CN108314734B

CN108314734B - anti-PD-1 monoclonal antibody and application thereof

Info

Publication number: CN108314734B
Application number: CN201810108897.8A
Authority: CN
Inventors: 邱郑; 王旻; 邢黎军; 徐祎凤; 王红
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2021-11-05
Anticipated expiration: 2038-01-31
Also published as: CN108314734A

Abstract

The invention relates to a novel anti-PD-1 monoclonal antibody and a variable region sequence thereof, belonging to the technical field of biology. The invention uses recombinant human PD-1 protein (rhEPD-1) as immunogen, immune BAL b/c mouse takes spleen cell of immune mouse and fuses it with myeloma cell sp2/0-Ag14, so as to obtain hybridoma cell strain capable of expressing anti-PD-1 antibody, and the cell strain can stably secrete single monoclonal antibody. The invention clones the H chain, L chain variable region nucleotide and amino acid sequence of anti-PD-1 monoclonal antibody. The anti-PD-1 monoclonal antibody can be specifically combined with PD-1, block the combination of PD1 and PD-L1 and restore the function of T cells. Can be used as an immune checkpoint inhibitor for the treatment of cancer and infectious diseases.

Description

anti-PD-1 monoclonal antibody and application thereof

The technical field is as follows:

the invention relates to a monoclonal antibody capable of specifically binding to PD-1, belonging to the field of biotechnology. In particular to a preparation method of the monoclonal antibody, a variable region sequence and application thereof.

Background art:

hybridoma technology (hybridoma technology, also known as monoclonal antibody technology, 1975 Koehler and Milstein demonstrated that the fusion of myeloma cells with splenocytes from immunized animals results in the formation of monoclonal antibodies with strong specificity for an antigen. The hybridoma technology utilizes the characteristic that myeloma cells can be continuously passaged in vitro and lymphocytes can secrete specific antibodies or factors, and the two cells are fused, so that successfully fused cells can have the main characteristics of the two cells.

The PD-1/PD-L1 immunotherapy improves the immune response by blocking the interaction between the two, and has the potential of treating various types of tumors and infectious diseases. Programmed death receptor-1 (PD-1) is a member of the CD28 superfamily, an important immunosuppressive molecule, and is mainly expressed on activated T cells and B cells. PD-1 and its ligand PD-Ls (mainly PD-L1 and PD-L2) combine and can inhibit proliferation, activation and relevant cytokine secretion of T cell, make the organism protect against the attack of autoimmune system. However, in the tumor microenvironment of an organism, the tumor cells highly express PD-L1, and after the PD-L1 is combined with PD-1 on T cells, the failure of the T cells is induced, the functions of the T cells are inhibited, the immune system cannot be effectively activated, and the immune escape of the tumor cells is caused. And monoclonal antibodies aiming at PD-1 and PD-L1 can block the combination of PD-1 and PD-L1, so that T cells are revived, and the immune response is enhanced. Currently, several anti-PD-1 and PD-L1 antibodies are marketed for the treatment of various tumors, such as Nivolumab (anti-PD-1 antibody), Keytruda (anti-PD-L1 antibody), and the like.

The invention takes recombinant human PD-1 extracellular domain (rhEPD-1) protein as antigen, obtains the high-affinity anti-PD-1 specific monoclonal antibody through screening and preparation of hybridoma technology, and verifies the high-efficiency biological activity through in vitro and in vivo experiments.

Disclosure of Invention

Object of the Invention

One of the purposes of the invention is to provide a preparation method of an anti-PD-1 monoclonal antibody;

the second purpose of the invention is to provide an anti-PD-1 monoclonal antibody;

the present invention also aims to provide the variable region amino acid sequence and nucleic acid sequence of the anti-PD-1 monoclonal antibody.

Technical scheme

The invention takes recombinant human PD-1 protein prepared in the early stage of a laboratory as immunogen, a Balb/c mouse is immunized for many times, when the serum titer meets the fusion requirement, spleen cells and sp2/0-Ag14 myeloma cells are taken for cell fusion, hybridoma cell strains capable of stably secreting anti-PD-1 antibodies are obtained by screening HAT selective culture medium, and the genes of heavy chain and light chain variable regions of the antibodies are extracted at the molecular level after subcloning and expanding culture; the monoclonal antibody can be obtained by injecting hybridoma cells into abdominal cavity of mouse, collecting ascites, and purifying with Protein A/G affinity chromatography column.

Advantageous effects

The invention successfully prepares the anti-PD-1 monoclonal antibody, which has good specificity and high binding rate and can block the biological binding of PD-1 and PD-L1. Can effectively restore the activity of lymphocytes in vitro and promote human peripheral blood lymphocytes (PBMC) to secrete IFN-gamma and IL-2, and shows the tumor inhibition effect of the monoclonal antibody on tumor-bearing mice in vivo experiments. In vivo and in vitro experiments show that the anti-PD-1 monoclonal antibody is a potential drug for tumor immunotherapy.

Description of the drawings:

FIG. 1 shows the results of SDS-PAGE detection of Protein A/G affinity column purified products. Lane M is protein Marker; lane 1 is the antibody protein in the eluate; FIG. 1A shows the result of non-reducing SDS-PAGE electrophoresis; FIG. 1B shows the result of reducing SDS-PAGE electrophoresis.

FIG. 2 is a schematic diagram of the results of monoclonal antibody subtype identification ELISA.

FIG. 3 flow cytometry detection of antibody binding to native PD-1 antigen on the cell surface of THP-1.

FIG. 4 and FIG. 5 are flow cytometric assays for specific binding of antibodies. FIG. 4 shows the detection of the binding of an antibody to a CHO cell line; FIG. 5 binding of the detection antibody to CHO cells stably expressing PD-1 (CHO-PD-1).

FIG. 6 flow cytometry detection of the binding of PD-L1 protein to PD-1 on the cell surface of THP-1.

FIG. 7 flow cytometry detection of anti-PD-1 monoclonal antibodies blocked the binding of PD-L1 to PD-1 on the cell surface.

Figure 8 ELISA experiments show binding curves of antibodies to PD-1 antigen protein.

FIG. 9. flow cytometry detects the concentration dependence of monoclonal antibody Pi binding to HEK293F-PD-1 cells.

FIG. 10 shows the measurement of the affinity of the monoclonal antibody Pi for the antigen by the detection of the intermolecular interaction force by the biofilm interference technique.

FIG. 11 shows the result of agarose electrophoresis of the gene products of heavy and light chain variable regions of the antibodies amplified by PCR, lane M being DNA Ladder;

lanes

1 and 2 are PCR amplified antibody heavy chain variable region gene products; lane 3 is the PCR amplified antibody light chain variable region gene product.

FIG. 12 and FIG. 13 examine the effect of anti-PD-1 monoclonal antibodies on the secretion of cytokines by PBMCs. FIG. 12 shows the detection of IFN- γ secretion changes in PBMCs; FIG. 13 shows the measurement of IL-2 secretion by PBMCs.

FIG. 14. in vivo pharmacodynamic experiments. Fig. 14a. is a tumor growth curve; fig. 14b is a graph of body weight change for different groups of mice; figure 14c. tumor weight results for different groups of mice.

The specific implementation mode is as follows:

the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the present invention. The monoclonal antibody and the simple modification of the preparation method thereof provided by the invention belong to the protection scope of the invention.

The experimental procedures used in the examples listed below are all conventional procedures unless otherwise specified.

Example 1 animal immunization

Recombinant human PD-1 protein (dissolved in PBS buffer, preliminarily identified, not emphasized in this patent, but not expanded) prepared in the early stage of the laboratory was mixed with Quick Antibody in equal volume, and then the mixture was intramuscularly injected into BAL b/c mice at 6-8 weeks, 50. mu.g/200. mu.l/mouse. The immunization procedure was performed according to Quick Antibody instructions. 3-5 times of immunization, after a period of time of each immunization, performing orbital bleeding (50 μ l) on the immunized mice by adopting a capillary blood-taking method, and measuring the serum antibody titer of the mice by adopting indirect ELISA and utilizing a confining liquid to dilute the serum in a gradient manner. The mouse with the highest specific antibody titer in serum was selected for cell fusion. The final detection result shows that the serum titer of the immune mice can reach more than 1: 100,000.

EXAMPLE 2 preparation of hybridoma 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 were revived and the cell state could be adjusted with 8-azaguanine. Two days before cell fusion, performing enlarged culture of sp2/0-Ag14, observing the state to make the cell approach logarithmic growth phase one day before fusion, and changing the liquid for treatment one day before fusion;

2) generally, when the serum titer of the immunized mice reaches 100,000, the fusion can be carried out. Selecting a mouse with the highest PD-1 specific antibody titer in serum, performing impact immunization 3-4 days before fusion, and injecting PD-1 protein into abdominal cavity or tail vein;

3) preparation of feeder layer cells (mouse peritoneal macrophages): feeder cells were plated one day prior to fusion, ICR mice of older week (e.g. over 8 weeks old) were selected, sacrificed by cervical dislocation, and peritoneal macrophages were extracted under sterile conditions. Cells were diluted to 1X 10⁵Each/ml of the sample was plated at a rate of 100. mu.l per well, labeled and placed at 37 ℃ in 5% CO₂The cell culture chamber of (1) was cultured overnight.

4) 1 hour before cell fusion, sp2/0-Ag14 cells were pretreated, resuspended sp2/0-Ag14 cells and centrifuged at 1,200rpm × 5min, and then the cells were resuspended in serum-free DMEM and counted, and the total cell number was generally in the range of 0.6-2 × 10⁷Standing at 4 deg.C.

5) The mice to be fused are subjected to orbital bleeding, serum is collected by the same operation, and the mice are stored at the temperature of minus 20 ℃ or minus 80 ℃ temporarily and can be used as positive control after fusion. The mice were sacrificed by cervical dislocation, soaked in 75% alcohol, and transferred to the cell house. Spleen was ground, filtered through a 70 μm mesh to prepare a single cell suspension, centrifuged at 2,000rpm × 5min, and the supernatant was discarded. And adding erythrocyte lysate into the cell sediment for treatment, carrying out centrifugal treatment after 5min of reaction, washing the cell sediment once by serum-free DMEM, finally carrying out heavy suspension, and counting the viable cells.

6) According to the counting result, sp20-Ag14 myeloma cells and spleen cells were mixed at a ratio of 1: 5, and after mixing, the supernatant was completely aspirated by centrifugation. Adding 650 μ l of PEG1450(SIGMA) pre-warmed at 37 deg.C slowly dropwise onto the cell precipitate, acting for 90s, and immediately adding serum-free DME pre-warmed at 37 deg.CM medium, mixing, centrifuging for 1,200 × 5min, resuspending the cell pellet in 20% FBS-DMEM medium containing HAT (SIGMA), spreading in 96-well plate with feeder cells, 200 μ l/well, and culturing at 37 deg.C and 5% CO₂Cultured in a cell culture box.

7) Cell status was observed periodically after fusion. On day four of fusion, half-changes were made, i.e., one-half of the medium (100. mu.l) in the culture well plate was changed out with fresh 20% FBS-HAT-DMEM medium; day 7-10 after the fusion, the cells were cultured in 20% FBS-DMEM medium containing HT (SIGMA).

2. Screening and subcloning of Positive fusion cells

The cells in each well were tested for antibody secretion by ELISA. The method comprises the steps of taking rhEPD-1 protein as an ELISA screening coating antigen, taking HRP-labeled Goat anti Mouse IgG as a detection antibody, selecting a hole with a high ELISA positive value, observing a viable hybridoma cell or a cell mass under a microscope, labeling, selecting the positive hole, cloning the cell in the hole by adopting a limiting dilution method, establishing a hybridoma cell strain capable of stably secreting a monoclonal antibody after two times of subcloning, and carrying out amplification culture.

EXAMPLE 3 preparation and purification of anti-PD-1 monoclonal antibody

1. Ascites collection

Intraperitoneal injection of 500. mu.l paraffin oil (SIGMA) was performed to female Bal b/c mice aged 8-10 weeks. One week later, the intraperitoneal injection inoculation is 1X 10⁶And (3) hybridoma cells with good growth state. The abdomen of the mouse is obviously bulged after about 7-10 days of inoculation of the hybridoma cells, ascites is collected at the moment, the mouse is centrifuged at 12,000rpm for 5min at 4 ℃, cell debris is removed, and the supernatant is collected for subsequent purification.

2. Antibody purification

Antibody purification was performed using ProteinA/G column packing (Abmart). The method comprises the following specific operations: the abdominal water supernatant is diluted to a certain volume by using a Binding Buffer, filtered and sterilized, and then loaded. And (2) after sample loading, washing by using a Binding Buffer, washing off non-specifically bound impurities, finally eluting, adding 5-10ml of Elution Buffer to elute the target protein, collecting the eluent, immediately adding a neutrallization Buffer to make the pH value neutral, washing the column filler by using the Binding Buffer, and storing by using 20% ethanol at 4 ℃.

3. Antibody purity and molecular weight determination

The collected eluate was subjected to SDS-PAGE to preliminarily identify the antibody, as shown in FIG. 1A and FIG. 1B.

EXAMPLE 4 characterization of monoclonal antibodies

1. Identification of antibody subtypes

The subtype of the obtained anti-PD-1 monoclonal antibody is identified by using 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, and specific experimental operations are shown in a kit instruction. As a result, as shown in FIG. 2, the heavy chain subtype of the anti-PD-1 monoclonal antibody obtained by us 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 detection of antibody binding to PD-1 on cell membranes

A human monocyte leukemia cell line (THP-1) expressing human PD-1 on the cell surface and Chinese hamster ovary Cells (CHO) CHO-PD-1 stably expressing PD-1 were selected, and the specificity of the anti-PD-1 monoclonal antibody was determined by flow cytometry. The THP-1 with good growth state is re-suspended with PBS and counted, the cell density is adjusted to 1-2X 10⁶And each cell/ml is subpackaged into 1.5ml EP tubes, an equal volume of cell suspension is added into each tube, generally 100-. The detection method of CHO-PD-1 cells is the same as that of THP-1, and the binding of the antibody to CHO and CHO-PD-1 cells is detected respectively, so that the specific binding with PD-1 is shown.

From the binding condition of flow cytometry (figure 3), the anti-PD-1 monoclonal antibody can be well bound with PD-1 on the surface of human cells, and the binding rate can reach 89.9%; FIG. 4 shows that Pi did not bind to untransfected CHO cells, and FIG. 5 illustrates that the monoclonal antibody Pi specifically binds to CHO cells stably expressing PD-1 (CHO-PD-1).

3. Monoclonal antibody Pi blocks PD-L1 binding to cell surface PD-1

PD-L1 is a ligand of PD-1, PD-L1 protein can be combined with PD-1 on the surface of the cell, and the combination rate of PD-L1 protein and THP-1 cell is 83.1% by flow detection as shown in figure 6. Based on the detection, the anti-PD-1 monoclonal antibody blocks the binding of PD-L1 to cell surface PD-1 by using a flow cytometry method. THP-1 cells were treated as before, 5. mu.g of PD-L1 protein was added to each group, and anti-PD-1 monoclonal antibodies Pi were added at concentrations of 0, 1, 10 and 50. mu.g/ml in this order for co-incubation, and binding of PD-L1 protein to THP-1 cells was detected by flow cytometry.

From the flow results (FIG. 7), the blocking effect of 1. mu.g/ml anti-PD-1 monoclonal antibody can reach 50%, and the binding of PD-L1 protein to the cell surface can be completely blocked by 10. mu.g/ml and 50. mu.g/ml.

ELISA detection of EC50 binding of monoclonal antibody Pi to PD-1

Recombinant human PD-1 protein is used as a solid phase antigen, 10 mu g/ml is coated overnight, 5% skimmed milk is sealed for 2 hours the next day, then a gradient dilution series of monoclonal antibodies is added, the reaction is carried out for 2 hours at room temperature, HPR marked goat anti-mouse secondary antibody is added after washing, and OD is detected₄₅₀The binding curve of the anti-PD-1 monoclonal antibody to the recombinant human PD-1 protein and the EC50 value are obtained by fitting through data processing and mapping analysis by using GraphPad Prism 7 software, and the result is shown in FIG. 8, which shows that the anti-PD-1 monoclonal antibody can be specifically bound with the recombinant human PD-1 protein, and the EC50 is 0.46 nmol/L.

5. Flow cytometry to detect the concentration dependence of monoclonal antibody Pi binding to HEK293F-PD-1 cells

HEK293F is human embryonic kidney cell, the cell surface of which does not express human PD-1 antigen, we transfect recombinant PD-1 recombinant plasmid by cell transfection method, so that HEK293F cell surface can express human PD-1 antigen, after culturing for 72 hours, the expression level of PD-1 reaches the maximum value, collect cells to prepare single cell suspension, and co-incubate with monoclonal antibody Pi diluted in gradient, detect the combination condition of Pi with HEK293F-PD-1 by flow cytometry, FlowJo software calculates the Mean Fluorescence Intensity (MFI) of combination, use GraphPad Prism 7 software to process data and map analysis, reflect the concentration dependence tolerance of combination with cells.

As can be seen from the experimental results of FIG. 9, Pi was very concentration-dependent on binding of HEK293F-PD-1 cells.

6. Determination of affinity of anti-PD-1 monoclonal antibody to antigen

The affinity of anti-PD-1 monoclonal antibodies to PD-1 was determined using a ForteBio Octet biomolecular interactor using biofilm interference technology (BLI). The antigen is first biotinylated, and the streptavidin biosensor is selected, with the biotin tag attached. Based on this property (SA), the affinity of the antigen and antibody is measured by anchoring biotinylated antigen to the probe. The procedure was set in order of equilibration, anchoring of antigen, equilibration, binding, dissociation, and affinity was determined using ForteBio Octet. The data were processed and analyzed using Octet data analysis software.

As can be seen from the experimental results of FIG. 10, the anti-PD-1 monoclonal antibody Pi measured by BLI has an affinity for PD-1 of 3.66X 10^-10nmol/L。

EXAMPLE 5 cloning of heavy and light chain variable region Gene of Positive hybridoma cell line

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

Hybridoma cell strains in the logarithmic growth phase are collected, and total RNA is extracted by using the RNAioso Plus of TaKaRa company, and the specific operation is shown in the product instruction. Adding 70 μ l RNase-free water to dissolve the precipitate, taking 10 μ l for nucleic acid electrophoresis at 200V/10 min. The rest was stored temporarily at-80 ℃ or the first cDNA strand was synthesized.

Total RNA was used as a template for reverse transcription to synthesize the first cDNA, and the reagents were purchased from Vazyme. The reaction system is shown in the specification, and the first cDNA synthesis reaction is carried out in a PCR instrument according to the reaction conditions of the specification. The product can be immediately subjected to PCR amplification reaction or stored at-20 ℃.

PCR amplification of heavy and light chain variable domain gene, degenerate primers designed according to the upstream and downstream gene sequences of heavy and light chain variable regions of hybridoma cell strain antibody in the reference, the primer sequences are as follows:

5’MH1：5’-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC-3’

5’MH2：5’-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TCW GG-3’

3’VH：5’-gga aga tct CTT GAC CAG GCA TCC TAG AGT CA-3’

5’Kappa：5’-Gg gag ctc GAY ATT GTG MTS ACM CAR WCT MCA-3’

3’Kappa：5’-Ggt gca tgc GGA TAC AGT TGG TGC AGC ATC-3’

(degenerate codon specification: R ═ a, g; Y ═ c, t; M ═ a, c; S ═ c, g; W ═ a, t).

The reaction system is 50 ul, and the reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃; 30s at 57 ℃; 45s at 72 ℃, 30 times of circulation and 7min at 72 ℃. 10 mul of PCR product is taken and subjected to nucleic acid electrophoresis for identification, and as shown in FIG. 11, the heavy-light chain variable domain gene with about 450bp is obtained by PCR amplification. The PCR product was purified using a gel recovery kit (biologies) and the procedure was as described in the kit.

As Taq Plus DNA Polymerase (Vazyme) is used in the PCR amplification reaction step, and adenine (A) is arranged at the 3 'end of the PCR product, the PCR product can be directly cloned to a T vector without carrying out a reaction of adding A at the 3' end. Ligation of PCR products

18-T vector, 1. mu. l T vector, 4. mu.l or 2. mu.l PCR product (up to 4. mu.l double distilled water) were mixed well, 5. mu.l Solution I was added, and ligation was performed overnight at 16 ℃.

The ligation product was transformed into competent JM109 by calcium chloride transformation: 10 μ l of the ligation product was transformed into 100 μ l of competent cell JM109, mixed and then stood in an ice-water bath for 30min, heat-shocked in a 42 ℃ water bath for 2min, immediately taken out of the ice bath for 1min, 1ml of non-resistant LB medium pre-warmed at 37 ℃ was added to each tube, and the mixture was shaken slowly at 37 ℃ and 150rpm for 1 h. Taking out, centrifuging at 4,000rpm × 5min, resuspending the precipitate with 100 μ l LB medium, coating ampicillin resistant plate, inverting, culturing in 37 deg.C constant temperature incubator for 16-20h, and observing the result.

Colonies were picked from the plates and cultured overnight in LB medium containing benzyl amine resistance. 5 μ l of the bacterial solution was collected at 12,000rpm × 2min, the supernatant was aspirated and the bacterial pellet was used as a template for colony PCR as described above. 10 μ l of colony PCR product was subjected to nucleic acid electrophoresis and positive clones with bands were picked for sequencing.

2. Gene sequencing and analysis of anti-PD-1 monoclonal antibody heavy and light chain variable domain

Sequencing results show that the hybridoma cell strain antibody variable domain gene is successfully obtained. By means of databases on NCBI's websites (https://www.ncbi.nlm.nih.gov/igblast/) Analyzing the heavy and light chain variable region gene of the anti-PD-1 monoclonal antibody, and the analysis result shows that: hybridoma cell line antibody heavy chain variable domain V_HThe nucleotide sequence and amino acid sequence of (a) are shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; hybridoma cell line light chain variable domain V_LThe nucleotide sequence and amino acid sequence of (a) 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.

Example 6 Effect of anti-PD-1 monoclonal antibodies on the secretion of cytokines from PBMCs

The 96-well plate was pre-coated with 2. mu.g/ml of CD3 antibody, 50. mu.l per well, incubated at 37 ℃ for 4 hours, and then washed 3 times with PBS. PBMCs were isolated from the blood of healthy subjects using lymphocyte isolate (from CEDARLANE) and processed according to the instructions of the product. After counting, according to 2X 10 of each hole⁵The individual cells were added to the above-described treated 96-well plate in a volume of 100. mu.l. At the same time, 5. mu.l of CD28 antibody was added to each well: (Mother liquor concentration of 20. mu.g/ml). The experimental groups were as follows: (1) PBMCs stimulated with only CD3 antibody and CD28 antibody; (2) stimulation with CD3 antibody and CD28 antibody, and addition of PBMCs co-incubated with PD-L1 protein; (3) CD3 antibody and CD28 antibody were stimulated with the addition of PBMCs of PD-L1 protein and Pi, with three duplicate wells per group. After 5 days of cell culture at 37 ℃ and 5% CO2, medium supernatants were removed from each well for cytokine assay, following the procedure of the commercially available human IFN-. gamma.and IL-2 detection kit (Council).

The results show (FIGS. 12 and 13) that PD-L1 inhibits lymphocyte activity and reduces secretion of cytokines IFN-. gamma.and IL-2. The anti-PD-1 monoclonal antibody blocks the binding of PD-L1 and PD-1 on the surface of lymphocytes, thereby restoring the cell vitality and promoting the secretion of IFN-gamma and IL-2.

Example 7 anti-PD-1 monoclonal antibody in tumor-bearing mouse model tumor-inhibiting action

Female Bal b/c mice, 6-8 weeks old, were selected and randomly divided into three groups. 5 mice per group, grouped including: (1) a control group, (2) an antibody low dose group, and (3) an antibody high dose group. The control group is administered with isotype control IgG, and the antibody high and low dose groups are respectively administered with 10mg/kg and 5mg/kg of anti-PD-1 monoclonal antibody Pi. The first administration on day 0, day 1, tumor inoculation, subcutaneous injection of CT26-PD-L1 mouse colon cancer cells stably transfected with human PD-L1, 2X 10⁵PD-L1 expressed on individual cells/mouse, transfected CT26 cells, has been shown to bind well to antibodies. Administering twice a week, administering via tail vein injection, observing tumor volume change every day, measuring with vernier caliper to obtain tumor volume data when tumor diameter reaches 1mm, and calculating tumor volume according to a.b²The calculation of/2 (a is the major diameter, b is the minor diameter) is carried out when the tumor volume of the control group reaches 1000mm³In the above, the mice were euthanized, the results of tumor-peeling, weighing and photographing are shown in fig. 14A, according to the results, in a CT26-PD-L1 mouse tumor model, according to the tumor growth curve, it can be seen that the administration group has a certain inhibition effect on tumor growth compared with the control group, and the inhibition effect of the high dose group is superior to that of the low dose group, and fig. 14B shows that there is no significant difference between the weight groups of the mice. MouseThe post-mortem tumor-shedding weighing results are shown in FIG. 14C, and the groups administered with the drug showed significant differences (P < 0.01 and P < 0.05) regardless of the low dose group and the high dose group compared with the control group.

Claims

1. Variable region sequences of an anti-PD-1 monoclonal antibody, characterized in that said antibody comprises a heavy chain variable domain VH and a light chain variable domain VL; wherein the nucleotide sequence of the heavy chain variable domain VH is SEQ ID NO: 1, amino acid sequence of SEQ ID NO: 2; 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 sequences are SEQ ID: 4. 6, 8; the nucleotide sequence of the light chain variable domain VL is SEQ ID NO: 9, amino acid sequence of SEQ ID NO: 10; 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 sequences are SEQ ID: 12. 14, 16.

2. An antibody or antibody fragment capable of binding to PD-1, wherein the heavy chain variable region comprises SEQ ID NO: 2; the light chain variable region comprises SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

3. A genetically engineered antibody that binds to PD-1, wherein the heavy chain variable region comprises SEQ ID NO: 2; the light chain variable region comprises SEQ ID NO: 10, the genetically engineered antibody comprises: human-murine chimeric antibodies; or a humanized antibody; or a functional fragment of an antibody, Fab; or is a single chain antibody; or an antibody functional fragment VH-L fused with a heavy chain variable region and a complete light chain; or a bispecific antibody; or the antibody-like functional fusion protein obtained by connecting, splicing and fusing the antibody, the antibody fragment and other various proteins or polypeptides.

4. A nucleic acid comprising a nucleotide sequence encoding a variable region of the anti-PD-1 monoclonal antibody of claim 1.

5. A composition comprising the antibody or antibody fragment or nucleic acid of any one of claims 1-4 and at least one pharmaceutically acceptable carrier.

6. Use of the antibody or antibody fragment or nucleic acid of any one of claims 1 to 4 in the preparation of an anti-tumor medicament, wherein the tumor is a tumor positive for PD-L1 expression.

7. Use of the antibody or antibody fragment or nucleic acid of any one of claims 1-4 in the preparation of a medicament for modulating immunity for the indication of an immune disorder resulting from aberrant activation of the PD-1/PD-L1 pathway.

8. Use of the antibody or antibody fragment or nucleic acid of any one of claims 1 to 4 in the preparation of a diagnostic agent whose detection target is a PD-1 molecule.