Monoclonal antibody with strong promotion of tumor cell phagocytosis and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a CD47 monoclonal antibody with strong phagocytosis of tumor cells, and also provides use of the antibody in preparation of an anti-tumor drug or a drug for fibrosis diseases. The novel anti-human CD47 monoclonal antibody has high affinity to human CD47, and has strong phagocytosis of tumor cells compared with the existing anti-human CD47 monoclonal antibody.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a CD47 monoclonal antibody with strong promotion of phagocytosis of tumor cells and use of the CD47 monoclonal antibody in preparation of an anti-tumor drug or a drug for fibrotic diseases. BACKGROUND

[0002] CD47 is a transmembrane protein expressed on almost all human cells. CD47 is also known as integrin-associated protein (IAP) and is a member of the immunoglobulin superfamily. CD47 is widely expressed on the surface of cells and can interact with SIRPα, thrombospondin-1 (TSP-1) and integrin to mediate a series of reactions such as apoptosis, proliferation and immunity.

[0003] CD47 releases signals to macrophages and dendritic cells (DCs) through interaction with SIRPα protects cells from attack by the immune system and plays a role in inhibiting effector T cells by promoting intratumoral vascular proliferation, promoting tumor cell expansion and growth. Cancer cells escape the host's immune surveillance through this pathway, and overexpression of CD47 is found to be associated with poor clinical outcomes. CD47 has also been identified as a cancer stem cell marker in leukemia and solid tumors (Jaiswal, et al., (2009) Cell, 138(2): 271-85; Chan, et al., (2009) Proc Natl Acad Sci USA, 106(33): 14016-21; Chan, et al., (2010) Curr Opin Urol, 20(5): 393-7; Majeti R, et al., (2011) Oncogene, 30(9): 1009-19). Therefore, CD47 blocking antibodies have been used for tumor treatment and have shown anti-tumor activity in multiple in vivo tumor models. In addition, these antibodies have been shown to synergize with other therapeutic antibodies, including rituximab and herceptin in tumor models.

[0004] Since then, CD47 has been further identified as a potential therapeutic target for treating pulmonary fibrosis, a disease that is incurable and life-threatening. In recent years, drug development targeting the tumor escape mechanism mediated by the CD47-SIRPα signaling axis has become a hot topic in tumor immunotherapy. However, the currently developed anti-human CD47 monoclonal antibodies still have the problems of low affinity or unclear target sites such as antigen epitopes. Therefore, there is an urgent need for a new type of therapeutic candidate CD47 antibody that can induce macrophages to phagocytose cancer cells and has high affinity. SUMMARY

[0005] The present application aims to solve the above problems in the prior art, and provides a novel anti-human CD47 monoclonal antibody with strong promotion of tumor cell phagocytosis, which has high affinity with human CD47 and strong promotion of tumor cell phagocytosis.

[0006] The present application provides a monoclonal antibody with strong promotion of tumor cell phagocytosis, which comprises a heavy chain variable region and a light chain variable region.

[0007] 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.

[0008] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 2.

[0009] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 4.

[0010] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 6.

[0011] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 12.

[0012] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 14.

[0013] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 16.

[0014] Preferably, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 8; and the light chain variable region amino acid sequence is shown in SEQ ID NO: 18.

[0015] Preferably, the heavy chain amino acid sequence is shown in SEQ ID NO: 10; and the light chain amino acid sequence is shown in SEQ ID NO: 20.

[0016] Preferably, the heavy chain and the light chain further comprise a constant region, which is a constant region of murine or human IgG, preferably a constant region of IgG4.

[0017] The present application further provides a nucleotide molecule encoding the above monoclonal antibody.

[0018] Preferably, the sequence of the nucleotide molecule is selected from SEQ ID NO: 7 and SEQ ID NO: 17.

[0019] The sequence of SEQ ID NO: 7 encodes the heavy chain variable region of the antibody;

[0020] The sequence of SEQ ID NO: 17 encodes the light chain variable region of the antibody.

[0021] The present application further provides an expression vector containing the nucleotide molecule.

[0022] The present application further provides a host cell containing the expression vector.

[0023] Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0024] The present application further provides a method for preparing the monoclonal antibody, comprising the following steps:

[0025] (1) preparing an expression vector containing a nucleotide molecule expressing the anti-human CD47 monoclonal antibody;

[0026] (2) transfecting a eukaryotic host cell with the expression vector obtained in step (1) and culturing;

[0027] (3) isolating and purifying to obtain the anti-human CD47 monoclonal antibody.

[0028] The present application further provides an antibody immunoconjugate, a bispecific molecule, a chimeric antigen receptor or a pharmaceutical composition comprising the monoclonal antibody.

[0029] Further, the pharmaceutical composition comprises a therapeutically effective amount of the novel anti-human CD47 monoclonal antibody, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0030] The present application further provides the use of the monoclonal antibody in the preparation of an anti-tumor drug or a drug for fibrotic diseases.

[0031] Preferably, the tumor is a hematological tumor or a solid tumor, including non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), ovarian cancer, fallopian tube cancer, colorectal cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, lung cancer, glioma and glioblastoma.

[0032] Preferably, the fibrotic disease includes angina pectoris, osteoarthritis, pulmonary fibrosis, asthma and bronchitis.

[0033] Advantages:

[0034] The novel anti-human CD47 monoclonal antibody of the present application has high affinity to human CD47, and has stronger promotion of phagocytosis of tumor cells and better CD47-SIRPα blocking activity compared to the existing anti-human CD47 monoclonal antibody. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 To capture ELISA assay the binding ability of the antibody to human CD47 protein;

[0036] Figure 2 To capture ELISA assay the binding ability of the antibody to cynomolgus monkey CD47 protein;

[0037] Figure 3 To flow cytometry to evaluate the binding of the antibody to 293F cells overexpressing human CD47 on the surface;

[0038] Figure 4 To ligand binding blocking ELISA;

[0039] Figure 5 To reference antibody blocking ELISA. DETAILED DESCRIPTION

[0040] TERMS

[0041] "Binding to CD47" or "binds to CD47" means capable of interacting with human CD47.

[0042] "Antigen binding site" refers to a three-dimensional space on an antigen that is occupied by an antibody or antigen-binding fragment of the present disclosure and around which an antibody or antigen-binding fragment of the present disclosure can make contact.

[0043] "Monoclonal antibody" refers to a preparation of antibody molecules of single amino acid composition, and not to the method by which it is produced. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such or other techniques known in the art.

[0044] "Affinity" refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, herein, "binding affinity" refers to the intrinsic binding affinity reflecting the 1 : 1 interaction between an antibody and an antigen. Affinity can be measured by common methods known in the art, including methods known in the art and described herein.

[0045] The term "compete" when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope means that the antigen binding proteins compete with each other as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) the 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 cells in the presence of the antigen binding protein being tested. Usually 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 the reference antigen binding protein; and antigen binding proteins that bind to an adjacent epitope in sufficient proximity to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder each other from binding.

[0046] Methods for producing and purifying antibodies and antigen binding fragments are well known and disclosed in the art, such as The Antibody Experimentation Guide, Cold Spring Harbor. For example, mice can be immunized with human CD47 or a fragment thereof, the resulting antibodies can be renatured, purified, and can be subjected to amino acid sequencing using conventional methods. Antigen binding fragments can likewise be prepared using conventional methods.

[0047] "Treatment" means the administration of 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 an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired therapeutic effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical detection method typically used by a physician or other health care professional to assess the severity or progression of the symptom.

[0048] "Effective amount" includes an amount that is sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. An effective amount also means an amount that is sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0049] "Pharmaceutical composition" means a mixture of one or more CD47 antibodies or antigen-binding fragments thereof described herein with other pharmaceutical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredients to a subject to enhance absorption into the body for the purpose of eliciting a biological activity.

[0050] The present application is further illustrated by the following examples. It is to be understood that these examples are merely illustrative of specific embodiments of the application and do not limit the scope of the application. Methods for experiments not specifically described in the examples were generally performed according to routine conditions, or according to the conditions recommended by the manufacturer. Reagents not specifically identified were purchased from commercial suppliers and were used according to the manufacturer's recommendations.

[0051] Example 1. Generation of specific anti-CD47 mouse monoclonal antibodies by fusion hybridoma technology

[0052] 1.1 Immunization of animals

[0053] Mice were immunized according to the general methods described 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.

[0054] To increase the immune response, the first immunization and the boost immunization used Freund's complete and incomplete adjuvant (Sigma, St. Louis, Mo., USA), respectively. Briefly, the preparation of the adjuvant-antigen mixture started with vortexing the adjuvant in a vial. The required amount of adjuvant was taken from the vial and put into 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 with the adjuvant, and the mixture was emulsified by vortexing for 2 min and repeated to form a water-in-oil solution. The adjuvant-antigen solution was then drawn into a suitable syringe for animal injection. Each animal was immunized, and then 2-3 times of boost immunization was performed according to the titer of the antisera. The animals with good titer were terminally immunized by intraperitoneal injection before fusion.

[0055] 1.2 Hybridoma fusion and screening

[0056] Prior to cell fusion, mouse myeloma cells (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured in log phase growth. Spleens from immunized mice were removed aseptically and fused with myeloma cells according to 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).

[0057] The fused "hybrid cells" were then distributed into 96-well cell plate culture media containing HAT. Surviving hybridoma cells were typically observed to grow out under a microscope 7-10 days after fusion. Two weeks after plating, the culture supernatant from each well was collected and screened for hybridomas using an ELISA method with recombinant human CD47-his protein antigen. Briefly, an ELISA plate was coated with human CD47-his protein (ACRO biosystems, cat# CD7-H5227, 2.0 μg / ml in PBS) overnight at 4°C. The plate was washed 4 times with PBST and blocked with blocking buffer (PBST with 5% skim milk). Diluted mouse immune serum (for determination of mouse serum titer) or hybridoma supernatant was added to each well and incubated at 37°C for 40 minutes. The plate was washed 4 times with PBST and detected with horseradish peroxidase-goat anti-mouse IgG (Jackson Immuno research, cat# 115-036-071) and the absorbance at 450 nm was determined for each well. Positive hybridomas that secreted antibodies that bound to human CD47-his were then selected and transferred to 24-well plates.

[0058] Hybridoma clones that produced antibodies with high specificity to bind human CD47 and with CD47 / SIRPa ligand blocking activity were subcloned by limiting dilution to ensure clonality of the cell line and then purified. Hybridoma clones that produced antibodies with high specificity to bind cell surface CD47 FACS and CD47 / SIRPa ligand blocking activity were subcloned to ensure clonality of the cell line and then purified the monoclonal antibodies.

[0059] Example 2 Determination of affinity of mouse anti-CD47 monoclonal antibodies using BIACORE surface plasmon resonance technology

[0060] Anti-CD47 mouse monoclonal antibodies (mAbs) produced by hybridoma clones in Example 1 were subjected to affinity kinetic characterization determination by Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0061] Briefly, goat anti-mouse IgG was covalently attached to a CM5 chip (carboxymethyl dextran coated chip) via primary amine using the standard amine coupling kit provided by Biacore. Unreacted portions of the biosensor surface were blocked with ethanolamine. Mouse anti-CD47 antibodies produced in Example 1 were purified and reference antibodies CC-9000 (Celgene) and Hu5F9-G4 (Forty Seven) were run on the chip at a concentration of 66.7 nM at a flow rate of 10 μL / min. Recombinant human CD47-his protein (Acro biosystems, cat# CD7-H5227, MW: 15.6 kDa) or cynomolgus CD47-his protein (Acro biosystems, cat# CD7-C52H1, MW: 15.8 kDa) in HBS EP buffer (provided by Biacore) were then run on the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were observed for 2 minutes and dissociation kinetics were observed for 10 minutes. Association and dissociation were fitted to a 1:1 Langmuir binding model curve using BIAevaluation software.

[0062] where k a ,k d and K D values are shown in Table 1.

[0063] Table 1. Kinetic parameters of mouse anti-CD47 monoclonal antibodies binding to human or cynomolgus CD47 by Biacore assay

[0064]

[0065] The binding K D value of the monoclonal antibody 1D5 of the present application to human CD47 was similar to the level of reference antibodies, indicating that it has high affinity to human CD47.

[0066] Example 3. Study of binding activity of mouse anti-CD47 monoclonal antibodies

[0067] The mouse anti-CD47 monoclonal antibodies (mAbs) produced by the hybridoma clones in Example 1 were further tested for their binding activity using the following methods.

[0068] 3.1. Capture ELISA-based assay to measure the binding capacity of antibodies

[0069] 96-well ELISA plates were coated with goat anti-mouse IgG Fc gamma fragment specific antibody (Jackson immuno Research, #115-006-071, 100 μΐ / well) at a final concentration of 2 μg / ml in PBS and incubated overnight at 4°C. The ELISA plates were washed 4 times with elution buffer (PBS + 0.05% v / v Tween-20, PBST) and then blocked with 200 μΐ / well of 5% w / v skimmed milk powder in PBST buffer for 2 hours at 37°C. The plates were washed again and incubated with 100 μΐ / well of different concentrations of CD47 mouse monoclonal antibodies for 40 minutes at 37°C, then washed the plates 4 times. The plates with captured CD47 antibodies were incubated with biotin-labeled human CD47 protein (ACRO Biosystems, cat# CD7-H5227) or monkey CYNO-CD47-HIS-BIO (ACRO Biosystems, cat No. #CD7-C52H1) (60 nM, 2.5% skimmed milk powder in PBST buffer, 100 μΐ / well) for 40 minutes at 37°C, washed the plates 4 times and incubated with streptavidin-conjugated horseradish peroxidase (diluted 1 : 10000 in PBST, Jackson Immuno Research, #016-030-084, 100 μΐ / well) for 40 minutes at 37°C. After the final wash, the plates were incubated with 100 μΐ / well of ELISA substrate TMB (Innoreagents, #TMB-S-002). The reaction was stopped with 50 μΐ / well of 1 M H2S04at 25°C within 15 minutes and the absorbance at 450 nm was measured. The results are shown in Figure 1. Figures 1-2 and Table 2.

[0070] Figure 1 and Figure 2 The results show that the antibody 1D5 of the present application has good binding capacity to both human and cynomolgus monkey CD47 proteins.

[0071] 3.2 Determination of the binding of CD47 monoclonal antibodies to 293F cell line overexpressing human CD47 on the surface using flow cytometry (FACS)

[0072] The stable cell line 293F overexpressing human CD47 on the surface was collected from cell culture flasks, washed twice and resuspended in PBS phosphate buffer containing 2% v / v fetal bovine serum (FACS buffer). 2 x 10 5Cells were incubated with FACS buffer containing different concentrations of CD47 antibody on ice for 40 minutes. Cells were washed 3 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 in FACS buffer, Jackson Immunoresearch, cat#115-116-072) secondary antibody was added. After incubation at 4°C for 40 minutes in the dark, cells were washed 3 times and then resuspended with FACS buffer. Fluorescence measurements were performed using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software to obtain the EC50 value, i.e. the antibody concentration value corresponding to 50% of the maximum fluorescence binding signal of CD47 antibody to CD47 overexpressing cells, the results of which are shown in Table 1. 50 Table 1. Binding activity of mouse anti-CD47 antibodies Figure 3 and Table 2.

[0073] Figure 3 The results show that the antibody 1D5 of the present application has a stronger binding ability to 293F cells overexpressing human CD47 on the surface.

[0074] Table 2. Binding activity of mouse anti-CD47 antibodies

[0075]

[0076] Example 4 Competitive functional blocking ability of mouse anti-CD47 monoclonal antibodies on CD47-SIRPa interaction

[0077] The blocking ability of antibodies on CD47-SIRPa interaction was detected using a competitive ELISA.

[0078] 4.1 Ligand blocking ELISA

[0079] The blocking ability of anti-CD47 antibodies of the present application on CD47-SIRPa interaction was detected using a competitive ELISA. Briefly, human SIRPa-his protein (Sino biological inc., cat#11612-H08H) was added at 200 ng / well on a 96-well microplate and incubated at 4°C overnight. The next day, the plate was washed with wash buffer (PBS + 0.05% Tween-20, PBST) and then blocked with PBST containing 5% w / v skimmed milk powder for 2 hours at 37°C. The plate was then washed again with wash buffer.

[0080] Human CD47-biotin (ACRO biosystems, cat# CD7-H5227) solution was used to dilute CD47 antibodies or reference antibodies (antibodies were serially diluted from 66.7 nM, 4-fold dilution). The antibody / CD47-biotin mixture was added to SIRPa-coated plates after incubation at room temperature for 40 minutes. After incubation at 37°C for 40 minutes, the plates were washed 4 times with wash buffer. Streptavidin-conjugated HRP was then added and incubated at 37°C for 40 minutes to detect the binding of biotin-labeled human CD47 to SIRPa on the bottom plate. The plates were washed again with wash buffer. Finally, TMB was added and the reaction was stopped with 1 M H2SO4, and the absorbance at 450 nm was measured. The data were analyzed using Graphpad Prism software to obtain IC 50 values, which are shown in Figure 4 and Table 3.

[0081] 4.2 Reference antibody blocking ELISA

[0082] The ability of the anti-CD47 antibodies of the present application to block the binding of reference antibody (Hu5F9-G4, Forty Seven) to human CD47 protein was determined using a competition ELISA. Briefly, CD47 reference antibody was coated on 96-well microplates with 1 μg / mL PBS and incubated at 4°C overnight. The next day, the plates were washed with wash buffer and blocked with 5% skim milk in PBST at 37°C for 2 hours. For blocking, biotin-labeled human CD47 (ACRO biosystems, cat# CD7-H5227) (10 nM in 2.5% skim milk in PBST) was mixed with antibodies (1.2 pM-100 nM, 5-fold serial dilution) and incubated at 25°C for 40 minutes. After washing the plates, the antibody / human CD47-biotin mixture (100 μl / well) was added to the Hu5F9-G4 plates and incubated at 37°C for 40 minutes. The plates were washed again with wash buffer, and 100 μl / well of SA-HRP was added and incubated at 37°C for 40 minutes to detect biotin-labeled human CD47 bound to the plates. The plates were finally washed with wash buffer. TMB was added and the reaction was stopped with 1 M H2SO4, and the absorbance at 450 nm was measured. The data were analyzed using Graphpad Prism software to obtain IC 50 values, which are shown in Figure 5 and Table 3.

[0083] As shown in Table 3, the antibodies of the present application can block human CD47-SIRPa interaction, and at the same time indicate that the antibodies of the present application have similar antigen binding epitopes to the reference antibody. Compared with the reference antibody, the antibody 1D5 of the present application has better CD47-SIRPa blocking activity.

[0084] Table 3. Ability of anti-CD47 antibodies to block CD47-SIRPa and CD47 reference antibody interaction

[0085]

[0086] Example 5 Mouse anti-CD47 monoclonal antibodies induce macrophage phagocytosis of tumor cells

[0087] The biological activity of anti-CD47 antibodies to induce macrophage phagocytosis of tumor cells was tested in vitro using cell experiments. 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), monocytes were seeded in RPMI 1640 + 10% FBS + 1% penicillin-streptomycin (Peprotech, 300-25-100) in the presence of human M-CSF. On day 2 and day 4, cells were washed and fresh cytokine-containing medium was replaced. On day 6, adherent cells were detached and washed twice with PBS.

[0088] MDMs were detached from the plate and plated in a 96-well plate overnight. Jurkat cells were collected for CFSE (5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester) (Sigma, 87444) labeling. Anti-CD47 mAbs were diluted accordingly. 100 uL of CFSE-labeled Jurkat tumor cells and diluted CD47 mAb mix were added to MDMs and incubated at 37°C for 4h. All cells were detached 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 values, as shown in Table 4.

[0089] Table 4 shows that the antibodies of the application are able to induce macrophage phagocytosis of tumor cells with lower EC 50 values than the two reference antibodies, showing a stronger phagocytosis of tumor cells than the reference antibodies.

[0090] Table 4. Ability of anti-CD47 antibodies to induce macrophage phagocytosis of tumor cells

[0091]

[0092]

[0093] Example 6 DNA cloning and sequencing, sequence analysis of anti-CD47 antibodies

[0094] Total RNA was extracted from the hybridoma cells of Example 1 using Trizol reagent (Invitrogen, catalog # 15596-018).

[0095] The procedure is briefly as follows: 5 x 106cells were collected by centrifugation into 1.5 ml microfuge tubes, and the supernatant was aspirated. One ml of Trizol reagent was added and the cells were lysed by pipetting several times, and incubating at 25°C for 5 minutes. Next, 0.2 ml of chloroform was added to each tube, and the tubes were shaken vigorously for 15 seconds and incubated at room temperature for 3 minutes. The tubes were then centrifuged at 12,000 g for 10 minutes at 4°C, and the aqueous phase was removed from the tubes and transferred to new 1.5 ml microfuge tubes. To the aqueous phase, 0.4 ml of isopropanol was added to precipitate the RNA. The tubes were mixed by hand and incubated at 25°C for 10 minutes, and then centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was discarded, and 1 ml of 75% ethanol was added to each tube. The tubes were again centrifuged at 7,500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The RNA pellet was dried at room temperature for 10 minutes, and then 30 to 50 μl of sterile DEPC-treated water was added to dissolve the RNA. 6

[0096] Next, the total RNA was converted to cDNA using the Taraka reverse transcription cDNA kit (catalog # 6110A). The reaction was set up as follows: 5 μl of total RNA + 0.5 μl Oligo(dT) + 8.5 μl RNase-free water (total of 14 μl) was pre-denatured at 65°C for 5 minutes, and then placed on ice for 2 minutes. Further additions were: 4 μl of 5x buffer + 1 μl of dNTP mix + 0.5 μl of RNase inhibitor + 1 μl of reverse transcriptase (total of 20.5 μl), mixed, and incubated at 40°C for 50 minutes, and then at 70°C for 10 minutes, to complete the cDNA synthesis. The cDNA was further poly-G tailed at the 3' end, using the following reaction: 5 μl of cDNA sample + 33.5 μl of ddH2O + 5 μl of 10x TdT buffer + 5 μl of CoCl2+ 1 μl of dGTP + 0.5 μl of terminal deoxynucleotidyl transferase (total volume of 50 μl), incubated at 37°C for 30 minutes, and then at 70°C for 10 minutes, to complete the poly-G tailing.

[0097] ​Further, the cDNA with tail was used as template to amplify the variable region of antibody. For amplifying the heavy chain variable region of antibody, the PCR reaction system was prepared as follows: 10x Taq enzyme buffer 5 μl + universal poly C primer (forward primer) 0.5 μl + mouse IgG1 reverse primer 0.5 μl + dNTP 1 μl + Taq polymerase 1 μl + cDNA 1 μl + ddH2O 41 μl. For amplifying the light chain variable region of antibody, the PCR reaction system was prepared as follows: 10x Taq enzyme buffer 5 μl + universal poly C primer (forward primer) 0.5 μl + mouse IgG kappa chain reverse primer 0.5 μl + dNTP 1 μl + Taq polymerase 1 μl + cDNA 1 μl + ddH2O 41 μl. The temperature cycle of PCR amplification of heavy chain and light chain variable region of antibody was as follows (in which steps 2 to 4 were repeated for 25 cycles):

[0098] 1) pre-denaturation 95 °C, 5 min;

[0099] 2) denaturation 95 °C, 20 sec;

[0100] 3) annealing 56 °C, 20 sec;

[0101] 4) extension 72 °C, 30 sec;

[0102] 5) preservation 25 °C, 60 min.

[0103] The PCR product was analyzed by 1% agarose gel electrophoresis, and the bands of DNA segments corresponding to the size (VH about 600 bp, VK about 500 bp) were cut off, and the DNA was extracted by QIAquick gel DNA recovery kit (catalog # 28704). The brief description was as follows: the gel was weighed, 3 times the gel volume of QG buffer was added, and then the gel was completely dissolved at 50 °C for 10 min. After 1 times the gel volume of isopropanol was added and mixed, the sample was moved to the QIA purification column, and centrifuged at 13000 rpm for 1 min. 750 μl of PE buffer was added to the column, and then centrifuged at 13000 rpm for 1 min. And again centrifuged at 13000 rpm to remove the residual liquid in the column. 30 μl of water was added and centrifuged at 13000 rpm for 1 min for elution to obtain the prepared DNA sample. The purified PCR product was sequenced to obtain the variable region sequence of antibody.

[0104] The sequence information of the cloned sequences of the application is shown in Table 5.

[0105] Table 5. Sequence information of anti-CD47 antibody

[0106]

[0107] NA: nucleotide; AA: amino acid. SEQUENCE LISTING <110> Bio-legend Biotech (Nanjing) Co., Ltd. <120> A monoclonal antibody with strong promotion of tumor cell phagocytosis and application thereof <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 ggcttcaaca ttaaagacta ctat 24 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 2 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 3 attgatccta aaaatggtga tact 24 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 4 Ile Asp Pro Lys Asn Gly Asp Thr 1 5 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 5 aatgcggggg gacgaggggg gtttgcttac 30 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <400> 6 Asn Ala Gly Gly Arg Gly Gly Phe Ala Tyr 1 5 10 <210> 7 <211> 351 <212> DNA <213> Artificial Sequence <400> 7 gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg 60 tcctgcacag cttctggctt caacattaaa gactactata tgtcctgggt gaggcagagg 120 cctgaacagg gcctggagtg gattggatgg attgatccta aaaatggtga tactgcatat 180 gacccgaagt tccagggcaa ggccactatg actgcagaca catcctccaa cacagcctac 240 ctgcagttca gcagcctgac atctgaggac actgccgact attactgtaa tgcgggggga 300 cgaggggggt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <400> 8 Glu Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gin Arg Pro Glu Gin Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Lys Asn Gly Asp Thr Ala Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gin Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Asp Tyr Tyr Cys 85 90 95 Asn Ala Gly Gly Arg Gly Gly Phe Ala Tyr Trp Gly Gin Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 9 <211> 1323 <212> DNA <213> Artificial Sequence <400> 9 gaggttcagc tgcagcagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg 60 TCCTGCACAG CTTCTGGCTT CAACATTAAA GACTACTATA TGTCTGGGTG AGGCAGAGG 120 CCTGAACAGG GCCTGGAGTG GATTGGATGG ATTGATCCTA AAAATGGTGA TACTGCATAT 180 GACCCGAAGT TCCAGGGCAA GGCCACTATG ACTGCAGACA CATCCTCCAA CACAGCCTAC 240 CTGCAGTTCA GCAGCCTGAC ATCTGAGGAC ACTGCCGACT ATTACTGTAA TGCGGGGGGA 300 CGAGGGGGGT TTGCTTACTG GGGCCAAGGG ACTCTGGTCA CTGTCTCTGC AGCCAAAACG 360 ACACCCCCAT CTGTCTATCC ACTGGCCCCT GGATCTGCTG CCCAAACTAA CTCCATGGTG 420 ACCCTGGGAT GCCTGGTCAA GGGCTATTTT CCTGAGCCAG TGACAGTGAC CTGGAACTCT 480 GGATCCCTGT CCAGCGGTGT GCACACCTTC CCAGCTGTCC TGCAGTCTGA CCTCTACACT 540 CTGAGCAGCT CAGTGACTGT CCCCTCCAGC ACCTGGCCCA GCAGAGACCT ACCTGCAAC 600 GTTGCCCACC CGGCCAGCAG CACCAAGGTG GACAAGAAAA TTGTGCCCAG GGATTGTGGT 660 TGTAAGCCTT Gcatatgtac AGTCCCAGAA GTATCATCTG TCTTcatctt CCCCCCAAAG 720 CCCAAGGATG TGCTCACCAT TACTCTGACT CTAAGGTCA 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 Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gin Arg Pro Glu Gin Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Lys Asn Gly Asp Thr Ala Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gin Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Asp Tyr Tyr Cys 85 90 95 Asn Ala Gly Gly Arg Gly Gly Phe Ala Tyr Trp Gly Gin Gly Thr Leu 100 105 110 Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Gly Ser Ala Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Gin 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 Gin 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 Gin Val Tyr Thr lie Pro Pro Pro Lys Glu Gin Met 340 345 350 Ala Lys Asp Lys Val Ser Leu Thr Cys Met lie Thr Asp Phe Phe Pro 355 360 365 Glu Asp lie Thr Val Glu Trp Gin Trp Asn Gly Gin Pro Ala Glu Asn 370 375 380 Tyr Lys Asn Thr Gin Pro lie Met Asp Thr Asp Gly Ser Tyr Phe Val 385 390 395 400 Tyr Ser Lys lie Asn Val Gin 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 agatctaatc agagcattgt acatagtaat ggatacactt atttagaa 48 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <400> 12 Arg Ser Asn Gin Ser lie Val His Ser Asn Gly Tyr Thr Tyr Leu Glu 1 5 10 15 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 aaagtttcca accgattttc t 21 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <400> 14 Lys 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 Gin Gly Ser His Val Pro Arg Thr 1 5 <210> 17 <211> 336 <212> DNA <213> Artificial Sequence <400> 17 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctaatca gagcattgta catagtaatg gatacactta tttagaatgg 120 atctcttgca gatctaatca gagcattgta catagtaatg gatacactta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct ataaagtttc caaccgattt 180 tacctgcaga aaccaggcca gtctccaaag ctcctgatct ataaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 cggacgttcg gtggaggcac caagctggaa 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 Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser Ile Val His Ser Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser Ile Val His Ser 20 25 30 20 25 30 Asn Gly Tyr Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Asn Gly Tyr 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 Lys Val Ser Asn Arg Phe Ser Gly Val Pro Pro Lys Leu Leu Ile Tyr Lys 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 Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 657 <212> DNA <213> Artificial Sequence <400> 19 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctaatca gagcattgta catagtaatg gatacactla tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct ataaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgc accaactgta 360 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 480 caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 540 agcagcaccc tcacgttgac taaggacgag tatgaacgac ataacagcta tacctgtgag 600 gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacagggg agagtgt 657 <210> 20 <211> 219 <212> PRT <213> Artificial Sequence <400> 20 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser Ile Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gin 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 Gin 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. A monoclonal antibody having a strong promotion of tumor cell phagocytosis, characterized by, The monoclonal antibody with strong promotion of tumor cell phagocytosis comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 8; The amino acid sequence of the light chain variable region is shown as SEQ ID NO:

18.

2. The monoclonal antibody having strong promotion of tumor cell phagocytosis according to claim 1, characterized by, The amino acid sequence of the heavy chain is shown as SEQ ID NO: 10; the amino acid sequence of the light chain is shown as SEQ ID NO:

20.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein The sequence of the nucleic acid molecule comprises SEQ ID NO: 7 and SEQ ID NO: 17; The sequence SEQ ID NO: 7 encodes the heavy chain variable region of the antibody; The sequence SEQ ID NO: 17 encodes the light chain variable region of the antibody.

5. An expression vector, characterized by, The expression vector contains the nucleic acid molecule according to claim 3 or 4.

6. A host cell, characterized in that, The host cell contains the expression vector according to claim 5.

7. The method for preparing a monoclonal antibody with strong tumor cell phagocytosis-promoting activity as described in claim 1 or 2, characterized in that, Comprising the following steps: Preparation of an expression vector containing a nucleic acid molecule expressing a monoclonal antibody according to claim 1 or 2; Transfecting the obtained expression vector into a eukaryotic host cell and culturing; Isolation and purification to obtain a monoclonal antibody with strong promotion of tumor cell phagocytosis.

8. A pharmaceutical composition comprising the monoclonal antibody according to claim 1 or 2.

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