RU2832773C1 - Cd47-targeted antibody and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to an anti-CD47 antibody or its antigen-binding fragment, as well as to a pharmaceutical composition and a kit containing it. Also disclosed is a nucleic acid coding said antibody or its fragment, as well as a vector and a cell containing it.

EFFECT: invention is effective for treating a CD47-positive tumour.

25 cl, 13 dwg, 4 tbl, 13 ex

Description

Field of technology to which the invention relates

The present invention relates to the technical field of biological medicinal products. In particular, the invention relates to an antibody targeting CD47 and its use, and more specifically relates to an anti-CD47 antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a cell, a composition, a use, and a kit.

Prior art

Cancer immunotherapy is a major development in the field of biological sciences in recent years. Immune checkpoint inhibitor therapy, such as T cell-based therapy, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, etc., and cell therapy using CAR-T, TCR-T, etc. are all the main immunotherapeutic approaches in recent years. These approaches are all related to the restoration of the function of all T cells without exception, in other words, to the improvement of the acquired immune system's ability. However, the path of using the immune checkpoint as a target and activating the function of T cells to improve the ability of the acquired immune system to defeat cancer is still full of twists and turns. However, the effect of the innate immune system in tumor immunotherapy has not been evident for a long time. In fact, in the entire tumor infiltration area, macrophages account for about 50% of tumor tissues. More importantly, the number of macrophages is inversely related to tumor prognosis, further indicating that macrophages have a very important effect on tumors. 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 surface of the same target. The absence of either signal is not sufficient to trigger phagocytosis. Increasing evidence suggests that CD47 is a type of “do not eat me” signal and inhibits macrophage phagocytosis by cross-linking with signal regulatory protein α (SIRPα) on the surface of macrophages. Tumor cells can also escape phagocytosis by macrophages by expressing CD47 (e.g., EP 2242512 and related documents cited therein).

CD47, also known as 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 multiple ligands including, but not limited to, SIRPα, SIRPγ, integrin, and thrombospondin-1 (TSP-1). SIRPα is primarily expressed on myeloid cells, including macrophages, myeloid dendritic cells (DCs), granulocytes, mast cells and their precursors, and hematopoietic stem cells. The CD47/SIRPα interaction transmits a “don’t eat me” signal and prevents autophagy. Analysis of patient tumors and corresponding adjacent normal (non-tumor) tissues shows that CD47 protein is overexpressed on malignant tumor cells, effectively helping them to evade innate immune surveillance and elimination. It has already been shown that blocking the CD47-SIRPα interaction with an anti-CD47 antibody effectively induces tumor cell phagocytosis in vitro and inhibits the growth of various hematological and solid tumors in vivo. Thus, CD47 is an effective target for cancer therapy, and its corresponding antagonist is required to obtain therapeutic agents for humans.

The essence of the invention

The monoclonal anti-CD47 antibody has a higher ability to target red blood cells and easily causes agglutination of red blood cells, so that the therapeutic effect of the corresponding antibody is significantly reduced, and side effects of drugs occur. The present invention is intended to provide an antibody targeting CD47 that can block the binding of CD47 to SIRPα and promote phagocytosis of macrophages. More importantly, the ability of this antibody to prevent red blood cell agglutination to a certain extent and good safety are also important.

The aim of the present invention is to create an anti-CD47 antibody or antigen-binding fragment thereof, wherein said antibody comprises the following complementarity determining regions (CDRs):

LCDR1 shown in SEQ ID NO:1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, LCDR2 shown in SEQ ID NO:2 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 and LCDR3 shown in SEQ ID NO:3 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3; and

HCDR1 shown in SEQ ID NO:10 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:12 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:12.

Another object of the present invention is to provide a nucleic acid, vector, cell or pharmaceutical composition relating to an anti-CD47 antibody or antigen-binding fragment thereof.

The present invention also relates to the use of an anti-CD47 antibody or antigen-binding fragment thereof and a nucleic acid, vector, cell or pharmaceutical composition related thereto for the production of a medicament for the treatment and/or prevention of a CD47-positive tumor.

The present invention also relates to the use of an anti-CD47 antibody or antigen-binding fragment thereof and a nucleic acid, vector or cell related thereto for the preparation of a kit for detecting CD47 or diagnosing a CD47-associated disease.

The present invention further provides a kit for detecting CD47, which comprises an anti-CD47 antibody or an antigen-binding fragment thereof.

The present invention further provides a kit for diagnosing a disease associated with CD47, which comprises an anti-CD47 antibody or an antigen-binding fragment thereof.

Brief description of the drawings

In order to more clearly describe specific embodiments of the present invention, the drawings necessary for their disclosure are briefly presented below. It should be understood that the drawings show only some embodiments of the present invention, but this should not be considered as limiting the scope of the present invention. Other drawings can also be obtained by those skilled in the art in accordance with these drawings without creative work.

Fig. 1 represents the mean fluorescence intensity of anti-CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to human CD47.

Fig. 2 represents the mean fluorescence intensity of anti-CD47 antibody (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to human CD47.

Fig. 3 represents the mean fluorescence intensity of anti-CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to monkey CD47.

Fig. 4 represents the mean fluorescence intensity of anti-CD47 antibody (7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to monkey CD47.

Fig. 5 shows the result of inhibition of CD47/SIRPα (human CD47) binding by anti-CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42 and isotype control hIgG4).

Fig. 6 shows the result of inhibition of CD47/SIRPα (human CD47) binding by anti-CD47 antibody (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, and 7A11H55).

Fig. 7 is the result of testing the ability of anti-CD47 antibody to stimulate macrophage phagocytosis of tumor cell.

Fig. 8 shows the result of testing the red blood cell (RBC) agglutination ability of CD47 antibody.

Fig. 9 is the result of testing the ability of anti-CD47 antibody to bind to human red blood cell.

Fig. 10 is the result of the binding assay of anti-CD47 antibody to human platelets.

Fig. 11 is the result of analysis of the effect of anti-CD47 antibody on erythrocyte phagocytosis by activated macrophage.

Fig. 12 shows the result of antitumor effect of anti-CD47 antibody in human B-lymphocyte subcutaneous xenograft model.

Fig. 13 is the result of the antitumor effect of anti-CD47 antibody on human malignant melanoma model.

Detailed description of incarnations

The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise indicated, the reagents, methods and devices used in the present description are conventional reagents, methods and devices in the art.

Unless otherwise indicated, reagents and materials used in the embodiments presented below are commercially available.

The present invention relates to an anti-CD47 antibody or antigen-binding fragment thereof, and said antibody comprises the following CDRs:

LCDR1 shown in SEQ ID NO:1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, LCDR2 shown in SEQ ID NO:2 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 and LCDR3 shown in SEQ ID NO:3 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3; and

HCDR1 shown in SEQ ID NO:10 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:12 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:12.

An important advantage of the antibody or its antigen-binding fragment is its high affinity for CD47.

An important advantage of the antibody or its antigen-binding fragment is that it has the ability to block the binding of CD47 to SIRPα.

An important advantage of an antibody or its antigen-binding fragment is its ability to stimulate a macrophage to phagocytose a tumor cell.

An important advantage of the antibody or its antigen-binding fragment is that it significantly reduces the agglomeration of red blood cells.

An important advantage of the antibody or its antigen-binding fragment is that it exhibits very weak or no binding to the red blood cell.

Due to the above properties, the antibody or antigen-binding fragment thereof can be preferably used as an antibody-based drug.

In the present invention, the technical term "antibody or antigen-binding fragment thereof" means a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments containing CDRs. "Antibody" specifically refers to a full-length antibody. The term "full-length antibody" includes a polyclonal antibody and a monoclonal antibody.

The term "antigen-binding fragment" means a substance comprising part or all of the CDRs of an antibody and lacking at least some of the amino acids present in the full-length chain, but still capable of specifically binding to an antigen. This type of fragment has biological activity because it binds to a target antigen and can compete with other antigen-binding molecules (including the full antibody) for binding to a given epitope. 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 has the function of blocking the binding of CD47 to its ligand and activating an immune cell, and in one aspect, this type of fragment is selected from Fab (consisting of a full light chain and Fd), Fv (consisting of VH and VL), ScFv (single chain antibody, VH and VL are linked by a linker peptide) or a single domain antibody (consisting of VH only). The fragment may be produced by recombinant nucleic acid technology or may be obtained by enzymatic lysis or chemical cleavage of antigen-binding molecules (including the whole antibody).

The term "complementarity determining region" or "CDR" refers to the hypervariable regions of the immunoglobulin heavy chain and light chain. There are three CDRs of the heavy chain and three CDRs of the light chain. In this specification, as appropriate, the terms "CDR" and "CDRs" are used to refer to a region containing one or more or even all of the essential amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment thereof for the antigen or epitope recognized by them. In another specific embodiment, the CDR region or simply CDR refers to the hypervariable region of the immunoglobulin heavy chain and light chain as defined by the International Immunogenetics Information System (IMGT).

In the present invention, the complementarity determining region of the heavy chain is designated as HCDR and the complementarity determining region of the light chain is designated as LCDR. Common CDR labeling methods used in the art include the Kabat numbering scheme, the Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for the immunoglobulin variable region. In their compilation, Sequences of Proteins of Immunological Interest, the amino acid sequences of the variable regions of the light (λ, κ) and heavy chains of antibodies, as well as the variable regions of the T cell receptors (α, β, γ, and δ) are aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered a widely accepted standard for numbering antibody residues. The present invention uses the Kabat annotation standard for labeling CDR regions, but CDR regions labeled in other ways are also within the scope of the present invention.

The terms "specific recognition", "selective binding", "selectively binding" and "specifically binding" or the like refer to the binding of an antibody or antigen-binding fragment thereof to a predetermined epitope on an antigen. Typically, the antibody or antigen-binding fragment thereof binds with an affinity (KD) of less than about 10 ^-6 M, such as less than about 10 ^-7 M, 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or less.

In the present invention, the object specifically recognized by the proposed antibody or antigen-binding fragment thereof may be CD47 obtained from various genera, such as human, mouse and monkey (e.g. cynomolgus macaque).

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

In some embodiments, the amino acid sequence of the light chain variable region is any one of SEQ ID NOs:5~9 or a sequence having at least 95% identity to any one of SEQ ID NOs:5~9.

In some embodiments, the amino acid sequence of the heavy chain variable region is any one of SEQ ID NOs:14~18 or a sequence having at least 95% identity to any one of SEQ ID NOs:14~18.

In some embodiments, the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:5, the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:15; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:17; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:18; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:8, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:9, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:7, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:8, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:9, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:9, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:18.

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

In some embodiments, the source of the constant region is a mouse, rabbit, sheep, monkey, or human.

In some embodiments, the antibody is any one or more CDR-grafted antibodies, a multimeric antibody, or a bispecific antibody.

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

In some embodiments, CD47 is human CD47, mouse CD47, or monkey CD47.

The present invention further relates to a nucleic acid, and the nucleic acid encodes an anti-CD47 antibody or an antigen-binding fragment thereof.

The nucleic acid is typically ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the coding sequence. DNA nucleic acid is preferably used when it is ligated into a vector.

In addition, since an antibody is a membrane protein, the nucleic acid typically includes a signal peptide sequence.

The present invention further relates to a vector which is a carrier of the above nucleic acid.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. Since a vector can express a protein encoded by the inserted polynucleotide, such a vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material element carried by it can be expressed in the host cell. A vector is well known to those skilled in the art and includes, but is not limited to: a plasmid; a phagemid; a cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC); a phage such as a λ phage or an M13 phage, and an animal virus. An animal virus that can be used as a vector includes, but is not limited to, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpesvirus (e.g., a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, and a papovavirus (e.g., SV40). In some embodiments, the vector described in the present invention comprises a regulatory element commonly used in genetic engineering, such as an enhancer, a promoter, an internal ribosome entry site (IRES), and other expression control elements (e.g., a transcription termination signal or a polyadenylation signal, and a polymeric U sequence).

The present invention further relates to a cell, and this cell comprises the above nucleic acid or the above vector. The nucleic acid encoding the anti-CD47 antibody or antigen-binding fragment thereof is used to bind to the vector, which is then expressed in a cell in which the corresponding antibody can be produced. The vector can be introduced into a eukaryotic cell, especially a mammalian cell, to construct and obtain cells capable of expressing the anti-CD47 antibody or antigen-binding fragment thereof described in the present invention.

As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and the culture obtained therefrom, regardless of the number of transfers. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. The scope of the invention includes mutant progeny that have the same function or biological activity as the original transformed cell, as determined by screening. Where another name is intended, this is clear from the context.

The present invention further relates to a pharmaceutical composition, and the pharmaceutical composition comprises an anti-CD47 antibody or antigen-binding fragment thereof, a nucleic acid, a vector or a cell.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and fungicidal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible to prolong the shelf life or effectiveness of the antibody.

The use of an anti-CD47 antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or composition in the preparation of a medicament for the treatment and/or prevention of a CD47-positive tumor is also within the scope of the present invention.

The medicinal product may have a reduced ability to agglutinate red blood cells and have reduced red blood cell binding activity.

The use of an anti-CD47 antibody or antigen-binding fragment thereof, nucleic acid, vector or cell for the manufacture of a kit for detecting CD47 or diagnosing a disease associated with CD47 is also within the scope of the present invention.

The present invention further provides a kit for detecting CD47, which comprises an anti-CD47 antibody or an antigen-binding fragment thereof.

The present invention further provides a kit for diagnosing a disease associated with CD47, and the kit comprises an anti-CD47 antibody or an antigen-binding fragment thereof.

The present invention provides the following beneficial effects.

The present invention provides an anti-CD47 antibody or antigen-binding fragment thereof. The antibody does not agglutinate red blood cells in vitro, and more remarkably, it exhibits very weak or no binding to red blood cells; the antibody can effectively block the binding of CD47 to SIRPα, and activate and mediate the phagocytic activity of macrophages against tumor cells; In addition, the antibody exhibits significant targeting specificity for CD47-positive tumor cells, has high affinity and strong specificity without causing agglutination of red blood cells and exhibiting extremely weak binding to human red blood cells and platelets, has good safety, and therefore, the anti-CD47 antibody or its antigen-binding fragment can be used as a promising target site in the tumor immunotherapy system, play an important role in the treatment of human tumors, and have a broad application prospect in obtaining a drug for the treatment of CD47-positive tumors.

Embodiments of the present invention are described in detail below.

Example 1. Screening of mouse monoclonal anti-CD47 antibodies

1. Construction of recombinant expression plasmid hCD47-ECD-HIS

The DNA sequence encoding the extracellular region of human CD47 (hCD47-ECD) followed by the 6×HIS-tag sequence was synthesized based on the template (CEJ 94640.1) from GenBank and then cloned into the expression vector pCDNA3.4 (Thermo Company) to generate a recombinant eukaryotic expression plasmid of extracellular full-length CD47 protein, which was named hCD47-ECD-HIS recombinant DNA plasmid.

2. Expression and purification of recombinant hCD47-ECD-HIS protein

Expression and purification of recombinant hCD47-ECD-HIS protein involves the following steps.

(1) The day before transient transfection, Expi293 cells (ThermoFisher Scientific; A14635) were passaged, inoculated into Dynamis medium (gibco; A2617502) at a density of 2*10 ⁶ in a 1 L shake flask (conning; 431147), placed in a cell culture shaker (Adolf Kuhner; ISF4-XC), and cultured at 37°C, 8% CO ₂ and 120 rpm.

On the day of transfection, Expi293 cells were counted using a cell counter (Countstar; IC1000), the cells were diluted and adjusted to 2.9× ¹⁰⁶ with fresh Dynamis culture solution; then the cells were ready for transfection; PEI:DNA=3:1; mixed homogeneously for 5 min, gently vortexed 20 times and kept for 15-30 min; DNA-PEI mixture was added to Expi293 cells, mixed homogeneously and placed in a cell culture shaker (Adolf Kuhner; ISF4-XC) and cultured at 37°C, 8% _CO2 and 120 rpm; and 4 hours after transfection, sandwich assay antibody (gibco; 15140122) and anticoagulant (gibco; 0010057) were added.

(2) Supernatant collection: The transfectomas were continuously cultured for 7 days and then the sample was collected. The sample was first centrifuged at a low speed of 1000 rpm×10 min at 4°C and then centrifuged at a high speed of 12000 rpm×30 min at 4°C; the cell culture supernatant was collected and filtered using a 0.22 μm filter.

(3) Purification using HisTrap affinity chromatography column: The supernatant was loaded onto a HisTrap affinity chromatography column at a flow rate of 1 mL/min. After completion of the loading, the chromatography column was washed with 5 column volumes of an equilibration solution containing 20 mM Tris-HCl and 150 mM NaCl, pH 8.0; then, the chromatography column was washed with 5 column volumes of an eluent containing 20 mM Tris-HCl, 150 mM NaCl, and 0-500 mM imidazole, pH 8.0, and the eluted peak was collected. The purified CD47-ECD protein was identified by SDS-PAGE.

3. Screening of mouse monoclonal anti-CD47 antibodies

The recombinant hCD47-ECD-HIS protein (hereinafter referred to as hCD47 antigen) purified in Step 2 was used to immunize BALB/C mice (purchased from Guangdong Experimental Animal Center). The specific method is as follows.

(1) Animal immunization: Purified hCD47 antigen was emulsified with Freund's complete adjuvant, 6-8 week-old BALB/C mice were immunized by subcutaneous or intraperitoneal injection, the immunization dose was 25 μg/mouse, and there were 5 mice in each group; the second immunization was performed 1 week later, the antigen was emulsified with Freund's incomplete adjuvant, the immunization dose was 25 μg/mouse. After 4 times immunization, tail blood was collected and diluted in gradient by enzyme-linked immunosorbent assay (ELISA) to determine serum titer; and the immunization was boosted three days before fusion, and the mouse with the highest antibody titer was selected for cell fusion.

(2) Cell fusion: Sp2/0 derived from BALB/C (ATCC® CRL-1581) was used as myeloma cells, and they were in the logarithmic growth phase at the time of fusion; the spleen of the immunized mouse was taken to prepare single-cell lymphocyte suspension; the mouse spleen lymphocytes and myeloma cells were mixed at a ratio of 1:5~1:10, 1ml of 50% PEG1500 (pH 8.0) was added dropwise at 37°C, DMEM incomplete medium and another stop solution were added. The HAT medium was suspended and mixed uniformly, the supernatant was removed by centrifugation, placed in a 96-well plate, and the plate was maintained in an incubator with a constant temperature of 37°C and 5% _CO2 . After one week of cultivation, the first replacement of the solution with HAT medium was carried out, and after another three days of cultivation, the second replacement of the solution with HAT medium was carried out.

(3) Screening and cloning: 2 weeks after fusion, the cell supernatant was taken for ELISA test to determine whether the cell supernatant had bound the purified recombinant CD47-ECD-His protein, and then the cells whose ELISA results were positive were screened, and a second ELISA test was performed for retesting; the cells in the positive wells were subjected to limiting dilution, the ELISA value was determined 7 days after each limiting dilution, and the monoclonal wells with higher positive OD280 value were selected for limiting dilution until the ELISA results of the entire 96-well plate became positive. Monoclonal strains with high positive values were selected.

(4) Preparation and purification of monoclonal antibody from cell supernatant: Positive clones were cultured in a T25 culture flask with DMEM medium containing 15% serum, and when the culture was expanded, they were centrifuged at 800 rpm for 5 min, the supernatant was removed, and the cells were transferred to a 500 ml shake flask, and serum-free medium (Hybridoma-SFM Complete DPM; Gibco; 12300-067) was added so that the cell density was about 3× ¹⁰⁵ cells/ml. After continuous 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 for 20 minutes, the supernatant was collected, then the supernatant was purified by affinity chromatography, and the antibody was purified by protein A affinity chromatography. Sample loading; flow-through wash: flow-through wash with 2.5 M phosphate buffer solution (PBS) pH 7.4 was carried out until the initial UV280 level became 0; elution: elution with 0.1 M citric acid solution pH 3.5, a volume of 2 ml was collected in each eluate tube, 100 μl of 1 M Tris solution was added to each tube; the collected solution was concentrated; The concentration of purified monoclonal antibody was determined and the purity of the antibody was confirmed by SDS-PAGE gel; the antibody was packaged (100 µl/tube) and stored at -80°C.

Example 2. Humanized design and combination with mouse monoclonal antibody

The amino acid sequences of the mouse antibody light chain CDRs are shown in SEQ ID NO:1~3:

7A11D3D3-LCDR1 7A11D3D3-LCDR2 7A11D3D3-LCDR3 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 KSSQSLLNSRTRKNYLA WASTES KQSYNLRT

The amino acid sequences of the CDRs of the mouse antibody heavy chain are shown in SEQ ID NO:10~12:

7A11D3D3-HCDR1 7A11D3D3-HCDR2 7A11D3D3-HCDR3 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SNWM MIHPSDSETRLNQKFKD GTTVVDAFAY

The amino acid sequence of the light chain variable region of the mouse antibody (>7A11D3D3-VL) is shown in SEQ ID NO:4.

The amino acid sequence of the variable region of the heavy chain of the mouse antibody (>7A11D3D3-VH) is shown in SEQ ID NO:13.

The humanized design was performed on the mouse antibody, 5 humanized sequences, 7A11-L01~05 respectively, were designed for the light chain of the mouse monoclonal antibody 7A11D3D3, the amino acid sequences of the variable regions of the humanized light chain (7A11-L01~05) are shown in SEQ ID NOs:5-9; and 5 humanized sequences, 7A11-H01~05 respectively, were designed for the heavy chain, the amino acid sequences of the humanized variable regions of the heavy chain (7A11-H01~05) are shown in SEQ ID NOs:14~18.

The above mentioned amino acid sequences were combined, and the obtained antibodies and the sequences corresponding to their heavy chain (VH) and light chain (VL) variable regions are presented in Table 1.

Table 1. Antibody and its corresponding variable heavy chain and variable light chain sequences Name of the antibody VH VL 7A11H11 SEQ ID NO:14 SEQ ID NO:5 7A11H12 SEQ ID NO:14 SEQ ID NO:6 7A11H22 SEQ ID NO:15 SEQ ID NO:6 7A11H32 SEQ ID NO:16 SEQ ID NO:6 7A11H42 SEQ ID NO:17 SEQ ID NO:6 7A11H52 SEQ ID NO:18 SEQ ID NO:6 7A11H14 SEQ ID NO:14 SEQ ID NO:8 7A11H15 SEQ ID NO:14 SEQ ID NO:9 7A11H33 SEQ ID NO:16 SEQ ID NO:7 7A11H34 SEQ ID NO:16 SEQ ID NO:8 7A11H35 SEQ ID NO:16 SEQ ID NO:9 7A11H55 SEQ ID NO:18 SEQ ID NO:9

Example 3. Obtaining anti-CD47 antibody

The humanized variable domain was fused with the secretion signal and the constant domains of human κ and FcIgG4S228P, cloned into a mammalian expression system, and transfected into 293 cells to produce a humanized mAb. The humanized variant was expressed as a full-length IgG molecule, secreted into the culture medium, and purified using protein A.

The humanized antibody and the positive control antibody Hu5F9-G4 (the sequence of Hu5F9-G4 has already been disclosed in US application US2015/0183874A1) were transiently expressed in Expi293 cells and purified.

For transient expression of the antibody in Expi293 cells, the pCDNA3.4 vector was used, and the heavy chain and light chain of the antibody were first cloned into separate pCDNA3.4 vectors, PEI chemical transfection reagent (purchased from Polysciences) was used, the pCDNA3.4 vectors with the heavy and light chain of the antibody molecule were transfected into Expi293 cells by chemical transfection, and the cells were transfected and cultured according to the protocol suggested by the manufacturer.

The day before transient transfection, Expi293 cells (ThermoFisher Scientific; A14635) were passaged, inoculated with Dynamis medium (gibco; A2617502) at a density of 2E6 in a 1 L shake flask (conning; 431147), placed in a cell culture shaker and cultured (Adolf Kuhner; ISF4-XC) at 37°C, 8% _CO2 and 120 rpm.

On the day of transfection, Expi293 cells were counted with a cell counter (Countstar; IC1000), the cells were diluted and adjusted to a density of 2.9E6 with fresh Dynamis culture solution; then the cells were ready for transfection; PEI:DNA=3:1 was mixed evenly for 5 min, gently vortexed 20 times and kept for 15~30 min; the DNA-PEI mixture was added to Expi293 cells, mixed evenly, placed in a cell culture shaker (Adolf Kuhner; ISF4-XC) and cultured at 37°C, 8% _CO2 and 120 rpm, and 4 hours after transfection, sandwich assay antibody (gibco; 15140122) and anticoagulant (gibco; 0010057) were added.

Purification of collected supernatant: Transfectomas were continuously cultured for 7 days and then the sample was collected. First, centrifuged at a low speed of 1000 rpm, 10 min and 4 °C (Xiangyi H2050R) and then centrifuged at a high speed of 12,000 rpm, 30 min and 4 °C; the cell culture supernatant was collected and filtered at 0.22 μm. The culture supernatants were used in a protein A-Sepharose column (GE Healthcare). The column was washed with PBS and eluted with elution buffer (0.1 M sodium citrate buffer, pH 3.0) to remove protein. The collected component was neutralized with 1 M Tris pH 9.0. Finally, the purified sample was dialyzed with PBS.

Example 4 Anti-CD47 Antibody Affinity Determination

1. Experimental method

The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human CD47 (hCD47) was determined using biofilm interferometry (ForteBio). The ForteBio affinity determination was performed according to a method known in the art (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs; 2013.5 (2): p. 270-8), which consists of the following: the sensor was used for offline equilibration for 30 minutes in the assay buffer and detected online for 60 seconds to establish a baseline. The purified antibody obtained in Example 3 was loaded in the AHC sensor (ForteBio) for affinity determination in the ForteBio mode, and then the sensor with the loaded antibody was exposed to 100 nM CD47 antigen for 5 minutes, after which the sensor was transferred to the assay dissociation buffer for 5 minutes and used for dissociation rate measurement. Kinetic analysis was performed with a 1:1 binding model.

2. Experimental result

The results of the affinity determination of anti-CD47 antibody are presented in Table 2, and the results showed that the anti-CD47 antibody has high affinity.

Table 2. Results of determination of affinity of anti-CD47 antibody Name of the antibody KD(M) Kon (1/Mс) 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

Example 5. Binding of anti-CD47 antibody to human CD47

1. Experimental method

The binding of the anti-CD47 antibody of the present invention to human CD47 was measured by a flow cytometry-based method. The specific steps were as follows.

The CCRF-CEM cell line (Cell Bank of China Academy of Sciences, Shanghai) expressing human CD47, which belongs to human acute lymphoblastic leukemia T lymphocytes, was used. CCRF-CEM cells (0.1× ¹⁰⁶ cells) were mixed with experimental antibodies (anti-CD47 antibody of the present invention and Hu5F9-G4 antibody) at different concentrations (the highest concentration was 30 μg/mL, it was a three-fold dilution, and there were 10 concentrations in total) in PBS containing 3% bovine serum albumin (BSA), and incubated on ice for 30 minutes. Then, the cells were washed at least twice, fluorescently labeled secondary goat anti-human IgG Fc PE antibody (1:500× dilution) was prepared with FCM buffer (1XPBS+3%BSA), added to the corresponding 96-well plate at 100 μl/well and incubated in a refrigerator at 4°C for 30 min. Then, the 96-well plate was taken out and centrifuged at 250 g for 5 min, after which the supernatant was carefully removed, FCM buffer was added at 200 μl/well and centrifuged again at 250 g for 5 min, and the supernatant was carefully removed. Cells were washed at least twice and resuspended using 100 µl/well 1×PBS and analyzed by flow cytometry and a concentration versus mean fluorescence intensity (MFI) curve was generated in GraphPad. Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The EC ₅₀ results of binding of anti-CD47 antibody to human CD47 are shown in Table 3, the mean fluorescence intensity of anti-CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to human CD47 is shown in Fig. 1, and the mean fluorescence intensity of anti-CD47 antibody (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to human CD47 is shown in Fig. 2. The results showed that the anti-CD47 antibody of the present invention and the positive control antibody have comparable specific binding ability to human CD47 at the cellular level.

Table 3: Results of determination of the ability of anti-CD47 antibody to bind to human CD47 Name of the antibody 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

Example 6. Binding of anti-CD47 antibody to monkey CD47

1. Experimental method

By transfecting the pCDNA3.4 vector carrying full-length monkey CD47, a stable CHO cell line (CHO-cynoCD47 cells) overexpressing monkey CD47 was obtained, and CHO-cynoCD47 cells (0.1× ¹⁰⁶ cells) were mixed with experimental antibodies (anti-CD47 antibody of the present invention and Hu5F9-G4 antibody) at different concentrations (the highest concentration was 10 μg/ml, it was a three-fold dilution, there were 10 concentrations in total) in PBS containing 3% BSA and incubated on ice for 30 minutes. The cells were then washed at least twice, fluorescently labeled secondary goat anti-human IgG Fc PE antibody (1:500× dilution) was prepared with FCM buffer (1XPBS+3%BSA), added to the corresponding 96-well plate at 100 μl/well and incubated in a refrigerator at 4°C for 30 min. The 96-well plate was removed and centrifuged at 250 g for 5 min, after which the supernatant was carefully removed, FCM buffer was added at 200 μl/well and centrifuged again at 250 g for 5 min, after which the supernatant was carefully removed. Cells were washed at least twice and resuspended with 1×PBS at 100 μl/well and analyzed by flow cytometry and a concentration-dependent curve was generated using GraphPad according to the median fluorescence intensity (MFI). Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The EC ₅₀ results of binding of CD47 antibody to monkey CD47 are shown in Table 4, the mean fluorescence intensity of anti-CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to monkey CD47 is shown in Fig. 3, and the mean fluorescence intensity of anti-CD47 antibody (7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and isotype control hIgG4) binding to monkey CD47 is shown in Fig. 4. The results showed that the antibody of the present invention and the positive control antibody have comparable specific binding ability to monkey CD47 at the cellular level.

Table 4. EC ₅₀ results of binding of anti-CD47 antibody to monkey CD47 Name of the antibody 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

Example 7. Anti-CD47 antibody blocks the interaction of SIRPα, a ligand for human CD47, with CD47

1. Experimental method

The ability of the anti-CD47 antibody of the present invention to block the binding of human CD47 to SIRPα was determined by flow cytometry. The specific steps were as follows:

Antibody dilution: FCM buffer (1XPBS+3%BSA) was used to dilute the anti-CD47 antibody of the present invention and the control antibody Hu5F9-G4 to 90 μg/ml, then the antibody was diluted to 10 concentrations in a 3-fold gradient, and the control hIgG4 subtype was diluted to 30 μg/ml, 1.1 μg/ml, and 0.04 μg/ml, and the hSIRP ligand α-mFC (AcroBiosystems) was diluted to 10 μg/ml.

CCRF-CEM cells (Cell Bank of Chinese Academy of Sciences, Shanghai) were added to a 96-well V-type plate at 0.1× ¹⁰⁶ cells/well, and hSIRPα-mFC binding was monitored under increasing conditions of anti-CD47 antibody. Bound SIRPα was detected using a second goat anti-mouse IgG Fc PE antibody (Biolegend). Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The results of the inhibition of CD47/SIRPα binding by the anti-CD47 antibody to human CD47 (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, Hu5F9-G4, and the isotype control hIgG4) are shown in Fig. 5 and the results of the inhibition of CD47/SIRPα binding by the anti-CD47 antibody to human CD47 (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, and 7A11H55) are shown in Fig. 6. The results showed that the anti-CD47 antibody of the present invention can significantly inhibit the binding of CD47 to SIRPα at the cellular level, and compared with the positive control antibody, the anti-CD47 antibody of the present invention exhibits comparable blocking ability.

Example 8. Detection of the ability of anti-CD47 antibodies to stimulate macrophages to phagocytose tumor cells

1. Experimental method

The ability of the anti-CD47 antibody of the present invention to stimulate macrophage phagocytosis of tumor cells was measured by a flow cytometry-based method. The specific steps were as follows:

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

Jurkat tumor cell line (Cell Bank of Chinese Academy of Sciences, Shanghai) with high expression of human CD47 was selected as the target cell type, and these target tumor cells were fluorescently labeled according to the instructions of the CellTraceTM CFSE kit. The labeled tumor cells were co-cultured with the above differentiated macrophages at a ratio of 1:1, simultaneously adding antibodies at final concentrations of 10 μg/mL, 1 μg/mL, and 0.1 μg/mL, and incubated at 37°C for 2 h. Cells were then washed at least twice and gently purged, allophycocyanin (APC)-labeled CD14 antibody (purchased from Biolegend; B259538) was added and incubated in PBS containing 0.1% BSA on ice (protected from light) for 30 min. Cells were washed at least twice and analyzed by flow cytometry. The phagocytic cell population was the population of cells that were CD14-positive living cells and also positive for the fluorescent dye CFSE (carboxyfluorescein diacetate succinimidyl ester). Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The results of measuring the ability of the anti-CD47 antibody to stimulate macrophage phagocytosis of tumor cells are shown in Fig. 7. The results showed that the anti-CD47 antibody of the present invention has the ability to stimulate phagocytosis of tumor cells by macrophages, and the ability to stimulate phagocytosis of tumor cells by macrophages is comparable to the ability of the positive control antibody Hu5F9-G4.

Example 9. Analysis of hemagglutination with anti-CD47 antibodies of human erythrocytes

1. Experimental method

An erythrocyte hemagglutination assay was performed to characterize the ability of anti-CD47 antibodies to agglutinate erythrocytes. Anti-CD47 antibodies were screened for erythrocyte agglutination by observing the ability of the antibodies not to precipitate human erythrocytes. The specific method was as follows.

Red blood cells were diluted to 2% in PBS and anti-CD47 antibody (sequential concentrations of 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) were added dropwise and incubated in a round-bottomed 96-well plate at 37°C for 2 hours. The presence of non-sedimented red blood cells indicated hemagglutination of red blood cells and non-sedimented red blood cells appeared as a haze compared to non-agglutinated red blood cells which formed distinct red dots. The Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The results of measuring the ability of the anti-CD47 antibody to agglutinate erythrocytes are shown in Fig. 8. The results showed that although the concentration of the anti-CD47 antibody reached 200 μg/ml, cell agglutination did not occur, and it was found that the anti-CD47 antibody of the present invention had an effect of significantly reducing the hemagglutination of erythrocytes. In the treatment of cancer, the side effects of the anti-CD47 antibody can be significantly reduced, and good safety is achieved.

Example 10. Analysis of binding of anti-CD47 antibody to human erythrocytes

1. Experimental method

Monoclonal anti-CD47 antibody has the property of binding to human red blood cells. For inhibitory anti-CD47 antibodies, there is a potential risk of decreased drug efficacy due to interference with red blood cells and non-tumor targets. If an antibody with low red blood cell binding activity is screened, the risk of off-target effects can be reduced and the safety of the antibody can be improved. The specific steps were as follows.

(1) Antibody dilution: FACS buffer was used to dilute anti-CD47 antibody to a stock concentration of 20 μg/mL in a volume of 180 μL, the antibody was diluted in a 3-fold gradient (60+120) to obtain 11 concentrations.

(2) Cell counting and seeding: RBCs were centrifuged at 250 g for 5 min, then the supernatant was discarded, the cell density was adjusted to 2E+06 with FACS buffer and evenly distributed in a 96-well V-shaped plate at 100 μl/well.

(3) The above diluted antibody was added to the cells at 100 μl/well and incubated at 2°C~8°C for 0.5 hour.

(4) The 96-well plate was taken out and centrifuged at 250 g for 5 minutes, after the supernatant was carefully removed, FACS buffer was added at 200 μl/well and centrifuged again at 250 g for 5 minutes, after which the supernatant was carefully removed.

(5) Fluorescently labeled goat anti-human IgG Fc secondary antibody PE (biolegend) (1:500 dilution) was prepared with FACS buffer and added to the corresponding 96-well plate at 100 μl/well, the cells were resuspended and incubated at 2°C~8°C for 30 min.

(6) The 96-well plate was taken out and centrifuged at 250 g for 5 minutes, after carefully removing the supernatant, FACS buffer was added at 200 μl/well and centrifuged again at 250 g for 5 minutes, and the supernatant was carefully removed.

(7) The pellet was resuspended with 1×PBS at 100 μl/well and analyzed by FACS. Data were analyzed by flow cytometer (Beckman, Cytoflex) and plotted using GraphPad Prism. Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The results of measuring the ability of the anti-CD47 antibody to bind to human erythrocytes are shown in Fig. 9. The results showed that, compared with the positive control antibody, the binding activity of the anti-CD47 antibody of the present invention to human erythrocytes was significantly reduced, and its safety as a drug was increased.

Example 11. Analysis of binding of anti-CD47 antibodies to platelets

1. Experimental method

In addition to the ability of CD47 monoclonal antibody to bind to human red blood cells, anti-CD47 monoclonal antibody has active platelet binding characteristics, and there are many side effects caused by platelet reduction. An antibody with low platelet binding can reduce the risk of off-target effects and improve its safety. The specific method was as follows.

Antibody dilution: The antibody was diluted to 20 μg/ml with FACS buffer, the volume was 240 μl.

Cell counting and seeding: Whole blood cells were diluted 20-fold and distributed evenly into a 96-well V-well plate at 100 µl/well.

The above diluted antibody was added to the cells at 100 μl/well and incubated at 2°C-8°C for 0.5 hour.

The 96-well plate was removed and centrifuged at 250 g for 5 min, after which the supernatant was carefully removed, FACS buffer was added at 200 μl/well and centrifuged again at 250 g for 5 min, after which the supernatant was carefully removed.

Fluorescently labeled secondary PE antibody (1:500 dilution) (goat anti-human IgG Fc PE; biolegend; 409304) was prepared with FACS buffer and added to the corresponding 96-well plate at 100 μl/well, and simultaneously 1 μl of APC anti-human CD61 antibody (biolegend; 336411) was added to each well, the cells were resuspended and incubated at 2°C-8°C for 30 min.

The 96-well plate was removed and centrifuged at 250 g for 5 min, after carefully removing the supernatant, FACS buffer was added at 200 μl/well and centrifuged again at 250 g for 5 min, and the supernatant was carefully removed.

The pellet was resuspended with 1xPBS at 200 µl/well and FACS detection was performed. Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The results of the anti-CD47 antibody-platelet binding assay are shown in Fig. 10. The results showed that the anti-CD47 antibody of the present invention has significantly lower platelet binding activity than the positive control antibody and exhibits better safety.

Example 12. Analysis of the effect of anti-CD47 antibodies on erythrocyte phagocytosis by activated macrophages

1. Experimental method

The detection method was the same as in Example 8, tumor cells were replaced with erythrocytes as target cells, and whether the anti-CD47 antibody of the present invention has an activating effect on the phagocytosis of erythrocytes by macrophages was determined. The Hu5F9-G4 antibody was used as a positive control.

2. Experimental result

The results of the analysis of the effect of anti-CD47 antibody on phagocytosis of erythrocytes by activated macrophages are shown in Fig. 11. The results showed that the anti-CD47 antibody of the present invention very weakly mediates phagocytosis of erythrocytes by macrophages, and the mediation effect is significantly lower than that of the positive control antibody, and demonstrates better safety.

Example 13. Study of antitumor activity in vivo

1. Experimental method

70 female NOD SCID mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were selected to establish a human subcutaneous B lymphocyte xenograft model (Raji) and a human malignant melanoma model (A375), and the antitumor activity of the anti-CD47 antibody of the present invention was evaluated. In these studies, the antibody analog Hu5F9-G4 and the antibody analog TJC-4 were used as positive control antibodies, and at the same time, hIgG4 was used as an isotype control antibody.

2. Experimental result

The antitumor effect of the anti-CD47 antibody on the human subcutaneous B-lymphocyte xenograft model is shown in Fig. 12; the results indicate that the anti-CD47 antibody of the present invention is significantly more effective than the positive control antibody in acting on the human subcutaneous B-lymphocyte xenograft.

The results of the antitumor effect of the anti-CD47 antibody in the human malignant melanoma model shown in Fig. 13 showed that, compared with the positive control antibody, the anti-CD47 antibody of the present disclosure is more advantageous in acting on human malignant melanoma.

The above embodiments are preferred embodiments of the present invention, but the variations of the present invention are not limited to the above embodiments and any other changes, modifications, substitutions, combinations, simplifications can be made without departing from the essence and principles of the present invention and are equivalent to the disclosed embodiments and all of them are included in the scope of the present invention.

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MODIFIED SEQUENCE LIST

<110> GUANGDONG FAPON BIOPHARMA INC.

<120> CD47 TARGETED ANTIBODY AND ITS APPLICATIONS

<130> PN212981FPSW

<150>CN202011544262.6

<151> 2020-12-23

<160> 18

<170> PatentIn version 3.5

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Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Thr Val Val Asp Ala Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 16

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Ser Asn

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Thr Val Val Asp Ala Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 17

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Ser Asn

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Thr Val Val Asp Ala Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 18

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Ser Asn

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Thr Val Val Asp Ala Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<---

Claims (29)

1. An anti-CD47 antibody or antigen-binding fragment thereof, wherein the antibody comprises the following complementarity determining regions (CDRs) as set forth in any of subparagraphs A), B) or C): A) HCDR1, HCDR2 and HCDR3 identical to the complementarity determining regions of the sequence SEQ ID NO:14, and LCDR1, LCDR2 and LCDR3 identical to the complementarity determining regions of the sequence SEQ ID NO:8, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined in accordance with Kabat numbering; B) LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:3, and HCDR1 as shown in SEQ ID NO:10, HCDR2 as shown in SEQ ID NO:11, and HCDR3 as shown in SEQ ID NO:12, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to Kabat numbering; or C) HCDR1, HCDR2 and HCDR3 identical to the complementarity determining regions of the sequence SEQ ID NO:18, and LCDR1, LCDR2 and LCDR3 identical to the complementarity determining regions of the sequence SEQ ID NO:6, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined in accordance with Kabat numbering. 2. An anti-CD47 antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody comprises at least one of a heavy chain variable region sequences and a light chain variable region sequences, wherein at least a portion of at least one of the heavy chain variable region sequences and the light chain variable region sequences are derived from at least one antibody that is an antibody derived from a mouse, a humanized antibody, an antibody derived from a primate, or a mutant thereof. 3. An anti-CD47 antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the light chain variable region is the sequence presented in any one of SEQ ID NOs:5-9. 4. An anti-CD47 antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the heavy chain variable region is the sequence presented in any one of SEQ ID NOs:14-18. 5. The anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, wherein the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO: 5, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO: 14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO: 6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO: 14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO: 6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO: 15; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO: 6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO: 16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:17; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:6, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:18; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:8, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:9, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:14; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:7, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:8, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:9, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:16; or the amino acid sequence of the variable region of the light chain is presented in SEQ ID NO:9, and the amino acid sequence of the variable region of the heavy chain is presented in SEQ ID NO:18. 6. An anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein said antibody has a constant region, wherein the heavy chain constant region is selected from the constant region of any of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is a κ or λ chain. 7. An anti-CD47 antibody or antigen-binding fragment thereof according to claim 6, wherein the species of the source of the constant region is selected from mouse, rabbit, sheep, monkey or human. 8. An anti-CD47 antibody or antigen-binding fragment thereof according to claim 7, wherein the light chain constant region is a human κ chain and the heavy chain constant region is a human IgG4 heavy chain constant region having the S228P mutation. 9. An anti-CD47 antibody or antigen-binding fragment thereof according to claim 8, wherein the constant region of the light chain is a human κ chain, and the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 8; and the constant region of the heavy chain is a constant region of a human IgG4 heavy chain having the S228P mutation, and the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 14. 10. An anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that said antibody is any one or more of a CDR-grafted antibody or a multimeric antibody. 11. An anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antigen-binding fragment is any one or more of F(ab')2, Fab, scFv and Fv. 12. An anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein CD47 is human CD47, mouse CD47 or monkey CD47. 13. A nucleic acid encoding an anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12. 14. An expression vector comprising a nucleic acid according to claim 13. 15. A transformed cell for expressing an anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12, containing the nucleic acid according to claim 13 or the expression vector according to claim 14. 16. A pharmaceutical composition for treating a CD47-positive tumor, wherein said pharmaceutical composition comprises an anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1-12, a nucleic acid according to claim 13, an expression vector according to claim 14 or a transformed cell according to claim 15, and a pharmaceutically acceptable carrier. 17. Use of an anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12, a nucleic acid according to claim 13, an expression vector according to claim 14, a transformed cell according to claim 15, or a composition according to claim 16 for the preparation of a medicament for the treatment of a CD47-positive tumor. 18. Use according to paragraph 17, where the medicinal product is capable of reducing the hemagglutination of erythrocytes. 19. Use according to claim 17, wherein the medicinal product has a reduced ability to bind red blood cells. 20. Use of an anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12, a nucleic acid according to claim 13, an expression vector according to claim 14, or a transformed cell according to claim 15 for the manufacture of a CD47 detection kit. 21. A kit for detecting CD47, comprising an anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12. 22. A kit for diagnosing a disease associated with CD47, comprising an anti-CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1-12. 23. An anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12, a nucleic acid according to claim 13, an expression vector according to claim 14, a transformed cell according to claim 15, or a composition according to claim 16 for use as a medicinal product for the treatment of a CD47-positive tumor. 24. An anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12, a nucleic acid according to claim 13, an expression vector according to claim 14, a transformed cell according to claim 15, or a composition according to claim 16 for use in a method of treating a CD47-positive tumor. 25. A method for treating a CD47-positive tumor, comprising: administering an anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1-12 or a composition according to claim 16 to a subject in need thereof.