CN115746135A - CD 47-targeted monoclonal antibody and application thereof in preparation of antitumor drugs - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a CD 47-targeted monoclonal antibody and application thereof in preparing antitumor drugs, and also provides a coding nucleic acid molecule, an expression vector, a host cell and a method for expressing the antibody of the antibody, and further provides a pharmaceutical composition containing the antibody and application thereof. The CD 47-targeted monoclonal antibody has high affinity with human CD47, and has stronger tumor cell phagocytosis promotion effect compared with the existing CD 47-targeted monoclonal antibody.

Description

CD 47-targeted monoclonal antibody and application thereof in preparation of antitumor drugs

Technical Field

The invention relates to the technical field of biomedicine, in particular to a CD 47-targeted monoclonal antibody and application thereof in preparing an anti-tumor medicament.

Background

CD47, also known as integrin-associated protein (IAP), is an anti-phagocytic receptor with multiple signaling pathways, belongs to the immunoglobulin superfamily, and is widely expressed on the surface of almost all normal cells. CD47 has an immunoglobulin variable N-terminal domain, five transmembrane domains, and a short C-terminal intracellular tail. The natural ligands for CD47 that are currently known are integrin, thrombospondin-1 (TSP-1), and Signal-regulatory protein alpha (SIRP alpha). The biological roles in which CD47 has been reported to participate include cell migration, adhesion, proliferation, apoptosis, and maintenance of immune homeostasis in the body.

Under normal physiological state, CD47-SIRP alpha plays an important role in maintaining organism tolerance and assisting immune response as an immune inspection point. In tumor tissues, macrophages achieve the purpose of clearing tumor cells through phagocytosis, but after CD47 highly expressed on the surfaces of the tumor cells is combined with SIRP alpha on the surfaces of phagocytic cells such as macrophages and dendritic cells, the phagocytosis of the macrophages is inhibited, and the immune escape is realized. Blocking a CD47-SIRP alpha signal channel, promoting phagocytosis of tumor cells by macrophages, promoting uptake of tumor cells by dendritic cells, facilitating antigen presentation, promoting cytokine secretion and the like.

In the field of cancer immunotherapy, CD47 is a promising target. However, the monoclonal antibodies targeting CD47 that have been developed at present still have problems of low affinity, unclear target of action such as epitope, and the like. Thus, there is a need for a therapeutic candidate CD47 antibody with higher affinity and better blocking activity.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a monoclonal antibody targeting CD47 and application thereof in preparing an anti-tumor medicament, wherein the monoclonal antibody has stronger affinity with human CD47 and stronger tumor cell phagocytosis promoting effect.

The invention provides a monoclonal antibody targeting CD47, which comprises a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises V _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises V _L CDR1、V _L CDR2 and V _L CDR3；

The V is _H The amino acid sequence of CDR1 is shown in SEQ ID NO:2 is shown in the specification;

the V is _H The amino acid sequence of CDR2 is shown in SEQ ID NO:4 is shown in the specification;

the V is _H The amino acid sequence of CDR3 is shown in SEQ ID NO:6 is shown in the specification;

the V is _L The amino acid sequence of CDR1 is shown in SEQ ID NO:12 is shown in the specification;

the V is _L The amino acid sequence of CDR2 is shown in SEQ ID NO:14 is shown in the figure;

the V is _L The amino acid sequence of CDR3 is shown in 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 CD 47-targeted 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 CD 47-targeted monoclonal antibody, which comprises the following steps:

(1) Preparing an expression vector containing a nucleotide molecule encoding the CD 47-targeted 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 CD 47-targeted monoclonal antibody.

The invention further provides bispecific molecules, antibody immunoconjugate conjugates, chimeric antigen receptors or pharmaceutical compositions comprising the above CD 47-targeting monoclonal antibodies.

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

The invention further provides application of the CD 47-targeted monoclonal antibody in preparation of antitumor drugs.

Preferably, the tumor is a hematological tumor or a solid tumor, including a hematological malignancy such as non-hodgkin lymphoma (NHL), acute Lymphocytic Leukemia (ALL), acute Myelogenous Leukemia (AML), and solid tumors such as colorectal cancer, ovarian cancer, breast cancer, fallopian tube cancer, bladder cancer, head and neck cancer, pancreatic cancer, lung cancer, and glioblastoma.

Has the beneficial effects that:

the CD 47-targeted monoclonal antibody has better affinity with human CD47, and simultaneously has stronger tumor cell phagocytosis promotion effect and better CD47-SIRP alpha blocking activity compared with the existing CD 47-targeted monoclonal antibody.

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;

FIG. 5 is a reference antibody blocking ELISA;

FIGS. 6A-6B are flow cytometry assays of the ability of antibodies to induce macrophages to phagocytose tumor cells.

Detailed Description

Term(s) for

"bind to CD47" or "bind to CD47" refers to the ability to interact with human CD47. By "specifically binds" is meant binds to human CD47 protein (and possibly CD47 protein from one or more non-human species) but does not substantially bind to non-CD 47 protein.

An "antigen binding site" refers to one or more fragments of an antibody that have the ability to specifically bind to an antigen (e.g., a CD47 protein).

"monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, exhibiting a single binding specificity and affinity for a particular epitope. Monoclonal antibodies or antigen-binding fragments thereof can be prepared, for example, by hybridoma techniques, recombinant DNA techniques, phage display techniques, synthetic techniques such as CDR grafting techniques, or a combination 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 common methods known in the art, including the prior art and methods 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 bound 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 antibody experimental guidelines of cold spring harbor. 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 can 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. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. 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). The immunogen was recombinant human CD47 protein (Sino biological inc., cat # 12283-H02H).

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 required amount of adjuvant was first added to an autoclaved 1.5mL microcentrifuge tube; then preparing the antigen in PBS or physiological saline with the concentration of 0.5-1.0 mg/ml; and finally, adding the calculated amount of antigen and the adjuvant into a micro centrifugal tube, slightly vortex and mixing for 2 minutes, and repeatedly emulsifying and uniformly mixing to form a water-in-oil solution, thereby obtaining the adjuvant-antigen mixed solution. Each animal was then immunized by injection with a syringe to aspirate the appropriate amount of adjuvant-antigen mixture. 2-3 boosts were performed according to antiserum titer. Animals with better titers were given a final boost by intraperitoneal injection before cell fusion.

1.2 hybridoma fusion and screening

Cells of a mouse myeloma cell line (SP 2/0-Ag14, ATCC # CRL-1581) were cultured to reach logarithmic growth phase before cell fusion. Immunized mouse splenocytes are prepared aseptically and fused with myeloma cells according to the methods described in the literature (Kohler G, and Milstein C, "continuous cells secreted antibody of predefined specificity," Nature, 256.

The fused "hybrid cells" were then distributed into 96-well cell plates containing DMEM/20% FCS/HAT medium. The growth of viable hybridoma cells is typically observed microscopically after 7-10 days post-fusion. Two weeks after cell plating, the supernatants of each well were tested by ELISA using recombinant human CD47-his protein. 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 per well and incubated for 40 min 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 monoclonality of the cell line, and then purified. Briefly, a protein a sepharose chromatography column was washed with 5-10 column volumes of PBS buffer. Cell supernatants of hybridoma monoclonal antibodies were passed through the chromatography column, which was then washed with PBS buffer until 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 immunoglobulin containing fractions were mixed and dialyzed overnight at 4 ℃ in PBS. Subsequently, the functional activity of the purified monoclonal antibodies was characterized in vitro as described below.

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

Affinity kinetic characterization assays for anti-CD 47 mouse-derived monoclonal antibodies (mAbs) generated by the hybridoma clones of example 1 were performed using 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. Unreacted groups on the surface of the biosensor are blocked by ethanolamine. The purified mouse-derived anti-CD 47 antibody produced in example 1, the reference antibodies CC-9000 (Celgene) and Hu5F9-G4 (Forty Seven) were flowed through the chip at a concentration of 66.7nM and a flow rate of 10. Mu.L/min. Then, the mixture was formulated into HBS-EP ⁺ 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 buffer (supplied by Biacore) was flowed through the chip at a flow rate of 30. Mu.L/min. The antigen-antibody binding kinetics were observed for 2 minutes and the dissociation kinetics for 10 minutes. Fit to 1:1Langmuir binding model. Wherein k is _a ，k _d And K _D The values are shown in Table 1.

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

Binding K of monoclonal antibody 1B4 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-derived anti-CD 47 monoclonal antibody

The mouse-derived anti-CD 47 monoclonal antibody produced by the hybridoma clone of example 1 was further tested for binding activity by the following method.

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

96-well plates were coated with a final concentration of 2. Mu.g/ml Fc gamma fragment-specific goat anti-mouse IgG antibody (Jackson immuno Research, cat #115-006-071, 100. Mu.l/well) in PBS and incubated overnight at 4 ℃. The ELISA plates were washed 4 times with elution buffer (PBS + 0.05%/v Tween-20, PBST), then 200. Mu.l/well of PBST buffer containing 5% w/v skim milk powder was added to the plates and blocked at 37 ℃ for 2 hours. The plates were washed again, 100. Mu.l/well of various concentrations of the CD47 mouse-derived monoclonal antibody were added, incubated at 37 ℃ for 40 minutes, and then washed 4 times. Biotin-labeled human CD47 protein (ACRO Biosystems, cat # CD 7-H5227) or monkey CYNO-CD47-HIS-BIO (ACRO Biosystems, cat # CD7-C52H 1) (60 nM, 2.5% nonfat dry milk PBST buffer, 100. Mu.l/well) was added to the plate containing the captured CD47 antibody, incubated at 37 ℃ for 40 minutes, the plate was washed 4 times, streptavidin-conjugated horseradish peroxidase (SA-HRP, 1, 10000 diluted in PBST buffer, jackson Immuno Research, cat #016-030-084, 100. Mu.l/well) was added, and incubated at 37 ℃ for 40 minutes. After final washing, 100. Mu.l/well of ELISA substrate TMB (Innorreagens, cat # TMB-S-002) was added to the plate. After 15 minutes incubation, 50. Mu.l/well of 1M H was added at room temperature ₂ SO ₄ The reaction was terminated 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 antibody 1B4 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)

The stable cell line 293F surface overexpressing human CD47 was collected from cell culture flasks, washed twice and resuspended in PBS (FACS buffer) containing 2% v/v fetal bovine serum. Add 2 xl 0 to 96 well plates ⁵ FACS buffer of individual cells/well and different concentrations of CD47 antibody, incubated on ice for 40 minutes. Wash 3 times with FACS buffer and continue to add 100. Mu.L/wellAffinity purification of F (ab') 2 fragment-specific goat anti-mouse IgG (1. After incubation at 4 ℃ for 40 min in the absence of light, 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 maximum fluorescent binding signal of the CD47 antibody and CD 47-overexpressing cells, are shown in fig. 3 and table 2.

The results in FIG. 3 show that the antibody 1B4 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 CD47-SIRP alpha 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, 96-well microplates were coated with 200 ng/well of human SIRP α -his protein (Nano biological inc., cat # 11612-H08H) and incubated overnight at 4 ℃. The following day, plates were washed with washing buffer (PBS +0.05% Tween-20, PBST) and blocked with 5% w/v skim milk powder in PBST for 2 hours at 37 ℃. The plates were then washed with wash buffer.

The CD47 antibody or reference antibody (starting concentration 66.7nm, 4-fold gradient dilution) was diluted with biotin labelled human CD47 (ACRO biosystems, cat # CD 7-H5227) solution and incubated at room temperature for 40 minutes before the antibody/CD 47 mixture was added to the sirpa coated plates. After incubation at 37 ℃ for 40 min, the plates were washed 4 times with wash buffer. SA-HRP was then added, incubated at 37 ℃ for 40 min, and the plate was washed with washing buffer. Finally, TMB was added, using 1M H ₂ SO ₄ The reaction was terminated, and the absorbance of each well at 450nm was measured using a microplate reader. Data were analyzed using Graphpad Prism software to obtain 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) to human CD47 protein was determined by a competition ELISA method. Briefly, 96-well microplates were coated with 1 μ g/mL of CD47 reference antibody in PBS solution and incubated overnight at 4 ℃. The next day, plates were washed with wash buffer and blocked with 5% nonfat dry milk in PBST 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,5 fold gradient dilution) and incubated at 25 ℃ for 40 min. After washing the plates, the antibody/human CD47 mixture (100. Mu.l/well) was added to the Hu5F9-G4 coated 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, and incubated at 37 ℃ for 40 minutes to detect biotin-labeled human CD47 bound to the plate. Final wash with wash buffer. Adding TMB, 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 arrive at 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 1B4 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 macrophages to phagocytose 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, cat # 17-1440-02). To differentiate PBMC into monocyte-derived macrophages (MDM), 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 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 harvested and labeled with CFSE (5 (6) -carboxyfluorescein diacetate succinimidyl ester) (Sigma, cat # 87444). 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 at 37 ℃ for 4h. All cells were isolated and washed once more with FACS buffer. Cell staining was performed with anti-human CD14 APC (eBioscience, cat # 17-0149-42) and CD14+ CFSE + cells were detected by flow cytometry (FACS). Data (percentage of CD14+ CFSE + cells to CD14+ cells) were analyzed using Graphpad Prism software to obtain EC ₅₀ Values and percent phagocytosis, determined as shown in Table 4 and FIGS. 6A-6B, respectively.

Table 4 and the results in FIGS. 6A-6B show that the antibodies of the present invention are capable of inducing macrophages to phagocytose tumor cells with their EC 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 antibody to induce macrophage phagocytosis of tumor cells

Example 6DNA 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, cat # 15596-018).

The procedure is briefly described below, and 5X 10 are collected by centrifugation ⁶ Into a 1.5ml centrifuge tube and the supernatant removed. 1ml of Trizol reagent was added and repeatedly blown 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 centrifuge tube was centrifuged at 12000g for 10 minutes at 4 ℃ and taken out, and the upper aqueous phase solution was sucked into a new 1.5ml centrifuge tube, and then 0.4ml of isopropyl alcohol was added for precipitation of RNA from the aqueous phase. The EP tube was mixed by hand and left at 25 ℃ for 10min, 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 drying the RNA precipitate at the bottom of the tube for 10 minutes at room temperature, adding 30-50ul of sterile DEPC treated water to dissolve the RNA sample.

Next, first strand cDNA was synthesized from the total RNA using a reverse transcription cDNA kit (Taraka, cat # 6110A). 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 incubated at 65 ℃ for 5 minutes and then on ice for 2 minutes. 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 RTase (20.5. Mu.l system in total) was 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 formulated 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 of terminal deoxynucleotidyl transferase TdT (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 the sequence of the heavy chain variable region of the amplified antibody, a PCR reaction system is prepared: 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. Temperature cycling for PCR amplification of variable regions of antibody heavy and light chainsAs follows (where steps 2 to 4 are 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 (cat # 28704). Briefly described as follows: the gel was weighed, 3 gel volumes of QG buffer were added, followed by incubation at 50 ℃ for 10min 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 tables 5-7.

TABLE 5 sequence information of anti-CD 47 antibodies

NA is nucleotide; AA is amino acid.

TABLE 6 amino acid sequence of anti-CD 47 antibody

TABLE 7 nucleotide sequences of anti-CD 47 antibodies

Claims (10)

1. A monoclonal antibody targeting CD47, wherein the antibody comprises a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises V _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises V _L CDR1、V _L CDR2 and V _L CDR3；

The V is _H The amino acid sequence of CDR1 is shown in SEQ ID NO:2 is shown in the specification;

the V is _H The amino acid sequence of CDR2 is shown in SEQ ID NO:4 is shown in the specification;

the V is _H The amino acid sequence of CDR3 is shown in SEQ ID NO:6 is shown in the specification;

the V is _L The amino acid sequence of CDR1 is shown in SEQ ID NO:12 is shown in the specification;

the V is _L The amino acid sequence of CDR2 is shown in SEQ ID NO:14 is shown in the figure;

the V is _L The amino acid sequence of CDR3 is shown in SEQ ID NO: shown at 16.

2. The monoclonal antibody targeting CD47 of claim 1, wherein 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.

3. The monoclonal antibody targeted to CD47 of 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 CD 47-targeting monoclonal antibody of any one of claims 1 to 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 a CD 47-targeting monoclonal antibody according to any one of claims 1 to 3, comprising the steps of:

preparing an expression vector containing a nucleotide molecule expressing the CD 47-targeting 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 CD 47-targeted monoclonal antibody.

9. A bispecific molecule, antibody immunoconjugate conjugate, chimeric antigen receptor or pharmaceutical composition comprising a CD 47-targeting monoclonal antibody of any one of claims 1 to 3.

10. Use of the CD 47-targeting monoclonal antibody of any one of claims 1-3 in the preparation of an anti-tumor medicament.

Images