CN109438576B

CN109438576B - Preparation and application of an anti-human CD47 monoclonal antibody

Info

Publication number: CN109438576B
Application number: CN201811350763.3A
Authority: CN
Inventors: 钱峰; 俞凯凯; 孙树洋; 吴穷; 张志愿; 孙磊; 王军; 顾子悦; 胡玉冬
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2021-08-20
Anticipated expiration: 2038-11-14
Also published as: CN109438576A

Abstract

The invention belongs to the field of antibodies, and particularly relates to a preparation method and application of an anti-human CD47 monoclonal antibody. The invention discloses a highly variable region sequence of the monoclonal antibody. The anti-human CD47 monoclonal antibody provided by the invention has good binding activity, can effectively recognize the expression of CD47 on the surface of a tumor cell, and can effectively recognize the recombinant human CD47 extracellular region protein at the protein level. Meanwhile, the epitope identification of the monoclonal antibody is clear, and the monoclonal antibody can be effectively applied to the diagnosis of CD47 target molecules and the preparation and development of anti-human CD47 monoclonal antibody medicines.

Description

Preparation and application of anti-human CD47 monoclonal antibody

Technical Field

The invention relates to the technical field of biology, in particular to preparation and application of an anti-human CD47 monoclonal antibody.

Background

CD47(Cluster of Differentiation 47), also known as integrin-associated protein, is widely expressed on normal cell surfaces and is one of the currently known immune checkpoints. CD47, when bound to thrombospondin-1 (thrombospondin-1) as a receptor, can affect cell-related functions including cell migration, adhesion, proliferation and apoptosis, and also modulate angiogenesis and inflammatory responses. When the protein is combined with a signal-regulatory protein alpha (SIRP alpha) on the surface of the macrophage as a ligand, the protein-regulatory protein alpha can transmit an inhibitory signal to inhibit the phagocytosis of related cells by the macrophage. Aged red blood cells in the blood circulation are more easily cleared by macrophages due to the reduced expression of CD 47. The research shows that the CD47 is widely expressed in various tissues and is highly expressed on the surface of tumor cells, and the clinical report shows that the high expression of CD47 in the tumor tissues is related to poor prognosis. The inhibitory signal generated when the highly expressed CD47 on the surface of the tumor cell is combined with SIRP alpha promotes the tumor cell to escape from phagocytosis of macrophages, thereby inducing the occurrence and development of tumors, and being one of the main molecules for guiding the tumor cell to escape from immune surveillance in the tumor microenvironment. The tumor cell phagocytosis function of macrophages is restored or strengthened by blocking an inhibitory signal path transmitted by the combination of CD47 on the surface of the tumor cells and SIRPa, and the macrophage-mediated cellular immune response can be promoted, thereby providing a new theory and method for tumor immunotherapy.

Human CD47 (encoded by gene CD 47) is a member of the immunoglobulin superfamily, and the extracellular domain structure of human CD47 and the crystal structure of CD47 and its ligand sirpa have been defined so far. CD47 consists of five transmembrane regions and an extracellular N-terminal IgV domain, the extracellular region comprising 139 amino acids, with four spliceosome forms depending on the length of the intracellular region, distributed in different tissues, with the most widely expressed spliceosome 2 being distributed in circulating and immune cells. The sirpa intracellular domain has a tyrosine-based Immunoreceptor Tyrosine Inhibition Model (ITIM) which, when bound to CD47, is phosphorylated and recruits inhibitory molecules such as SHP-1 and the like to prevent immune cell activation.

CD47 plays a crucial role in tumor immune evasion mechanisms. The existing research results already confirm that the CD47 is involved in the generation and development of tumors and is considered as an important target point of tumor immunotherapy. High expression of CD47 has been found in human hematological tumors including acute and chronic myeloid leukemia and non-hodgkin's lymphoma and solid tumors including sarcomas, colon cancer, renal cancer, multiple myeloma and including bladder cancer, lung cancer, etc., by flow cytometry and immunohistochemical analysis. Therefore, the analysis of the expression of CD47 in tumor diseases has extremely important clinical significance for the prognosis evaluation of tumors.

In recent years, the development of drugs aiming at the tumor escape mechanism mediated by CD47 and a ligand SIRP alpha signal axis becomes the hot door of tumor immunotherapy besides PD-1/PD-L1, and the development of CD47 antibody currently enters the preclinical stage. However, the monoclonal antibody against human CD47 currently being developed has the disadvantages of low affinity, large side effects, and unclear target of action, such as epitope.

Disclosure of Invention

The invention provides a preparation method of anti-human CD47 monoclonal antibody and application thereof, aiming at solving the main technical problems that the existing anti-CD 47 monoclonal antibody has low affinity and the target point of action such as epitope is not clear.

The first aspect of the present invention provides an anti-human CD47 monoclonal antibody, wherein the anti-human CD47 antibody comprises a heavy chain variable region and a light chain variable region, and the anti-CD 47 antibody has one or more of the following technical characteristics:

<1> the complementarity determining region of the heavy chain variable region comprises CDRH1 having an amino acid sequence shown in SEQ ID No. 1;

<2> the complementarity determining region of the heavy chain variable region comprises CDRH2 having an amino acid sequence shown in SEQ ID No. 2;

<3> the complementarity determining region of the heavy chain variable region comprises CDRH3 having an amino acid sequence shown in SEQ ID No. 3;

<4> the complementarity determining region of the light chain variable region includes CDRL1 having an amino acid sequence shown in SEQ ID No. 4;

<5> the complementarity determining region of the light chain variable region includes CDRL2 having an amino acid sequence shown in SEQ ID No. 5;

<6> the complementarity determining region of the light chain variable region includes CDRL3 whose amino acid sequence is shown in SEQ ID No. 6.

A CDR (complementary determining region) generally refers to a region of an antibody that can sterically complement an antigenic determinant. The variability in antibodies is typically not evenly distributed throughout the variable region of the antibody, and monoclonal antibodies typically have 3 hypervariable regions (HVRs) in both the heavy and light chain variable regions, which are typically sterically complementary to an antigenic determinant, so the hypervariable regions are also referred to as Complementarity Determining Regions (CDRs), i.e., the heavy chain variable region typically includes three complementarity determining regions, namely CDRH1, CDRH2 and CDRH3, and the light chain variable region typically includes three complementarity determining regions, namely CDRL1, CDRL2 and CDRL 3.

In one embodiment, the complementarity determining regions of the heavy chain variable region of the anti-human CD47 monoclonal antibody include CDRH1 having an amino acid sequence shown in SEQ ID No.1, CDRH2 having an amino acid sequence shown in SEQ ID No.2, and CDRH3 having an amino acid sequence shown in SEQ ID No. 3; and/or the complementarity determining region of the light chain variable region of the anti-human CD47 monoclonal antibody comprises a CDRL1 with an amino acid sequence shown as SEQ ID No.4, a CDRL2 with an amino acid sequence shown as SEQ ID No.5 and a CDRL3 with an amino acid sequence shown as SEQ ID No. 6.

In one embodiment, the complementarity determining regions of the heavy chain variable region of the anti-human CD47 monoclonal antibody include CDRH1 having an amino acid sequence shown in SEQ ID No.1, CDRH2 having an amino acid sequence shown in SEQ ID No.2, and CDRH3 having an amino acid sequence shown in SEQ ID No. 3.

In one embodiment, the complementarity determining regions of the light chain variable region of the anti-human CD47 monoclonal antibody include CDRL1 with the amino acid sequence shown in SEQ ID No.4, CDRL2 with the amino acid sequence shown in SEQ ID No.5, and CDRL3 with the amino acid sequence shown in SEQ ID No. 6.

In one embodiment, the complementarity determining regions of the heavy chain variable region of the anti-human CD47 monoclonal antibody include CDRH1 having an amino acid sequence shown in SEQ ID No.1, CDRH2 having an amino acid sequence shown in SEQ ID No.2, and CDRH3 having an amino acid sequence shown in SEQ ID No.3, and the complementarity determining regions of the light chain variable region include CDRL1 having an amino acid sequence shown in SEQ ID No.4, CDRL2 having an amino acid sequence shown in SEQ ID No.5, and CDRL3 having an amino acid sequence shown in SEQ ID No. 6.

In one embodiment, the heavy chain variable region and the light chain variable region of the anti-human CD47 monoclonal antibody may further include a framework region, and the framework region may be located between the complementarity determining regions or located at two ends of the complementarity determining regions.

In one embodiment, the heavy chain variable region of the anti-human CD47 monoclonal antibody has the amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO. 8.

In a second aspect, the invention provides a nucleotide molecule encoding a monoclonal antibody against human CD47 as described above.

In one embodiment, the nucleotide sequence of the heavy chain of the anti-human CD47 monoclonal antibody encoded by the nucleotide molecule is shown as SEQ ID NO. 9, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 10.

In a third aspect, the present invention provides an expression vector comprising a nucleotide molecule as described above. The expression vector of the present invention is generally referred to various commercially available expression vectors well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

In a fourth aspect, the invention provides a host cell comprising an expression vector or a gene as described above, wherein the exogenous nucleotide molecule is integrated. Any cell suitable for expression of an expression vector may be used as a host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Further examples are BL21(DE3) cells and 293F, COS, CHO cells and the like.

The fifth aspect of the present invention provides a method for preparing the above-mentioned monoclonal antibody against human CD47, comprising the following steps:

culturing the host cell as described above under conditions suitable for expression of the antibody, thereby expressing the monoclonal antibody, and purifying and isolating the monoclonal antibody.

The host cell expresses the anti-human CD47 monoclonal antibody under a proper expression condition or obtains a hybridoma cell which stably secretes the anti-human CD47 monoclonal antibody by a hybridoma technology.

The host cells used in the present invention are available in the prior art, and can be obtained directly from commercial sources, and the culture medium used in the culture can be various conventional media, and those skilled in the art can select an appropriate medium according to experience and culture the appropriate medium under conditions suitable for the growth of the host cells. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The sixth aspect of the invention provides the use of the anti-CD 47 antibody as described above in the preparation of an anti-tumor therapeutic agent, or in the preparation of a diagnostic agent for tumors.

The tumor treatment medicine promotes the function of macrophage for phagocytizing tumor cells by blocking CD47 so as to kill the tumor cells.

The tumor treatment drug can be a drug which takes a CD47 antigen functionally expressed on the surface of a tumor cell as a target, binds or acts on a CD47 antigen, and thereby treats and/or prevents tumors. Such tumors include, but are not limited to: lung cancer, gastric cancer, cervical cancer, B lymphoma.

The medicine for treating the tumor can be used for tumor cells with high expression of CD47, macrophages are added after the anti-human CD47 monoclonal antibody and the tumor cells are incubated together for phagocytosis, and the using concentration of the medicine is adjusted according to different cells. The conventional dose is 20-50. mu.g antibody/10⁵And (4) tumor cells.

The tumor diagnosis medicine can be used for detecting by taking a CD47 antigen functionally expressed on the surface of a tumor cell as a target, and the anti-human CD47 monoclonal antibody is used as a primary antibody and a fluorescent secondary antibody, such as a FITC-coupled goat anti-mouse antibody, for combined use, and the use concentration of the anti-human CD47 monoclonal antibody is adjusted according to different cells. The conventional dose is 10-100. mu.g antibody/10⁶And (4) cells.

The seventh aspect of the invention provides a target antigen of a monoclonal antibody against human CD47, which is characterized in that the epitope amino acid sequence of the target antigen is shown as SEQ ID NO. 11.

The eighth aspect of the present invention provides a method for identifying an antigen targeted by the above-described anti-human CD47 monoclonal antibody. The identification method comprises the following steps: identifying an epitope of the target antigen may specifically be:

a) the amplified antigen fragment truncation can be used for obtaining the antigen fragment truncation nucleotide molecules by using a molecular cloning technology such as a PCR method or an artificial complete sequence synthesis method.

b) The antigen fragment truncation is constructed into an expression type prokaryotic vector such as pGEX-4T-1 by utilizing a molecular cloning technology, and then is transformed into escherichia coli such as BL21 for expression by using a chemical transformation mode.

c) Taking the antigen fragment truncation body expressed in the b) to carry out protein immune imprinting, and analyzing the antigen epitope recognized by the antibody according to the result.

The reagent consumables used in the invention are all available on the market.

The invention has the beneficial effects that: the anti-human CD47 monoclonal antibody provided by the invention has good binding activity, can effectively recognize the expression of CD47 on the surface of a tumor cell, and can effectively recognize the recombinant human CD47 extracellular region protein at the protein level. Meanwhile, the epitope identification of the monoclonal antibody is clear, and the monoclonal antibody can be effectively applied to the diagnosis of CD47 target molecules and the preparation and development of anti-human CD47 monoclonal antibody medicines.

Drawings

FIG. 1 shows the results of investigation of the expression conditions of the target protein.

FIG. 2 shows the purification results of the target protein.

FIG. 3 shows the purity test result of the renaturation of the target protein.

FIG. 4 is a flow cytometry assay for CD47 antigen expression on the surface of tumor cells.

FIG. 5 shows the IPTG induction of the expression of the target protein and GST.

FIG. 6 shows the result of identifying epitope by Western blot.

FIG. 7 shows the result of IPTG induction of target protein expression.

FIG. 8 shows the result of further identifying epitope by Western blot.

FIG. 9 shows the in vitro anti-tumor effect of the monoclonal antibody.

Detailed Description

The term "monoclonal antibody (mab)" as used herein 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. Monoclonal antibodies are directed against a single antigenic spot with high specificity. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

The terms "antibody" and "immunoglobulin" as used herein are heterotetrameric glycoproteins of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain is composed of a variable domain (VL) at one end and a constant domain at the other end: the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acids form the interface between the variable regions of the light and heavy chains.

The term "variable" as used herein means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called the Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are substantially in a p-fold configuration, connected by three CDRs forming a concatemeric loop, and in some cases may form part of a p-fold structure. The CDRs in each chain are held closely together by the FR regions and form the antigen-binding site of the antibody with the CDRs of the other chain, and the constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, e.g., participation in antibody-dependent cytotoxicity of the antibody.

Monoclonal antibodies can be obtained by various methods well known to those skilled in the art. For example, monoclonal antibodies can be made by the hybridoma method (first proposed by Kohler et al, Nature,256:495 (1975)), or by recombinant DNA methods (U.S. Pat. No.4,816,567). Monoclonal antibodies can also be identified, for example, by Clackson et al, Nature, 352: 624-: 581-597(1991) from phage antibody library.

The resulting monoclonal antibodies can be identified by conventional means. The binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro tuberculosis assay analysis.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and its several details are capable of modifications and variations in various respects, all without departing from the spirit of the invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Experimental materials:

balb/c female mice at 6-8 weeks, supplied by Shanghai Slek laboratory animals, Inc.;

cell line: MKN-45 cells (human gastric cancer cell line), A549 cells (human lung adenocarcinoma cell line), Hela cells (human cervical cancer cell line), Raji cells (B lymphoma cell line) and 293F cell line (human embryonic kidney cell line), SP2/0 (mouse myeloma cell line); wherein the MKN-45 cells and the Hela cells are cultured in RPMI1640 medium which contains necessary nutrient fetal bovine serum (10%) required by cell growth and a certain content of double antibodies (1%, penicillin and streptomycin solution); the cell lines from other sources are all cultured by using a DMEM medium, and the rest are the same as the components and the proportion; cell culture media (including DMEM and RPMI 1640), fetal bovine serum, 0.25% pancreatin, penicillin and streptomycin solutions (100 ×, double antibody), FreeStyle MAX transfection reagent and 293F FreeStyle medium, ECL luminophores were all from seemer flyer (Thermo, china).

Sandwich enzyme-linked immunosorbent assay (ELISA) reagents:

antigen coating buffer (PH9.60.05M carbonate buffer): 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, and distilled water is added to the mixture to reach 1000 ml; wash buffer (PH7.4PBS): 0.15 g of potassium dihydrogen phosphate 0.2M; disodium hydrogen phosphate dodecahydrate 2.9 g, sodium chloride 8.0 g, potassium chloride 0.2 g, 0.05% Tween 200.5 ml, add distilled water to 1000 ml; sealing liquid: 2g of Bovine Serum Albumin (BSA), and adding a washing buffer to 100 ml; stop solution (2M sulfuric acid): 178.3ml of distilled water, 21.7ml of concentrated sulfuric acid (98%) is added dropwise; substrate buffer (PH5.0 phosphate and citrate): 25.7ml of 0.2M disodium hydrogen phosphate dodecahydrate (28.4 g/L), 24.3ml of 0.1M citric acid (19.2 g/L), and 50ml of distilled water; TMB (tetramethylbenzidine) use solution: 0.5ml of TMB solution (10mg of TMB dissolved in 5ml of absolute ethanol), 10ml of substrate buffer (pH5.0), 0.75% H₂O₂32 μ l; HRP-goat anti-mouse IgG antibody was purchased from Jackson as Elisa secondary antibody. ELISA was coated with plates from Biofil.

And others: trizol RNA extraction reagent, Gel/PCR purification kit of Sigma company, FAGCK001, FAVORGEN; plasmid DNA miniprep kit, fapide 300, FAVORGEN; ReverTra Ace qPCR RT Kit, FSQ-101, TOYOBO; KOD-neo-pl μ s high fidelity PCR enzyme, TOYOBO; skim milk powder, BD; bovine serum albumin BSA, V900933, Sigma company; DH5 α competence, BL21 competence, shanghai geoonly biotechnology limited; kanamycin sulfate, Ni-NTA agarose gravity column, biological engineering (Shanghai); recombinant mouse colony stimulating factor, near shore protein, prepared into 100 mug/ml mother liquor and packed in a refrigerator at-80 deg.c. Freund's complete adjuvant, Freund's incomplete adjuvant, PEG1500, Sigma Co; 8-azaguanine, Sigma for stable selection culture of the SP2/0 cell line; BCA assay kit, pecan corporation; mouse immunoglobulin typing kit, BD Pharmingen. Reagents such as methanol, absolute ethanol, isopropanol, chloroform, 30% hydrogen peroxide solution, dimethyl sulfoxide, sodium chloride, glycine, Tris, SDS, urea and the like were purchased from the national pharmaceutical group.

EXAMPLE 1 preparation of recombinant human CD47 extracellular region antigen

Extracting RNA in MKN-45 cells by using a Trizol method: 1X10 a night in advance⁶Putting MKN45 seeds in a 3cm culture dish, discarding culture medium supernatant the next day, rinsing with 1xPBS, digesting with 1ml Trizol, centrifuging at 12000 Xg in a refrigerated high-speed centrifuge at 4 ℃ for 5 minutes, carefully taking out the tube after centrifugation, slightly tilting, sucking the uppermost layer of liquid into another sterilized 1.5ml centrifuge tube without RNase by using a gun head, adding chloroform according to the volume ratio of 1:5 of chloroform to Trizol, and fully and uniformly mixing the liquid by turning upside down to make it milk white; standing at room temperature for 3 minutes, centrifuging at 12000 Xg in a refrigerated high-speed centrifuge at 4 deg.C for 15 minutes, carefully taking out the tube after centrifugation, wherein layering is observed in the tube, adding the upper aqueous phase (containing RNA) into another sterilized centrifuge tube without RNase, which is marked with corresponding name, to avoid sucking the middle layer as much as possible, and preventing RNA from being polluted by protein; then carefully adding isopropanol into the tube according to the volume ratio of 500 mul of isopropanol in each milliliter of Trizol, turning the tube upside down for about ten times to fully mix the liquid evenly, then standing the mixture for 10 minutes at room temperature, and then adding the mixture into the tubeCentrifuging at 4 deg.C for 10min at 12000 Xg in a refrigerated centrifuge, and decanting the supernatant; then adding 75% ethanol according to the proportion that 1ml of 75% ethanol (prepared by DEPC water, namely 7.5ml of absolute ethanol is mixed with 1.5ml of DEPC water, and then placing the mixture in a refrigerator for precooling at 4 ℃) is added into each ml of Trizol, and the tube wall is flicked to suspend the sediment, and the sediment is in a white feather shape; the tube was then centrifuged at 7500 Xg for 5 minutes at 4 ℃; after centrifugation, the supernatant was aspirated, the tube was inverted on a dust-free paper and left at room temperature for 15 minutes to ensure that the solvent was evaporated as clean as possible, and finally 50. mu.l of DEPC was added to the centrifuge tube to dissolve the precipitate, and the RNA concentration was determined using a Nanodrop.

Total mRNA extracted from MKN-45 cells was reverse transcribed using the reverta Ace qPCR RT kit to obtain human CD47 cDNA: polymerase Chain Reaction (PCR) forward primers were designed using primer5 software: GATATCATATGCAGCTACTATTTAATAAAACAAAA (SEQ ID NO.12) and a reverse primer: GATATCTCGAGTTATGGAGAAAACCATGAAACAAC (SEQ ID NO.13) amplified the human CD47 extracellular domain protein. Analyzing the purity of the product by nucleic acid electrophoresis; the PCR product was subjected to rapid product recovery and purification using Gel/PCR purification kit, then BamHI and Xho I cleavage of pET28a (+) vector (Novagen, Darmstadt, Germany) and the target gene were performed, and ligation of the vector and the target gene was performed at 4 ℃ overnight. And (3) performing transformation on the next day, selecting about 10 single colonies to an LB plate with corresponding resistance and grid number, culturing for about 3 hours, performing colony PCR detection, and using the PCR product as a PCR template of a positive control. PCR positive clones were cultured overnight in a 37 ℃ bacterial shaker. The next day, plasmid extraction was performed with a plasmid DNA miniprep kit, and positive cloning was verified by restriction enzyme digestion and sequencing. After verification, the plasmid was transformed into BL21 competent cells. The transformed colonies were inoculated into 10ml of LB medium containing 50. mu.g/ml kanamycin and cultured overnight at 37 ℃ at 220 rpm. The following day, the reaction mixture was mixed with 10ml of LB in a volume of 1:100 to 5 new 50ml tubes (labeled "Ct 1", "1", "2", "3", "4") were seeded with cells. These tubes were incubated at 37 ℃ and 220rpm for about 3 hours until OD600 values were between 0.5 and 0.8. The 5 labeled tubes were then incubated under 5 different conditions: induction was carried out at 37 ℃ for 5 hours with isopropyl-. beta. -D-thiogalactopyranoside (IPTG) (0.5mM and 0.1mM) and at 16 ℃ for 16 hours with IPTG (0.5mM and 0.1). The culture was incubated at 37 ℃ for 5 hours without IPTG induction. The cells were centrifuged at 4,000rpm at 4 ℃ for 20 minutes to remove the upper layer, and the precipitate was resuspended in 2ml of phosphate buffered saline (PBS, pH7.4) and subjected to ultrasonic lysis. The lysate was then centrifuged at 13,000g for 10 minutes at 4 ℃ for supernatant. Dissolving 40 μ l of lysate and supernatant in 10 μ l of 5X sample buffer, and decocting at 99 deg.C for 10 min; 20 μ L of the mixture was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie Brilliant blue for 1 hour, and then destained overnight using a destaining solution containing 45% (v/v) methanol and 10% (v/v) acetic acid to evaluate the optimal expression conditions for the target protein, and the results are shown in FIG. 1. As shown in FIG. 1, "T" represents lysate, "S" represents supernatant, the expression level of the target protein is high when the target protein is induced with IPTG 0.5mM at 16 ℃, and most of the target protein is not in the supernatant and needs to be purified from inclusion bodies.

For the abundantly expressed CD47 antigen: the overnight bacterial cultures (8ml) were all inoculated into 800ml fresh medium and induced with 0.5mM IPTG for 16 hours at 16 ℃. The inclusion body pellet from the cell lysate was washed 3 times with PBS, then denatured by denaturing buffer (8M urea, 5mM EDTA, 20mM Tris, pH8.0), lysis denaturation of the inclusion bodies was promoted using ultrasonication and vigorous shaking, and then the supernatant was collected as solubilized inclusion body protein by centrifugation at 13,000g for 10 minutes at 4 ℃. Purifying denatured CD47 extracellular region protein by using a pre-loaded Ni-NTA agarose gravity column, slowly flowing protein supernatant through the gravity column, and collecting flowing liquid; then eluted and collected with 1ml of 20mM, 1ml of 50mM and 10ml of 300mM imidazole solution (dissolved in PBS), respectively. After purification, 40. mu.l of each eluent was dissolved in 10. mu.l of 5 Xloading buffer and boiled at 99 ℃ for 10 min; SDS-PAGE was performed to analyze the size and purity of the target protein in the eluate, and the results are shown in FIG. 2. As shown in fig. 2, the bands of samples 2 to 7 are relatively single and of high purity.

Renaturation of the CD47 antigen: the relatively pure eluted samples were pooled and adjusted to a concentration of 0.5 mg/ml using the BCA assay kit, and then transferred to dialysis bags. Folding renaturation of CD47ECD was started by dropwise addition of 1L of buffer A (6M urea, 5mM EDTA, 20mM Tris, pH8.0) using a constant flow pump (flow rate: 1 ml/min); after 24h, 1L of buffer B (5mM EDTA, 20mM Tris, pH8.0) is added dropwise; removing half of the liquid after 24 hours, adding 1L of buffer B, and standing for 24 hours; half of the liquid was discarded and 1L of buffer C (5mM EDTA, 20mM Tris, pH8.0) was added and left to stand for 12 hours, then all the liquid was discarded and 1L of buffer C (5mM EDTA, 20mM Tris, pH8.0) was added for 12 hours, followed by low-speed stirring with a magnetic stirrer. Then, samples were collected and centrifuged at 13,000rpm at 4 ℃ for 20 minutes to remove protein aggregation precipitates, and then SDS-PAGE was performed to analyze the size and purity of the proteins, and the results are shown in FIG. 3. As shown in FIG. 3, the size of the protein "2" after renaturation is identical to that of the protein "1" before renaturation, and the purity is high. The protein solution was concentrated using a 3kDa Amicon centrifugal filter. BCA assay kit was used to determine protein concentration. Then the recombinant human CD47 protein after purification and renaturation is subpackaged at-80 ℃ for storage.

EXAMPLE 2 immunization of mice with recombinant human CD47 extracellular region antigen

For the first immunization, 50. mu.g of PBS solution containing recombinant human CD47 antigen was ultrasonically emulsified with equal volume of Freund's complete adjuvant in a total volume of 200. mu.l, and Balb/c female mice were injected subcutaneously at multiple points. 5 mice were immunized each time. A second boost immunization was performed 3 weeks later: the antigen amount of 25 mu g of each mouse and an equal volume of Freund's incomplete adjuvant are prepared into a total volume of 200 mu l, and ultrasonic emulsification and subcutaneous multi-point injection are carried out. A third immunization was performed two weeks later: 25 mu g of antigen per mouse and equivalent volume of Freund incomplete adjuvant are prepared into 200 mu l of total volume for ultrasonic emulsification and subcutaneous multi-point injection. All mice were sera taken after three immunizations. Obtaining of mouse serum: one week after the third immunization, the mice were fixed, a small portion of the tail was excised, tail blood was collected in a 1.5ml EP tube at about 100. mu.L, centrifuged at 11,000rpm for 5min, and the supernatant was removed for measurement by the double antibody sandwich Elisa method to detect the titer in the mouse serum.

The night before the experiment, CD47 antigen solution with the concentration of 10 mug/ml (dissolved in coating solution) is coated overnight at 4 degrees in 100 mug/hole; the next day, discarding the antigen coating solution, filling each well with PBS washing buffer solution, standing for 5min, patting dry, and washing for 3 times; adding 200 mul of confining liquid into each hole, and incubating for 1h at 37 ℃; discarding the blocking solution, filling each hole with PBS washing buffer solution, standing for 5min, patting dry, and washing for 3 times; adding the sample to be detected, diluting the serum by PBS according to the proportion of 200, 2000, 20000 and 200000 times, adding 100 μ l of the sample to be detected in each hole, and using PBS as a blank control. Incubating for 2h at 37 ℃; discarding serum samples, filling each hole with PBS washing buffer solution, standing for 5min, patting dry, and washing for 3 times; HRP-goat anti-mouse antibody was added and diluted 1: 5000. Incubating for 1h at 37 ℃ and 100 mul/well; discarding the antibody solution, filling each hole with PBS washing buffer solution, standing for 5min, patting dry, and washing for 3 times; the ready-to-use TMB color developing solution is placed in a dark place at room temperature for 15min at a concentration of 100 mu l/hole to be blue; add 50. mu.l of stop solution into each well, stop the reaction, develop yellow color, and measure the absorbance at 450nm with microplate reader, the results are shown in Table 1. As shown in Table 1, the test was positive when the absorbance was greater than 0.2 compared to the blank control, and the titer of serum antibodies in

mice

1, 2, 4, and 5 was greater than 20,000. One week after blood collection, mice with an antibody titer greater than 20,000 were shock immunized and intraperitoneally injected with PBS containing 25 μ g of antigen.

TABLE 1 determination of serum antibody titers after three immunizations of mice

Example 3 fusion and selection of hybridoma cells

3 days after the challenge immunization, spleens were prepared for cell fusion. The sacrificed mice were immersed in 70% alcohol and placed on a dissecting plate, the spleens of the mice were taken out from the upper left of the abdominal cavity, and placed in a 60mm diameter petri dish containing 3ml of complete DMEM medium; the culture dish containing the spleen is transferred to an ultra-clean bench, and a set of sterile scissors forceps is used for carefully peeling off fat and other tissues on the surface of the spleen; spleen was transferred to a cell filtration sieve, which was placed in a 100mm petri dish containing 10ml of dmem; 2ml of DMEM was injected into the spleen at various locations using a 3ml syringe with a 26G needle; by means of sterile scissorsShearing spleen filled with DMEM for several times; the spleen was expelled with a 3ml syringe plug until only fibrous tissue remained on the surface of the sieve, and the tissue crushed through the sieve was collected in a sterile petri dish below; rinsing the sieve once with 2ml of complete DMEM medium; transferring the spleen cell suspension into a 15ml centrifuge tube, and blowing and beating the spleen cell suspension by using a 5ml pipette for several times to separate lumps; standing the suspension at room temperature for 3 min; transferring 95% cell suspension to a 15ml centrifuge tube; the number of viable cells was detected using trypan blue staining and the proportion of viable cells was recorded (red cells were omitted, which were significantly smaller in size than nucleated cells); transfer 1x10⁸Live splenocytes were transferred to a 15ml centrifuge tube; the hybridoma cells were rinsed 2 times with complete DMEM medium, centrifuged at 200g for 5min, and resuspended in 5ml of complete medium; adding splenocytes into a 50ml centrifuge tube of myeloma cells, and adding DMEM; centrifuging at room temperature for 5min at 200 g; resuspending the cell pellet with 50ml DMEM as above, and centrifuging; placing the centrifuge tube in a small beaker, placing in a 37 deg.C water bath kettle, warm-bathing for 2min, and flicking the tube bottom to loosen the precipitate. Spleen cells and myeloma cells were fused using sterile PEG solution: adding 1ml of PEG solution preheated to 37 deg.C within 1min, and gently mixing (adding PEG solution while rotating the tube); centrifuging at 100g within 2min (total centrifugation time is 2 min); 4.5ml of complete DMEM was added dropwise over 3 min; adding 5ml of complete DMEM within 2min and then adding the complete DMEM; after centrifugation at 100g for 5min at room temperature, the supernatant was removed and the cell pellet was resuspended in 35ml HAT, a selection medium containing hypoxanthine (H), aminopterin (A) and thymidine (T) (containing 20% fetal bovine serum); the cell suspension was placed at 37 ℃ in 8% CO₂In an incubator with 98% humidity, cell plating and culture are carried out after at least 30 min: the central 60 of the 96-well plate was filled with 100. mu.l of cell suspension per well and the peripheral 30 wells with 100. mu.l of sterile 1xPBS per well. The 96-well plate was incubated at 37 ℃ with 8% CO₂Cultivation in an incubator at 98% relative humidity, this is day 1. On day 5 of culture, 100. mu.l of HAT medium was added to each well. The hybridoma cells were cultured for 2 weeks in HAT selection medium, and after two weeks, they were changed to HT medium, which was used during the first two rounds of limiting dilution clonal culture of hybridoma cells.

On day 7 of culture, 100. mu.l of old medium was discarded from each well, 100. mu.l of fresh medium was added, and the procedure was repeated every other day until the cell confluence in each well reached 10% to 50%, at which time the detection of antibodies by sandwich ELISA was initiated.

Wells with positive cells were placed in 24-well plates for expanded culture and a first round of limiting dilution was performed: blowing and resuspending the cells in each positive hole, and measuring the density by using a blood counting chamber; taking out the required cell amount, and gradually diluting the cell suspension, wherein the dilution multiple does not exceed 100 times each time until the minimum concentration is 8 cells/ml; first, 100. mu.l of sterile 1xPBS was added to 30 wells surrounding a 96-well plate. Then, the cell suspension with 8/ml concentration was added to 100. mu.l/well in the upper 50 of the 60 wells and to 80/ml concentration in the bottom 10 wells by using a discharging gun at 37 ℃ and 8% CO₂Cultivation in 98% relative humidity, this is day 1. Adding 100 mul of culture medium into each hole on day 6, if necessary, replacing the culture medium every other day, discarding 100 mul of old culture medium, and adding 100 mul of fresh culture medium; when the confluency of cells reached 10% -50%, wells with only single clonal cell clusters were selected and the supernatant was tested for specific antibodies by sandwich ELISA. Obtaining monoclonal hybridoma cells after 3 rounds of limiting dilution and monoclonal culture, carrying out amplification culture on the obtained monoclonal cell strains, and freezing and storing the monoclonal cell strains in liquid nitrogen.

EXAMPLE 4 preparation and purification of ascites antibodies

Preparation of ascites antibody: taking Balb/c female mice of 6-8 weeks, and injecting 0.8ml Freund's incomplete adjuvant into each mouse abdominal cavity by using a 22G needle before injecting hybridoma cells into the abdominal cavity; one week later, collecting the hybridoma cells cultured to the logarithmic phase, lightly blowing the cells, transferring the cell suspension into a 50ml centrifuge tube at 1500rpm, and centrifuging for 5min at room temperature; discarding the supernatant, resuspending the cells into 50ml sterile PBS, and centrifuging at 1500rpm at room temperature for 5 min; removing the supernatant, and repeatedly washing twice; resuspending the cells in 5ml PBS and trypan blue cells counting cell viability; regulation of viable cell concentration to 2.5X10 with PBS⁶Perml, each mouse was injected intraperitoneally with 2ml of the cell suspension for 1 to 2 weeks, and ascites production was observed daily(ii) a condition; collecting the peritoneal fluid, centrifuging the peritoneal fluid at 4 deg.C for 10min, 3000 rpm. Collecting supernatant, removing precipitate, collecting abdominal cavity liquid, and storing in refrigerator at 4 deg.C; detecting the titer of the monoclonal antibody in the peritoneal fluid by a sandwich ELISA method; diluting with binding solution at a ratio of 1:10, and filtering with 0.45 μm filter for sterilization.

Purification of ascites antibody: diluting the abdominal cavity liquid with binding solution at a ratio of 1:10, filtering with 0.45 μm filter, and purifying with gravity column preloaded with 1ml Protein G agarose gel; before purification, washing and stabilizing the column material by using 5ml of water and 5ml of Binding b [ mu ] ffer respectively, then adding diluted peritoneal fluid, and collecting flow liquid for analysis; eluting with eluent, collecting 10ml in 1ml tube, and adding 60 μ l 1M tris-HCL (pH9.0) into each tube for neutralization; after purification, 40. mu.l of each eluent was dissolved in 10. mu.l of 5 Xloading buffer and boiled at 99 ℃ for 10 min; performing SDS-PAGE analysis on the size and purity of the target protein in the eluate; the purer eluted samples were pooled and then transferred to dialysis bags for dialysis against 1L of 1XPBS, and dialysis continued once after 12 h. And collecting dialyzed samples, determining the protein concentration by using a BCA kit, and subpackaging at-80 ℃ for storage.

Example 5 subtype identification of murine anti-human CD47 monoclonal antibody

Firstly, an enzyme-labeled micropore plate is coated with a corresponding antigen specifically bound by a monoclonal antibody by using 1XPBS buffer solution, each hole is 100 mu l of the enzyme-labeled micropore plate and 100ng of the enzyme-labeled micropore plate, the enzyme-labeled micropore plate is placed at 4 ℃ for 12 hours, and then coating solution is thrown off and washed once by using PBS-T. Blocking with 5% BSA blocking solution at 37 deg.C for 2 hr; adding a row of ascites antibodies (diluted 15,000 times) to be tested into each of 8 wells in total, wherein each well is 100 mu l, and incubating at 37 ℃ for 30 minutes; then absorbing the sample solution and washing with PBS-T for 5 times, adding 100 microliter of each of 6 enzyme markers (Goat Anti-Mo microlise Ig (G1\ G2a \ G2b \ G3\ M \ A \ kappa \ lambda) × HRP) for 1 hole and 12 holes (or 6 holes), incubating at 37 ℃ for 30 minutes, continuously absorbing the enzyme-labeled antibody solution and washing with PBS-T for 5 times, and then adding TMB color developing solution at 37 ℃ for shading and developing for 20 minutes; the reaction was stopped with 2M sulfuric acid and judged by a microplate reader with 450nm dual wavelength readings, the results are shown in Table 2. As can be seen from Table 2, the heavy chain of this monoclonal antibody belongs to the IgG2b subtype, and the light chain belongs to the kappa chain.

TABLE 2 subtype identification of murine anti-human CD47 monoclonal antibody

Subtype of cell	OD
		IgG1	0.501
IgG2a	0.95
		IgG2b	1.616
IgG3	0.161
		IgM	0.099
IgA	0.057
		κ	1.352
λ	0.047

Example 6 detection of tumor cell surface CD47

After culturing MKN-45, A549, Hela and Raji cell lines until the logarithmic growth phase, collecting 2X10⁶Adding intoMu.g of the purified antibody was incubated at 4 ℃ for half an hour, then FITC-goat anti-mouse IgG secondary antibody was added, incubated at 4 ℃ for half an hour, then 1XPBS was added for washing, and flow cytometry analysis was performed, the results are shown in FIG. 4. As shown in FIG. 4, the dashed line represents the normal mouse IgG negative control, the solid line represents the purified monoclonal antibody, and a significant right shift in fluorescence intensity after incubation of the monoclonal antibody is seen, indicating the recognition of the CD47 antigen on the surface of tumor cells by the monoclonal antibody.

EXAMPLE 7 sequencing of murine anti-human CD47 monoclonal antibody

RNA from hybridoma cell lines was extracted and reverse transcribed into cDNA according to the method described in example 1. According to the scFv antibody library construction method described by Zhou et al, Nucleic Acids Res, 22(5):888-9 (1994), multiple pairs of primers are designed to carry out PCR amplification on antibody variable regions, and heavy chain variable regions and light chain variable regions of the murine anti-human CD47 egg clone antibody are obtained after sequencing. After alignment, 3 CDRs and 4 framework regions were identified. Wherein the amino acid sequence of the heavy chain complementarity determining region is CDRH1: GYTFTDYW (SEQ ID NO:1), CDRH2: IDTSDSYT (SEQ ID NO:2), CDRH3: ASGHYSFTY (SEQ ID NO:3), and the amino acid sequence of the light chain complementarity determining region is CDRL 1: QSLVHSNGNTY (SEQ ID NO:4), CDRL2: KVSN (SEQ ID NO:5), CDRL3: SQSTHVPWT (SEQ ID NO: 6).

Example 8 construction and affinity analysis of human murine chimeric antibody against human CD47

Respectively carrying out PCR amplification on the heavy chain variable region and the light chain variable region by using a PCR method; respectively inserting the heavy chain variable region and the light chain variable region into a vector containing a human IgG light heavy chain constant region by utilizing a fusion pc and homologous recombination method; the recombinant plasmid is transformed and amplified, and 293F cell line is cultured to logarithmic growth period, and 293F cell transfection is prepared. Cells were passaged 24h before transfection, at a density of 5X10⁵～6x10⁵293F Freestyle Medium in 100ml volume flasks shaken at 130rpm on a cell shaker at 37 ℃ in 8% CO₂Medium culture; trypan blue staining before transfection determines the number of living cells, and the activity must be more than 95%; filtering the cells by using a 40-micron cell filter membrane to obtain uniform single-cell suspension; dilute the cells in pre-warmed 293F Freestyle MediumCell, at 1x10⁶The density of each/ml was inoculated into a new flask, 30ml in volume. After gently inverting FreeStyle MAX reagent several times to mix well, 50. mu.g of plasmid was added and made up to 1.5ml with OptiPRO SFM medium and gently mixed well. Another 5ml centrifuge tube was filled with 50. mu.l FreeStyle MAX reagent and OptiPRO SFM medium to 1.5ml, and gently mixed. Immediately, the diluted FreeStyle MAX solution was added to the plasmid dilution solution, the tip was completely immersed in the plasmid solution, and the transfection reagent solution was slowly released with stirring. Mixing by gentle vortex, and standing at room temperature for 10min to form DNA-liposome complex; 3ml of DNA-liposome solution was added drop wise to the cell shake flask while gently shaking the flask. The shaking was continued at 130rpm, 37 ℃ and 8% CO₂Culturing the cells in a culture environment. After 5-7 days of transfection, collecting cell suspension, centrifuging to remove precipitates, filtering supernate by a filter membrane of 0.45 mu m, and adding a gravity column preloaded with 1ml of Protein G agarose gel for purification in the same way as mouse abdominal cavity fluid antibody purification; the purified antibody was subjected to affinity analysis by sandwich ELISA, and the results are shown in Table 3. As shown in table 3, B6H12 is a commercially available monoclonal antibody against human CD47, and 1X, 1/10X, and 1/100X respectively represent the antibody stock solution obtained by purification, the liquid obtained by dilution by 10 times, and the liquid obtained by dilution by 100 times, and the OD values indicate that the chimeric antibody has high binding activity to the recombinant human CD47 antigen.

TABLE 3 detection of the binding Activity of the chimeric antibodies to the recombinant human CD47 antigen

Group of	OD
		PBS	0.6712
B6H12	1.6584
		1X	2.6392
1/10X	2.5981
		1/100X	2.5495

Example 9 epitope identification of anti-human CD47 monoclonal antibody

The construction of a truncated body is carried out on the amino acid sequence of the extracellular region 19-139 of the human CD47, which is 121 amino acids in total: fragment 1 comprises the amino acid sequence 19-79, fragment 2 comprises the amino acid sequence 49-109, fragment 3 comprises the amino acid sequence 79-139; designing upstream and downstream primers, and constructing the primers into a pGEX-4T-1 vector. After the sequence to be tested is correct, transforming to BL21 strain for expression: three successfully constructed recombinant plasmids and empty vectors are transformed into BL21, and monoclonal shake bacteria is selected for overnight expression; taking the bacteria liquid in the following day according to the proportion of 1:100 volume ratio was inoculated into 5ml of LB medium, and medium culture was continued. After about 2.5h, 1M IPTG was added to a final concentration of 0.3mM and the culture was continued for 5 h. Then centrifugally collecting thalli precipitates, and meanwhile, centrifugally collecting thalli precipitates from bacterial suspension which is not induced by IPTG, and respectively re-suspending with 1ml of 1 XPBS; after 40. mu.l of the resuspension solution was added to 10. mu.l of 5 XLoading buffer and denatured at 99 ℃ for 10 minutes, SDS-PAGE was performed to determine whether the target protein was expressed, and the results are shown in FIG. 5. As shown in FIG. 6, "1", "2", "3", "G" represent

fragments

1, 2, 3 and GST empty vector control, respectively, "-" and "+" represent no IPTG induction and IPTG induction, respectively, indicating that IPTG successfully induces the expression of the target protein and GST.

The epitope identification of the anti-human CD47 monoclonal antibody was performed by protein immunoblotting (Western blot): samples of overexpressed protein with three truncations and GST empty vector control protein were run on SDS-PAGE: and (5) after glue running and bonding, carrying out film transfer, and sealing after the film transfer is finished. Then diluting and purifying the obtained antibody as a primary antibody according to the dilution ratio of 1:1000, incubating the primary antibody, and then shaking the primary antibody at 4 ℃ overnight; taking out the membrane the next day, washing the membrane with 1XTNE membrane washing solution for 3 times, each time for 10 minutes; incubating corresponding secondary antibody after membrane washing is finished, preparing goat anti-mouse IgG secondary antibody marked by HRP according to a dilution ratio of 1:5000, and incubating at room temperature for 2 hours; then the membrane was washed with 1XTNE washing solution for 5 minutes 1 time. Development was then carried out, the results being shown in FIG. 6. As shown in FIG. 6, "P", "G", 1 "," 2 "and" 3 "represent the extracellular domain antigen of CD47, GST empty vector control and

fragments

1, 2 and 3, respectively, and a clear band was observed in fragment 3 as a result of development, so that the monoclonal antibody recognized the major recognition fragment 3, i.e., 79-139 amino acid sequence.

Further determination of the epitope: constructing a truncation aiming at 61 amino acids in the 61-121 amino acid sequence of the human CD47 extracellular region: fragment 1 comprises the amino acid sequence 19-79, fragment 2 comprises the amino acid sequence 49-109, fragment 3 comprises the amino acid sequence 79-139, fragment 4 comprises the amino acid sequence 19-79, fragment 5 comprises the amino acid sequence 19-79, fragment 6 comprises the amino acid sequence 19-79, fragment 7 comprises the amino acid sequence 19-79, fragment 8 comprises the amino acid sequence 19-79, fragment 9 comprises the amino acid sequence 19-79, and fragment 10 comprises the amino acid sequence 19-79; designing upstream and downstream primers, and constructing the upstream and downstream primers into a pGEX-4T-1 vector. After the sequence to be detected is correct, the strain is transformed to BL21 strain for expression, and SDS-PAGE analysis is carried out to determine whether target protein expression is carried out, and the result is shown in figure 7. As shown in FIG. 7, lanes "1" to "10" represent fragments 1 to 10, respectively, "-" and "+" represent induction without IPTG and induction with IPTG, respectively, indicating that IPTG successfully induces the expression of the target protein. Western blot was performed on the expressed samples, and the antibodies obtained by dilution and purification at a dilution ratio of 1:1000 were used as primary antibodies, and the results of development are shown in FIG. 8. As shown in FIG. 8, the monoclonal antibody recognizes fragments 1 to 8, but does not recognize

fragments

9 and 10, and the epitope amino acid sequence of the monoclonal antibody, that is, SEQ ID NO. 11, can be deduced.

Example 10 analysis of antitumor Activity of anti-human CD47 monoclonal antibody

Macrophage isolation was first performed from Balb/c mice: collecting leg bone of Balb/c mouse, flushing bone marrow collected cells with 1XPBS, adding M-CSF with final concentration of 50ng/ml to stimulate for 7 days, and inducing to obtain macrophage derived from bone marrow. One day before the phagocytosis experiment, wells 1X10^5 cells were plated into 24-well plates.

After culturing the Raji cell line to a logarithmic growth phase, 2X10 was collected⁵Cells were labeled with CFSE dye at a final concentration of 2. mu.M for 30min and washed once with 1 XPBS. Then 20-50 mug of purified antibody is added, cell suspension is added into BMDM after half a hour of incubation at 37 ℃, after 1-2 hours of incubation, system supernatant is discarded, 1XPBS is added for washing once, cells in a collection culture dish are added with anti-mouse F4/80-APC flow type antibody for half an hour of incubation at 4 ℃, and flow cytometry analysis is carried out, and the result is shown in figure 9. As shown in FIG. 9, with normal mouse IgG as a negative control, it can be seen that the macrophages after incubation with the monoclonal antibody have a significant right shift in the CFSE green fluorescence range, indicating that the purified monoclonal antibody has the ability of promoting phagocytosis of tumor cells by macrophages.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to persons skilled in the art to which the invention pertains are intended to be covered by the scope of the invention.

Sequence listing

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Claims

1. The monoclonal antibody against human CD47 comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody against human CD47 is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

2. A nucleotide molecule encoding the anti-human CD47 monoclonal antibody of claim 1.

3. The nucleotide molecule of claim 2, wherein the nucleotide sequence of said nucleotide molecule encoding the heavy chain of the anti-human CD47 monoclonal antibody is set forth in SEQ ID NO. 9, and the nucleotide sequence encoding the variable region of the light chain is set forth in SEQ ID NO. 10.

4. An expression vector comprising the nucleotide molecule of claim 2 or 3.

5. A host cell comprising the expression vector or gene of claim 4 having incorporated therein an exogenous nucleotide molecule of claim 2 or 3.

6. A method for preparing an anti-human CD47 monoclonal antibody according to claim 1, comprising the steps of:

culturing the host cell of claim 5 under conditions suitable for expression of said antibody, thereby expressing said monoclonal antibody, and purifying and isolating said monoclonal antibody.

7. Use of the monoclonal antibody against human CD47 according to claim 1 for the preparation of a medicament for promoting phagocytosis of Raji cells by macrophages.