CN110724670B

CN110724670B - Hybridoma cell strain 3H4, antibody and application thereof

Info

Publication number: CN110724670B
Application number: CN201911050232.7A
Authority: CN
Inventors: 郭志刚; 黄子祥; 杨洋; 李梦晗
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: 2021-04-13
Anticipated expiration: 2039-10-31
Also published as: CN110724670A

Abstract

The invention belongs to the field of biotechnology, and particularly discloses a hybridoma cell strain 3H4, an anti-human CD70 monoclonal antibody generated by the hybridoma cell strain and application of the anti-human CD70 monoclonal antibody.

Description

Hybridoma cell strain 3H4, antibody and application thereof

Technical Field

The invention belongs to a biological genetic engineering antibody technology, and particularly relates to a hybridoma cell strain 3H4, an anti-human CD70 monoclonal antibody generated by the same and application thereof.

Background

CD70 is a type ii transmembrane glycoprotein and also belongs to the TNF family. Human CD70 is a trimer of 193 amino acids in which the extramural 155 amino acid sequence shares some homology with other members of the TNF ligand superfamily. The cytokine receptor CD27 is a member of the tumor necrosis factor receptor (TFNR) superfamily, which plays a role in cell growth and differentiation as well as apoptosis. The ligand for CD27 is CD70, which belongs to the tumor necrosis factor family of ligands.

Normally, CD70 is only transiently expressed on the surface of activated T and B lymphocytes and mature DC cells, and is not normally expressed on other non-lymphoid normal tissues or cells. However, in many hematologic and solid tumors, CD70 is constitutively expressed on the surface of tumor cells, including renal cell carcinoma, metastatic breast cancer, brain tumors, leukemia, lymphoma, and nasopharyngeal carcinoma, among others. CD27 is expressed primarily on mature T lymphocytes, memory B lymphocytes, developmental center B cells, and NK cells, as well as on some hematologic tumor cells expressing CD 27. CD70 interacts with CD27 to allow the intracellular segment of CD27 to bind to TRAF2/5, thereby activating NF-KB and c-Jun kinase signaling pathways, causing cell proliferation, survival, differentiation and the like. In addition, CD70 binding to CD27 may also cause the intracellular segment of CD27 to bind to Siva, thereby causing Caspase-mediated apoptosis.

CD70, constitutively expressed on the surface of tumor cells, promotes immune escape from tumors mainly by four possible mechanisms. 1. Increasing the number of immunosuppressive regulatory T cells; 2. inducing T cell apoptosis; 3. inducing T cell depletion; 4. promote the proliferation and survival of the tumor cells co-expressing CD 27. Based on the characteristics, the CD70 has the following advantages as an anti-tumor therapeutic target: 1. a wider range of indications; 2. the specificity is strong, the toxic effect is small, and the kit is suitable for selecting target spots of CART; the CD70 antibody can mediate endocytosis and thus be an excellent target for ADCs. Currently, no antibody against CD70 is available on the market, and basically, the antibody is mainly used as ADC in different clinical periods.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the prior art, the invention provides a hybridoma cell strain 3H4, an anti-human CD70 monoclonal antibody generated by the hybridoma cell strain, a preparation method of the anti-human CD70 monoclonal antibody, and an amino acid sequence and a nucleic acid sequence of a variable region of the anti-human CD70 monoclonal antibody. The invention further provides pharmaceutical application of the monoclonal antibody.

The technical scheme is as follows: the hybridoma cell strain 3H4 is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: C2019258, address: wuhan university in Wuhan City, preservation time: 2019, 10 and 29.

The anti-human CD70 monoclonal antibody produced by the hybridoma cell strain.

The CDRH1 of the heavy chain variable region of the anti-human CD70 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 CD70 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 CD70 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.

Preferably, the anti-human CD70 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 CD70 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 CD70 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 CD70 is also within the protection scope of the invention. The diseases include tumors, cancers, and the like.

The invention takes a commercial recombinant human CD70-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 3H4 capable of stably secreting an anti-human CD70 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 activity.

Has the advantages that: the invention successfully prepares a hybridoma cell strain 3H4, generates an anti-human CD70 monoclonal antibody through the hybridoma cell strain, has good specificity and high affinity, can be combined with human and cynomolgus monkey CD70 with high affinity on a cellular level, and 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 CD70 and cynomolgus monkey CD70 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 CD70 overexpressing stable cell lines, wherein in FIG. 1, the A histogram represents CHO-K1 mother cells, the B histogram represents human CD70 overexpressing cell lines, in FIG. 2, the A histogram represents CHO-K1 mother cells, and the B histogram represents cynomolgus monkey CD70 overexpressing cell lines (the abscissa is fluorescence intensity and the ordinate is cell number);

FIG. 3: the indirect ELISA reaction titer results of antiserum and human CD70 recombinant protein of 3 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: 3 immunized mice combined flow assay results of antiserum and 786-0 renal cancer cell line on day 7 after 3 immunization, wherein the A column represents PBS control group, and the B, C, D column represents #1, #2, #3 mouse antiserum (fluorescence intensity on abscissa and cell number on ordinate), respectively;

FIG. 5: the purified anti-human CD70 monoclonal antibody denaturalizes and reduces SDS-PAGE electrophoresis results;

FIG. 6: identifying an ELISA detection result of an anti-human CD70 monoclonal antibody subtype;

FIG. 7: the purified anti-human CD70 monoclonal antibody and 786-0 renal cancer cell line combined flow detection results, wherein the A bar chart represents a PBS control group, the B bar chart represents a commercial mouse anti-human CD70 monoclonal antibody, and the C bar chart represents the purified mouse anti-human CD70 monoclonal antibody (the abscissa is fluorescence intensity and the ordinate is cell number).

Detailed Description

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

Example 1: construction of human CD70 and cynomolgus monkey CD70 overexpression CHO-K1 stable cell line

1) Shuttle vector construction

Human CD70 protein sequence

NP_001243CD70 antigen isoform 1[Homo sapiens]

MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPL ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDG IYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRL TPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP

21,118Da, 193aa, 1-17 are endodomains, 18-38 are transmembrane helices, and 39-193 are ectodomains.

Cynomolgus monkey CD70 protein sequence

XP_005587773PREDICTED:CD70 antigen[Macaca fascicularis]

MNGPRKNEVEREIGRSGGEGLGTGNSVAHPRPLPGPSGNHLHPLCELQT GSSWREFPLANRSSPSPRPAGHPQRGAGWSPDKLRQVDAQEPREGAAVAFLPF PAALCAPLAPPALAEVIAAAMPEEGSGCAVRRRPYACVLRAAVVPLVAGLAIC LVVCVQRLSRAQQQLPLESLGWDIAELQLNHTGPQQDPRLYWQGGPALGRSF LRGPELDKGQLRIRRDGIYMVHIQVTLAICSSTSTSRHHHPTTLAVGICSPASRS ISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP

33,971Da，316aa。

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 correct recombinant plasmid was sequenced, amplified in clonobacteria DH5 alpha (Wuhanbo), and plasmid extraction was performed using an endotoxin-free plasmid macroextraction kit (Wuhanbo). After extraction, the molecular weight was determined by nucleic acid electrophoresis, and the quality of the plasmid was evaluated using an ultramicro spectrophotometer. The ratio of A260 to A280 is 1.7-1.9, the ratio of OD260 to 230 is >20, and the concentration is higher than 0.5 mu g/mu l, so that the plasmid is high in quality.

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 CD70 monoclonal antibody, and detecting the expression quantity of the constructed cell strain relative to the CD70 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) Adding 50 μ l of commercial mouse anti-human CD70 monoclonal antibody (Abcam ab77868, 10 μ g/ml), mixing, and reacting at room temperature in the dark 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 detection result shows that finally, a stable cell line with high expression of human CD70 and cynomolgus monkey CD70 is screened out, and is respectively shown as (1) and (2) in figure 2, wherein, (1) is high expression human CD70, and (2) is high expression cynomolgus monkey CD 70.

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

Selecting 3 Balb/c mice (Beijing sbefu) with the age of 6-8 weeks for breeding in the same cage, and numbering the mice #1, #2 and #3 respectively. 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, recombinant human CD70-mFc fusion protein immunogen (Acro Biosystems CDL-H525a) 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 enzyme plates were coated with recombinant human CD70-hFc fusion protein (Acro Biosystems TN7-H526x), 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 ImmunoResearch 115. sup. 035. sup. 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 3 mice all reached 1:256000 (see FIG. 3), indicating that immunization of mice with the immunogen produced high affinity antibodies that bound human CD70 at the recombinant protein level.

The binding activity of antiserum to CD70 on human cell membranes was measured by flow cytometry, as described in example 1 for indirect immunofluorescent-labeled sample preparation, using a human renal carcinoma cell line 786-0 (Beijing Beiner), which was shown to highly express CD70 on cell membranes. The results of the assay showed that 3 mice had all antisera that bound 786-0 cell membrane CD70, while #1 had the highest binding activity (FIG. 4), indicating that immunization of mice with the immunogen produced high affinity antibodies that bound human CD70 at the cellular level.

After the third immunization, the #1 mouse with the highest binding activity with CD70 on the cell membrane of the human is selected at an interval of 28 days for the fourth immunization, the immunization is carried out without using an adjuvant and the tail vein injection is adopted, the immunization dose is 25 mug 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 cells in each well were tested for antibody secretion by indirect ELISA. Recombinant human CD70-hFc fusion protein is used as ELISA screening coating antigen, goat anti-mouse IgG (Fab) secondary antibody marked by HRP is used as detection antibody, and a hole which reacts positively with the recombinant human CD70-hFc fusion protein is selected. And then detecting the binding activity of the clone supernatants in the wells with human CD70 and cynomolgus monkey CD70 overexpression CHO-K1 cell lines by adopting flow cytometry, finally selecting a clone which has higher binding activity with the two cell lines, observing a viable hybridoma cell or cell mass under a microscope, marking, cloning the cells in the wells by adopting a limiting dilution method, establishing a hybridoma cell strain stably secreting the monoclonal antibody after twice subcloning, and carrying out expanded culture. The hybridoma cell strain is named as 3H4 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2019258 and the address: wuhan university in Wuhan City, preservation time: 2019, 10 and 29.

Example 4 preparation and purification of anti-human CD70 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 6x 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 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 CD70 monoclonal antibody

1. Identification of antibody subtypes

The subtype of the obtained anti-human CD70 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 CD70 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 CD70 on cell membranes

A cell line 786-0 with high cell surface expression of CD70 was selected, and the binding of the purified anti-human CD70 monoclonal antibody to CD70 on the cell membrane was examined by flow cytometry in accordance with the indirect immunofluorescent-labeled sample preparation method described in example 1. As shown in the results of fig. 7, the anti-human CD70 monoclonal antibody obtained by us can bind to CD70 on the cell membrane with an affinity better than that of the control commercial antibody.

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 CD70 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 Total RNA was isolated from the pooled hybridoma cell lines and then purified using the First Strand cDNA Synthesis KitAnd Ig-3'constant region primer, then using Ig-5' primers and NovaTaq DNA Polymerase to perform PCR amplification by using the first cDNA strand as a template, Cloning the obtained PCR amplification product into a Cloning Vector by using Vector Cloning Kit, screening, and isolating DNA for gene sequencing.

2. Gene sequencing and analysis of heavy and light chain variable domain of anti-human CD70 monoclonal antibody

In GenBank, the results of alignment sequencing and mouse antibody nucleic acid sequences show that the variable region sequences of the light chain and the heavy chain of the antibody have 100 percent of homology with the submitted mouse IgG variable region sequences, 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 antibody 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 NO：1：

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGA GACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCA ATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTG GATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAGGGAC GGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAA CAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTATAGACCCCATA AGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO：2：

QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWM GWINTETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCAIDPIRY FDVWGAGTTVTVSS

SEQ ID NO：3：

GACTATTCAATGCAC

SEQ ID NO；4：

DYSMH

SEQ ID NO：5：

TGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAG GGA

SEQ ID NO：6：

WINTETGEPTYADDFKG

SEQ ID NO：7：

GACCCCATAAGGTACTTCGATGTC

SEQ ID NO：8：

DPIRYFDV

SEQ ID NO：9：

GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGA GAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAG CATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATG CAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCA GGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGG AGTTATTACTGTCAACATTTTTGGAGTACTCCATTCACGTTCGGCTCGGGGA CAAAGTTGGAAATAAAA

SEQ ID NO：10：

DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVY NAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPFTFGSGT KLEIK

SEQ ID NO：11：

CGAGCAAGTGGGAATATTCACAATTATTTAGCA

SEQ ID NO：12：

RASGNIHNYLA

SEQ ID NO：13：

AATGCAAAAACCTTAGCAGAT

SEQ ID NO：14：

NAKTLAD

SEQ ID NO：15：

CAACATTTTTGGAGTACTCCATTCACG

SEQ ID NO：16：

QHFWSTPFT

Sequence listing

<110> Zhejiang Landun pharmaceuticals Co., Ltd

<120> hybridoma cell strain 3H4, antibody and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

caggtgcagc tgaatcagtc aggacctggc ctagtgcagc cctcacagac cctgtccatc 60

acctgcacag tctctggttt ctcattaacc acctatggtg tacactggat tcgccagcct 120

ccaggaaagg gtctggagtg gctgggagtg atatggagtg gtggaagcac agactatgac 180

actactttca tatccagact gagcatcagc aaggacagct ccaagagcca agttttcttt 240

aaaataaaca gtctgcaagc tgatgacaca gccatgtact actgtgccag aaatacctac 300

tatggtaacc tttatgcttt ggactattgg ggtcaaggaa cctcagtcac cgtctcctca 360

<210> 2

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Val Gln Leu Asn Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asp Thr Thr Phe Ile

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Ile Asn Ser Leu Gln Ala Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Asn Thr Tyr Tyr Gly Asn Leu Tyr Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 3

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

acctatggtg tacac 15

<210> 4

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Thr Tyr Gly Val His

1 5

<210> 5

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtgatatgga gtggtggaag cacagactat gacactactt tcatatcc 48

<210> 6

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asp Thr Thr Phe Ile Ser

1 5 10 15

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aatacctact atggtaacct ttatgctttg gactat 36

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asn Thr Tyr Tyr Gly Asn Leu Tyr Ala Leu Asp Tyr

1 5 10

<210> 9

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gacattgtgc tgacacagtc tcctgcttcc ttagttgtat ctctggggca gggggccacc 60

atctcttgca aggccagcga aagtgtcagt gcatctggct atagttttat gcactggtac 120

caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga gtttccgctc 300

acgttcggtg cagggaccaa gctggagctg aaa 333

<210> 10

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

Gln Gly Ala Thr Ile Ser Cys Lys Ala Ser Glu Ser Val Ser Ala Ser

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaggccagcg aaagtgtcag tgcatctggc tatagtttta tgcac 45

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Lys Ala Ser Glu Ser Val Ser Ala Ser Gly Tyr Ser Phe Met His

1 5 10 15

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cttgcatcca acctagaatc t 21

<210> 14

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 15

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cagcacagta gggagtttcc gctcacg 27

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln His Ser Arg Glu Phe Pro Leu Thr

1 5

Claims

1. The hybridoma cell strain 3H4 is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2019258.

2. The monoclonal antibody against human CD70 produced by the hybridoma cell line of claim 1.

3. The anti-human CD70 monoclonal antibody of claim 2, wherein the CDRH1 of the heavy chain variable region 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.

4. The monoclonal antibody against human CD70 according to claim 2 or 3, wherein the heavy chain variable region is SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

5. The monoclonal antibody against human CD70, according to claim 2 or 3, wherein the light chain variable region is SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

6. The anti-human CD70 monoclonal antibody of claim 2 or 3, wherein the heavy chain constant region is of IgG1 subtype and the light chain constant region is of Kappa subtype.

7. A nucleic acid encoding the anti-human CD70 monoclonal antibody of claim 2 or 3, wherein the heavy chain variable region has the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO: shown at 9.

8. A detection reagent comprising the monoclonal antibody of claim 2 or 3.