CN115746133A - Anti-human CD47 monoclonal antibody and application thereof - Google Patents

Anti-human CD47 monoclonal antibody and application thereof Download PDF

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CN115746133A
CN115746133A CN202111038072.1A CN202111038072A CN115746133A CN 115746133 A CN115746133 A CN 115746133A CN 202111038072 A CN202111038072 A CN 202111038072A CN 115746133 A CN115746133 A CN 115746133A
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陈明久
马志清
彭则羽
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Boaoxin Biotechnology Nanjing Co ltd
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Abstract

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

Description

Anti-human CD47 monoclonal antibody and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to an anti-human CD47 monoclonal antibody, a composition containing the antibody and related application.
Background
CD47 belongs to the immunoglobulin superfamily, is widely expressed on the surface of cells, can be combined with a plurality of proteins, including integrin, thrombospondin-1 (TSP-1), and is involved in a plurality of physiological and pathological processes, and mediates a series of reactions such as apoptosis, proliferation, immunity and the like. CD47 is highly expressed on various solid tumor cells and hematological malignancies, and its expression level is positively correlated with disease progression. This broad expression suggests that CD47, or one of the important targets in the field of tumor therapy, could be used for the treatment of multiple types of cancer.
SIRP α is a protein belonging to the SIRP receptor family. The Chinese language of SIRP is called Signal regulatory proteins (SIRP), and is a receptor family encoded by a gene cluster located at the position of 20p13 of chromosome, and the family includes five members of SIRP alpha, SIRP beta 1, SIRP gamma, SIRP beta 2 and SIRP delta. SIRP α is not widely expressed in humans, but is mainly expressed on the cell surface of myeloid cells (monocytes, macrophages, granulocytes, and myeloid DC cells, etc.), and also expressed in neuronal cells of the nervous system. The CD47 can inhibit the phagocytosis of tumor cells by macrophages after being combined with SIRP alpha.
In recent years, many pharmaceutical companies have focused on the development of CD47 target drugs. However, the anti-human CD47 monoclonal antibodies developed so far have the problems of low affinity or unclear target of action such as epitope. Therefore, there is a need for a therapeutic candidate CD47 antibody that induces macrophages to phagocytose cancer cells and has a high affinity.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides an anti-human CD47 monoclonal antibody and application thereof, wherein the monoclonal antibody has higher affinity with human CD47 and stronger tumor cell phagocytosis promotion effect.
The invention provides an anti-human CD47 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3;
the amino acid sequence of the CDR-H1 is shown as SEQ ID NO:2 is shown in the specification;
the amino acid sequence of the CDR-H2 is shown as SEQ ID NO:4 is shown in the specification;
the amino acid sequence of the CDR-H3 is shown as SEQ ID NO:6 is shown in the specification;
the amino acid sequence of the CDR-L1 is shown as SEQ ID NO:12 is shown in the specification;
the amino acid sequence of the CDR-L2 is shown as SEQ ID NO:14 is shown in the figure;
the amino acid sequence of the CDR-L3 is shown as SEQ ID NO: shown at 16.
Preferably, the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO:8 is shown in the specification; the variable region amino acid sequence of the light chain is shown as SEQ ID NO:18, respectively.
Preferably, the heavy chain amino acid sequence is as set forth in SEQ ID NO:10 is shown in the figure; the light chain amino acid sequence is shown as SEQ ID NO: shown at 20.
Preferably, both the heavy and light chains also comprise a constant region that is a constant region of murine or human IgG, preferably IgG 4.
The invention further provides a nucleotide molecule for encoding the anti-human CD47 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 anti-human CD47 monoclonal antibody, which comprises the following steps:
(1) Preparing an expression vector containing a nucleotide molecule for expressing the anti-human CD47 monoclonal antibody;
(2) Transfecting eukaryotic host cells by using the expression vector obtained in the step (1) and culturing;
(3) Separating and purifying to obtain the anti-human CD47 monoclonal antibody.
The invention further provides an antibody immune coupling conjugate, a bispecific molecule, a chimeric antigen receptor or a pharmaceutical composition comprising the anti-human CD47 monoclonal antibody.
Further, 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.
The invention further provides the application of the anti-human CD47 monoclonal antibody in preparing anti-tumor drugs or drugs for treating fibrotic diseases.
Preferably, the tumor is a hematological or solid tumor, including non-hodgkin's lymphoma (NHL), acute Lymphoblastic Leukemia (ALL), acute Myeloblastic Leukemia (AML), ovarian cancer, fallopian tube cancer, colorectal cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, lung cancer, glioma, and glioblastoma.
Preferably, the fibrotic disease includes angina, osteoarthritis, pulmonary fibrosis, asthma and bronchitis.
Has the advantages that:
the anti-human CD47 monoclonal antibody has higher affinity with human CD47, has stronger tumor cell phagocytosis promotion effect compared with the existing anti-human CD47 monoclonal antibody, can induce macrophages to phagocytose tumor cells, and has EC (specific cytokine inhibitory factor) 50 Values lower than the two reference antibodies showed a stronger tumor cell phagocytosis than the reference 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;
figure 5 is a reference antibody blocking ELISA.
Detailed Description
Term(s) for
"bind to CD47" or "bind to CD47" refers to the ability to interact with human CD47.
An "antigen binding site" refers to a three-dimensional spatial site that is not contiguous on an antigen and is recognized by an antibody or antigen binding fragment herein.
"monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and not to the method of production thereof. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or a combination of such or other techniques known in the art.
"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by conventional methods known in the art, including those known in the art and described herein.
The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between antigen binding proteins as determined by the following assay: in such assays, the antigen binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., CD47 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be detected is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds a contiguous epitope sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder binding from occurring.
Methods for producing and purifying antibodies and antigen-binding fragments are well known and disclosed in the art, such as the Cold spring harbor antibody Experimental guidelines. For example, mice can be immunized with human CD47 or a fragment thereof, and the resulting antibodies can be renatured, purified, and 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 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 the symptoms of any particular disease (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease state, age, and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
By "pharmaceutical composition" is meant a mixture comprising one or more of the CD47 antibodies or antigen-binding fragments thereof described herein and other pharmaceutical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and exert biological activity.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. 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 mouse monoclonal antibody specific against CD47 by the hybridoma technique
1.1 animal immunization
Mice were immunized according to methods commonly used in the literature (E Harlow, D.Lane, antibody: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1998). Recombinant human CD47 protein (Sino biological inc., cat # 12283-H02H) was used as immunogen.
To increase the immune response, freund's complete adjuvant and freund's incomplete adjuvant (Sigma, st. Louis, mo., USA) were used for the prime and boost, respectively. Briefly, the adjuvant-antigen mixture is prepared by first gently mixing the adjuvant in a vial using a vortex method. The desired amount of adjuvant was removed from the vial and placed in an autoclaved 1.5mL microcentrifuge tube. The antigen is prepared in PBS or physiological saline at a concentration of 0.5-1.0 mg/ml. Adding calculated amount of antigen and adjuvant into a microcentrifuge tube, gently stirring for 2 min, and repeatedly emulsifying and uniformly mixing to form water-in-oil solution. The adjuvant-antigen solution is then drawn into an appropriate syringe for injection into the animal. Each animal was immunized and then boosted 2-3 times according to the antiserum titer. Animals with good titers were terminally immunized by intraperitoneal injection prior to fusion.
1.2 hybridoma fusion and screening
Prior to cell fusion, mouse myeloma cells (SP 2/0-Ag14, ATCC # CRL-1581) were cultured in logarithmic growth phase. Following sacrifice of the mouse sterile environment, spleens were harvested and fused with myeloma cells according to the method described by Kohler G and Milstein C, in Continuous cultures of fused cells and characterization of predefined specificity, "Nature, 256.
The fused "hybrid cells" were then dispensed into 96-well cell plate media containing HAT. The growth of viable hybridoma cells is typically observed microscopically after 7-10 days post-fusion. Two weeks after cell plating, culture supernatants from each well were collected and screened for hybridomas using recombinant human CD47-his protein antigen by ELISA. Briefly, ELISA plates were coated with human CD47-his protein (ACRO biosystems, cat # CD7-H5227, 2.0. Mu.g/ml in PBS) overnight at 4 ℃. The plate was washed 4 times with PBST and blocked with blocking buffer (5% nonfat dry milk in PBST). Diluted mouse immune serum (for determination of mouse serum titers) or hybridoma supernatants were added to each well and incubated for 40 minutes at 37 ℃. The plate was washed 4 times with PBST, detected with horseradish peroxidase-goat anti-mouse IgG (Jackson Immuno research, cat # 115-036-071) and the absorbance of each well at 450nm determined. Positive hybridomas secreting antibodies that bind to human CD47-his were then selected and transferred to 24-well plates.
Hybridoma clones producing antibodies that bind human CD47 with high specificity and have CD 47/sirpa ligand blocking activity were subcloned by limiting dilution to ensure clonality of the cell line, and then purified. The antibodies produced by the hybridoma clones with high specific cell surface CD47 FACS binding and CD 47/sirpa ligand blocking activity were subcloned to ensure clonality of the cell lines, and then the monoclonal antibodies were purified.
Example 2 determination of the affinity of mouse anti-CD 47 monoclonal antibody Using BIACORE surface plasmon resonance
anti-CD 47 mouse monoclonal antibodies (mAbs) produced by the hybridoma clones of example 1 were subjected to an affinity kinetic characterization assay by the Biacore T200 system (GE healthcare, pittsburgh, pa., USA).
Briefly, goat anti-mouse IgG was covalently attached to a CM5 chip (carboxymethyldextran coated chip) via primary amines using a standard amine coupling kit supplied by Biacore. The unreacted portion of the biosensor surface is blocked with ethanolamine. The mouse anti-CD 47 antibody produced in example 1 was purified, and the reference antibodies CC-9000 (Celgene) and Hu5F9-G4 (Forty Seven) were flowed onto the chip at a concentration of 66.7nM and a flow rate of 10. Mu.L/min. Then, recombinant human CD47-his protein (Acro biosystems, cat # CD7-H5227, MW:15.6 kDa) or cynomolgus monkey CD47-his protein (Acro biosystems, cat # CD7-C52H1, MW:15.8 kDa) in HBS EP buffer (supplied by Biacore) was flowed onto the chip at a flow rate of 30. Mu.L/min. Antigen-antibody binding kinetics were observed for 2 minutes and dissociation kinetics for 10 minutes. A Langmuir binding model curve with binding and dissociation of 1 was fitted using BIA evaluation software.
Wherein k is a ,k d And K D The values of (A) are shown in Table 1.
Table 1.Biacore determination of kinetic parameters of mouse anti-CD 47 monoclonal antibody binding to human or cynomolgus monkey CD47
Figure BDA0003248039060000061
Binding K of monoclonal antibody 1H10 of the invention to human CD47 D Values were similar to the reference antibody, indicating high affinity for human CD47.
Example 3 binding Activity study of mouse anti-CD 47 monoclonal antibody
Mouse anti-CD 47 monoclonal antibodies (mAbs) generated by the hybridoma clones of example 1 were further tested for binding activity using the following method.
3.1 determination of the binding Capture-based ELSIA
96-well ELISA plates were coated with goat anti-mouse IgG Fc gamma fragment specific antibody (Jackson immuno Research, #115-006-071, 100. Mu.l/well) at a final concentration of 2. Mu.g/ml in PBS and incubated overnight at 4 ℃. Elution buffer for ELISA plates (PBS +0.05% v/v Tween-2)0,PBST) was washed 4 times, and then blocked at 37 ℃ for 2 hours by adding 200. Mu.l/well of 5% w/v skimmed milk powder PBST buffer. The plates were washed again and incubated with different concentrations of CD47 murine monoclonal antibody at 37 ℃ for 40 minutes at 100. Mu.l/well, followed by 4 additional washes. The microplate containing the captured CD47 antibody was incubated with biotin-labeled human CD47 protein (ACRO Biosystems, cat # CD 7-H5227) or monkey CYNO-CD47-HIS-BIO (ACRO Biosystems, cat No. # CD7-C52H 1) (60nM, 2.5% nonfat dry milk PBST buffer, 100. Mu.l/well) at 37 ℃ for 40 minutes, the plate was washed 4 times and incubated with streptavidin-conjugated horseradish peroxidase (diluted 1,10000 with PBST, # 016-030-084, 100. Mu.l/well) at 37 ℃ for 40 minutes. After final washing, the plates were incubated with 100. Mu.l/well of ELISA substrate TMB (innoreagens, # TMB-S-002). 50. Mu.l/well of 1M H over 15 min 2 SO 4 The reaction was terminated at 25 ℃ and the absorbance at 450nm was measured, the results of which are shown in FIGS. 1-2 and Table 2.
The results in FIGS. 1 and 2 show that the antibody 1H10 of the present invention has a better binding ability to both 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 with human CD47 overexpressed on the surface was collected from the cell culture flask, washed twice and resuspended in PBS phosphate buffer (FACS buffer) containing 2% v/v fetal bovine serum. 2 xl 0 per well in 96-well plates 5 Individual cells were incubated on ice for 40 minutes with FACS buffer containing different concentrations of CD47 antibody. Cells were washed 3 times with FACS buffer and R-Phoerythrin affinity purified F (ab') 2 Fragment goat anti-mouse IgG specific F (ab') 2 Fragments (FACS buffer diluted at 1:1000, jackson Immunoresearch, cat # 115-116-072) secondary antibody. After incubation at 4 ℃ for 40 min in the dark, the cells were washed 3 times and then resuspended in FACS buffer. Fluorescence measurements were performed using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software to derive EC for antibody-bound cells 50 Concentration value, i.e.the value at which the CD47 antibody reaches 50% of the maximum fluorescence binding signal with CD 47-overexpressing cellsThe corresponding antibody concentration values, the results of the assay are shown in FIG. 3 and Table 2.
The results in FIG. 3 show that the antibody 1H10 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
Figure BDA0003248039060000071
Figure BDA0003248039060000081
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, human SIRP α -his protein (Nano biological inc., cat # 11612-H08H) was added at 200 ng/well to a 96-well microplate and incubated overnight at 4 ℃. The following day, plates were washed with washing buffer (PBS +0.05% Tween-20, PBST) and blocked with PBST containing 5% w/v skimmed milk powder for 2 hours at 37 ℃. The plates were then washed with wash buffer.
The CD47 antibody or reference antibody (antibody starting at 66.7nM, 4-fold serial 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 plates. After incubation at 37 ℃ for 40 min, the plates were washed 4 times with wash buffer. Streptavidin-conjugated HRP was then added and incubated at 37 ℃ for 40 minutes to detect binding of biotin-labeled human CD47 to the bottom plate sirpa. The plate was washed with wash buffer. Finally, TMB was added, using 1M H 2 SO 4 The reaction was terminated and the absorbance at 450nm was measured. Data were analyzed using Graphpad Prism software to generate IC 50 Value, concrete nodeThe results are shown in FIG. 4 and Table 3.
4.2 reference antibody blocking ELISA
The ability of the anti-CD 47 antibody of the present invention to block the binding of a reference antibody (Hu 5F9-G4, forty Seven) -human CD47 protein was determined by a competitive ELISA method. Briefly, the CD47 reference antibody was coated onto 96-well microplates with 1. Mu.g/mL PBS and incubated overnight at 4 ℃. The next day, plates were washed with wash buffer and blocked with PBST containing 5% nonfat dry milk for 2 hours at 37 ℃. For blocking, biotin-labeled human CD47 (ACRO biosystems, cat # CD 7-H5227) (10 nM, PBST containing 2.5% skim milk powder) was mixed with antibody (1.2 pM-100nM, serial 5-fold dilutions) and incubated at 25 ℃ for 40 min. After washing the plates, the antibody/human CD 47-biotin mixture (100. Mu.l/well) was added to the Hu5F9-G4 plates and incubated at 37 ℃ for 40 min. The plate was washed again with washing buffer, 100. Mu.l/well of SA-HRP was added, followed by incubation at 37 ℃ for 40 minutes to detect biotin-labeled human CD47 bound to the plate. Final wash with wash buffer. Adding TMB, 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 1H10 of the invention has better CD47-SIRP alpha 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
Figure BDA0003248039060000091
Example 5 mouse anti-CD 47 monoclonal antibody induces macrophages to phagocytose tumor cells
In vitro cell experiment is adopted to detect the bioactivity of CD47 resisting antibody in inducing macrophage to phagocytize tumor cell. Human Peripheral Blood Mononuclear Cells (PBMC) were extracted from fresh human blood using Ficoll (GE Healthcare, 17-1440-02). To differentiate PBMC into monocyte-derived macrophages (MDM), monocytes were inoculated with RPMI 1640+10% FBS +1% penicillin-streptomycin (Peprotech, 300-25-100) in the presence of human M-CSF. On days 2 and 4, the cells were washed and replaced with fresh medium containing cytokines. On day 6, adherent cells were isolated and washed 2 times with PBS.
MDMs were isolated from plates and placed in 96-well plates overnight. Jurkat cells were harvested for CFSE (5 (6) -carboxyfluorescein N-hydroxysuccinimide ester) (Sigma, 87444) labeling. anti-CD 47 mab was diluted accordingly. To the MDM, 100uL of CFSE-labeled Jurkat tumor cells and diluted mixture of CD47 mab were added and incubated at 37 ℃ for 4h. All cells were isolated and washed once with FACS buffer. Cell staining was performed with anti-human CD14 APC (eBioscience, 17-0149-42), and CD14+ CFSE + cells were detected with flow cytometry (FACS). Data (percentage of CD14+ CFSE + cells) were analyzed using Graphpad Prism software to obtain EC 50 The values and measurement results are shown in Table 4.
Table 4 results show that the antibody of the present invention can induce macrophages to phagocytose tumor cells, the EC of which 50 Values lower than the two reference antibodies showed a stronger tumor cell phagocytosis than the reference antibody.
TABLE 4 ability of anti-CD 47 antibodies to induce phagocytosis of tumor cells by macrophages
Figure BDA0003248039060000092
Figure BDA0003248039060000101
Example 6 DNA cloning and sequencing, sequence analysis of anti-CD 47 antibody
Total RNA was extracted from the hybridoma cells of example 1 using Trizol reagent (Invitrogen, catalog # 15596-018).
The procedure is briefly described below, and 5X 10 are collected by centrifugation 6 The cells were transferred to a 1.5ml centrifuge tube and the supernatant was blotted dry. 1ml of Trizol reagent was added and the process repeatedAfter several strokes, the cells were left at 25 ℃ for 5 minutes for lysis. Subsequently, 0.2ml of chloroform solution was added to each tube, and the tube was vigorously shaken for 15 seconds and then left at room temperature for 3 minutes. Then, the tube was centrifuged at 12000g for 10 minutes at 4 ℃ and then removed, and the upper aqueous phase solution was aspirated into a new 1.5ml tube, and then 0.4ml of isopropanol was added for RNA precipitation from the aqueous phase. The EP tube was manually mixed and left at 25 ℃ for 10min, then centrifuged at 12000g at 4 ℃ for 10min, and the supernatant was discarded. 1ml of 75% ethanol was added thereto, and the mixture was centrifuged again at 7500rpm at 4 ℃ for 5min, and the supernatant was discarded. After the bottom RNA pellet was dried at room temperature for 10 minutes, 30 to 50ul of sterile DEPC-treated water was added to dissolve the RNA sample.
Next, the reverse transcription cDNA kit (catalog # 6110A) from Taraka was used to convert the total RNA into cDNA. The experimental system was prepared as follows: mu.l of total RNA + 0.5. Mu.l Oligo (dT) + 8.5. Mu.l RNase-free water (14. Mu.l total) were pre-denatured at 65 ℃ for 5min and then on ice for 2 min. Further, 4. Mu.l of 5 Xbuffer + 1. Mu.l of dNTP mix + 0.5. Mu.l of RNase inhibitor + 1. Mu.l of reverse transcriptase (20.5. Mu.l system in total) were added thereto, mixed well, incubated at 40 ℃ for 50 minutes, and then at 70 ℃ for 10 minutes to complete cDNA synthesis. The cDNA was further added with poly-G at the 3' end, and the reaction system was prepared as follows: mu.l of cDNA sample + 33.5. Mu.l of ddH 2 O + 5. Mu.l of 10 XTdT buffer + 5. Mu.l of CoCl 2 + 1. Mu.l dGTP + 0.5. Mu.l of terminal deoxynucleotidyl transferase (50 ul total volume), 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. The temperature cycles for PCR amplification of the antibody heavy and light chain variable regions were as follows (where steps 2 to 4 were 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 (VH about 600bp, VK about 500 bp) of corresponding sizes were excised, and DNA was extracted using QIAquick's gel DNA recovery kit (catalog # 28704). Briefly described as follows: the gel was weighed, 3 gel volumes of QG buffer were added, followed by incubation at 50 ℃ for 10 minutes until the gel was completely dissolved. After adding isopropanol of 1 gel volume and mixing, the sample was transferred to a QIA purification column and centrifuged at 13000rpm for 1 minute. 750. Mu.l of PE buffer was added to the column, followed by centrifugation at 13000rpm for 1 minute. And centrifuged again at 13000rpm to remove liquid residue from the column. 30. Mu.l of water was added and centrifuged at 13000rpm for 1 minute to elute, and the prepared DNA sample was obtained and the purified PCR product was sequenced to obtain the variable region sequence of the antibody.
Sequence information for clones of the invention is shown in Table 5.
TABLE 5 sequence information of anti-CD 47 antibodies
Figure BDA0003248039060000111
NA is nucleotide; AA is amino acid.
Sequence listing
<110> Boaoxin Biotechnology (Nanjing) Ltd
<120> anti-human CD47 monoclonal antibody and use thereof
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<170> SIPOSequenceListing 1.0
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ggcttcaaaa ttaaagacta ctat 24
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly Phe Lys Ile Lys Asp Tyr Tyr
1 5
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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attggtcctg aaaacggtga tact 24
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<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ile Gly Pro Glu Asn Gly Asp Thr
1 5
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aatgcggggg gacgaggggg gtttccttac 30
<210> 6
<211> 10
<212> PRT
<213> Artificial Sequence (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 (Artificial Sequence)
<400> 7
gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcacgttg 60
tcctgcacag cttctggctt caaaattaaa gactactata tgaactgggt gaaacagagg 120
cctgaacagg gcctggagtg gattggatgg attggtcctg aaaacggtga tactgagaat 180
ggcccgaagt tccaggacaa ggccactatg actgcaaata catcctccaa cacagccttc 240
ctgcaactca gcagcctgac acctgaagac actgccttct attactgtaa tgcgggggga 300
cgaggggggt ttccttactg gggccagggg actcaggtca ctgtctctac a 351
<210> 8
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Thr Leu Ser Cys Thr Ala Ser Gly Phe Lys 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 Gly Pro Lys Phe
50 55 60
Gln Asp Lys Ala Thr Met Thr Ala Asn Thr Ser Ser Asn Thr Ala Phe
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Pro 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 Gln
100 105 110
Val Thr Val Ser Thr
115
<210> 9
<211> 1323
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcacgttg 60
tcctgcacag cttctggctt caaaattaaa gactactata tgaactgggt gaaacagagg 120
cctgaacagg gcctggagtg gattggatgg attggtcctg aaaacggtga tactgagaat 180
ggcccgaagt tccaggacaa ggccactatg actgcaaata catcctccaa cacagccttc 240
ctgcaactca gcagcctgac acctgaagac actgccttct attactgtaa tgcgggggga 300
cgaggggggt ttccttactg gggccagggg actcaggtca 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 (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 Lys 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 Gly Pro Lys Phe
50 55 60
Gln Asp Lys Ala Thr Met Thr Ala Asn Thr Ser Ser Asn Thr Ala Phe
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Pro 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 Gln
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 (Artificial Sequence)
<400> 11
agatctagtc aggacattgt tcatattaat ggaaaaacct atttagaa 48
<210> 12
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Arg Ser Ser Gln Asp Ile Val His Ile Asn Gly Lys Thr Tyr Leu Glu
1 5 10 15
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
agagtttcca accgattttc t 21
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Arg Val Ser Asn Arg Phe Ser
1 5
<210> 15
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tttcaaggtt cacatgttcc tcggacg 27
<210> 16
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Phe Gln Gly Ser His Val Pro Arg Thr
1 5
<210> 17
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gatattttgt tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
ttctcttgca gatctagtca ggacattgtt catattaatg gaaaaaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acagagtttc caaccgattt 180
tctggggtcc cagacagatt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300
cggacgttcg ggggaggcac caagctggaa atcaaa 336
<210> 18
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
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 Asp 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
<210> 19
<211> 657
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gatattttgt tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
ttctcttgca gatctagtca ggacattgtt catattaatg gaaaaaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acagagtttc caaccgattt 180
tctggggtcc cagacagatt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300
cggacgttcg ggggaggcac caagctggaa atcaaacggg ctgatgctgc accaactgta 360
tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420
ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 480
caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 540
agcagcaccc tcacgttgac taaggacgag tatgaacgac ataacagcta tacctgtgag 600
gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacagggg agagtgt 657
<210> 20
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Asp 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 Asp 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 (10)

1. An anti-human CD47 monoclonal antibody, wherein said 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 as SEQ ID NO:2 is shown in the specification;
the amino acid sequence of the CDR-H2 is shown as SEQ ID NO:4 is shown in the specification;
the amino acid sequence of the CDR-H3 is shown as SEQ ID NO:6 is shown in the specification;
the amino acid sequence of the CDR-L1 is shown as SEQ ID NO:12 is shown in the specification;
the amino acid sequence of the CDR-L2 is shown as SEQ ID NO:14 is shown in the figure;
the amino acid sequence of the CDR-L3 is shown as SEQ ID NO: shown at 16.
2. The anti-human CD47 monoclonal antibody 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 anti-human CD47 monoclonal antibody 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 anti-human CD47 monoclonal antibody according to 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 producing an anti-human CD47 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 anti-human CD47 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 anti-human CD47 monoclonal antibody.
9. An antibody immunoconjugate, bispecific molecule, chimeric antigen receptor or pharmaceutical composition comprising the anti-human CD47 monoclonal antibody of any one of claims 1 to 3.
10. Use of the anti-human CD47 monoclonal antibody according to any one of claims 1 to 3 for the preparation of an antitumor drug or a drug for fibrotic diseases.
CN202111038072.1A 2021-09-06 2021-09-06 Anti-human CD47 monoclonal antibody and application thereof Pending CN115746133A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111038072.1A CN115746133A (en) 2021-09-06 2021-09-06 Anti-human CD47 monoclonal antibody and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111038072.1A CN115746133A (en) 2021-09-06 2021-09-06 Anti-human CD47 monoclonal antibody and application thereof

Publications (1)

Publication Number Publication Date
CN115746133A true CN115746133A (en) 2023-03-07

Family

ID=85332706

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111038072.1A Pending CN115746133A (en) 2021-09-06 2021-09-06 Anti-human CD47 monoclonal antibody and application thereof

Country Status (1)

Country Link
CN (1) CN115746133A (en)

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