Anti-programmed death receptor 1 antibody G09 and its application

Abstract

The present invention discloses an anti-programmed death receptor 1 antibody G09 and its application. The amino acid sequence of the CDR1 of the heavy chain of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.1; the amino acid sequence of the CDR2 of the heavy chain of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.2; the amino acid sequence of the CDR3 of the heavy chain of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.3. The anti-programmed death receptor 1 antibody G09 provided by the present invention has a strong affinity, can block the binding of PD-1 and PD-L1, enhance the function of immune cells, and improve the ability of T cells to eliminate tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular immunology and tumor immunotherapy, and relates to an anti-programmed death receptor 1 antibody G09 and an application thereof. Background Art

[0002] In the immune system, T cell-mediated cellular immunity is the main force in eliminating tumors. T cells need to be activated to exert anti-tumor effects. The activation amplitude and quality of T cells depend on the balance between activation signals and inhibitory signals. Activating antibodies and inhibitory antibodies send activation signals and inhibitory signals respectively. Immune checkpoints can be considered as the brake system of immune cells. They refer to a series of molecules expressed on immune cells that can regulate the degree of immune activation, so that the activation level of T cells remains within the normal range and prevents damage to healthy tissues of the body. Programmed death 1 (PD-1), also known as CD279, is widely expressed on the surface of immune cells. It is an important immunosuppressive molecule belonging to the immunoglobulin superfamily CD28 / B7. It is a type I transmembrane glycoprotein composed of 288 amino acids and is an immunosuppressive receptor with at least two ligands, PD-L1 and PDL2.

[0003] 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 PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, it can inhibit T lymphocytes, leading to downregulation of T cell proliferation, and even inducing T cell apoptosis, promoting tumor immune escape. Therefore, blocking the PD-1 / PD-L1 negative regulatory pathway can activate the function of the immune system and kill tumor cells. At present, there are many monoclonal antibodies targeting PD-1 on the market, including Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Cindilimab, etc. For example, CN113861295A discloses an anti-PD-1 antibody or its antigen-binding fragment, nucleic acid molecules encoding them, methods for preparing them, and pharmaceutical compositions containing them, but 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, one-tenth of ordinary antibodies, which allows them to penetrate into tissues and cells, especially the blood-brain barrier. Second, they are highly stable. Nano antibodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90°C. Third, they are simple in structure. Nano antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs, making them suitable for large-scale production. Fourth, they are highly specific. The alpaca's immune system can produce antibodies that are highly specific and affinity for specific antigens, and have weak immunogenicity to humans. Nano antibodies have a low risk of causing an immune response.

[0005] 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 the focus of cancer immunotherapy. Therefore, it is urgent to provide an alpaca antibody with strong stability, simple structure and high specificity for the preparation of antibody drugs or cell drugs. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides an anti-programmed death receptor 1 antibody G09 and its application, which can penetrate into tissues and cells, especially penetrate the blood-brain barrier, has strong stability, can maintain stability under extreme temperature and pH conditions, can still maintain biological activity at high temperatures up to 90°C, has a simple structure and high specificity.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides an anti-programmed death receptor 1 antibody G09, wherein the amino acid sequence of CDR1 of the heavy chain of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.1; the amino acid sequence of CDR2 of the heavy chain of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.2; and the amino acid sequence of CDR3 of the heavy chain of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.3.

[0009] SEQ ID NO. 1: ASGIINSIDDMA.

[0010] SEQ ID NO. 2: ARITSGLSTNYAD.

[0011] SEQ ID NO. 3: NREIRGSSGTWYPLHY.

[0012] There is a special antibody lacking light chains in the blood of camelids and cartilaginous fish, namely heavy chain antibodies. Compared with ordinary antibodies, it contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single domain antibody (also known as nano antibody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity. The anti-programmed death receptor 1 antibody G09 provided by the present invention is small in size, can penetrate into tissues and cells, especially can penetrate the blood-brain barrier, has strong stability, can maintain stability under extreme temperature and pH conditions, can still maintain biological activity at high temperatures up to 90°C, has a simple structure, and has high specificity.

[0013] The present invention uses PD-1 protein to immunize alpacas, and after determining the titer of PD-1 specific antibodies in alpacas serum by ELISA, separates alpacas peripheral blood mononuclear cells (PBMC), extracts RNA and reverse transcribes 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. Then, the yeast display library is positively and negatively screened with biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeasts that bind to PD-1 protein. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to PD-1 is detected by ELISA. After the positive antibodies are expressed and purified, their affinity for binding to PD-1 protein is detected. Antibody clones with strong affinity to PD-1 are selected to detect the ability to block the binding of PD-1 and PD-L1. The results show that alpaca antibodies have high specificity and can block the binding of PD-1 and PD-L1, enhance the function of immune cells, and enhance the ability of T cell immunity to eliminate tumors.

[0014] Preferably, the amino acid sequence of the heavy chain variable region of the anti-programmed death receptor 1 antibody G09 includes the sequence shown in SEQ ID NO.4.

[0015] SEQ ID NO.4: DVQLVESGGGLVQPGGSLRLSCAASGIINSIDDMAWYRQAPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGTWYPLHYWGQGTQVTVSS.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-programmed death receptor 1 antibody G09 described in the first aspect.

[0017] Preferably, the nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.5.

[0018] SEQ ID NO.5: GACGTCCAGTTAGTAGAAAGCGGAGGAGGTTTTGGTGCAGCCTGGTGGTTCTCTACGTTTGTCGTGCGCCGCTTCGGGAATTATCAATAGTATCGATGATATGGCCTGGTATCGTCAAGCTCCGGGAAAACAGAGGGAGTTGGTGGCCAGGATCACTTCAGGATTGTCAACGAACTACGCTG ACTCAGTCAAAGGGCGCTTCACGATCAGCAGGGATAACGCCAAGAATACAGTATATCTGCAAATGAATTCGCTTAAGCCTGAAGACACGGCCGTCTATTACTGTAACAGAGAAATACGGGGATCCAGTGGGACCTGGTACCCACTCCACTATTGGGGTCAGGGAACCCAGGTAACGGTTTCAAGT.

[0019] In a third aspect, the present invention provides a heavy chain antibody, comprising the anti-programmed death receptor 1 antibody G09 described in the first aspect and a human immunoglobulin crystallizable segment Ig Fc.

[0020] Preferably, the Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

[0021] Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.6.

[0022] SEQ ID NO.6: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] Preferably, the amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.7.

[0024] SEQ ID NO.7: DVQLVESGGGLVQPGGSLRLSCAASGIINSIDDMAWYRQAPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGTWYPLHYWGQGTQVTVSSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0025] In a fourth aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0026] Preferably, the expression vector contains the gene encoding the heavy chain antibody described in the third aspect.

[0027] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains the expression vector described in the fourth aspect.

[0028] In a sixth aspect, the present invention provides a pharmaceutical composition, comprising the anti-programmed death receptor 1 antibody G09 described in the first aspect and immune cells.

[0029] Preferably, the pharmaceutical composition comprises the heavy chain antibody and immune cells described in the third aspect.

[0030] Preferably, the pharmaceutical composition comprises the host cells and immune cells described in the fifth aspect.

[0031] Preferably, the immune cells include T cells.

[0032] In the seventh aspect, the present invention provides the use of any one of the anti-programmed death receptor 1 antibody G09 described in the first aspect, the nucleic acid molecule described in the second aspect, the heavy chain antibody described in the third aspect, the expression vector 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 tumor treatment drugs.

[0033] Preferably, the tumor includes any one of colorectal cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, bile duct cancer, lung cancer or nasopharyngeal cancer, or a combination of at least two of them.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The antibody provided by the present invention is small in size, one tenth of the size of ordinary antibodies, and can penetrate into tissues and cells, especially the blood-brain barrier.

[0036] (2) The antibodies provided by the present invention have strong stability. Nanobodies can maintain stability under extreme temperature and pH conditions and can still maintain biological activity at high temperatures up to 90°C.

[0037] (3) The antibody structure provided by the present invention is simple, and nanobodies are easier to modify, such as humanization and multivalent construction, and the production cost is low, which is suitable for large-scale production.

[0038] (4) The antibodies provided by the present invention 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 nanoantibodies causing immune responses is low, and they can block the binding of PD-1 and PD-L1 and enhance the function of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are the electrophoresis results after amplification of the VHH region of the alpaca antibody, where Figure A is the result of the first round of PCR amplification of the heavy chain antibody fragment, and Figure B is the result of the second round of PCR amplification of the VHH region fragment.

[0040] Figure 2 The figure shows the enrichment of yeast library before and after sorting detected by flow cytometry.

[0041] Figure 3 Figure 3 shows the specificity of recombinant single domain antibodies detected by flow cytometry.

[0042] Figure 4 Detection of EC of recombinant single domain antibodies by flow cytometry 50 picture.

[0043] Figure 5 This is the concentration-effect curve of alpaca antibodies tested on CHO-K1-PD-1 cells.

[0044] Figure 6 IC of recombinant single domain antibodies for ELISA 50 The result graphs, wherein A is the blocking curve graph of 195-1-G09, B is the blocking curve graph of the negative control antibody, and C is the blocking curve graph of the positive control antibody. DETAILED DESCRIPTION

[0045] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] Reagents used in the following examples:

[0048] Streptavidin PE (eBioscience, CAT# 12-4317-87);

[0049] PE-anti-Human IgG antibody (eBioscience, CAT# 12-4998-82);

[0050] PBS (bico, CAT# 14190-250);

[0051] Adjuvant (GERBU, CAT# 3030);

[0052] HRP-ProteinA (Boster, CAT# BA1080);

[0053] HRP-Streptavidin (Boster, CAT# BA1088);

[0054] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0055] DNA fragment recovery kit (TakaRa, Cat# 9761);

[0056] Goat anti-Llama IgG (H+L) antibody [HRP] (Novus, AT# NB7242);

[0057] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0058] Nivolumab (MedChemExpress, HY-P9903).

[0059] Example 1 Alpaca immunization and antibody titer determination

[0060] (1) Alpaca immunization and serum titer testing

[0061] Two alpacas (98# and 195#) were immunized with PD-1 protein, injected on both sides of the alpaca's cervical lymph nodes, and about 1000 μg of emulsified antigen was injected on each side. Immunization was performed once every 3 weeks for 4 times. 5 mL of peripheral blood was collected before immunization, two weeks after the second immunization, the third immunization, and the fourth immunization, respectively, and centrifuged at 800×g for 10 min to collect the upper serum. 100 μl / well of 1 μg / mL PD-1-His recombinant protein was added to a 96-well plate and coated overnight at 4°C; then the plate was discarded and washed 5 times with PBST (containing 0.05% Tween-20); 200 μl / well of blocking solution was added and incubated at 37°C for 2 h; after discarding the blocking buffer, the plate was washed 5 times with PBST; the serum collected above was graded diluted with PBS, and 100 μL of graded diluted serum was added to the 96-well ELISA plate, incubated at 25°C for 60 min, and the control well was PBS; the liquid in the well was discarded and washed 5 times with PBST; 100 μl of HRP anti-llama IgG (H+L) antibody (1:50000 dilution) was added and incubated at 25°C for 60 min; after discarding the liquid in the well, the plate was washed 5 times with PBST; 100 μl / well of TMB colorimetric solution was added; incubated at 25°C in the dark for 15 min; 50 μL / well stop solution; read the OD in the well using a microplate reader 450 value.

[0062] "1:X" represents the antibody titer (X is the maximum dilution multiple at which the antibody can be detected). The titer test results of the serum of the 98# alpaca after immunization are shown in Table 1, and the titer test results of the serum of the 195# alpaca after immunization are shown in Table 2. The binding titers of the sera of the two alpacas before immunization with the PD-1 protein were both lower than 1:2000, indicating that the content of anti-PD-1 specific antibodies contained therein was extremely low. The binding titers of the sera of the 98# and 195# alpacas after the fourth immunization with the PD-1 protein were significantly higher than 1:64K, indicating that the content of anti-PD-1 specific antibodies in the serum of the alpacas after multiple immunizations was significantly increased, and the immunization was successful, which can be used for the construction of yeast display libraries.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067] Example 2 Construction of single domain antibody yeast display library

[0068] (1) Cloning of VHH antibody fragments

[0069] 100 mL of peripheral blood was collected from alpacas and PBMC was separated. RNA was extracted and reverse transcribed to obtain cDNA. Using PBMC cDNA as a template, primers were used to PCR amplify the alpaca heavy chain antibody sequence. Among them, the upstream primer binds to the signal peptide of the VHH antibody ORF, and the sequence is shown in SEQ ID NO.8; the downstream primer binds to the CH2 region, and the sequence is shown in SEQ ID NO.9. The PCR product was analyzed using 1% agarose electrophoresis. The electrophoresis results are shown in Figure 1 As shown in Figure A, a fragment with a molecular weight of about 750 bp was recovered.

[0070] SEQ ID NO. 8: GTCCTGGCTGCTCTTCTACAAGG.

[0071] SEQ ID NO.9: GGTACGTGCTGTTGAACTGTTCC.

[0072] The first round PCR product was used as a template to amplify the heavy chain antibody VHH fragment with primers. 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.10; the downstream primer binds to the hinge region and FR4 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC, and the sequence is shown in SEQ ID NO.11. The PCR product was analyzed using 1% agarose electrophoresis. The electrophoresis results are shown in Figure 1 As shown in Figure B, the recovered fragments with a molecular weight of about 450 bp are the VHH fragment library.

[0073] SEQ ID NO. 10: ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG.

[0074] SEQ ID NO. 11: GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG.

[0075] (2) Detection of electroporation of library vectors and library capacity and diversity

[0076] The yeast surface display vector pYDisplay and the VHH fragment library were digested with SfiI. The pYDisplay vector fragment of 5000 bp was separated and recovered using 1% agarose gel. The yeast competent strain frozen at -80°C was streaked onto a YPD solid medium plate in advance and cultured at 30°C for 5 days. The yeast competent strain was inoculated into 50 mL YPD medium and cultured at 30°C for 2 days. The linearized vector fragment and PCR product were mixed and transformed by electroporation; the yeast competent strain after electroporation was transferred to a culture bottle and cultured at 30°C for 60 min. Take 20 μl of the resuspension, dilute 5000 times with SDCAA, aspirate 100 μL, apply it to the SDCAA plate, calculate the reservoir capacity after 3 days of culture, and continue to culture the rest of the bacterial solution for 24 h. The remaining bacterial liquid was collected into a 50 mL centrifuge tube, centrifuged at 3000×g for 5 min, and the supernatant was discarded; 10 mL SDCAA was added for resuspending, and 50% glycerol: resuspending solution was mixed in a ratio of 1:1, and frozen at -80°C. The results showed that the library capacity of the single-domain antibody yeast display library obtained from 98# alpaca PBMC was approximately 2.4×10 9 The single-domain antibody yeast display library obtained from 195# alpaca PBMC has a capacity of approximately 1.2×10 9 .

[0077] Example 3 Panning of yeast display library

[0078] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0079] In order to screen antibodies with high affinity for PD-1, take 10 mL of yeast library (about 2×10 8 Yeast cells) to a 50 mL centrifuge tube, centrifuge at 3000 × g for 5 min, and remove the supernatant; resuspend the yeast library with 0.5% PBSA (PBS + 0.5% BSA), transfer to a 1.5 mL centrifuge tube, centrifuge at 3000 × g for 5 min, and discard the supernatant; wash again with 0.5% PBSA. Prepare 2 centrifuge tubes for negative selection of empty magnetic beads and 1 centrifuge tube for positive selection. Add 1 mL of 0.5% PBSA to each centrifuge tube; resuspend the aliquoted streptavidin magnetic beads by pipetting thoroughly, and transfer 10 μL of magnetic beads to a 1.5 mL centrifuge tube; put the centrifuge tube in a bag, fix it on a rotating mixer, and rotate and incubate at 4°C for 5 min; place the centrifuge tube on a magnetic stand for 5 min, and remove the supernatant. Then add 1 mL of 0.5% PBSA again, rotate and incubate at 4°C for another 5 min, and remove the supernatant.

[0080] (2) Panning of yeast display library

[0081] Negative panning with empty magnetic beads: Place the centrifuge tube on the magnetic rack, transfer the washed yeast solution to the empty magnetic bead tube, put it in a bag, and incubate at 4°C for 60 min; Place the empty magnetic bead tube with yeast on the magnetic rack for 10 min, aspirate the yeast solution and transfer it to a new empty magnetic bead tube, and incubate at 4°C for 30 min; After the incubation, place it on the magnetic rack for 15 min, aspirate the yeast solution and transfer it to a new empty magnetic bead tube.

[0082] Biotin-PD-1-His magnetic separation: Add 100 μL of 50 μg / mL biotin-PD-1-His protein solution (0.5% PBSA dilution) to the centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 60 minutes; then add 1 mL of 0.5% PBSA, let it stand for 5 minutes, keep the centrifuge tube on the magnetic rack, discard the supernatant, and add 1 mL of 0.5% PBSA. Remove the centrifuge tube from the magnetic rack, blow and mix, place the centrifuge tube on the magnetic rack again, let it stand for 5 minutes, keep the centrifuge tube on the magnetic rack, and discard the supernatant; repeat the above washing steps once again to obtain positive selection magnetic beads coated with biotin-PD-1-His. Add the positive selection magnetic beads bound to biotin-PD-1-His to the yeast cells that have completed negative selection, and rotate and incubate at 4°C for 60 minutes. After the 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 solution; remove the 1.5 mL centrifuge tube from the magnetic stand, add 1 mL of 0.5% PBSA buffer to the centrifuge tube, gently blow the magnetic beads, and transfer them to a sterile 1.5 mL centrifuge tube. Place the centrifuge tube on the magnetic stand and let it stand at 25°C for 5 min, discard the supernatant; repeat the wash twice. After the wash, resuspend the magnetic beads and adhered yeast cells with 1 mL SDCAA medium, pipette 20 μl of the resuspension into 180 μL SDCAA medium and apply it to two plates, each with a volume of 100 μL, and then pipette 5 μL of the resuspension into 95 μL SDCAA medium and apply it to one plate.

[0083] Flow cytometry of yeast after selection: The yeast after the above two steps of selection was incubated with PD-1 protein containing His tag at 4°C for 60 min; then centrifuged to remove the supernatant, added anti-His tag and anti-V5 tag flow cytometry antibodies (the yeast expression vector contains V5 tag to indicate whether the expression vector has been successfully transferred into yeast), and incubated at 4°C for 60 min; then centrifuged to remove the supernatant, and resuspended the cells in 1 mL PBS; then centrifuged to remove the supernatant, resuspended the cells in 500 μL PBS, and analyzed by flow cytometry. The results are as follows Figure 2As shown, 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.

[0084] Selection of monoclonal clones: Pick a yeast monoclonal clone and inoculate it 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 min; then centrifuged and discarded the supernatant, and the precipitate was resuspended with a solution containing a biotin-coupled anti-His tag flow antibody, and incubated at 4°C for 60 min; then centrifuged and discarded the supernatant, and the precipitate was resuspended with a solution containing PE-streptavidin, and incubated at 4°C for 60 min; 1mL PBS was resuspended in the cells, and then centrifuged and discarded the supernatant, and 500μl PBS was resuspended in the cells for flow analysis. Finally, the monoclonal clone 195-1-G09 with a high positive rate was selected. 0.5 μL of the supernatant of the 195-1-G09 bacterial solution was used as a template for PCR amplification and testing (the remaining bacterial solution was stored at -20°C).

[0085] Example 4 Detection of expression of recombinant single domain antibodies and binding to target proteins

[0086] The PCR products of the 195-1-G09 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were connected, and then the purified products were transiently transfected into HEK293 cells. The harvested cell culture supernatant contained the recombinant single domain antibody 195-1-G09.

[0087] The culture supernatant containing 195-1-G09 was mixed with 5×10 5 CHO-K1 or CHO-K1-PD-1 cells were incubated at 25°C for 60 min. The cells were washed three times with PBS. 100 μL of PE-labeled anti-human IgG antibody was added and incubated at 25°C for 45 min. After washing the cells three times with PBS, the cells were resuspended in 500 μL PBS and analyzed by flow cytometry.

[0088] The results are as follows Figure 3 As shown, the expression supernatant of the negative control group did not significantly bind to CHO-K1 or CHO-K1-PD-1; the positive control antibody only significantly bound to CHO-K1-PD-1, 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 antibody detection cells. 195-1-G09 did not bind to CHO-K1 cells, but significantly bound to CHO-K1-PD-1, and most CHO-K1-PD-1 cells expressing PD-1 protein could be detected, indicating that 195-1-G09 has high affinity and specificity.

[0089] Example 5 Purification and determination of half effective concentration (EC) of recombinant antibodies 50 )

[0090] Transiently transfect 195-1-G09 antibody expression plasmid into 293F cells and culture in a shaking flask. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash Protein A magnetic beads twice with 30 mL PBS, 0.1M NaOH, and PBS in sequence. Add Protein A magnetic beads to the 293F cell shaking flask according to the required sample volume. Incubate in an incubator at 120 rpm and 25°C for 60 min. Collect Protein A magnetic beads with a magnetic separation rack and transfer to a 50 mL centrifuge tube. Use 30 mL PBS and ddH 2 After washing twice, resuspend with 1 mL elution buffer. After incubation at 25°C for 5 min, collect the magnetic beads with a magnetic stand and transfer them to a 15 mL centrifuge tube. After eluting the Protein A magnetic beads twice, combine the eluates and adjust the pH of the antibody solution with neutralization buffer. The eluted sample is dialyzed with PBS at least 100 times the volume of the sample, first dialyzed at 25°C for 2 h, changed the solution once, and then dialyzed at 8°C for 16 h. Finally, the protein concentration was determined, and the sample was filtered with a 0.22 μm sterile filter membrane and aliquoted, stored in a -80°C refrigerator for use.

[0091] PD-1 protein was diluted to 2 μg / mL with coating solution, and 100 μl / well was pipetted into 96-well plates, and coated at 4°C for more than 16 h. Washed 5 times with PBST, 200 μl / well of blocking solution was added, and blocked at 25°C for 2 h. Washed 5 times with PBST, different concentrations of 195-1-G09 candidate antibodies (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) were added, and incubated at 25°C for 60 min. Washed 5 times with PBST, HRP-Protein A was diluted 1:50000, and 100 μL / well was added to the ELISA plate, and incubated at 25°C for 45 min. Washed 5 times with PBST, 100 μL of TMB color development solution was added to each well, and color was developed at room temperature and protected from light for 10 min. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[0092] 195-1-G09 Antibody EC 50 The test results are shown in Table 3 and Figure 4 As shown, the EC value of 195-1-G09 for CHO-K1-PD-1 50The value was 0.012 μg / mL, indicating that 195-1-G09 had a higher affinity.

[0093] Table 3

[0094]

[0095] Example 6 Detection of the blocking function of recombinant antibodies

[0096] In a 96-well plate, add 100 μl of cell suspension, including 2×10 4 Effector cells Jurkat-PD-1-luciferase (expressing PD-1 molecules, which can inhibit the expression of luciferase 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 Jurkat-PD-1-luciferase cell fluorescent protein). Then, 100 μl of 195-1-G09 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 h of co-culture, 20 μl of One-Glo reagent was added to each well, and the fluorescence value was read in an ELISA reader.

[0097] result Figure 5 As shown, the maximum induction effect value of 195-1-G09 is 1.98 (the difference between the maximum fluorescence value and the fluorescence value of 0 μg / mL), indicating that 195-1-G09 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-luciferase cells to express luciferase, and can be used to develop immune-enhancing drugs.

[0098] Example 7 Detection of half inhibitory concentration (IC 50 )

[0099] PD-1 protein was diluted to 2 μg / mL with coating solution, and 100 μl / well was pipetted into 96-well plates and coated overnight at 4°C. Washed 5 times with PBST, and 200 μL / well of blocking solution was added, and blocked at 25°C for 2 h. Washed 5 times with PBST, and 25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0.1 μg / mL, 0.03 μg / mL, and 0 μg / mL of 195-1-G09 candidate antibody and anti-PD-1 positive control antibody Nivolumab were added, respectively, and incubated at 25°C for 15 min. Biotin-PD-L1 protein (final concentration 4 μg / mL) was added and incubated at 25°C for 45 min. Wash 5 times with PBST, dilute streptavidin-HRP at 1:10000, add 100 μl / well to the ELISA plate, and incubate at 25°C for 45 min. After washing 5 times with PBST, add 100 μL TMB colorimetric solution to each well and color for 10 min at room temperature in the dark. Add 50 μL stop solution to each well, and read the absorbance at 450 nm on an ELISA reader.

[0100] The results are shown in Table 4 and Figure 6 As shown, Figure 6 Figure A is the blocking curve of 195-1-G09, Figure B is the blocking curve of negative control antibody, and Figure C is the blocking curve of positive control antibody. IC 50 The IC value of 195-1-G09 antibody was 0.2 μg / mL. 50 The value was 0.1 μg / mL, which was significantly lower than the positive control antibody, indicating that the blocking ability of 195-1-G09 antibody was stronger than that of the positive control antibody Nivolumab.

[0101] Table 4

[0102]

[0103] In summary, the anti-programmed death receptor 1 antibody G09 provided by the present invention is small in size, can penetrate into tissues and cells, especially can penetrate the blood-brain barrier, has strong stability, can maintain stability under extreme temperature and pH conditions, can still maintain biological activity at high temperatures up to 90°C, has a simple structure, can be modified, and has high specificity. Compared with the prior art antibodies, the blocking ability of the 195-1-G09 antibody is significantly improved.

[0104] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-programmed death receptor 1 single domain antibody G09, characterized in that: The amino acid sequence of CDR1 of the anti-programmed death receptor 1 single domain antibody G09 is shown in SEQ ID NO.1; The amino acid sequence of CDR2 of the anti-programmed death receptor 1 single domain antibody G09 is shown in SEQ ID NO.2; The amino acid sequence of CDR3 of the anti-programmed death receptor 1 single domain antibody G09 is shown in SEQ ID NO.

3.

2. The anti-programmed death receptor 1 single domain antibody G09 according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-programmed death receptor 1 single domain antibody G09 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 single domain antibody G09 according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.

5.

5. A heavy chain antibody, characterized in that: The heavy chain antibody comprises the anti-programmed death receptor 1 single domain antibody G09 according to claim 1 or 2 and the crystallizable segment Ig Fc of human immunoglobulin.

6. The heavy chain antibody according to claim 5, characterized in that The Ig Fc includes any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4; The Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.

6.

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

8. A host cell, characterized in that The host cell contains the expression vector according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-programmed death receptor 1 single domain antibody G09 according to claim 1 or 2, and further comprises immune cells.

10. Use of any one of the anti-programmed death receptor 1 single domain antibody G09 according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the heavy chain antibody according to claim 5 or 6, the expression vector according to claim 7, the host cell according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a tumor therapeutic drug, characterized in that: The tumor is selected from any one or a combination of at least two of colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, bile duct cancer, lung cancer or nasopharyngeal cancer.

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