An anti-human CD47 monoclonal antibody, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an anti-human CD47 monoclonal antibody, its preparation method, and its applications. It also provides the encoding nucleic acid molecule, expression vector, host cell, and method for expressing the antibody, as well as pharmaceutical compositions comprising the antibody of this invention and their uses. The anti-human CD47 monoclonal antibody of this invention has a high affinity for human CD47 and exhibits a stronger pro-tumor cell phagocytic activity compared to existing anti-human CD47 monoclonal antibodies.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-human CD47 monoclonal antibody, its preparation method, and its application. Background Technology

[0002] CD47 is a widely expressed membrane surface receptor with a molecular weight of 50 kDa, belonging to the immunoglobulin superfamily. CD47 possesses a type V Ig-like extracellular domain, a five-transmembrane region, and a short intracellular tail. CD47 can bind to various proteins, including integrins and thrombospondin-1 (TSP-1), and participates in various physiological and pathological processes, including cell migration, T cell and dendritic cell activation, axonal development, apoptosis, proliferation, and inflammation. Furthermore, CD47 can also bind to the signal-regulatory protein α chain (SIRPα). Studies have shown that CD47 acts as a "don't eat me" signal in the CD47-SIRPα signaling pathway. Under normal physiological conditions, it can maintain the immune tolerance of the body's own cells. However, under pathological conditions, CD47 is highly expressed on the surface of various hematologic malignancies and solid tumor cells. By binding to SIRPα receptors on phagocytes (macrophages, dendritic cells, etc.), it initiates a series of inhibitory signals. These inhibitory signals can be transduced into the phagocytes, ultimately causing the phagocytes to lose their phagocytic function and become unable to engulf and eliminate malignant proliferating cells.

[0003] In recent years, CD47 has become a very popular target among numerous immunotherapy regimens. However, currently developed anti-human CD47 monoclonal antibodies still suffer from problems such as low affinity and unclear target sites, such as antigenic epitopes. Therefore, there is an urgent need for a therapeutic candidate CD47 antibody with high affinity and good blocking activity. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-human CD47 monoclonal antibody, its preparation method and application. This monoclonal antibody has a high affinity for human CD47 and a strong effect on promoting tumor cell phagocytosis.

[0005] This invention provides an anti-human CD47 monoclonal antibody, the antibody comprising a heavy chain variable region and a light chain variable region;

[0006] The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0007] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 2;

[0008] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 4;

[0009] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 6;

[0010] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 12;

[0011] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 14;

[0012] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 16.

[0013] Preferably, 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.

[0014] Preferably, the heavy chain amino acid sequence is as shown in SEQ ID NO: 10; the light chain amino acid sequence is as shown in SEQ ID NO: 20.

[0015] Preferably, both the heavy chain and the light chain further include a constant region, which is a constant region of mouse or human IgG, preferably a constant region of IgG4.

[0016] The present invention further provides a nucleotide molecule encoding the above-mentioned anti-human CD47 monoclonal antibody.

[0017] Preferably, the sequence of the nucleotide molecule is selected from SEQ ID NO: 7 and SEQ ID NO: 17;

[0018] Sequence SEQ ID NO: 7 encodes the heavy chain variable region of the antibody described;

[0019] The sequence SEQ ID NO: 17 encodes the light chain variable region of the antibody described.

[0020] The present invention further provides an expression vector containing the aforementioned nucleotide molecules.

[0021] The present invention further provides a host cell containing the aforementioned expression vector.

[0022] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0023] This invention further provides a method for preparing the above-mentioned anti-human CD47 monoclonal antibody, comprising the following steps:

[0024] (1) Prepare an expression vector containing a nucleotide molecule expressing the aforementioned anti-human CD47 monoclonal antibody;

[0025] (2) Transfect eukaryotic host cells with the expression vector obtained in step (1) and culture them;

[0026] (3) Separate and purify to obtain anti-human CD47 monoclonal antibody.

[0027] The present invention further provides antibody immunoconjugates, bispecific molecules, chimeric antigen receptors, or pharmaceutical compositions comprising the above-mentioned anti-human CD47 monoclonal antibody.

[0028] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of the anti-human CD47 monoclonal antibody, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0029] The present invention further provides the application of the aforementioned anti-human CD47 monoclonal antibody in the preparation of anti-tumor drugs or drugs for fibrotic diseases.

[0030] Preferably, the tumor is a hematologic malignancy or a solid tumor, including non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute medulloblastic leukemia (AML), ovarian cancer, fallopian tube cancer, colorectal cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, lung cancer, glioma, and glioblastoma.

[0031] Preferably, the fibrotic diseases include angina pectoris, osteoarthritis, pulmonary fibrosis, asthma, and bronchitis.

[0032] Beneficial effects:

[0033] The anti-human CD47 monoclonal antibody of the present invention has a good affinity for human CD47, and compared with existing anti-human CD47 monoclonal antibodies, it also has a stronger tumor cell phagocytic effect and a better CD47-SIRPα blocking activity. Attached Figure Description

[0034] Figure 1 To capture the binding ability of antibodies to human CD47 protein using ELISA;

[0035] Figure 2 To capture the binding ability of antibodies to cynomolgus monkey CD47 protein by ELISA;

[0036] Figure 3 To evaluate the binding of antibodies to 293F cells overexpressing human CD47 by flow cytometry;

[0037] Figure 4Ligand binding blockade ELISA;

[0038] Figure 5 The reference antibody was used to block the ELISA. Detailed Implementation

[0039] the term

[0040] "Binding with CD47" or "binding with CD47" refers to the ability to interact with human CD47.

[0041] "Antigen binding site" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment in this article.

[0042] "Monoclonal antibody" refers to a preparation of an antibody molecule consisting of a single amino acid, not to the method of its production. Monoclonal antibodies or their antigen-binding fragments 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 techniques known in the art.

[0043] "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 stated, "binding affinity" herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by methods commonly known in the art, including those known in the prior art and those described herein.

[0044] The term "competition" in the context of competing antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) for the same epitope refers to competition between antigen-binding proteins, as determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or its immunologically functional fragment) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., CD47 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein is competing with another. Competitive inhibition is measured by measuring the amount of a label bound to a solid surface or cell in the presence of the antigen-binding protein being tested. Typically, the antigen-binding protein being tested is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, the two epitopes spatially preventing each other from binding.

[0045] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and publicly available in the prior art, such as the Cold Spring Harbor Guide to Antibody Laboratory Techniques. For example, mice can be immunized with human CD47 or fragments thereof, and the resulting antibodies can be refolded, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

[0046] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing a CD47 antibody or its antigen-binding fragment, to a patient who has symptoms of one or more diseases. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also called the "therapeutic effective dose") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms.

[0047] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0048] "Pharmaceutical composition" refers to a mixture containing one or more CD47 antibodies or their antigen-binding fragments described herein, along with other pharmaceutical components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0049] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Reagents not specifically named are commercially available, conventional reagents.

[0050] Example 1: Obtaining a mouse monoclonal antibody specifically against CD47 using fusion hybridoma technology.

[0051] 1.1 Animal Immunization

[0052] Mice were immunized according to the method generally described in the literature (E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998). Recombinant human CD47 protein (Sino Biological Inc., cat#12283-H02H) was used as the immunogen.

[0053] To enhance the immune response, Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma, St. Louis, Mo., USA) were used for the initial immunization and booster immunizations, respectively. In short, the adjuvant-antigen mixture was prepared by first gently mixing the adjuvant in a vial using a vortex method. The required amount of adjuvant was then transferred from the vial to a sterile 1.5 mL microcentrifuge tube. The antigen was prepared in PBS or physiological saline at a concentration of 0.5–1.0 mg / mL. The calculated amount of antigen was added to the microcentrifuge tube along with the adjuvant, and the mixture was gently stirred for 2 minutes, repeating the emulsification process to form a water-in-oil solution. The adjuvant-antigen solution was then aspirated into an appropriate syringe and injected into the animal. Each animal was immunized, followed by 2–3 booster immunizations based on the antiserum titer. Animals with good titers were given a final immunization via intraperitoneal injection before fusion.

[0054] 1.2 Hybridoma fusion and screening

[0055] Prior to cell fusion, mouse myeloma cells (SP2 / 0-Ag14, ATCC#CRL-1581) were in logarithmic growth phase. Following the method described by Kohler G and Milstein C in “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, 256:495-497 (1975), immunized mice were sacrificed, their spleens were harvested in a sterile environment, and fused with myeloma cells.

[0056] The fused "hybrid cells" were then aliquoted into 96-well cell culture media containing HAT. Viable hybridoma cells were typically observed growing under a microscope 7-10 days after fusion. Two weeks after cell seeding, the culture supernatant from each well was collected, and hybridomas were screened using ELISA with recombinant human CD47-his protein antigen. In short, the ELISA plate was coated overnight at 4°C with human CD47-his protein (ACRO biosystems, cat#CD7-H5227, 2.0 μg / ml in PBS). The plate was washed four times with PBST and then blocked with blocking buffer (PBST containing 5% skim milk). Diluted mouse immune serum (for determining mouse serum titer) or hybridoma supernatant was added to each well, and the plate was incubated at 37°C for 40 minutes. The plates were washed four times with PBST, and the absorbance at 450 nm was measured using horseradish peroxidase-goat anti-mouse IgG (Jackson Immuno Research, CAT #115-036-071). Positive hybridomas secreting antibodies binding to human CD47-his were then selected and transferred to 24-well plates.

[0057] Hybridoma clones producing antibodies with high specificity for binding to human CD47 and CD47 / SIRPα ligand blocking activity were subcloned using limiting dilution to ensure cell line clonality, and then purified. Antibodies from hybridoma clones exhibiting high specificity for binding to cell surface CD47 FACS and CD47 / SIRPα ligand blocking activity were subcloned to ensure cell line clonality, and then the monoclonal antibodies were purified.

[0058] Example 2: Determination of affinity of mouse anti-CD47 monoclonal antibody using BIACORE surface plasmon resonance technology

[0059] The anti-CD47 mouse monoclonal antibodies (mAbs) generated from the hybridoma clone in Example 1 were characterized for affinity kinetics using a Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

[0060] In short, goat anti-mouse IgG was covalently linked to a CM5 chip (carboxymethyl dextran-coated chip) via a primary amine using a standard amine conjugation kit provided by Biacore. Unreacted portions of the biosensor surface were blocked with ethanolamine. The mouse anti-CD47 antibody generated in Example 1 was purified, and reference antibodies CC-9000 (Celgene) and Hu5F9-G4 (Forty Seven) were flowed onto the chip at a concentration of 66.7 nM and a flow rate of 10 μL / min. 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 (provided by Biacore) was flowed onto the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were observed for 2 minutes, and dissociation kinetics for 10 minutes. The Langmuir binding model curve with a binding-to-dissociation ratio of 1:1 was fitted using BIA evaluation software.

[0061] Where k a ,k d and K D The values ​​are shown in Table 1.

[0062] Table 1. Kinetic parameters of mouse anti-CD47 monoclonal antibody binding to human or cynomolgus monkey CD47 as determined by Biacore.

[0063]

[0064] The binding K of the monoclonal antibody 1A5 of the present invention to human CD47 D The value is similar to that of the reference antibody, indicating that it has a high affinity for human CD47.

[0065] Example 3: Study on the binding activity of mouse anti-CD47 monoclonal antibody

[0066] The mouse anti-CD47 monoclonal antibodies (mAbs) generated from the hybridoma clone in Example 1 were further tested for their binding activity using the following method.

[0067] 3.1 Antibody binding ability was determined based on capture ELISA.

[0068] 96-well ELISA plates were coated with 100 μl / well of goat anti-mouse IgG Fcγ fragment-specific antibody (Jackson Immunological Research, #115-006-071, 2 μg / ml) prepared in PBS and incubated overnight at 4°C. The plates were washed four times with elution buffer (PBS + 0.05% v / v Tween-20, PBST), then blocked at 37°C for 2 hours with 200 μl / well of 5% w / v skim milk PBST buffer. The plates were washed again and incubated at 37°C for 40 minutes with 100 μl / well of CD47 mouse monoclonal antibody at different concentrations, followed by four more washes. The ELISA plate containing the CD47 capture antibody was incubated with biotin-labeled human CD47 protein (ACRO Biosystems, cat#CD7-H5227) or monkey CYNO-CD47-HIS-BIO (ACROBiosystems, cat No.#CD7-C52H1) (60 nM, 2.5% skim milk powder PBST buffer, 100 μl / well) at 37°C for 40 minutes. The plate was then washed four times and incubated with streptavidin-conjugated horseradish peroxidase (1:10000 dilution with PBST, Jackson Immuno Research, #016-030-084, 100 μl / well) at 37°C for 40 minutes. After the final wash, the plate was incubated with 100 μl / well of ELISA substrate TMB (Innoreagents, #TMB-S-002). The reaction was terminated within 15 minutes by adding 50 μl / well of 1M H₂SO₄ at 25 °C, and the absorbance at 450 nm was measured. The results are shown in the figure. Figure 1-2 And Table 2.

[0069] Figure 1 and Figure 2 The results showed that the antibody 1A5 of the present invention has good binding ability to both human and cynomolgus monkey CD47 protein.

[0070] 3.2 Flow cytometry (FACS) was used to determine the binding of CD47 monoclonal antibody to the 293F cell line overexpressing human CD47.

[0071] Stable cell line 293F, overexpressing human CD47, was collected from cell culture flasks, washed twice, and resuspended in PBS phosphate-buffered saline (FACS buffer) containing 2% v / v fetal bovine serum. 2 × 10⁶ cells were then placed in each well of a 96-well plate. 5Cells were incubated on ice for 40 minutes with FACS buffer containing different concentrations of CD47 antibody. Cells were washed three times with FACS buffer, and 100 μL / 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 incubation at 4°C in the dark for 40 minutes, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS CantoII-HTS instrument. Data were analyzed using Graphpad Prism software to determine the EC50 of antibody-bound cells. 50 The concentration value, i.e., the antibody concentration corresponding to 50% of the maximum fluorescence binding signal when CD47 antibody reaches cells overexpressing CD47, is shown in the figure below. Figure 3 And Table 2.

[0072] Figure 3 The results showed that the antibody 1A5 of the present invention had a stronger binding ability to 293F cells overexpressing human CD47.

[0073] Table 2. Binding activity of mouse anti-CD47 antibody

[0074]

[0075] Example 4: Competitive functional blocking ability of mouse anti-CD47 monoclonal antibody against CD47-SIRPα interaction

[0076] The ability of the antibody to block CD47-SIRPα interaction was detected using a competitive ELISA.

[0077] 4.1 Ligand Blocking ELISA

[0078] The blocking ability of the anti-CD47 antibody of this invention against CD47-SIRPα interaction was detected using a competitive ELISA. Briefly, human SIRPα-his protein (Sino Biological Inc., cat#11612-H08H) was added at 200 ng / well to a 96-well microplate and incubated overnight at 4°C. The next day, the plates were washed with washing buffer (PBS + 0.05% Tween-20, PBST) and then blocked with PBST containing 5% w / v skim milk powder at 37°C for 2 hours. The plates were then washed again with washing buffer.

[0079] Dilute the CD47 antibody or reference antibody with human CD47-biotin (ACRO biosystems, cat#CD7-H5227) solution (antibody starts at 66.7 nM and is serially diluted 4-fold), incubate at room temperature for 40 minutes, then add the antibody / CD47-biotin mixture to a SIRPα-coated plate. After incubation at 37°C for 40 minutes, wash the plate four times with washing buffer. Then add streptavidin-bound HRP and incubate at 37°C for 40 minutes to detect the binding of biotin-labeled human CD47 to SIRPα on the substrate. Wash the plate again with washing buffer. Finally, add TMB and terminate the reaction with 1M H2SO4, measuring the absorbance at 450 nm. Analyze the data using GraphpadPrism software to obtain the IC50 values. 50 Values, see details in the results. Figure 4 And Table 3.

[0080] 4.2 Reference antibody blocking ELISA

[0081] The ability of the anti-CD47 antibody of this invention to block the binding of the reference antibody (Hu5F9-G4, Forty Seven) to human CD47 protein was determined using a competitive ELISA method. In short, the CD47 reference antibody was coated onto a 96-well microplate with PBS containing 1 μg / mL and incubated overnight at 4°C. The next day, the plates were washed with washing buffer and blocked with PBST containing 5% skim milk powder at 37°C for 2 hours. For blocking, biotin-labeled human CD47 (ACRO biosystems, cat#CD7-H5227) (10 nM, PBST containing 2.5% skim milk powder) was mixed with the antibody (1.2 pM–100 nM, serially diluted 5 times), and then incubated at 25°C for 40 minutes. After washing, the antibody / human CD47-biotin mixture (100 μl / well) was added to the Hu5F9-G4 plate and incubated at 37°C for 40 minutes. The plate was washed again with washing buffer, and 100 μl of SA-HRP was added to each well. The plate was then incubated at 37°C for 40 minutes to detect the biotin-labeled human CD47 bound to the plate. A final wash with washing buffer was performed. TMB was added, and the reaction was terminated with 1 M H₂SO₄. The absorbance at 450 nm was measured. The data were analyzed using Graphpad Prism software to determine the IC₂ value. 50 Values, see details in the results. Figure 5 And Table 3.

[0082] As shown in Table 3, the antibody of the present invention can block human CD47-SIRPα interaction, and also indicates that the antibody of the present invention has an antigen-binding epitope similar to that of the reference antibody. Compared with the reference antibody, antibody 1A5 of the present invention has better CD47-SIRPα blocking activity.

[0083] Table 3. Ability of anti-CD47 antibodies to block the interaction between CD47-SIRPα and CD47 reference antibody

[0084]

[0085]

[0086] Example 5: Mouse anti-CD47 monoclonal antibody induces macrophages to phagocytose tumor cells.

[0087] In vitro cell experiments were used to detect the bioactivity of anti-CD47 antibody-induced macrophage phagocytosis of tumor cells. Human peripheral blood mononuclear cells (PBMCs) were extracted from fresh human blood using Ficoll (GE Healthcare, 17-1440-02). To differentiate PBMCs into monocyte-derived macrophages (MDMs), the monocytes were seeded in the presence of human M-CSF with RPMI 1640 + 10% FBS + 1% penicillin-streptomycin (Peprotech, 300-25-100). On days 2 and 4, the cells were washed and replaced with fresh culture medium containing cytokines. On day 6, adherent cells were isolated and washed twice with PBS.

[0088] MDMs were isolated from the plate and incubated overnight in 96-well plates. Jurkat cells were collected for CFSE (5(6)-carboxyfluorescein N-hydroxysuccinimide) (Sigma, 87444). Anti-CD47 monoclonal antibody was diluted accordingly. 100 μL of the CFSE-labeled Jurkat tumor cells and diluted CD47 monoclonal antibody mixture was added to the MDM and incubated at 37°C for 4 h. All cells were isolated and washed once with FACS buffer. Cells were stained with anti-human CD14 APC (eBioscience, 17-0149-42), and CD14+CFSE+ cells were detected by 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.

[0089] Table 4 shows that the antibody of the present invention can induce macrophages to phagocytose tumor cells, and its EC50... 50 The value was lower than two reference antibodies, indicating a stronger pro-tumor cell phagocytosis effect than the reference antibody.

[0090] Table 4. Ability of anti-CD47 antibody to induce macrophages to phagocytose tumor cells

[0091]

[0092] Example 6: DNA cloning and sequencing, sequence analysis of anti-CD47 antibody

[0093] Total RNA was extracted from the hybridoma cells of Example 1 using Trizol reagent (Invitrogen, catalog #15596-018).

[0094] The process is briefly described below: Centrifuge and collect 5 × 10⁵ samples. 6 Cells were transferred to 1.5 ml centrifuge tubes, and the supernatant was aspirated. 1 ml of Trizol reagent was added, and the tubes were repeatedly pipetted several times before incubating at 25°C for 5 minutes to lyse the cells. Immediately afterwards, 0.2 ml of chloroform solution was added to each tube, and the tubes were vigorously vortexed for 15 seconds, then incubated at room temperature for 3 minutes. The tubes were then centrifuged at 12000 g for 10 minutes at 4°C. The upper aqueous phase was transferred to a new 1.5 ml centrifuge tube, and 0.4 ml of isopropanol was added to precipitate RNA from the aqueous phase. The EP tubes were manually mixed and incubated at 25°C for 10 minutes, then centrifuged at 12000 g for 10 minutes at 4°C, and the supernatant was discarded. 1 ml of 75% ethanol was added, and the tubes were centrifuged again at 7500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The RNA precipitate at the bottom of the tube was allowed to dry at room temperature for 10 minutes, and then 30 to 50 μL of sterile DEPC-treated water was added to dissolve the RNA sample.

[0095] Next, the total RNA was converted into cDNA using Taraka's reverse transcription cDNA kit (catalog #6110A). The experimental system was prepared as follows: 5 μl total RNA + 0.5 μl Oligo(dT) + 8.5 μl RNase-free water (total 14 μl). The mixture was first pre-denatured at 65°C for 5 min, then placed on ice for 2 min. Next, 4 μl 5× buffer + 1 μl dNTP mixture + 0.5 μl RNase inhibitor + 1 μl reverse transcriptase (total 20.5 μl) were added, mixed well, and incubated at 40°C for 50 min, followed by incubation at 70°C for 10 min to complete cDNA synthesis. The cDNA was further poly-G added to the 3' end. The reaction system was prepared as follows: 5 μl cDNA sample + 33.5 μl ddH2O + 5 μl 10×TdT buffer + 5 μl CoCl2 + 1 μl dGTP + 0.5 μl terminal deoxynucleotidyl transferase (total volume 50 μl). The mixture was incubated at 37 °C for 30 min, and then at 70 °C for 10 min to complete the poly-G tailing.

[0096] Further, the tailed cDNA was used as a template for gene amplification of the antibody variable region. For amplifying the antibody heavy chain variable region sequence, the PCR reaction system was prepared as follows: 5 μl 10×Taq enzyme buffer + 0.5 μl universal poly C primer (forward primer) + 0.5 μl mouse IgG1 reverse primer + 1 μl dNTP + 1 μl Taq polymerase + 1 μl cDNA + 41 μl ddH2O. For amplifying the antibody light chain variable region sequence, the PCR reaction system was prepared as follows: 5 μl 10×Taq enzyme buffer + 0.5 μl universal poly C primer (forward primer) + 0.5 μl mouse IgG kappa chain reverse primer + 1 μl dNTP + 1 μl Taq polymerase + 1 μl cDNA + 41 μl ddH2O. The temperature cycling for PCR amplification of the antibody heavy and light chain variable regions was as follows (steps 2 to 4 were repeated 25 times):

[0097] 1) Pre-denaturation at 95℃ for 5 minutes;

[0098] 2) Denaturation at 95℃ for 20 seconds;

[0099] 3) Anneal at 56℃ for 20 seconds;

[0100] 4) Extend to 72℃ for 30 seconds;

[0101] 5) Store at 25℃ for 60 minutes.

[0102] PCR products were analyzed by 1% agarose gel electrophoresis, and corresponding DNA bands (VH approximately 600 bp, VK approximately 500 bp) were excised. DNA extraction was performed using the QIAquick Gel DNA Recovery Kit (catalog #28704). The procedure is briefly described as follows: The gel was weighed, and 3 volumes of QG buffer were added. The gel was then incubated at 50°C for 10 minutes until completely dissolved. 1 volume of isopropanol was added and mixed thoroughly. The sample was then transferred to a QIA purification column and centrifuged at 13000 rpm for 1 minute. 750 μl of PE buffer was added to the column, and the column was centrifuged again at 13000 rpm for 1 minute. A second centrifugation at 13000 rpm was performed to remove any remaining liquid. 30 μl of water was added, and the column was eluted by centrifugation at 13000 rpm for 1 minute to obtain the prepared DNA sample. The purified PCR product was sequenced to obtain the variable region sequence of the antibody.

[0103] The sequence information of the clones of this invention is shown in Table 5.

[0104] Table 5. Sequence information of anti-CD47 antibody

[0105]

[0106] NA: Nucleotide; AA: Amino acid. sequence list <110> Bio-Sens Biotechnology (Nanjing) Co., Ltd. <120> An anti-human CD47 monoclonal antibody, its preparation method and application <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <400> 1 ggcttcaaca ttaaagacta ctat 24 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 attggtcctg aaaatggtga tact 24 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <400> 4 Ile Gly Pro Glu Asn Gly Asp Thr 1 5 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 aatgcggggg gacgaggggg gtttccttac 30 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <400> 6 Asn Ala Gly Gly Arg Gly Gly Phe Pro Tyr 1 5 10 <210> 7 <211> 351 <212> DNA <213> Artificial Sequence <400> 7 gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcacgttg 60 tcctgcacag cttctggctt caacattaaa gactactata tgaattgggt gaagcagagg 120 cctgaacagg gcctggagtg gattggatgg attggtcctg aaaatggtga tactgaaaat 180 gacccgaggt tccagggcaa ggccactatg actgcagaca catcctccaa cacagccttc 240 ctgcaactca gcagcctgac atctgaggac actgccttct attactgtaa tgcgggggga 300 cgaggggggt ttccttactg gggccagggg actctggtca ctgtctctac a 351 <210> 8 <211> 117 <212> PRT <213> 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 Thr Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Gly Pro Glu Asn Gly Asp Thr Glu Asn Asp Pro Arg Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Asn Ala Gly Gly Arg Gly Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Thr 115 <210> 9 <211> 1323 <212> DNA <213> Artificial Sequence <400> 9 gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcacgttg 60 tcctgcacag cttctggctt caacattaaa gactactata tgaattgggt gaagcagagg 120 cctgaacagg gcctggagtg gattggatgg attggtcctg aaaatggtga tactgaaaat 180 gacccgaggt tccagggcaa ggccactatg actgcagaca catcctccaa cacagccttc 240 ctgcaactca gcagcctgac atctgaggac actgccttct attactgtaa tgcgggggga 300 cgaggggggt ttccttactg gggccagggg actctggtca ctgtctctac 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 <400> 10 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Gly Pro Glu Asn Gly Asp Thr Glu Asn Asp Pro Arg Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Asn Ala Gly Gly Arg Gly Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Thr 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 <400> 11 agatctagtc agaacattgt tcatattaat ggaaaaacct atttagaa 48 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <400> 12 Arg Ser Ser Gln Asn Ile Val His Ile Asn Gly Lys Thr Tyr Leu Glu 1 5 10 15 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 agagtttcca accgattttc t 21 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <400> 14 Arg Val Ser Asn Arg Phe Ser 1 5 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <400> 15 tttcaaggtt cacatgttcc tcggacg 27 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Phe Gln Gly Ser His Val Pro Arg Thr 1 5 <210> 17 <211> 336 <212> DNA <213> Artificial Sequence <400> 17 gatattttgt tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 gatattttgt tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 ttctcttgca gatctagtca gaacattgtt catattaatg gaaaaaccta tttagaatgg 120 ttctcttgca gatctagtca gaacattgtt catattaatg gaaaaaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acagagtttc caaccgattt 180 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acagagtttc caaccgattt 180 tctggggtcc cagacagatt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 tctggggtcc cagacagatt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tattactgtt ttcaaggttc acatgttcct 300 agcagagtgg aggctgagga tctgggagtt tattactgtt ttcaaggttc acatgttcct 300 cggacgttcg gtggaggcac caagttggaa atcaaa 336 cggacgttcg gtggaggcac caagttggaa atcaaa 336 <210> 18<210> 18 <211> 112<211> 112 <212> PRT<212> PRT <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 18<400> 18 Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 1 5 10 15 Asp Gln Ala Ser Phe Ser Cys Arg Ser Ser Gln Asn Ile Val His Ile Asp Gln Ala Ser Phe Ser Cys Arg Ser Ser Gln Asn Ile Val His Ile 20 25 30 20 25 30 Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 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 <400> 19 gatattttgt tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 ttctcttgca gatctagtca gaacattgtt catattaatg gaaaaaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acagagtttc caaccgattt 180 tctggggtcc cagacagatt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tattactgtt ttcaaggttc acatgttcct 300 cggacgttcg gtggaggcac caagttggaa 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 <400> 20 Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Phe Ser Cys Arg Ser Ser Gln Asn Ile Val His Ile 20 25 30 Asn Gly Lys Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Arg 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 Lys 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

1. An anti-human CD47 monoclonal antibody, 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 CDR-H1 is shown as SEQ ID NO: 2; The amino acid sequence of CDR-H2 is shown as SEQ ID NO: 4; The amino acid sequence of CDR-H3 is shown as SEQ ID NO: 6; The amino acid sequence of CDR-L1 is shown as SEQ ID NO: 12; The amino acid sequence of CDR-L2 is shown as SEQ ID NO: 14; The amino acid sequence of CDR-L3 is shown as SEQ ID NO:

16.

2. The anti-human CD47 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 8; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

18.

3. The anti-human CD47 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown as SEQ ID NO: 10; and the amino acid sequence of the light chain is shown as SEQ ID NO:

20.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human CD47 monoclonal antibody according to any one of claims 1-3.

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

6. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule according to claim 4 or 5.

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

8. The method for preparing the anti-human CD47 monoclonal antibody according to any one of claims 1 to 3, characterized in that, Comprising the following steps: Preparation of an expression vector comprising a nucleic acid molecule expressing the anti-human CD47 monoclonal antibody according to any one of claims 1-3; Transfection of the obtained expression vector into a eukaryotic host cell and culture; Isolation and purification to obtain the anti-human CD47 monoclonal antibody.

9. A pharmaceutical composition comprising the anti-human CD47 monoclonal antibody according to any one of claims 1-3.

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