Antibody A3 against programmed death receptor 1 and its application

Abstract

The present invention relates to an anti-programmed death receptor 1 antibody A3 and its use. The anti-programmed death receptor 1 antibody A3 comprises a heavy chain variable region, wherein the heavy chain variable region includes 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; and the amino acid sequence of the CDR3 includes the sequence shown in SEQ ID NO.3. The antibody of the present invention has high affinity and strong ability to block the binding of PD-1 and PD-L1, and can be used to prepare immune-enhancing drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an anti-programmed death receptor 1 antibody A3 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 downregulating the immune system's response to human cells and by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.

[0003] Currently, 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 a focus of cancer immunotherapy. Alpaca antibodies offer numerous advantages over conventional antibodies, so relevant technologies are being used to prepare anti-PD-1 alpaca antibodies, which can be formulated into antibody drugs or cell-based therapeutics for cancer treatment. Therefore, there is an urgent need to provide an alpaca antibody targeting PD-1 for use in the preparation of antibody drugs or cell-based therapeutics. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims 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 an adjuvant and immunized with alpacas. The titer of PD-1-specific antibodies in alpaca serum is detected by ELISA. After confirming that it is sufficiently high, the alpaca peripheral blood mononuclear cells are isolated, 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 using 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 PD-1 protein, and the recombinant single-domain antibody VHH sequence in the monoclonal yeast is amplified and named 195-5-A3. The recombinant single-domain antibody 195-5-A3 VHH sequence is connected to the CMV promoter sequence and 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 expression and purification, the positive antibodies were tested for their affinity for the PD-1 protein. Antibody clones with strong affinity for PD-1 were selected and tested for their ability to block the binding of PD-1 and PD-L1. The results showed that alpaca antibodies had high specificity and could simultaneously block the binding of PD-1 and PD-L1, 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 comprising 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 this application has the advantage of being small in size, and it can penetrate into tissues and cells and can penetrate the blood-brain barrier.

[0027] (2) The antibodies prepared in this application have a simple structure and are therefore easier to modify, such as humanization and multivalent construction, and the production cost of the antibodies is low, making them suitable for large-scale production.

[0028] (3) The antibodies prepared in this application are highly specific. The immune system of alpacas is able to produce 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 figure shows the results of flow cytometry detection of the specificity of recombinant single-domain antibodies.

[0031] Figure 3 This is a graph showing the EC50 results of ELISA testing of recombinant single-domain antibodies.

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

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

[0034] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely 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 both sides of the alpaca near the cervical lymph nodes, with 2 injections on each side and 500 μg of antigen 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, two weeks after the second immunization, the third immunization, and the fourth immunization, respectively. The blood was centrifuged at 800 × g for 10 minutes, and the upper serum was collected.

[0048] The PD-1-His recombinant protein was diluted with sterile PBS to a final concentration of 1 μg / mL. 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 five times with PBST containing 0.05% Tween 20, 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 five 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 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 enzyme-linked microplate 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 its serum, which allowed for subsequent experiments. 100 mL of peripheral blood was collected, and PBMCs were separated using lymphocyte separation medium, followed by RNA extraction. PrimeScript TM cDNA was obtained by reverse transcription using the II 1st Strand cDNA Synthesis Kit. 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'GTCCTGGCTGCTCTTCTACAAGG 3'; the downstream primer binds to the CH2 region; the primer sequence is 5'GGTACGTGCTGTTGAACTGTTCC 3'). Electrophoresis was performed on 1% agarose gel to recover and isolate the target fragment of approximately 750 bp. The first-round PCR product was then used as a template to amplify the heavy chain antibody VHH fragment using specific primers. Electrophoresis was performed on 1% agarose gel to isolate the target fragment of approximately 450 bp. The target band was recovered using a gel recovery kit and stored at -80°C (preservative solution containing 1 / 10 volume 3M sodium acetate, 1 μg / 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 has a wide range of applications in nanoantibody screening.

[0062] Take 10mL yeast library (about 2×10 8 Yeast cells) at 3000×g, centrifuge for 5 minutes, and discard the supernatant. Simultaneously, dilute sterile 5% PBSA (PBS + 5% BSA) to a concentration of 0.5%. Resuspend the yeast library in 1 mL of 0.5% PBSA, centrifuge at 3000×g for 5 minutes, and discard the supernatant. Wash once with 0.5% PBSA. Prepare three 1.5 mL centrifuge tubes (two for negative panning with empty magnetic beads and one for positive panning), and add 1 mL of 0.5% PBSA to each tube. Resuspend the streptavidin affinity magnetic beads and pipette 10 μL of the beads into a 1.5 mL centrifuge tube. Incubate with rotation at 4°C for 5 minutes. Place the centrifuge tube on a magnetic stand for 5 minutes and discard the supernatant. Then, add 1 mL of 0.5% PBSA again, incubate with rotation at 4°C for 5 minutes, and discard the supernatant.

[0063] (2) Yeast display library selection

[0064] Negative panning with empty magnetic beads: Place the centrifuge tube on a magnetic rack, add the washed yeast solution to the centrifuge tube containing empty magnetic beads, and incubate with rotation at 4°C for 60 minutes; Place the empty magnetic bead centrifuge tube containing yeast on a magnetic rack for 10 minutes, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube, and incubate with rotation at 4°C for 30 minutes. After the incubation is completed, place it on a magnetic rack for 15 minutes, aspirate the yeast solution and add it to 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 with rotation at 4°C for 60 minutes; add 1 mL of 0.5% PBSA, let it stand for 5 minutes, and keep the centrifuge tube on the magnetic stand. Remove the supernatant, add 1 mL of 0.5% PBSA, remove the centrifuge tube from the magnetic stand, mix well, place the centrifuge tube on the magnetic stand again, let it stand for 5 minutes, and keep the centrifuge tube on the magnetic stand. Use a pipette to aspirate the supernatant; repeat the above washing steps once to obtain positive selection magnetic beads coated with Biotin-PD-1-His.

[0066] Add Biotin-PD-1-His-bound positive panning beads to the negatively panned yeast cells and incubate with rotation at 4°C for 60 minutes. After incubation, place the tube on a magnetic stand and let it stand at room temperature for 15 minutes. While keeping the tube on the magnetic stand, remove the unbound yeast using a pipette. Remove the 1.5mL tube from the magnetic stand and add 1mL of sterile 0.5% PBSA buffer. Gently pipette the beads and transfer them to a sterile 1.5mL tube. Place the tube on a magnetic stand and let it stand at room temperature for 5 minutes. Discard the supernatant. Repeat the wash process two more times, following aseptic procedures throughout. After washing, resuspend the beads and attached yeast cells in 1mL of SDCAA medium. Pipette 20μL of the resuspension into 180μL of SDCAA medium and spread on two plates, each with a volume of 100μL. Pipette 5μL of the resuspension into 95μL of SDCAA medium and spread on 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 PD-1 protein containing a His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, added flow cytometry antibodies for anti-His tag and anti-V5 tag, incubated at 4°C for 60 minutes, centrifuged to remove the supernatant, and resuspended the cells with 1mL PBS. The supernatant was then centrifuged to remove the supernatant, and the cells were resuspended with 500μL 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 number of yeasts 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] Single clone selection: After overnight culture, single clones were selected and inoculated into culture medium to induce expression. 48 hours later, the cells were incubated with His-tagged PD-1 protein at 4°C for 60 minutes with rotation. The supernatant was removed by centrifugation, and the pellet was resuspended in a solution containing a biotin-conjugated anti-His tag flow cytometry antibody. The pellet was incubated at 4°C with rotation for 60 minutes, and the supernatant was removed by centrifugation. The pellet was resuspended in a solution containing PE-Streptavidin and incubated at 4°C with rotation for 60 minutes. The cells were resuspended in 1 mL of PBS, the supernatant was removed by centrifugation, and the cells were resuspended in 500 μL of PBS for flow cytometry analysis. The single clone 195-5-A3 with a high positive rate was selected. 195-5-A3 was lysed with 0.2% SDS (incubated at 95°C for 10 minutes), centrifuged, and 0.5 μL of the supernatant was used as template for PCR amplification and sent for analysis (the remaining cell suspension was stored at -20°C).

[0069] Example 4

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

[0071] To detect the binding level of the 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 and linked it to the CMV promoter sequence and IgG1 Fc sequence. After transient transfection into HEK293 cells, the harvested cell culture supernatant contained the 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 5Incubate CHO-K1-PD-1 cells at room temperature for 1 hour. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Add 100 μL of PE-conjugated anti-human IgG antibody (1:500 dilution) and incubate at room temperature for 45 minutes in the dark. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Resuspend the cells in 500 μL of PBS for flow cytometry analysis.

[0073] The results are as follows Figure 2 As shown, the supernatant of the negative control group showed no significant binding 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. CHO-K1-PD-1 cells do 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 PD-1 protein expression in the majority of CHO-K1-PD-1 cells, demonstrating good affinity and specificity.

[0074] Example 5

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

[0076] In the fields of immunology and biomedicine, antibody affinity is a key parameter for evaluating antibody performance. Affinity refers to the ability of an antibody to bind to an antigen and forms the basis of the interaction between the two. EC50 (half-maximal effect concentration) is a measure of antibody affinity, representing the antigen concentration required for antibody-antigen binding to achieve half its maximal effect. Understanding the EC50 affinity of an antibody is crucial for understanding antibody function, optimizing antibody therapies, 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, cultured in shake flasks, and affinity purified using Protein A magnetic beads. The Protein A magnetic beads were washed twice with 30 mL of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer, respectively. The appropriate volume of Protein A magnetic beads (calculated at 20 mg IgG / mL Protein A magnetic beads) was added to the 293F cell shake flask. The cells were incubated in a shaking incubator at 120 rpm at room temperature for 4 hours. The Protein A magnetic beads were collected using a magnetic separation rack, washed twice with 30 mL of PBS buffer and deionized water, and then resuspended in 1 mL of elution buffer. After incubation at room temperature for 5 minutes, the beads were collected using a magnetic separation rack and eluted twice. The eluates were combined and the pH of the solution was adjusted with neutralization buffer. The eluted sample was dialyzed against 100 times the sample volume of PBS at 18°C ​​for 2 h, with the solution changed once, followed by 14 h at 8°C. Finally, the protein concentration was determined, and the sample was filtered through a 0.22 μm sterile filter, aliquoted, and stored at -80°C until use.

[0078] PD-1 protein was diluted to 2 μg / mL in coating buffer and pipetted into a 96-well microtiter plate at 100 μL / well. Coating was allowed to proceed overnight at 4°C. After washing five times with PBST, 200 μL / well of blocking buffer was added and blocked at room temperature for 2 hours. After washing five times with PBST, various concentrations of the 195-5-A3 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) were added and incubated at room temperature for 60 minutes. After washing five times with PBST, HRP-Protein A was diluted 1:50,000 and 100 μL / well was added to the microtiter plate and incubated at room temperature for 45 minutes. After washing five times with PBST, 100 μL of TMB colorimetric solution was added to each well and developed for 10 minutes at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at a wavelength of 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, with 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 were 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). 100 μL of 195-5-A3 at different concentrations was then added to the corresponding wells, resulting in final concentrations of 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] To determine the half-inhibitory concentration of 195-5-A3 in blocking PD-1 and PD-L1 binding, PD-1 protein was diluted to 2 μg / mL in coating buffer and pipetted into a 96-well microtiter plate at 100 μL / well. The plates were coated overnight at 4°C. After washing five times with PBST, 200 μL / well of blocking buffer was added and blocked at room temperature for 2 hours. After washing five times with PBST, different concentrations of the 195-5-A3 candidate antibody (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, and 0 μg / mL) and the anti-PD-1 positive control antibody nivolumab were added and incubated at room temperature for 15 minutes. Biotin-PD-L1 protein (final concentration 4 μg / mL) was added and incubated at room temperature for 45 minutes. Wash the plate five times with PBST, dilute the secondary antibody (Streptavidin-HRP) at a 1:10,000 dilution, add 100 μL / well to the microplate, and incubate at room temperature for 45 minutes. Wash the plate five times with PBST, add 100 μL of TMB colorimetric solution to each well, and develop the color for 10 minutes 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.

[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 comparable to that of the positive control antibody Nivolumab, indicating that at a certain concentration, the blocking ability of 195-5-A3 is comparable to that of the positive control antibody Nivolumab.

[0089] Table 4

[0090]

[0091] The applicant states that while the present invention illustrates the anti-programmed death receptor 1 antibody A3 and its applications through the aforementioned embodiments, the present invention is not limited to these embodiments, nor does it necessarily rely on these embodiments for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for raw materials in the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure 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, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection 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 appropriate 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 VHH antibody A3, characterized in that The anti-programmed death receptor 1 VHH 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 is the sequence shown in SEQ ID NO.1; The amino acid sequence of the CDR2 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the CDR3 is the sequence shown in SEQ ID NO.

3.

2. The anti-programmed death receptor 1 VHH antibody A3 according to claim 1, characterized in that The amino acid sequence of the anti-programmed death receptor 1 VHH 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 VHH 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 VHH antibody A3 according to claim 1 or 2.

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

9. Use of the anti-programmed death receptor 1 VHH 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, wherein the target of the detection reagent is PD-1.

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