A nanobody against programmed death molecule 1 and its application

Abstract

The present invention relates to a nanobody against programmed death molecule 1 and its application. The amino acid sequence of the nanobody comprises CDR1 shown in SEQ ID No.1 and CDR3 shown in SEQ ID No.2; the amino acid sequence of the nanobody further comprises CDR2 shown in SEQ ID No.3 or SEQ ID No.4. The nanobody prepared by the present invention has high specificity, can block the binding of PD-1 and PD-L1, and enhance the function of immune cells.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and particularly relates to a nanobody against programmed death 1 and its application. Background Art

[0002] In the immune system, T cell-mediated cellular immunity is the "main army" for eliminating tumors. T cells need to be activated to exert their anti-tumor effects. The activation amplitude and quality of T cells depend on the balance between activation signals and inhibitory signals. Activating antibodies send activation signals like the accelerator of a car, and inhibitory antibodies send inhibitory signals like stepping on the brake. Immune checkpoints can be considered as the braking system of immune cells, which refer 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 the normal range and preventing damage to healthy tissues of the body.

[0003] Programmed death 1 (PD-1), which is widely expressed on the surface of immune cells, is an important immunosuppressive molecule. PD-1 belongs to the immunoglobulin superfamily CD28 / B7 and is a type I transmembrane glycoprotein composed of 288 amino acids, serving as an immunosuppressive receptor. It has at least two ligands, PD-L1 and PD-L2. Under normal circumstances, PD-1 on the surface of T cells can inhibit the function of T lymphocytes, thereby inhibiting autoimmune responses and preventing the occurrence of autoimmune diseases. However, in tumors, after the PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, it can, through the inhibitory effect on T lymphocytes, lead to a decrease in T cell proliferation and even induce T cell apoptosis, promoting tumor immune escape. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate the immune system function and kill tumor cells. Currently, there are already various PD-1 monoclonal antibodies on the market, including Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Cindilimab, etc. These PD-1 monoclonal antibodies have achieved successful applications in clinical practice. However, these antibodies are all ordinary antibodies.

[0004] The research carriers of ordinary antibodies are mainly mice and rabbits. Compared with ordinary antibodies, alpaca antibodies have many advantages: First, they are small in size, being one-tenth of ordinary antibodies, which enables them to penetrate into tissues and cells, especially to penetrate the blood-brain barrier. Second, they have strong stability. Nanobodies can maintain stability under extreme temperature and pH conditions and still maintain biological activity at a high temperature of up to 90 degrees. Third, they have a simple structure. Nanobodies are easier to modify, such as humanization, multivalency construction, etc., and have low production costs, making them suitable for large-scale production. Fourth, they have high specificity. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens and have weak immunogenicity to humans. The risk of nanobodies causing immune reactions is relatively low.

[0005] Therefore, how to prepare nanobodies against programmed death molecule 1 has become an urgent problem to be solved at present. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a nanobody against programmed death molecule 1 and its application. The alpaca antibody prepared by the present invention has high specificity, can block the binding of PD-1 and PD-L1, and enhance the function of immune cells.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a nanobody against programmed death molecule 1, and the amino acid sequence of the nanobody includes CDR1 shown in SEQ ID No.1 and CDR3 shown in SEQ ID No.2.

[0009] The amino acid sequence of the nanobody further includes CDR2 shown in SEQ ID No.3 or SEQ ID No.4.

[0010] SEQ ID No.1: GTIFSMND.

[0011] SEQ ID No.2: NREIRGSSGSWYPLHY.

[0012] SEQ ID No.3: IDSGLSA.

[0013] SEQ ID No.4: IDSALSA.

[0014] In the present invention, nanobodies are obtained after immunizing alpacas with PD-1. They are small in size and highly specific. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and have weak immunogenicity to humans. After experimental detection, the antibody has high affinity and high specificity, and is used in the scientific research field. For example, it can be used for theoretical research on immune checkpoints and for screening more drugs for treating diseases with high expression of PD-1, etc.

[0015] Preferably, the amino acid sequence of the nanobody comprises the sequence shown in SEQ ID No.5 or SEQ ID No.6.

[0016] SEQ ID No.5:

[0017] QVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSGLSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSP.

[0018] SEQ ID No.6:

[0019] EVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSALSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSS.

[0020] In the second aspect, the present invention provides a recombinant antibody, which comprises the sequence after fusion of the nanobody against programmed death molecule 1 described in the first aspect and a human Ig Fc fragment.

[0021] Preferably, the human Ig Fc comprises any one or at least two combinations of IgG1, IgG2, IgG3 or IgG4.

[0022] Preferably, the sequence of the human Ig Fc fragment is as shown in SEQ ID No.7.

[0023] SEQ ID No.7:

[0024] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0025] Preferably, the sequence of the recombinant antibody is as shown in SEQ ID No.8 or SEQ ID No.9.

[0026] SEQ ID No.8:

[0027] QVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSGLSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSPAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0028] SEQ ID No.9:

[0029] EVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSALSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0030] In the present invention, the recombinant antibody constructed with a nanobody, a human Ig Fc fragment, and a promoter has a strong ability to significantly block the binding of PD-1 to PD-L1, can effectively promote the expression of Luciferase by Jurkat-PD-1-Luciferase, and still has a strong blocking ability at a relatively low concentration.

[0031] In a third aspect, the present invention provides a nucleic acid molecule encoding the nanobody against programmed death molecule 1 described in the first aspect or the recombinant antibody described in the second aspect.

[0032] In a fourth aspect, the present invention provides an expression vector containing the nucleic acid molecule described in the third aspect; and after transfection, transduction, or transformation of a host cell with the expression vector, the host cell expresses the nanobody against programmed death molecule 1 described in the first aspect or the recombinant antibody described in the second aspect.

[0033] Preferably, the expression vector further includes a promoter.

[0034] Preferably, the promoter includes any one or a combination of at least two of EF1a, PGK1, Ubc, human beta actin, CAG, or SV40.

[0035] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the nanobody against programmed death molecule 1 described in the first aspect or the recombinant antibody described in the second aspect.

[0036] Preferably, the pharmaceutical composition further includes immune cells.

[0037] In a seventh aspect, the present invention provides an application of the nanobody against programmed death molecule 1 described in the first aspect, the recombinant antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug for blocking PD-1.

[0038] Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] 1. The nanobody against programmed death molecule 1 in the present invention is small in size, strong in stability, and simple in structure. Through experimental detection, it has high specificity. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and has weak immunogenicity to humans. The risk of the nanobody causing an immune response is relatively low.

[0040] 2. The recombinant antibody prepared in the present invention has high affinity and strong ability to block the binding of PD-1 to PD-L1, and can effectively promote Jurkat-PD-1-Luciferase to express Luciferase, and can be used for the research and development of immune-enhancing drugs. Description of the Drawings

[0041] Figure 1 Flow cytometry detection chart for detecting the specificity of recombinant single-domain antibody 195-1-D08.

[0042] Figure 2 Flow cytometry detection chart for detecting the specificity of recombinant single-domain antibody 195-1-G06.

[0043] Figure 3 Result chart of ELISA for detecting the EC50 of recombinant single-domain antibody 195-1-D08.

[0044] Figure 4 Result chart of ELISA for detecting the EC50 of recombinant single-domain antibody 195-1-G06.

[0045] Figure 5 Result chart of the blocking function detection of recombinant antibody 195-1-D08.

[0046] Figure 6 Result chart of the blocking function detection of recombinant antibody 195-1-G06.

[0047] Figure 7 Result chart of ELISA for detecting the IC50 of recombinant single-domain antibody 195-1-D08. Among them, Figure A is the blocking curve of 194-1-D08, Figure B is the blocking curve of the negative control antibody, and Figure C is the blocking curve of the positive control antibody.

[0048] Figure 8 Result graph for detecting the IC50 of the recombinant single-domain antibody 195-1-G06 by ELISA. Among them, Figure A is the blocking curve of 194-1-D08, Figure B is the blocking curve of the negative control antibody, and Figure C is the blocking curve of the positive control antibody.

[0049] Reagents used in the following examples:

[0050] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82);

[0051] PBS (bico, CAT# 14190-250);

[0052] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (Novus, AT#NB7242);

[0053] Nivolumab (MedChemExpress, HY-P9903);

[0054] Streptavidin PE (eBioscience, CAT# 12-4317-87);

[0055] Adjuvant (GERBU, CAT# 3030);

[0056] HRP-ProteinA (Boster, CAT# BA1080);

[0057] HRP-Streptavidin (Boster, CAT# BA1088);

[0058] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0059] DNA Fragment Recovery kit (TakaRa, CAT# 9761);

[0060] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805). Detailed implementation method

[0061] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific embodiments. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of the claimed protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0062] Example 1

[0063] Alpaca immunization and antibody titer determination

[0064] The alpacas were immunized with PD-1 protein. Approximately 1000 μg of emulsified antigen was injected on both sides of the cervical lymph nodes, and immunization was carried out once every 3 weeks for 4 times. 5 mL of peripheral blood was collected before immunization, and after the second, third, and fourth immunizations. The blood was centrifuged at 800 g for 10 min, and the upper serum was collected. 100 μl / well of 1 μg / mL PD-1-His protein was added to a 96-well plate and coated overnight at 4°C; then the supernatant was discarded, and the plate was washed 5 times with PBST buffer (PBS buffer containing 0.05% Tween-20); 200 μl / well of blocking buffer was added and incubated at 37°C for 2 h; after discarding the blocking buffer, the plate was washed 5 times with PBST buffer; the collected serum was serially diluted with PBS buffer, and 100 μL of the serially diluted serum was added to the 96-well ELISA plate and incubated at 25°C for 60 min. The control well was PBS buffer. The liquid in the wells was discarded, and the plate was washed 5 times with PBST buffer. 100 μl of HRP anti-camelid IgG (H+L) antibody (diluted 1:50000) was added and incubated at 25°C for 60 min. After discarding the liquid in the wells, the plate was washed 5 times with PBST buffer; 100 μl / well of TMB chromogenic solution was added; and incubated at 25°C in the dark for 15 min. 50 μL / well of stop solution was added; the OD450 value in the wells was read using an ELISA reader.

[0065] The binding titer of the serum before alpaca immunization with PD-1 protein was lower than 1:2000, indicating that the content of anti-PD-1 specific antibodies in it was extremely low. The binding titer of the serum after the fourth immunization with PD-1 protein was significantly higher than 1:64,000, indicating that the content of anti-PD-1 specific antibodies in alpaca serum was significantly increased after multiple immunizations and could be used for the construction of a yeast display library.

[0066] Example 2

[0067] Construction of a single-domain antibody yeast display library

[0068] (1)Cloning of VHH antibody fragment: 100 mL of alpaca peripheral blood was collected and PBMC was isolated. RNA was extracted and reverse transcribed into cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit. Using PBMC cDNA as a template, the alpaca heavy chain antibody sequence was amplified by PCR. Among them, the upstream primer binds to the signal peptide of the VHH antibody ORF, and the sequence is shown in SEQ ID NO.10; the downstream primer binds to the CH2 region, and the sequence is shown in SEQ ID NO.11. The target fragment with a size of about 750 bp was recovered by 1% agarose gel electrophoresis.

[0069] SEQ ID NO.10: GTCCTGGCTGCTCTTCTACAAGG.

[0070] SEQ ID NO.11: GGTACGTGCTGTTGAACTGTTCC.

[0071] Using the first-round PCR product as a template, the heavy chain antibody VHH fragment was amplified by the second-round PCR. Among them, 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.12; the downstream primer binds to the hinge region and the FR4 region, and the 5' end contains the SfiI restriction enzyme site GGCCACGAAGGCC, and the sequence is shown in SEQ ID NO.13. The target fragment with a molecular weight of about 450 bp was recovered by 1% agarose gel electrophoresis to obtain the VHH fragment library.

[0072] SEQ ID NO.12: ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG.

[0073] SEQ ID NO.13: GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG.

[0074] (2)Electroporation of the library vector, library capacity, and diversity: Digest the yeast surface display vector pYDisplay and the VHH fragment library with SfiI. Recover the 5000 bp pYDisplay vector fragment by electrophoresis. Streak the yeast competent cells stored at -80°C onto a YPD solid medium plate and culture at 30°C for 5 days. Inoculate the yeast competent cells into 50 mL of YPD medium and culture on a shaker at 30°C for 2 days. Mix the linearized vector fragment and the PCR product and perform electroporation; transfer the electroporated yeast competent cells to a culture flask and culture on a shaker at 30°C for 1 h. Take 20 μl of the resuspended solution, dilute it 5000-fold with SDCAA medium, pipette 100 μL, spread it on an SDCAA plate, and calculate the library capacity after culturing for 3 days. Continue to culture the remaining bacterial solution for 1 day. Centrifuge the remaining bacterial solution at 3000 g for 5 min, discard the supernatant; add 10 mL of SDCAA medium to resuspend, mix with 50% glycerol:resuspended solution = 1:1, and store at -80°C. The results showed that the library capacity of the single-domain antibody yeast display library obtained from alpaca PBMC was approximately 1.2×10 9 .

[0075] Example 3

[0076] Panning of the yeast display library

[0077] (1)Pretreatment of streptavidin magnetic beads and yeast cells: To screen for antibodies with high affinity for PD-1, take 10 mL of the yeast library (about 2×10 8 yeast cells), centrifuge at 3000 g for 5 min, and discard the supernatant. Resuspend the yeast library with 0.5% PBSA (PBS + 0.5% BSA), transfer it to a 1.5 mL centrifuge tube, centrifuge at 3000 g for 5 min, discard the supernatant after centrifugation, and repeat the washing once. Add 1 mL of 0.5% PBSA to three 1.5 mL centrifuge tubes, and then add 10 μL of streptavidin magnetic beads. Place the centrifuge tubes in a bag, fix them to a rotary mixer, and incubate with rotation at 4°C for 5 min. Place the centrifuge tubes on a magnetic rack for 5 min, aspirate and discard the supernatant. Subsequently, add 1 mL of 0.5% PBSA again, incubate with rotation at 4°C for 5 min, and discard the supernatant.

[0078] (2)Negative panning of the yeast display library with empty magnetic beads: Transfer the washed yeast cell suspension to an empty magnetic bead tube, place it in a bag, and incubate with rotation at 4°C for 60 min; place the empty magnetic bead tube containing the incubated yeast on a magnetic rack for 10 min, aspirate the yeast cell suspension and transfer it to a new empty magnetic bead tube, and incubate with rotation at 4°C for 30 min. After the incubation, place it on a magnetic rack for 15 min, aspirate the yeast cell suspension and transfer it to a new empty magnetic bead tube.

[0079] (3)Biotin-PD-1-His magnetic panning of yeast display library: Add 100 μL of 50 μg / mL biotin-PD-1-His protein solution (diluted with 0.5% PBSA buffer) into a centrifuge tube containing streptavidin magnetic beads. Place the centrifuge tube in a bag and incubate with rotation at 4°C for 60 min. Then add 1 mL of 0.5% PBSA buffer, let it stand for 5 min, and 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, pipette to mix well, then place the centrifuge tube back on the magnetic stand, let it stand for 5 min, and keep the centrifuge tube on the magnetic stand. Discard the supernatant. Repeat the above washing steps once again to obtain the positively panned magnetic beads coated with biotin-PD-1-His. Add the positively panned magnetic beads conjugated with biotin-PD-1-His to the yeast cells that have completed negative panning, and incubate with rotation at 4°C for 60 min. After incubation, place it on the magnetic stand and let it stand at 25°C for 15 min; keep the centrifuge tube on the magnetic stand and discard the yeast cell suspension. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 1 mL of 0.5% PBSA buffer to the centrifuge tube, gently pipette the magnetic beads, and transfer them to a sterile 1.5 mL centrifuge tube. Place the centrifuge tube on the magnetic stand, let it stand at 25°C for 5 min, discard the supernatant; repeat the washing two more times. After washing, resuspend the magnetic beads and the adhered yeast cells in 1 mL of SDCAA medium. Pipette 20 μl of the resuspended solution into 180 μL of SDCAA medium and plate on two plates, with a plating volume of 100 μL per plate. Then pipette 5 μL of the resuspended solution into 95 μL of SDCAA medium and plate on one plate.

[0080] (4)Flow cytometry analysis of the panned yeast cells: Incubate the yeast cells panned through the above two steps with PD-1 protein containing His tag at 4°C with rotation for 60 min. Then centrifuge to remove the supernatant, add flow cytometry antibodies against His tag and V5 tag (the yeast expression vector contains V5 tag to indicate whether the expression vector has been successfully transferred into yeast cells), and incubate with rotation at 4°C for 60 min. Then centrifuge to remove the supernatant, add 1 mL of PBS buffer to resuspend the cells. Then centrifuge to remove the supernatant, add 500 μL of PBS buffer to resuspend the cells, and perform flow cytometry analysis. The results show that compared with before panning, the yeast antibody display library after panning has a 35% increase in yeast cells that can bind to PD-1 protein.

[0081] (5)Selection of monoclonal antibodies: Pick yeast monoclonal antibodies and inoculate them into the medium for induced expression. After 48 h, incubate the bacterial solution with the His-tagged PD-1 protein at 4 °C with rotation for 60 min. Then centrifuge to discard the supernatant, resuspend the precipitate with a solution containing a biotin-conjugated anti-His-tag flow antibody, and incubate at 4 °C with rotation for 60 min. Then centrifuge to discard the supernatant, resuspend the precipitate with a solution containing PE-streptavidin, and incubate at 4 °C with rotation for 60 min. Then centrifuge to discard the supernatant, resuspend the cells with 500 μl PBS, and perform flow cytometry analysis. Finally, two monoclonal antibodies with high positive rates were selected and named 195-1-D08 and 195-1-G06, respectively. By Sanger sequencing, the CDR1 sequences of both 195-1-D08 and 195-1-G06 were SEQ ID No.1, and the CDR3 sequences were both SEQ ID No.2. The CDR2 sequence of 195-1-D08 was SEQ ID No.3, and the CDR2 sequence of 195-1-G06 was SEQ ID No.4. The amino acid sequence of 195-1-D08 was SEQ ID No.5, and the amino acid sequence of 195-1-G06 was SEQ ID No.6.

[0082] Example 4

[0083] Expression of recombinant single-domain antibodies and detection of binding to target proteins

[0084] To express the recombinant single-domain antibodies 195-1-D08 and 195-1-G06, the VHH sequences of candidates 195-1-D08 and 195-1-G06, the CMV promoter sequence, and the IgG1 Fc sequence were amplified separately using a PCR instrument. Take 50 μL of the PCR product, add 1 / 10 volume of 10× loading buffer, and perform electrophoresis analysis using 1% agarose. The band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band sizes of the VHHs of 195-1-D08 and 195-1-G06 were 500 bp and 450 bp, respectively. Cut the target bands from the gel and purify the PCR products. Use a PCR instrument to ligate the VHH sequences of 195-1-D08 and 195-1-G06 to the MV promoter and the IgG1 Fc sequence, respectively. Then take 50 μL of the PCR product, add 1 / 10 volume of 10× loading buffer, perform electrophoresis analysis using 1% agarose, cut the target bands from the gel, and purify the PCR products. After transiently transfecting the obtained PCR products into HEK293 cells, the cell culture supernatants harvested contained the recombinant single-domain antibodies 195-1-D08 and 195-1-G06, respectively.

[0085] Detect the binding specificity of 195-1-D08 and 195-1-G06. Incubate the culture supernatants containing 195-1-D08 and 195-1-G06 respectively with 3×10 5 CHO-K1 or CHO-K1-PD-1 cells at room temperature for 1 h. After centrifugation at 800 g for 5 min at room temperature, discard the supernatant and wash the cells 3 times with PBS. Add 100 μL of PE-labeled Anti-human IgG antibody (diluted 1:500), and incubate in the dark at room temperature for 45 min. After centrifugation at 800 g for 5 min at room temperature, discard the supernatant and wash the cells 3 times with PBS. Resuspend the cells with 500 μL of PBS and perform flow cytometry analysis.

[0086] The results of 195-1-D08 are as Figure 1 shown. The expression supernatant of the negative control group did not show significant binding to either CHO-K1 or CHO-K1-PD-1; the positive control antibody showed significant binding to CHO-K1-PD-1 but not to CHO-K1 cells, indicating that CHO-K1 cells do not express the PD-1 protein and CHO-K1-PD-1 cells express the PD-1 protein, which can be used as the detection cells for alpaca antibodies. 195-1-D08 did not bind to CHO-K1 cells, showed significant binding to CHO-K1-PD-1, and most of the CHO-K1-PD-1 cells expressing the PD-1 protein could be detected, indicating that 195-1-D08 has good affinity and specificity.

[0087] The results of 195-1-G06 are as Figure 2 shown. 195-1-G06 did not bind to CHO-K1 cells, showed significant binding to CHO-K1-PD-1, and most of the CHO-K1-PD-1 cells expressing the PD-1 protein could be detected, indicating that 195-1-G06 has good affinity and specificity.

[0088] Example 5

[0089] Purification of recombinant antibody and determination of half maximal effective concentration (EC50)

[0090] To determine the EC50 values of 195-1-D08 antibody and 195-1-G06 antibody, the expression plasmids expressing recombinant heavy-chain alpaca antibodies were transiently transfected into 293F cells and cultured with shaking in a shake flask for antibody expression and purification. Since the target recombinant antibody contains a human IgG fragment, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30 mL of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20 mg IgG / mL Protein A magnetic beads) to the 293F cell shake flask according to the sample requirement. Incubate at 120 rpm at room temperature for 2 h in a shaking incubator. Collect the Protein A magnetic beads with a magnetic separator and transfer them to a 50 mL centrifuge tube. After washing twice with 30 mL of PBS buffer and deionized water respectively, resuspend with 1 mL of elution buffer. Incubate at room temperature for 5 min, then collect the magnetic beads with a magnetic separation rack and transfer the magnetic beads to a 15 mL centrifuge tube. Repeat the elution of the Protein A magnetic beads twice, combine the eluates, and add neutralization buffer to adjust the solution pH. Dialyze the eluted sample with PBS at least 100 times the volume of the sample, first dialyze at 20 °C for 2 h, change the solution once, and then dialyze at 6 °C for 15 h. Finally, measure the protein concentration, filter the sample with a 0.22 μm sterile filter membrane, aliquot, and store at -80 °C in the refrigerator for later use.

[0091] Dilute PD-1 protein to 2 μg / mL with coating buffer, pipette 100 μL / well into a 96-well ELISA plate, and coat overnight at 4 °C. Wash 5 times with PBST, add 200 μL / well of blocking solution, and block at room temperature for 2 h. Wash 5 times with PBST, add different concentrations of 195-1-D08 candidate antibodies, which are 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, 0 μg / mL, and incubate at room temperature for 60 min. Wash 5 times with PBST, dilute HRP-Protein A at 1:50000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB chromogenic solution to each well and develop color at room temperature in the dark for 10 min. Add 50 μL of stop solution to each well and read the absorbance at a wavelength of 450 nm on an ELISA reader.

[0092] The results are shown in Table 1 and Figure 3 as follows. The EC50 value of 195-1-D08 for CHO-K1-PD-1 is 0.018 μg / mL, indicating that 195-1-D08 has a high affinity.

[0093] The results are shown in Table 2 andFigure 4 As shown, the EC of 195-1-G06 for CHO-K1-PD-1 50 value is 0.083 μg / mL, indicating that 195-1-G06 has a high affinity.

[0094] Table 1 Absorbance values at OD450nm of 195-1-D08 at different dilution concentrations

[0095]

[0096] Table 2 Absorbance values at OD450nm of 195-1-G06 at different dilution concentrations

[0097]

[0098] Example 6

[0099] Detection of the blocking function of recombinant antibodies

[0100] To detect the function of 195-1-D08 and 195-1-G06 in blocking the binding of PD-1 to PD-L1, 100 μL of cell suspension was added to a 96-well plate, with each well containing 2×10 4 effector cells Jurkat-PD-1-Luciferase (expressing the PD-1 molecule, which can inhibit the expression of the Luciferase fluorescent protein after binding to PD-L1) and 8×10 4 target cells CHO-K1-PD-L1 (expressing the PD-L1 molecule, which can bind to PD-1 and inhibit the expression of the Luciferase fluorescent protein in Jurkat-PD-1-Luciferase cells). Subsequently, 100 μL of different concentrations of 195-1-D08 and 195-1-G06 were independently 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, 0 μg / mL. After co-culturing for 18 h, 20 μL of One-Glo reagent was added to each well, and the Luciferase fluorescence value was read.

[0101] Results Figure 5As shown, the maximum induction effect value of 195-1-D08 is 2.2 (the difference between the maximum Luciferase fluorescence value and the Luciferase fluorescence value at 0 μg / mL), indicating that 195-1-D08 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 for the research and development of immune-enhancing drugs.

[0102] Results Figure 6 As shown, the maximum induction effect value of 195-1-G06 is 2.0 (the difference between the maximum fluorescence value and the fluorescence value at 0 μg / mL), indicating that 195-1-G06 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 for the research and development of immune-enhancing drugs.

[0103] Example 7

[0104] Recombinant antibody half-inhibitory concentration (IC50) experiment

[0105] To detect the half-inhibitory concentration of 195-1-D08 and 195-1-G06 in blocking the binding of PD-1 and PD-L1, dilute the PD-1 protein to 2 μg / mL with coating buffer, pipette 100 μL per well into a 96-well microplate, and coat overnight at 4°C. Wash 5 times with PBST, add 200 μL per well of blocking solution, and block at room temperature for 2 h. Wash 5 times with PBST, and independently add different concentrations of 195-1-D08 and 195-1-G06, which are 25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0 μg / mL) and the anti-PD-1 positive control antibody Nivolumab, and incubate at room temperature for 15 min. Add Biotin-PD-L1 protein (4 μg / mL), and incubate at room temperature for 45 min. Wash 5 times with PBST, dilute the secondary antibody (Streptavidin-HRP) at 1:10000, add 100 μL per well to the microplate, and incubate at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB chromogenic solution per well, and develop color in the dark at room temperature for 10 min. Add 50 μL of stop solution to each well, and read the absorbance at a wavelength of 450 nm on a microplate reader.

[0106] The results are shown in Table 3 and Figure 7As shown, the IC50 value of 195-1-D08 is 0.24 μg / mL, and the IC50 value of the positive control antibody is 0.3 μg / mL. The IC50 value of 195-1-D08 is lower than that of the positive control antibody Nivolumab. Meanwhile, when the antibody concentration is only 0.034 μg / ml, the absorbance values of the 195-1-D08 treatment group (2.207 and 2.168) are still significantly lower than those of the control group without antibody addition (3.079 and 3.092); on the contrary, there is no significant difference in the absorbance values of the positive control antibody at this concentration (1.626 and 1.669) and the control group without antibody addition (1.703 and 1.602). This result indicates that the blocking ability of 195-1-D08 is stronger than that of the positive control antibody Nivolumab.

[0107] The results are shown in Table 4 and Figure 8 As shown, the IC50 value of 195-1-G06 is 0.29 μg / mL, and the IC50 value of the positive control antibody is 0.3 μg / mL. The IC50 value of 195-1-G06 is comparable to that of the positive control antibody Nivolumab, indicating that the blocking ability of 195-1-G06 is comparable to that of the positive control antibody Nivolumab. (The above results are from the same batch of experiments, so the control group results in Table 3 and Figure 7 are the same as those in Table 4 and Figure 8 are the same)

[0108] Table 3 Blocking ability of 195-1-D08 at different concentrations

[0109]

[0110] Table 4 Blocking ability of 195-1-G06 at different concentrations

[0111]

[0112] In summary, the present invention immunizes alpacas with PD-1 protein. After determining the titer of PD-1 specific antibodies in alpaca serum by ELISA, alpaca peripheral blood mononuclear cells (PBMC) are isolated, RNA is extracted and reverse transcribed to obtain cDNA. Using specific primers for alpaca single domain antibodies, the VHH sequence is amplified and cloned into a yeast expression plasmid to construct a yeast display library. Then, the yeast display library is subjected to positive and negative screening using biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich the yeast cells that bind to PD-1 protein. Monoclonal colonies are picked from the enriched product, and the specific binding of the candidate antibodies to PD-1 is detected by ELISA. After the positive antibodies are expressed and purified, their binding affinity to PD-1 protein is detected. Antibody clones with strong affinity for PD-1 are selected, and their ability to block the binding of PD-1 and PD-L1 is detected. The results show that the alpaca antibodies have high specificity, and their effect of blocking the binding of PD-1 and PD-L1 is stronger than that of the prior art, enhancing the function of immune cells.

[0113] The applicant declares that the above description is only a specific embodiment 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 conceived within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A nanobody against programmed death molecule 1, characterized in that, The amino acid sequence of the nanobody comprises CDR1 shown in SEQ ID No.1 and CDR3 shown in SEQ ID No.2; The amino acid sequence of the nanobody further comprises CDR2 shown in SEQ ID No.3 or SEQ ID No.

4.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody comprises the sequence shown in SEQID No.5 or SEQ ID No.

6.

3. A recombinant antibody, characterized in that, The recombinant antibody comprises the sequence after fusion of the nanobody against programmed death molecule 1 as claimed in claim 1 or 2 and a human Ig Fc fragment.

4. The recombinant antibody according to claim 3, wherein, The human Ig Fc comprises any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4; The sequence of the human Ig Fc fragment is as shown in SEQ ID No.

7.

5. The recombinant antibody according to claim 3, wherein The sequence of the recombinant antibody is as shown in SEQ ID No.8 or SEQ ID No.

9.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody against programmed death molecule 1 as claimed in claim 1 or 2 or the recombinant antibody as claimed in any one of claims 3-5.

7. An expression vector, characterized in that, The expression vector contains the nucleic acid molecule as claimed in claim 6; and after transfection, transduction or transformation of a host cell, the host cell expresses the nanobody against programmed death molecule 1 as claimed in claim 1 or 2 or the recombinant antibody as claimed in any one of claims 3-5.

8. The expression vector according to claim 7, wherein The expression vector further comprises a promoter; The promoter comprises any one or a combination of at least two of EF1a, PGK1, Ubc, human beta actin, CAG, CMV or SV40.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanobody against programmed death molecule 1 as claimed in claim 1 or 2 or the recombinant antibody as claimed in any one of claims 3-5; The pharmaceutical composition further comprises immune cells.

10. Use of the nanobody against programmed death molecule 1 as claimed in claim 1 or 2, the recombinant antibody as claimed in any one of claims 3-5, the nucleic acid molecule as claimed in claim 6, the expression vector as claimed in claim 7 or 8, and the pharmaceutical composition as claimed in claim 9 in the preparation of a drug for treating a disease by blocking PD-1; The disease is ovarian cancer.

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