Antibody D10 for resisting CD279 and application thereof

Abstract

The invention relates to an antibody D10 for resisting CD279 and application of the antibody D10. The amino acid sequence of the antibody D10 comprises a CDR1 shown as SEQ ID No. 1, a CDR2 shown as SEQ ID No. 2 and a CDR3 shown as SEQ ID No. 3. The antibody D10 and CD279 protein have high affinity and specificity, and experiments prove that the antibody can effectively restore the anti-tumor activity of T cells by blocking a PD-1 / PD-L1 pathway, and enhance the anti-tumor ability of the T cells.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-CD279 antibody D10 and applications thereof. Background Art

[0002] CD279, also known as programmed death receptor 1 (PD-1), is an important immunosuppressive molecule. It belongs to the immunoglobulin superfamily and is a membrane protein with 288 amino acid residues. It was originally cloned from the apoptotic mouse T cell hybridoma 2B4.11. Immune regulation targeting PD-1 is of great significance for anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant 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 bind to initiate programmed cell death of T cells, allowing tumor cells to gain immune escape. PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 inhibitors are a new class of drugs that block PD-1, which can activate the immune system to attack tumors and are used to treat certain types of cancer.

[0003] Immune checkpoints refer to programmed death receptors and their ligands. Immune checkpoint blockade therapy based on programmed death receptors and their ligands inhibits the binding of programmed death receptors and their ligands, thereby increasing the host immune system's aggressiveness against tumor cells. Immune checkpoints can be considered the brake system of immune cells, which refers to a series of molecules expressed on immune cells that can regulate the degree of immune activation, keeping the activation level of T cells within a normal range and preventing damage to the body's healthy tissues.

[0004] Single domain antibody is a naturally missing light chain antibody in the peripheral blood of alpacas. It contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate as artificially modified single chain antibody fragments (scFv). More importantly, the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody.

[0005] Therefore, how to prepare anti-CD279 nanoantibodies has become a problem that needs to be solved urgently. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an anti-CD279 antibody D10 and its application. The antibody has high affinity and specificity to the CD279 protein. Experiments have shown 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 this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an anti-CD279 antibody D10, 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 invention creatively constructs an anti-CD279 antibody D10, which includes three complementary determining regions. Antibody D10 is obtained by amplifying the VHH fragment of the CD279 antibody in alpaca serum. It is a single-domain antibody. The antibody fragment is smaller and easier to penetrate into tissues and cells, has higher stability, and has been proven experimentally to have high specificity for CD279 protein, and can be used for subsequent research on drugs and other aspects.

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

[0014] SEQ ID No.4:

[0015] QVQLVESGGGLVQPGGSLRLSCATSGSIFSLNDMVWYRQAPGKQRELVA RITNNLSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGS SGTWYPLHYWGQGTQVSVSS.

[0016] In a second aspect, the present invention provides a recombinant anti-CD279 antibody, wherein the recombinant antibody comprises the anti-CD279 antibody D10 described in the first aspect and an Ig Fc fragment.

[0017] In the present invention, an antibody composed of anti-CD279 antibody D10 and Ig Fc fragment is constructed. The recombinant antibody has been experimentally verified to have high affinity and strong blocking effect, and is used in the field of scientific research, for example, to explore the mechanism of immunosuppression points, to screen blocking drugs related to immunosuppression points, and so on.

[0018] Preferably, the Ig Fc fragment is derived from any one of human, mouse or monkey sources, or a combination of at least two of them.

[0019] Preferably, the Ig Fc comprises any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4.

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

[0021] SEQ ID No.5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

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

[0024] SEQ ID No.6:

[0025] QVQLVESGGGLVQPGGSLRLSCATSGSIFSLNDMVWYRQAPGKQRELVARITNNLSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSS GTWYPLHYWGQGTQVSVSSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

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

[0027] In a fourth aspect, the present invention provides an expression vector, which contains the nucleic acid molecule described in the third aspect; and after transfecting, transducing or transforming a host cell, the expression vector enables the host cell to express the anti-CD279 antibody D10 described in the first aspect or the recombinant anti-CD279 antibody described in 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 the fifth aspect, a use of the anti-CD279 antibody D10 described in the first aspect, the recombinant anti-CD279 antibody described in the second aspect, the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect in the preparation of an anti-PD-1 drug.

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

[0032] 1. In the present invention, the anti-CD279 antibody D10 is obtained by amplifying the VHH fragment of the CD279 antibody in alpaca serum. It is a single-domain antibody. The antibody fragment is smaller and easier to penetrate into tissues and cells, has higher stability, and has been proven to have high specificity for CD279 protein through experiments.

[0033] 2. In the present invention, the antibody D10 has a simple structure and is easier to modify, and has a low production cost, and is suitable for large-scale production. Alpaca-derived antibodies have a low risk of causing immune responses because the alpaca's immune system can produce antibodies with high specificity and affinity for specific antigens and have weak immunogenicity to humans.

[0034] 3. In the present invention, it has been experimentally verified that the antibody D10 has a high affinity and a strong blocking effect, and is used in the field of scientific research, for example, to explore the mechanism of immunosuppression points, to screen blocking drugs related to immunosuppression points, and so on. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Flow cytometry analysis of the recombinant single domain antibody.

[0036] Figure 2 ELISA for detecting EC of recombinant single domain antibodies50 Result graph.

[0037] Figure 3 This is the result of the recombinant antibody blocking function test.

[0038] Figure 4 This is a graph showing the IC50 results of ELISA testing of recombinant single domain antibodies. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only 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 shall be subject to the claims.

[0040] 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 both sides of the cervical lymph nodes. Immunization was performed once every 3 weeks, for a total of 4 times. 5 mL of peripheral blood was collected before immunization, after the second, third and fourth immunizations, centrifuged at 800g for 10 minutes, and the upper serum was collected. 1 μg / mL of PD-1-His protein was added to a 96-well plate at 100 μl / well and coated overnight at 4°C; then the supernatant was discarded, and PBST buffer (PBS buffer containing 0.05% Tween-20) was used to wash 5 times; 200 μl / well of blocking buffer was added, and incubated at 37°C for 2 hours. After discarding the blocking buffer, the plate was washed 5 times with PBST buffer. The serum collected above was gradiently diluted with PBS buffer, and 100 μL of gradient diluted serum was added to the 96-well ELISA plate, and incubated at 25°C for 60 minutes. The control well was PBS buffer. The liquid in the well was discarded and washed 5 times with PBST buffer. Add 100 μl HRP anti-llama IgG (H+L) antibody (1:50,000 dilution) and incubate at 25°C for 60 min. After discarding the liquid in the wells, wash the plate 5 times with PBST buffer. Add 100 μl / well TMB colorimetric solution and incubate at 25°C in the dark for 15 min. Add 50 μL / well stop solution and read the OD450 value in the wells using an ELISA reader.

[0054] "1:X" is used to represent the antibody titer (X refers to the maximum dilution multiple at which the antibody can be detected). The binding titer of the serum of the alpaca before immunization with the PD-1 protein was lower 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 the PD-1 protein was significantly higher than 1:64,000, indicating that the content of anti-PD-1 specific antibodies in the alpaca serum 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 alpacas and PBMC was isolated. RNA was extracted and cloned using PrimeScript TMII 1st Strand cDNA Synthesis Kit was used to reverse transcribe cDNA. PBMC cDNA was used as a template to PCR amplify the alpaca heavy chain antibody sequence. The upstream primer was bound to the signal peptide of the VHH antibody ORF, and the sequence was shown in SEQ ID NO.7; the downstream primer was bound to the CH2 region of the VHH antibody ORF, and the sequence was shown in SEQ ID NO.8. The target fragment 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 product was used as a template to perform a second round of PCR to amplify the heavy chain antibody VHH fragment. The upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC, and the sequence is shown in SEQ ID NO.9; the downstream primer binds to the antibody hinge region and FR4 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC, and the sequence is shown in SEQ ID NO.10. The target fragment with a molecular weight of about 450 bp was recovered by electrophoresis to obtain a VHH fragment library.

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

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

[0063] (2) Detection of electroporation of library vectors and library capacity and diversity: Use SfiI to digest the yeast surface display vector pYDisplay and the VHH fragment library. Recover the 5000 bp pYDisplay vector fragment by electrophoresis. Streak the yeast competent strain frozen at -80°C onto a YPD solid culture medium plate and culture at 30°C for 5 days. Inoculate the yeast competent strain into 50 mL YPD culture medium and culture at 30°C on a shaker for 2 days. Mix the linearized vector fragment and PCR product and perform electroporation transformation; transfer the yeast competent strain after electroporation to a culture bottle and culture at 30°C on a shaker for 1 hour. Take 20 μl of the resuspension and dilute it 5000 times with SDCAA culture medium. Pipette 100 μL and apply it to the SDCAA plate. Calculate the library capacity after 3 days of culture. After the remaining bacterial solution was cultured for 1 day, the remaining bacterial solution was collected by centrifugation at 3000g for 5 min, and the supernatant was discarded; 10 mL of SDCAA medium was added for resuspending, and 50% glycerol: resuspending solution was mixed 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 by alpaca PBMC was about 1.2×10 9 .

[0064] Example 3

[0065] Panning of yeast display libraries

[0066] (1) Pretreatment of streptavidin magnetic beads and yeast cells: To screen antibodies with high affinity for PD-1, 10 mL of yeast library (about 2 × 10 8 Yeast cells), 3000g, centrifuge for 5min, and remove the supernatant. Resuspend the yeast library with 0.5% PBSA buffer (PBS buffer + 0.5% BSA), transfer to a 1.5mL centrifuge tube, 3000g, centrifuge for 5min, discard the supernatant, and repeat the wash once. Add 1mL 0.5% PBSA buffer to 3 1.5mL centrifuge tubes, and then add 10μL streptavidin magnetic beads. Put the centrifuge tubes in a bag, fix them to the rotating mixer, and rotate and incubate at 4℃ for 5min. Place the centrifuge tubes on the magnetic stand for 5min and discard the supernatant. Then add 1mL 0.5% PBSA buffer again, rotate and incubate at 4℃ for another 5min, and discard the supernatant.

[0067] (2) Empty magnetic beads negative panning yeast display library: Transfer the washed yeast solution to an empty magnetic bead tube, place it in a bag, and incubate at 4°C with rotation for 60 min. Place the empty magnetic bead tube with yeast on a magnetic rack for 10 min, transfer the yeast solution to a new empty magnetic bead tube, and incubate at 4°C with rotation for 30 min. After the incubation, place it on a magnetic rack for 15 min, transfer the yeast solution to a new empty magnetic bead tube.

[0068] (3) Biotin-PD-1-His magnetic panning yeast display library: Add 100 μL of 50 μg / mL biotin-PD-1-His protein solution (diluted with 0.5% PBSA buffer) to the centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 60 minutes. Then add 1 mL of 0.5% PBSA buffer, let it stand for 5 minutes, keep the centrifuge tube on the magnetic stand, discard the supernatant, and add 1 mL of 0.5% PBSA buffer. Remove the centrifuge tube from the magnetic stand, blow and mix, place the centrifuge tube on the magnetic stand again, let it stand for 5 minutes, keep the centrifuge tube on the magnetic stand, and discard the supernatant. After repeating the above washing steps once again, the positive selection magnetic beads coated with biotin-PD-1-His are obtained. Add the positive selection magnetic beads bound to biotin-PD-1-His to the yeast cells that have completed negative selection, and incubate at 4°C for 60 minutes. After the incubation, place it on a magnetic stand and let it stand at 25°C for 15 minutes; keep the centrifuge tube on the magnetic stand and discard the yeast solution. Remove the 1.5mL centrifuge tube from the magnetic stand, add 1mL of 0.5% PBSA buffer to the centrifuge tube, gently blow the magnetic beads, and transfer them to a sterile 1.5mL centrifuge tube. Place the centrifuge tube on a magnetic stand, let it stand at 25°C for 5 minutes, discard the supernatant; repeat the wash twice. After the wash, resuspend the magnetic beads and adhered yeast cells with 1mL SDCAA medium, pipette 20μl of the resuspension into 180μL SDCAA medium and apply two plates, each with a volume of 100μL, and then pipette 5μL of the resuspension into 95μL SDCAA medium and apply one plate.

[0069] (4) Flow cytometry detection of yeast after selection: The yeast after the above two steps of selection was incubated with the PD-1 protein containing the His tag at 4°C for 60 minutes. The supernatant was then removed by centrifugation, and anti-His tag and anti-V5 tag flow cytometry antibodies were added (the yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into the yeast), and the cells were incubated at 4°C for 60 minutes. The supernatant was then removed by centrifugation, and 1 mL of PBS buffer was added to resuspend the cells. The supernatant was then removed by centrifugation, and 500 μL of PBS buffer was added to resuspend the cells for flow cytometry analysis. The results showed that compared with before selection, the number of yeast that could bind to the PD-1 protein in the yeast antibody display library after selection increased by 35%.

[0070] (5) Selection of single clones: Pick a yeast single clone and inoculate it into the culture medium to induce expression. After 48 hours, incubate the bacterial solution with the PD-1 protein containing the His tag at 4°C for 60 minutes. Then centrifuge and discard the supernatant, resuspend the precipitate with a solution containing a biotin-conjugated anti-His tag flow antibody, and incubate at 4°C for 60 minutes. Then centrifuge and discard the supernatant, resuspend the precipitate with a solution containing PE-streptavidin, and incubate at 4°C for 60 minutes. Then centrifuge and discard the supernatant, resuspend the cells in 500μl PBS, and perform flow analysis.

[0071] Finally, a single clone with a high positive rate was selected and named 195-1-D10. The 195-1-D10 yeast single clone was lysed using 0.2% SDS (incubated at 95°C for 10 min), centrifuged, and 0.5 μL of the bacterial supernatant was taken as a template for PCR amplification and sent for testing. The remaining bacterial solution was stored at -20°C. After Sanger sequencing, 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 is SEQID No.4.

[0072] Example 4

[0073] Expression of recombinant single domain antibodies and detection of binding to target proteins

[0074] In order to express the recombinant single domain antibody 195-1-D10, the VHH sequence, CMV promoter sequence and IgG1 Fc sequence of the candidate 195-1-D10 were amplified by PCR instrument, 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. 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, CMV promoter and IgG1 Fc sequence of 195-1-D10 were connected using a PCR instrument, and then 50 μL of the PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The target band was cut out 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 tested. The culture supernatant containing 195-1-D10 was mixed with 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 3 times with PBS. 100μl PE-labeled Anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature for 45 minutes in the dark. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500μL 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 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 was able to detect that most CHO-K1-PD-1 cells expressed PD-1 protein, indicating that 195-1-D10 had good affinity and specificity.

[0077] Example 5

[0078] Purification and half effective concentration (EC 50 ) determination

[0079] To determine the EC of 195-1-D10 antibody 50 , transiently transfect the expression plasmid encoding the recombinant single domain antibody into 293F cells, and shake the flask to culture for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30mL of PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20mg IgG / 1mL Protein A magnetic beads) to the 293F cell shake flask according to the required amount of the sample. Incubate in a shaking incubator at 120rpm and 4°C overnight.

[0080] Collect the Protein A beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. Wash twice with 30mL of PBS buffer and deionized water, and resuspend with 1mL of elution buffer. After incubation at room temperature for 5 minutes, collect the beads with a magnetic separation rack and transfer them to a 15mL centrifuge tube. After eluting the Protein A beads twice, combine the eluates and add neutralization buffer to adjust the pH of the solution. The eluted sample is dialyzed with at least 100 times the volume of the sample with PBS, first dialyzed at 25℃ for 2h, changed the solution once, and then dialyzed at 8℃ for 16h. Finally, determine the protein concentration and filter the sample with a 0.22μm sterile filter membrane and aliquot it, and store it in a -80℃ refrigerator for use.

[0081] Use coating solution to dilute PD-1 protein to 2μg / mL, pipette 100μL / well into 96-well ELISA plate, and coat overnight at 4℃. Wash 5 times with PBST, add 200μL / well of blocking solution, and block at room temperature for 2h. Wash 5 times with PBST, add different concentrations of 195-1-D10 candidate antibody, which are 10μg / mL, 3.3μg / mL, 1.1μg / mL, 0.37μg / mL, 0.12μg / mL, 0.04μg / mL, 0.014μg / mL and 0μg / mL, and incubate at room temperature for 60min. Wash 5 times with PBST, dilute HRP-Protein A at 1:50000, add 100μL / well into ELISA plate, and incubate at room temperature for 45min. Wash 5 times with PBST, add 100μL of TMB colorimetric solution to each well, and color for 10min at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

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

[0083] Table 1

[0084]

[0085] Example 6

[0086] Recombinant antibody blocking function test

[0087] In order to detect the function of 195-1-D10 in blocking the binding of PD-1 and PD-L1, 100 μL of cell suspension was added to a 96-well plate, including 2×10 4 Effector cells 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 Target cells CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of Luciferase fluorescent protein in Jurkat-PD-1-Luciferase cells). Then, 100 μL of 195-1-D10 of different concentrations was 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. After 18 hours of co-culture, 20 μL of One-Glo reagent was added to each well to read the Luciferase fluorescence value.

[0088] result Figure 3 As shown, the maximum induction effect value of 195-1-D10 is 2.0 (the difference between the maximum Luciferase fluorescence value and the 0 μg / mL Luciferase fluorescence value), indicating that 195-1-D10 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-Luciferase to express Luciferase, and can be used to develop 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 blocking the binding of PD-1 and PD-L1, the PD-1 protein was diluted to 2 μg / mL with the coating solution, and 100 μL / well was pipetted into a 96-well ELISA plate and coated overnight at 4°C. Washed 5 times with PBST, 200 μL / well of blocking solution was added, and blocked at room temperature for 2 hours. Washed 5 times with PBST, and different concentrations of 195-1-D10 candidate antibodies were added, namely 25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL and 0 μg / mL, and incubated at room temperature for 15 minutes. Add Biotin-PD-L1 protein (4 μg / mL) and incubate at room temperature for 45 minutes. Washed 5 times with PBST, the secondary antibody (Streptavidin-HRP) was diluted 1:10000, and 100 μL / well was added to the ELISA plate and incubated at room temperature for 45 minutes. Wash 5 times with PBST, add 100 μL TMB colorimetric solution to each well and color for 10 min at room temperature in the dark, add 50 μL stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[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 and PD-1, and the blocking ability is strong.

[0093] Table 2

[0094]

[0095]

[0096] In summary, the present invention uses PD-1 protein to immunize alpacas. After determining the titer of PD-1 specific antibodies in alpaca serum by ELISA, alpaca peripheral blood mononuclear cells (PBMC) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single domain antibody specific primers are used to amplify the VHH sequence and clone it into a yeast expression plasmid to construct a yeast display library.

[0097] Then, the yeast display library was positively and negatively screened with biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeast that bind to PD-1 protein. Monoclones were picked from the enriched products, and the specific binding of candidate antibodies to PD-1 was detected by ELISA. After the positive antibodies were expressed and purified, their affinity for binding to PD-1 protein was tested. Antibody clones with strong affinity to PD-1 were selected to test their ability to block the binding of PD-1 and PD-L1. The results showed that alpaca antibodies have high specificity and can block the binding of PD-1 and PD-L1 at the same time, enhancing the function of immune cells.

[0098] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-CD279 antibody D10, characterized in that: The amino acid sequence of the antibody D10 includes CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.2 and CDR3 shown in SEQ ID No.

3.

2. The antibody D10 according to claim 1, characterized in that The amino acid sequence of the antibody D10 includes the sequence shown in SEQ ID No.

4.

3. A recombinant anti-CD279 antibody, characterized in that: The recombinant antibody comprises the anti-CD279 antibody D10 according to claim 1 or 2 and an Ig Fc fragment.

4. The anti-CD279 recombinant antibody according to claim 3, characterized in that: The Ig Fc fragment is derived from any one of human, mouse or monkey sources, or a combination of at least two of them; The Ig Fc includes any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4.

5. The anti-CD279 recombinant antibody according to claim 3, characterized in that: The sequence of the Ig Fc fragment is shown as SEQ ID No.

5.

6. The anti-CD279 recombinant antibody according to claim 3, characterized in that: The sequence of the recombinant antibody is shown as SEQ ID No.

6.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD279 antibody D10 according to claim 1 or 2 or the recombinant anti-CD279 antibody according to any one of claims 3-6.

8. An expression vector, characterized in that The expression vector contains the nucleic acid molecule of claim 7; and after transfecting, transducing or transforming the host cell, the expression vector causes the host cell to express the anti-CD279 antibody D10 of claim 1 or 2 or the recombinant anti-CD279 antibody of any one of claims 3-6.

9. The expression vector according to claim 8, characterized in that The expression vector further comprises a promoter; The promoter includes any one of CMV, EF1a, PGK1, Ubc, human beta actin, CAG, SV40 or CMV, or a combination of at least two thereof.

10. Use of the anti-CD279 antibody D10 according to claim 1 or 2, the recombinant anti-CD279 antibody according to any one of claims 3 to 6, the nucleic acid molecule according to claim 7 or the expression vector according to claim 8 or 9 in the preparation of an anti-PD-1 drug.

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