CN115703833A - Monoclonal antibody with strong tumor cell phagocytosis promoting effect and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, particularly relates to a CD47 monoclonal antibody with strong tumor cell phagocytosis promoting effect, and further provides application of the antibody in preparation of anti-tumor medicines or medicines for treating fibrotic diseases. The novel anti-human CD47 monoclonal antibody has higher affinity with human CD47, and has stronger tumor cell phagocytosis promotion effect compared with the existing anti-human CD47 monoclonal antibody.

Description

Monoclonal antibody with strong tumor cell phagocytosis promoting effect and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a CD47 monoclonal antibody with strong tumor cell phagocytosis promoting effect, and an application thereof in preparation of anti-tumor medicines or medicines for treating fibrotic diseases.

Background

CD47 is a transmembrane protein that is expressed on almost all human cells. CD47, also known as integrin-associated protein (IAP), is a member of the immunoglobulin superfamily. CD47 is widely expressed on the cell surface, and can interact with SIRP alpha, thrombospondin (thrombospondin-1, TSP-1) and integrin to mediate a series of reactions such as apoptosis, proliferation, immunity and the like.

CD47 releases signals to macrophages through interaction with SIRP alpha

And Dendritic Cells (DCs) that protect cells from the immune system and promote tumor cell expansion and growth by promoting vascular proliferation in tumors, inhibiting effector T cells. Cancer cells evade immune surveillance by the host through this pathway and CD47 overexpression was found to be associated with poor clinical outcome. CD47 has also been identified as a cancer stem Cell marker in leukemias and solid tumors (Jaiswal, et al., (2009) Cell,138 (2): 271-85 Chan, et al., (2009) Proc Natl Acad Sci USA,106 (33): 14016-21 Chan, et al., (2010) Curr Opin Urol,20 (5): 393-7, majeti R, et al., (2011) Oncogene,30 (9): 1009-19. Thus, CD47 blocking antibodies have been used for tumor therapy and have shown anti-tumor activity in a number of in vivo tumor models. In addition, these antibodies have been shown to react with other therapeutic antibodiesThe body, including the synergistic effect of rituximab and herceptin in tumor models.

Since then, CD47 was further identified as a potential therapeutic target for the treatment of pulmonary fibrosis, an incurable and life-threatening disease. In recent years, drug development aiming at a tumor escape mechanism mediated by CD47 and a SIRP alpha signal axis of a ligand thereof becomes a hot door of tumor immunotherapy. However, the anti-human CD47 monoclonal antibodies developed so far have problems of low affinity or unclear target of action such as epitope. Therefore, there is a need for a novel candidate CD47 antibody with high affinity that can induce macrophages to phagocytose cancer cells.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a novel anti-human CD47 monoclonal antibody with strong tumor cell phagocytosis promotion effect, and the monoclonal antibody has high affinity with human CD47 and strong tumor cell phagocytosis promotion effect.

The invention provides a monoclonal antibody with strong tumor cell phagocytosis promotion effect, which comprises a heavy chain variable region and a light chain variable region;

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;

the amino acid sequence of the CDR-H1 is shown as SEQ ID NO:2 is shown in the specification;

the amino acid sequence of the CDR-H2 is shown as SEQ ID NO:4 is shown in the specification;

the amino acid sequence of the CDR-H3 is shown as SEQ ID NO:6 is shown in the specification;

the amino acid sequence of the CDR-L1 is shown as SEQ ID NO:12 is shown in the specification;

the amino acid sequence of the CDR-L2 is shown as SEQ ID NO:14 is shown in the figure;

the amino acid sequence of the CDR-L3 is shown as SEQ ID NO: shown at 16.

Preferably, the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO:8 is shown in the specification; the variable region amino acid sequence of the light chain is shown as SEQ ID NO:18, respectively.

Preferably, the heavy chain amino acid sequence is as set forth in SEQ ID NO:10 is shown in the figure; the light chain amino acid sequence is shown as SEQ ID NO: shown at 20.

Preferably, both the heavy and light chains also comprise a constant region that is a constant region of murine or human IgG, preferably IgG 4.

The invention further provides a nucleotide molecule for encoding the monoclonal antibody.

Preferably, the sequence of the nucleotide molecule is selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:17;

sequence SEQ ID NO:7 encodes the heavy chain variable region of said antibody;

sequence SEQ ID NO:17 encodes the light chain variable region of said antibody.

The invention further provides an expression vector containing the nucleotide molecule.

The invention further provides a host cell containing the expression vector.

Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell.

The invention further provides a preparation method of the monoclonal antibody, which comprises the following steps:

(1) Preparing an expression vector containing a nucleotide molecule for expressing the anti-human CD47 monoclonal antibody;

(2) Transfecting eukaryotic host cells by using the expression vector obtained in the step (1) and culturing;

(3) Separating and purifying to obtain the anti-human CD47 monoclonal antibody.

The invention further provides antibody immunoconjugate conjugates, bispecific molecules, chimeric antigen receptors or pharmaceutical compositions comprising the above monoclonal antibodies.

Further, the pharmaceutical composition comprises a therapeutically effective amount of the novel anti-human CD47 monoclonal antibody, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The invention further provides application of the monoclonal antibody in preparation of anti-tumor drugs or drugs for treating fibrotic diseases.

Preferably, the tumor is a hematological or 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.

Preferably, the fibrotic disease includes angina, osteoarthritis, pulmonary fibrosis, asthma and bronchitis.

Has the advantages that:

the novel anti-human CD47 monoclonal antibody has higher affinity with human CD47, has stronger tumor cell phagocytosis promotion effect compared with the existing anti-human CD47 monoclonal antibody, and also has better CD47-SIRP alpha blocking activity.

Drawings

FIG. 1 is a capture ELISA assay for the binding capacity of antibodies to human CD47 protein;

FIG. 2 is a capture ELISA assay for antibody binding to cynomolgus monkey CD47 protein;

FIG. 3 is a flow cytometry assay for antibody binding to 293F cells overexpressing human CD47 on the surface;

FIG. 4 is a ligand binding blocking ELISA;

figure 5 is a reference antibody blocking ELISA.

Detailed Description

Term(s) for

"bind to CD47" or "bind to CD47" refers to the ability to interact with human CD47.

An "antigen binding site" refers to a three-dimensional spatial site that is not contiguous on an antigen and is recognized by an antibody or antigen binding fragment herein.

"monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and not to the method of production thereof. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or combinations of these or other techniques known in the art.

"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). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by conventional methods known in the art, including those known in the art and described herein.

The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between antigen binding proteins as determined by the following assay: in such assays, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., CD47 or 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 that binds to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be detected is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds a contiguous epitope sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder binding from occurring.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and disclosed in the art, such as the Cold spring harbor antibody Experimental guidelines. For example, mice can be immunized with human CD47 or a fragment thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods.

By "treating" is meant administering an internal or external therapeutic agent, such as a composition comprising a CD47 antibody or antigen-binding fragment thereof, to a patient having one or more symptoms of a disease. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

By "pharmaceutical composition" is meant a mixture comprising one or more of the CD47 antibodies or antigen-binding fragments thereof described herein and other pharmaceutical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 obtaining of a mouse monoclonal antibody specific against CD47 by the fusion hybridoma technique

1.1 animal immunization

Mice were immunized according to methods commonly used in the literature (E Harlow, D.Lane, antibody: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1998). Recombinant human CD47 protein (Sino biological inc., cat # 12283-H02H) was used as immunogen.

To increase the immune response, freund's complete adjuvant and freund's incomplete adjuvant (Sigma, st. Louis, mo., USA) were used for the prime and boost, respectively. Briefly, the adjuvant-antigen mixture is prepared by first gently mixing the adjuvant in a vial using a vortex method. The desired amount of adjuvant was removed from the vial and placed in an autoclaved 1.5mL microcentrifuge tube. The antigen is prepared in PBS or physiological saline at a concentration of 0.5-1.0 mg/ml. Adding calculated amount of antigen and adjuvant into a microcentrifuge tube, gently stirring for 2 min, and repeatedly emulsifying and uniformly mixing to form water-in-oil solution. The adjuvant-antigen solution is then drawn into an appropriate syringe for injection into the animal. Each animal was immunized and then boosted 2-3 times according to the antiserum titer. Animals with good titers were terminally immunized by intraperitoneal injection prior to fusion.

1.2 hybridoma fusion and screening

Prior to cell fusion, mouse myeloma cells (SP 2/0-Ag14, ATCC # CRL-1581) were cultured in logarithmic growth phase. Following sacrifice of the mouse sterile environment, spleens were harvested and fused with myeloma cells according to the method described by Kohler G and Milstein C, in Continuous cultures of fused cells and characterization of predefined specificity, "Nature, 256.

The fused "hybrid cells" were then dispensed into 96-well cell plate media containing HAT. The growth of viable hybridoma cells is typically observed microscopically after 7-10 days post-fusion. Two weeks after cell plating, culture supernatants from each well were collected and hybridoma screened by ELISA using recombinant human CD47-his protein antigen. Briefly, ELISA plates were coated with human CD47-his protein (ACRO biosystems, cat # CD7-H5227, 2.0. Mu.g/ml in PBS) overnight at 4 ℃. The plate was washed 4 times with PBST and blocked with blocking buffer (5% nonfat dry milk in PBST). Diluted mouse immune serum (for determination of mouse serum titers) or hybridoma supernatants were added to each well and incubated for 40 minutes at 37 ℃. The plate was washed 4 times with PBST, detected with horseradish peroxidase-goat anti-mouse IgG (Jackson Immuno research, cat # 115-036-071) and the absorbance was determined for each well at 450 nm. Positive hybridomas secreting antibodies that bind to human CD47-his were then selected and transferred to 24-well plates.

Hybridoma clones producing antibodies that bind human CD47 with high specificity and have CD 47/sirpa ligand blocking activity were subcloned by limiting dilution to ensure clonality of the cell line, and then purified. Antibodies produced by hybridoma clones with high specific cell surface CD47 FACS binding and CD 47/sirpa ligand blocking activity were subcloned to ensure clonality of the cell lines, and then the monoclonal antibodies were purified.

Example 2 determination of affinity of mouse anti-CD 47 monoclonal antibody Using BIACORE surface plasmon resonance technique

The anti-CD 47 mouse monoclonal antibodies (mAbs) generated by the hybridoma clone of example 1 were subjected to an affinity kinetic characterization assay by the Biacore T200 System (GE healthcare, pittsburgh, pa., USA).

Briefly, goat anti-mouse IgG was covalently linked to a CM5 chip (carboxymethyldextran coated chip) via primary amines using a standard amine coupling kit supplied by Biacore. The unreacted portion of the biosensor surface is blocked with ethanolamine. The mouse anti-CD 47 antibody produced in example 1 was purified, and the reference antibodies CC-9000 (Celgene) and Hu5F9-G4 (Forty Seven) were flowed onto the chip at a concentration of 66.7nM and a flow rate of 10. Mu.L/min. Then, 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 (supplied by Biacore) was flowed onto the chip at a flow rate of 30. Mu.L/min. Antigen-antibody binding kinetics were observed for 2 minutes and dissociation kinetics for 10 minutes. The Langmuir binding model curve for binding and dissociation of 1:1 was fitted using BIA evaluation software.

Wherein k is _a ,k _d And K _D The values of (A) are shown in Table 1.

Table 1 biacore determination of kinetic parameters of mouse anti-CD 47 monoclonal antibody binding to human or cynomolgus monkey CD47

Binding K of monoclonal antibody 1D5 of the present invention to human CD47 _D Values were similar to the reference antibody, indicating high affinity for human CD47.

Example 3 binding Activity study of mouse anti-CD 47 monoclonal antibody

Mouse anti-CD 47 monoclonal antibodies (mAbs) generated by the hybridoma clones of example 1 were further tested for binding activity using the following method.

3.1 determination of the binding Capacity of antibodies based on Capture ELSIA

96-well ELISA plates were coated with goat anti-mouse IgG Fc gamma fragment specific antibody (Jackson immuno Research, #115-006-071, 100. Mu.l/well) at a final concentration of 2. Mu.g/ml in PBS and incubated overnight at 4 ℃. ELISA plates were washed 4 times with elution buffer (PBS +0.05% v/v Tween-20, PBST), then blocked by adding 200. Mu.l/well of 5%w/v skim milk powder PBST buffer at 37 ℃ for 2 hours. The plates were washed again and incubated with different concentrations of CD47 murine monoclonal antibody at 100. Mu.l/well for 40 minutes at 37 ℃ before washing the plates 4 times. The microplate containing the captured CD47 antibody was incubated with biotin-labeled human CD47 protein (ACRO Biosystems, cat # CD 7-H5227) or monkey CYNO-CD47-HIS-BIO (ACRO Biosystems, cat No. # CD7-C52H 1) (60nM, 2.5% nonfat dry milk PBST buffer, 100. Mu.l/well) at 37 ℃ for 40 minutes, the plate was washed 4 times and incubated with streptavidin-conjugated horseradish peroxidase (diluted 1,10000 with PBST, #016-030-084, 100. Mu.l/well) at 37 ℃ for 40 minutes. After final washing, the plates were incubated with 100. Mu.l/well of ELISA substrate TMB (Inoregens, # TMB-S-002). 50 mul/well 1M H in 15 minutes ₂ SO ₄ The reaction was terminated at 25 ℃ and the absorbance at 450nm was measured, the results of which are shown in FIGS. 1-2 and Table 2.

The results in FIGS. 1 and 2 show that the antibody 1D5 of the present invention has a good binding ability to human and cynomolgus monkey CD47 proteins.

3.2 determination of the binding of the CD47 monoclonal antibody to the 293F cell line with human CD47 overexpression on the surface by flow cytometry (FACS)

Collection of surface-overexpressed human CD4 from cell culture flasks7, washed twice and resuspended in PBS phosphate buffer (FACS buffer) containing 2%v/v fetal bovine serum. 2 xl 0 per well in 96-well plates ⁵ Individual cells were incubated on ice for 40 minutes with FACS buffer containing different concentrations of CD47 antibody. The cells were washed 3 times with FACS buffer and the addition of 100. Mu.L/well of R-Phyerythrin was continued for affinity purification of F (ab') ₂ Fragment goat anti-mouse IgG specific F (ab') ₂ Fragments (FACS buffer diluted at 1:1000, jackson Immunoresearch, cat # 115-116-072) secondary antibody. After incubation at 4 ℃ for 40 min in the dark, the cells were washed 3 times and then resuspended in FACS buffer. Fluorescence measurements were performed using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software to derive EC for antibody-bound cells ₅₀ The concentration values, i.e., the antibody concentration values corresponding to 50% of the maximal fluorescent binding signal of the CD47 antibody to CD 47-overexpressing cells, are shown in fig. 3 and table 2.

The results in FIG. 3 show that the antibody 1D5 of the present invention has a stronger binding ability to 293F cells overexpressing human CD47 on their surface.

TABLE 2 binding Activity of mouse anti-CD 47 antibodies

Example 4 competitive functional blockade of the CD47-SIRP α interaction by mouse anti-CD 47 monoclonal antibodies

The blocking ability of the antibodies to the CD47-SIRP alpha interaction was tested by competition ELISA.

4.1 ligand blocking ELISA

The ability of the anti-CD 47 antibodies of the invention to block the CD47-SIRP alpha interaction was tested using a competition ELISA. Briefly, human SIRP α -his protein (Nano biological inc., cat # 11612-H08H) was added at 200 ng/well to a 96-well microplate and incubated overnight at 4 ℃. The next day, plates were washed with wash buffer (PBS +0.05% Tween-20, PBST) and blocked with PBST containing 5%w/v skim milk powder for 2 hours at 37 ℃. The plates were then washed with wash buffer.

Human CD 47-rawThe CD47 antibody or reference antibody (antibody starting at 66.7nM, 4-fold serial dilutions) was diluted in a biotin (ACRO biosystems, cat # CD 7-H5227) solution, incubated at room temperature for 40 minutes, and the antibody/CD 47-biotin mixture was added to the SIRP α -coated plate. After incubation at 37 ℃ for 40 min, the plates were washed 4 times with wash buffer. Streptavidin-conjugated HRP was then added and incubated at 37 ℃ for 40 minutes to detect binding of biotin-labeled human CD47 to the bottom plate sirpa. The plate was washed with wash buffer. Finally, TMB was added using 1M H ₂ SO ₄ The reaction was terminated and the absorbance at 450nm was measured. Data were analyzed using Graphpad Prism software to generate IC ₅₀ The values, specific results are shown in fig. 4 and table 3.

4.2 reference antibody blocking ELISA

The ability of the anti-CD 47 antibody of the present invention to block the binding of a reference antibody (Hu 5F9-G4, forty Seven) -human CD47 protein was determined by a competitive ELISA method. Briefly, the CD47 reference antibody was coated onto 96-well microplates with 1. Mu.g/mL PBS and incubated overnight at 4 ℃. The next day, plates were washed with wash buffer and blocked with PBST containing 5% nonfat dry milk for 2 hours at 37 ℃. For blocking, biotin-labeled human CD47 (ACRO biosystems, cat # CD 7-H5227) (10 nM, PBST containing 2.5% skim milk powder) was mixed with antibody (1.2 pM-100nM, serial 5-fold dilutions) and incubated at 25 ℃ for 40 min. After washing the plates, the antibody/human CD 47-biotin mixture (100. Mu.l/well) was added to the Hu5F9-G4 plates and incubated at 37 ℃ for 40 min. The plate was washed again with washing buffer, 100. Mu.l/well of SA-HRP was added, followed by incubation at 37 ℃ for 40 minutes to detect biotin-labeled human CD47 bound to the plate. Final wash with wash buffer. Adding TMB, and reacting with 1M H ₂ SO ₄ The reaction was terminated and the absorbance at 450nm was measured. Data were analyzed using Graphpad Prism software to generate IC ₅₀ The values, specific results are shown in fig. 5 and table 3.

As can be seen from table 3, the antibodies of the invention were able to block the human CD 47-sirpa interaction, while showing that the antibodies of the invention have similar antigen binding epitopes to the reference antibody. The antibody 1D5 of the invention has better CD 47-SIRPa blocking activity compared to the reference antibody.

TABLE 3 ability of anti-CD 47 antibodies to block the interaction of CD47-SIRP alpha and CD47 reference antibodies

Example 5 mouse anti-CD 47 monoclonal antibody induces macrophage phagocytosis of tumor cells

In vitro cell experiments are adopted to detect the biological activity of the anti-CD 47 antibody for inducing macrophages to phagocytose tumor cells. Human Peripheral Blood Mononuclear Cells (PBMC) were extracted from fresh human blood using Ficoll (GE Healthcare, 17-1440-02). To differentiate PBMC into monocyte-derived macrophages (MDM), monocytes were inoculated with RPMI 1640+10% FBS +1% penicillin-streptomycin (Peprotech, 300-25-100) in the presence of human M-CSF. On days 2 and 4, the cells were washed and replaced with fresh medium containing cytokines. On day 6, adherent cells were isolated and washed 2 times with PBS.

MDMs were isolated from plates and placed in 96-well plates overnight. Jurkat cells were collected for CFSE (5 (6) -carboxyfluorescein N-hydroxysuccinimide ester) (Sigma, 87444) labeling. anti-CD 47 mab was diluted accordingly. To the MDM, 100uL of CFSE-labeled Jurkat tumor cells and diluted mixture of CD47 mab were added and incubated at 37 ℃ for 4h. All cells were isolated and washed once with FACS buffer. Cell staining was performed with anti-human CD14 APC (eBioscience, 17-0149-42), and CD14+ CFSE + cells were detected with flow cytometry (FACS). Data (percentage of CD14+ CFSE + cells) were analyzed using Graphpad Prism software to obtain EC ₅₀ The values and measurement results are shown in Table 4.

Table 4 results show that the antibody of the present invention can induce macrophages to phagocytose tumor cells, the EC of which ₅₀ Values lower than the two reference antibodies showed a stronger tumor cell phagocytosis than the reference antibody.

TABLE 4 ability of anti-CD 47 antibodies to induce phagocytosis of tumor cells by macrophages

Example 6 DNA cloning and sequencing, sequence analysis of anti-CD 47 antibody

Total RNA was extracted from the hybridoma cells of example 1 using Trizol reagent (Invitrogen, catalog # 15596-018).

The procedure is briefly described below, and 5X 10 are collected by centrifugation ⁶ The cells were transferred to a 1.5ml centrifuge tube and the supernatant was blotted dry. 1ml of Trizol reagent was added and repeatedly aspirated several times, and then left at 25 ℃ for 5 minutes for cell lysis. Subsequently, 0.2ml of chloroform solution was added to each tube, and the tube was vigorously shaken for 15 seconds and then left at room temperature for 3 minutes. Then, the tube was centrifuged at 12000g for 10 minutes at 4 ℃ and then removed, and the upper aqueous phase solution was aspirated into a new 1.5ml tube, and then 0.4ml of isopropanol was added for RNA precipitation from the aqueous phase. The EP tube was manually mixed and left at 25 ℃ for 10min, then centrifuged at 12000g at 4 ℃ for 10min, and the supernatant was discarded. 1ml of 75% ethanol was added thereto, and the mixture was centrifuged again at 7500rpm at 4 ℃ for 5min, and the supernatant was discarded. After the bottom RNA pellet was dried at room temperature for 10 minutes, 30 to 50ul of sterile DEPC-treated water was added to dissolve the RNA sample.

Next, the reverse transcription cDNA kit (catalog # 6110A) from Taraka was used to convert the total RNA into cDNA. The experimental system was prepared as follows: mu.l of total RNA + 0.5. Mu.l Oligo (dT) + 8.5. Mu.l RNase-free water (14. Mu.l total) were pre-denatured at 65 ℃ for 5min and then on ice for 2 min. Further, 4. Mu.l of 5 Xbuffer + 1. Mu.l of dNTP mix + 0.5. Mu.l of RNase inhibitor + 1. Mu.l of reverse transcriptase (20.5. Mu.l system in total) were added thereto, mixed well, incubated at 40 ℃ for 50 minutes, and then at 70 ℃ for 10 minutes to complete cDNA synthesis. The cDNA was further added with poly-G at the 3' end, and the reaction system was prepared as follows: mu.l of cDNA sample + 33.5. Mu.l of ddH ₂ O + 5. Mu.l of 10 XTdT buffer + 5. Mu.l of CoCl ₂ + 1. Mu.l dGTP + 0.5. Mu.l terminal deoxynucleotidyl transferase (total volume 50 ul), incubated at 37 ℃ for 30 minutes and then at 70 ℃ for 10min to complete poly-G tailing.

Further, gene amplification of the antibody variable region was carried out using the tailed cDNA as a template. For amplifying antibody heavy chainsChanging the sequence of the region, preparing a PCR reaction system: 10 XTaq enzyme buffer 5. Mu.l + Universal Poly C primer (Forward primer) 0.5. Mu.l + mouse IgG1 reverse primer 0.5. Mu.l + dNTP 1. Mu.l + Taq polymerase 1. Mu.l + cDNA 1. Mu.l + ddH ₂ O41. Mu.l. For the sequence of the amplified antibody light chain variable region, a PCR reaction system is prepared: 10 XTaq enzyme buffer 5. Mu.l + Universal Poly C primer (Forward primer) 0.5. Mu.l + mouse IgG kappa chain reverse primer 0.5. Mu.l + dNTP 1. Mu.l + Taq polymerase 1. Mu.l + cDNA 1. Mu.l + ddH ₂ O41. Mu.l. The temperature cycles for PCR amplification of the antibody heavy and light chain variable regions were as follows (with steps 2 to 4, repeated for 25 cycles):

1) Pre-denaturation at 95 deg.C for 5min;

2) Denaturation at 95 ℃ for 20sec;

3) Annealing at 56 deg.C for 20sec;

4) Extension 72 ℃ for 30sec;

5) Storing at 25 deg.C for 60min.

The PCR products were analyzed by 1% agarose gel electrophoresis, bands of DNA fragments (about 600bp for VH and about 500bp for VK) of corresponding sizes were excised, and DNA was extracted using QIAquick's gel DNA recovery kit (catalog # 28704). Briefly described as follows: the gel was weighed, 3 gel volumes of QG buffer were added, followed by incubation at 50 ℃ for 10 minutes until the gel was completely dissolved. After adding isopropanol of 1 gel volume and mixing, the sample was transferred to a QIA purification column and centrifuged at 13000rpm for 1 minute. 750. Mu.l of PE buffer was added to the column, followed by centrifugation at 13000rpm for 1 minute. And centrifuged again at 13000rpm to remove liquid residue from the column. The mixture was centrifuged at 13000rpm in 30. Mu.l of water for 1 minute to elute the DNA sample prepared, and the purified PCR product was sequenced to obtain the variable region sequence of the antibody.

Sequence information for clones of the invention is shown in Table 5.

TABLE 5 sequence information of anti-CD 47 antibodies

NA is nucleotide; AA is amino acid.

Sequence listing

<110> Boaoxin Biotechnology (Nanjing) Ltd

<120> monoclonal antibody with strong tumor cell phagocytosis promoting effect and application thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggcttcaaca ttaaagacta ctat 24

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Phe Asn Ile Lys Asp Tyr Tyr

1 5

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attgatccta aaaatggtga tact 24

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ile Asp Pro Lys Asn Gly Asp Thr

1 5

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatgcggggg gacgaggggg gtttgcttac 30

<210> 6

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asn Ala Gly Gly Arg Gly Gly Phe Ala Tyr

1 5 10

<210> 7

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg 60

tcctgcacag cttctggctt caacattaaa gactactata tgtcctgggt gaggcagagg 120

cctgaacagg gcctggagtg gattggatgg attgatccta aaaatggtga tactgcatat 180

gacccgaagt tccagggcaa ggccactatg actgcagaca catcctccaa cacagcctac 240

ctgcagttca gcagcctgac atctgaggac actgccgact attactgtaa tgcgggggga 300

cgaggggggt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351

<210> 8

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Lys Asn Gly Asp Thr Ala Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Asp Tyr Tyr Cys

85 90 95

Asn Ala Gly Gly Arg Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 9

<211> 1323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg 60

tcctgcacag cttctggctt caacattaaa gactactata tgtcctgggt gaggcagagg 120

cctgaacagg gcctggagtg gattggatgg attgatccta aaaatggtga tactgcatat 180

gacccgaagt tccagggcaa ggccactatg actgcagaca catcctccaa cacagcctac 240

ctgcagttca gcagcctgac atctgaggac actgccgact attactgtaa tgcgggggga 300

cgaggggggt ttgcttactg gggccaaggg actctggtca ctgtctctgc agccaaaacg 360

acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 420

accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 480

ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 540

ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 600

gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 660

tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 720

cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 780

agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 840

gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 900

cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 960

gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1020

caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1080

tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1140

ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1200

tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 1260

gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 1320

aaa 1323

<210> 10

<211> 441

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Lys Asn Gly Asp Thr Ala Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Asp Tyr Tyr Cys

85 90 95

Asn Ala Gly Gly Arg Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu

115 120 125

Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys

130 135 140

Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser

145 150 155 160

Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp

180 185 190

Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr

195 200 205

Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys

210 215 220

Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val

245 250 255

Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe

260 265 270

Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu

275 280 285

Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala

305 310 315 320

Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg

325 330 335

Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met

340 345 350

Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro

355 360 365

Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn

370 375 380

Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val

385 390 395 400

Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr

405 410 415

Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu

420 425 430

Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 11

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agatctaatc agagcattgt acatagtaat ggatacactt atttagaa 48

<210> 12

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Arg Ser Asn Gln Ser Ile Val His Ser Asn Gly Tyr Thr Tyr Leu Glu

1 5 10 15

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aaagtttcca accgattttc t 21

<210> 14

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 15

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tttcaaggtt cacatgttcc tcggacg 27

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Phe Gln Gly Ser His Val Pro Arg Thr

1 5

<210> 17

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctaatca gagcattgta catagtaatg gatacactta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct ataaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300

cggacgttcg gtggaggcac caagctggaa atcaaa 336

<210> 18

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser Ile Val His Ser

20 25 30

Asn Gly Tyr Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 657

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctaatca gagcattgta catagtaatg gatacactta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct ataaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300

cggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgc accaactgta 360

tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420

ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 480

caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 540

agcagcaccc tcacgttgac taaggacgag tatgaacgac ataacagcta tacctgtgag 600

gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacagggg agagtgt 657

<210> 20

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser Ile Val His Ser

20 25 30

Asn Gly Tyr Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims (10)

1. A monoclonal antibody with strong tumor cell phagocytosis promotion effect, which comprises a heavy chain variable region and a light chain variable region;

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;

the amino acid sequence of the CDR-H1 is shown as SEQ ID NO:2 is shown in the specification;

the amino acid sequence of the CDR-H2 is shown as SEQ ID NO:4 is shown in the specification;

the amino acid sequence of the CDR-H3 is shown as SEQ ID NO:6 is shown in the specification;

the amino acid sequence of the CDR-L1 is shown as SEQ ID NO:12 is shown in the specification;

the amino acid sequence of the CDR-L2 is shown as SEQ ID NO:14 is shown in the figure;

the amino acid sequence of the CDR-L3 is shown as SEQ ID NO: shown at 16.

2. The monoclonal antibody having enhanced tumor cell phagocytosis according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO:8 is shown in the specification; the variable region amino acid sequence of the light chain is shown as SEQ ID NO:18, respectively.

3. The monoclonal antibody having enhanced tumor cell phagocytosis according to claim 1, wherein the heavy chain amino acid sequence is as set forth in SEQ ID NO:10 is shown in the figure; the light chain amino acid sequence is shown as SEQ ID NO: shown at 20.

4. A nucleotide molecule encoding the monoclonal antibody of any one of claims 1-3.

5. The nucleotide molecule of claim 4, wherein the sequence of the nucleotide molecule is selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:17;

sequence SEQ ID NO:7 encodes the heavy chain variable region of said antibody;

sequence SEQ ID NO:17 encodes the light chain variable region of said antibody.

6. An expression vector comprising the nucleotide molecule of claim 4 or 5.

7. A host cell comprising the expression vector of claim 6.

8. The method for preparing monoclonal antibody with enhanced tumor cell phagocytosis according to any one of claims 1 to 3, comprising the steps of:

preparing an expression vector containing a nucleotide molecule for expressing the monoclonal antibody according to any one of claims 1 to 3;

transfecting the obtained expression vector to eukaryotic host cells and culturing;

separating and purifying to obtain the monoclonal antibody with strong tumor cell phagocytosis promoting effect.

9. An antibody immunoconjugate, bispecific molecule, chimeric antigen receptor or pharmaceutical composition comprising the monoclonal antibody of any one of claims 1-3.

10. Use of the monoclonal antibody according to any one of claims 1 to 3 for the preparation of an antitumor medicament or a medicament for fibrotic diseases.

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