An antibody d10 against cd279 and uses thereof

Abstract

The application relates to an anti-CD279 antibody D10 and application thereof, the amino acid sequence of the antibody D10 comprising CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2 and CDR3 shown in SEQ ID No. 3. The antibody D10 has high affinity and specificity to the CD279 protein, and experiments prove that the antibody can effectively restore T cell anti-tumor activity by blocking the PD-1 / PD-L1 pathway, and enhance the T cell anti-tumor ability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular to an anti-CD279 antibody D10 and application thereof. BACKGROUND

[0002] CD279, also known as programmed death receptor 1 (PD-1), is an important immunosuppressive molecule, which is an immunoglobulin superfamily and a 288-amino acid residue membrane protein. It is cloned from the 2B4.11 hybridoma of apoptotic mouse T cells. The immune regulation targeting PD-1 is of great significance for anti-tumor, anti-infection, anti-autoimmune disease and organ transplantation survival. Its ligand PD-L1 can also be used as a target, and the corresponding antibody can also play the same role. PD-1 and PD-L1 binding initiates the programmed death of T cells, and tumor cells obtain immune escape. PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes the apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces the apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 inhibitors are a new class of drugs that block PD-1 and can activate the immune system to attack tumors and treat certain types of cancer.

[0003] The immune checkpoint refers to the programmed death receptor and its ligand. The immune checkpoint blockade therapy based on the programmed death receptor and its ligand improves the aggressiveness of the host immune system against tumor cells by inhibiting the binding of the programmed death receptor and its ligand. The immune checkpoint can be considered as the brake system of the immune cells, which refers to a series of molecules expressed on the immune cells and capable of regulating the degree of immune activation, so that the activation level of T cells is maintained within a normal range to prevent damage to healthy tissues of the body.

[0004] Single-domain antibody is a natural antibody lacking light chain in peripheral blood of a llama, which only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but is not easy to stick together or even aggregate into a block like artificially modified single-chain antibody fragments (scFv). More importantly, the VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity with antigens.

[0005] Therefore, how to prepare anti-CD279 nanobodies has become a problem to be solved at present. SUMMARY

[0006] To solve the above technical problems, the present application provides an anti-CD279 antibody D10 and application thereof, which has high affinity and specificity with CD279 protein. Experiments prove that the antibody can effectively restore T cell tumor activity by blocking the PD-1 / PD-L1 pathway, thereby activating T cells.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides an antibody D10 against CD279, wherein the amino acid sequence of the antibody D10 comprises CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2 and CDR3 shown in SEQ ID No. 3.

[0009] SEQ ID No. 1: ATSGSIFSLND.

[0010] SEQ ID No. 2: ITNNLST.

[0011] SEQ ID No. 3: NREIRGSSGTWYPLHY.

[0012] The present application creatively constructs an antibody D10 against CD279, which comprises three complementarity determining regions, and the antibody D10 is obtained by VHH fragment amplification of the antibody against CD279 in the serum of a llama, and is a single-domain antibody. The antibody fragment is small, and is more easily permeable to the inside of tissues and cells, has high stability, and has high specificity for CD279 protein through experiments, and can be used for subsequent research in the field of drugs and the like.

[0013] Preferably, the amino acid sequence of the antibody D10 comprises the sequence shown in SEQ ID No. 4.

[0014] SEQ ID No. 4:

[0015] QVQLVESGGGLVQPGGSLRLSCATSGSIFSLNDMVWYRQAPGKQRELVARITNNLSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGTWYPLHYWGQGTQVSVSS.

[0016] In a second aspect, the present application provides a recombinant antibody against CD279, which comprises the antibody D10 against CD279 in the first aspect and an Ig Fc fragment.

[0017] In the present application, the antibody composed of the antibody D10 against CD279 and the Ig Fc fragment is constructed, and the recombinant antibody has high affinity and strong blocking effect through experiments, and is used in the field of scientific research, for example, can be used for mechanism research of immune suppression points, and for screening of blocking drugs related to immune suppression points and the like.

[0018] Preferably, the Ig Fc fragment source includes any one or a combination of at least two of human, murine or monkey origin.

[0019] Preferably, the Ig Fc includes any one or a combination of at least two of IgGl, IgG2, IgG3 or IgG4.

[0020] Preferably, the sequence of the Ig Fc fragment is as shown in SEQ ID No. 5.

[0021] SEQ ID No. 5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] Preferably, the sequence of the recombinant antibody is as shown in SEQ ID No. 6.

[0024] SEQ ID No. 6:

[0025] QVQLVESGGGLVQPGGSLRLSCATSGSIFSLNDMVWYRQAPGKQRELVARITNNLSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGTWYPLHYWGQGTQVSVSSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0026] In a third aspect, the present application provides a nucleic acid molecule encoding the anti-CD279 antibody D10 of the first aspect or the anti-CD279 recombinant antibody of the second aspect.

[0027] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid molecule of the third aspect; and the expression vector, after transfecting, transducing or transforming a host cell, enables the host cell to express the anti-CD279 antibody D10 of the first aspect or the anti-CD279 recombinant antibody of the second aspect.

[0028] Preferably, the expression vector further comprises a promoter;

[0029] Preferably, the promoter comprises any one or a combination of at least two of CMV, EF1a, PGK1, Ubc, human beta actin, CAG or SV40;

[0030] In a fifth aspect, the anti-CD279 antibody D10 of the first aspect, the anti-CD279 recombinant antibody of the second aspect, the nucleic acid molecule of the third aspect or the expression vector of the fourth aspect is used in the preparation of a medicament for anti-PD-1.

[0031] Compared with the prior art, the present application has at least the following beneficial effects:

[0032] 1. In the present application, the anti-CD279 antibody D10 is obtained by amplifying the VHH fragment of the antibody to CD279 in the serum of a llama, which is a single-domain antibody. The antibody fragment is smaller and more easily penetrates into the interior of tissues and cells, has higher stability, and has been proven to have high specificity for CD279 protein through experiments.

[0033] 2. In the present application, the structure of the antibody D10 is simple and easy to modify, and the production cost is low, which is suitable for large-scale production. The antibody from a llama has high specificity and affinity to a specific antigen due to the immune system of a llama, and has weak immunogenicity to humans, which has a lower risk of causing an immune response.

[0034] 3. In the present application, the antibody D10 has been verified to have high affinity and strong blocking effect through experiments, and is used in the field of scientific research, such as being used for exploring the mechanism of immune suppression points, for screening blocking drugs related to immune suppression points, etc. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Flow cytometry detection diagram for detecting the specificity of the recombinant single-domain antibody.

[0036] Figure 2 EC50 of the recombinant single-domain antibody detected by ELISA50 The result image.

[0037] Figure 3 This is a graph showing the results of the recombinant antibody blocking function test.

[0038] Figure 4 The result of ELISA detection of the IC50 of recombinant single-domain antibody. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0040] The reagents used in the following examples:

[0041] DNA fragment recovery kit (TakaRa, CAT#9761);

[0042] PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#6210B);

[0043] PE-anti-human IgG (eBioscience, CAT#12-4998-82);

[0044] PBS (bico, CAT#14190-250);

[0045] Nivolumab (MedChemExpress, HY-P9903).

[0046] Streptavidin PE (eBioscience, CAT#12-4317-87);

[0047] Adjuvant (GERBU, CAT#3030);

[0048] HRP-ProteinA (Boster, CAT#BA1080);

[0049] HRP-Streptavidin (Boster, CAT#BA1088);

[0050] THE TM V5 Tag antibody [iFluor 647], mAb (GenScript, CAT#A01805);

[0051] Example 1

[0052] Alpaca immunization and antibody titer determination

[0053] Alpacas were immunized with PD-1 protein, and about 1000 μg of emulsified PD-1 protein was injected into each of the two sides of the cervical lymph nodes. The immunization was performed once every 3 weeks, and a total of 4 immunizations were performed. 5 mL of peripheral blood was collected before immunization, after the second, third and fourth immunization, respectively, and centrifuged at 800 g for 10 min to collect the upper serum. 1 μg / mL of PD-1-His protein was added to a 96-well plate at 100 μl / well, and the plate was coated at 4°C overnight. Then the supernatant was discarded, and the plate was washed 5 times with PBST buffer (0.05% Tween-20 in PBS buffer). 200 μl / well of blocking buffer was added, and the plate was incubated at 37°C for 2 h. After the blocking buffer was discarded, the plate was washed 5 times with PBST buffer. The collected serum was diluted with PBS buffer, and 100 μL of the diluted serum was added to a 96-well enzyme-labeled plate, and the plate was incubated at 25°C for 60 min. The control wells were PBS buffer. The liquid in the wells was discarded, and the plate was washed 5 times with PBST buffer. 100 μl of HRP anti-lama IgG (H+L) antibody (1:50000 dilution) was added, and the plate was incubated at 25°C for 60 min. After the liquid in the wells was discarded, the plate was washed 5 times with PBST buffer. 100 μl / well of TMB developing solution was added, and the plate was incubated at 25°C for 15 min in the dark. 50 μL / well of stop solution was added, and the OD450 value in the wells was read using an enzyme-labeled instrument.

[0054] The antibody titer (X refers to the maximum dilution factor at which the antibody can be detected) is represented by "1:X". The binding titer of the serum of the alpaca before immunization with PD-1 protein was less than 1:2000, indicating that the content of anti-PD-1 specific antibodies contained therein was extremely low. In comparison, the binding titer of the serum after the fourth immunization with PD-1 protein was significantly higher than 1:64,000, indicating that the content of anti-PD-1 specific antibodies in the serum of the alpaca was significantly increased after multiple immunizations.

[0055] Example 2

[0056] Construction of a single-domain antibody yeast display library

[0057] (1) Cloning of VHH antibody fragments: 100 mL of peripheral blood was collected from an alpaca and PBMCs were isolated, and RNA was extracted, and PrimeScript TMII 1st Strand cDNA Synthesis Kit. The cDNA was obtained by reverse transcription. The llama heavy chain antibody sequence was amplified by PCR using the PBMC cDNA as template. The upstream primer was combined with the signal peptide of the VHH antibody ORF, and the sequence was shown as SEQ ID NO. 7; the downstream primer was combined with the CH2 region of the VHH antibody ORF, and the sequence was shown as SEQ ID NO. 8. The target fragment with a size of about 750 bp was recovered by electrophoresis.

[0058] SEQ ID NO. 7: GTCCTGGCTGCTCTTCTACAAGG.

[0059] SEQ ID NO. 8: GGTACGTGCTGTTGAACTGTTCC.

[0060] The first round of PCR products were used as templates to perform the second round of PCR to amplify the heavy chain antibody VHH fragment. The upstream primer was combined with the antibody FR1 region, and the 5' end contained the Sfil enzyme cutting site GGCCCAGCCGGCC, and the sequence was shown as SEQ ID NO. 9; the downstream primer was combined with the antibody hinge region and FR4 region, and the 5' end contained the Sfil enzyme cutting site GGCCACGAAGGCC, and the sequence was shown as SEQ ID NO. 10. The target fragment with a size of about 450 bp was recovered by electrophoresis, and the VHH fragment library was obtained.

[0061] SEQ ID NO. 9: ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAG CTGGTGGAGTCTGG.

[0062] SEQ ID NO. 10: GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGT GGTGCG.

[0063] (2) Detection of the electrotransformation of library vectors and library capacity and diversity: The yeast surface display vector pYDisplay and VHH fragment library were digested with Sfil. The 5000 bp pYDisplay vector fragments were recovered by electrophoresis. The yeast competent strain stored at -80°C was streaked onto YPD solid medium plates and incubated at 30°C for 5 days. The yeast competent was inoculated into 50 mL of YPD medium and incubated at 30°C for 2 days. After mixing the linearized vector fragments and PCR products, electroshock transformation was performed; the yeast competent after electroshock was transferred to a culture bottle and incubated at 30°C for 1 h. 20 μl of the resuspension was diluted 5000-fold with SDCAA medium. 100 μl was taken and plated on SDCAA plates. After 3 days of incubation, the library capacity was calculated. The remaining bacterial solution was further incubated for 1 day, then centrifuged at 3000g for 5 min to collect the remaining bacterial solution, and the supernatant was discarded; 10 mL of SDCAA medium was added for resuspension, mixed with 50% glycerol at a ratio of 1:1, and stored at -80°C. The results showed that the library capacity of the single-domain antibody yeast display library obtained from the llama PBMC was about 1.2 x 10 9 .

[0064] Example 3

[0065] Screening of the yeast display library

[0066] (1) Pretreatment of streptavidin magnetic beads and yeast cells: In order to screen antibodies with high affinity to PD-1, 10 mL of the yeast library (about 2 x 10 8 Yeast cells) were centrifuged at 3000g for 5 min, and the supernatant was discarded. The yeast library was resuspended with 0.5% PBSA buffer (PBS buffer + 0.5% BSA), transferred to a 1.5 mL centrifuge tube, centrifuged at 3000g for 5 min, and the supernatant was discarded after repeating the washing once. 1 mL of 0.5% PBSA buffer was added to three 1.5 mL centrifuge tubes, and 10 μl of streptavidin magnetic beads were added. The centrifuge tubes were placed in a bag and fixed to a rotary mixer for incubation at 4°C for 5 min. The centrifuge tubes were placed on a magnetic stand for 5 min, and the supernatant was aspirated. Then 1 mL of 0.5% PBSA buffer was added again, and the rotary incubation was performed at 4°C for another 5 min, and the supernatant was discarded.

[0067] (2) Negative selection of the yeast display library with empty magnetic beads: The washed yeast solution was transferred to an empty magnetic bead tube and placed in a bag for incubation at 4°C for 60 min. The empty magnetic bead tube with the incubated yeast was placed on a magnetic stand for 10 min, and the yeast solution was transferred to a new empty magnetic bead tube and incubated at 4°C for 30 min. After the incubation, the yeast solution was transferred to a new empty magnetic bead tube after being placed on a magnetic stand for 15 min.

[0068] (3) Biotin-PD-1-His magnetic panning yeast display library: 100 μL of 50 μg / mL biotin-PD-1-His protein solution (diluted with 0.5% PBSA buffer) was added to a centrifuge tube containing streptavidin magnetic beads, the centrifuge tube was placed in a bag, and rotation incubation was performed at 4°C for 60 min. Then 1 mL of 0.5% PBSA buffer was added, and the centrifuge tube was left to stand for 5 min, and the supernatant was discarded while the centrifuge tube was left to stand on the magnetic stand. 1 mL of 0.5% PBSA buffer was added. The centrifuge tube was removed from the magnetic stand, and the mixture was mixed by blowing. The centrifuge tube was again placed on the magnetic stand, and the supernatant was discarded while the centrifuge tube was left to stand on the magnetic stand. After the above washing step was repeated once, the biotin-PD-1-His coated positive panning magnetic beads were obtained. The biotin-PD-1-His coated positive panning magnetic beads were added to the yeast cells after negative panning, and rotation incubation was performed at 4°C for 60 min. After incubation, the mixture was left to stand on the magnetic stand at 25°C for 15 min; the centrifuge tube was left to stand on the magnetic stand, and the yeast cell solution was discarded. The 1.5 mL centrifuge tube was removed from the magnetic stand, and 1 mL of 0.5% PBSA buffer was added to the centrifuge tube. The magnetic beads were gently blown and transferred to a sterile 1.5 mL centrifuge tube. The centrifuge tube was placed on the magnetic stand, and the supernatant was discarded after the mixture was left to stand at 25°C for 5 min. The washing step was repeated twice. After washing, the magnetic beads and the adhered yeast cells were resuspended in 1 mL of SDCAA medium, and 20 μL of the resuspension was taken and plated on two plates, each with a volume of 100 μL. 5 μL of the resuspension was taken and plated on one plate.

[0069] (4) Flow cytometry detection of panned yeast cells: The yeast cells after the above two-step panning were rotationally incubated with the His-tagged PD-1 protein at 4°C for 60 min. Then the supernatant was removed by centrifugation, and anti-His-tag and anti-V5-tag flow cytometry antibodies (the yeast expression vector contains a V5 tag to indicate whether the expression vector is successfully transferred into the yeast cells) were added, and rotation incubation was performed at 4°C for 60 min. Then the supernatant was removed by centrifugation, and the cells were resuspended in 1 mL of PBS buffer. Then the supernatant was removed by centrifugation, and the cells were resuspended in 500 μL of PBS buffer, and flow cytometry analysis was performed. The results showed that the yeast cells capable of binding to the PD-1 protein in the antibody display library of the panned yeast cells increased by 35% compared with before panning.

[0070] (5) Selection of the monoclonal: the yeast monoclonal was picked and inoculated into the culture medium to induce expression. After 48 h, the bacteria solution was incubated with the His-tagged PD-1 protein at 4°C for 60 min. Then, the supernatant was discarded by centrifugation, the precipitate was resuspended with a solution containing biotin-coupled anti-His-tag flow cytometry antibody, and incubated at 4°C for 60 min. Then, the supernatant was discarded by centrifugation, the precipitate was resuspended with a solution containing PE-streptavidin, and incubated at 4°C for 60 min. Then, the supernatant was discarded by centrifugation, 500 μl of PBS was used to resuspend the cells, and flow cytometry analysis was performed.

[0071] Finally, a monoclonal with a high positive rate was selected and named 195-1-D10. The 195-1-D10 yeast monoclonal was lysed using 0.2% SDS (incubated at 95°C for 10 min), centrifuged, and 0.5 μL of the supernatant was taken as a template for PCR amplification and sequencing. The remaining bacteria solution was stored at -20°C. Sanger sequencing showed that the CDR1 amino acid sequence of 195-1-D10 was SEQ ID No. 1, the CDR2 amino acid sequence was SEQ ID No. 2, and the CDR3 amino acid sequence was SEQ ID No. 3. The amino acid sequence of 195-1-D10 was SEQ ID No. 4.

[0072] Example 4

[0073] Expression of the recombinant single-domain antibody and detection of binding to the target protein

[0074] To express the recombinant single-domain antibody 195-1-D10, the VHH sequence of the candidate 195-1-D10, the CMV promoter sequence, and the IgG1 Fc sequence were amplified using a PCR instrument. 50 μL of the PCR product was added with 1 / 10 volume of 10×loading buffer, and electrophoresis analysis was performed using 1% agarose. The band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band size of VHH was 500 bp. The target band was cut from the gel, and the PCR product was purified. The VHH sequence of 195-1-D10, the CMV promoter, and the IgG1 Fc sequence were ligated using a PCR instrument. Then, 50 μL of the PCR product was added with 1 / 10 volume of 10×loading buffer, and electrophoresis analysis was performed using 1% agarose. The target band was cut from the gel, and the PCR product was purified. After the obtained PCR product was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant single-domain antibody 195-1-D10.

[0075] The binding specificity of 195-1-D10 was detected. 3×10 5CHO-K1 or CHO-K1-PD-1 cells were incubated at room temperature for 1 hour. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. 100 μl of PE-labeled anti-human IgG antibody (1:500 dilution) was added, and the cells were incubated at room temperature in the dark for 45 minutes. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0076] The results are as follows Figure 1 As shown, the expression supernatant of the negative control group did not significantly bind to either CHO-K1 or CHO-K1-PD-1; the positive control antibody significantly bound to CHO-K1-PD-1 but not to CHO-K1 cells, indicating that CHO-K1 cells do not express PD-1 protein, while CHO-K1-PD-1 cells express PD-1 protein and can be used as detection cells for alpaca antibodies. 195-1-D10 did not bind to CHO-K1 cells but significantly bound to CHO-K1-PD-1, and most CHO-K1-PD-1 cells expressed PD-1 protein, indicating that 195-1-D10 has good affinity and specificity.

[0077] Example 5

[0078] Purification and half-maximal effect concentration (EC50) of recombinant antibodies 50 ) Measurement

[0079] To determine the EC of 195-1-D10 antibody 50 The expression plasmid encoding the recombinant single-domain antibody was transiently transfected into 293F cells and cultured in shake flasks for antibody expression and purification. Since the target recombinant antibody contains a human IgG fragment, affinity purification was performed using Protein A magnetic beads. Protein A magnetic beads were washed twice sequentially with 30 mL of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer, respectively. The appropriate volume of Protein A magnetic beads was added to the 293F cell shake flask according to the required sample volume (calculated at 20 mg IgG / 1 mL Protein A magnetic beads). The cells were incubated overnight at 4°C and 120 rpm.

[0080] Protein A beads were collected using a magnetic separator and transferred to 50 mL centrifuge tubes. The beads were washed twice with 30 mL PBS buffer and deionized water, and then resuspended in 1 mL elution buffer. After incubation at room temperature for 5 min, the beads were collected again using a magnetic separator and transferred to 15 mL centrifuge tubes. The Protein A beads were eluted twice more, and the eluates were combined. Neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with at least 100 times its volume of PBS, first at 25 °C for 2 h, then with one buffer change, followed by dialyzing at 8 °C for 16 h. Finally, the protein concentration was determined, and the sample was filtered through a 0.22 μm sterile filter and aliquoted. The samples were stored at -80 °C for later use.

[0081] Dilute PD-1 protein to 2 μg / mL using coating buffer, and add 100 μL / well to each well of a 96-well microplate. Coat overnight at 4°C. Wash 5 times with PBST, add 200 μL / well of blocking buffer, and block at room temperature for 2 h. Wash 5 times with PBST, and add different concentrations of 195-1-D10 candidate antibody (10 μg / mL, 3.3 μg / mL, 1.1 μg / mL, 0.37 μg / mL, 0.12 μg / mL, 0.04 μg / mL, 0.014 μg / mL, and 0 μg / mL), and incubate at room temperature for 60 min. Wash 5 times with PBST, dilute HRP-Protein A 1:50000, add 100 μL / well to each well, and incubate at room temperature for 45 min. Wash 5 times with PBST, and add 100 μL of TMB chromogenic buffer to each well for 10 min at room temperature in the dark. Add 50 μL of stop solution to each well and read the absorbance at 450 nm wavelength on a microplate reader.

[0082] The results are shown in Table 1 and Figure 2 As shown, 195-1-D10 is associated with the EC of CHO-K1-PD-1. 50 The value was 0.0042 μg / mL, indicating that 195-1-D10 has a high affinity.

[0083] Table 1

[0084]

[0085] Example 6

[0086] Recombinant antibody blocking function test

[0087] To test the function of 195-1-D10 in blocking the binding of PD-1 to PD-L1, 100 μL of cell suspension was added to each well of a 96-well plate, with 2 × 10⁶ cells per well. 4 One effector cell, Jurkat-PD-1-Luciferase (expressing PD-1 molecules, which can inhibit the expression of Luciferase fluorescent protein after binding to PD-L1), and 8 × 104 CHO-K1-PD-L1 cells (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of Luciferase fluorescent protein of Jurkat-PD-1-Luciferase cells). Then 100 μL of 195-1-D10 of different concentrations were added to the corresponding wells, so that the final concentrations in the corresponding wells were 120 μg / mL, 40 μg / mL, 13.3 μg / mL, 4.4 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.16 μg / mL, 0.05 μg / mL, 0.02 μg / mL and 0 μg / mL, respectively. After 18 h of co-culture, 20 μL of One-Glo reagent was added to each well, and the Luciferase fluorescence value was read.

[0088] Results Figure 3 As shown in the figure, the maximum induction effect value of 195-1-D10 was 2.0 (the difference between the maximum Luciferase fluorescence value and the Luciferase fluorescence value at 0 μg / mL), indicating that 195-1-D10 had strong ability to block the binding of PD-1 to PD-L1 and could effectively promote the expression of Luciferase by Jurkat-PD-1-Luciferase, which could be used for the development of immune-enhancing drugs.

[0089] Example 7

[0090] Recombinant antibody half-inhibitory concentration (IC50) experiment

[0091] In order to detect the half-inhibitory concentration of 195-1-D10 for blocking the binding of PD-1 to PD-L1, PD-1 protein was diluted to 2 μg / mL with coating solution, 100 μL / well was taken into a 96-well enzyme-labeled plate, and was coated at 4°C overnight. PBST was washed 5 times, 200 μL / well of blocking solution was added, and was blocked at room temperature for 2 h. PBST was washed 5 times, and 195-1-D10 candidate antibodies of different concentrations, i.e., 25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL and 0 μg / mL, respectively, were added, and were incubated at room temperature for 15 min. Biotin-PD-L1 protein (4 μg / mL) was added, and was incubated at room temperature for 45 min. PBST was washed 5 times, the secondary antibody (Streptavidin-HRP) was diluted at 1:10000, 100 μL / well was added to the enzyme-labeled plate, and was incubated at room temperature for 45 min. PBST was washed 5 times, 100 μL of TMB color developing liquid was added to each well, and was developed at room temperature for 10 min in the dark. 50 μL of stop solution was added to each well, and the absorbance was read on an enzyme-labeled instrument at a wavelength of 450 nm.

[0092] The results are shown in Table 2 and Figure 4As shown, the IC50 value of 195-1-D10 is 0.68 μg / mL, indicating that 195-1-D10 can block the binding of PD-L1 to PD-1, and the blocking ability is strong.

[0093] Table 2

[0094]

[0095]

[0096] In summary, the present application uses PD-1 protein to immunize a llama. After determining the titer of PD-1 specific antibodies in the serum of the llama by ELISA, the peripheral blood mononuclear cells (PBMC) of the llama are isolated, RNA is extracted and cDNA is obtained by reverse transcription. The VHH sequence is amplified using llama single domain antibody specific primers, and cloned into a yeast expression plasmid to construct a yeast display library.

[0097] The yeast display library is then positively and negatively screened using biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeast bacteria that bind to PD-1 protein. Single clones are picked from the enrichment product, and the specific binding of the candidate antibodies to PD-1 is detected by ELISA. After the positive antibodies are expressed and purified, the affinity of the antibodies to PD-1 protein is detected. Antibody clones with strong affinity to PD-1 are selected, and the ability to block the binding of PD-1 and PD-L1 is detected. The results show that the specificity of the llama antibodies is high, and the antibodies can block the binding of PD-1 and PD-L1, and enhance the function of immune cells.

[0098] The applicant states that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A recombinant antibody against CD279, characterized in that, The sequence of the recombinant antibody is shown as SEQ ID No.

6.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD279 recombinant antibody of claim 1.

3. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 2; and the expression vector, after transfecting, transducing or transforming a host cell, enables the host cell to express the anti-CD279 recombinant antibody of claim 1.

4. The expression vector of claim 3, wherein, The expression vector further comprises a promoter. The promoter is any one of EF1a, PGK1, Ubc, human beta actin, CAG, SV40 or CMV.

Images