CN115772221B - Monoclonal antibody capable of inducing macrophages to phagocytose cancer cells, and preparation method and application thereof - Google Patents

Abstract

The present invention, belonging to the field of biomedicine, provides a monoclonal antibody capable of inducing macrophages to phagocytose cancer cells. It also provides a nucleic acid molecule encoding the antibody, an expression vector, a host cell, and a method for preparing the antibody. It also provides a pharmaceutical composition comprising the antibody and its use. The anti-human CD47 monoclonal antibody of the present invention has a high affinity for human CD47 and, compared to existing anti-human CD47 monoclonal antibodies, exhibits a stronger effect in promoting tumor cell phagocytosis.

Description

Monoclonal antibody capable of inducing macrophages to phagocytose cancer cells, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a CD47 monoclonal antibody capable of inducing macrophages to phagocytose cancer cells, and a preparation method and application thereof.

Background

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

CD47, through interaction with sirpa, will release a "do not eat me" signal to macrophagesAnd Dendritic Cells (DCs), protect cells from the immune system, and also act by promoting proliferation of blood vessels within the tumor, inhibiting effector T cells, and promoting tumor cell expansion and growth. Cancer cells evade immune surveillance of the host by this approach, CD47 overexpression was found to be associated with poor clinical outcome. CD47 has also been identified as a cancer stem cell marker (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). in leukemia and solid tumors and thus CD47 blocking antibodies have been used in tumor therapy and have demonstrated anti-tumor activity in a number of in vivo tumor models. Furthermore, these antibodies have been demonstrated to act synergistically with other therapeutic antibodies, including rituximab and herceptin, in tumor models.

Researchers at the university of Stanford medical school reverse pulmonary fibrosis, a non-cure and life threatening disease, by administering anti-CD 47 antibodies to mutant mice, CD47 being further identified as a potential therapeutic target for the treatment of pulmonary fibrosis.

In recent years, drug development against tumor escape mechanisms mediated by CD47 and its ligand sirpa signaling axis has been the subject of heat in tumor immunotherapy. However, the anti-human CD47 monoclonal antibodies developed so far have problems of low affinity or ambiguous action targets such as epitopes.

Thus, there is a need for therapeutic candidate CD47 antibodies that induce macrophages to phagocytose cancer cells with higher affinity.

Disclosure of Invention

The invention aims to solve the defects in the prior art, and provides a monoclonal antibody capable of inducing macrophages to phagocytose cancer cells, a preparation method and application thereof, wherein the monoclonal antibody has higher affinity with human CD47 and has stronger tumor cell phagocytosis promoting effect.

The present invention provides a monoclonal antibody capable of inducing macrophages to phagocytose cancer cells, said antibody comprising 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;

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

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

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

The amino acid sequence of the CDR-L2 is shown as SEQ ID NO. 14;

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

Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8 and the light chain variable region has the amino acid sequence shown in SEQ ID NO. 18.

Preferably, the heavy chain amino acid sequence is shown as SEQ ID NO. 10, and the light chain amino acid sequence is shown as SEQ ID NO. 20.

Preferably, both the heavy and light chains further comprise a constant region which is a constant region of a murine or human IgG, preferably an IgG4 constant region.

The invention further provides a nucleotide molecule 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;

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

SEQ ID NO. 17 encodes the light chain variable region of the 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 capable of inducing macrophages to phagocytose cancer cells, which comprises the following steps:

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

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

(3) And (3) separating and purifying to obtain the monoclonal antibody capable of inducing macrophages to phagocytose cancer cells.

The invention further provides antibody immunoconjugates, bispecific molecules, chimeric and antigen receptors or pharmaceutical compositions comprising said monoclonal antibodies capable of inducing macrophages to phagocytose cancer cells.

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

The invention further provides application of the monoclonal antibody in preparing an anti-tumor drug or a drug for treating fibrosis diseases.

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.

Preferably, the fibrotic diseases include angina pectoris, osteoarthritis, pulmonary fibrosis, asthma and bronchitis.

The beneficial effects are that:

The monoclonal antibody capable of inducing macrophages to phagocytize cancer cells provided by the invention has higher affinity with human CD47, and has stronger tumor cell phagocytosis promoting effect compared with the existing anti-human CD47 monoclonal antibody, and in addition, the monoclonal antibody provided by the invention also has better CD47-SIRP alpha blocking activity.

Drawings

FIG. 1 is a capture ELISA assay for antibody binding 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 evaluation of antibody binding to 293F cells surface overexpressing human CD 47;

FIG. 4 is a ligand binding blocking ELISA;

FIG. 5 is a reference antibody blocking ELISA.

Detailed Description

Terminology

"Bind to CD47" or "bind to CD47" means to interact with human CD 47.

An "antigen binding site" refers to a discrete, three-dimensional spatial site on an antigen that 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 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 technology such as CDR grafting, or a combination of such or other technologies 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). Unless otherwise indicated, herein "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by common methods known in the art, including methods known in the art and described herein.

The term "compete" 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 that competition between antigen binding proteins is determined by an assay in which 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 bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competition 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 neighboring epitopes that are sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes spatially interfere with each other for binding to occur.

Methods for producing and purifying antibodies and antigen binding fragments are well known and disclosed in the art, such as the guidelines for antibody experimentation in cold spring harbor. For example, the mouse may be immunized with human CD47 or a fragment thereof, the resulting antibody may be renatured, purified, and amino acid sequenced by 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 to 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 extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom 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 a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses 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 is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on such factors as the condition to be treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"Pharmaceutical composition" means a mixture comprising one or more of the CD47 antibodies or antigen-binding fragments thereof described herein, and other pharmaceutical components, such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1 obtaining a mouse monoclonal antibody specific for anti-CD 47 by fusion hybridoma technique

1.1 Immunization of animals

Mice were immunized according to the method common in 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 first immunization and the boost, respectively. Briefly, the preparation of the adjuvant-antigen mixture was first gently mixed with the adjuvant in a vial using a vortexing method. The required amount of adjuvant was removed from the vial and placed into an autoclaved 1.5mL microcentrifuge tube. The antigen is prepared in PBS or physiological saline with concentration of 0.5-1.0 mg/ml. The calculated amount of antigen is added into a microcentrifuge tube together with the adjuvant, gently stirred for 2 minutes, and repeatedly emulsified and uniformly mixed to form a water-in-oil solution. The adjuvant-antigen solution is then inhaled into a suitable syringe for animal injection. Each animal was immunized and then boosted 2-3 times according to the anti-serum titer. Animals with good titers were terminally protected 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 the logarithmic growth phase. The mice were sacrificed in a sterile environment to remove spleen and fused with myeloma cells according to the method described by Kohler G and MILSTEIN C at "Continuous cultures of fused cells secreting antibody of predefined specificity,"Nature,256:495-497(1975).

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

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

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

Anti-CD 47 mouse monoclonal antibodies (mAbs) generated by the hybridoma clones of example 1 were assayed by affinity kinetic characterization via the Biacore T200 system (GE HEALTHCARE, pittsburgh, PA, USA).

Briefly, goat anti-mouse IgG was covalently linked to CM5 chip (carboxymethyl dextran coated chip) via primary amine using standard amine coupling kit provided by Biacore. The unreacted portion of the biosensor surface was blocked with ethanolamine. The mouse anti-CD 47 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 66.7nM, a flow rate of 10. Mu.L/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 (Biacore) was then flowed onto the chip at a flow rate of 30. Mu.L/min. Antigen-antibody binding kinetics were observed for 2 min and dissociation kinetics were observed for 10 min. The BIA evaluation software was used to fit a Langmuir binding model curve of binding to dissociation of 1:1.

Wherein the values of K _a,k_d and K _D are shown in table 1.

TABLE 1 Biacore determination of kinetic parameters of mouse anti-CD 47 monoclonal antibodies binding to human or cynomolgus monkey CD47

The binding K _D value of the monoclonal antibody 1D2 of the invention to human CD47 and the similar level of the reference antibody indicate that the monoclonal antibody has high affinity to human CD 47.

EXAMPLE 3 investigation of the binding Activity of mouse anti-CD 47 monoclonal antibody

Mouse anti-CD 47 monoclonal antibodies (mAbs) produced by the hybridoma clones of example 1 were further tested for binding activity as follows.

3.1 Determination of antibody binding Capture ELSIA

96-Well ELISA plates were coated with goat anti-mouse IgG Fcgamma fragment specific antibody (Jackson immuno Research, #115-006-071,100 μl/well) in PBS at a final concentration of 2 μg/ml and incubated overnight at4 ℃. ELISA plates were washed 4 times with elution buffer (PBS+0.05% v/v Tween-20, PBST) and then blocked at 37℃for 2 hours with 200. Mu.l/well of 5% w/v skimmed milk powder PBST buffer. Plates were again washed and incubated with different concentrations of 100 μl/well of CD47 murine monoclonal antibody for 40 minutes at 37 ℃ before washing the plates 4 more times. The elisa plate 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) (60 nm,2.5% nonfat dry milk PBST buffer, 100 μl/well) for 40 min at 37 ℃, and the plate was washed 4 more times and incubated with streptavidin-conjugated horseradish peroxidase (1:10000 dilution with PBST, jackson Immuno Research, #016-030-084,100 μl/well) for 40 min at 37 ℃. After final washing, the plates were incubated with 100. Mu.l/well ELISA substrate TMB (Innoreagents, # TMB-S-002). The reaction was stopped with 50. Mu.l/well 1M H ₂SO₄ at 25℃in 15 minutes and the absorbance at 450nm was determined, the results of which are shown in FIGS. 1-2 and Table 2.

The results in fig. 1 and 2 show that the antibody 1D2 of the present invention has a better binding ability to both human and cynomolgus monkey CD47 protein.

3.2 Determination of binding of CD47 monoclonal antibodies to 293F cell lines surface overexpressing human CD47 by flow cytometry (FACS)

The stable cell line 293F, with surface over-expression of human CD47, was harvested from the cell culture flask, washed twice and resuspended in PBS phosphate buffer (FACS buffer) containing 2% v/v fetal bovine serum. 2×l0 ⁵ cells per well in 96-well plates were incubated with FACS buffer containing different concentrations of CD47 antibody on ice for 40 min. Cells were washed 3 times with FACS buffer and 100. Mu.L/well of R-Phycoerythrin affinity purified F (ab ') ₂ fragment goat anti-mouse IgG specific F (ab') ₂ fragment (diluted 1:1000 with FACS buffer, jackson Immunoresearch, cat# 115-116-072) secondary antibody was added. After 40 min incubation at 4 ℃ in the dark, the cells were washed 3 times and then resuspended in FACS buffer. Fluorescence measurements were performed using Becton Dickinson FACS Canto II-HTS equipment. The data were analyzed using GRAPHPAD PRISM software to obtain EC ₅₀ concentration values for antibody-bound cells, i.e., the antibody concentration values corresponding to CD47 antibodies and cells that overexpressed CD47 reaching 50% of the maximum fluorescent binding signal, as determined in fig. 3 and table 2.

The results in FIG. 3 show that the antibody 1D2 of the present invention binds more strongly to 293F cells surface overexpressing human CD 47.

TABLE 2 binding Activity of mouse anti-CD 47 antibodies

Example 4 competitive function blocking ability of mouse anti-CD 47 monoclonal antibodies to CD47-SIRP alpha interactions

The blocking ability of the antibodies to CD 47-sirpa interactions was tested using a competition ELISA.

4.1 Ligand blocking ELISA

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

The CD47 antibody or reference antibody (antibody starting at 66.7nM, serial 4-fold dilutions) was diluted with human CD 47-biotin (ACRO biosystems, cat#cd 7-H5227) solution, incubated at room temperature for 40 minutes, and then the antibody/CD 47-biotin mixture was added to the sirpa-coated plate. After incubation at 37 ℃ for 40 minutes, 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 floor sirpa. The plate was then washed with wash buffer. Finally, TMB was added and the reaction was stopped with 1M H ₂SO₄ and the absorbance at 450nm was determined. Analysis of the data using GRAPHPAD PRISM software gave IC ₅₀ values, with specific results shown in fig. 4 and table 3.

4.2 Reference antibody blocking ELISA

The ability of the anti-CD 47 antibodies of the invention to block binding of the reference antibody (Hu 5F9-G4, forty Seven) -human CD47 protein was determined by competition ELISA. Briefly, the CD47 reference antibody was coated on 96-well microplates with 1 μg/mL PBS and incubated overnight at 4 ℃. The next day, plates were washed with wash buffer and blocked with PBST containing 5% nonfat milk powder for 2 hours at 37 ℃. At blocking, biotin-labeled human CD47 (ACRO biosystems, cat#CD7-H5227) (10 nM, PBST with 2.5% nonfat milk powder) was mixed with antibody (1.2 pM-100nM, serial 5-fold dilution) and then incubated at 25℃for 40 min. After plate washing, the antibody/human CD 47-biotin mixture (100. Mu.l/well) was added to the Hu5F9-G4 plate and incubated at 37℃for 40 min. Plates were washed again with wash buffer, 100 μl/well of SA-HRP was added, and incubated at 37℃for 40 min to detect biotin-labeled human CD47 bound to the plates. Final washing was performed with wash buffer. TMB was added and the reaction was stopped with 1M H ₂SO₄, and the absorbance at 450nm was measured. Analysis of the data using GRAPHPAD PRISM software gave IC ₅₀ values, with specific results shown in fig. 5 and table 3.

As can be seen from table 3, the antibodies of the present invention were able to block human CD 47-sirpa interactions, while demonstrating that the antibodies of the present invention have similar antigen binding epitopes as the reference antibodies. Compared with a reference antibody, the antibody 1D2 has better CD 47-SIRPalpha blocking activity.

TABLE 3 ability of anti-CD 47 antibodies to block interaction of CD 47-SIRPalpha and CD47 reference antibodies

EXAMPLE 5 Induction of macrophage phagocytosis of tumor cells by mouse anti-CD 47 monoclonal antibody

In vitro cell experiments were used to detect the bioactivity of anti-CD 47 antibodies in inducing phagocytic tumor cells by macrophages. Human Peripheral Blood Mononuclear Cells (PBMC) were extracted from human fresh blood using Ficoll (GE HEALTHCARE, 17-1440-02). To differentiate PBMC into mononuclear-derived macrophages (monocyte-derived macrophages, MDM), the 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 fresh medium containing cytokines was changed. 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. 100uL of CFSE labeled Jurkat tumor cells and diluted mixture of CD47 mab were added to MDM and incubated for 4h at 37 ℃. 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 by flow cytometry (FACS). The data (percentage of cd14+cfse+ cells) were analyzed using GRAPHPAD PRISM software to give EC ₅₀ values and the assay results are shown in table 4.

The results in table 4 show that the antibodies of the invention are capable of inducing macrophages to phagocytose tumor cells, and that their EC ₅₀ values are lower than those of the two reference antibodies, showing a stronger pro-tumor cell phagocytosis than the reference antibodies.

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

EXAMPLE 6 DNA cloning and sequencing, sequence analysis of anti-CD 47 antibodies

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

Briefly, the procedure is as follows, and 5X 10 ⁶ cells were collected by centrifugation into a 1.5ml centrifuge tube and the supernatant was blotted dry. 1ml of Trizol reagent was added and left at 25℃for 5 minutes after repeated pipetting for lysing cells. Next, 0.2ml of chloroform solution was added to each tube, and the mixture was left at room temperature for 3 minutes after shaking vigorously for 15 seconds. Then, the tube was centrifuged at 12000g for 10 minutes at 4℃and removed, and the upper aqueous solution was aspirated into a new 1.5ml tube, and 0.4ml of isopropyl alcohol was added for precipitating RNA from the aqueous phase. After the EP tube was manually homogenized and left at 25℃for 10min, 12000g was centrifuged at 4℃for 10min, and the supernatant was discarded. 1ml of 75% ethanol was added and centrifuged at 7500rpm at 4℃again for 5min, and the supernatant was discarded. After 10 minutes of drying of the RNA pellet at room temperature, 30 to 50ul of sterile DEPC treated water was added to dissolve the RNA sample.

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

Further, gene amplification of the antibody variable region was performed using the tailed cDNA as a template. For amplification of antibody heavy chain variable region sequences, a PCR reaction system was prepared of 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 amplification of antibody light chain variable region sequences, a PCR reaction system was prepared of 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 are as follows (where steps 2 to 4, 25 cycles are repeated):

1) Pre-denaturing at 95 ℃ for 5min;

2) Denaturation 95 ℃,20sec;

3) Annealing at 56 ℃,20sec;

4) Extending at 72 ℃ for 30sec;

5) Stored at 25 ℃ for 60min.

The PCR products were analyzed by 1% agarose gel electrophoresis, bands of DNA segments of the corresponding size (about 600bp for VH and about 500bp for VK) were excised, and DNA extraction was performed using the QIAquick gel DNA recovery kit (catalog # 28704). Briefly described, the gel was weighed, 3 gel volumes of QG buffer were added, and then incubated at 50℃for 10 minutes until the gel was completely dissolved. After adding 1 gel volume of isopropanol and mixing, the sample was transferred to QIA purification column and centrifuged at 13000rpm for 1 min. 750 μl PE buffer was added to the column, followed by centrifugation at 13000rpm for 1 minute. And centrifuged again at 13000rpm to remove liquid residue in the column. After adding 30. Mu.l of water and centrifuging at 13000rpm for 1 minute, eluting to obtain a prepared DNA sample, and sequencing the purified PCR product to obtain the variable region sequence of the antibody.

The sequence information of the clones of the invention is shown in Table 5.

TABLE 5 sequence information for anti-CD 47 antibodies

NA is nucleotide, AA is amino acid

Sequence listing

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<212> PRT

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<400> 2

Gly Tyr Lys Phe Thr Asp Tyr Asn

1 5

<210> 3

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<212> DNA

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atttatcctt ataatattag tagt 24

<210> 4

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<400> 4

Ile Tyr Pro Tyr Asn Ile Ser Ser

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Ala Arg Gly Gly Trp Arg Ala Met Asp Tyr

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gaggtccagc ttcagcagtc aggacctgag ctggtgaaac ctggggcctc agtgaggata 60

tcctgcaaga cttctggata taaattcact gactacaata tacactgggt gaagcagagc 120

catggaaaga gccttgaata tattggatat atttatcctt ataatattag tagtgcctac 180

aaccagaagt tcaagagcaa ggccacagtg actgtagaca attcctccag cacatcctac 240

atggaactcc gcagcctgac atctgaggac tctgcagtct attactgtgc aagggggggc 300

tggagggcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351

<210> 8

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

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Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Lys Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Ile Ser Ser Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Lys Ala Thr Val Thr Val Asp Asn Ser Ser Ser Thr Ser Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Trp Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 9

<211> 1323

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

gaggtccagc ttcagcagtc aggacctgag ctggtgaaac ctggggcctc agtgaggata 60

tcctgcaaga cttctggata taaattcact gactacaata tacactgggt gaagcagagc 120

catggaaaga gccttgaata tattggatat atttatcctt ataatattag tagtgcctac 180

aaccagaagt tcaagagcaa ggccacagtg actgtagaca attcctccag cacatcctac 240

atggaactcc gcagcctgac atctgaggac tctgcagtct attactgtgc aagggggggc 300

tggagggcta tggactactg gggtcaagga acctcagtca ccgtctcctc 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 Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Lys Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Ile Ser Ser Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Lys Ala Thr Val Thr Val Asp Asn Ser Ser Ser Thr Ser Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Trp Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser 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

agatctagtc agaacattgt ccatactaat ggatacacct atttagcg 48

<210> 12

<211> 16

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

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

1 5 10 15

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

aaggtttcca 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 gtggacg 27

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

Phe Gln Gly Ser His Val Pro Trp Thr

1 5

<210> 17

<211> 336

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

gctgttttga tgacccaaag tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

ctctcttgca gatctagtca gaacattgtc catactaatg gatacaccta tttagcgtgg 120

tacctgcaga ggccaggcca gtctccaaag ctcctgatct acaaggtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

tggacgttcg gtggaggcac caagctggaa atcaaa 336

<210> 18

<211> 112

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

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

1 5 10 15

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

20 25 30

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

gctgttttga tgacccaaag tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

ctctcttgca gatctagtca gaacattgtc catactaatg gatacaccta tttagcgtgg 120

tacctgcaga ggccaggcca gtctccaaag ctcctgatct acaaggtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

tggacgttcg 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

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

1 5 10 15

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

20 25 30

Asn Gly Tyr Thr Tyr Leu Ala Trp Tyr Leu Gln Arg 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 Trp 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 (9)

1. A monoclonal antibody capable of inducing macrophages to phagocytose cancer cells, characterized in that the antibody 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 in SEQ ID NO: 2; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 4; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 6; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 12; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 14; The amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 16. 2. The monoclonal antibody capable of inducing macrophages to phagocytose cancer cells according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 18. 3. The monoclonal antibody capable of inducing macrophages to phagocytose cancer cells according to claim 1, wherein the heavy chain amino acid sequence is as shown in SEQ ID NO: 10; and the light chain amino acid sequence is as shown in SEQ ID NO: 20. 4. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 3. 5. The nucleic acid molecule according to claim 4, wherein the sequence of the nucleic acid molecule comprises 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. 6. An expression vector, characterized in that it contains the nucleic acid molecule according to claim 4 or 5. 7. A host cell, characterized in that the host cell contains the expression vector according to claim 6. 8. The method for preparing the monoclonal antibody capable of inducing macrophages to phagocytose cancer cells according to any one of claims 1 to 3, characterized in that it comprises the following steps: preparing an expression vector containing a nucleic acid molecule expressing the monoclonal antibody; The obtained expression vector is transfected into eukaryotic host cells and cultured; The monoclonal antibody that can induce macrophages to phagocytize cancer cells is isolated and purified. 9. A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 3.