CN109160949B - Mouse anti-human PD-1 monoclonal antibody and application - Google Patents

Abstract

Translated fromChinese

本发明涉及一种鼠抗人PD‑1单克隆抗体及应用，本发明通过小鼠杂交瘤单克隆抗体筛选及RT‑PCR法克隆Ig可变区基因，共获得1株稳定分泌抗人PD‑1抗体的杂交瘤及其可变区序列，本发明提供的鼠抗人PD‑1单克隆抗体与人PD‑1蛋白具有高亲和性，能以1.9x10^‑8M的Kd值与人PD‑1结合。且结合特异性强，不与人CTLA‑4蛋白及人PD‑1阴性的细胞(如Raji细胞)产生交叉反应。能通过Western Blot的方法特异性结合线性化人PD‑1蛋白。基于这些特性这此单克隆抗体既可用于人PD‑1蛋白的检测，也能够单独或与其它方法联合应用在肿瘤免疫治疗中。

The present invention relates to a mouse anti-human PD-1 monoclonal antibody and its application. The invention uses mouse hybridoma monoclonal antibody screening and RT-PCR method to clone Ig variable region genes, and a total of 1 strain that stably secretes anti-human PD-1 is obtained. 1 The hybridoma of the antibody and the variable region sequence thereof, the mouse anti-human PD-1 monoclonal antibody provided by the present invention has high affinity with the human PD-1 protein, and can interact with human PD with a Kd value of 1.9×^10-8 M ‑1 combined. And the binding specificity is strong, and it does not cross-react with human CTLA-4 protein and human PD-1-negative cells (such as Raji cells). It can specifically bind to the linearized human PD-1 protein by the method of Western Blot. Based on these properties, this monoclonal antibody can be used for the detection of human PD-1 protein, and can also be used alone or in combination with other methods in tumor immunotherapy.

Description

Mouse anti-human PD-1 monoclonal antibody and application

Technical Field

The invention relates to the technical field of immunology, in particular to a mouse anti-human PD-1 monoclonal antibody and application thereof.

Background

Tumor immunotherapy has recently received much attention and is the focus of the tumor therapy field. Among them, targeting immune checkpoints is becoming one of the most promising approaches to treating cancer patients. The immune check point molecules belong to the members of the B7/CD28 and TNF/TNFR superfamily, are immune co-stimulating or co-inhibiting molecules, and influence the immune response of the organism by cooperatively regulating the activity of lymphocytes in real time. PD-1, also known as CD279, is a member of the B7-CD28 receptor family. PD-1 is a 55kD type I surface transmembrane glycoprotein receptor, consisting of 288 amino acids. PD-1 is combined with its ligand PD-L1 or PD-L2, so that two tyrosine signal motifs in the cytoplasmic region of PD-1 are phosphorylated, thereby activating downstream signal pathways, finally destroying sugar metabolism of T cells and generation of signal factors such as IL-2, and leading the T cells to lose immune function. Therefore, the blocking of the interaction between PD-1 and the ligand thereof can improve the immunocompetence of the tumor specific T cell and is beneficial to the immune system to eliminate the tumor cell, so that PD-1 becomes a hot target for developing tumor immunotherapy drugs. At present, a plurality of Anti-PD-1/PD-L1 antibodies are rapidly developed in clinical research of tumor immunotherapy. For example, Pembrolizumab and Nivolumab have been FDA approved for advanced melanoma, and Nivolumab has also been FDA approved in the United states for treatment of advanced squamous non-small cell lung cancer. In addition, MPDL3280A (anti-PD-L1 monoclonal antibody), Avelumab (anti-PD-L1 monoclonal antibody) and the like have also entered various advanced clinical studies covering various tumor species such as non-small cell cancer, melanoma, bladder cancer and the like. However, data from clinical trials show that only a fraction of patients respond to treatment. Nivolumab, for example, is a PD-1 drug developed by BMS. In the current, late-stage study conducted, only 3 of 5 cancer groups, treatment promoted tumor shrinkage, including 18% in 72 lung cancer patients, less than one-third in 98 melanoma patients, and 27% in 33 renal cancer patients. lambrolizumab was a PD-1 antibody developed by Merck and the results of its phase IB study showed that only 51% of 85 cancer patients also exhibited objective anti-tumor responses and only 9% showed complete responses.

At present, the mechanism of non-response of most patients to treatment is not clear, and a new anti-PD-1 antibody is still needed to provide a new idea for treatment.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel mouse anti-human PD-1 monoclonal antibody and a variable region sequence thereof, the antibody has high affinity and strong specificity with PD-1, and the variable region sequence thereof can be used for constructing a genetic engineering antibody.

The technical scheme adopted by the invention is as follows:

the invention provides a mouse anti-human PD-1 monoclonal antibody, which comprises a heavy chain variable region sequence and a light chain variable region sequence;

the heavy chain variable region sequence comprises three of the following sequences in the CDR region:

GYTFTSNTIT(CDRH1、SEQ ID NO:1)

YINPNSDFTNYNEKFRD(CDRH2、SEQ ID NO:2)

DYYGGSYYAMDY(CDRH3、SEQ ID NO:3)；

the light chain variable region sequence comprises three of the following sequences in the CDR region:

TADSSVPSSYLH(CDRL1、SEQ ID NO:4)

STSNLAS(CDRL2、SEQ ID NO:5)

HQYHRSPLT(CDRL3、SEQ ID NO:6)。

specifically, the invention provides a mouse anti-human PD-1 monoclonal antibody, which comprises a heavy chain variable region sequence and a light chain variable region sequence; the heavy chain variable region sequence is as follows: (SEQ ID NO:7)

QVKLQQSGAELARPGASVKMSCMASGYTFTSNTITWVKQRPGQGLEWIGYINPNSDFTNYNEKFRDKATLTADRSSSTAYMQLSSLTSEDSAVYYCARDYYGGSYYAMDYWGQGTSVTVSS

The light chain variable region sequence is as follows: (SEQ ID NO:8)

QIVLTQSPAIMSASLGERVTMTCTADSSVPSSYLHWYQQKPGSSPKFWIYSTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAGTKLELKRA。

The invention also provides a mouse anti-human PD-1 hybridoma cell strain 2F6 with the preservation number of CGMCC No. 15697.

The invention also provides a mouse anti-human PD-1 monoclonal antibody generated by the hybridoma cell strain 2F6 of the mouse anti-human PD-1 with the preservation number of CGMCC No. 15697.

The invention also provides a variable region sequence of the mouse anti-human PD-1 monoclonal antibody, which comprises a heavy chain variable region sequence and a light chain variable region sequence; the heavy chain variable region sequence is as follows: (SEQ ID NO:7)

QVKLQQSGAELARPGASVKMSCMASGYTFTSNTITWVKQRPGQGLEWIGYINPNSDFTNYNEKFRDKATLTADRSSSTAYMQLSSLTSEDSAVYYCARDYYGGSYYAMDYWGQGTSVTVSS

The light chain variable region sequence is as follows: (SEQ ID NO:8)

QIVLTQSPAIMSASLGERVTMTCTADSSVPSSYLHWYQQKPGSSPKFWIYSTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAGTKLELKRA。

The invention also provides a nucleotide molecule, wherein the nucleotide molecule codes the mouse anti-human PD-1 monoclonal antibody.

The nucleotide sequence of the heavy chain variable region of the nucleotide molecule coding mouse anti-human PD-1 monoclonal antibody is as follows: (SEQ ID NO:9)

CAGGTGAAGCTTCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCATGGCTTCTGGCTACACTTTTACTAGTAACACGATAACCTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAACAGTGATTTTACTAATTACAATGAGAAGTTCAGGGACAAGGCCACATTGACTGCAGACAGATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGATTACTACGGTGGTTCCTACTATGCTATGGACTACTGGGGTCAGGGAACTTCAGTCACCGTCTCCTCA；

The nucleotide sequence of the light chain variable region of the nucleotide molecule coding mouse anti-human PD-1 monoclonal antibody is as follows: (SEQ ID NO:10)

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCGACTCAAGTGTACCTTCCAGTTACTTGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAATTCTGGATTTATAGTACATCCAATCTGGCTTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAACTGAAACGGGCT。

The invention also provides application of the mouse anti-human PD-1 monoclonal antibody in a kit for detecting PD-1 protein.

The invention also provides a pharmaceutical composition, which comprises the mouse anti-human PD-1 monoclonal antibody and a pharmaceutically acceptable carrier.

The invention also provides the application of the mouse anti-human PD-1 monoclonal antibody in preparing medicaments for treating tumors, infectious diseases, autoimmune diseases or immune rejection.

The hybridoma cell strain 2F6 has the number of CGMCC No.15697, and the preservation addresses of the China general microbiological culture Collection center are as follows: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: year 2018, 5 month and 4 days.

The invention has the following beneficial effects:

the invention clones Ig variable region genes by mouse hybridoma monoclonal antibody screening and RT-PCR method, obtains 1 hybridoma which stably secretes anti-human PD-1 antibody and variable region sequence thereof, and identifies the antibody binding specificity by the methods of flow cytometry, Western Blot and the like.

The mouse anti-human PD-1 monoclonal antibody provided by the invention has high affinity with human PD-1 protein, and can be used for inhibiting the activity of the human PD-1 monoclonal antibody by 1.9x10^-8The Kd value of M binds to human PD-1. And has strong binding specificity, and does not generate cross reaction with human CTLA-4 protein and human PD-1 negative cells (such as Raji cells). Can be specifically combined with the linearized human PD-1 protein by a Western Blot method. Based on the characteristics, the monoclonal antibody can be used for detecting the human PD-1 protein, can be independently or jointly applied to tumor immunotherapy with other methods, and can be effectively applied to the preparation of medicines for treating tumors, infectious diseases, autoimmune diseases, immune rejection resistance and the like.

Drawings

FIG. 1 shows antibodies 2F6 and SP2/0 (hPD-1)⁺) Graph for analysis of affinity constants of cells.

FIG. 2 is a diagram of: the cross reaction of the antibody 2F6 and a PD-1 family member CTLA-4 is detected by flow cytometry, and the detected cell is a 3T3 cell over-expressing CTLA-4.

In fig. 2, the following are in sequence: left: negative control; the method comprises the following steps: CTLA-4 commercial antibody, positive control; and (3) right: 2F 6.

FIG. 3 is a flow cytometry assay for cross-reactivity of antibody 2F6 with Raji (human PD-1 negative cells).

In fig. 3, the following are in order: left: negative control; the method comprises the following steps: a commercial antibody to PD-1; and (3) right: 2F 6.

FIG. 4 shows FACS detection of binding of antibody 2F6 to Jurkat cells (human PD-1 positive cells).

FIG. 5: the Western Blot method detects the binding specificity of the antibody to human PD-1 in the total protein of Jurkat cells. And (3) loading sequence: left: marker; and (3) right: jurkat cell Total protein.

FIG. 6 shows FACS method to examine the competitive ability of antibody 2F6 to bind to Jurkat cell surface hPD-1 protein with commercial anti-hPD-1 antibody.

Sequentially comprises the following steps: the upper part 1: negative control; the upper 2: PD-1 commercial antibody 2.5ul (Positive control)

The following 1: 2F6(1nM) + 2.5ul of the PD-1 commercial antibody;

the following 2: 2F6(10nM) + 2.5ul of the PD-1 commercial antibody;

the following 3: 2F6(100nM) + 2.5ul of the commercial antibody PD-1.

FIG. 7 FACS method detection of antibody 2F6 specific binding to PD-1 protein on PBMC surface after activation (left to right: isotype control, commercial antibody to PD-1, 2F 6).

FIG. 8 LDH assay detects the enhancement of the ability of antibody 2F6 to kill target cells by PBMC.

Sequentially comprises the following steps: a: FACS method detection of PD-1 expression changes before and after PBMC activation: left, unactivated PBMC; right, PBMC after activation.

FACS method detects the expression change of PD-L1 before and after IFN-gamma induction of HepG2 cells: a, uninduced HepG2 cells; b, IFN-gamma induced 24h HepG2 cells.

Killing rate of HepG2 cells by PBMC in different dose groups of 2F 6.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

1. Mouse hybridoma monoclonal antibody screening

Immunizing a Balb/c mouse by taking human PD-1 protein as an immunogen in an intraperitoneal injection mode, performing boosting immunization respectively at 3 rd week and 5 th week after primary immunization, taking tail blood of the mouse at 8 th day after the boosting immunization, standing at room temperature for 1 hour, centrifuging at 4 ℃, 12000rpm for 10 minutes, collecting serum, and diluting the serum into different concentrations by PBS: 1:200,1:400,1:800,1:1600,1:3200,1:6400,1: 12800. jurkat cells were collected, washed 1 time with PBS, counted and1X 10 cells were used for each sample⁶Adding 100 mu l of serum with different dilutions into the cells, using the serum of the non-immune mouse to replace the antiserum in the negative control group, incubating for 1 hour at 4 ℃, and washing twice with PBS; adding 2 mul of PE-labeled rat anti-mouse IgG antibody, and incubating for 40 minutes at 4 ℃ in a dark place; the cells are resuspended in 500 mul PBS buffer solution, and the binding percentage and fluorescence intensity of the antibody and the cells in the serum are detected by a flow cytometer; the average fluorescence intensity is more than twice of the negative control to be effective titer, and when the titer is higher than 6400, the fusion can be carried out. 3 days before the fusion, the immunized mice were subjected to shock immunization by means of tail vein injection of immunogen. Taking successfully immunized mouse spleen cells and myeloma SP2/0 cells for cell fusion (in a ratio of 10: 1), adding 50% PEG into spleen cells and myeloma cell masses which are uniformly mixed and the supernatant is discarded within 1min under the environment of 37 ℃ water bath during fusion, shaking the mixture in the water bath at 37 ℃ for 1min, and adding 10ml of serum-free 1640 medium within 2 min. Centrifuge at 800rpm for 6min, discard supernatant, resuspend cells in HAT-containing 1640 medium and pipette into 96-well plates (2.5X 10)⁷Cells/plate). At 37 deg.C, 5% CO₂The cells are cultured under conditions.

When the clones were large enough in the fusion plates, 100. mu.l of supernatant per well was taken with 2X10⁵Co-incubation of Jurkat cells, detection, method and detection effectThe prices are the same. And taking the positive wells with the average immunofluorescence intensity more than twice that of the negative wells, and carrying out the next cloning culture. And expanding the hybridoma clones with positive screening from a 96-well plate to a 24-well plate for culturing for 3-5 days, performing culture supernatant screening detection again, performing subclone culture of the next step after positive detection, and freezing and storing the rest cells. Collecting hybridoma cells in a 24-well plate, counting the cells, and adjusting the cell density to 10 cells/mL; cells were plated in 96-well plates at 37 ℃ with 5% CO in 100. mu.l/well₂Culturing an incubator; culturing for about 10 days to form visible clone, selecting a hole with only a single clone, sucking culture supernatant, selecting positive clone, expanding to a 24-hole plate for culturing, detecting the supernatant again, selecting the positive clone to perform a second round of subclone culture, generally performing multiple rounds of subclone culture until all detection holes are positive, thus obtaining a stable hybridoma cell strain, and preserving the hybridoma cell strain to the China general microbiological culture Collection center to obtain the mouse anti-human PD-1 hybridoma cell strain 2F6 with the preservation number of CGMCC No. 15697. Selecting a positive hybridoma culture supernatant, and detecting the subtype of the antibody by using antibody subtype detection test paper, wherein the monoclonal antibody is numbered as 2F6, is a murine IgG1 subtype, and has a kappa chain as a light chain.

Variable region sequence:

antibody 2F6 murine IgG1 type

Heavy chain:

QVKLQQSGAELARPGASVKMSCMASGYTFTSNTITWVKQRPGQGLEWIGYINPNSDFTNYNEKFRDKATLTADRSSSTAYMQLSSLTSEDSAVYYCARDYYGGSYYAMDYWGQGTSVTVSS(SEQ ID NO:7)

CDRH1:GYTFTSNTIT(SEQ ID NO:1)

CDRH2:YINPNSDFTNYNEKFRD(SEQ ID NO:2)

CDRH3:DYYGGSYYAMDY(SEQ ID NO:3)

light chain:

QIVLTQSPAIMSASLGERVTMTCTADSSVPSSYLHWYQQKPGSSPKFWIYSTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAGTKLELKRA(SEQ ID NO:8)

CDRL1:TADSSVPSSYLH(SEQ ID NO:4)

CDRL2:STSNLAS(SEQ ID NO:5)

CDRL3:HQYHRSPLT(SEQ ID NO:6)

2. preparation and purification of ascites

Hybridoma cells were washed with sterile PBS solution at 5x10⁶0.5 ml/cell mass was injected intraperitoneally into liquid paraffin-primed Balb/c mice. Ascites was collected after 7 to 10 days at room temperature 3000rpm for 10min, and the supernatant was collected. The antibody is roughly purified by saturated ammonium sulfate with the final concentration of 33%, and the method comprises the steps of taking 1 part of ascites, adding 1 part of PBS, dropwise adding 1 part of saturated ammonium sulfate while stirring, standing overnight at 4 ℃, centrifuging at 10000rpm for 10min to remove supernatant, dissolving precipitate by using a small amount of PBS, dialyzing by using PBS at 4 ℃ to remove salt for 24h, and changing the solution for 3 times. The crude purified antibody was further purified by 1ml of Protein G purification pre-packed column using AKTA Protein purification system according to the purification manual provided by GE. The obtained pure antibody product is used for subsequent antibody detection and functional experiments.

3. Monoclonal antibody potency detection

PE is labeled straight 2F 6. The labeled antibody was used at final concentrations of 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6nM, 25nM, 3.2nM, 1.6nM, 0.8nM, 0.4nM, 0.2nM, 0.1nM, 0.05nM, 0.025nM, 0.0125nM, 0.0061nM, 0.003nM to 2.5X10 nM, respectively⁵SP2/0 cells (transfected PD-1) were co-incubated at room temperature for 30min, taking care to keep out of light. Centrifugation is carried out at 1800rpm for 10min, supernatant is discarded, cells are washed by PBS and repeated three times, the cells are resuspended in 400 μ l of PBS, fluorescence intensity is measured by FACS, and Mean value is counted. Kd values of the antibodies were calculated using the data analysis software GraphPad Prism 5. FIG. 1 shows antibodies 2F6 and SP2/0 (hPD-1)⁺) FIG. analysis of affinity constants of cells, Kd value of antibody 2F6 was 1.9X10^-8M。

Cloning of Ig variable region genes by RT-PCR

1) Total RNA extraction, single-stranded cDNA synthesis:

total RNA from 2F6 hybridoma cell line was extracted by Trizol (kit from Invitrogen) and reversed to a cDNA library using M-MLV reverse transcriptase (from Invitrogen).

Heavy chain framework region upstream primer

P1：5’SAGGTGMAGCTKCASSARTCWGG3’(SEQ ID NO:11)

Heavy chain variable region downstream primer

P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’(SEQ ID NO:12)

Light chain leader peptide upstream primer

P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’(SEQ ID NO:13)

Light chain variable region downstream primer

P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’(SEQ ID NO:14)

A PCR reaction system (50. mu.l) was prepared as follows:

2 μ l of cDNA; 2. mu.l of upstream primer (10. mu.M); 2. mu.l of downstream primer (10. mu.M); d NTP mix 2. mu.l; pfu DNA polymerase (5U/. mu.l) 1. mu.l; 5 mul of 10 Xpfu Buffer II; ddH2O make up to 50. mu.l. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; the following cycle was repeated 35 times: 30s at 95 ℃, 30s at 58 ℃ and 1min at 72 ℃; finally, extension was carried out at 72 ℃ for 10 min. The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated to pMD19-T (simple) vector (Takara) as follows: VL PCR product/VH PCR product each 70ng, pMD19-T (simple)vector 1. mu.l, Solution I ligation reaction 5. mu.l; ddH₂O make up to 10. mu.l and ligate overnight at 4 ℃. The ligation products were transformed into E.coli DH5 alpha competent bacteria, cultured overnight at 37 ℃, single colonies were picked up, shaken at 37 ℃ for 2 hours, and then subjected to PCR identification of bacterial liquid, using cDNA of the corresponding antibody as a positive control. The reaction system (25. mu.l) was prepared as follows: bacterial liquid: 1 μ l, forward primer (10 μ M): 1 mul; downstream primer (10 μ M): 1 mul; dNTP mix (2.5 Mm each) 2. mu.l; taq DNA polymerase (5U/. mu.l): 0.5 mul; 10 × Taq Buffer (Mg2+ plus): 2.5 mul; water was added to 25. mu.l. The reaction conditions were as before. The PCR positive clone of the selected strain is expanded and cultured, and the positive clone plasmid is extracted by a plasmid extraction kit (Takara company) and is checked and sequenced. At least 5 clone samples were tested per chain of each antibody until the sequencing results were identical for at least three samples. The variable region sequences of the heavy chain and the light chain of the antibody 2F6 are successfully cloned and meet the sequence characteristics of a typical antibody variable region.

5. Cross-reactivity with the PD-1 family member CTLA-4: antibodies were mixed at a final concentration of 100nM with 2X10⁵CTLA-4 infected 3T3 cells (GFP positive) were incubated with CTLA-4 commercial antibody (purchased from BD) as a positive control. Incubating for 1 hour at 4 ℃ and washing twice with PBS; 100ul of the resuspended cells, 1 ul of APC-labeled anti-mouse IgG antibody is added, and incubation is carried out for 40 minutes at 4 ℃ in the dark; cells were resuspended in 500. mu.l PBS buffer and examined by FACS. The results are shown in FIG. 2, and it can be seen that antibody 2F6 has no cross-reactivity with human CTLA-4.

6. Cross-reactivity with human PD-1 negative cells:

antibodies were mixed at a final concentration of 100nM with 2X10⁵Individual Raji cells (PD-1 negative) were incubated with a commercial PD-1 antibody as a control. Incubating for 1 hour at 4 ℃ and washing twice with PBS; 100ul of the resuspended cells, 2 ul of PE-labeled rat anti-mouse IgG1 antibody was added, and incubation was carried out for 40 minutes at 4 ℃ in the dark; cells were resuspended in 500. mu.l PBS buffer and examined by FACS. The results are shown in FIG. 3. Antibody 2F6 was seen to have no cross-reactivity with Raji cells.

7. Specifically bind to Jurkat cells highly expressing human PD-1

1) FACS detection of antibody binding to Jurkat surface PD-1 protein: antibodies were raised to final concentrations of 100nM, 10nM, 1nM, 0.1nM and 1X 10nM, respectively⁶Jurkat cells were incubated at room temperature for 40min and washed twice with PBS; 100ul of the resuspended cells, 2 ul of PE-labeled rat anti-mouse IgG1 antibody was added, and incubation was carried out for 40 minutes at room temperature in the dark; cells were resuspended in 500. mu.l PBS buffer and examined by FACS. The results are shown in FIG. 4: the antibody 2F6 can be effectively combined with Jurkat cells, and has strong affinity.

2) The Western Blot method detects the binding specificity of the antibody to human PD-1 in the total protein of Jurkat cells. Jurkat cells were collected, washed twice with precooled PBS, added with 100. mu.l of precooled RIPA lysate (strong) and 1. mu.l of PMSF (100mM), transferred into a 1.5ml LEP tube, ice-washed, and shaken in a horizontal shaker for 30 minutes; after centrifugation at 12,000rpm at 4 ℃ for 15 minutes, the supernatant was collected and protein was quantified by BCA method. The sample was loaded at a protein content of 40 ug/well, subjected to SDS-PAGE gel electrophoresis and membrane transfer, and then the NC membrane was added to a blocking solution containing 5% skim milk (PBST), and blocked at room temperature for 2 hours. Antibody 2F6 was diluted to 4. mu.g/ml with 5% skim milk, and after blocking, the diluted antibody was added and incubated overnight with gentle shaking at 4 ℃. Primary antibody was removed, PBST washed 3 times for 10 minutes each, gently shaken at room temperature; diluting HRP coupled secondary antibody (goat anti-mouse) with confining liquid according to a ratio of 1:3000, dropwise adding secondary antibody working solution into an NC membrane, and gently shaking at room temperature for 2 hours; the secondary antibody was removed and washed 3 times with PBST for 10 minutes each, with gentle shaking at room temperature. The NC membrane was exposed using an ECL substrate developing solution, a fluorescence/chemiluminescence imaging analyzer, and the results are shown in FIG. 5. The sample above each well was an equal amount of total Jurkat cell protein (40 ug); the antibody incubation concentrations used were all 4. mu.g/ml. It can be seen that the antibody 2F6 can specifically bind to the linearized human PD-1 protein. No obvious miscellaneous band, and on the other hand, the specificity of the antibody binding to the human PD-1 is verified.

FACS method to detect the competitive relationship of 2F6 with commercial anti-hPD-1 antibody.

Diluting antibody 2F6 with PBS to final concentration of 100nM, 10nM and 1nM, simultaneously adding 2.5ul of commercial PD-1 antibody (PE direct standard) into each well, mixing antibody 2F6 with different dilution times, and mixing with 2 × 10⁵Jurkat cells were incubated for 30min at room temperature in the dark. Centrifugation is carried out at 1800rpm for 10min, supernatant is discarded, cells are washed by PBS and repeated three times, the cells are resuspended in 400 μ l of PBS, fluorescence intensity is measured by FACS, and Mean value is counted. The results are shown in FIG. 6: it can be seen that antibody 2F6 completely competed with the commercial anti-hPD-1 antibody.

FACS method for detecting specific binding of antibody 2F6 to activated PBMC

Normal human Peripheral Blood Mononuclear Cells (PBMC) were isolated from normal human platelet-rich white membranes using Ficoll-Hypaque. After PBMC were activated with 1. mu.g/ml of the elicited CD3 antibody, and 1. mu.g/ml of the CD28 antibody for 24h, antibody 2F6 was administered at a final concentration of 10nM and1X 10⁶PBMC cells were incubated, and 2. mu.l of commercial PD-1 antibody and1X 10 antibody were taken as positive control⁶Incubating PBMC cells with a final volume of 100 μ l, incubating at room temperature for 40min, and washing twice with PBS; 100ul of the resuspended cells, 2 ul of PE-labeled rat anti-mouse IgG1 antibody is added, and the cells are incubated for 40 minutes at room temperature in the dark; cells were resuspended in 500. mu.l PBS buffer and examined by FACS. The results are shown in FIG. 7, and it can be seen that 2F6 is effective in recognizing PD-1 protein expressed on the surface of PBMC membrane after activation. LDH method for detecting enhancement effect of antibody 2F6 on target cell killing capacity of PBMC

Taking HepG2 cells in logarithmic growth phase, counting, and adjusting cell density to 2 × 10⁵Inoculating to T75 culture mediumThe flask is maintained at 15 ml/flask, and IFN-gamma is added to the final concentration of 700 units/ml after the cells are attached to the wall. 5% CO at 37 ℃₂And performing conventional culture in an incubator for 24 h. At the same time, PBMC were activated in the same manner as in example 9. HepG2 pretreated by IFN-gamma for 24 hours and PBMC activated for 24 hours are taken respectively, and the expression conditions of PD-L1 and PD-1 are detected by flow cytometry. The results are shown in FIGS. 8A and 8B, and it can be seen that HepG2 was treated, and that PBMC expressed PD-L1 and PD-1, respectively.

Taking IFN-gamma induced HepG2 cells, counting and adjusting the cell density to 1 × 10⁵Inoculating to 96-well plate (100 ul/well), and standing at 37 deg.C with 5% CO₂Culturing in an incubator by a conventional method. After all HepG2 cells were attached to the bottom of the well, the medium was aspirated and the activated PBMC cell suspension (200. mu.l/well) was added at an effective target ratio of 5: 1. IL-2 was added to each well at a final concentration of 150 units/ml, and 2F6 was added to the respective experimental wells at a final concentration of 0. mu.g/ml, 2.5. mu.g/ml, 5. mu.g/ml, with three parallel wells per group. Control wells were designed as per the instructions. Centrifuging at 250g for 4min to ensure that target cells are fully contacted; placing the reaction plate at 37 ℃ with 5% CO₂In the incubator, the target cells were co-cultured for 12 hours. 45 minutes before the supernatant was obtained, 10. mu.l of lysate was added to the wells for maximum release of target cells and control of medium volume. After lysis was complete, centrifugation was carried out at 250g for 4 min. The supernatant was collected and LDH was measured according to the protocol of Promega CytoToX96 nonradioactive cytotoxic assay kit and the kill rate was calculated. The results are shown in FIG. 8C, and the killing rates of the three dose groups of 0. mu.g/ml, 2.5. mu.g/ml and 5. mu.g/ml are 18.07 + -3.60%, 47.70 + -2.77% (P)<0.05)，52.50±0.81％(P<0.001)。

It can be seen that the antibody 2F6 can significantly improve the killing level of the PBMC to the HepG2 cells under the dosage of 2.5 mu g/ml and 5 mu g/ml.

Claims (9)

Translated fromChinese

1.一种鼠抗人PD-1单克隆抗体，其特征在于：其包含重链可变区序列和轻链可变区序列；1. A mouse anti-human PD-1 monoclonal antibody, characterized in that: it comprises a heavy chain variable region sequence and a light chain variable region sequence;所述重链可变区序列的CDR区三个序列，分别如SEQ ID NO:1、SEQ ID NO:2、SEQ IDNO:3所示；The three sequences of the CDR region of the heavy chain variable region sequence are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively;所述轻链可变区序列的CDR区三个序列，分别如SEQ ID NO:4、SEQ ID NO:5、SEQ IDNO:6所示。The three sequences of the CDR region of the light chain variable region sequence are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively.2.一种鼠抗人PD-1单克隆抗体，其特征在于：其包含重链可变区序列和轻链可变区序列；重链可变区序列如SEQ ID NO:7所示；轻链可变区序列如SEQ ID NO:8所示。2. A mouse anti-human PD-1 monoclonal antibody, characterized in that: it comprises a heavy chain variable region sequence and a light chain variable region sequence; the heavy chain variable region sequence is shown in SEQ ID NO: 7; The chain variable region sequence is shown in SEQ ID NO:8.3.保藏号为CGMCC No.15697的鼠抗人PD-1的杂交瘤细胞株2F6。3. The mouse anti-human PD-1 hybridoma cell line 2F6 with the deposit number of CGMCC No. 15697.4.一种鼠抗人PD-1单克隆抗体，其特征在于：由保藏号为CGMCC No.15697的鼠抗人PD-1的杂交瘤细胞株2F6产生的鼠抗人PD-1单克隆抗体。4. A mouse anti-human PD-1 monoclonal antibody, characterized in that: a mouse anti-human PD-1 monoclonal antibody produced by the mouse anti-human PD-1 hybridoma cell line 2F6 with a deposit number of CGMCC No. 15697 .5.一种核苷酸分子，其特征在于：所述的核苷酸分子编码权利要求2所述鼠抗人PD-1单克隆抗体。5. A nucleotide molecule, wherein the nucleotide molecule encodes the mouse anti-human PD-1 monoclonal antibody of claim 2.6.根据权利要求5所述的核苷酸分子，其特征在于：所述的核苷酸分子编码鼠抗人PD-1单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:9所示，所述的核苷酸分子编码鼠抗人PD-1单克隆抗体的轻链可变区的核苷酸序列为如SEQ ID NO:10所示。6. The nucleotide molecule according to claim 5, wherein the nucleotide sequence of the nucleotide molecule encoding the heavy chain variable region of the mouse anti-human PD-1 monoclonal antibody is as SEQ ID NO. As shown in SEQ ID NO: 9, the nucleotide sequence of the nucleotide molecule encoding the light chain variable region of the mouse anti-human PD-1 monoclonal antibody is as shown in SEQ ID NO: 10.7.权利要求1，2或4所述的鼠抗人PD-1单克隆抗体在制备检测PD-1蛋白试剂盒中的应用。7. The application of the mouse anti-human PD-1 monoclonal antibody of claim 1, 2 or 4 in the preparation of a kit for detecting PD-1 protein.8.一种药物组合物，其特征在于：所述组合物包括权利要求1，2或4所述的鼠抗人PD-1单克隆抗体和药学上可接受的载体。8. A pharmaceutical composition, characterized in that: the composition comprises the mouse anti-human PD-1 monoclonal antibody of claim 1, 2 or 4 and a pharmaceutically acceptable carrier.9.权利要求1，2或4所述鼠抗人PD-1单克隆抗体在制备治疗肿瘤、感染性疾病、自身免疫性疾病或抗免疫排斥的药物中的应用。9. Use of the mouse anti-human PD-1 monoclonal antibody of claim 1, 2 or 4 in the preparation of a medicament for treating tumors, infectious diseases, autoimmune diseases or anti-immune rejection.

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