CN110734897A

CN110734897A - Hybridoma cell line 12G6, antibody and application thereof

Info

Publication number: CN110734897A
Application number: CN201911050228.0A
Authority: CN
Inventors: 郭志刚; 黄子祥; 赵行琦
Original assignee: Zhejiang Blueshield Pharmacy Co ltd
Current assignee: Zhejiang Blueshield Pharmacy Co ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2020-01-31

Abstract

The invention discloses hybridoma cell strains 12G6 and an anti-human SIRP α monoclonal antibody generated by the same, wherein the high-affinity anti-human SIRP α specific monoclonal antibody capable of blocking the interaction of CD47-SIRP α is prepared by screening a SIRP α recombinant protein with biological activity as an antigen through a hybridoma technology, so that the antibody has potential value in tumor immunotherapy.

Description

Hybridoma cell line 12G6, antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to hybridoma cell lines 12G6, a monoclonal antibody which is generated by the hybridoma cell lines and can be specifically and high-affinity combined with human SIRP α, a preparation method of the monoclonal antibody, a variable region sequence of the monoclonal antibody, and application of the monoclonal antibody.

Background

Under normal conditions, the immune system can recognize and eliminate tumor cells in a tumor microenvironment, but in order to survive and grow, the tumor cells can adopt different strategies, so that the immune system of a human body is inhibited and can not normally kill the tumor cells, and thus the tumor cells can survive in each stage of anti-tumor immune response.

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.

Signal regulatory protein α (SIRP α, signal regulatory protein α), a member of the immunoglobulin superfamily membrane proteins, α, is typical inhibitory receptors in the SIRP family, which is specifically expressed on myeloid cells such as macrophages, dendritic cells, and neural cell membrane surfaces, which is transmembrane proteins, the extracellular domain contains 3 Ig-like domains, and the terminal domain of its amino group can bind to the terminal domain of the amino group of CD47 to function, the intracellular domain of signal regulatory protein α is a typical tyrosine inhibitory receptor sequence, activated by its own linked Src tyrosine phosphatase (SH2), Src tyrosine phosphatase (SH2) includes two parts, SHP-1 and SHP-2, the downstream signal pathway is regulated by phosphorylation process CD47 is a natural ligand of SIRP α, the CD 638-P6 signal pathway plays a role in various physiological mechanisms, the SIRP α signal pathway plays a role in the early stage of tumor phagocytosis of the SIREP α, which is designed to prevent tumor cells from developing anti-tumor phagocytosis, thus the early stage of the SIREP 354642 antibody which can prevent tumor cell phagocytosis.

Disclosure of Invention

The invention aims at the prior art and provides hybridoma cell strains 12G6, an anti-human SIRP α monoclonal antibody generated by the hybridoma cell strains, a preparation method of the monoclonal antibody, an amino acid sequence and a nucleic acid sequence of a variable region of the monoclonal antibody, and pharmaceutical application of the monoclonal antibody.

The technical scheme is as follows; the hybridoma cell strain 12G6 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2019261, address: wuhan university in Wuhan City, preservation time: 2019, 10 and 29.

The hybridoma cell strain 12G6 generates an anti-human SIRP α monoclonal antibody.

The anti-human SIRP α monoclonal antibody has the amino acid sequence shown in SEQ ID NO. 4 as CDRH1, the amino acid sequence shown in SEQ ID NO. 6 as CDRH2, the amino acid sequence shown in SEQ ID NO. 8 as CDRH3, the amino acid sequence shown in SEQ ID NO. 12 as CDRL1, the amino acid sequence shown in SEQ ID NO. 14 as CDRL2 and the amino acid sequence shown in SEQ ID NO. 16 as CDRL3 in a heavy chain variable region.

, the heavy chain variable region of the anti-human SIRP α monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2, and the light chain variable region thereof is the amino acid sequence shown in SEQ ID NO. 10.

The anti-human SIRP α monoclonal antibody also comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgM or IgA subtypes and a light chain constant region selected from Kappa or lambda subtypes.

preferably, the anti-human SIRP α monoclonal antibody further comprises a heavy chain constant region selected from the group consisting of the IgG2b subtype and a light chain constant region selected from the group consisting of the Kappa subtype.

The invention also discloses nucleic acids for encoding the anti-human SIRP α monoclonal antibody, wherein the heavy chain variable region is shown as SEQ ID NO. 1, and the light chain variable region is shown as SEQ ID NO. 9.

, pharmaceutical compositions, detection reagents or kits comprising the above anti-human SIRP α monoclonal antibodies are also within the scope of the invention.

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

The invention takes commercial recombinant human SIRP α -hFc fusion Protein as immunogen, immunizes Balb/c mice, when the serum titer meets the fusion requirement, takes spleen cells and SP2/0-Ag14 myeloma cells for cell fusion, obtains hybridoma cell strain 12G6 capable of stably secreting anti-human SIRP α antibody through HAT selective medium screening, after subcloning and expanding culture, transfers and sequences antibody heavy chain and light chain variable region genes on molecular level.

The invention successfully prepares the hybridoma cell strain 12G6, generates the anti-human SIRP α monoclonal antibody through the hybridoma cell strain, has good specificity and high affinity, can combine human and cynomolgus monkey SIRP α with high affinity on the cellular level, and simultaneously can block the combination of CD47 and SIRP α, and the anti-human SIRP α monoclonal antibody is a potential drug for tumor immunotherapy.

Drawings

FIG. 1 is a schematic diagram of the structure of a shuttle vector used in the construction of a stable cell line of human SIRP α and cynomolgus SIRP α overexpression CHO-K1, wherein MCS is a multiple cloning site, and a target gene is inserted into the position;

FIG. 2 flow assay results of human and cynomolgus SIRP α overexpressing stable cell lines, in which (1) bar graph A represents CHO-K1 mother cells, bar graph B represents human SIRP α overexpressing cell line finally selected for subsequent experiments, in (2) bar graph A represents CHO-K1 mother cells, bar graph B represents cynomolgus SIRP α overexpressing cell line finally selected for subsequent experiments (fluorescence intensity on abscissa, cell number on ordinate);

FIG. 3 shows the results of the indirect ELISA reaction titers of antiserum and human SIRP α recombinant protein of 2 immunized mice on day 7 after the 3 rd immunization (the abscissa is the dilution factor of antiserum in thousands, and the ordinate is the optical density value (OD) at a wavelength of 450 nm);

FIG. 4 shows the results of flow-based assay of antisera from 2 immunized mice on day 7 after the 3 rd immunization in combination with an overexpression cell line of human SIRP α, in which the A bar chart represents a PBS blank control group, the B bar chart represents the #2 mouse antisera, and the C bar chart represents the #1 mouse antisera (fluorescence intensity on the abscissa and cell number on the ordinate);

FIG. 5 shows the SDS-PAGE results of the denaturing reduction of the purified anti-human SIRP α monoclonal antibody;

FIG. 6 shows the result of ELISA detection for anti-human SIRP α monoclonal antibody subtype identification;

FIG. 7 shows the results of flow-based assays of the binding of purified anti-human SIRP α monoclonal antibody to human and cynomolgus SIRP α overexpressing stable cell lines, wherein the A, B, C histograms represent the binding signals to CHO-K1 blast cell line, cynomolgus SIRP α overexpressing cell line, human SIRP α overexpressing cell line, respectively (the abscissa is fluorescence intensity and the ordinate is cell number);

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

Detailed Description

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

Example 1 construction of human SIRP α and Macaca fascicularis SIRP α overexpressing CHO-K1 Stable cell lines

1) Shuttle vector construction

Human SIRP α protein sequence

NP_542970tyrosine-protein phosphatase non-receptor type substrate1isoform 1precursor[Homo sapiens]

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

54,967Da, 504aa, 1-30 are signal peptides, 31-373 are extracellular domains, 374-.

Macaca fascicularis SIRP α protein sequence

XP_015313155PREDICTED:tyrosine-protein phosphatase non-receptor typesubstrate 1isoform X2[Macaca fascicularis]

MEPAGPAPGRLGPLLCLLLTASCAWSGVLGEEELQVIQPEKSVSVAAGDSATLNCTVSSLIPVGPIQWFRGAGPGRELIYNLKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVICEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVTKFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRDDVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPRAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

54,841Da，503aa。

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 (Beijing Beiner) cells, and after 3 days of culture, puromycin (Wuhanbo' er, working concentration 2.5. mu.g/ml) was added, and culture was continued for 3 days (after which the cell culture was entirely carried out using 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

The commercial mouse anti-human SIRP α monoclonal antibody is used for carrying out simple immunofluorescence staining, and the expression quantity of the constructed cell strain relative to the non-transfected control cell SIRP α protein is detected, the indirect immunofluorescence labeling 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 SIRP α monoclonal antibody (R & D SYSTEMS MAB4546, 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) Add 50. mu.l of 20-fold diluted APC-labeled goat anti-mouse IGG fluorescent secondary antibody (R)&D SYSTEMS F0101B，2.5μl/2.5×10⁵) 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 test results show that two stable cell strains with high expression of human SIRP α and cynomolgus SIRP α are finally screened, and as shown in (1) and (2) in figure 2, the cell strain with the highest expression level of SIRP α is selected for subsequent experiments.

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

Selecting 2 Balb/c mice (Beijing sbefu) with the age of 6-8 weeks, feeding the mice in a cage, respectively numbering #1 and #2, collecting blood of about 0.05ml of each mouse through a tail vein 4 days before first immunization, placing the mice for half an hour at 4 ℃, centrifuging the mice at 10000rpm and 4 ℃ for 10min, separating serum to serve as negative control serum for later experiments, fully mixing recombinant human SIRP α -hFc fusion protein immunogen (Acro Biosystems SIA-H5251) and equal volume Freund's complete adjuvant (SIGMA) during first immunization, carrying out ultrasonic emulsification completely, adopting subcutaneous two-point and intraperitoneal-two-point injection, carrying out immunization at an immunization dose of 50 mu g of immunogen for each mouse, carrying out second immunization and third immunization at intervals of 14 days and 35 days respectively, and carrying out two times of immunization for the first immunization, wherein the two times of immunization are different from the Freund's complete adjuvant to the Freund's incomplete adjuvant (SIGMA), simultaneously adjusting the immunization dose to 25 mu g, collecting blood of the tail vein 7 days after the third immunization, placing the mice through the tail vein for 4 min, separating blood serum through ELISA and carrying out flow-type centrifugation at 4 ℃ for measuring the serum at 4 rpm.

The antiserum titer was determined by indirect ELISA method, wherein the indirect ELISA method comprises coating the ELISA plate with recombinant human SIRP α -his fusion protein (Acro Biosystems SIA-H5225), 0.5. mu.g/ml, 50. mu.l/well, overnight at 4 ℃, pouring off the coating solution the next day, washing the plate 3 times with 0.05% PBST, adding 0.5% BSA (SIGMA) for blocking, 200. mu.l/well, 37 ℃, 1H, discarding the blocking solution, washing the plate 3 times with 0.05% PBST, adding 10 dilutions of the antiserum (IgG) diluted at a magnification of 1:1000, blank PBS, positive control of the preimmune serum (PBS), 50. mu.l/well, 37 ℃, 1 h.0.05% PBST for 3 times, adding 1:20000 diluted goat anti-mouse IgG (secondary antibody) labeled with horseradish peroxide (Jackson 115. rearch), reading the antibody (ELISA antibody binding to the ELISA plate) at room temperature of 50. mu.35. mu. mu.25, and adding the antibody after the washing the plate 3 times with PBS, adding the ELISA plate 3, the antibody (ELISA plate) for binding to the test reagent, and reading the antibody (ELISA plate) at room temperature, and the test result showed that the antibody binding to the high affinity of the ELISA plate 3 times of the ELISA plate 3, wherein the ELISA plate is observed by using a high affinity test kit is observed in the ELISA plate, and the ELISA plate is observed by ELISA plate (ELISA plate) after the detection result showed that the detection result showed by ELISA.

The detection result shows that the antiserum of 2 mice can be combined with the human SIRP α on the cell membrane, while the binding activity of the #1 mouse is higher (as shown in figure 4), which indicates that the mouse immunized by using the immunogen generates a high-affinity antibody capable of combining with the human SIRP α at a cellular level.

And selecting the #1 mouse with the highest binding activity with the SIRP α on the cell membrane of the human body for the fourth immunization at an interval of 28 days after the third 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.

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) SP2/0-Ag14 myeloma cells (Beijing Beiner) were revived at weeks prior to fusion, two days prior to cell fusion, SP2/0-Ag14 was expanded and allowed to be in logarithmic growth phase 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 pretreated SP2/0-Ag14 cells and splenocytes uniformly, centrifuging at 1000rpm × 5min, discarding the supernatant, washing the mixed cells twice with serum-free RPMI1640 basic medium (GIBCO), pouring out the supernatant for times, slowly dripping 1ml of PEG1450(SIGMA) pre-warmed at 37 ℃ onto the cell sediment, acting for 90s, immediately adding serum-free RPMI1640 basic medium pre-warmed at 37 ℃, finishing within 5 minutes, centrifuging for 800 × 5min after mixing, resuspending the cell sediment in 10% FBS Royacel + hybridoma supplement (ROCHE) -RPMI1640 medium containing HAT (SIGMA), spreading uniformly in 96-well plates, and culturing at 80 μ l/well 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 using recombinant human SIRP α -his fusion protein as an ELISA screening coating antigen, using goat anti-mouse IgG (Fab) secondary antibody marked by HRP as a detection antibody, selecting a hole which is positive in reaction with the recombinant human SIRP α -his fusion protein, then using flow cytometry to detect the binding activity of clone supernatants in the holes with human SIRP α and cynomolgus SIRP α overexpression CHO-K1 cell strains, selecting clone supernatants with high binding capacity with the two cell strains, using flow cytometry to detect the binding condition of the clone supernatants for blocking CD47 protein and cell surface SIRP α, finally selecting clones which can better block the binding of CD47 protein and cell surface SIRP α, observing a hybridoma cell or cell cluster with survival under a microscope, marking, using a limiting dilution method to clone cells in the hole, establishing a stable secretory antibody secreting hybridoma cell strain after two times of subclones, preserving the hybridoma cell strain with survival or cell cluster under a microscope, performing culture on a Wuhan-Mikan-Mitsu-Mitsui-Han-Mitsui culture collection center, preserving the hybridoma cell strain for number of the Wuhan-Hao-Haemon culture, preserving the Wuhan cell strain, preserving the Wuhan-Hao-K-7 hybrid cell.

Example 4 preparation and purification of anti-human SIRP α monoclonal antibody

1. Preparation of ascites

3 balb/c female mice (Beijing sbefu) with the age of 10-16 weeks are respectively taken, and each mouse is injected with 0.25ml of ascites to prepare the medicine about the abdominal cavity 12-18 days in advanceAdjuvant (Beijing bo olong). Taking the growth logarithmic phase hybridoma cell count, taking 6 x 10⁶The cells were washed twice with 10ml sterile PBS, centrifuged at 1000rpm for 5min, and finally adjusted to 2 x 10 cell concentration with sterile PBS⁶And (2) injecting 0.5ml of cell suspension into the left and right abdominal cavities of the mice respectively, massaging the abdominal cavities to uniformly distribute the cells, obviously raising the abdominal cavities of the mice after about 10-14 days and collecting ascites, collecting the ascites for times every other day like , collecting the ascites for three times, collecting the supernatant (absorbing the uppermost layer of grease as much as possible by using a pipette) after centrifuging for 5min at 2000rpm, storing the supernatant at 4 ℃ for a short time and storing the supernatant at-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 SIRP α monoclonal antibodies

1. Identification of antibody subtypes

The subtype of the obtained anti-human SIRP α monoclonal antibody is identified by a Mouse monoclonal antibody subtype identification kit (Proteintech), specific antibodies aiming at Mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, Kappa light chain and lambda light chain are pre-coated on an enzyme label plate, the specific experimental operation is described in a kit instruction, the result is shown in figure 6, and the heavy chain subtype of the obtained anti-human SIRP α monoclonal antibody is IgG2b, and the light chain subtype is 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 antibody and SIRP α on cell membrane

Referring to the indirect immunofluorescent-labeled sample preparation method described in example 1, flow cytometry was used to detect the binding of the purified anti-human SIRP α monoclonal antibody to human and cynomolgus SIRP α -overexpressed SIRP α on cell membranes, as shown in FIG. 7, the results show that the anti-human SIRP α monoclonal antibody obtained by us can bind to human SIRP α on cell membranes with higher affinity, while the binding ability to cynomolgus SIRP α is slightly weaker than the binding ability to human SIRP α.

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

SIRP α is a ligand of CD47, CD47 protein can be combined with SIRP α on the cell surface by flow detection, and based on the result, a flow cytometry method is used for detecting that an anti-human SIRP α monoclonal antibody blocks the combination of CD47 and SIRP α on the cell surface.A human SIRP α overexpression cell strain is treated according to the indirect immunofluorescent labeling sample preparation method described in example 1, 1 mu g of CD47-hFc protein (Acro Biosystems CD7-H5256) is added into each group, and an anti-human SIRP α monoclonal antibody is added into the group for co-incubation, and the flow cytometry is used for detecting the combination of the CD47-hFc protein and the human SIRP α overexpression cell strain.from the flow result (FIG. 8), the anti-human SIRP α monoclonal antibody can better block the combination of the CD47 protein and the SIRP α on the cell surface.

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 SIRP α 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, th cDNA Strand was synthesized using First Strand cDNA Synthesis Kit and Ig-3'constant region primer, PCR amplification was performed using Ig-5' primers and NovaTaq DNApolymerase using th cDNA Strand as a template, and the resulting PCR amplification product was cloned into a cloning Vector using Vector cloning Kit and subjected to screening, and DNA was isolated for gene sequencing.

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

In GenBank, the results of alignment and sequencing with the nucleic acid sequences of the mouse antibodies show that the homology of the variable region sequences of the light chains and the heavy chains of the antibodies and the submitted variable region sequences of the mouse IgG exceeds 96 percent, and the gene sequences obtained by sequencing are determined to be the sequences of the mouse antibodies. 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：

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGATTTGAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACAAATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGACAGGACTGCCTTTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCAACATATTTCTGTGCAAGAGGGCATCACTATGGTAACTACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

SEQ ID NO2：

QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMHWVKQAPGKDLKWMGWINTYTGEPTNADDFKGRFAFSLETSDRTAFLQINNLKNEDTATYFCARGHHYGNYAWFAYWGQGTLVTVSA

SEQ ID NO3：

AACTATGGAATGCAC

SEQ ID NO4：

NYGMH

SEQ ID NO5：

TGGATAAACACCTACACTGGAGAGCCAACAAATGCTGATGACTTCAAGGGA

SEQ ID NO6：

WINTYTGEPTNADDFKG

SEQ ID NO7：

GGGCATCACTATGGTAACTACGCCTGGTTTGCTTAC

SEQ ID NO8：

GHHYGNYAWFAY

SEQ ID NO9：

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTCTTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAAGTACTGATTTATTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCGCAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGTAGTGTTCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAT

SEQ ID NO10：

DIVMTQSHKFMSTSVGDRVSITCKASQDVSLAVAWYQQKPGQSPKVLIYWASTRHTGVPDRFAGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIN

SEQ ID NO11：

AAGGCCAGTCAGGATGTGAGTCTTGCTGTAGCC

SEQ ID NO12：

KASQDVSLAVA

SEQ ID NO13：

TGGGCATCCACCCGGCACACT

SEQ ID NO14：

WASTRHT

SEQ ID NO15：

CAGCAACATTATAGCACTCCGTGGACG

SEQ ID NO16：

QQHYSTPWT

Sequence listing

<110> Zhejiang Landun pharmaceuticals Co., Ltd

<120> hybridoma cell line 12G6, antibody and application thereof

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<213> Artificial Sequence (Artificial Sequence)

<400>1

cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctggata taccttcaca aactatggaa tgcactgggt gaagcaggct 120

ccaggaaagg atttgaagtg gatgggctgg ataaacacct acactggaga gccaacaaat 180

gctgatgact tcaagggacg gtttgccttc tctttggaaa cctctgacag gactgccttt 240

ttgcagatca acaacctcaa aaatgaggac acggcaacat atttctgtgc aagagggcat 300

cactatggta actacgcctg gtttgcttac tggggccaag ggactctggt cactgtctct 360

gca 363

<210>2

<211>121

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met His Trp Val Lys Gln Ala Pro Gly Lys Asp Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Asn Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Asp Arg Thr Ala Phe

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Gly His His Tyr Gly Asn Tyr Ala Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ala

115 120

<210>3

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aactatggaa tgcac 15

<210>4

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Asn Tyr Gly Met His

1 5

<210>5

<211>51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tggataaaca cctacactgg agagccaaca aatgctgatg acttcaaggg a 51

<210>6

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Asn Ala Asp Asp Phe Lys

1 5 10 15

Gly

<210>7

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gggcatcact atggtaacta cgcctggttt gcttac 36

<210>8

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Gly His His Tyr Gly Asn Tyr Ala Trp Phe Ala Tyr

1 5 10

<210>9

<211>321

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgagt cttgctgtag cctggtatca acaaaaacca 120

gggcaatctc ctaaagtact gatttattgg gcatccaccc ggcacactgg agtccctgat 180

cgcttcgcag gcagtggatc tgggacagat tatactctca ccatcagtag tgttcaggct 240

gaagacctgg cactttatta ctgtcagcaa cattatagca ctccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa t 321

<210>10

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

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

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Leu Ala

20 25 30

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

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ala Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn

100 105

<210>11

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

aaggccagtc aggatgtgag tcttgctgta gcc 33

<210>12

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Lys Ala Ser Gln Asp Val Ser Leu Ala Val Ala

1 5 10

<210>13

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

tgggcatcca cccggcacac t 21

<210>14

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Trp Ala Ser Thr Arg His Thr

1 5

<210>15

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

cagcaacatt atagcactcc gtggacg 27

<210>16

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Gln Gln His Tyr Ser Thr Pro Trp Thr

1 5

Claims

1. The hybridoma cell line 12G6 is characterized in that the preservation number of the hybridoma cell line is CCTCC NO: C2019261.

2. The monoclonal antibody against human SIRP α produced by the hybridoma cell line 12G6 of claim 1.

3. The anti-human SIRP α monoclonal antibody according to claim 2, wherein the CDRH1 of the heavy chain variable region is the amino acid sequence shown by SEQ ID NO. 4, the CDRH2 is the amino acid sequence shown by SEQ ID NO. 6, the CDRH3 is the amino acid sequence shown by SEQ ID NO. 8, the CDRL1 of the light chain variable region is the amino acid sequence shown by SEQ ID NO. 12, the CDRL2 is the amino acid sequence shown by SEQ ID NO. 14, and the CDRL3 is the amino acid sequence shown by SEQ ID NO. 16.

4. The anti-human SIRP α monoclonal antibody according to claim 2 or 3, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO. 2.

5. The anti-human SIRP α monoclonal antibody according to claim 2 or 3, wherein the light chain variable region is the amino acid sequence shown in SEQ ID NO. 10.

6. The anti-human SIRP α monoclonal antibody according to claim 2 or 3, further comprising 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.

7, nucleic acids encoding the anti-human SIRP α monoclonal antibody of claim 2 or 3, characterized in that its heavy chain variable region is shown in SEQ ID NO. 1 and its light chain variable region is shown in SEQ ID NO. 9.

A pharmaceutical composition of comprising the monoclonal antibody of claim 2 or 3.

9, A detection reagent or kit comprising the monoclonal antibody of claim 2 or 3.

10. Use of the monoclonal antibody of claim 2 or 3 in the preparation of a medicament for treating a disease associated with abnormal or excessive, uncontrolled expression of human SIRP α.