CN115894692A - Antibody targeting CD47 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a CD 47-targeted antibody 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 a biological ligand SIRPa thereof, and effectively recognize and bind to CD47 positive tumor cells. Meanwhile, the CD47 antibody does not cause significant hemagglutination and red blood cell binding of human blood at high concentration.

Description

Antibody targeting CD47 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a CD 47-targeted antibody and application thereof.

Background

Up to now, immunotherapy is considered as the most promising systemic tumor treatment compared to conventional anticancer treatment strategies, and plays an indispensable role in improving the therapeutic effect. Emerging cancer immunotherapy includes cancer vaccines, CAR-T cell therapy, cytokine therapy, immune checkpoint inhibitors, and tumor-targeting monoclonal antibodies. Among them, monoclonal antibodies have become a key 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 technologies applied to the fields of antibody discovery, target discovery and protein engineering, is one of the technical means for developing therapeutic antibodies in vitro, and realizes the progress from a prokaryotic expression system to a eukaryotic expression system. In the antibody engineering, the characteristics of a yeast eukaryotic expression system are utilized to express antibody library proteins on the surface of yeast cells, a magnetic bead and flow cell sorting technology is utilized to carry out screening based on antibody affinity and display level to obtain an antibody sequence with high affinity or high stability, various framework antibodies such as single-chain variable scFv (single-chain variable region), antibody binding region fragment Fab (antibody binding region fragment), full-length IgG (immunoglobulin G) and camelid single-domain antibody variable region VHH (variable domain human H) can be obtained according to design, meanwhile, a eukaryotic system is utilized to carry out post-translational modification on the antibodies, and the antibody characteristics are closer to those produced by mammalian cells.

The leukocyte differentiation antigen CD47 (cluster of differentiation 47), also known as 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 its structure comprises an extracellular variable region interacting with a corresponding ligand, a transmembrane region formed by a highly hydrophobic transmembrane segment, and a hydrophilic carboxyl-terminal intracellular region, and belongs to a member of the immunoglobulin superfamily. CD47 and its ligands not only modulate 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 regulatory protein alpha (SIRP alpha) is a specific ligand of the protein, and is mainly expressed on the surfaces of phagocytic cells such as macrophages and dendritic cells. The CD47/SIRP alpha interaction can transmit an inhibitory signal to inhibit the phagocytosis of relevant cells by macrophages.

Numerous studies have shown that CD47 is overexpressed in different types of tumors, that high levels of CD47 expression are associated with therapeutic response and prognosis of cancer progression, and that tumor cells transmit "do not eat me" signals to macrophages through CD 47/sirpa interactions, leading to innate immune escape of tumor cells, inducing further development and progression of tumors. Therefore, the CD47/SIRP alpha axis mediated inhibitory signal is blocked, the phagocytosis of the tumor cells by macrophages is restored, and a new idea and means are provided for tumor immunotherapy.

However, due to the widespread expression of CD47, the potential problem of anti-CD 47 antibodies as anti-cancer drugs may produce off-target effects, such as anemia. CD47 is a key regulator of erythrocyte renewal, and CD47 targeting antibodies generally have higher targeting binding property with erythrocytes, and can accelerate erythrocyte clearance and cause hemolytic anemia, for example, CD47 targeting agents (i.e. Hu5F9-G4 and TTI-621) can cause acute anemia and thrombocytopenia in human. Therefore, the monoclonal antibody of anti-CD 47 with high activity and low toxic and side effect has important significance for tumor immunotherapy.

Disclosure of Invention

The purpose of the invention is as follows: the present invention provides a novel antibody which has not only a good affinity for CD47 but also weak erythrocyte agglutination.

The invention provides a brand-new CD47 antibody or an antigen binding fragment thereof and a preparation method thereof, the provided CD47 antibody can identify and specifically bind to CD47, can be used for preparing a medicament for treating diseases related to CD47 abnormal expression or excess expression, and does not cause obvious hemagglutination reaction; or used for preparing products for detecting CD47 expression by Western blot, ELISA and flow cytometry.

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

VH CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 8-10;

VH CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 11-13;

VH CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 14-16;

VL CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 17-19;

VL CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20-22;

VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23-26.

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

。

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

。

an engineered antibody comprising heavy and light chain amino acid sequences identical to the amino acid sequences of claim 3; the genetic engineering antibody comprises: human-murine chimeric antibodies; or a humanized antibody; or a functional fragment of an antibody, fab; or is a single chain antibody; or an antibody functional fragment VH-L fused with a heavy chain variable region and a complete light chain; or a functional fragment of an antibody comprising an arrangement, tandem or combination of one or more CDRs of a heavy and light chain; or the antibody-like functional fusion protein obtained by connecting, splicing and fusing the antibody, the antibody functional fragment and other various proteins or polypeptides.

The anti-CD 47 antibody or antigen-binding fragment thereof or the engineered antibody, wherein the antibody has a constant region and the heavy chain constant region is selected from any one of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is a kappa or lambda chain. A nucleotide sequence 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 polypeptide.

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

Advantageous effects

The invention provides a series of full-length antibodies or single-chain antibodies, which have good CD47 resisting effect, weak or no erythrocyte aggregation effect and good application prospect in clinic.

Drawings

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

FIG. 2 is a flow cytometry assay of the ability of CD47 antibodies to bind to CD 47-expressing tumor cells.

FIG. 3 shows the results of CD47 antibody induced agglutination of red blood cells at different concentrations.

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

Detailed Description

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

The term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised 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. Specifically, the Complementarity Determining Regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. mabs contain antigen-binding sites that immunoreact with specific epitopes of an antigen.

The term "single chain antibody" (scFv) refers to an antibody in which the variable region of the antibody heavy chain (VH) and the variable region of the antibody light chain (VL) are linked by a 15-20 amino acid linker.

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

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein may be performed using molecular biology, biochemistry, cell culture, recombinant DNA techniques, and techniques common in the relevant art.

The reagents required for the following embodiments are commercially available (commercially available) without specific reference.

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-tagged hCD47 recombinant protein. When the vaccine is used for the first time, 100ug of antigen protein is mixed and emulsified with Freund's complete adjuvant according to the proportion of 1:1, and the vaccine is completed by subcutaneous multipoint injection. Four boosts were performed after that, each booster was separated by 14 days, the amount of antigen was halved, mixed and emulsified with Freund's incomplete adjuvant at the ratio of 1:1, the immunization was completed by intraperitoneal injection, and after each booster, a part of the tail of the mouse was cut off, tail vein blood of the mouse was collected and serum was isolated, and the serum titer of the mouse was measured by ELISA.

2) The hFc label hCD47 recombinant protein is diluted to 1 mu g/ml by using antigen coating solution, added to the enzyme standard strip after ultraviolet activation in an amount of 100 mu l/hole, provided with multiple holes, and provided with a PBS group as a negative control, and coated at 4 ℃ overnight. Absorbing and removing the coating solution, adding PBST to wash each hole for 5min each time, and washing for 6 times; adding 5% skimmed milk sealing solution into each hole, and sealing at 37 deg.C for 2 hr; removing the sealing liquid by suction, and washing each hole by the added PBST for 5min and 6 times each time; adding mouse serum diluted by PBS in different proportions into each hole, and incubating for 2h at 37 ℃; the mouse serum was aspirated, and the wells were washed with added PBST for 5min each time for 6 times; adding diluted HRP-goat anti-mouse IgG antibody into each hole, and incubating for 1.5h at 37 ℃; the secondary antibody was discarded, and the wells were washed with the added PBST 5min each time for 6 times; adding a freshly prepared TMB color developing solution into each hole, and developing for 15-30min at 37 ℃ in a dark place; stop solution is added into each hole to stop the color reaction, and the light absorption value of each hole under the wavelength of 450nm is measured to measure the serum titer of the immunized mice.

Example 2: screening and preparation of CD47 antibody

1. Extraction of mouse spleen Total RNA

1) According to the result of ELISA, selecting the mouse with the highest serum titer after the boosting immunization, separating the spleen of the mouse after the neck is cut off, preparing a spleen tissue single cell suspension, removing red blood cells by using a red blood cell lysate, adding a TRizol reagent, repeatedly blowing and beating the cells, and fully cracking the cells.

2) Adding 1/5 volume of chloroform into the lysate, and standing at room temperature for 2-3min; centrifuging at 12000g for 10-15min at 4 deg.C. Carefully absorbing the upper aqueous phase into a new centrifugal tube, adding 1/2 volume of isopropanol, reversing, uniformly mixing, and standing at room temperature for 10min; centrifuge at 12000g for 10min at 4 ℃. Discarding the supernatant, adding equal volume of 75% ethanol to wash the precipitate, and standing at room temperature for air drying for 5-10min. Add 50. Mu.l RNase-free water to dissolve the RNA.

2.RT-PCR

Designing a specific primer according to a mouse antibody variable region nucleic acid sequence, carrying out PCR amplification on mouse heavy chain variable region (VH) and light chain variable region (VL) genes by taking a cDNA first chain as a template, obtaining a DNA fragment of a mouse single-chain antibody (scFv) through overlap PCR, and purifying the scFv fragment by gel recovery after 1% agarose gel electrophoresis.

Construction of CD47 Yeast antibody library and antibody screening

1) Library construction: ecoRI and XhoI restriction enzymes are used for carrying out enzyme digestion on the yeast vector pYD1 overnight at 37 ℃, the linearized vector is recovered after gel electrophoresis of 1% agarose gel, and is electrically transformed and together transferred into a saccharomyces cerevisiae strain EBY100 with purified scFv gene fragments, the saccharomyces cerevisiae strain EBY is cultured overnight at 30 ℃ and 220rpm, so that the single-chain antibody is displayed on the surface of a yeast cell, a corresponding single-chain antibody library is constructed, and the library is named DEM139.

2) Magnetic bead fractionSelecting: determination of OD of yeast liquid after electrotransformation ₆₀₀ Inoculating the strain in an induction culture medium at a proper density, and performing induction culture at 20 ℃ and 220rpm overnight; taking the post-induction yeast, washing with 1% PBSA, adding the hFc-labeled CD47 antigen protein after biotin labeling, incubating at room temperature for 20min, washing the yeast with pre-cooled 1% PBSA, placing on a magnetic frame, sorting with streptavidin magnetic beads, collecting the yeast with an amplification medium, and culturing at 30 ℃ and 220rpm overnight.

3) Flow type separation: determining OD of yeast liquid after magnetic bead sorting ₆₀₀ Inoculating the strain in an induction medium at a suitable density, inducing and culturing overnight at 20 deg.C and 220rpm, and measuring OD of the strain ₆₀₀ Sucking proper amount of yeast, washing with PBS, centrifuging at 3000rpm for 5min, removing supernatant, re-suspending yeast with biotin-labeled hFc-labeled 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 Alexa

And (3) incubating the 488-labeled mouse anti-V5 tag antibody at 4 ℃ for 30min, washing with precooled PBS, centrifuging at 3000rpm for 5min, removing supernatant, adding PBS to resuspend cells, sorting yeast cells with APC (adenosine monophosphate) and 488 double positive into an amplification culture medium according to a fluorescence intensity circle gate, performing overnight culture at 30 ℃ and 220rpm, performing induced culture again, and performing second alternate sorting.

4) Library sequencing: and (3) carrying out amplification culture on the yeast after two-turn sorting, extracting yeast plasmids, sequencing the library, and selecting 50 monoclonals with better growth states for sequencing.

Preparation of CD47 antibody

1) Construction of eukaryotic expression vectors: according to the library sequencing result, selecting 4 single-chain antibody sequences with incompletely identical CDR sequences, designing primers, splicing an antibody light chain variable region and a human Ig kappa chain constant region through overlap PCR, splicing an antibody heavy chain variable region and a human IgG4 constant region, converting each single-chain antibody into a full-length antibody, splicing antibody light and heavy chain genes into a mammal expression vector through a homologous recombination method, and obtaining the corresponding CD47 full-length antibody eukaryotic expression vector.

2) Combinations of VH and VL for 4 full length IgG4 antibodies are shown in table 1; the C end of VH is connected with CH with the sequence as SEQ ID NO. 27 to form the heavy chain of the antibody; the C end of VL is connected with CL with the sequence shown as SEQ ID NO. 28 to form the light chain of the antibody; the variable region of the heavy chain of an antibody is represented by "D" + antibody heavy chain number + "-VH", e.g., D06-VH; the variable region of the light chain of the antibody is represented by "D" + the antibody light chain number + "-VL", e.g., D04-VL; igG4 full-length antibodies are designated by "D" + antibody heavy chain number + antibody light chain number + "-G4", e.g., D0604-G4 (antibody heavy chain consisting of D06-VH (SEQ ID NO: 1) and CH having the sequence shown in SEQ ID NO:27, and antibody light chain consisting of D04-VL (SEQ ID NO: 4) and CL having the sequence shown in SEQ ID NO: 28), and the 4 full-length antibody numbers selected are abbreviated as D0604-G4, D0643-G4, D2510-G4, and D3705-G4.

3) The recombinant plasmids of light and heavy chains of the coded CD47 antibody and a positive control antibody Hu5F9-G4 (the Hu5F9-G4 sequence is disclosed in US20150183874A 1) are transfected into HEK293 cells according to the proportion of 2:1, after 5 days of culture, supernatant is centrifugally collected at 4 ℃, the CD47 antibody is obtained after purification by a protein A column, and the molecular weight and purity of the antigen and the type of the antibody are respectively detected by SDS-PAGE, SEC-HPLC and Western blot.

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

Table 1: full-length antibody formed by combining CD47 antibody heavy chain variable region and light chain variable region

Table 2: CDR regions of antibodies

Example 3: determination of the binding Capacity and affinity of the CD47 antibody to the hCD47 molecule

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 enzyme standard strip at a rate of 100. Mu.l/well, duplicate wells were set, and PBS group was set as a negative control, and 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, a gradient of diluted CD47 antibody and control antibody Hu5F9 (maximum concentration 200nM, total 8 concentration gradients) was added and incubated for 2h at 37 ℃. PBST washing 3 times, adding HRP labeled goat anti-human IgG secondary antibody, incubating at 37 ℃ for 1h, washing with PBST 6 times, adding TMB developing solution, incubating for 15min in dark place, and terminating the developing reaction. 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, for each CD47 antibody at the protein level that was effective in binding hCD47 protein.

Table 3: determination of affinity of CD47 antibody to hCD47 molecule

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 blocking CD47 and SIRP alpha by CD47 antibody

The hFc label hCD47 recombinant protein is diluted to 1 mu g/ml by using antigen coating solution, added to the enzyme standard strip after ultraviolet activation in an amount of 100 mu l/hole, provided with multiple holes, and provided with a PBS group as a negative control, and coated at 4 ℃ overnight. Washed 3 times with PBST, added with 5% skim milk, and blocked at 37 ℃ for 2h. Washed 3 times with PBST, added with gradient diluted CD47 antibody and control antibody Hu5F9 (maximum concentration 300nM, total 10 concentration gradients) and a final concentration of 2 μ g/ml of biotinylated mFc-tagged SIRP α recombinant protein, and incubated for 1h at 37 ℃. Washing PBST for 3 times, adding HRP-labeled streptavidin, incubating at 37 ℃ for 1h, washing with PBST for 6 times, adding TMB color development solution, incubating for 15min in dark place, and terminating the color development reaction. The absorbance at 450nm was read on a microplate reader and the IC50 of each antibody was calculated and the results are shown in table 4 for CD47 antibodies that effectively blocked the interaction of CD47 with sirpa.

Table 4: assay results for CD47 antibodies blocking the binding of CD47 to SIRP alpha

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 CD47 expressing tumor cells

Jurkat cells highly expressing CD47 were cultured in 1640 medium containing 10% FBS at 37 ℃ under 5% CO2 conditions. When the cell growth state is good, counting, and taking 2 × 10 per tube ⁵ Centrifuging the cells at 1500rpm at 4 deg.C for 5min, and removing the culture medium; resuspend the cells with 100. Mu.l of 2-percent BSA in PBS, and block for 20min at 4 ℃; centrifuging at 1500rpm and 4 deg.C for 5min, and removing supernatant; resuspend the cells with 100. Mu.l CD47 antibody at 100nM and incubate for 1h at 4 ℃; centrifuging at 1500rpm and 4 deg.C for 5min, and removing primary antibody; resuspend the washed cells with 500. Mu.l PBS, centrifuge at 1500rpm at 4 ℃ for 5min, and discard the supernatant; with 100. Mu.l of diluted Alexa

The 488-labeled goat anti-human IgG antibody is used for resuspending the cells, incubating at 4 ℃ for 30min, centrifuging at 1500rpm at 4 ℃ for 5min, and absorbing and discarding the secondary antibody; resuspend the washed cells with 500. Mu.l PBS, centrifuge at 1500rpm at 4 ℃ for 5min, aspirate the supernatant, and repeat once; 250ul PBS was added to resuspend the cells, filtered through a 200 mesh cell screen and tested on a flow cytometer. The result is shown in fig. 2, the CD47 antibody of the present invention can effectively bind to the tumor cells with high expression of CD47, and has good tumor cell targeting ability.

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

Collecting fresh blood of healthy human donor, separating lymphocyte with lymphocyte separating medium, centrifuging, sucking 500 μ l of erythrocyte, adding 3ml PBS to wash the cell, centrifuging at 2000rpm for 5min at room temperature, and washing for three times; resuspending and diluting erythrocytes with PBS to 6% erythrocyte suspension, adding to 96-well plate, and performing erythrocyte plating with 50 μ l cell suspension per well; setting the highest incubation final concentration of each CD47 antibody to 300nM, and serially diluting the antibody with PBS 2-fold for 12 concentration gradients; 50 μ l of each concentration gradient of CD47 antibody should be added to each well, incubated at 37 ℃ for 4h, and taken out to observe the agglutination of the red blood cells in each well. As shown in FIG. 3, among the tested CD47 antibodies, the antibodies D3705-G4 (300-9.375 nM) and HU5F9-G4 induced erythrocyte agglutination at high concentrations (300-1.172 nM). Antibodies D0604-G4, D0643-G4 showed slight agglutination of erythrocytes at high concentrations (300-150 nM), while D2510-G4 showed no obvious agglutination of erythrocytes even at the highest concentrations.

Example 7: detection of the binding Capacity of CD47 antibodies to human Red Blood Cells (RBC)

Pipetting 20. Mu.l/tube of a suspension of red blood cells having a cell density of 6%, resuspending the cells in PBS containing 2% BSA, blocking at 4 ℃ for 20min, at 1500rpm, centrifuging at 4 ℃ for 5min, and discarding the supernatant; setting the highest incubation final concentration of each CD47 antibody to be 200nM, continuously diluting the antibody with PBS by 2 times, obtaining 10 concentration gradients in total, resuspending red blood cells by100 mu l per tube, and incubating the antibody for 1h at 4 ℃; the cells were washed once with 500. Mu.l PBS resuspension; with 100. Mu.l of diluted Alexa

The 488-labeled goat anti-human IgG antibody is used for resuspending the cells, incubating at 4 ℃ for 30min, centrifuging at 1500rpm at 4 ℃ for 5min, and absorbing and discarding the secondary antibody; the cells were washed twice with 500. Mu.l PBS, and 1ml PBS was added to resuspend the cells, which were filtered through a 200 mesh cell screen and examined on a flow cytometer. The results are shown in FIG. 4, and the tested CD47 antibodies, antibodies D0604-G4, D2510-G4 and Hu5F9-G4, all showed the ability to bind to human erythrocytes to different degrees, and the ability was dose-dependent. In particular, D0604-G4 and D2510-G4 showed lower binding to red blood cells, suggesting a lower potential for erythrocytic toxicity compared to Hu5F 9-G4. />

Claims (8)

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

1) VH CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 8-10;

2) VH CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 11-13;

3) VH CDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 14-16;

4) VL CDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 17-19;

5) VL CDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20-22;

6) VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23-26.

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

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

4. an engineered antibody comprising heavy and light chain amino acid sequences identical to the amino acid sequences of claim 3; the genetic engineering antibody comprises: human-murine chimeric antibodies; or a humanized antibody; or a functional fragment of an antibody, fab; or is a single chain antibody; or an antibody functional fragment VH-L fused with a heavy chain variable region and a complete light chain; or a functional fragment of an antibody comprising an arrangement, tandem or combination of one or more CDRs of the heavy and light chains; or the antibody-like functional fusion protein obtained by connecting, splicing and fusing the antibody, the antibody functional fragment and other various proteins or polypeptides.

5. The anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-3 or the engineered antibody of claim 4, wherein the antibody has a constant region and the heavy chain constant region is selected from any one of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is a kappa or lambda chain.

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

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

8. Use of the anti-CD 47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the genetically engineered antibody according to claim 4 for the preparation of an anti-tumor medicament.

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