Antibody targeting CD47 and its applications

Abstract

The present invention relates to the field of biomedical science, specifically to an antibody targeting CD47 and its application. The provided anti-CD47 antibody can specifically bind to tumor cells, block signals of human SIRPα and human CD47, and promote phagocytosis of tumor cells by macrophages. Since the antibody has high affinity, strong specificity, and excellent safety, it does not cause red blood cell agglutination and exhibits very weak binding to RBCs and platelets.

Description

Technology Field

[0001] The present invention belongs to the field of biomedical technology. Specifically, it relates to an antibody targeting CD47 and its applications, and more specifically, to an anti-CD47 antibody or its antigen-binding fragment, nucleic acid, vector, cell, composition, application, and kit. Background Technology

[0002] Cancer immunotherapy is a major recent development in the field of life sciences. Immune checkpoint inhibitor therapies based on T-cell antibodies such as CTLA4, PD-1, and PD-L1, as well as cell therapies like CAR-T and TCR-T, have all been popular immunotherapies in recent years. Without exception, these therapies focus on methods to restore T-cell function—that is, methods to enhance the capabilities of the adaptive immune system. However, the path to conquering cancer by activating T-cell function through targeting immune checkpoints to improve the adaptive immune system still faces many twists and turns. Yet, the role of the innate immune system in tumor immunotherapy has long been overlooked. In fact, macrophages account for approximately 50% of the tumor tissue within the total area of ​​tumor invasion; more importantly, the number of macrophages is inversely proportional to the tumor's prognosis, which represents a significant advancement in the role macrophages play in tumors. The exercise of phagocytosis by macrophages requires the simultaneous action of two signals: one is the activation of the “eat me” signal on the surface of the target cell, and the other is the inactivation of the “do not eat me” signal on the same target surface. The absence of either signal is insufficient to initiate phagocytosis. Increasing evidence shows that CD47 acts as a type of “do not eat me” signal by interacting with Signal Regulatory Protein α (SIRPα) on the surface of macrophages to inhibit phagocytosis. Tumor cells can also evade macrophage phagocytosis through the expression of CD47 (see, e.g., EP2242512 and related literature cited herein).

[0003] CD47, also known as an integrin-associated protein (IAP), is a 50 kDa membrane protein with an amino-terminal immunoglobulin domain and a carboxy-terminal multiple transmembrane domain. It interacts with various ligands, including but not limited to single regulatory protein α (SIRPα), SIRPγ, integrins, and thrombospondin-1 (TSP-1). SIRPα is primarily expressed in myeloid cells, including macrophages, myeloid dendritic cells (DCs), granulocytes, mast cells, and their precursors, including hematopoietic stem cells. CD47 / SIRPα interactions transmit a "don't eat me" signal and prevent autophagy. Analysis of patient tumors and matching adjacent normal (non-tumor) tissues revealed that the CD47 protein is overexpressed in cancer cells, which helps effectively evade innate immune surveillance and elimination. Blocking the interaction between CD47-SIRPα and anti-CD47 antibodies has been shown to effectively induce phagocytosis of tumor cells in vitro and inhibit the growth of various hematological and solid tumors in vivo. Therefore, CD47 is an effective target for cancer treatment, and appropriate antagonists are required for the manufacture of human therapeutic agents. The problem to be solved

[0004] Since CD47 monoclonal antibodies have a high target binding affinity to red blood cells, easily cause red blood cell agglutination, significantly reduce the therapeutic effect of the antibody, and can cause drug side effects, the present invention aims to provide an antibody targeting CD47 that can block the binding of CD47 and SIRPα and promote the phagocytic activity of macrophages, and in particular can prevent red blood cell agglutination to some extent and has excellent safety. means of solving the problem

[0005] The object of the present invention is an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the antibody comprises: LCDR1 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 1 or SEQ ID NO. 1; LCDR2 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 2 or SEQ ID NO. 2; and LCDR3 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 3.

[0006] The present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof comprising HCDR1 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 10 or SEQ ID NO. 10, HCDR2 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 11 or SEQ ID NO. 11, and HCDR3 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 12 or SEQ ID NO. 12.

[0007] Another object of the present invention is to provide a nucleic acid, vector, cell, or pharmaceutical composition associated with the anti-CD47 antibody or its antigen-binding fragment.

[0008] The present invention also relates to the application of the anti-CD47 antibody or its antigen-binding fragment, and its related nucleic acid, vector, cell, or pharmaceutical composition in the manufacture of drugs for treating and / or preventing CD47-positive tumors.

[0009] The present invention also relates to the application of the anti-CD47 antibody or its antigen-binding fragment, and its related nucleic acid, vector, or cell, in the manufacture of a kit for detecting CD47 or diagnosing CD47-related diseases.

[0010] The present invention also provides a kit for detecting CD47 comprising the anti-CD47 antibody or an antigen-binding fragment thereof.

[0011] The present invention also provides a kit for diagnosing CD47-related diseases comprising the anti-CD47 antibody or an antigen-binding fragment thereof. Brief explanation of the drawing

[0012] Hereinafter, in order to more clearly explain the technical solution means of the embodiments of the present invention, the drawings intended for use in the embodiments are briefly described. It should be understood that the following drawings represent only some embodiments of the present invention and should not be construed as limiting the scope, and that a person skilled in the art can obtain other related drawings based on these drawings without having to strive to create an inventive step. Figure 1 shows the average fluorescence intensity of CD47 antibodies (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to human CD47. Figure 2 shows the average fluorescence intensity of CD47 antibodies (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to human CD47. Figure 3 shows the average fluorescence intensity of CD47 antibodies (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to monkey CD47. Figure 4 shows the average fluorescence intensity of CD47 antibodies (7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to monkey CD47. Figure 5 shows the results of CD47 / SIRPα binding inhibition by CD47 antibodies against human CD47 (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42 and hIgG4-isotype control). Figure 6 shows the results of CD47 / SIRPα binding inhibition by CD47 antibodies (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55) against human CD47. Figure 7 shows the results of measuring the ability of the CD47 antibody to promote the phagocytosis of macrophages against tumor cells. Figure 8 shows the results of measuring the RBC agglutination ability of the CD47 antibody. Figure 9 shows the results of measuring the binding ability of CD47 antibodies to human red blood cells. Figure 10 shows the results of analyzing the binding of CD47 antibodies to human platelets. Figure 11 shows the results of an analysis showing that CD47 antibodies activate the phagocytic activity of macrophages against red blood cells. Figure 12 shows the antitumor results of a CD47 antibody against a human B lymphocyte subcutaneous xenograft tumor model. Figure 13 shows the antitumor results of a CD47 antibody against a human malignant melanoma model. Specific details for implementing the invention

[0013] The present invention will be further described below with specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are ordinary reagents, methods, and equipment in the art.

[0014] Unless otherwise specified, all reagents and materials used in the following examples are available on the market.

[0015] The present invention relates to an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the antibody is,

[0016] LCDR1 represented by SEQ ID NO. 1 or an amino acid sequence having at least 95% homology with SEQ ID NO. 1, LCDR2 represented by SEQ ID NO. 2 or an amino acid sequence having at least 95% homology with SEQ ID NO. 2, and LCDR3 represented by SEQ ID NO. 3 or an amino acid sequence having at least 95% homology with SEQ ID NO. 3;

[0017] It includes HCDR1 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 10 or SEQ ID NO. 10, HCDR2 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 11 or SEQ ID NO. 11, and HCDR3 represented by an amino acid sequence having at least 95% homology with SEQ ID NO. 12 or SEQ ID NO. 12.

[0018] An important advantage of the above antibody or its antigen-binding fragment is that it has a high affinity for CD47.

[0019] An important advantage of the above antibody or its antigen-binding fragment is that it has the activity of blocking the binding of CD47 and SIRPα.

[0020] An important advantage of the above antibody or its antigen-binding fragment is that it has the ability to promote the phagocytosis of macrophages against tumor cells.

[0021] An important advantage of the above antibody or its antigen-binding fragment is that it has the effect of significantly reducing red blood cell agglutination.

[0022] An important advantage of the above antibody or its antigen-binding fragment is that it exhibits a very weak level of binding to red blood cells or does not bind at all.

[0023] Due to the above characteristics, the antibody or its antigen-binding fragment can preferably be used as an antibody drug.

[0024] In the present invention, the technical term "antibody or antigen-binding fragment thereof" refers to a protein that binds to a specific antigen, and generally means any protein and protein fragment containing a complementarity determining region (CDR region). "Antibody" specifically means a full-length antibody. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies.

[0025] The term “antigen-binding fragment” is a substance comprising part or all of the CDR of an antibody that is missing at least some of the amino acids present in the full chain but is still capable of specifically binding to an antigen. Such fragments have biological activity because they bind to a target antigen and can bind to a given epitope in competition with other antigen-binding molecules (including complete antibodies). In some embodiments, the antigen-binding fragment has the function of specifically recognizing and binding to CD47. In some embodiments, the antigen-binding fragment is a fragment that blocks CD47 from binding to its ligand and activates the function of immune cells, wherein in one aspect, such fragments are selected from Fab (composed of a complete light chain and Fd), Fv (composed of VH and VL), ScFv (a single-chain antibody formed by linking VH and VL by a linker peptide), or a single-domain antibody (composed of VH alone). The fragments may be produced by recombinant nucleic acid technology or by enzymatic or chemical degradation of antigen-binding molecules (including complete antibodies).

[0026] The term "complementary determining region" or "CDR" refers to the supervariable regions of the heavy and light chains of the immunoglobulin. There are three heavy chain CDRs and three light chain CDRs. Herein, depending on the context, the terms "CDR" and "CDRs" refer to regions containing one, multiple, or all major amino acid residues that act on the binding affinity of the antibody or its antigen-binding fragment and the antigen or epitope recognized by it. In other specific embodiments, the CDR region or CDR refers to the supervariable regions of the heavy and light chains of the immunoglobulin defined by the IMGT.

[0027] In the present invention, the complementation determining region of the heavy chain is denoted as HCDR, and the complementation determining region of the light chain is denoted as LCDR. Commonly used CDR notation methods in the art include the Kabat numbering scheme, the Chothia and Lesk numbering schemes, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. first proposed a standardized numbering scheme for immunoglobulin variable regions. In their compilation of "Sequences of Proteins of Immunological Interest," the light chain (λ,κ) variable region, the amino acid sequence of the antibody heavy chain, and the variable region of the T cell receptor (α,β,γ,δ) are aligned and numbered. With the accumulation of sequences over the past few decades, the KABATMAN database has been established, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to indicate the CDR area, but the CDR area indicated by other methods also falls within the scope of protection of the present invention.

[0028] The terms "specific recognition," "selective binding," "selectively binding," and "specifically binding," or similar expressions thereof, refer to the binding of an antibody or its antigen-binding fragment to an epitope on a predetermined antigen. Generally, an antibody or its antigen-binding fragment is about 10 -6 Less than M, for example, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M or about 10 -10 Affinity less than M or lower (K D Combines with ).

[0029] In the present invention, the provided antibody or its antigen-binding fragment can specifically recognize CD47 of various species, such as humans, mice, and monkeys (e.g., cynomolgus monkeys).

[0030] In some embodiments, the antibody comprises at least one of a heavy chain variable region sequence and a light chain variable region sequence, and at least a portion of at least one of the heavy chain variable region sequence and the light chain variable region sequence is derived from at least one of a mouse-derived antibody, a humanized antibody, a primate-derived antibody, or a mutant thereof.

[0031] In some embodiments, the amino acid sequence of the light chain variable region is represented by any one of SEQ ID NOs 5 to 9 or is a sequence having at least 95% homology with any one of SEQ ID NOs 5 to 9.

[0032] In some embodiments, the amino acid sequence of the heavy chain variable region is represented by any one of SEQ ID NOs 14 to 18 or is a sequence having at least 95% homology with any one of SEQ ID NOs 14 to 18.

[0033] In some embodiments, the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 5 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 15; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 17; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 18; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 8 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; Or the amino acid sequence of the light chain variable region is indicated by SEQ ID NO. 9 and the amino acid sequence of the heavy chain variable region is indicated by SEQ ID NO. 14; or the amino acid sequence of the light chain variable region is indicated by SEQ ID NO. 7 and the amino acid sequence of the heavy chain variable region is indicated by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is indicated by SEQ ID NO. 8 and the amino acid sequence of the heavy chain variable region is indicated by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is indicated by SEQ ID NO. 9 and the amino acid sequence of the heavy chain variable region is indicated by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is indicated by SEQ ID NO. 9 and the amino acid sequence of the heavy chain variable region is indicated by SEQ ID NO. 18.

[0034] In some embodiments, the antibody has a constant region, the heavy chain constant region is any one selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is a κ chain or a λ chain.

[0035] In some embodiments, the dependent origin of the invariant region is selected from a mouse, rabbit, sheep, monkey, or human.

[0036] In some embodiments, the antibody is one or more of a CDR transplant antibody, a multimeric antibody, or a bispecific antibody.

[0037] In some embodiments, the antigen-binding fragment is one or more of F(ab')2, Fab, scFv, Fv, and a single-domain antibody.

[0038] In some embodiments, the CD47 is a human CD47, a mouse CD47, or a monkey CD47.

[0039] The present invention also relates to a nucleic acid encoding the anti-CD47 antibody or its antigen-binding fragment.

[0040] Nucleic acids are generally RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is positioned in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if a promoter or enhancer influences the transcription of a coding sequence, the promoter or enhancer is operably linked to said coding sequence. It is preferable to use DNA nucleic acids when linked to a vector.

[0041] In addition, since antibodies are membrane proteins, their nucleic acids generally contain signal peptide sequences.

[0042] The present invention also relates to a vector carrying the nucleic acid.

[0043] The term "vector" refers to a nucleic acid transport device into which a polynucleotide can be inserted. If the vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. Since a vector can be introduced into a host cell by transformation, transduction, or transfection, it enables the genetic material element carried by the vector to be expressed in the host cell. Vectors are known to those skilled in the art and include plasmids; pasmids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); Phages such as phages or M13 phages; and animal viruses, etc., are included but not limited thereto. Animal viruses that may be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). In some embodiments, the vector according to the present invention includes regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly U sequences, etc.).

[0044] The present invention also relates to a cell that carries the nucleic acid or contains the vector. A corresponding antibody can be obtained by linking a nucleic acid encoding an anti-CD47 antibody or its antigen-binding fragment to a vector and then expressing it in a cell. By introducing the vector into eukaryotic cells, particularly mammalian cells, cells capable of expressing the anti-CD47 antibody or its antigen-binding fragment according to the present invention can be constructed.

[0045] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include offspring. Accordingly, the terms "transformer" and "transformed cell" include the primary subject cell and the culture derived therefrom, regardless of the number of transfers. It should also be understood that due to intentional or unintended mutations, not all offspring may have exactly the same DNA content. Mutant offspring are included that possess the same function or biological activity as that screened in the originally transformed cell. Where a different name is intended, it is clear from the context.

[0046] The present invention also relates to a pharmaceutical composition comprising the anti-CD47 antibody or its antigen-binding fragment, the nucleic acid, the vector, or the cell.

[0047] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may include any and all physiologically suitable solvents, dispersion media, coatings, antimicrobial agents, antifungal agents, isotonic agents, and absorption retardants, etc., used to extend the shelf life or efficacy of the antibody.

[0048] The application of the anti-CD47 antibody or its antigen-binding fragment, the nucleic acid, the vector, the cell, or the composition in the manufacture of a drug for treating and / or preventing CD47-positive tumors must also fall within the scope of protection of the present invention.

[0049] The above drug can reduce red blood cell aggregation and reduce binding activity to red blood cells.

[0050] In the manufacture of a kit for detecting CD47 or diagnosing CD47-related diseases, the application of the anti-CD47 antibody or its antigen-binding fragment, the nucleic acid, the vector, or the cell must also fall within the scope of protection of the present invention.

[0051] The present invention also provides a kit for detecting CD47, said kit comprising the anti-CD47 antibody or an antigen-binding fragment thereof.

[0052] The present invention also provides a kit for diagnosing CD47-related diseases, wherein the kit comprises the anti-CD47 antibody or an antigen-binding fragment thereof.

[0053] The present invention has the following beneficial effects.

[0054] The present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the antibody does not cause red blood cell agglutination in vitro and, in particular, exhibits a very weak level of binding to red blood cells or does not bind at all; effectively blocks the binding of CD47 and SIRPα, can activate and mediate the phagocytic activity of macrophages toward tumor cells, exhibits significant target specificity toward CD47-positive tumor cells, has high affinity and strong specificity, does not cause red blood cell agglutination, exhibits very weak binding to human red blood cells and platelets, and has excellent safety. Therefore, the anti-CD47 antibody or an antigen-binding fragment thereof can be used as a very promising target in tumor immunotherapy systems, plays a powerful role in human tumor treatment, and has broad application prospects in the manufacture of drugs for the treatment of CD47-positive tumors.

[0055] Hereinafter, embodiments of the present invention will be described in detail together with examples.

[0056] Screening of anti-CD47 mouse monoclonal antibodies

[0057] 1. Construction of hCD47-ECD-HIS recombinant expression plasmid

[0058] Using a sequence provided by GenBank as a template (CEJ 95640.1), the full-length coding DNA sequence of the human CD47 extracellular region (hCD47-ECD) was synthesized, six HIS tag sequences were added to the 3' end, and the expression vector pCDNA3.4 (thermo) was cloned through the double digestion sites of EcolI at the 5' end and HindIII at the 3' end, and the recombinant eukaryotic expression plasmid of the CD47 extracellular full-length protein, namely the hCD47-ECD-HIS recombinant plasmid DNA, was constructed.

[0059] 2. Expression and Purification of hCD47-ECD-HIS Recombinant Protein

[0060] The expression and purification of the hCD47-ECD-HIS recombinant protein includes the following steps.

[0061] (1) One day before transient transfection, Expi293 (ThermoFisher Scientific; A14635) cells were subcultured and Dynamis medium (gibco; A2617502) was used 2 Х 10 6 The cells were inoculated into a 1L shaking flask (conning; 431147) at a density of [value], placed in a cell culture shaker (Adolf Kuhner; ISF4-XC), and cultured at 37°C, 8% CO2, and 120 rpm.

[0062] On the day of transfection, Expi293 cells were counted using a cell counter (Countstar; IC1000) and diluted with fresh Dynamis medium to a cell density of 2.9 x 10⁻⁶ 6The mixture was adjusted to prepare for transfection; PEI:DNA = 3:1 was mixed uniformly for 5 minutes, and both were gently mixed uniformly 20 times and left for 15 to 30 minutes. The DNA-PEI mixture was added to Expi293 cells, mixed uniformly, placed in a cell culture shaker (Adolf Kuhner; ISF4-XC), and cultured at 37°C, 8% CO2, and 120 rpm; 4 hours after transfection, a dual antibody (gibco; 15140122) and an anticoagulant (gibco; 0010057) were added.

[0063] (2) Obtaining supernatant: After transfection and culture for 7 consecutive days, samples were collected, and the cell culture supernatant was collected by first centrifuging at low speed of 1000 rpm at 4°C for 10 minutes (Xiangyi H2050R), and then centrifuging at high speed of 12000 rpm at 4°C for 30 minutes, and filtered through 0.22 μm.

[0064] (3) HisTrap affinity chromatography column purification: The supernatant was loaded onto the HisTrap affinity chromatography column at a rate of 1 mL / min. After loading was complete, the chromatography column was rinsed with 5 column volumes of 20 mM Tris-HCl, 150 mM NaCl pH 8.0 equilibrium solution; the chromatography column was rinsed with 5 column volumes of 20 mM Tris-HCl, 150 mM NaCl, 0–500 mM imidazole pH 8.0 eluent, and the elution peaks were collected. The purified CD47-ECD protein was identified by polyacrylamide gel electrophoresis (SDS-PAGE).

[0065] 3. Screening of anti-CD47 mouse monoclonal antibodies

[0066] The hCD47-ECD-HIS recombinant protein (hereinafter abbreviated as hCD47 antigen) obtained by purification in Step 2 was used for the immunization of BALB / C mice (purchased from the Guangdong Experimental Animal Center). The specific method is as follows.

[0067] (1) Animal Immunization: Purified hCD47 antigen was emulsified with a complete Freund adjuvant and 6-8 week old BALB / C mice were immunized with an immunizing dose of 25 μg / mice using subcutaneous or intraperitoneal injection, with 5 mice per group; a second immunization was performed at 1-week intervals with an immunizing dose of 25 μg / mice, emulsified with an incomplete Freund adjuvant. After 4 immunizations, blood was collected from the tail and serum titers were measured by gradient dilution using the ELISA method; 3 days before fusion, immunity was boosted, and the mouse with the highest antibody titer was selected to perform cell fusion.

[0068] (2) Cell Fusion: Sp2 / 0 (ATCC® CRL-1581) derived from BALB / C was used as the myeloma cells, and were in the logarithmic growth phase at the time of fusion; the spleen of an immunized mouse was taken to prepare a single-cell lymphocyte suspension; mouse spleen lymphocytes and myeloma cells were mixed in a ratio of 1:5 to 1:10, 1 mL of 50% PEG1500 (pH 8.0) at 37°C was added dropwise, incomplete medium DMEM and the remaining stop solution were added, the supernatant was removed by centrifugation, HAT medium was added to suspend the cells, and the mixture was uniformly mixed and plated in a 96-well plate, and cultured in an incubator at 37°C and 5% CO2. After culturing for 1 week, the medium was replaced with HT medium for the first time, and after culturing for 3 days, the medium was replaced with HT medium for the second time.

[0069] (3) Screening and Cloning: After 2 weeks of fusion, the cell supernatant was taken and an ELISA test was performed to detect the binding status of the cell supernatant and the purified CD47-ECD-His recombinant protein. Cells with positive ELISA results were screened, and a second ELISA test was performed to detect the results again.

[0070] Cells in positive wells were reduced to limit dilutions, and ELISA values ​​were measured on day 7 after each limit dilution. Single clone wells with high OD280 positive values ​​were selected and reduced to limit dilutions until the entire 96-well plate was found to be positive. Single clones with high positive values ​​were selected.

[0071] (4) Preparation and purification of monoclonal antibodies from cell supernatant: Positive clones were cultured in T25 culture flasks using DMEM medium containing 15% serum; during extended culture, centrifugation was performed at 800 rpm / min for 5 minutes, and after removing the supernatant, the cells were transferred to a 500 mL shaking flask, and serum-free medium (Hybridoma-SFM Complete DPM; Gibco; 12300-067) was added until the cell density was approximately 3 x 10⁻¹⁰ 5 The concentration was adjusted to cells / mL. After continuing culture for 1–2 weeks, when the cell death rate reached 60–70%, the cell suspension was collected and centrifuged at a high speed of 8000 rpm / min for 20 minutes. The supernatant was collected and purified by affinity chromatography, and the antibody was purified using Protein A affinity chromatography. Samples were loaded; rinsed with pH 7.4, 2.5 M PBS until the UV280 baseline was 0; eluted with pH 3.5, 0.1 M citric acid solution; elution tubes were collected in 2 mL portions; 100 μL of 1 M Tris was added to each tube; the collected solution was concentrated; the concentration of the purified monoclonal antibody was measured and confirmed using an SDS-PAGE gel; aliquots (100 μL / tube) were taken and stored at -80°C.

[0072] Humanization Design and Combination of Mouse Monoclonal Antibodies

[0073] The amino acid sequences of the mouse antibody light chain CDR are indicated by sequence numbers 1 to 3.

[0074]

[0075] The amino acid sequences of the mouse antibody heavy chain CDR are represented by sequence numbers 10 to 12.

[0076]

[0077] The amino acid sequence of the mouse antibody light chain variable region (>7A11D3D3-VL) is denoted by SEQ ID NO. 4. The amino acid sequence of the mouse antibody heavy chain variable region (>7A11D3D3-VH) is denoted by SEQ ID NO. 13.

[0078] Humanization design is performed on mouse antibodies to design five humanization sequences, each 7A11-L01~05, for the light chains of the 7A11D3D3 mouse monoclonal antibody, and the amino acid sequences of the humanized light chain variable regions (7A11-L01~05) are indicated by SEQ ID NOs 5~9; five humanization sequences, each 7A11-H01~05, are designed for the heavy chains, and the amino acid sequences of the humanized heavy chain variable regions (7A11-H01~05) are indicated by SEQ ID NOs 14~18.

[0079] The antibody obtained by combining the above amino acid sequences and the sequences corresponding to its heavy chain variable region (VH) and light chain variable region (VL) are shown in Table 1.

[0080] Antibody and sequences corresponding to its heavy chain variable region and light chain variable region Antibody names VH VL 7A11H11 Sequence No. 14 Sequence No. 5 7A11H12 Sequence No. 14 Sequence number 6 7A11H22 Sequence number 15 Sequence number 6 7A11H32 Sequence number 16 Sequence number 6 7A11H42 Sequence number 17 Sequence number 6 7A11H52 Sequence number 18 Sequence number 6 7A11H14 Sequence No. 14 Sequence number 8 7A11H15 Sequence No. 14 Sequence number 9 7A11H33 Sequence number 16 Sequence number 7 7A11H34 Sequence number 16 Sequence number 8 7A11H35 Sequence number 16 Sequence number 9 7A11H55 Sequence number 18 Sequence number 9

[0081] production of CD47 antibodies

[0082] The humanization variable domain, secretory signal, and human κ and human FcIgG4S228P constant domains were combined, cloned into a mammalian expression system, and transfected into 293 cells to produce humanized mAbs. The humanized variants were expressed as full-length IgG molecules, secreted into the medium, and purified using Protein A.

[0083] The humanized antibody and the positive control antibody Hu5F9-G4 (the sequence of Hu5F9-G4 is disclosed in U.S. Patent US2015 / 0183874A1) were transiently expressed and purified in Expi293 cells.

[0084] For the transient expression of antibodies in Expi293 cells, the heavy and light chains of the antibody were first cloned into separate pCDNA3.4 vectors using the vector pCDNA3.4. Then, the pCDNA3.4 vectors containing the heavy and light chains of the antibody molecule were transfected into Expi293 cells according to the chemical transfection method using PEI (purchased from Polysciences) chemical transfection reagent, and the cultured Expi293 cells were transiently transfected according to the instructions provided by the manufacturer.

[0085] One day before transient transfection, Expi293 (ThermoFisher Scientific; A14635) cells were subcultured and inoculated into a 1L shaking flask (conning; 431147) at a density of 2E6 using Dynamis medium (gibco; A2617502), placed in a cell culture shaker (Adolf Kuhner; ISF4-XC), and cultured at 37°C, 8% CO2, and 120 rpm.

[0086] On the day of transfection, Expi293 cells were counted using a cell counter (Countstar; IC1000) and diluted with fresh Dynamis medium to adjust the cell density to 2.9E6; preparation for transfection was made; PEI:DNA = 3:1 was mixed uniformly for 5 minutes, and both were gently mixed uniformly 20 times and left for 15 to 30 minutes. The DNA-PEI mixture was added to Expi293 cells, mixed uniformly, placed in a cell culture shaker (Adolf Kuhner; ISF4-XC), and cultured at 37°C, 8% CO2, and 120 rpm; 4 hours after transfection, a dual antibody (gibco; 15140122) and an anticoagulant (gibco; 0010057) were added.

[0087] Collection and Purification of Supernatant: Samples were collected after continuous transfection and culture for 7 days. The cells were first centrifuged at low speed (1000 rpm) at 4°C for 10 minutes (Xiangyi H2050R), followed by high speed (12000 rpm) at 4°C for 30 minutes to collect the cell culture supernatant, which was then filtered through a 0.22 μm sieve. The culture supernatant was applied to a Protein A Sepharose column (GE Healthcare). The column was washed with PBS, and the protein was eluted using elution buffer (0.1 M sodium citrate buffer, pH 3.0). The collected components were neutralized with 1 M Tris pH 9.0. Finally, the purified samples were dialyzed with PBS.

[0088] CD47 antibody affinity measurement

[0089] 1. Experimental Method

[0090] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human CD47 (hCD47) was measured using a biolayer interferometer (ForteBio). ForteBio affinity was measured according to a conventional method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs; 2013.5(2): p.270-8). Specifically, a baseline was established by equilibrating the sensor offline in the analysis buffer for 30 minutes and then detecting it online for 60 seconds. The purified antibody obtained in Example 3 was loaded onto the AHC sensor (ForteBio) to measure ForteBio affinity online. After exposing the sensor containing the loaded antibody to 100 nM CD47 antigen for 5 minutes, the sensor was transferred to the analysis buffer and dissociated for 5 minutes to measure the dissociation rate. A kinetic analysis was performed using a 1:1 binding model.

[0091] 2. Experimental Results

[0092] The results of measuring the affinity of the CD47 antibody are shown in Table 2, and the results show that the CD47 antibody has high affinity.

[0093] CD47 antibody affinity measurement results Antibody names KD(M) kon(1 / Ms) kd(1 / s) 7A11H11 5.90E-10 5.90E-10 5.90E-10 7A11H12 4.59E-10 4.59E-10 4.59E-10 7A11H14 5.62E-10 5.62E-10 5.62E-10 7A11H15 2.12E-10 1.48E+06 3.13E-04 7A11H35 8.81E-10 8.81E-10 8.81E-10 7A11H22 7.40E-10 7.40E-10 7.40E-10 7A11H33 3.60E-10 3.60E-10 3.60E-10 7A11H42 3.09E-10 3.09E-10 3.09E-10

[0094] Binding of CD47 antibody and human CD47

[0095] 1. Experimental Method

[0096] The binding of the CD47 antibody of the present invention to human CD47 was measured using a measurement method based on flow cytometry. The specific steps are as follows.

[0097] The human CD47-expressing cancer cell line CCRF-CEM (Shanghai Cell Bank, Chinese Academy of Sciences) belongs to human acute lymphoblastic leukemia T lymphocytes. CCRF-CEM cells (0.1×10⁻⁶ 6 Experimental antibodies (the CD47 antibody and Hu5F9-G4 antibody of the present invention) at different concentrations (maximum concentration 30 μg / mL, 3-fold dilution, total of 10 concentrations) were incubated on ice in PBS containing 3% bovine serum albumin (BSA) for 30 minutes. Then, the cells were washed at least twice, and the PE Goat anti-human IgG Fc (1:500x dilution) fluorescent secondary antibody was prepared with FCM buffer (1XPBS+3%BSA), added to the corresponding 96-well plate at a rate of 100 μL / well, and incubated in a 4°C refrigerator for 30 minutes. The 96-well plate was removed and centrifuged at 250g for 5 minutes, and the supernatant was carefully removed. Then, 200μL / well of FCM buffer was added and centrifuged again at 250g for 5 minutes, and the supernatant was carefully removed. The cells were washed at least twice, resuspended in 100μL / well of 1xPBS, analyzed via flow cytometry, and a concentration-dependent curve was fitted using GraphPad according to the MFI. Hu5F9-G4 antibody was used as the positive control antibody.

[0098] 2. Experimental Results

[0099] The EC50 results of CD47 antibodies binding to human CD47 are shown in Table 3, and the average fluorescence intensity of CD47 antibodies binding to human CD47 (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and hIgG4-isotype control) is shown in Fig. 1, and the average fluorescence intensity of CD47 antibodies binding to human CD47 (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) is shown in Fig. 2, and the results show that the CD47 antibodies of the present invention, compared to positive control antibodies, [attach] to human CD47 at the cellular level It demonstrates that it possesses significant specific binding ability.

[0100] Results of measuring the binding ability of CD47 antibodies and human CD47 Antibody names EC50(nM) 7A11H11 0.8218 7A11H12 0.7157 7A11H22 0.8202 7A11H32 0.7172 7A11H42 0.7269 7A11H52 0.9148 7A11H14 0.9334 7A11H15 0.7234 7A11H33 0.7598 7A11H34 1.195 7A11H35 0.8623 7A11H55 1.209 Hu5F9-G4 0.7289

[0101] Conjugation of CD47 antibody and monkey CD47

[0102] 1. Experimental Method

[0103] Produce a stable CHO cell line (CHO-cynoCD47 cells) overexpressing monkey CD47 by transfecting with a pCDNA3.4 vector carrying whole-length monkey CD47, and CHO-cynoCD47 cells (0.1X10 6Experimental antibodies (the CD47 antibody and Hu5F9-G4 antibody of the present invention) at different concentrations (maximum concentration 10 μg / mL, 3-fold dilution, total 10 concentrations) were incubated on ice in PBS containing 3% bovine serum albumin (BSA) for 30 minutes. Then, the cells were washed at least twice, and the PE Goat anti-human IgG Fc (1:500x dilution) fluorescent secondary antibody was prepared with FCM buffer (1XPBS+3% BSA), added to the corresponding 96-well plate at a rate of 100 μL / well, and incubated in a 4°C refrigerator for 30 minutes. The 96-well plate was removed and centrifuged at 250g for 5 minutes, and the supernatant was carefully removed. Then, 200μL / well of FCM buffer was added and centrifuged again at 250g for 5 minutes, and the supernatant was carefully removed. The cells were washed at least twice, resuspended in 100μL / well of 1xPBS, analyzed via flow cytometry, and a concentration-dependent curve was fitted using GraphPad according to the MFI. Hu5F9-G4 antibody was used as the positive control antibody.

[0104] 2. Experimental Results

[0105] The EC50 results of CD47 antibodies binding to monkey CD47 are shown in Table 4, and the average fluorescence intensity of CD47 antibodies binding to monkey CD47 (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4, and hIgG4-isotype control) is shown in Fig. 3, and the average fluorescence intensity of CD47 antibodies binding to monkey CD47 (7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4, and hIgG4-isotype control) is shown in Fig. 4, and the results indicate that the antibodies of the present invention are significantly specific to monkey CD47 at the cellular level compared to the positive control antibodies. It demonstrates that it possesses binding ability.

[0106] EC50 results of CD47 antibodies binding to monkey CD47 Antibody names EC50(nM) 7A11H11 8.932 7A11H12 7.353 7A11H22 8.546 7A11H32 17.12 7A11H42 5 7A11H52 4.506 7A11H14 4.957 7A11H15 7.271 7A11H33 4.847 7A11H34 8.609 7A11H35 4.379 7A11H55 4.616 Hu5F9-G4 5.123

[0107] Blocking of the interaction between human CD47 ligand SIRPα and CD47 by CD47 antibodies

[0108] 1. Experimental Method

[0109] The ability of the CD47 antibody of the present invention to block the binding of human CD47 and SIRPα was measured through flow cytometry analysis. The specific steps are as follows.

[0110] Antibody dilution: The CD47 antibody of the present invention and the control antibody Hu5F9-G4 were diluted to 90 μg / mL in FCM buffer (1XPBS+3%BSA), then diluted to 10 concentrations in a 3-fold gradient, the subtype control hIgG4 was diluted to 30 μg / mL, 1.1 μg / mL, and 0.04 μg / mL, and the ligand hSIRPα-mFC (AcroBiosystems) was diluted to 10 μg / mL.

[0111] CCRF-CEM (Shanghai Cell Bank of the Chinese Academy of Sciences) cells in 0.1 x 10⁻⁶ V-bottom 96-well plates 6 hSIRPα-mFC binding was monitored under conditions where the amount of CD47 antibody was increased after adding to the cells / well. The bound SIRPα was determined using the PE Goat anti-mouse IgG Fc secondary antibody (Biolegend). The Hu5F9-G4 antibody was used as the positive control antibody.

[0112] 2. Experimental Results

[0113] The results of CD47 / SIRPα binding inhibition by CD47 antibodies against human CD47 (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, Hu5F9-G4 and hIgG4-isotype control) are shown in Fig. 5, and the results of CD47 / SIRPα binding inhibition by CD47 antibodies against human CD47 (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55) are shown in Fig. 6. The results show that the CD47 antibodies of the present invention can significantly inhibit and block the binding of CD47 and SIRPα at the cellular level, and that the CD47 antibodies of the present invention exhibit significant blocking ability compared to positive control antibodies.

[0114] Detection of the ability of CD47 antibodies to promote the phagocytosis of macrophages against tumor cells

[0115] 1. Experimental Method

[0116] The ability of the CD47 antibody to promote the phagocytosis of macrophages against tumor cells was measured using a flow cytometry-based measurement method. The specific steps are as follows.

[0117] Fresh blood was collected from a donor to isolate peripheral blood mononuclear cells (PBMCs), CD14-positive mononuclear cells were isolated from PBMCs using hCD14 magnetic beads (Miltenyi / 130-050-201), and a complete medium containing rhGM-CSF (R&D; 7954-GM-010) with a final rhGM-CSF concentration of 50 ng / mL and a CD14-positive mononuclear cell concentration of 5E5 / mL was prepared and added to a cell culture dish at a rate of 20 mL / dish; the dish was transferred to a cell incubator at 5% CO2 and 37°C, half of which was replaced with fresh medium every 3 days; and the culture was continued for 4 days (containing 50 ng / mL GM-CSF). On the 8th day, the macrophage supernatant was pipetted into a 15 mL centrifuge tube, pre-cooled DPBS was added simultaneously, and the cells were collected directly using a cell scraper.

[0118] The human CD47-expressing tumor cell line Jurkat (Shanghai Cell Bank, Chinese Academy of Sciences) was selected as the target cell type, and the target tumor cells were fluorescently labeled according to the instructions of the CellTrace™ CFSE kit. The labeled tumor cells were co-cultured with the aforementioned differentiated macrophages in a 1:1 ratio, and simultaneously incubated with antibodies at final concentrations of 10 μg / mL, 1 μg / mL, and 0.1 μg / mL at 37°C for 2 hours. Subsequently, the cells were washed at least twice, carefully blown out, and allophycocyanin (APC)-labeled CD14 antibody (purchased from Biolegend, B259538) was added. The cells were then incubated on ice (avoided of light) in PBS containing 0.1% BSA for 30 minutes. The cells were washed at least twice and analyzed by flow cytometry. The phagocytic cell population is CD14 positive in living cells and is also positive for the fluorescent dye CFSE (carboxyfluorescein diacetate, succinimidyl ester). The Hu5F9-G4 antibody was used as a positive control antibody.

[0119] 2. Experimental Results

[0120] The results of measuring the ability of the CD47 antibody to promote the phagocytosis of macrophages against tumor cells are shown in Figure 7, and the results show that the CD47 antibody of the present invention has the ability to promote the phagocytosis of macrophages against tumor cells and that the ability to promote the phagocytosis of macrophages against tumor cells is equivalent to the ability of the positive control antibody Hu5F9-G4.

[0121] Analysis of CD47 antibodies against human red blood cell (RBC) agglutination

[0122] 1. Experimental Method

[0123] The RBC agglutination ability of CD47 antibodies was characterized by performing red blood cell agglutination analysis. CD47 antibodies were screened for RBC agglutination by observing the antibody's ability to avoid the precipitation of human RBCs. The specific method is as follows.

[0124] Human red blood cells were diluted to 2% in PBS and incubated in a round-bottom 96-well plate at 37°C for 2 hours with titrated CD47 antibody (concentrations were sequentially 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 1.5625 μg / mL, 0.78125 μg / mL, 0.390625 μg / mL, 0.195313 μg / mL, and 0.097656 μg / mL). Evidence of red blood cell agglutination was demonstrated by the presence of unprecipitated red blood cells, which appeared faint compared to the clear red dots formed by the precipitation of unagglutinated red blood cells. Hu5F9-G4 antibody was used as a positive control antibody.

[0125] 2. Experimental Results

[0126] The results of measuring the RBC agglutination ability of the CD47 antibody are shown in Figure 8, and the results show that no cell agglutination occurred when the CD47 antibody concentration reached 200 μg / mL. This demonstrates that the CD47 antibody of the present invention has a function of significantly reducing red blood cell agglutination, and that the CD47 antibody can significantly reduce side effects in the clinical treatment of cancer and has excellent safety.

[0127] Analysis of CD47 antibody binding to human red blood cells (RBCs)

[0128] 1. Experimental Method

[0129] CD47 monoclonal antibodies have the characteristic of binding to human red blood cells; however, in the case of CD47 antibody inhibitors, their efficacy is interfered with by red blood cells, posing a potential risk of tumor off-targeting. If antibodies with low binding activity to red blood cells can be screened, the risk of off-targeting can be reduced and safety can be enhanced. The specific steps are as follows.

[0130] (1) Antibody dilution: CD47 antibody was diluted with FACS buffer to 11 concentrations, with an initial concentration of 20 μg / mL, a volume of 180 μL, and a 3-fold gradient dilution (60+120).

[0131] (2) Cell counting and plating: RBC cells were centrifuged at 250g for 5 minutes, the supernatant was removed, the cell density was adjusted to 2E+06 with FACS buffer, and 100μL / tube was evenly distributed into a V-bottom 96-well plate.

[0132] (3) The above diluted antibody was added to the cells at a rate of 100 μL / well and cultured at 2°C to 8°C for 0.5 hours.

[0133] (4) Remove the 96-well plate, centrifuge at 250g for 5 minutes, carefully remove the supernatant, add 200μL / well of FACS buffer, centrifuge again at 250g for 5 minutes, and carefully remove the supernatant.

[0134] (5) PE goat anti-human IgG Fc (biolegend) fluorescent secondary antibody (1:500 dilution) was prepared with FACS buffer, 100 μL / well was added to the corresponding 96-well plate, cells were resuspended, and cultured at 2°C to 8°C for 30 minutes.

[0135] (6) Remove the 96-well plate, centrifuge at 250g for 5 minutes, carefully remove the supernatant, add 200μL / well of FACS buffer, centrifuge again at 250g for 5 minutes, and carefully remove the supernatant.

[0136] (7) The sample was resuspended in 100 μL / well of 1x PBS and detected by FACS. Data were analyzed using a flow cytometer (Beckman, cytoflex) and graphs were created using GraphPad Prism. Hu5F9-G4 antibody was used as a positive control antibody.

[0137] 2. Experimental Results

[0138] The results of measuring the binding ability of the CD47 antibody to human red blood cells are shown in Fig. 9, and the results show that the binding activity of the CD47 antibody of the present invention to human red blood cells is significantly reduced compared to a positive control antibody, and safety is improved when used as a drug.

[0139] Analysis of CD47 antibody and platelet binding

[0140] 1. Experimental Method

[0141] Just as CD47 monoclonal antibodies bind to human red blood cells, they possess active properties that bind to platelets, and they are associated with many side effects due to thrombocytopenia. Antibodies with low binding to platelets can reduce off-target risks and improve safety. The specific methods are as follows.

[0142] Antibody dilution: The antibody was diluted with FACS buffer to 20 μg / mL, in a volume of 240 μL.

[0143] Cell counting and plating: Whole blood cells were diluted 20-fold and evenly distributed at 100 μL / tube into a V-bottom 96-well plate.

[0144] The above diluted antibody was added to cells at a rate of 100 μL / well and cultured at 2°C to 8°C for 0.5 hours.

[0145] The 96-well plate was removed, centrifuged at 250g for 5 minutes, and the supernatant was carefully removed. Then, 200μL / well of FACS buffer was added and the plate was centrifuged again at 250g for 5 minutes, and the supernatant was carefully removed.

[0146] PE fluorescent secondary antibody (1:500 dilution) (PE goat anti-human IgG Fc; biolegend; 409304) was prepared in FACS buffer and added to the corresponding 96-well plate at a rate of 100 μL / well, while simultaneously adding 1 μL of APC anti-human CD61 (biolegend; 336411) to each well, resuspending the cells, and incubating at 2°C–8°C for 30 minutes.

[0147] The 96-well plate was removed, centrifuged at 250g for 5 minutes, and the supernatant was carefully removed. Then, 200μL / well of FACS buffer was added and the plate was centrifuged again at 250g for 5 minutes, and the supernatant was carefully removed.

[0148] The sample was resuspended in 200 μL / well of 1xPBS and detected by FACS. Hu5F9-G4 antibody was used as a positive control antibody.

[0149] 2. Experimental Results

[0150] The results of the binding analysis of the CD47 antibody and platelets are shown in Fig. 10, and the results show that the binding activity of the CD47 antibody of the present invention to platelets is significantly lower than that of the positive control antibody, indicating superior safety.

[0151] Analysis of CD47 antibody activation against macrophage phagocytosis of red blood cells

[0152] 1. Experimental Method

[0153] The detection method was the same as in Example 8, where red blood cells were replaced with tumor cells as target cells, and whether the CD47 antibody of the present invention activated the phagocytic activity of macrophages against red blood cells was detected. The Hu5F9-G4 antibody was used as a positive control antibody.

[0154] 2. Experimental Results

[0155] The results of analyzing the activation of the phagocytic activity of macrophages against red blood cells by the CD47 antibody are shown in Figure 11, and the results show that the CD47 antibody of the present invention has an extremely low degree of mediation of the phagocytic activity of macrophages against red blood cells, and the mediation activity is significantly lower than that of the positive control antibody, showing superior safety.

[0156] Antitumor activity analysis

[0157] 1. Experimental Method

[0158] Seventy female NOD SCID mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected to establish a human B lymphocyte subcutaneous xenograft tumor model (Raji) and a human malignant melanoma model (A375) to evaluate the antitumor activity of the CD47 antibody of the present invention. Hu5F9-G4 antibody and TJC-4 antibody were used as positive control antibodies, and hIgG4 was used as an isotype control antibody.

[0159] 2. Experimental Results

[0160] The antitumor results of the CD47 antibody against a human B lymphocyte subcutaneous xenograft tumor model are shown in Fig. 12, and the results show that the CD47 antibody of the present invention is significantly superior to the positive control antibody in the anti-human B lymphocyte subcutaneous xenograft tumor effect.

[0161] The antitumor results of the CD47 antibody against a human malignant melanoma model are shown in Fig. 13, and the results show that the CD47 antibody of the present invention is significantly superior to the positive control antibody in the anti-human malignant melanoma effect.

[0162] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments, and any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principles of the present invention are all equivalent alternatives and should all be included within the scope of protection of the present invention.

Claims

Claim 1 As an anti-CD47 antibody or an antigen-binding fragment thereof, said antibody comprises: A) HCDR1, HCDR2, and HCDR3 are identical to the complementarity determining region of SEQ ID NO. 14, LCDR1, LCDR2, and LCDR3 are identical to the complementarity determining region of SEQ ID NO. 8, and said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by Kavat numbering; B) HCDR1, HCDR2, and HCDR3 are identical to the complementarity determining region of SEQ ID NO. 14, LCDR1, LCDR2, and LCDR3 are identical to the complementarity determining region of SEQ ID NO. 6, and said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by Kavat numbering; or C) HCDR1, HCDR2, and HCDR3 are identical to the complementarity determining region of SEQ ID NO. 17, LCDR1, LCDR2, and LCDR3 are identical to the complementarity determining region of SEQ ID NO. 6, and said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by Kavat numbering; an anti-CD47 antibody or an antigen-binding fragment thereof characterized by including a complementarity determining region (CDRs). Claim 2 An anti-CD47 antibody or an antigen-binding fragment thereof, wherein, in claim 1, the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, and at least a portion of the heavy chain variable region sequence and the light chain variable region sequence is derived from at least one of a mouse-derived antibody, a humanized antibody, a primate-derived antibody, or a mutant thereof. Claim 3 An anti-CD47 antibody or its antigen-binding fragment, characterized in that, in paragraph 2, the amino acid sequence of the light chain variable region is represented by any one of SEQ ID NOs 5 to 9. Claim 4 An anti-CD47 antibody or its antigen-binding fragment, characterized in that, in paragraph 2, the amino acid sequence of the heavy chain variable region is represented by any one of SEQ ID NOs 14 to 18. Claim 5 In paragraph 2, the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 5 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 15; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 17; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 6 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 18; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 8 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; An anti-CD47 antibody or an antigen-binding fragment thereof, characterized in that the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 9 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 14; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 7 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 8 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 9 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO. 16; or the amino acid sequence of the light chain variable region is denoted by SEQ ID NO. 9 and the amino acid sequence of the heavy chain variable region is denoted by SEQ ID NO.

18. Claim 6 An anti-CD47 antibody or an antigen-binding fragment thereof, characterized in that, in claim 1, the antibody has a constant region, the heavy chain constant region is any one selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; and the light chain constant region is a κ chain or a λ chain. Claim 7 In claim 6, the anti-CD47 antibody or its antigen-binding fragment is characterized in that the dependent origin of the invariant region is selected from a mouse, rabbit, sheep, monkey, or human. Claim 8 An anti-CD47 antibody or its antigen-binding fragment, characterized in that, in claim 6, the light chain constant region is a human κ chain and the heavy chain constant region is a human IgG4 having an S228P mutant. Claim 9 An anti-CD47 antibody or an antigen-binding fragment thereof, characterized in that, in paragraph 2, the light chain constant region is a human κ chain and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 8, the heavy chain constant region is a human IgG4 having an S228P mutant, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.

14. Claim 10 An anti-CD47 antibody or an antigen-binding fragment thereof, characterized in that, in claim 1, the antibody is one or more of a CDR transplant antibody, a multimeric antibody, or a bispecific antibody. Claim 11 An anti-CD47 antibody or its antigen-binding fragment according to claim 1, characterized in that the antigen-binding fragment is F(ab')2, Fab, scFv, or Fv. Claim 12 An anti-CD47 antibody or an antigen-binding fragment thereof, characterized in that, in claim 1, the CD47 is human CD47, mouse CD47, or monkey CD47. Claim 13 A nucleic acid characterized by encoding an anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. Claim 14 A vector characterized by containing nucleic acid according to Clause 13. Claim 15 Isolated cell characterized by comprising a nucleic acid according to paragraph 13 or a vector containing said nucleic acid. Claim 16 A pharmaceutical composition comprising an anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, a nucleic acid encoding said anti-CD47 antibody or the antigen-binding fragment thereof, a vector comprising said nucleic acid or isolated cells comprising said nucleic acid or said vector, wherein the pharmaceutical composition is characterized by treating and / or preventing CD47-positive tumors, reducing red blood cell agglutination, or reducing the binding activity of said pharmaceutical composition to red blood cells. Claim 17 A pharmaceutical composition characterized by further comprising a pharmaceutically acceptable carrier in claim 16. Claim 18 A kit for detecting CD47 characterized by comprising an anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. Claim 19 A kit for diagnosing a CD47-positive tumor characterized by comprising an anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. Claim 20 An anti-CD47 antibody according to any one of claims 1 to 12 or an antigen-binding fragment thereof used as a drug capable of treating and / or preventing CD47-positive tumors, reducing red blood cell aggregation, or reducing the binding activity of a pharmaceutical composition to red blood cells. Claim 21 An anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 used in a method for treating and / or preventing CD47-positive tumors. Claim 22 delete Claim 23 delete

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