CN115894692B - An antibody targeting CD47 and its application - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibody targeting CD47 and application thereof. The anti-human CD47 antibody provided by the invention can effectively bind to recombinant CD47 protein, block the binding of CD47 and biological ligand SIRPa thereof, and effectively identify and bind to CD47 positive tumor cells. Meanwhile, the CD47 antibody does not cause obvious human blood erythrocyte hemagglutination reaction and red blood cell combination at high concentration.

Description

CD 47-targeting antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an antibody targeting CD47 and application thereof.

Background

To date, immunotherapy is considered as the most promising systemic tumor treatment method compared to conventional anticancer treatment strategies, and plays an indispensable role in improving the therapeutic effect. Emerging cancer immunotherapies include cancer vaccines, CAR-T cell therapies, cytokine therapies, immune checkpoint inhibitors, and tumor-targeted monoclonal antibodies. Among them, monoclonal antibodies have become a critical and effective therapeutic modality in cancer treatment due to their ability to specifically target molecules.

The yeast surface display technology is a set of general technology applied to the fields of antibody discovery, target spot discovery and protein engineering, and is used as one of technical means for developing therapeutic antibodies in vitro, thereby realizing the progress from a prokaryotic expression system to a eukaryotic expression system. In antibody engineering, antibody library proteins are expressed on the surface of yeast cells by utilizing the characteristics of a yeast eukaryotic expression system, and are screened based on antibody affinity and display level by utilizing a magnetic bead and flow cell sorting technology to obtain antibody sequences with high affinity or high stability, so that antibodies with various framework forms, such as single-chain variable region scFv, antibody binding region fragment Fab, full-length IgG and camelid single-domain antibody variable region VHH, can be obtained according to the design, and meanwhile, the antibodies can be subjected to post-translational modification by utilizing a eukaryotic system, and the characteristics of the antibodies are more similar to those produced by mammalian cells.

The leukocyte differentiation antigen CD47 (cluster of differentiation 47), also known as integrin-associated protein (Integrin-associated Protein, IAP), is a glycosylated transmembrane protein that is widely expressed on the surface of a variety of cells. CD47 has a molecular weight of 45-55kDa and a structure comprising an extracellular variable region interacting with the corresponding ligand, a transmembrane region formed by a highly hydrophobic transmembrane segment and a hydrophilic carboxy-terminal intracellular region, and belongs to the immunoglobulin superfamily members. CD47 and its ligands not only regulate immune responses, but also mediate various pathophysiological processes such as neutrophil chemotaxis and nervous system development, and play a regulatory role in immune tolerance and T cell activation. Signal regulator protein alpha (SIRPalpha) is a specific ligand thereof, and is mainly expressed on the surfaces of phagocytic cells such as macrophages and dendritic cells. The interaction of CD 47/SIRPalpha can transmit an inhibitory signal to inhibit phagocytosis of relevant cells by macrophages.

A large number of researches show that the over-expression of CD47 in different types of tumors, the high expression level of the CD47 is related to the treatment response and prognosis of cancer exacerbation, and tumor cells transmit signals of ' don't eat me ' to macrophages through CD47/SIRP alpha interaction, so that the inherent immune escape of the tumor cells is caused, and the further occurrence and development of the tumors are induced. Therefore, the method can block the inhibitory signal mediated by the CD47/SIRP alpha axis, restore the phagocytosis of the macrophage to the tumor cells, and provide a new thought and means for tumor immunotherapy.

However, due to the broad expression of CD47, the potential problem with anti-CD 47 antibodies as anti-cancer drugs may create off-target effects, such as anemia. CD47 is a key regulator of erythrocyte turnover, and CD 47-targeted antibodies generally have high targeted binding to erythrocytes, which may accelerate erythrocyte clearance and lead to hemolytic anemia, as CD 47-targeted formulations (i.e., hu5F9-G4 and TTI-621) can lead to acute anemia and thrombocytopenia in humans. Therefore, the obtained monoclonal antibody with high activity and low toxic and side effect against CD47 has important significance for tumor immunotherapy.

Disclosure of Invention

The invention aims to provide a brand-new antibody which has good affinity with CD47 and weak erythrocyte agglutination.

The invention provides a brand-new CD47 antibody or antigen binding fragment thereof and a preparation method thereof, and the provided CD47 antibody can recognize and specifically bind to CD47, can be used for preparing medicines for treating related diseases with abnormal or excessive expression of CD47 without causing obvious hemagglutination reaction, or can be used for preparing products for detecting the expression of CD47 by Western blot, ELISA and flow cytometry.

An anti-human CD47 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds human CD47 and comprises VH CDR1, VH CDR2, and VH CDR3 of the heavy chain variable region and VL CDR1, VL CDR2, and VL CDR of the light chain variable region, wherein:

VH CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO 8-10;

VH CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO 11-13;

VH CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO 14-16;

VL CDR1 comprises any of the amino acid sequences shown in SEQ ID NOS.17-19;

VL CDR2 comprises any of the amino acid sequences shown in SEQ ID NOS.20-22;

VL CDR3 comprises any of the amino acid sequences shown in SEQ ID NOS.23-26.

An anti-human CD47 antibody or antigen-binding fragment thereof, characterized in that:

the anti-human CD47 antibody or the antigen binding fragment thereof is characterized in that:

a genetically engineered antibody having the amino acid sequences of heavy and light chains identical to those of claim 3, which comprises a human-mouse chimeric antibody, a humanized antibody, a functional fragment Fab of an antibody, a single-chain antibody, a functional fragment VH-L of an antibody obtained by fusing a heavy chain variable region and a complete light chain, a functional fragment of an antibody obtained by arranging, concatenating or combining one or more CDRs of a heavy chain and a light chain, or a functional fusion protein of an antibody-like body obtained by connecting, splicing or fusing the above-mentioned antibody and functional fragment of an antibody with other various proteins or polypeptides.

The anti-CD 47 antibody or the antigen binding fragment thereof or the genetically engineered antibody is characterized in that the antibody has a constant region, a heavy chain constant region is selected from any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD, and a light chain constant region is a kappa or lambda chain. Nucleotide sequences encoding said anti-CD 47 antibody or antigen-binding fragment thereof or said genetically engineered antibody.

An expression vector comprising a nucleotide sequence encoding said nucleotide sequence.

The anti-CD 47 antibody or antigen binding fragment thereof or the genetic engineering antibody is applied to the preparation of anti-tumor drugs.

Advantageous effects

The invention provides a series of full-length antibodies or single-chain antibodies, which have good anti-CD 47 effect, weak or no erythrocyte aggregation effect and good application prospect in clinic.

Drawings

FIG. 1 is an indirect ELISA for detecting binding of CD47 antibodies to recombinant human CD47 antigen. Wherein, A is the EC50 fitting curve of antibodies D0604-G4 and D0643-G4, and B is the EC50 fitting curve of antibodies D2510-G4, D3705-G4 and positive control antibody Hu5F 9-G4.

FIG. 2 is a flow cytometry measurement of the binding capacity of CD47 antibodies to tumor cells expressing CD 47.

FIG. 3 shows the results of CD47 antibodies inducing red blood cell agglutination at various concentrations.

FIG. 4 is a flow cytometry measurement of binding activity of CD47 antibodies to red blood cells.

Detailed Description

The present invention provides antibodies that specifically bind CD47. In some embodiments, the antibodies of the invention are capable of specifically binding to human CD47. Herein, this written antibody is collectively referred to as CD47 antibody.

The term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. It contains only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the Complementarity Determining Regions (CDRs) of a monoclonal antibody are the same in all molecules of the population. mabs contain antigen binding sites that are capable of immunoreacting with a particular epitope of an antigen.

The term "single chain antibody" (scFv) refers to an antibody made from an antibody heavy chain variable region (VH) and a light chain variable region (VL) joined by a 15-20 amino acid linker.

The term "complementarity determining regions" or "CDRs" refers to the highly variable regions of the heavy and light chains of an immunoglobulin. There are three heavy chain CDRs and three light chain CDRs. Here, the terms "CDR" and "CDRs" are used to refer to regions comprising one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen binding fragment thereof to an antigen or epitope recognized by it, depending on the circumstances. In another specific embodiment, CDR regions or CDRs refer to the highly variable regions of the heavy and light chains of IMGT-defined immunoglobulins.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques of molecular biology, biochemistry, cell culture, recombinant DNA technology, and related art conventional techniques that are conventional in the art.

The reagents required for the following embodiments are commercially available (commercially available) unless otherwise specified.

EXAMPLE 1 immunization of mice

1) Balb/c healthy female mice with the age of 6-8 weeks were selected and immunized five times with His tag hCD47 recombinant protein. In the first immunization, 100ug of antigen protein is mixed and emulsified with Freund's complete adjuvant according to the ratio of 1:1, and the immunization is completed by subcutaneous multipoint injection. Four booster immunizations were then performed, each time at 14 days intervals, the antigen dose was halved, emulsified by mixing with Freund's incomplete adjuvant in a 1:1 ratio, the immunization was completed by intraperitoneal injection, and after each boost, part of the mouse tail was cut off, the mouse tail venous blood was collected and serum was isolated, and the mouse serum titer was determined by ELISA.

2) The hFc-tagged hCD47 recombinant protein was diluted to 1. Mu.g/ml with antigen coating solution, added to the UV-activated ELISA strips at 100. Mu.l/well, duplicate wells were set, and PBS set as negative control, coated overnight at 4 ℃. Absorbing and discarding coating liquid, adding PBST to wash each hole for 5min each time and 6 times, adding 5% of skim milk sealing liquid into each hole, sealing at 37 ℃ for 2h, absorbing and discarding sealing liquid, adding PBST to wash each hole for 5min each time and 6 times, adding PBS to dilute mouse serum at different proportions, incubating at 37 ℃ for 2h, absorbing and discarding mouse serum, adding PBST to wash each hole for 5min each time and 6 times, adding diluted HRP-goat anti-mouse IgG antibody into each hole, incubating at 37 ℃ for 1.5h, absorbing and discarding secondary antibody, adding PBST to wash each hole for 5min each time and 6 times, adding freshly prepared TMB developing liquid into each hole, developing at 37 ℃ in dark place for 15-30min, adding a stop solution into each hole, stopping developing reaction, and measuring the absorbance value of each hole at a wavelength of 450nm to measure the serum titer of the immunized mice.

EXAMPLE 2 screening and preparation of CD47 antibodies

1. Extraction of total RNA from mouse spleen

1) According to ELISA results, selecting mice with highest serum titer after enhancing immunity, killing the mice after neck breakage, separating spleen of the mice, preparing spleen tissue single cell suspension, removing red blood cells by using red blood cell lysate, adding TRizol reagent, repeatedly blowing the cells, and fully lysing the cells.

2) Adding 1/5 volume chloroform into the above lysate, standing at room temperature for 2-3min, and centrifuging at 12000g for 10-15min at 4deg.C. Carefully aspirate the upper aqueous phase into a fresh centrifuge tube, add 1/2 volume of isopropanol, mix upside down and then place at room temperature for 10min, centrifuge at 12000g for 10min at 4 ℃. The supernatant was discarded, the precipitate was washed with an equal volume of 75% ethanol, and air dried at room temperature for 5-10min. Add 50. Mu.l RNase-free water to dissolve RNA.

2.RT-PCR

According to the nucleic acid sequence of the variable region of the mouse antibody, a specific primer is designed, a cDNA first chain is used as a template, genes of a variable region (VH) and a variable region (VL) of a heavy chain of the mouse are amplified by PCR, DNA fragments of single-chain antibodies (scFv) of a mouse source are obtained by overlap PCR, and the scFv fragments are purified by gel recovery after 1% agarose gel electrophoresis.

Construction of CD47 Yeast antibody library and antibody screening

1) Library construction by digestion of the yeast vector pYD1 overnight at 37 ℃ with EcoRI and XhoI restriction enzymes, gel recovery of the linearized vector after 1% agarose gel electrophoresis, co-transformation with the purified scFv gene fragment, and overnight culture at 220rpm at 30 ℃ with the purified scFv gene fragment in saccharomyces cerevisiae strain EBY100, to display single chain antibodies on the surface of yeast cells, to construct a corresponding single chain antibody library, and designation of the library as DEM139.

2) Magnetic bead separation, namely, measuring OD ₆₀₀ of yeast liquid after electrotransformation, inoculating the yeast liquid into an induction culture medium at a proper density, performing induction culture at 20 ℃ and 220rpm overnight, taking the induced yeast, washing the induced yeast by using 1% PBSA, adding the hFc tag CD47 antigen protein marked by biotin, incubating the yeast for 20min at room temperature, washing the yeast by using precooled 1% PBSA, placing the yeast on a magnetic frame, using streptavidin magnetic bead separation, collecting the yeast by using an amplification culture medium, and culturing the yeast at 30 ℃ and 220rpm overnight.

3) Flow sorting, namely measuring OD ₆₀₀ of yeast liquid after magnetic bead sorting, inoculating the yeast liquid into an induction culture medium at a proper density, carrying out induction culture at 20 ℃ and 220rpm overnight, measuring OD ₆₀₀ of the yeast liquid, absorbing proper amount of yeast, washing with PBS, centrifuging at 3000rpm for 5min, removing supernatant, re-suspending yeast with biotin-labeled hFc tag CD47 antigen protein, incubating at room temperature for 20min, washing yeast with precooled PBS, centrifuging at 3000rpm for 5min, removing supernatant, adding diluted APC-labeled streptavidin and Alexa488-Labeled mouse anti-V5 tag antibody, incubated at 4 ℃ for 30min, washed with pre-chilled PBS, centrifuged at 3000rpm for 5min, the supernatant removed, PBS resuspended cells added, APC and 488-double positive yeast cells sorted according to fluorescent intensity gate into amplification medium, cultured at 30 ℃ overnight at 220rpm, and subjected to a second alternate sorting after re-induction culture.

4) And (3) library sequencing, namely taking two alternate-type sorted yeasts for amplification culture, extracting yeast plasmids, carrying out library sequencing, and sequencing 50 monoclonal antibodies with better growth states.

Preparation of CD47 antibodies

1) And (3) constructing a eukaryotic expression vector, namely selecting single-chain antibody sequences with the 4 CDR sequences not completely identical according to the library sequencing result, designing primers, splicing an antibody light chain variable region and a human Ig kappa chain constant region through overlapPCR, splicing an antibody heavy chain variable region and a human IgG4 constant region, converting each single-chain antibody into a full-length antibody, and splicing an antibody light chain gene and a heavy chain gene into a mammal expression vector through a homologous recombination method to obtain the corresponding CD47 full-length antibody eukaryotic expression vector.

2) The combination of VH and VL of 4 IgG4 full-length antibodies is shown in Table 1, the C-terminal of VH is connected with CH with the sequence shown as SEQ ID NO:27 to form the heavy chain of the antibody, the C-terminal of VL is connected with CL with the sequence shown as SEQ ID NO:28 to form the light chain of the antibody, the heavy chain variable region of the antibody is shown as "D" + antibody heavy chain number+ "-VH", such as D06-VH, the light chain variable region of the antibody is shown as "D" + antibody light chain number+ "-VL", such as D04-VL, the IgG4 full-length antibody is shown as "D" + antibody heavy chain number + antibody light chain number+ "-G4", such as D0604-G4 (the heavy chain of the antibody is composed of D06-VH (SEQ ID NO: 1) and CH with the sequence shown as SEQ ID NO: 27), and the light chain of the antibody is composed of D04-VL (SEQ ID NO: 4) and CL with the sequence shown as SEQ ID NO:28, and the 4 full-length antibodies obtained are abbreviated as D0604-G4, D0643-G4-G.20 and D3704-G.5.

3) The recombinant plasmids of light and heavy chains, which encode the CD47 antibody and a positive control antibody Hu5F9-G4 (the sequence of Hu5F9-G4 is disclosed in U.S. Pat. No. 3, 20150183874A 1), are transfected into HEK293 cells according to the proportion of 2:1, and supernatant is collected by centrifugation at 4 ℃ after 5 days of culture, and the CD47 antibody is obtained after purification by protein A column, and the antigen molecular weight, purity and antibody type are detected by SDS-PAGE, SEC-HPLC and Western blot respectively.

The results show that the molecular weight of the antibodies is 150-160kd and the type is IgG.

TABLE 1 full length antibodies formed by the CD47 antibody heavy chain variable region and light chain variable region regions

TABLE 2 CDR regions of antibodies

Example 3 determination of binding Capacity and affinity of CD47 antibodies to hCD47 molecules

Binding of CD47 antibodies to recombinant human CD47 antigen was detected using ELISA. The mFc-tagged hCD47 recombinant protein was diluted to 1. Mu.g/ml with antigen coating solution, added to the UV-activated ELISA strips at 100. Mu.l/well, duplicate wells were set, and PBS set as negative control, coated overnight at 4 ℃. Washed 3 times with PBST, added with 5% skim milk, and blocked at 37 ℃ for 2h. Washed 3 times with PBST, gradient diluted CD47 antibody and control antibody Hu5F9 (maximum concentration 200nM, total of 8 concentration gradients) were added and incubated for 2h at 37 ℃. PBST was washed 3 times, HRP-labeled goat anti-human IgG secondary antibody was added, incubated at 37℃for 1h, washed 6 times with PBST, TMB chromogenic solution was added, incubated for 15min in the dark, and the chromogenic reaction was terminated. The absorbance at 450nm was read on a microplate reader and the EC50 was calculated, and the results are shown in fig. 1 and table 3 that each CD47 antibody was able to bind hCD47 protein efficiently at the protein level.

TABLE 3 affinity assay results of CD47 antibodies with hCD47 molecules

Antibody numbering	EC50(nM)
		D0604-G4	0.3457
D0643-G4	0.0853
		D3705-G4	0.2218
D2510-G4	2.6910
		Hu5F9-G4	0.0570

。

Example 4 determination of the binding of CD47 antibody to SIRPalpha by CD47 blocking

The hFc-tagged hCD47 recombinant protein was diluted to 1. Mu.g/ml with antigen coating solution, added to the UV-activated ELISA strips at 100. Mu.l/well, duplicate wells were set, and PBS set as negative control, coated overnight at 4 ℃. Washed 3 times with PBST, added with 5% skim milk, and blocked at 37 ℃ for 2h. Washing 3 times with PBST, adding gradient diluted CD47 antibody and control antibody Hu5F9 (maximum concentration 300nM, total 10 concentration gradients) with a final concentration of 2 μg/ml of biotin-labeled mFc-tagged sirpa recombinant protein and incubating for 1h at 37 ℃. PBST was washed 3 times, HRP-labeled streptavidin was added, incubated for 1h at 37℃and 6 times with PBST, TMB chromogenic solution was added, incubated for 15min in the dark, and the chromogenic reaction was terminated. The absorbance at 450nm was read on a microplate reader and the IC50 of each antibody was calculated, resulting in the CD47 antibody being effective in blocking CD47 interaction with sirpa as shown in table 4.

TABLE 4 determination of the effect of CD47 antibodies on blocking the binding of CD47 to SIRPalpha

Antibody numbering	IC50(nM)
		D0604-G4	8.594
D0643-G4	5.196
		D3705-G4	5.025
D2510-G4	41.090
		Hu5F9-G4	0.456

Example 5 determination of the binding Capacity of CD47 antibodies to CD 47-expressing tumor cells

Jurkat cells highly expressing CD47 were cultured using 1640 medium containing 10% fbs at 37 ℃ under 5% co 2. When the cell growth state was good, 2X 10 ⁵ cells were counted per tube, centrifuged at 1500rpm at 4℃for 5min, medium was aspirated off, 100. Mu.l of PBS containing 2% BSA was used to resuspend the cells, blocked at 4℃for 20min, 1500rpm at 4℃for 5min, supernatant was aspirated off, 100. Mu.l of CD47 antibody at 100nM was used to resuspend the cells, 4℃for 1h, 1500rpm at 4℃for 5min, primary antibody was aspirated off, 500. Mu.l of PBS was used to resuspend the washed cells, 1500rpm at 4℃for 5min, supernatant was aspirated off, 100. Mu.l of diluted Alexa was used488 Labeled goat anti-human IgG antibody resuspended cells, incubated at 4℃for 30min, centrifuged at 1500rpm for 5min at 4℃and the secondary antibody was blotted off, washed cells were resuspended with 500. Mu.l PBS, centrifuged at 1500rpm for 5min at 4℃and the supernatant was blotted off and repeated once, resuspended cells were added with 250ul PBS, filtered through a 200 mesh cell screen and detected on-line using a flow cytometer. As shown in FIG. 2, the CD47 antibody of the invention can be effectively combined with tumor cells with high expression of CD47, and has good tumor cell targeting capability.

Example 6 detection of the Effect of CD47 antibodies on human Red Blood Cell (RBC) agglutination

Collecting fresh blood of a healthy human donor, separating lymphocytes by using lymphocyte separation liquid, sucking 500 mu l of red blood cells after centrifugation, adding 3ml of PBS to wash the cells, centrifuging at 2000rpm for 5min at room temperature, washing three times, re-suspending the red blood cells with PBS and diluting the red blood cells into 6% red blood cell suspension, adding the red blood cell suspension into a 96-well plate, performing red blood cell plating, setting the highest incubation final concentration of each CD47 antibody to 300nM, continuously diluting the red blood cells with PBS for 2 times, adding 12 concentration gradients in total, adding 50 mu l of each concentration gradient of CD47 antibody into each well correspondingly, taking out and observing the agglutination condition of the red blood cells in each well. The results are shown in FIG. 3, in the CD47 antibodies tested, antibodies D3705-G4 (300-9.375 nM) and HU5F9-G4 induced hemagglutination at high concentrations (300-1.172 nM). Antibodies D0604-G4, D0643-G4 had slight hemagglutination at high concentrations (300-150 nM), whereas D2510-G4 showed no significant hemagglutination even at the highest concentrations.

Example 7 detection of binding Capacity of CD47 antibody to human Red Blood Cells (RBC)

Mu.l of a 6% cell density suspension was aspirated per tube, the cells were resuspended in PBS containing 2% BSA, blocked at 4℃for 20min, spun at 1500rpm, at 4℃for 5min, the supernatant was aspirated, the highest incubation final concentration of each CD47 antibody was set to 200nM and serial 2-fold dilutions were made with PBS for 10 concentration gradients, 100. Mu.l of red blood cells were resuspended per tube, incubated at 4℃for 1h, the washed cells were resuspended once with 500. Mu.l of PBS, 100. Mu.l of diluted Alexa were used488 Labeled goat anti-human IgG antibody resuspended cells, 4℃incubation for 30min,1500rpm,4℃centrifugation for 5min, secondary antibody blotted off, washed twice with 500. Mu.l PBS, 1ml PBS was added to resuspend cells, filtered through a 200 mesh cell screen and detected on-machine using a flow cytometer. The results are shown in FIG. 4, where antibodies D0604-G4, D2510-G4 and Hu5F9-G4 all showed the ability to bind human blood erythrocytes to varying degrees and this ability was dose dependent. In particular, D0604-G4 and D2510-G4 showed lower binding to red blood cells, suggesting that there may be lower erythrocyte toxic side effects compared to Hu5F 9-G4.

Claims (7)

1. An anti-human CD47 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment specifically binds human CD47 and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region consists of VL CDR1, VL CDR2, and VL CDR3

Wherein:

D0604-G4 is the amino acid sequence shown in SEQ ID NO 8, 11 and 14 for VH CDR1, VH CDR2 and VH CDR3 in sequence, and the amino acid sequence shown in SEQ ID NO 18, 21 and 23 for VL CDR1, VL CDR2 and VL CDR3 in sequence;

D0643-G4 is the amino acid sequence shown in ID NO 8, 11 and 14 in sequence, and VL CDR1, VL CDR2 and VL CDR3 are the amino acid sequences shown in SEQ ID NO 17, 20 and 26 in sequence;

D2510-G4, wherein the VH CDR1, the VH CDR2 and the VH CDR3 are the amino acid sequences shown in ID NO 9, 12 and 15 in sequence, and the VL CDR1, the VL CDR2 and the VL CDR3 are the amino acid sequences shown in SEQ ID NO 17, 20 and 25 in sequence;

D3705-G4 the amino acid sequences shown in SEQ ID NOS 10, 13 and 16 for VH CDR1, VH CDR2 and VH CDR3 in order, and the amino acid sequences shown in SEQ ID NOS 19, 22 and 24 for VL CDR1, VL CDR2 and VL CDR3 in order.

2. An anti-human CD47 antibody or antigen-binding fragment thereof according to claim 1, wherein:

The heavy chain variable region of D0604-G4 is the amino acid sequence shown in SEQ ID NO. 1, and the light chain variable region is the amino acid sequence shown in SEQ ID NO. 4;

the heavy chain variable region of D0643-G4 is the amino acid sequence shown in SEQ ID NO. 1, and the light chain variable region is the amino acid sequence shown in SEQ ID NO. 7;

The heavy chain variable region of D2510-G4 is the amino acid sequence shown in SEQ ID NO. 2, and the light chain variable region is the amino acid sequence shown in SEQ ID NO. 6;

The heavy chain variable region of D3705-G4 is the amino acid sequence shown in SEQ ID NO. 3, and the light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

3. The anti-CD 47 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody has a constant region, a heavy chain constant region is selected from any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD, and a light chain constant region is a kappa or lambda chain.

4. A genetically engineered antibody comprising the anti-human CD47 antibody or an antigen-binding fragment thereof according to claim 2, wherein the genetically engineered antibody comprises a human-mouse chimeric antibody, or a humanized antibody, or a functional fragment Fab of an antibody, or a single-chain antibody.

5. A nucleotide sequence encoding the anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-3 or the genetically engineered antibody of claim 4.

6. An expression vector comprising a nucleotide sequence encoding the sequence of claim 5.

7. The use of an anti-CD 47 antibody or antigen-binding fragment thereof according to any one of claims 1-3 or a genetically engineered antibody according to claim 4 for the preparation of an anti-tumor drug, wherein the tumor is a CD47 high expressing tumor selected from leukemia, lymphoma, lung cancer, breast cancer, colorectal cancer, melanoma or ovarian cancer.