Antibody A3 for resisting programmed death receptor 1 and application thereof

Abstract

The invention relates to an antibody A3 for resisting a programmed death receptor 1 and application thereof, the antibody A3 for resisting the programmed death receptor 1 comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 comprises a sequence as shown in SEQ ID NO. 1; the amino acid sequence of the CDR2 comprises a sequence as shown in SEQ ID NO. 2; the amino acid sequence of the CDR3 comprises a sequence as shown in SEQ ID NO. 3. The antibody disclosed by the invention is high in affinity and strong in capability of blocking the combination of PD-1 and PD-L1, and can be used for preparing immune-enhanced drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an antibody A3 against programmed death receptor 1 and an application thereof. Background Art

[0002] PD-1 (programmed death receptor 1), also known as CD279 (cluster of differentiation 279), is an important immunosuppressive molecule. It regulates the immune system and promotes self-tolerance by down-regulating the immune system's response to human cells, as well as by suppressing T-cell inflammatory activity. This prevents autoimmune diseases, but it also prevents the immune system from killing cancer cells.

[0003] At present, the use of PD-1 antibodies to block the PD-1 / PD-L1 pathway to restore T cell tumor activity, activate T cells, and kill tumor cells has become the focus of cancer immunotherapy. Alpaca antibodies have many advantages over ordinary antibodies, so the application of relevant technologies to prepare anti-PD-1 alpaca antibodies, made into antibody drugs or cell drugs, can be used for cancer treatment. Therefore, it is urgent to provide an alpaca antibody with PD-1 as a therapeutic target for the preparation of antibody drugs or cell drugs. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide an anti-programmed death receptor 1 antibody A3 and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-programmed death receptor 1 antibody A3, wherein the anti-programmed death receptor 1 antibody A3 comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3;

[0007] The amino acid sequence of the CDR1 includes the sequence shown in SEQ ID NO.1;

[0008] The amino acid sequence of the CDR2 includes the sequence shown in SEQ ID NO.2;

[0009] The amino acid sequence of the CDR3 includes the sequence shown in SEQ ID NO.3.

[0010] SEQ ID NO.1: GGSLDDYA.

[0011] SEQ ID NO. 2: GVARITSGLST.

[0012] SEQ ID NO. 3: NREIRGSGIWYPLDY.

[0013] In this application, PD-1 protein is emulsified with adjuvant and immunized with alpacas. The titer of PD-1 specific antibodies in alpaca serum is detected by ELISA. After confirming that it is high enough, the alpaca peripheral blood mononuclear cells 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. The yeast display library is then positively and negatively screened with biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeast that binds to PD-1 protein. A single yeast clone is picked from the yeast that binds to the PD-1 protein, and the recombinant single-domain antibody VHH sequence in the monoclonal yeast is amplified, named 195-5-A3, and the recombinant single-domain antibody 195-5-A3 VHH sequence is connected to the CMV promoter sequence and the IgG1 Fc sequence using a PCR instrument. After transient transfection into HEK293 cells, the harvested cell culture supernatant contains the recombinant single-domain antibody 195-5-A3. ELISA was used to test the specific binding of candidate antibodies to PD-1. After the positive antibodies were expressed and purified, their affinity for binding to the PD-1 protein was tested. Antibody clones with strong affinity for 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.

[0014] Preferably, the amino acid sequence of the anti-programmed death receptor 1 antibody A3 includes the sequence shown in SEQ ID NO.4.

[0015] SEQ ID NO.4:

[0016] MAQLQLVESGGGVVQPGGSLRLTCAASGGSLDDYAIGWFRQAPGKERE GVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREI RGSSGIWYPLDYWGQGTQVTVSSEPKTPKPQPAAASGHEG.

[0017] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-programmed death receptor 1 antibody A3 described in the first aspect.

[0018] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0019] In a fourth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the third aspect.

[0020] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains the expression vector described in the third aspect.

[0021] In a sixth aspect, the present invention provides a pharmaceutical composition, comprising the anti-programmed death receptor 1 antibody A3 described in the first aspect.

[0022] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0023] In the seventh aspect, the present invention provides a use of the anti-programmed death receptor 1 antibody A3 described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant lentivirus described in the fourth aspect, the host cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a detection reagent or an immune-enhancing drug.

[0024] Preferably, the target of the detection reagent is PD-1.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The antibody prepared in the present application has the advantage of being small in size and can penetrate into tissues and cells and the blood-brain barrier.

[0027] (2) The antibody prepared in the present application has a simple structure and is therefore easier to modify, such as humanization, multivalent construction, etc., and the production cost of the antibody is low, making it suitable for large-scale production.

[0028] (3) The antibodies prepared in the present application are highly specific. The immune system of alpacas is capable of producing antibodies with high specificity and affinity for specific antigens, and the risk of causing an immune response is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of flow cytometry detection of yeast library enrichment before and after sorting.

[0030] Figure 2 This is a graph showing the results of flow cytometry testing of the specificity of the recombinant single domain antibody.

[0031] Figure 3 This is the result graph of ELISA detection of EC50 of recombinant single domain antibody.

[0032] Figure 4 It is a recombinant antibody blocking function test.

[0033] Figure 5 The figure is the result graph of ELISA detection of IC50 of recombinant single domain antibody, wherein Figure A is the blocking curve of 195-5-A3, Figure B is the blocking curve of negative control antibody, and Figure C is the blocking curve of positive control antibody. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0035] Reagents used in the following examples:

[0036] Streptavidin PE(eBioscience,CAT#12-4317-87);

[0037] PE-anti-human IgG (eBioscience, CAT#12-4998-82);

[0038] Adjuvant immune adjuvant (GERBU, CAT#3030);

[0039] HRP-ProteinA(Boster,CAT#BA1080);

[0040] HRP-Streptavidin (Boster, CAT#BA1088);

[0041] PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#6210B);

[0042] DNA fragment recovery kit (TakaRa, CAT#9761);

[0043] Goat anti-Llama IgG(H+L)) Secondary Antibody[HRP](Novus,AT#NB7242);

[0044] THE TM V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805).

[0045] Example 1

[0046] Alpaca immunization and antibody titer determination

[0047] Two healthy alpacas (numbered 98# and 195#) were selected and immunized with PD-1 protein. The emulsified antigen was injected on the left and right sides near the cervical lymph nodes of the alpacas, with 2 injections on each side, and 500 μg of antigen was injected at each point. Immunization was performed once every 3 weeks, for a total of 4 immunizations. After each immunization, the alpacas were observed for 30 minutes to confirm that they were in good condition and had no discomfort symptoms. 5 mL of peripheral blood was collected before immunization, the second immunization, the third immunization, and two weeks after the fourth immunization, respectively, and centrifuged at 800×g for 10 minutes to collect the upper serum.

[0048] The PD-1-His recombinant protein was diluted with sterile PBS to a final concentration of 1 μg / mL, and the diluted recombinant protein was added to the ELISA plate (the amount of recombinant protein added was 100 μL / well), and coated overnight at 4°C; after removing the antigen coating solution, the plate was washed 5 times with PBST containing 0.05% Tween 20, and then mixed with the blocking solution (the amount of blocking solution added was 200 μL / well), blocked at 37°C for 2 hours, and after removing the blocking solution, the plate was washed 5 times with PBST. Serum was diluted with PBS in a gradient manner, and then added to a 96-well ELISA plate (100 μL / well). The control wells were filled with an equal volume of PBS and incubated at room temperature for 1 hour. The liquid in the wells was removed and washed 5 times with PBST. 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50,000 dilution) was added and incubated at room temperature for 1 hour. After shaking off the liquid in the wells, the plate was washed 5 times with PBST. 100 μL / well of TMB colorimetric solution was added. The wells were incubated at room temperature in the dark for 15 minutes, and 50 μL / well of stop solution was added. The OD450 value in the wells was read using an ELISA reader.

[0049] The results are shown in Tables 1 and 2. The binding titers of the sera of the two alpacas after four immunizations to the PD1 protein were significantly higher than those of the sera before immunization, indicating that the content of PD1-specific antibodies in the sera of the two alpacas was significantly increased after multiple rounds of immunization and can be used for the construction of yeast display libraries.

[0050] Table 1

[0051]

[0052]

[0053] Table 2

[0054]

[0055] Example 2

[0056] (1) VHH antibody fragment cloning

[0057] After four immunizations, the alpaca had a high level of anti-PD-1 specific antibodies in the serum, which was suitable for subsequent experiments. 100 mL of peripheral blood was collected, PBMCs were separated using lymphocyte separation solution, and RNA was subsequently extracted. PrimeScript TM II 1stStrand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, the alpaca heavy chain antibody sequence was amplified (the upstream primer binds to the signal peptide of the VHH antibody ORF, the primer sequence is 5'GTCCTGGCTGCTCTTCTACAAGG3', the downstream primer binds to the CH2 region, the primer sequence is 5'GGTACGTGCTGTTGAACTGTTCC 3'). Use 1% agarose for electrophoresis to recover and separate the target fragment with a molecular weight of about 750bp. Then use the first round PCR product as a template and use specific primers to amplify the heavy chain antibody VHH fragment. Use 1% agarose for electrophoresis to separate the target fragment with a molecular weight of about 450bp. Use a gel recovery kit to recover the target band and store the target fragment at -80℃ (the storage solution contains 1 / 10 volume of 3M sodium acetate, 1μg / μL glycogen, and 80% anhydrous ethanol).

[0058] Example 3

[0059] Panning of yeast display libraries

[0060] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0061] Yeast cell surface display technology is a eukaryotic protein expression system. Its basic principle is to fuse the exogenous target protein gene (nanoantibody sequence) with a specific vector gene sequence and then introduce it into yeast cells. The mechanism of protein transport from yeast cells to the membrane surface is used to immobilize and express the target protein on the yeast cell surface. It is widely used in nanoantibody screening.

[0062] Take 10mL yeast library (about 2×10 8 Yeast cells) at 3000×g, centrifuge for 5min, and remove the supernatant; at the same time, dilute sterile 5% PBSA (PBS+5% BSA) to a concentration of 0.5%; resuspend the yeast library with 1mL 0.5% PBSA, centrifuge at 3000×g for 5min, and discard the supernatant; wash once with 0.5% PBSA. Prepare 3 1.5mL centrifuge tubes (2 centrifuge tubes for negative selection of empty magnetic beads, 1 centrifuge tube for positive selection), add 1mL 0.5% PBSA to each centrifuge tube; resuspend the streptavidin affinity magnetic beads, pipette 10μL of magnetic beads into the 1.5mL centrifuge tube; rotate and incubate at 4℃ for 5 minutes; place the centrifuge tube on the magnetic stand for 5min, and discard the supernatant; then add 1mL 0.5% PBSA again, rotate and incubate at 4℃ for 5 minutes, and discard the supernatant.

[0063] (2) Yeast display library screening

[0064] Negative selection with empty magnetic beads: Place the centrifuge tube on a magnetic rack, add the washed yeast solution to the centrifuge tube containing the empty magnetic beads, and incubate at 4°C with rotation for 60 minutes; Place the empty magnetic bead centrifuge tube with yeast on a magnetic rack for 10 minutes, pipette the yeast solution into a new empty magnetic bead centrifuge tube, and incubate at 4°C with rotation for 30 minutes. After the incubation, place it on a magnetic rack for 15 minutes, pipette the yeast solution into a new empty magnetic bead centrifuge tube.

[0065] Biotin-PD-1-His magnetic separation: add 100 μL of 50 μg / mL Biotin-PD-1-His protein solution (biotin-coupled, diluted with 0.5% PBSA) to a centrifuge tube containing streptavidin magnetic beads, and incubate at 4°C for 60 minutes; add 1 mL of 0.5% PBSA, let stand for 5 minutes, and keep the centrifuge tube on the magnetic rack, remove the supernatant, add 1 mL of 0.5% PBSA, remove the centrifuge tube from the magnetic rack, mix well, place the centrifuge tube on the magnetic rack again, let stand for 5 minutes, and keep the centrifuge tube on the magnetic rack, and use a pipette to remove the supernatant; repeat the above washing steps once to obtain positive selection magnetic beads coated with Biotin-PD-1-His.

[0066] Add the positive selection magnetic beads bound to Biotin-PD-1-His to the yeast cells that have completed negative selection, rotate and incubate at 4°C for 60 minutes, and after the incubation, place it on a magnetic stand and let it stand at room temperature for 15 minutes; keep the centrifuge tube on the magnetic stand and use a pipette to absorb the unbound yeast solution; remove the 1.5mL centrifuge tube from the magnetic stand, add 1mL sterile 0.5% PBSA buffer, gently blow the magnetic beads, and then transfer them to a sterile 1.5mL centrifuge tube, place the centrifuge tube on the magnetic stand, let it stand at room temperature for 5 minutes, and discard the supernatant; repeat the washing twice, and perform the whole process in accordance with the aseptic operation requirements. After washing, resuspend the magnetic beads and adhered yeast cells with 1mL SDCAA medium, and draw 20μL of the resuspension into 180μL SDCAA medium for plate coating, and coat 2 plates in total, each plate has a volume of 100μL, and then draw 5μL of the resuspension into 95μL SDCAA medium to coat one plate.

[0067] After panning, yeast flow cytometry was used to detect whether the yeast expression vector contained a V5 tag to indicate whether the expression vector was successfully transferred into the yeast. The yeast that had undergone negative panning with empty magnetic beads and magnetic separation with Biotin-PD-1-His was incubated with the PD-1 protein containing the His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, added flow cytometry antibodies with anti-His tag and anti-V5 tag, incubated at 4°C for 60 minutes, centrifuged to remove the supernatant, resuspended the cells with 1 mL of PBS, then centrifuged to remove the supernatant, and resuspended the cells with 500 μL of PBS for flow cytometry analysis. The results are shown in Figure 2. Figure 1 As shown by Figure 1 It can be seen that the yeast capable of binding to PD-1 protein in the yeast antibody display libraries of 98# alpaca and 195# alpaca increased by 38% and 35% respectively before and after sorting, and subsequent experiments can be carried out.

[0068] Monoclonal selection: After overnight culture, monoclones were selected and inoculated into the culture medium to induce expression. After 48 hours, the bacterial solution was incubated with the PD-1 protein containing the His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, and the precipitate was resuspended with a solution containing a biotin-conjugated anti-His tag flow antibody, incubated at 4°C for 60 minutes, then centrifuged to remove the supernatant, and the precipitate was resuspended with a solution containing PE-Streptavidin, incubated at 4°C for 60 minutes, and the cells were resuspended with 1mL PBS, then centrifuged to remove the supernatant, and the cells were resuspended with 500μL PBS for flow analysis. Finally, the monoclonal 195-5-A3 with a high positive rate was selected. 195-5-A3 was lysed with 0.2% SDS (incubated at 95°C for 10min), centrifuged, and 0.5μL 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).

[0069] Example 4

[0070] Expression of recombinant single domain antibodies and detection of binding to target proteins

[0071] In order to detect the binding level of recombinant single domain antibody 195-5-A3 to target cells, we amplified the recombinant single domain antibody 195-5-A3 VHH sequence using a PCR instrument, connected it with the CMV promoter sequence and IgG1 Fc sequence, and transiently transfected it into HEK293 cells. The harvested cell culture supernatant contained recombinant single domain antibody 195-5-A3.

[0072] The binding specificity of 195-5-A3 was tested. The culture supernatant containing 195-5-A3 was mixed with 3×10 5 CHO-K1 cells or 3×10 5CHO-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 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 3 times with PBS. The cells were resuspended in 500μL PBS for flow cytometry analysis.

[0073] The results are as follows Figure 2 As shown, the supernatant of the negative control group did not bind significantly 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. 195-5-A3 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 195-5-A3 had good affinity and specificity.

[0074] Example 5

[0075] Purification of recombinant antibodies and determination of half effective concentration (EC50)

[0076] In the field of immunology and biomedicine, antibody affinity is one of the important parameters for evaluating antibody performance. Affinity refers to the ability of an antibody to bind to an antigen, which is the basis of the interaction between the antibody and the antigen. EC50 (half-maximal effect concentration) is one of the indicators reflecting antibody affinity, which refers to the antigen concentration required when the antibody binds to the antigen to achieve half of the maximum effect. Understanding the EC50 affinity of an antibody is of great significance for understanding the function of the antibody, optimizing antibody therapy, and predicting drug efficacy.

[0077] To determine the EC50 value of the 195-5-A3 antibody, the expression plasmid was transiently transfected into 293F cells, shaken and cultured, and affinity purified using ProteinA magnetic beads. Wash the ProteinA magnetic beads twice with 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of ProteinA magnetic beads (calculated as 20mg IgG / mL ProteinA magnetic beads) to the 293F cell shaker. Incubate at room temperature for 4 hours at 120rpm in a shaking incubator. Collect the ProteinA magnetic beads with a magnetic separation rack, wash them twice with 30mL PBS buffer and deionized water, and resuspend them with 1mL elution buffer. After incubation at room temperature for 5min, collect the magnetic beads with a magnetic separation rack, repeat the elution of the ProteinA magnetic beads twice, combine the eluates, and add neutralization buffer to adjust the pH of the solution. The eluted sample was dialyzed with PBS 100 times the sample volume, first dialyzed at 18°C ​​for 2 hours, then the solution was changed once and dialyzed at 8°C for 14 hours. Finally, the protein concentration was determined and the sample was filtered with a 0.22 μm sterile filter membrane and packaged, and stored in a -80°C refrigerator for use.

[0078] 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 2 hours. Wash 5 times with PBST, add different concentrations of 195-5-A3 candidate antibody (concentrations are 10μg / mL, 3.3μg / mL, 1.1μg / mL, 0.37μg / mL, 0.12μg / mL, 0.04μg / mL, 0.014μg / mL, 0μg / mL), and incubate at room temperature for 60min. Wash 5 times with PBST, dilute HRP-ProteinA at 1:50000, add 100μL / well to the ELISA plate, and incubate at room temperature for 45min. Wash 5 times with PBST, add 100μL 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.

[0079] The results are shown in Table 3 and Figure 3 As shown, the EC50 value of 195-5-A3 for CHO-K1-PD-1 was 0.1 μg / mL, indicating that 195-5-A3 had a high affinity.

[0080] Table 3

[0081]

[0082] Example 6

[0083] Recombinant antibody blocking function test

[0084] To detect the effect of 195-5-A3 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 8×10 4 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 195-5-A3 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, respectively. After 18 hours of co-culture, 20 μL of One-Glo reagent was added to each well, and the Luciferase fluorescence value was read.

[0085] result Figure 4 As shown, the maximum induction effect value of 195-5-A3 is 2.07 (the difference between the maximum Luciferase fluorescence value and the 0 μg / mL Luciferase fluorescence value), indicating that 195-5-A3 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.

[0086] Example 7

[0087] In order to detect the half-inhibitory concentration of 195-5-A3 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. Wash 5 times with PBST, add 200 μL / well of blocking solution, and block at room temperature for 2 hours. Wash 5 times with PBST, add different concentrations (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0 μg / mL) of 195-5-A3 candidate antibody and anti-PD-1 positive control antibody Nivolumab, and incubate at room temperature for 15 minutes. Add Biotin-PD-L1 protein (final concentration 4ug / mL) and incubate at room temperature for 45 minutes. Wash 5 times with PBST, dilute the secondary antibody (Streptavidin-HRP) at 1:10000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 45 minutes. Wash 5 times with PBST, add 100 μL TMB colorimetric solution to each well and color for 10 minutes at room temperature in the dark. Add 50 μL stop solution to each well, and read the absorbance at 450 nm on the ELISA reader.

[0088] The results are shown in Table 4 and Figure 5 As shown, the IC50 value of 195-5-A3 is 0.29 μg / mL, the IC50 value of the positive control antibody is 0.3 μg / mL, and the IC50 value of 195-5-A3 is equivalent to that of the positive control antibody Nivolumab, indicating that at a certain concentration, the blocking ability of 195-5-A3 is equivalent to that of the positive control antibody Nivolumab.

[0089] Table 4

[0090]

[0091] The applicant declares that the present invention illustrates an anti-programmed death receptor 1 antibody A3 and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0092] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0093] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An anti-programmed death receptor 1 antibody A3, characterized in that: The anti-programmed death receptor 1 antibody A3 comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3; The amino acid sequence of the CDR1 includes the sequence shown in SEQ ID NO.1; The amino acid sequence of the CDR2 includes the sequence shown in SEQ ID NO.2; The amino acid sequence of the CDR3 includes the sequence shown in SEQ ID NO.

3.

2. The anti-programmed death receptor 1 antibody A3 according to claim 1, characterized in that The amino acid sequence of the anti-programmed death receptor 1 antibody A3 includes the sequence shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-programmed death receptor 1 antibody A3 according to claim 1 or 2.

4. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 3.

5. A recombinant lentivirus, characterized in that: The recombinant lentivirus contains the expression vector according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 4.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-programmed death receptor 1 antibody A3 according to claim 1 or 2.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. Use of the anti-programmed death receptor 1 antibody A3 according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the recombinant lentivirus according to claim 5, the host cell according to claim 6 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a detection reagent or an immune-enhancing drug.

10. The use according to claim 9, characterized in that: The target of the detection reagent is PD-1.

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