CN110724672B - Hybridoma cell strain 105D11, antibody and application thereof - Google Patents

Abstract

The invention discloses a hybridoma cell strain 105D11 and an anti-human CD47 monoclonal antibody generated by the same, wherein the high-affinity anti-human CD47 specific monoclonal antibody capable of blocking the interaction of CD47-SIRP α is prepared by taking CD47 recombinant protein with biological activity as an antigen through hybridoma technology screening, and meanwhile, in vitro experiments prove that macrophages can be promoted to phagocytose tumor cells and erythrocyte aggregation cannot be caused, so the antibody has potential value in tumor immunotherapy.

Description

Hybridoma cell strain 105D11, antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain 105D11, a monoclonal antibody which is generated by the hybridoma cell strain and can be specifically and high-affinity combined with human CD47, a preparation method and a variable region sequence of the hybridoma cell strain, and pharmaceutical application of the hybridoma cell strain and the monoclonal antibody.

Background

Normally, the immune system can recognize and eliminate tumor cells in the tumor microenvironment, but for survival and growth, the tumor cells can adopt different strategies, so that the immune system of the human body is inhibited and the tumor cells cannot be normally killed, thereby surviving in each stage of the anti-tumor immune response. The tumor-immune cycle is divided into the following seven links: 1. tumor antigen release; 2. tumor antigen presentation; 3. priming and activating effector T cells; 4. migration of T cells to tumor tissue; 5. tumor tissue T cell infiltration; 6. t cells recognize tumor cells; 7. eliminating tumor cells. Abnormalities in any of these links can lead to failure of the anti-tumor-immune cycle and immune escape. Different tumors can inhibit the effective recognition and killing of tumor cells by the immune system through the abnormality of different links, thereby generating immune tolerance and even promoting the occurrence and development of tumors. Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restarting and maintaining tumor-immune circulation and restoring normal anti-tumor immune response of the body. Including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, small molecule inhibitors, and the like. In recent years, the tumor immunotherapy is well-known, and has now demonstrated strong antitumor activity in the treatment of solid tumors such as melanoma, non-small cell lung cancer, renal cancer and prostate cancer, and many tumor immunotherapy drugs have been approved by FDA in the united states for clinical application. Because of its excellent curative effect and innovativeness, immunotherapy for tumors is judged by the journal of science in 2013 as the most important scientific breakthrough every year.

In recent years, the most known immune checkpoint inhibitors are PD1-PDL1 antibody drugs and the like, tumor cells escape from immune monitoring by up-regulating the expression of surface PDL1 protein, PD1-PDL1 belongs to an immune checkpoint pathway in acquired immunity, and the immune checkpoint pathway in acquired immunity, namely CD47-SIRP α, is found to play a crucial role in tumor immune escape in recent years.

CD47, also known as Integrin Associated Protein (IAP), is widely expressed on the surface of many normal and tumor cells and interacts with Signal regulatory protein α (Signal regulated protein α α), thrombospondin (TSP 1) and integrins (integrins) to mediate a series of responses such as apoptosis, proliferation, immunity etc. CD47 is a 5-time transmembrane protein with a molecular mass of around 50kDa belonging to the immunoglobulin superfamily and an extracellular N-terminus is an IgV domain CD47 was first identified as a tumor antigen for human ovarian cancer in the 19 th century 80 s and then CD47 is expressed in a variety of human tumor types including acute myelogenous leukemia (NHL), Chronic Myelogenous Leukemia (CML), acute lymphoblastic leukemia (mll), non-hodgkin lymphoma (NHL), Multiple Myeloma (MM), bladder cancer and other solid tumors including CD47, which are involved in the early phagocytosis pathway of CD-9 b-t receptor macrophages, although CD-9 b β -t.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the prior art, the invention provides a hybridoma cell strain 105D11, an anti-human CD47 monoclonal antibody generated by the hybridoma cell strain, a preparation method of the anti-human CD47 monoclonal antibody, an amino acid sequence and a nucleic acid sequence of a variable region of the anti-human CD47 monoclonal antibody, and pharmaceutical application of the anti-human CD47 monoclonal antibody.

The technical scheme is as follows: the hybridoma cell strain 105D11 is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: C2019260, address: wuhan university in Wuhan City, preservation time: 2019, 10 and 29.

The hybridoma cell strain 105D11 disclosed by the invention produces an anti-human CD47 monoclonal antibody.

The CDRH1 of the heavy chain variable region of the anti-human CD47 monoclonal antibody is SEQ ID NO: 4, CDRH2 is SEQ ID NO: 6, CDRH3 is SEQ ID NO: 8; the CDRL1 of the light chain variable region is SEQ ID NO: 12, CDRL2 is SEQ ID NO: 14, CDRL3 is SEQ ID NO: 16.

Further, the heavy chain variable region of the anti-human CD47 monoclonal antibody is SEQ ID NO: 2, and the light chain variable region thereof is SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

The anti-human CD47 monoclonal antibody further comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM or IgA subtypes and a light chain constant region selected from the group consisting of Kappa or lambda subtypes.

Further preferably, the anti-human CD47 monoclonal antibody further comprises a heavy chain constant region selected from the IgG1 subtype and a light chain constant region selected from the Kappa subtype.

The invention also discloses a nucleic acid for encoding the anti-human CD47 monoclonal antibody, wherein the heavy chain variable region of the nucleic acid is shown as SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: shown at 9.

Further, a pharmaceutical composition, a detection reagent or a kit comprising the above-mentioned anti-human CD47 monoclonal antibody is also within the scope of the present invention.

The application of the monoclonal antibody in preparing the medicine for treating diseases related to abnormal or excessive and uncontrolled expression of the CD47 is also within the protection scope of the invention.

The invention takes a commercial recombinant human CD47-mFc fusion protein with bioactivity as an immunogen to immunize a Balb/c mouse, when the serum titer meets the fusion requirement, spleen cells and SP2/0-Ag14 myeloma cells are taken to carry out cell fusion, a hybridoma cell strain 105D11 capable of stably secreting an anti-human CD47 monoclonal antibody is obtained by screening an HAT selective culture medium, and after subcloning and amplification culture, the genes of antibody heavy chain and light chain variable regions are regulated and sequenced on the molecular level. The monoclonal antibody can be obtained by injecting hybridoma cells into the abdominal cavity of a mouse, collecting ascites, purifying by an ammonium sulfate precipitation method and a Protein G affinity chromatography column, and identifying the antibody characteristics and verifying the in vitro biological function activity.

The invention successfully prepares the hybridoma cell strain 105D11, and produces the anti-human CD47 monoclonal antibody through the hybridoma cell strain, the antibody has good specificity and high affinity, can be combined with human and cynomolgus monkey CD47 with high affinity on a cellular level, and can block the combination of CD47 and SIRP α, can effectively promote the phagocytosis of tumor cells by macrophages in vitro, and can not cause erythrocyte agglutination, and the anti-human CD47 monoclonal antibody is a potential drug for tumor immunotherapy.

Drawings

FIG. 1: the structural schematic diagram of a shuttle vector used in the construction of a stable cell line of human CD47 and cynomolgus monkey CD47 overexpression CHO-K1 is shown, MCS is a multiple cloning site, and a target gene is inserted into the position;

FIG. 2: flow assay results of human and cynomolgus monkey CD47 overexpressing stable cell lines, wherein in fig. 1, the a bar chart represents a human CD47 overexpressing negative cell line, the B bar chart represents a human CD47 overexpressing positive cell line, in fig. 2, the a bar chart represents a cynomolgus monkey CD47 overexpressing negative cell line, and the B bar chart represents a cynomolgus monkey CD47 overexpressing positive cell line (the abscissa is fluorescence intensity, and the ordinate is cell number);

FIG. 3: the indirect ELISA reaction titer results of antiserum and human CD47 recombinant protein of 5 immunized mice on day 7 after 3 immunization (the abscissa is the dilution factor of the antiserum, the unit is thousand, and the ordinate is the Optical Density (OD) value under the wavelength of 450 nm);

FIG. 4: the antiserum of 5 immunized mice combines with the over-expressed cell strains of human and cynomolgus monkey CD47 after 3 days of immunization to form a flow detection result, the figure (1) to (5) are respectively the antiserum of the #1 to the #5 mice, in the figure (1) to (5), a bar chart A represents CHO-K1 mother cells, a bar chart B represents cynomolgus monkey CD47 over-expressed CHO-K1 cell strains, and a bar chart C represents human CD47 over-expressed CHO-K1 cell strains (the abscissa is fluorescence intensity, and the ordinate is the number of the cells after homogenization);

FIG. 5: the result of the electrophoresis of the purified anti-human CD47 monoclonal antibody by a denaturing non-reducing SDS-PAGE;

FIG. 6: identifying an ELISA detection result of an anti-human CD47 monoclonal antibody subtype;

FIG. 7: the purified anti-human CD47 monoclonal antibody and human and cynomolgus monkey CD47 overexpression stable cell strain combined flow detection results, wherein A, B, C bar charts respectively represent the combined signals with CHO-K1 blast cell strain, human CD47 overexpression cell strain and cynomolgus monkey CD47 overexpression cell strain (the abscissa is fluorescence intensity, and the ordinate is the number of homogenized cells);

FIG. 8 shows the flow-based detection result of the purified anti-human CD47 monoclonal antibody for blocking the binding of SIRP α recombinant protein to CD47 on human CD47 overexpressing stable cell membrane, wherein the A histogram represents the binding signal of SIRP α recombinant protein to CD47 on human CD47 overexpressing stable cell membrane in the presence of anti-human CD47 monoclonal antibody, and the B histogram represents the binding signal of SIRP α recombinant protein to CD47 on human CD47 overexpressing stable cell membrane in the absence of anti-human CD47 monoclonal antibody (the abscissa represents fluorescence intensity and the ordinate represents the number of cells after homogenization);

FIG. 9: the anti-CD 47 monoclonal antibody erythrocyte coagulation test results show that the antibody concentration is decreased three times from 10 mug/ml to 9 concentrations, the first action is positive control antibody Hu5F9-IgG4, and the second action is the antibody 105D11F2 in the invention;

FIG. 10: the result of detecting the titer of the anti-CD 47 monoclonal antibody by macrophage phagocytosis reaction is shown in the left graph, which is a positive control antibody Hu5F9-IgG4, and in the right graph, which is an antibody 105D11F2 (the abscissa is the antibody concentration, the unit is mu g/ml, and the ordinate is the macrophage phagocytosis efficiency).

Detailed Description

The present application will be described in detail with reference to specific examples.

Example 1: construction of human CD47 and cynomolgus monkey CD47 overexpression CHO-K1 stable cell line

1) Shuttle vector construction

Human CD47 protein sequence

NP_001768leukocyte surface antigen CD47 isoform 1precursor[Homosapiens]

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

35,214Da, 323aa, 1-18 are signal peptides, 19-141,198-207,257-268 are extracellular domains, 142-162,177-197,208-228,236-256,269-289 are transmembrane helices, 163-176,229-235,290-323 are intracellular domains.

Cynomolgus monkey CD47 protein sequence

XP_005548289PREDICTED:leukocyte surface antigen CD47 isoform X1[Macaca fascicularis]

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

35,187Da，323aa。

The whole genes encoding the above two proteins were synthesized separately and cloned into pUC57 vector (Kinzhi, Suzhou), which was inserted into the MCS region of lentiviral over-expression vector (Wuhanber, see FIG. 1) by restriction enzyme digestion.

2) Preparation of viral packaging plasmids

The recombinant plasmid with correct sequencing is transferred into a clone bacterium DH5 α (Wuhanbo) for amplification, an endotoxin-free plasmid macroextraction kit (Wuhanbo) is used for plasmid extraction, nucleic acid electrophoresis is used for determining the molecular weight after extraction, and meanwhile, an ultramicro spectrophotometer is used for evaluating the quality of the plasmid, wherein the A260/A280 ratio is 1.7-1.9, the OD260/230 ratio is more than 20, and the concentration is more than 0.5 mug/microliter and is the high-quality plasmid.

At the same time, the corresponding viral packaging helper plasmids were prepared in the same manner.

3) Virus package

The main procedure was to add all the above plasmids and transfection reagents for transfection when the density of HEK293T cells (Beijing Beiner) reached 80% confluence, as described with strict reference to transfection reagent (Nanjing Novozam). And replacing a fresh culture medium after 6h of transfection, and collecting a culture solution containing virus particles 48-72h after transfection.

4) CHO-K1 cell virus infection and pressure screening

The virus solution was added to CHO-K1 cells (Beijing Beiner), and after 3 days of culture, puromycin (Wuhanbo' er, working concentration 2.5. mu.g/ml) was added and the culture was continued for 3 days (after which the cell culture was entirely carried out using the antibiotic-containing medium), and passaging was carried out.

5) Culture of monoclonal

Cells were diluted to 1 cell per well, and monoclonal cells were picked and cultured with medium containing working concentration of puromycin for 2-3 weeks. And (5) subculturing, gradually expanding and culturing to a T25 cell culture flask, and preserving the seeds.

6) Flow assay

And (3) carrying out simple immunofluorescence staining by using a commercial mouse anti-human CD47 monoclonal antibody, and detecting the expression quantity of the constructed cell strain relative to the CD47 protein of an untransfected control cell. The indirect immunofluorescent labeled sample preparation method comprises the following steps:

a) cultured cells were well digested with 0.25% pancreatin (GIBCO) (excess digestion otherwise prone to floc) and gently pipetted with PBS to prepare single cell suspensions.

b) Cells were washed 1 time with 10ml PBS, centrifuged at 1000rpm for 5min, and cells were suspended in 1ml PBS and counted.

c) Take 2.5X 10⁵The cells were washed 1 time with 1ml PBS in a 1.5ml centrifuge tube, centrifuged at 2000rpm for 5min and the supernatant was discarded.

d) Mu.l of commercial mouse anti-human CD47 monoclonal antibody (Novusbio NBP2-31106, 10. mu.g/ml) was added thereto, mixed well, and reacted at room temperature with exclusion of light for 30 min.

e) The cells were washed 1 time with 1ml PBS, centrifuged at 2000rpm for 5min and the supernatant was discarded.

f) 50. mu.l of 20-fold diluted FITC-labeled goat anti-mouse IGG fluorescent secondary antibody (Jackson ImmunoResearch 115-096-062, 2.5. mu.l/2.5X 10)⁵) Mixing, and reacting at room temperature in dark for 20 min.

g) The cells were washed 1 time with 1ml PBS, centrifuged at 2000rpm for 5min and the supernatant was discarded.

h) Add 200. mu.l PBS to re-suspend into single cell suspension, and detect on machine with flow cytometer.

The detection results show that finally, a stable cell line with high expression of human CD47 and cynomolgus monkey CD47 is screened respectively as shown in (1) and (2) in figure 2.

Example 2: animal immunization and antiserum ELISA titer and flow cytometry determination

5 Balb/c mice (Beijing sbefu) with the age of 6-8 weeks are selected and bred in the same cage, and are respectively numbered as #1, #2, #3, #4 and # 5. 4 days before the first immunization, about 0.05ml of blood is collected from each mouse through the tail vein, the mouse is placed at 4 ℃ for half an hour, then the mouse is centrifuged at 10000rpm and 4 ℃ for 10min, and the separated serum is used as the negative control serum of the later experiment. When in first immunization, immunogen of recombinant human CD47-mFc fusion protein (Acro Biosystems CD7-H52A5) is fully mixed with equal volume of Freund's complete adjuvant (SIGMA), ultrasonic emulsification is complete, subcutaneous two-point injection and intraperitoneal two-point injection are adopted, the immunization dose is 50 mu g of immunogen per mouse, and the injection volume is 0.2 ml. The second and third immunizations were performed at intervals of 14 days and 35 days, respectively, and the two immunizations were distinguished from the first immunization in that Freund's complete adjuvant was changed to Freund's incomplete adjuvant (SIGMA), and the immunization dose was adjusted to 25. mu.g. On day 7 after the third immunization, each mouse was bled by tail vein at about 0.05ml, left at 4 ℃ for half an hour, centrifuged at 10000rpm at 4 ℃ for 10min, and serum was separated to detect antiserum ELISA titer and flow cytometry.

An indirect ELISA method is adopted for detecting the titer of the antiserum, wherein the indirect ELISA method comprises the following steps: the ELISA plate was coated with recombinant human CD47-mFc fusion protein at 0.5. mu.g/ml, 50. mu.l/well, overnight at 4 ℃. The next day, the coating solution was decanted, plates were washed 3 times with 0.05% PBST, blocked with 0.5% BSA (SIGMA), 200. mu.l/well, 37 ℃ for 1 h. The blocking solution was discarded, the plates were washed 3 times with 0.05% PBST, and then 10 dilutions of the antiserum diluted at a rate of 1:1000 were added, the blank control was PBS, the preimmune serum was negative control, 50. mu.l/well, 37 ℃ and 1 hour. After 3 washes of the 0.05% PBST manual plate, a 1:20000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (Fab) secondary antibody (Jackson ImmunoResearch115-035-072) was added at 50. mu.l/well at 37 ℃ for 1 h. After washing the plate 5 times with 0.05% PBST, the chromogenic substrate TMB (England, Huzhou) was added at 50. mu.l/well at room temperature for 10min, and the reaction was stopped by adding 1M sulfuric acid. The OD450nm reading on the microplate reader showed that the antiserum titers of 5 mice reached 1:64000, 1:256000, 1:256000, 1:512000 (as in FIG. 3), respectively, indicating that immunization of mice with the immunogen produced high affinity antibodies that bind to human CD47 at the recombinant protein level.

The binding activity of antiserum to CD47 on human and cynomolgus monkey cell membranes was determined by flow cytometry, as described in example 1 for indirect immunofluorescent-labeled sample preparation, using the cell line CHO-K1 cell line over-expressed in human and cynomolgus monkey CD47 selected in example 1. The results of the assay showed that the antisera from 5 mice bound to different degrees to human and cynomolgus CD47 on the cell membrane, whereas the combined binding activity of the #1 mouse and both was higher (see fig. 4), indicating that immunization of mice with the immunogen produced high affinity antibodies that bound human CD47 at the cellular level.

And selecting the #1 mouse with higher comprehensive binding activity with CD47 on the cell membranes of the human and the cynomolgus monkey at an interval of 28 days after the third immunization for the fourth immunization, wherein the immunization is carried out without using an adjuvant and by tail vein injection, the immunization dose is 25 mu g of immunogen, and the injection volume is 0.2 ml. Cell fusion was performed 4 days after this immunization interval.

EXAMPLE 3 preparation of hybridoma monoclonal cell lines

1. Cell fusion

The cell fusion is carried out by the polyethylene glycol method. The specific operation is as follows:

1) one week prior to fusion, SP2/0-Ag14 myeloma cells (Beijing Beiner) were revived. Two days prior to cell fusion, SP2/0-Ag14 was expanded to be in log phase growth on the day of fusion.

2) Half an hour before cell fusion, pretreating SP2/0-Ag14 cells, resuspending SP2/0-Ag14 cells, counting, and collecting 2-3 × 10 cells⁷The cells were placed in a 37 ℃ water bath for use.

3) The mice to be fused are subjected to heart blood sampling, serum is collected by the same operation, and the serum is stored at the temperature of minus 20 ℃ and can be used as a positive control for screening after fusion. The mice were sacrificed by cervical dislocation, soaked in 75% alcohol, and transferred to the cell house. The spleen was ground and filtered through a 70 μm mesh to make a single cell suspension.

4) Mixing the pretreated SP2/0-Ag14 cells and splenocytes, centrifuging at 1000rpm for 5min, and discarding the supernatant. The mixed cells were washed twice with serum-free RPMI1640 basal medium (GIBCO) and the supernatant was decanted off the last time. 1ml of PEG1450(SIGMA) pre-warmed at 37 ℃ is slowly dripped on the cell sediment for 90s, serum-free RPMI1640 basic culture medium pre-warmed at 37 ℃ is immediately added within 5 minutes, the mixture is evenly mixed and centrifuged for 800X 5min, and the cell sediment is resuspended in 10% FBS (Royacel) + hybridoma supplement (ROCHE) -RPMI1640 culture medium containing HAT (SIGMA), evenly paved in a 96-well plate, 80 mu l per well and cultured in a cell culture box at 37 ℃ and 5% CO 2.

5) Cell status was observed periodically after fusion. And (3) changing the culture solution on day 5-7 of fusion, namely, changing the whole culture medium in the culture well plate by using a fresh 10% FBS + hybridoma supplement-RPMI 1640 culture medium containing HAT, and continuously culturing for 5-7 days at a rate of 120 mu l per well.

2. Screening and subcloning of Positive fusion cells

The method comprises the steps of detecting the antibody secretion condition of cells in each hole by indirect ELISA, selecting a hole which is positive in reaction with recombinant human CD47-mFc fusion protein by using recombinant human CD47-mFc fusion protein as an ELISA screening coating antigen and HRP-labeled goat anti-mouse IgG (Fab) secondary antibody as a detection antibody, detecting the binding activity of clone supernatants in the holes with human CD47 and cynomolgus monkey CD47 overexpression CHO-K1 cell strains, selecting clone supernatants with high binding capacity with the two cell strains, detecting the condition that the clone supernatants block the binding of SIRP α protein and cell surface CD47 by using flow cytometry, finally selecting a clone which can better block the binding of the SIRP α protein and the cell surface CD47, observing a viable hybridoma cell or a cell cluster under a microscope, labeling, cloning the cells in the holes by using a limiting dilution method, establishing a hybridoma cell strain which stably secretes monoclonal antibodies after two times of cloning, performing culture and preserving the hybridoma cell strain in Wuhan-Mikan-Han-Mitsu-Kong (national center) No. 7: Wookanji-wu-shi-C culture collection center, preservation No. 7: 2019-wu.

Example 4 preparation and purification of anti-human CD47 monoclonal antibody

1. Preparation of ascites

3 balb/c female mice (Beijing sbefu) with the age of 10-16 weeks are respectively taken, and the abdominal cavity of each mouse is injected with 0.25ml of ascites respectively for preparing the adjuvant (Beijing Boolong) 12-18 days in advance. Taking the growth logarithmic phase hybridoma cell count, taking 6 x 10⁶The individual cells were washed twice with 10ml sterile PBS, centrifuged at 1000rpm for 5min and finally sterilized PBS adjusted cell concentration to 2 x 10⁶And each cell per ml, then 0.5ml of cell suspension is injected into the abdominal cavity of the mouse respectively from left to right, and the abdomen is massaged to ensure that the cells are uniformly distributed. After about 10-14 days, the abdomen of the mouse obviously rises and ascites begins to be collected, generally once every other day and three times. Ascites was collected each time, centrifuged at 2000rpm for 5min and the supernatant (uppermost layer of oil was removed as much as possible with a pipette) was collected and stored at 4 ℃ for a short time and-20 ℃ for a long time.

2. Antibody purification

1) Centrifuging the above retained ascites at 14000rpm for 10min to remove cell debris and particulate impurities.

2) The supernatant was transferred and filtered with filter paper, and the filtrate was collected and measured for volume.

3) An equal volume of saturated ammonium sulfate was slowly added to the filtrate with stirring to a final concentration of 1: 1.

4) The solution was gently stirred on a magnetic stirrer at room temperature for 2 hours and then dispensed into a high-speed centrifuge tube and allowed to stand overnight at 4 ℃ to allow the protein to precipitate sufficiently.

5) Taking out the supernatant on the next day, directly centrifuging at 10000rpm for 30min, discarding the supernatant, and keeping the precipitate for air drying.

6) The precipitate was dissolved by adding 0.5 volume of PBS to the filtrate, and then concentrated by ultrafiltration and centrifugation.

7) The concentrate was diluted by adding twice the volume of the filtrate of binding buffer and filtered through a 0.45 μm filter.

8) The filtrate was collected and affinity purified using protein G pre-packed columns (Henzhou Tiandi and Co.) according to the manufacturer's instructions.

9) The eluate was collected and desalted and concentrated by an ultrafiltration centrifuge tube (Millipore) having a molecular weight cut-off of 50 KD.

3. Determination of antibody concentration and purity

The antibody concentration of the desalted and concentrated antibody was determined by the Bradford method, and the purity of the antibody was preliminarily determined by SDS-PAGE electrophoresis (see FIG. 5).

Example 5 characterization of anti-human CD47 monoclonal antibody

1. Identification of antibody subtypes

The subtype of the obtained anti-human CD47 monoclonal antibody is identified by a Mouse monoclonal antibody subtype identification kit (Proteitech), a specific antibody aiming at Mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, kappa light chain and lambda light chain is pre-coated on an enzyme label plate, and the specific experimental operation is shown in a kit instruction book. As shown in FIG. 6, the heavy chain subtype of the anti-human CD47 monoclonal antibody was IgG1 and the light chain subtype was Kappa.

The antibodies of the invention may be recombinantly expressed as other isotypes, such as IgG2, IgG3, IgG4, IgM, and IgA.

2. Flow cytometry for detecting reactivity of antibodies with CD47 on cell membranes

With reference to the indirect immunofluorescent-labeled sample preparation method described in example 1, flow cytometry was used to detect the binding of purified anti-human CD47 monoclonal antibody to CD47 on the human and cynomolgus monkey CD47 overexpressed cell membranes. As shown in the results of FIG. 7, the monoclonal antibody against human CD47 obtained by us can bind to human and cynomolgus monkey CD47 on the cell membrane with higher affinity.

3. Flow cytometry detection of antibody blocking SIRP α protein from binding with cell surface CD47

SIRP α is a ligand of CD47, CD47 protein can be combined with SIRP α on the cell surface, and flow detection is shown in FIG. 8, on the basis of the detection, the anti-human CD47 monoclonal antibody can be used for blocking the combination of SIRP α and CD47 on the cell surface, human CD47 overexpression cell strains are treated according to the sample preparation method of indirect immunofluorescence labeling described in example 1,1 mu g of SIRP α -hFc protein (Acro Biosystems SIA-H5251) is added into each group, meanwhile, the anti-human CD47 monoclonal antibody is added into the co-incubation, and the combination of the SIRP α -hFc protein and the human CD47 overexpression cell strain is detected by flow cytometry, from the flow result (FIG. 8), the anti-human CD47 monoclonal antibody can better block the combination of the SIRP α protein and the CD47 on the cell surface.

4. Hemagglutination test of anti-human CD47 monoclonal antibody

The nature of erythrocyte agglutination is an antigen-antibody reaction. Acting as antigens are specific proteins or glycolipids, called agglutinants, embedded in the red cell membrane. The anti-CD 47 antibody binds to the CD47 membrane protein expressed on red blood cells,and bridges are formed between the red blood cells, so that the agglutination phenomenon occurs. Lymphoprep was used in this example^TM(Axis-Shield PoCAS, Cat # AS1114547) Red blood cells were separated from pooled healthy human blood by density gradient centrifugation, then resuspended in PBS and diluted to the appropriate density. The red blood cell suspension was added to a 96-well cell culture plate, and a completely diluted anti-human CD47 monoclonal antibody (control positive antibody: Hu5F9-G4 of FORTY SEVEN, antibody No. 105D11F2 in the present invention) was added to the corresponding well of the culture plate. Incubation was performed at room temperature for about 1 hour. And observing the agglutination condition of the red blood cells under different test concentrations, and photographing and storing by using a gel imaging system. The results are shown in FIG. 9, the positive control antibody can mediate the agglutination of human erythrocytes, while the antibody of the present invention has no obvious effect of mediating the agglutination of human erythrocytes at the tested concentration.

5. Macrophage phagocytosis reaction detection of anti-human CD47 monoclonal antibody potency

The experiment is carried out by taking human promyelocytic acute leukemia cell HL-60 (Beijing Beiner) as a target cell, inducing macrophage from human monocyte as an effector cell, and taking Hu5F9-G4 (Nanjing Kingkunshi) as a positive reference, during the experiment, PKH26 dye (SIGMA MINI26-1KT) and APC-AntiCD11b antibody are used for marking specific markers CD11b on HL-60 cell membrane and macrophage respectively, the PKH26 dye with red fluorescent dye with long lipid tail can be stably combined to a lipid region of phagocytosis of HL-60 cell, so that the HL-60 cell has fluorescent marker, the APC-AntiCD11b antibody can be specifically combined with CD b on macrophage, so that the cell has specific fluorescent signal, and the number of the cells with the specific fluorescent signal is counted by detecting the fluorescent signal carried by the cell, and the number of the cells with PKH26 and APC-AntiCD11 positive signals under the experiment condition that the specific marker CD b on the macrophage phagocytosis and macrophage positive markers are calculated as follows:

1) the target Cells were collected by centrifugation, the supernatant was discarded, the Cells were diluted and resuspended (density less than 1E7Cells/mL) using DPBS (GIBCO), and appropriate amount of PKH26 dye working solution was added for incubation staining.

2) The stained target Cells were resuspended at 2E5 Cells/mL density in complete medium in a cell culture incubator (37 ℃/5% CO)₂) The culture was continued overnight.

3) Target Cells that had completed PKH26 staining were collected by centrifugation, resuspended Cells using 1640 complete medium and the cell suspension density was adjusted to 2E5 Cells/mL.

4) The target cell suspension was added to a 96-Well cell culture plate at 50. mu.L per Well (10000 Cells/Well).

5) The diluted test sample and the positive reference working solution were added to the corresponding wells of the plate at 50. mu.L per well, and the plate was transferred to a cell incubator and incubated for about 0.5 hour.

6) Effector Cells were harvested by digestion and centrifugation, resuspended using 1640 complete medium and cell suspension density adjusted to 5E5 Cells/mL.

7) The effector cell suspension was added to 100. mu.L per Well of the cell culture plate (50000Cells/Well) where incubation was completed. Transfer the plates to the cell incubator for an additional incubation period of about 1 hour.

8) Preparing a working solution for detecting the antibody.

The macrophage phagocytosis experiment group is marked by using a CD11b detection antibody;

the macrophage QC assay was labeled with detection antibodies CD11b (Miltenyi Biotec 130-091 241), CD14(Miltenyi Biotec 130-.

9) After the cell culture plate is incubated, centrifuging the culture plate, discarding the incubation supernatant, adding the working solution for detecting the antibody into the culture hole, and incubating for 15min at 4 ℃.

10) The plates were centrifuged and after discarding the incubation supernatant, the cells were resuspended using DPBS.

11) Cells were collected using a flow cytometer and the fluorescent signal was read.

Experimental data was analyzed by FlowJo and the rate of Phagocytosis was calculated by the following formula,% Phagocytosis by MDMs ═ PKH26+ and CD11b + Cells/All PKH26+ Cells × 100%. GraphPad Prism 6 was used to fit a four parameter curve to obtain the EC50 of the antibody, the results are shown in FIG. 10, both positive reference (Hu5F9-G4) and the antibody of the invention are effective in blocking the interaction of CD47 with SIPR α and inducing macrophages to phagocytose target Cells, and the Phagocytosis effect is dose-effect dependent with EC50 of about 0.002749 μ G/ml and 0.005394 μ G/ml, respectively.

EXAMPLE 6 cloning of heavy and light chain variable region Gene of hybridoma cell line

1. Extraction, amplification and preliminary identification of heavy and light chain variable region gene of anti-human CD47 monoclonal antibody

After the positive hybridoma cell line in example 3 was expanded, cells in the logarithmic growth phase were collected, and the heavy and light chain variable region genes of the antibody of the present invention were subjected to cloning and sequencing using the entire set of reagents for cloning the variable region genes of the murine Novagen antibody according to the instructions thereof. The specific method route is as follows: using Straight A' s^TMmRNA Isolation Kits were isolated from the collected hybridoma cell lines for total RNA, followed by Synthesis of the First cDNA Strand using First Strand cDNA Synthesis Kit and Ig-3'constant region primer, PCR amplification using Ig-5' primers and NovaTaq DNApolymerase using the First cDNA Strand as a template, cloning of the resulting PCR amplification product into a cloning Vector using Vector cloning Kit, screening, Isolation of DNA and gene sequencing.

2. Gene sequencing and analysis of heavy and light chain variable domain of anti-human CD47 monoclonal antibody

In GenBank, the results of alignment sequencing and mouse antibody nucleic acid sequences show that the homology of the variable region sequences of the light chain and the heavy chain of the antibody and the submitted mouse IgG variable region sequences exceeds 93 percent, and the gene sequences obtained by sequencing are determined to be mouse antibody sequences. The amino acid sequences of the variable regions of the light chain and the heavy chain of the antibody, and the division of the CDR region and the FR region are obtained by utilizing the IMGT/V-QUEST and ABYSIS software analysis. The analysis result shows that: the nucleotide sequence and the amino acid sequence of the variable domain VH of the heavy chain of the hybridoma cell strain antibody are shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; the nucleotide sequence and the amino acid sequence of the variable domain VL of the light chain of the hybridoma cell strain are shown as SEQ ID NO: 9 and SEQ ID NO: 10 is shown in the figure; the heavy chain variable domain VH comprises in sequence the hypervariable regions CDRH1, CDRH2 and CDRH3, said nucleotide sequences being in sequence SEQ ID NO: 3. 5 and 7, the amino acid sequence is SEQ ID NO: 4. 6, 8; the light chain variable domain VL comprises the hypervariable regions CDRL1, CDRL2 and CDRL3 in sequence, and the nucleotide sequences are SEQ ID NO: 11. 13 and 15, the amino acid sequence is SEQ ID NO: 12. 14, 16.

Antibody variable region nucleic acid and amino acid sequences

SEQ ID NO1：

CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTCCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGTTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACGAGGCCTCGAGTGGATTGGAAGGATTGATTCTTCCGATAGTGAAACTCACTACAATCAAAAGTTCAAGGACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATCCAACTCAGCAGCCTGACATCTGAGGATTCTGCGGTCTATTACTGTGCAAGACAGGGGGAAGGATTACGACGGTCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCATTGTCTCTGCA

SEQ ID NO2：

QVQLQQPGAELVKPGAPVKLSCKASGYTFTSYWMNWVKQRPGRGLEWIGRIDSSDSETHYNQKFKDKATLTVDKSSSTAYIQLSSLTSEDSAVYYCARQGEGLRRSWFPYWGQGTLVIVSA

SEQ ID NO3：

AGTTACTGGATGAAC

SEQ ID NO4：

SYWMN

SEQ ID NO5：

AGGATTGATTCTTCCGATAGTGAAACTCACTACAATCAAAAGTTCAAGGAC

SEQ ID NO6：

RIDSSDSETHYNQKFKD

SEQ ID NO7：

CAGGGGGAAGGATTACGACGGTCCTGGTTTCCTTAC

SEQ ID NO8：

QGEGLRRSWFPY

SEQ ID NO9：

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGATACTAATGTAGTCTGGTATCAACAGAAACCAGGGCAGTCTCCTAAAATACTGATTTATTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCTCAGGCAGTGGATCTGGGACAGATTTCACTCTCATCATCAGCAATGTGCAGTCTGAAGACTTGTCAGGATATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGGAGGGGGGACCCAGCTGGAAATAAAA

SEQ ID NO10：

DIVMTQSQKFMSTSVGDRVSVTCKASQNVDTNVVWYQQKPGQSPKILIYSASYRYSGVPDRFSGSGSGTDFTLIISNVQSEDLSGYFCQQYNSYPYTFGGGTQLEIK

SEQ ID NO11：

AAGGCCAGTCAGAATGTGGATACTAATGTAGTC

SEQ ID NO12：

KASQNVDTNVV

SEQ ID NO13：

TCGGCATCCTACCGGTACAGT

SEQ ID NO14：

SASYRYS

SEQ ID NO15：

CAGCAATATAACAGCTATCCGTACACG

SEQ ID NO16：

QQYNSYPYT

Sequence listing

<110> Zhejiang Landun pharmaceuticals Co., Ltd

<120> hybridoma cell line 105D11, antibody and application thereof

<160>16

<170>SIPOSequenceListing 1.0

<210>1

<211>363

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggctcc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agttactgga tgaactgggt gaagcagagg 120

cctggacgag gcctcgagtg gattggaagg attgattctt ccgatagtga aactcactac 180

aatcaaaagt tcaaggacaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atccaactca gcagcctgac atctgaggat tctgcggtct attactgtgc aagacagggg 300

gaaggattac gacggtcctg gtttccttac tggggccaag ggactctggt cattgtctct 360

gca 363

<210>2

<211>121

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

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

1 5 10 15

Pro Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln ArgPro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Ser Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Glu Gly Leu Arg Arg Ser Trp Phe Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Ile Val Ser Ala

115 120

<210>3

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

agttactgga tgaac 15

<210>4

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Ser Tyr Trp Met Asn

1 5

<210>5

<211>51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aggattgatt cttccgatag tgaaactcac tacaatcaaa agttcaagga c 51

<210>6

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Arg Ile Asp Ser Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210>7

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

cagggggaag gattacgacg gtcctggttt ccttac 36

<210>8

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Gln Gly Glu Gly Leu Arg Arg Ser Trp Phe Pro Tyr

1 5 10

<210>9

<211>321

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60

gtcacctgca aggccagtca gaatgtggat actaatgtag tctggtatca acagaaacca 120

gggcagtctc ctaaaatact gatttattcg gcatcctacc ggtacagtgg agtccctgat 180

cgcttctcag gcagtggatc tgggacagat ttcactctca tcatcagcaa tgtgcagtct 240

gaagacttgt caggatattt ctgtcagcaa tataacagct atccgtacac gttcggaggg 300

gggacccagc tggaaataaa a 321

<210>10

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ile Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Gly Tyr Phe Cys Gln Gln TyrAsn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys

100 105

<210>11

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

aaggccagtc agaatgtgga tactaatgta gtc 33

<210>12

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Lys Ala Ser Gln Asn Val Asp Thr Asn Val Val

1 5 10

<210>13

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

tcggcatcct accggtacag t 21

<210>14

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210>15

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

cagcaatata acagctatcc gtacacg 27

<210>16

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

Claims (7)

1. The hybridoma cell strain 105D11 is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2019260.

2. The monoclonal antibody against human CD47 produced by the hybridoma cell line 105D11 of claim 1.

3. An anti-human CD47 monoclonal antibody, wherein the heavy chain variable region is SEQ ID NO: 2, and the light chain variable region thereof is SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

4. The anti-human CD47 monoclonal antibody according to claim 3, wherein the heavy chain constant region is selected from the group consisting of IgG2, IgG3, IgG4, IgM or IgA subtypes and the light chain constant region is selected from the group consisting of lambda subtypes.

5. A nucleic acid encoding the anti-human CD47 monoclonal antibody of claim 2 or 3, wherein the heavy chain variable region is as set forth in SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: shown at 9.

6. A pharmaceutical composition comprising the monoclonal antibody of claim 2.

7. A test kit comprising the monoclonal antibody of claim 2.

Images