to the antigen alone or in combination with another antibody domain (e.g., a VL domain) or a region thereof. An exemplary functional region of a VH domain is a region comprising CDR1, CDR2, and / or CDR3 of a VH domain.

[0085] As used herein, a functional region of a VL domain is at least a portion of a complete VL domain that retains at least a portion of the binding specificity of the complete VL domain (e.g., by retaining one or more CDRs of the complete VL domain), such that the functional region of the VL domain binds antigen alone or in combination with another antibody domain (e.g., a VH domain) or a region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2, and / or CDR3 of a VL domain.

[0086] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond.

[0087] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. "Isolated" nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture media when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesized. The exemplary isolated nucleic acid molecules provided herein include the isolated nucleic acid molecules of the antibody or Fab provided by encoding.

[0088] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or peptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: 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 Pres S, 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 to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0089] 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, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0090] As used herein, "host cell" is a cell that is used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, HeLa cells, HEK cells such as HEK 293 cells.

[0091] As used herein, "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

[0092] As used herein, "expression vector" includes vectors capable of expressing DNA, which is 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 replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.

[0093] As used herein, "treating" an individual suffering from a disease or condition means that the symptoms of the individual are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and / or cure. Prevention refers to preventing potential diseases and / or preventing symptoms from worsening or disease development. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided and any composition provided herein.

[0094] As used herein, "therapeutic effect" refers to the effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0095] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after being applied to a subject. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. Similarly, as used herein, "prophylactic effective amount" or "prophylactic effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that will have the expected preventive effect when applied to a subject, for example, preventing or delaying the occurrence or recurrence of a disease or symptom, reducing the likelihood of the occurrence or recurrence of a disease or symptom. A completely preventive effective dose does not have to occur by administering one dose, and may occur only after administering a series of doses. Therefore, a preventive effective dose may be administered in one or more administrations.

[0096] As used herein, the term "subject" refers to a mammal, such as a human.

[0097] Anti-SIRPα antibodies of the present invention

[0098] The present invention provides an anti-SIRPα antibody with high affinity for human SIRPα protein. The antibody can effectively inhibit the binding of SIRPα to its ligand CD47, thereby blocking CD47 / SIRPα downstream signal transduction, promoting the phagocytosis of tumor cells by macrophages, and thus achieving the clearance of tumor cells. Once the anti-SIRPα antibody described herein binds to the SIRPα protein on the cell surface, it can cause endocytosis, resulting in a decrease in the expression of SIRPα protein on the cell surface, thereby further reducing the signal transmission between CD47 and SIRPα.

[0099] The anti-SIRPα antibodies or antigen-binding fragments thereof of the present invention comprise substitutions, insertions or deletions. The anti-SIRPα antibodies of the present invention include modifications to the light chain variable region, the heavy chain variable region, the light chain or the heavy chain, and the amino acid sequence after modification is different from the amino acid sequence from which the antibody is derived. For example, an amino acid sequence derived from the same specified protein may be similar to the starting sequence, for example, having a certain percentage identity, for example, it may have a percentage identity of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% with the starting sequence.

[0100] In the present invention, "identity" refers to the percentage of bases (or amino acids) in the two sequences being compared that are identical when the sequences are aligned between two peptides or between two nucleic acid molecules. The 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) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP.

[0101] In certain embodiments, amino acid modifications may be introduced into the Fc region of an antibody provided herein, and the amino acid modifications may be one or more, thereby generating Fc variants. Fc variants may comprise a human Fc region sequence comprising amino acid modifications at one or more amino acid positions.

[0102] "Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies, nanobodies, and epitope-binding fragments of any of the above.

[0103] In some embodiments, the antibodies of the invention 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 a second or more binding specificities.

[0104] In certain embodiments, the antibody can be further modified to add functional components, and suitable parts for antibody derivatization include, but are not limited to, the following examples, PEG, dextran, protein, lipid, therapeutic agent or toxin. Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or connection with other proteins.

[0105] Tumor treatment methods and antibody uses

[0106] The anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention and pharmaceutical compositions comprising the same can be used for diagnosis, prognosis, treatment or inhibition of cancer. The present invention relates to providing a method for treating cancer in a subject by administering the anti-SIRPα antibodies or fragments thereof of the present invention to a subject in need thereof. The therapeutic compounds of the present invention include, but are not limited to, the antibodies of the present invention (including variants and derivatives of the present invention) and nucleic acids or polynucleotides encoding the antibodies of the present invention (including variants and derivatives of the present invention).

[0107] The present invention also provides a combination therapy, comprising the anti-SIRPα antibody of the present invention and at least one other therapeutic agent used in combination, wherein the other therapeutic agent includes but is not limited to a chemotherapeutic agent, a radiotherapeutic agent, and a biomacromolecule drug. In one embodiment, the biomacromolecule drug is a monoclonal antibody drug targeting tumor cell surface antigens, including anti-CD20 antibodies (such as zebetozumab, rituximab), cetuximab, and trastuzumab.

[0108] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including but not limited to intraperitoneal, intravenous, subcutaneous, intranasal, intramuscular injection. Antibodies and variants thereof or compositions can be administered by any convenient route, such as by bolus injection or infusion, by absorption through epithelial or mucocutaneous membranes.

[0109] Anti-SIRPα antibody sequences according to the present invention

[0110] Table 1: Amino acid sequence numbers of VH, VH-CDR1, VH-CDR2, VH-CDR3, VL, VL-CDR1, VL-CDR2, VL-CDR3 of the antibodies of the present invention

[0111]

[0112] Table 2: VH and VL DNA sequence numbers of the antibodies of the present invention

[0113] Antibody ID VH DNA sequence numbering VL DNA sequence numbering #4 2 7 #11 12 17 #13 22 27 #14 32 37

[0114] Table 3: Sequence information of the present invention

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Table 4#14 Antibody Heavy Chain and Light Chain Amino Acid Sequences

[0121]

[0122]

[0123] Example 1: Preparation of anti-SIRPα mouse monoclonal antibody

[0124] This example describes a method for preparing mouse anti-human SIRPα monoclonal antibodies using hybridoma technology. First, the extracellular SIRPα protein (Uniprot: P78324) was expressed as an immunogen, and an Fc tag was added to the C-terminus of the extracellular SIRPα protein amino acid sequence (Glu31-Arg370), and then cloned into the V152 vector (provided by Huaan Monoclonal Antibody) to obtain V152-SIRPαECD-Fc. It was transiently transfected into 293 cells (source: ATCC), and the cell culture fluid was collected after 5 days, and the supernatant was purified with protein A (manufacturer: Solarbio, catalog number: I8090). To prepare anti-human SIRPα mouse monoclonal antibodies, 4-week-old BAL B / c mice (provided by Huaan Monoclonal Antibody) were first immunized with 100 μg of SIRPα protein. On the 14th and 28th days after the first immunization, the immunized mice were re-immunized with 50 μg of SIRPα protein. The ELISA method was used to detect the titer of mouse serum after immunization. SIRPα was diluted into PBS (manufacturer: Zhongshan Jinqiao, catalog number: ZLI-9062) at a concentration of 1 μg / ml and coated on a microplate at 4°C overnight. Then, the plate was blocked with 1% BSA-PBS blocking solution at 37°C for 1 hour; after washing the plate with PBST, the serum dilution from the immunized mouse was added to the plate and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled goat anti-mouse IgG (manufacturer: Abcam, catalog number: Ab205719, 1:10000 dilution) was added and reacted at 37°C for 0.5 hours. After washing the plate, TMB solution (manufacturer: Huzhou Yingchuang, catalog number: TMB-S-00) was added, and the reaction was carried out at room temperature in the dark for 5 minutes, and then 2N H2SO4 was added to terminate the reaction. The absorbance was detected at a wavelength of 450nm on an enzyme reader. On the 42nd day after immunization, mice with sufficient titer of anti-SIRPα antibodies were boosted with 50 μg SIRPα protein. After 3-5 days, the mice were killed, spleen cells were collected, and the cells were washed 2-3 times by centrifugation with IMDM (manufacturer: Shanghai Yuanpei, catalog number: L610KJ) basal medium, and then mixed with mouse myeloma cells SP2 / 0 (provided by Huaan Monoclonal Antibody) at a ratio of 1:1. PEG (manufacturer: Roche, catalog number: 25771700) was added to the mixed cells and gently stirred, and then stood at 37°C for 30s. The fused cells were diluted into IMEM selective medium containing 15% fetal bovine serum (manufacturer: Zhejiang Tianhang Biology, catalog number: 11011-8611) and 1×HAT (manufacturer: Sigma, catalog number: H0262-1VL), and 200 μl per well were added to a 96-well cell culture plate and placed in a 5% CO2, 37°C incubator. After 10-14 days, anti-SIRPα antibodies in the hybridoma supernatant were detected by ELISA experiment.Eleven different hybridoma clones were identified, including 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, and were used for further analysis.

[0125] Example 2: Binding of mouse antibodies to SIRPα

[0126] The binding ability of mouse antibodies to SIRPα protein was detected by ELISA method, and the specific method was as described in Example 1, wherein the initial concentration of mouse antibodies was 1 μg / ml, and then PBS was used for 3-fold gradient dilution to obtain a total of 7 gradients. The results are shown in Table 5.

[0127] Table 5: EC50 values ​​of binding activity of mouse monoclonal antibodies to SIRPα

[0128] Antibody ID clone ELISA EC50(pM) mAb#4 10F11-5-6 4.22 mAb#6 1B6-1-1 9.15 mAb#11 27A11-1-8 5.47 mAb#12 14B11-5-4 8.67 mAb#13 31D4-4-5 6.77 mAb#14 7C2-3-8 5.69 mAb#15 30C1-6-4 10.31 mAb#16 4A3-5-1 10.64 mAb#17 6H1-8-6 11.93 mAb#18 9A12-5 8.05 mAb#19 10F4-15 8.66

[0129] Flow cytometry (FACS) was used to analyze the binding of mouse monoclonal antibodies to SIRPα on the membrane of human myeloid leukemia mononuclear cells (THP-1, source: Chinese Academy of Sciences Cell Bank) that endogenously express SIRPα. THP-1 cells were reacted with different concentrations of mouse monoclonal antibodies (the highest concentration was 1 μg / ml, 3-fold dilution, a total of 7 concentration points) at 4°C for 30 minutes. After washing the cells twice, FITC-labeled goat anti-mouse IgG (manufacturer: Jackson, catalog number: 115-095-003, 1:1000 dilution) was added and reacted at 4°C in the dark for 30 minutes. After washing the cells twice, the cells were detected using a flow cytometer BD C6. The results are shown in Table 6.

[0130] Table 6: EC50 values ​​of mouse monoclonal antibodies binding to THP-1 cells

[0131] Antibody ID clone FACS EC50 (pM) mAb#4 10F11-5-6 139.22 mAb#6 1B6-1-1 231.81 mAb#11 27A11-1-8 125.84 mAb#12 14B11-5-4 211.43 mAb#13 31D4-4-5 144.27 mAb#14 7C2-3-8 128.72 mAb#15 30C1-6-4 128.70 mAb#16 4A3-5-1 232.4 mAb#17 6H1-8-6 544.21 mAb#18 9A12-5 212.34 mAb#19 10F4-15 220.11

[0132] Example 3: Blocking effect of mouse anti-SIRPα antibody on the binding of SIRPα to CD47

[0133] According to the results of Example 2, mAb#4, mAb#11, mAb#13 and mAb#14 are the most active molecules based on the binding ability of mouse monoclonal antibodies to SIRPα and to THP-1 cells. Therefore, mAb#4, mAb#11, mAb#13 and mAb#14 were selected to detect whether these mouse monoclonal antibodies can block the binding of SIRPα to CD47. CD47-Fc (manufacturer: Acrobiosystems, catalog number: CD7-H5256) was coated on a 96-well ELISA plate and incubated at 4°C overnight; after washing the plate, 1% BSA-PBST blocking solution was added and blocked at 37°C for 1h. Different concentrations of antibodies (66.7nM starting, 3-fold dilution, a total of 7 concentrations) and biotin-labeled SIRPα (manufacturer: Acrobiosystems, catalog number: CDA-H82F2) were added to the plate and incubated at 37°C for 1 hour. After washing, HRP-labeled streptavidin (manufacturer: Abcam, catalog number: ab7403) was added and reacted at 37°C for 0.5 hours. After washing, TMB solution was added and reacted at room temperature in the dark for 30 minutes. 2N H2SO4 was added to terminate the reaction and the absorbance at 450nm was detected by an enzyme marker. IgG (synthesized by GenScript) was used as an experimental control. The results are shown in Figure 1 , showing that these mouse antibodies can inhibit the binding of SIRPα to CD47 to varying degrees.

[0134] Example 4: Humanization of murine antibodies

[0135] According to the results of Example 2 and Example 3, mAb#4, mAb#11, mAb#13 and mAb#14 were selected for humanization, and the heavy chain variable regions and light chain variable regions of these antibodies were compared with the available human IgG gene sequence database to identify the best matching human germline Ig gene sequence. Specifically, the human germline IgG heavy chains selected by mAb#4, mAb#11, mAb#13 and mAb#14 were IGHV1-69-2*01, IGHV2-26*01, IGHV7-4-1*01, IGHV4-31*02, respectively, and the human germline IgG light chains selected by mAb#4, mAb#11, mAb#13 and mAb#14 were IGKV4-1*01, IGKV1-9*01, IGKV4-1*01, IGKV4-1*01, respectively. The CDR regions of the heavy chains of mAb#4, mAb#11, mAb#13 and mAb#14 were transplanted onto the framework sequences of the matching heavy chain variable region genes, and the CDR regions of the light chains of mAb#4, mAb#11, mAb#13 and mAb#14 were transplanted onto the framework sequences of the matching light chain variable region genes. The new humanized molecules were named #4, #11, #13 and #14, respectively. After construction, the sequences of the heavy chain variable region, light chain variable region and CDR region of each humanized antibody obtained are shown in Table 3. The heavy chain and light chain sequences of #14 antibody are shown in Table 4. The heavy chain constant region and light chain constant region of #4, #11 and #13 antibodies are consistent with the heavy chain constant region and light chain constant region of #14 antibody. A person skilled in the art can fully know the complete amino acid sequences of the heavy and light chains of #4, #11 and #13 antibodies based on the above disclosed information.

[0136] The antibody of the present invention was expressed and purified in 293 cells (source: ATCC). First, the heavy chain and light chain coding sequences of the antibody were cloned into the V152 vector (provided by Huaan Monoclonal Antibody), and then the V152 vector with the heavy chain and light chain coding sequences of the antibody molecule was transferred into the 293 cells using the transfection reagent PEI (manufacturer: Polyscience, catalog number: 23966-2). Prepare plasmid DNA and transfection reagent in a biological safety cabinet, mix the plasmid DNA and PEI (mass ratio: 0.15:1.75) and let stand for 10 minutes, then gently pour the mixture of DNA and transfection reagent into the 293 cells and mix. After culturing for 5 days at 37°C and 5% CO2, the cell culture supernatant was centrifuged at 3000rpm for 10 minutes. Take the supernatant and purify the supernatant with protein A (manufacturer: Solarbio, catalog number: I8090) to make the antibody purity >95%.

[0137] Example 5: Binding of humanized antibodies to SIRPα protein

[0138] The binding of humanized antibodies and SIRPα protein was detected by ELISA. SIRPα (manufacturer: Acrobiosystems, catalog number: SIA-H5225) was diluted into PBS at a concentration of 1 μg / ml and coated on a microplate at 4°C overnight. Then, it was blocked with 1% BSA-PBS blocking solution at 37°C for 1 hour; after washing the plate with PBST, the humanized antibody was diluted to different concentrations (starting at 66.7 nM, 3-fold dilution, a total of 11 concentrations) and added to the plate and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled goat anti-human IgG (manufacturer: Abcam, catalog number Ab98595, 1:10000 dilution) was added and reacted at 37°C for 0.5 hours. After washing the plate, TMB solution was added and reacted at room temperature in the dark for 5 minutes, and then 2N H2SO4 was added to terminate the reaction. The absorbance was detected at a wavelength of 450nm on an enzyme reader. Results are shown in Figure 2 The EC50 values ​​of #4, #11, #13 and #14 antibodies binding to SIRPα were 1.833nM, 0.4642nM, 0.9517nM and 0.6831nM, respectively, indicating that these humanized molecules all have high binding activity with SIRPα.

[0139] Example 6: Blocking effect of humanized anti-SIRPα antibody on the binding of SIRPα to CD47

[0140] The blocking effect of humanized anti-SIRPα antibodies #4, #11, #13 and #14 on the binding of SIRPα to CD47 was detected according to the method described in Example 3. The antibody concentration started from 66.7 nM and was diluted 3 times, with a total of 11 concentrations. The results are shown in Figure 3 The IC50 values ​​of antibodies #4, #11, #13 and #14 were 6.03 nM, 0.2086 nM, 1.5 nM and 0.1315 nM, respectively, indicating that these humanized antibodies can effectively block the binding of SIRPα to CD47, among which #11 and #14 have stronger blocking effects than other humanized antibodies.

[0141] KWAR23, 18D5 and 1H9 were used as controls. KWAR23 was generated according to the sequence disclosed in US2018037652 (SEQ ID NO: 1, SEQ ID NO: 2), 18D5 was generated according to the sequence disclosed in US20190127477 (SEQ ID: 24, SEQ ID NO: 31), and 1H9 was generated according to the sequence disclosed in US20190119396 (SEQ ID: 7, SEQ ID NO: 8). Because human SIRPα protein is highly polymorphic in the extracellular region, common genotypes include V1, V2, and V8, the blocking effect of #14 antibody and the above control antibodies on the binding of common SIRPα genotypes to CD47 was further compared (the SIRPα proteins without genotypes in other examples were all V1).

[0142] The blocking effect of different antibodies on the binding of SIRPα (V1) to CD47 was detected according to the method described in Example 3. The antibody concentration started from 83.4 nM and was diluted 2-fold for a total of 9 concentrations. The results are as follows Figure 4A As shown, the IC50 value of #14 antibody is 3.404 nM, and the IC50 value of KWAR23 is 13.54 nM, indicating that the blocking effect of #14 antibody on the binding of SIRPα (V1) and CD47 is stronger than that of KWAR23. Figure 4B The IC50 values ​​of #14 antibody, 18D5 and 1H9 were 4.4 nM, 4.1 nM, and 4.2 nM, respectively, indicating that the blocking activities of #14 antibody, 18D5 and 1H9 on the binding of SIRPα (V1) and CD47 were similar.

[0143] The blocking effect of different antibodies on the binding of SIRPα(V2) to CD47 was detected by referring to the method described in Example 3. The biotin-labeled SIRPα used was SIRPα(V2) (manufacturer: Acrobiosystems, catalog number: SI2-H82W8). The antibody concentration started from 200 nM and was diluted 3 times for a total of 9 concentrations. The results are as follows: Figure 4C As shown, #14 antibody, KWAR23 and 1H9 can all block the binding of SIRPα(V2) to CD47, while 18D5 cannot block the binding of SIRPα(V2) to CD47. The blocking activity of #14 antibody is stronger than that of 1H9. The IC50 value of #14 antibody is 7.8nM, and the IC50 value of KWAR23 is 6.4nM, indicating that the blocking activity of #14 antibody and KWAR23 on SIRPα(V2) is similar.

[0144] The blocking effect of different antibodies on the binding of SIRPα (V8) to CD47 was detected by referring to the method described in Example 3. The biotin-labeled SIRPα used was SIRPα (V8) (manufacturer: Acrobiosystems, catalog number: SI8-H82W6). The antibody concentration started from 200 nM and was diluted 3 times for a total of 9 concentrations. The results are as follows: Figure 4D As shown, #14 antibody, KWAR23 and 1H9 can all block the binding of SIRPα(V8) to CD47, while 18D5 cannot block the binding of SIRPα(V8) to CD47. The blocking activity of #14 antibody is stronger than that of 1H9. The IC50 value of #14 antibody is 11.5nM, and the IC50 value of KWAR23 is 10.8nM, indicating that the blocking activity of #14 antibody and KWAR23 on SIRPα(V8) is similar.

[0145] Based on the above results, antibody #14 can block the binding of common SIRPα genotypes (V1, V2 and V8) to CD47. Its blocking activity against SIRPα (V1) is stronger than that of KWAR23, and its blocking activity against SIRPα (V2) and SIRPα (V8) is stronger than that of 18D5 and 1H9.

[0146] Example 7: Binding kinetics of antibodies to SIRPα

[0147] The antibody-antigen binding kinetics was tested using Octet Red96 (manufacturer: ForteBio) as the test instrument, and H1S1K biosensor (manufacturer: ForteBio) was selected as the test sensor, which can be used to directly capture the antigen, and then the sensor is immersed in the analyte sample (antibody). This experiment ran five steps: 1. Baseline (100s), 2. Loading (capture antigen SIRPα) (500s), 3. Baseline (100s), 4. Association (binding antibody) (500s), 5. Dissociation (antibody dissociation) (1000s). After the test was completed, the sensor was regenerated by alternating immersion with regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS) for 5 seconds, and a total of five cycles. The running buffer in this experiment was PBS.

[0148] Sample processing: The antigen SIRPα protein (manufacturer: Acrobiosystems, catalog number: SIA-H5225) was diluted to a working concentration of 2.5 μg / mL using running buffer, and the analyte samples (#14 antibody and KWAR23) were gradiently diluted to five working concentrations: 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml. Data analysis was performed using Octet Data Analysis (version 7.0 or the latest) to calculate the response signal value (the coupled analyte sample signal minus the blank analyte sample signal), and the data was fitted using a 1:1 binding model.

[0149] Binding kinetics fitting diagram is shown in Figure 5 The binding constant, dissociation constant and equilibrium constant are shown in Table 7. It can be seen that the #14 antibody has a very high affinity, which is comparable to the positive control antibody KWAR23.

[0150] Table 7: Binding kinetics of #14 antibody and KWAR23 to SIRPα

[0151] Sample name Kon Koff KD(M) #14 <![CDATA[1.33×10 6 ]]> <![CDATA[2.89×10 -5 ]]> <![CDATA[2.173×10 -11 ]]> KWAR23 <![CDATA[2.90×10 6 ]]> <![CDATA[1.48×10 -4 ]]> <![CDATA[5.094×10 -11 ]]>

[0152] Example 8: The promoting effect of the anti-SIRPα antibody of the present invention on macrophage phagocytosis of tumor cells. PBMC cells (manufacturer: Allcells, catalog number: PB004F-C) were cultured with 1640 basal medium (manufacturer: Gibco, catalog number: 22400-089) at 5% CO2 and 37°C for 2 to 3 hours, non-adherent cells were gently aspirated, and induction medium (80ng / ml M-CSF) was added to start induction. Fresh culture medium containing sufficient cytokines was replaced every 3 days. Macrophages were obtained on the 7th day of culture. Raji cells (source: Chinese Academy of Sciences Cell Bank) were fluorescently labeled according to the instructions of CFSE reagent (manufacturer: Abcam, catalog number: AB113853). The labeled target cells Raji cells and the above-mentioned differentiated macrophages were mixed evenly in a ratio of 3:1, and anti-CD20 antibody (Zuberitamab, CAS RN: 2251143-19-6, WHO Drug Information, Vol33, No.4, 2019, 914-915), concentration: 0.05 μg / ml, 0.5 μg / ml, manufacturer: Borui Bio), or a combination of anti-CD20 antibody (concentration: 0.05 μg / ml, 0.5 μg / ml) and #14 antibody (concentration: 10 μg / ml), or a combination of anti-CD20 antibody (concentration: 0.05 μg / ml, 0.5 μg / ml) and KWAR23 antibody (concentration: 10 μg / ml) were added, and cultured at 37°C, 5% CO2 for 4 hours. After washing the cells with PBS, anti-CD14-APC (manufacturer: BD Biosciences, catalog number: 555399) was added and incubated at 4°C in the dark for 30 min. After washing the cells, flow cytometry analysis was performed, and the phagocytosis rate (ADCP) was calculated according to the following formula: phagocytosis rate (%) = (APC+CFSE) positive cell ratio / APC positive cell ratio*100%. Fig. 6A It shows that antibody #14 can enhance the phagocytic ability of anti-CD20 antibody on Raji cells.

[0153] At the same time, the promotion effect of #14 antibody and anti-Her2 antibody on macrophage phagocytosis of Her2-highly expressed SK-BR-3 cells was investigated. According to the above method, the labeled SK-BR-3 cells (source: Chinese Academy of Sciences Cell Bank) and differentiated macrophages were mixed evenly at a ratio of 2:1, and anti-Her2 antibody (concentration: 0.5μg / ml, manufacturer: Borui Bio) or a combination of anti-Her2 antibody (concentration: 0.5μg / ml) and #14 antibody (concentration: 10μg / ml) were added at the same time, and cultured at 37°C, 5% CO2 for 2 hours, and the phagocytic rate was calculated by flow cytometry analysis. Figure 6B It showed that antibody #14 could enhance the phagocytic ability of anti-Her2 antibody on SK-BR-3 cells.

[0154] Example 9: Anti-SIRPα antibodies of the present invention induce endocytosis of SIRPα on the cell surface

[0155] After THP-1 cells (source: Chinese Academy of Sciences Cell Bank) were reacted with 10 μg / ml antibody at 4°C for 30 minutes, the cells were washed twice and divided into two parts, one part of the cells was incubated at 37°C for 4 hours, and the other part of the cells was continued at 4°C as control cells. After washing the cells twice, FITC-labeled goat anti-human IgG (manufacturer: Abcam, catalog number: ab97224) was added and reacted at 4°C in the dark for 30 minutes. After washing the cells, flow cytometry analysis was performed, and the endocytosis rate was calculated based on the reduction of cell surface fluorescence at 37°C relative to 4°C. The endocytosis rate was calculated according to the following formula: endocytosis rate (%) = (4°C fluorescence intensity MFI-37°C fluorescence intensity MFI) / 4°C fluorescence intensity MFI*100%.

[0156] Table 8 shows that the internalization rate of antibody #14 is 52.9%, while the internalization rate of KWAR23 is 34.7%, indicating that antibody #14 can more effectively reduce the expression level of SIRPα on the cell surface.

[0157] Table 8: Internalization rate of SIRPα on THP-1 cell membrane by #14 and KWAR23

[0158] sample Internalization rate (%) #14 52.9 KWAR23 34.7

[0159] Example 10: Anti-SIRPα antibodies of the present invention inhibit tumor growth

[0160] The anti-tumor efficacy of antibody #14 was evaluated in the hCD47-MC38 mouse colon cancer tumor model. hCD47-MC38 cells (Source: Southern Model Organisms) were inoculated into hSIRPα / hCD47 dual humanized C57BL / 6 mice (Source: Southern Model Organisms). The tumor volume grew to 150mm 3 Around 200 μg / mouse, the mice were intraperitoneally injected with #14 antibody (200 μg / mouse), and the control group was given an equal amount of PBS solution, and the administration was performed twice a week. Figure 7 As shown, compared with the control group, #14 antibody can sustainably inhibit tumor growth.

[0161] The anti-tumor efficacy of the combination of antibody #14 and anti-CD20 antibody (Zebetuzumab, source: Hisun BioRich) in lymphoma models was evaluated. Raji-Luc cells (source: Biocytogen) were inoculated into the tail vein of B-NDG-hSIRPΑ mice (source: Biocytogen), and the average imaging signal intensity reached 1×10 6At about 1:1 p / sec, mice were administered with the indicated doses of IgG control (provided by Biocytogen, 200 μg / mouse), #14 antibody alone (200 μg / mouse), anti-CD20 antibody alone (2 μg / mouse), and #14 antibody (200 μg / mouse) plus anti-CD20 antibody (2 μg / mouse). Figure 8 It showed that #14 antibody and anti-CD20 antibody could exert synergistic anti-tumor effects.

Claims

1. Use of an anti-SIRPα antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating tumors in combination with one or more other cancer therapeutic agents, wherein: The anti-SIRPα antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs, namely VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises three CDRs, namely VL CDR1, VLCDR2 and VL CDR3; wherein, VH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3, 13, 23 or 33, VH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, 14, 24 or 34, VH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5, 15, 25 or 35, VL CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, 18, 28 or 38, VL CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, 19, 29 or 39, and VL CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, 20, 30 or 40; The other cancer therapeutic agent is an anti-Her2 antibody.

2. The use according to claim 1, wherein: The anti-SIRPα antibody or antigen-binding fragment thereof comprises VHCDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein, The amino acid sequence of VH CDR1 is shown in SEQ ID NO:3; The amino acid sequence of VH CDR2 is shown in SEQ ID NO:4; The amino acid sequence of VH CDR3 is shown in SEQ ID NO:5; The amino acid sequence of VL CDR1 is shown in SEQ ID NO:8; The amino acid sequence of VL CDR2 is shown in SEQ ID NO:9; The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 10; or, The amino acid sequence of VH CDR1 is shown in SEQ ID NO:13; The amino acid sequence of VH CDR2 is shown in SEQ ID NO:14; The amino acid sequence of VH CDR3 is shown in SEQ ID NO:15; The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 18; The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 19; The amino acid sequence of VL CDR3 is shown in SEQ ID NO:20; or, The amino acid sequence of VH CDR1 is shown in SEQ ID NO:23; The amino acid sequence of VH CDR2 is shown in SEQ ID NO:24; The amino acid sequence of VH CDR3 is shown in SEQ ID NO:25; The amino acid sequence of VL CDR1 is shown in SEQ ID NO:28; The amino acid sequence of VL CDR2 is shown in SEQ ID NO:29; The amino acid sequence of VL CDR3 is shown in SEQ ID NO:30; or, The amino acid sequence of VH CDR1 is shown in SEQ ID NO:33; The amino acid sequence of VH CDR2 is shown in SEQ ID NO:34; The amino acid sequence of VH CDR3 is shown in SEQ ID NO:35; The amino acid sequence of VL CDR1 is shown in SEQ ID NO:38; The amino acid sequence of VL CDR2 is shown in SEQ ID NO:39; The amino acid sequence of VL CDR3 is shown in SEQ ID NO:

40.

3. The use according to claim 1 or 2, wherein: The amino acid sequence of the heavy chain variable region of the anti-SIRPα antibody or its antigen-binding fragment is as shown 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 with the above sequence; the amino acid sequence of the light chain variable region is as shown 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 with the above sequence.

4. The use according to any one of claims 1 to 3, wherein: The anti-SIRPα antibody or its antigen-binding fragment further comprises 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 λ chain or a κ chain constant region; further preferably, the antibody or its antigen-binding fragment is of IgG1, IgG2, IgG3 or IgG4 type; more preferably, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody or its antigen-binding fragment.

5. The use according to claim 4, wherein: The heavy chain amino acid sequence of the anti-SIRPα antibody is shown in SEQ ID NO:41, and the light chain amino acid sequence is shown in SEQ ID NO:

42.

6. The use according to any one of claims 1 to 5, wherein the anti-Her2 antibody is trastuzumab.

7. The use according to any one of claims 1 to 6, wherein: The tumor is a blood tumor or a solid tumor. Preferably, the tumor is leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer or glioma.

8. The use according to any one of claims 1 to 7, wherein: The tumor is a CD47-positive tumor.

9. The use according to any one of claims 1 to 7, wherein the other cancer therapeutic agents further include chemotherapeutic agents, radiotherapeutic agents and biomacromolecule drugs; preferably, the biomacromolecule drug is a monoclonal antibody drug targeting tumor cell surface antigens; more preferably, the monoclonal antibody drug is an anti-CD20 antibody or cetuximab.

10. The use according to claim 9, wherein the anti-CD20 antibody is zebeziuzumab or rituximab.

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