A monoclonal antibody with CD47 binding activity and its application

Abstract

The present invention belongs to the field of biomedicine and specifically relates to a monoclonal antibody with CD47 binding activity. It also provides the use of the antibody in the preparation of anti-tumor drugs or drugs for fibrotic diseases. The monoclonal antibody with CD47 binding activity described in the present invention has a high affinity for human CD47 and exhibits a stronger phagocytic effect on tumor cells compared to existing anti-human CD47 monoclonal antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a monoclonal antibody with CD47 binding activity, and its use in preparing anti-tumor drugs or drugs for fibrotic diseases. Background Art

[0002] Cluster of differentiation 47 (CD47) is an immunomodulatory molecule overexpressed on tumor cells. Its molecular structure consists of an N-terminal extracellular variable domain, five hydrophobic transmembrane helices, and a very short C-terminal intracellular signaling sequence. Unlike immune checkpoints such as PD-1 that act directly on T cells, CD47 primarily inhibits macrophage phagocytosis by binding to signal-regulatory protein α (SIRPα).

[0003] After CD47 binds to SIRPα, it causes tyrosine phosphorylation of ITIMs (immunoreceptor tyrosine inhibitory motifs). Phosphorylated ITIMs recruit and activate protein tyrosine phosphatases SHP-1 / 2, inhibiting downstream myosin IIA and preventing macrophage phagocytosis. CD47-SIRPα also inhibits the maturation of dendritic cells, thereby inhibiting their production of cytokines and preventing the presentation of tumor antigens to CD8 + T cells. CD47 is overexpressed in solid tumors and hematologic malignancies, including breast cancer, non-small cell lung cancer, and gastric cancer, and is often associated with a poor prognosis.

[0004] The concentration of IgG4 in plasma is 1%-4%, and CD47 antibodies prepared from this subtype have limited erythrocyte toxicity. Magrolimab (Hu5F9-G4), originally developed by Forty Seven (now acquired by Gilead), combines the humanized 5F9 variable fragment with an IgG4 backbone to reduce its erythrocyte cytotoxicity. Magrolimab is currently the most advanced humanized anti-CD47 monoclonal antibody, and a Phase III clinical trial combining it with azacitidine for the treatment of myelodysplastic syndromes is ongoing (NCT04313881).

[0005] In recent years, drug development targeting immune escape mediated by the CD47-SIRPα signaling pathway has become a hot topic in tumor immunotherapy. However, currently developed anti-human CD47 monoclonal antibodies suffer from low affinity or unclear target epitopes. Therefore, there is an urgent need for novel therapeutic CD47 antibody candidates with high affinity that can induce macrophage phagocytosis of cancer cells. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies of the above-mentioned prior art and to provide a novel monoclonal antibody with CD47 binding activity, which has good CD47-SIRPα blocking activity and strong tumor cell phagocytosis-promoting effect.

[0007] The present invention provides a monoclonal antibody having CD47 binding activity, wherein the antibody comprises a heavy chain variable region and a light chain variable region;

[0008] The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3;

[0009] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 2;

[0010] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 4;

[0011] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 6;

[0012] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 12;

[0013] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 14;

[0014] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 16.

[0015] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.

[0016] Preferably, the heavy chain amino acid sequence is shown in SEQ ID NO: 10; and the light chain amino acid sequence is shown in SEQ ID NO: 20.

[0017] Preferably, both the heavy chain and the light chain further comprise a constant region, which is a constant region of mouse or human IgG, preferably a constant region of IgG4.

[0018] The present invention further provides a nucleotide molecule encoding the above monoclonal antibody.

[0019] Preferably, the sequence of the nucleotide molecule is selected from SEQ ID NO: 7 and SEQ ID NO: 17;

[0020] The sequence SEQ ID NO: 7 encodes the heavy chain variable region of the antibody;

[0021] The sequence SEQ ID NO: 17 encodes the light chain variable region of the antibody.

[0022] The present invention further provides an expression vector containing the nucleotide molecule.

[0023] The present invention further provides a host cell containing the expression vector.

[0024] Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0025] The present invention further provides a method for preparing the above-mentioned monoclonal antibody, comprising the following steps:

[0026] (1) preparing an expression vector containing a nucleotide molecule for expressing the monoclonal antibody;

[0027] (2) transfecting eukaryotic host cells with the expression vector obtained in step (1) and culturing the cells;

[0028] (3) Isolation and purification to obtain a monoclonal antibody with CD47 binding activity.

[0029] The present invention further provides an antibody immunoconjugate, a bispecific molecule, a chimeric antigen receptor or a pharmaceutical composition comprising the above monoclonal antibody.

[0030] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of the monoclonal antibody having CD47 binding activity, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0031] The present invention further provides the use of the above monoclonal antibody in the preparation of anti-tumor drugs or drugs for fibrotic diseases.

[0032] Preferably, the tumor is a hematological tumor or a solid tumor, including non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), ovarian cancer, fallopian tube cancer, colorectal cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, lung cancer, glioma and glioblastoma.

[0033] Preferably, the fibrotic disease includes angina pectoris, osteoarthritis, pulmonary fibrosis, asthma and bronchitis.

[0034] Beneficial effects:

[0035] The monoclonal antibody with CD47 binding activity of the present invention has a higher affinity with human CD47, and compared with existing anti-human CD47 monoclonal antibodies, it has better CD47-SIRPα blocking activity and stronger tumor cell phagocytosis-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Capture ELISA was used to determine the antibody's ability to bind to human CD47 protein;

[0037] Figure 2 The antibody's binding ability to cynomolgus monkey CD47 protein was determined by capture ELISA;

[0038] Figure 3 Flow cytometry was used to evaluate the binding of antibodies to 293F cells overexpressing human CD47.

[0039] Figure 4 for ligand binding blocking ELISA;

[0040] Figure 5 for reference antibody blocking ELISA;

[0041] Figures 6A-6B Flow cytometry was used to evaluate the ability of antibodies to induce macrophages to phagocytose tumor cells. DETAILED DESCRIPTION

[0042] the term

[0043] "Binding to CD47" or "binding to CD47" means being able to interact with human CD47.

[0044] "Antigen binding site" refers to one or more segments of an antibody that have the ability to specifically bind to an antigen (eg, CD47 protein).

[0045] "Monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition that exhibits a single binding specificity and affinity for a particular epitope, and does not refer to the method by which it is produced. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or a combination of these or other techniques known in the art.

[0046] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by common methods known in the art, including those known in the art and described herein.

[0047] The term "competition" when used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) that compete for the same epitope means competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be tested (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., CD47 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen-binding protein being tested. Typically, the antigen-binding protein being tested is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein that the two epitopes sterically hinder each other from binding.

[0048] Methods for producing and purifying antibodies and antigen-binding fragments are well known and disclosed in the art, such as the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. For example, mice can be immunized with human CD47 or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

[0049] "Treatment" means administering a therapeutic agent, such as a composition comprising a CD47 antibody or antigen-binding fragment thereof, to a patient who has one or more symptoms of a disease. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect on the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test commonly used by a physician or other health care professional to assess the severity or progression of such symptoms.

[0050] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0051] A "pharmaceutical composition" refers to a mixture containing one or more CD47 antibodies or antigen-binding fragments thereof described herein and other pharmaceutical ingredients, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0052] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Reagents for which specific sources are not specified are conventional reagents purchased from the market.

[0053] Example 1 Obtaining a mouse monoclonal antibody specific for CD47 by fusion hybridoma technology

[0054] 1.1 Animal immunization

[0055] Mice were immunized according to the commonly used method in the literature (E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), using recombinant human CD47 protein (Sino biological inc., cat# 12283-H02H) as the immunogen.

[0056] To increase the immune response, Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma, St. Louis, Mo., USA) were used for the first immunization and booster immunization, respectively. Briefly, the preparation of the adjuvant-antigen mixture was first performed by gently mixing the adjuvant in a vial using a vortex method. The required amount of adjuvant was removed from the vial and placed in an autoclaved 1.5 mL microcentrifuge tube. The antigen was prepared in PBS or normal saline at a concentration of 0.5-1.0 mg / ml. The calculated amount of antigen was added to the microcentrifuge tube together with the adjuvant, gently stirred for 2 minutes, and the emulsification was repeated to form an oil-in-water solution. Then, an appropriate amount of the adjuvant-antigen mixture was injected into each animal for immunization. Two to three booster immunizations were performed according to the antiserum titer. Before cell fusion, animals with good antiserum titers were boosted by intraperitoneal injection.

[0057] 1.2 Hybridoma fusion and screening

[0058] Mouse myeloma cell line (SP2 / 0-Ag14, ATCC #CRL-1581) cells were cultured and allowed to reach the logarithmic growth phase before cell fusion. They were fused with myeloma cells according to the method described in the literature (Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibodies of predefined specificity," Nature, 256:495-497 (1975).).

[0059] The fused "hybrid cells" were then distributed into 96-well cell plates containing DMEM / 20% FCS / HAT medium. Surviving hybridoma cells can usually be observed growing under a microscope 7-10 days after fusion. Two weeks after cell plating, the supernatant from each well was tested by ELISA using recombinant human CD47-his protein. Briefly, the ELISA plate was coated with human CD47-his protein (ACRO biosystems, cat#CD7-H5227, 2.0 μg / ml in PBS) at 4°C overnight. The plate was washed four times with PBST and then blocked with blocking buffer (PBST containing 5% skim milk powder). Diluted mouse immune serum (for determination of mouse serum titer) or hybridoma supernatant was added to each well and incubated at 37°C for 40 minutes. The plate was washed four times with PBST and detected with horseradish peroxidase-goat anti-mouse IgG (Jackson Immuno Research, cat# 115-036-071). The absorbance of each well was measured at 450 nm. Positive hybridomas secreting antibodies that bind to human CD47-his were then selected and transferred to a 24-well plate.

[0060] Hybridoma clones that produce antibodies that bind to human CD47 with high specificity and have CD47 / SIRPα ligand blocking activity were subcloned by limiting dilution and then purified. Briefly, the protein A agarose column was washed with 5-10 column volumes of PBS buffer. The cell supernatant of the hybridoma monoclonal antibody was passed through the column, and then the column was washed with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1M glycine-HCl, pH 2.7) and immediately collected into a 1.5ml tube containing neutral buffer (1M Tris-HCl, pH 9.0). The fractions containing immunoglobulins were mixed and dialyzed in PBS at 4°C overnight. Subsequently, the functional activity of the purified monoclonal antibodies was characterized in vitro as described below.

[0061] Example 2 Determination of the affinity of mouse anti-CD47 monoclonal antibodies using BIACORE surface plasmon resonance technology

[0062] The anti-CD47 mouse monoclonal antibodies (mAbs) produced by the hybridoma clones in Example 1 were subjected to affinity kinetic characterization using the Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0063] Briefly, goat anti-mouse IgG was covalently linked to a CM5 chip (carboxymethyl dextran coated chip) via primary amines using a standard amine coupling kit provided by Biacore. Unreacted portions of the biosensor surface were blocked with ethanolamine. The mouse anti-CD47 antibody produced in Example 1 was purified, and reference antibodies CC-9000 (Celgene) and Hu5F9-G4 (Forty Seven) were flowed onto the chip at a concentration of 66.7 nM and a flow rate of 10 μL / min. Recombinant human CD47-his protein (Acro biosystems, cat#CD7-H5227, MW: 15.6 kDa) or cynomolgus monkey CD47-his protein (Acro biosystems, cat#CD7-C52H1, MW: 15.8 kDa) in HBS EP buffer (provided by Biacore) was then flowed onto the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were observed for 2 minutes, and dissociation kinetics were observed for 10 minutes. The Langmuir binding model curve with a binding and dissociation ratio of 1:1 was fitted using BIAcore evaluation software.

[0064] where k a , k d and K D The values ​​of are shown in Table 1.

[0065] Table 1. Kinetic parameters of mouse anti-CD47 monoclonal antibody binding to human or cynomolgus monkey CD47 determined by Biacore

[0066]

[0067] The binding K of the monoclonal antibody 1H4 of the present invention to human CD47 D The values ​​were in the same order of magnitude as those of the reference antibody, indicating that it had a higher affinity for human CD47.

[0068] Example 3 Study on Binding Activity of Mouse Anti-CD47 Monoclonal Antibodies

[0069] The binding activity of the mouse anti-CD47 monoclonal antibody generated in Example 1 was further tested by capture ELISA and flow cytometry (FACS).

[0070] 3.1 Determination of Antibody Binding Capacity Based on Capture ELSIA

[0071] A 96-well ELISA plate was coated with a goat anti-mouse IgG antibody specific for Fcγ fragments (Jackson ImmunoResearch, cat#115-006-071, 100 μl / well) in PBS at a final concentration of 2 μg / ml and incubated overnight at 4°C. The plate was washed four times with elution buffer (PBS + 0.05% v / v Tween-20, PBST), then blocked at 37°C for 2 hours with 200 μl / well of 5% w / v skim milk powder in PBST. The plate was then washed again, and 100 μl / well of various concentrations of CD47 mouse monoclonal antibody were added. The plate was incubated at 37°C for 40 minutes, and then washed four more times. ELISA plates containing capture CD47 antibodies were incubated with biotinylated human CD47 protein (ACRO Biosystems, cat#CD7-H5227) or monkey CYNO-CD47-HIS-BIO (ACRO Biosystems, cat#CD7-C52H1) (60 nM, 2.5% nonfat dry milk in PBST buffer, 100 μl / well) at 37°C for 40 minutes. The plates were then washed four times and incubated with streptavidin-conjugated horseradish peroxidase (SA-HRP, 1:10,000 diluted in PBST buffer, Jackson Immuno Research, cat#016-030-084, 100 μl / well) at 37°C for 40 minutes. After the final wash, the plates were incubated with 100 μl / well of the ELISA substrate TMB (Innoreagents, cat#TMB-S-002). After 15 minutes, the reaction was terminated with 50 μl / well 1M H2SO4 at 25°C and the absorbance at 450 nm was measured. Figure 1 、 Figure 2 and Table 2.

[0072] Figure 1 and Figure 2 The results showed that the antibody 1H4 of the present invention has good binding ability to both human and cynomolgus monkey CD47 proteins.

[0073] 3.2 FACS determination of the binding activity of anti-CD47 monoclonal antibodies to 293F cells overexpressing human CD47

[0074] Stable 293F cell lines overexpressing human CD47 were collected from cell culture flasks, washed twice, and resuspended in PBS phosphate buffered saline (FACS buffer) containing 2% v / v fetal bovine serum. 2×10 5Cells were incubated on ice for 40 minutes with FACS buffer containing different concentrations of CD47 antibodies. The cells were washed three times with FACS buffer and 100 μL / well of R-phycoerythrin affinity purified F(ab')2 fragment-specific goat anti-mouse IgG (1:1000 dilution in FACS buffer, Jackson Immunoresearch, cat#115-116-072) was added. After incubation at 4°C in the dark for 40 minutes, the cells were washed three times and then resuspended in FACS buffer. Fluorescence measurements were performed using a Becton Dickinson FACS Canto II-HTS device. Data were analyzed using Graphpad Prism software to obtain the EC value of antibody-bound cells. 50 The concentration value is the antibody concentration value corresponding to the maximum fluorescence binding signal of 50% when CD47 antibody binds to cells overexpressing CD47. The measurement results are shown in Figure 3 and Table 2.

[0075] Figure 3 The results showed that the antibody 1H4 of the present invention has a stronger ability to bind to 293F cells that overexpress human CD47 on their surface.

[0076] Table 2. Binding activity of mouse anti-CD47 antibodies

[0077]

[0078] Example 4 Competitive Functional Blockade of CD47-SIRPα Interaction by Mouse Anti-CD47 Monoclonal Antibodies

[0079] Competition ELISA was used to detect the blocking ability of antibodies on CD47-SIRPα interaction.

[0080] 4.1 Ligand blocking ELISA

[0081] The ability of the anti-CD47 antibodies of the present invention to block the CD47-SIRPα interaction was tested using a competition ELISA. Briefly, human SIRPα-his protein (Sino Biological Inc., cat# 11612-H08H) was added to a 96-well microplate at 200 ng / well and incubated overnight at 4°C. The next day, the plates were washed with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with PBST containing 5% w / v skim milk powder at 37°C for 2 hours. The plates were then washed with wash buffer.

[0082] Dilute the CD47 antibody or reference antibody (antibody starts at 66.7nM and is diluted 4-fold continuously) with biotin-labeled human CD47 (ACRO biosystems, cat#CD7-H5227) solution, incubate at room temperature for 40 minutes, and then add the antibody / CD47-biotin mixture to the SIRPα-coated plate. After incubation at 37°C for 40 minutes, wash the plate 4 times with washing buffer. Then add SA-HRP and incubate at 37°C for 40 minutes to detect the binding of biotin-labeled human CD47 to the bottom plate SIRPα. Wash the plate again with washing buffer. Finally, add TMB, stop the reaction with 1M H2SO4, and measure the absorbance at 450nm with a microplate reader. Use Graphpad Prism software to analyze the data and obtain IC 50 For specific results, see Figure 4 and Table 3.

[0083] 4.2 Reference Antibody Blocking ELISA

[0084] The ability of the anti-CD47 antibodies of the present invention to block the binding of a reference antibody (Hu5F9-G4, Forty Seven) to human CD47 protein was determined using a competitive ELISA. Briefly, a 96-well microplate was coated with 1 μg / mL of the CD47 reference antibody in PBS and incubated overnight at 4°C. The next day, the plate was washed with wash buffer and blocked with 5% skim milk powder in PBST at 37°C for 2 hours. For blocking, biotinylated human CD47 (ACRO biosystems, cat#CD7-H5227) (10 nM in PBST containing 2.5% skim milk powder) was mixed with the antibody (1.2 pM to 100 nM, serially diluted 5-fold) and incubated at 25°C for 40 minutes. After washing, the antibody / human CD47 mixture (100 μl / well) was added to the plate containing Hu5F9-G4 and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer, and 100 μl / well of SA-HRP was added. The plate was then incubated at 37°C for 40 minutes to detect biotinylated human CD47 bound to the plate. A final wash was performed with wash buffer. TMB was added, and the reaction was terminated with 1 M H2SO4. The absorbance at 450 nm was measured. The data were analyzed using Graphpad Prism software to obtain the IC 50 For specific results, see Figure 5 and Table 3.

[0085] As can be seen from Table 3, the antibodies of the present invention are able to block the human CD47-SIRPα interaction, and also indicate that the antibodies of the present invention have similar antigen binding epitopes as the reference antibody. Compared with the reference antibody, the antibody 1H4 of the present invention has better CD47-SIRPα blocking activity.

[0086] Table 3. Ability of anti-CD47 antibodies to block CD47-SIRPα and CD47 reference antibody interactions

[0087]

[0088] Example 5 Mouse anti-CD47 monoclonal antibody induces macrophages to phagocytose tumor cells

[0089] In vitro cell experiments were used to detect the biological activity of anti-CD47 antibodies in inducing macrophages to phagocytose tumor cells. Human peripheral blood mononuclear cells (PBMCs) were extracted from fresh human blood using Ficoll (GE Healthcare, cat#17-1440-02). In order to differentiate PBMCs into monocyte-derived macrophages (MDMs), monocytes were inoculated with RPMI 1640 + 10% FBS + 1% penicillin-streptomycin (Peprotech, cat#300-25-100) in the presence of human M-CSF. On days 2 and 4, the cells were washed and replaced with fresh culture medium containing cytokines. On day 6, adherent cells were separated and washed twice with PBS.

[0090] MDMs were separated from the plate and placed in a 96-well plate overnight. Jurkat cells were collected and labeled with CFSE (5(6)-carboxyfluorescein N-hydroxysuccinimide ester) (Sigma, cat#87444). Anti-CD47 monoclonal antibody was diluted. 100uL of CFSE-labeled Jurkat tumor cells and diluted CD47 monoclonal antibody mixture were added to MDM and incubated at 37°C for 4h. All cells were separated and washed once with FACS buffer. Cells were stained with anti-human CD14 APC (eBioscience, cat#17-0149-42) and CD14+CFSE+ cells were detected by flow cytometry (FACS). Data were analyzed using Graphpad Prism software (the percentage of CD14+CFSE+ cells to CD14+ cells) to obtain EC 50 The values ​​and phagocytic percentages are shown in Tables 4 and Figures 6A-6B .

[0091] Table 4 and Figures 6A-6B The results showed that the antibody of the present invention can induce macrophages to phagocytize tumor cells, and its EC 50 The values ​​were lower than those of the two reference antibodies, indicating a stronger tumor cell phagocytosis-promoting effect than the reference antibodies.

[0092] Table 4. Ability of anti-CD47 antibodies to induce macrophage phagocytosis of tumor cells

[0093]

[0094] Example 6 DNA cloning and sequencing, sequence analysis of anti-CD47 antibodies

[0095] Total RNA was extracted from the hybridoma cells of Example 1 using Trizol reagent (Invitrogen, cat# 15596-018).

[0096] The process is briefly described as follows: 5×10 6 Transfer the cells to a 1.5ml centrifuge tube and remove the supernatant. Add 1ml of Trizol reagent and pipette repeatedly several times, then place at 25℃ for 5 minutes to lyse the cells. Subsequently, add 0.2ml of chloroform solution to each tube, shake vigorously for 15 seconds and place at room temperature for 3 minutes. Centrifuge the tube at 12000g at 4℃ for 10 minutes, remove the centrifuge tube, aspirate the upper aqueous phase solution into a new 1.5ml centrifuge tube, and add 0.4ml of isopropanol to precipitate RNA from the aqueous phase. Manually mix the EP tube and place it at 25℃ for 10 minutes, then centrifuge at 12000g at 4℃ for 10 minutes and discard the supernatant. Add 1ml of 75% ethanol and centrifuge again at 7500rpm at 4℃ for 5 minutes, then discard the supernatant. After the RNA precipitate at the bottom of the tube is dried at room temperature for 10 minutes, 30 to 50ul of sterile DEPC-treated water is added to dissolve the RNA sample.

[0097] Next, total RNA was converted to cDNA using Taraka's Reverse Transcription cDNA Kit (cat#6110A). The experimental system was prepared as follows: 5 μl of total RNA, 0.5 μl of Oligo(dT), and 8.5 μl of RNase-free water (14 μl total) were pre-denatured at 65°C for 5 minutes, followed by 2 minutes on ice. Furthermore, 4 μl of 5× buffer, 1 μl of dNTP mix, 0.5 μl of RNase inhibitor, and 1 μl of reverse transcriptase (20.5 μl total) were added, mixed, and incubated at 40°C for 50 minutes, followed by 70°C for 10 minutes to complete cDNA synthesis. The cDNA was further added with poly-G at the 3' end. The reaction system was prepared as follows: 5μl of cDNA sample + 33.5μl of ddH2O + 5μl of 10×TdT buffer + 5μl of CoCl2 + 1μl of dGTP + 0.5μl of terminal deoxynucleotidyl transferase (total volume 50ul), incubated at 37°C for 30 minutes, then incubated at 70°C for 10 minutes to complete the poly-G tailing.

[0098] Furthermore, the tailed cDNA was used as a template for gene amplification of the antibody variable region. For amplifying the antibody heavy chain variable region sequence, the PCR reaction system was prepared: 5 μl of 10× Taq enzyme buffer + 0.5 μl of universal poly C primer (forward primer) + 0.5 μl of mouse IgG1 reverse primer + 1 μl of dNTP + 1 μl of Taq polymerase + 1 μl of cDNA + 41 μl of ddH2O. For amplifying the antibody light chain variable region sequence, the PCR reaction system was prepared: 5 μl of 10× Taq enzyme buffer + 0.5 μl of universal poly C primer (forward primer) + 0.5 μl of mouse IgG kappa chain reverse primer + 1 μl of dNTP + 1 μl of Taq polymerase + 1 μl of cDNA + 41 μl of ddH2O. The temperature cycle for PCR amplification of the antibody heavy and light chain variable regions is as follows (steps 2 to 4 are repeated 25 cycles):

[0099] 1) Pre-denaturation at 95°C for 5 minutes;

[0100] 2) Denaturation at 95°C for 20 seconds;

[0101] 3) Annealing at 56°C for 20 seconds;

[0102] 4) Extension at 72°C for 30 seconds;

[0103] 5) Store at 25°C for 60 minutes.

[0104] The PCR products were analyzed by 1% agarose gel electrophoresis, and DNA segments of corresponding sizes were cut out (VH approximately 600 bp, VK approximately 500 bp). DNA was extracted using the QIAquick gel DNA recovery kit (cat#28704). The procedure is as follows: the gel was weighed, 3 times the volume of QG buffer was added, and then incubated at 50°C for 10 minutes until the gel was completely dissolved. After adding 1 times the volume of isopropanol to mix, the sample was transferred to a QIA purification column and centrifuged at 13,000 rpm for 1 minute. 750 μl of PE buffer was added to the column, and then centrifuged at 13,000 rpm for 1 minute. Centrifuge again at 13,000 rpm to remove residual liquid in the column. 30 μl of water was added and centrifuged at 13,000 rpm for 1 minute for elution to obtain the prepared DNA sample. The purified PCR product was sequenced to obtain the variable region sequence of the antibody.

[0105] The sequence information of the clones of the present invention is shown in Tables 5 to 7.

[0106] Table 5. Sequence information of anti-CD47 antibodies

[0107]

[0108]

[0109] NA: nucleotide; AA: amino acid.

[0110] Table 6. Amino acid sequences of anti-CD47 antibodies

[0111]

[0112]

[0113] Table 7. Nucleotide sequences of anti-CD47 antibodies

[0114]

[0115]

[0116]

Claims

1. A monoclonal antibody having CD47 binding activity, characterized in that: The monoclonal antibody having CD47 binding activity comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

18.

2. The monoclonal antibody having CD47 binding activity according to claim 1, wherein The heavy chain amino acid sequence is shown in SEQ ID NO: 10; the light chain amino acid sequence is shown in SEQ ID NO:

20.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, wherein The sequence of the nucleic acid molecule includes SEQ ID NO: 7 and SEQ ID NO: 17; The sequence SEQ ID NO: 7 encodes the heavy chain variable region of the antibody; The sequence SEQ ID NO: 17 encodes the light chain variable region of the antibody.

5. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 3 or 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 5.

7. The method for preparing a monoclonal antibody having CD47 binding activity according to claim 1 or 2, wherein: The following steps are included: preparing an expression vector containing a nucleic acid molecule for expressing the monoclonal antibody according to claim 1 or 2; The obtained expression vector is transfected into eukaryotic host cells and cultured; The monoclonal antibody with CD47 binding activity was obtained by separation and purification.

8. A pharmaceutical composition comprising the monoclonal antibody according to claim 1 or 2.

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