Anti-programmed death receptor 1 antibody H1 and application thereof

Abstract

The invention discloses an anti-programmed death receptor 1 antibody H1 and application thereof. The amino acid sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain of the anti-programmed death receptor 1 antibody H1 respectively comprise the sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The PD-1 protein is used for immunizing alpaca, and the obtained programmed death receptor 1 antibody H1 is high in affinity and can block combination of PD-1 and PD-L1, enhance the function of immune cells and improve the tumor killing capacity of T cells.

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 H1 and an application thereof. Background Art

[0002] PD-1 (programmed death 1) is a receptor protein on the surface of immune cells T cells. It is a very important immune checkpoint. It interacts with a protein PD-L1 expressed on the surface of tumor cells. PD-L1 (programmed cell death-Ligand 1) is the ligand of PD-1, which is related to the suppression of the immune system and can transmit inhibitory signals. Once PD-1 and PD-L1 are combined, they will transmit a negative regulatory signal to T cells, inducing T cells to enter a resting state, reducing the proliferation of lymph node T cells, making them unable to recognize cancer cells, and reducing the proliferation of T cells themselves or causing apoptosis, effectively relieving the body's immune response. 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. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate the immune system function and kill tumor cells.

[0003] Currently, there are many monoclonal antibodies targeting PD-1 on the market, including Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), Cemiplimab (Cemiplimab), Toripalimab (Toripalimab), Cindilimab (Cindilimab), etc. For example, CN113227142A discloses an antibody or an antigen-binding fragment thereof that binds to PD-1, which comprises a heavy chain variable region of CDR1, 2 and 3, and a light chain variable region of CDR1, 2 and 3, but these antibodies are all common antibodies.

[0004] 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

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides an anti-programmed death receptor 1 antibody H1 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, can be modified, and has high specificity.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-programmed death receptor 1 antibody H1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the anti-programmed death receptor 1 antibody H1 include the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0008] SEQ ID NO.1:TASGSIFKTSA.

[0009] SEQ ID NO.2:ITNNLST.

[0010] SEQ ID NO. 3: NREIRGSSGTWYPLHY.

[0011] There is a special antibody lacking a light chain in the blood of camelids and chondrichthyes, namely a heavy chain antibody. 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 a nano antibody) consisting only of a 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 H1 of the present invention is small in size, can penetrate into tissues and cells, and in particular 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.

[0012] 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, alpacas peripheral blood mononuclear cells (PBMCs) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody specific primers are used to amplify the VHH sequence and clone it into a yeast expression plasmid to construct a yeast library. The yeast library is then positively and negatively screened with biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeast that binds to PD-1 protein. 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 at the same time, thereby enhancing the function of immune cells.

[0013] Preferably, the amino acid sequence of the heavy chain variable region of the anti-programmed death receptor 1 antibody H1 includes the sequence shown in SEQ ID NO.4.

[0014] SEQ ID NO.4: MAQVQFVEPGGGLSQPGGSLTLSCTASGSIFKTSAMGWYRQAPGKQ RELVARITNNLSTNYADSVKGRFTISRDNAKNTGYLQMNRLKPEDTAVYYCNREIRGSSGTWYPLHYWGQGTQVSVSSAHHS.

[0015] In a second aspect, the present invention provides a heavy chain antibody, comprising the anti-programmed death receptor 1 antibody H1 described in the first aspect and a human immunoglobulin crystallizable segment Ig Fc.

[0016] Preferably, the Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

[0017] Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.5.

[0018] Preferably, the amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.6.

[0019] SEQ ID NO.5:EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0020] SEQ ID NO.6: MAQVQFVEPGGGLSQPGGSLTLSCTASGSIFKTSAMGWYRQAPGKQ RELVARITNNLSTNYADSVKGRFTISRDNAKNTGYLQMNRLKPEDTAVYYCNREIRGSSGTWYPLHYWGQGTQVSVSSAHHSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0021] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-programmed death receptor 1 antibody H1 described in the first aspect.

[0022] Preferably, the nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.7.

[0023] SEQ ID NO.7:ATGGCCCAGGTTCAATTCGTGGAGCCGGGAGGGGGTCTGTCACAA CCAGGTGGTTCACTAACTTTATCGTGTACGGCCAGCGGTTCTATCTTCAAAACCTCCGCCATGGGCTGGTATCGTCAAGCGCCAGGAAAACAGAGAGAATTGGTCGCCCGTATCACTAACAATTTGAGCACGAACTACGCTGATTCCGTCAAGGGGCGCTTTACTATTAG CCGGGACAACGCTAAAAATACGGGCTACCTCCAGATGAATAGGCTCAAGCCGGAAGACACTGCAGTATATTATTGTAACCGTGAGATTCGTGGTTCAAGTGGCACCTGGTATCCCTTACACTATTGGGGACAGGGAACGCAGGTGTCTGTATCCTCCGCTCATCATTCT.

[0024] In a fourth aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the third aspect.

[0025] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains the expression vector described in the fourth aspect.

[0026] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the anti-programmed death receptor 1 antibody H1 described in the first aspect and immune cells.

[0027] Preferably, the immune cells include T cells.

[0028] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0029] Preferably, the auxiliary materials include any one or a combination of at least two of a carrier, a surfactant, a disintegrant, a coating material, an excipient, a solubilizer, a diluent, a pH regulator, a binder, a wetting agent, a colorant, an emulsifier, an antibacterial agent, a solubilizer, an osmotic pressure regulator, a filler, an antioxidant or a buffer.

[0030] In the seventh aspect, the present invention provides the use of any one of the anti-programmed death receptor 1 antibody H1 described in the first aspect, the heavy chain antibody described in the second aspect, the nucleic acid molecule 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.

[0031] 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.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The antibody of 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.

[0034] (2) The antibodies of 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.

[0035] (3) The antibody structure of the present invention is simple, and the nano-antibody is easier to be modified, such as humanized, multivalent construction, etc., and the production cost is low, which is suitable for large-scale production.

[0036] (4) The antibodies of the present invention are highly specific. 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. They 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the amplification results 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.

[0038] Figure 2 The figure shows the enrichment of yeast library before and after sorting detected by flow cytometry.

[0039] Figure 3 Figure 3 shows the specificity of recombinant single domain antibodies detected by flow cytometry.

[0040] Figure 4 ELISA for detecting EC of recombinant single domain antibodies 50 Result graph.

[0041] Figure 5 This is a graph showing the results of the recombinant antibody blocking function test.

[0042] Figure 6 This is the blocking curve of 195-5-H1 antibody. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] Reagents used in the following examples:

[0046] Streptavidin PE (eBioscience, CAT#12-4317-87);

[0047] PE-anti-Human IgG antibody (eBioscience, CAT#12-4998-82);

[0048] PBS buffer (bico, CAT#14190-250);

[0049] Adjuvant immune adjuvant (GERBU, CAT#3030);

[0050] HRP-Streptavidin (Boster, CAT#BA1088);

[0051] HRP-ProteinA (Boster, CAT#BA1080);

[0052] DNA fragment recovery kit (TakaRa, CAT#9761);

[0053] PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#6210B);

[0054] THE TM V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0055] Goat anti-Llama IgG(H+L)Secondary Antibody[HRP](Novus,AT#NB7242);

[0056] Nivolumab (MedChemExpress, HY-P9903).

[0057] Example 1 Alpaca immunization and antibody titer determination

[0058] (1) Alpaca immunization and serum titer testing

[0059] Two alpacas, 98# and 195#, were immunized with PD-1 protein. Each time, the alpacas were injected at two points near the cervical lymph nodes, with about 500 μg of emulsified antigen injected at each point. Immunization was performed once every three weeks for a total of four times. 5 mL of peripheral blood was collected before immunization, two weeks after the second immunization, the third immunization, and the fourth immunization, and centrifuged at 1,000×g for 20 minutes to collect the upper serum. Use sterile PBS buffer to dilute the PD-1-His tag recombinant protein to a final concentration of 1μg / mL, add 1μg / mL PD-1-His tag recombinant protein to a 96-well plate at 100μL / well, and coat overnight at 4℃; then aspirate the antigen coating solution, wash 3 times with PBST buffer (containing 0.05% Tween-20); add 200μL / well of blocking solution and block at 37℃ for 3h; after aspirating the blocking buffer, wash the plate 3 times with PBST buffer; use PBS buffer to gradient dilute the above collected serum, add 100μL of gradient diluted serum to the 96-well plate, and let it stand at 25℃ for 1h, with PBS buffer as the control well; aspirate the liquid in the well, and wash 3 times with PBST buffer; add 100μL HRP anti-llama IgG (H+L) antibody (1:50,000 dilution), incubate at 25°C for 2 hours; discard the liquid in the wells, wash the plate 5 times with PBST buffer; add 100 μL / well TMB colorimetric solution; incubate at 25°C in the dark for 25 minutes; add 50 μL / well stop solution; read the OD in the wells using an ELISA reader 450 The titer test results of the serum from the 98# alpaca immunization are shown in Table 1, and the titer test results of the serum from the 195# alpaca immunization are shown in Table 2.

[0060] Table 1

[0061]

[0062]

[0063] Table 2

[0064]

[0065] The antibody titer is expressed as "1:X", where X is the maximum dilution multiple at which the antibody can be detected. The binding titer of the serum of the two alpacas before immunization with the PD-1 protein was lower than 1:2000, indicating that the content of anti-PD-1 specific antibodies contained in them was extremely low. The binding titer of the serum after the fourth immunization with the PD-1 protein exceeded 64K, indicating that the content of anti-PD-1 specific antibodies in the serum of the alpacas increased significantly after multiple immunizations, indicating that the immunization was successful.

[0066] Example 2 Construction of single domain antibody yeast display library

[0067] (1) VHH antibody fragment cloning

[0068] After confirming that the alpaca serum contains anti-PD-1 antibodies, 100 mL of peripheral blood was collected, PBMC was separated using lymphocyte separation fluid, and RNA was extracted. Reverse transcription was performed using a cDNA synthesis kit to obtain cDNA, and the alpaca heavy chain antibody sequence was amplified using specific primers. Among them, the upstream primer binding site is in the signal peptide of the VHH antibody open reading frame (the primer sequence is SEQ ID NO.8: GTCCTGGCTGCTCTTCTACAAGG), and the downstream primer binding site is in the CH2 region (the primer sequence is SEQ ID NO.9: GGTACGTGCTGTTGAACTGTTCC). The electrophoresis results are as follows Figure 1 As shown in Figure A

[0069] After recovering the target fragment with a molecular weight of about 750bp, a secondary amplification was performed with specific primers to obtain the heavy chain antibody VHH fragment. Among them, the upstream primer binding site is in the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC (primer sequence is SEQ ID NO.10: ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG), and the downstream primer binding site is in the hinge region and FR4 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC (primer sequence is SEQ ID NO.11: GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG). The electrophoresis results are as follows. Figure 1 As shown in Figure B, it shows that the heavy chain antibody VHH fragment was successfully amplified, and the target fragment with a molecular weight of about 450 bp was recovered as the VHH fragment library.

[0070] (2) Electroporation of library vectors and detection of library capacity and diversity

[0071] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI enzyme, and the pYDisplay vector fragment was separated by electrophoresis. The 5,000 bp vector fragment was cut and recovered by gel. The yeast competent strain was streaked onto a YPD solid culture medium plate, activated at 30°C for 7 days, and the monoclonal yeast competent strain was inoculated into 50 mL YPD culture medium and cultured at 250 rpm and 30°C for 3 days. The linearized vector fragment and PCR product were mixed and added to the electroporation cup for electroporation transformation; then the yeast competent strain was transferred to a shake flask and cultured at 220 rpm and 30°C for 1 hour. Take 20 μL of the resuspension, dilute 5000 times with SD-CAA, take 100 μL, apply it to the SD-CAA plate for 3 days, calculate the storage capacity, and continue to culture the rest of the bacterial solution overnight. The remaining bacterial liquid was collected into a 50 mL centrifuge tube, centrifuged at 3,000 × g for 15 min, the supernatant was discarded, 10 mL SD-CAA was added for resuspending, and the mixture was mixed with 50% glycerol: resuspending liquid in a ratio of 1:1, and stored at -80°C.

[0072] The single domain antibody yeast display library obtained from 98# alpaca PBMC has a library capacity of 2.4×10 9 The single domain antibody yeast display library obtained from 195# alpaca PBMC has a capacity of 1.2×10 9 The Sanger sequencing results showed that the yeast library had large sequence differences, no repeated sequences, good diversity, and was suitable for subsequent experiments.

[0073] Example 3 Panning of yeast display library

[0074] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0075] Take 10mL yeast library (about 2×10 8Yeast cells) were transferred to a 50 mL centrifuge tube, centrifuged at 5,000 × g for 15 min, and the supernatant was removed; at the same time, sterile 5% PBSA buffer (PBS buffer + 5% BSA) was diluted to 0.5% PBSA buffer; the yeast library was resuspended with 1 mL 0.5% PBSA buffer, added to a 1.5 mL centrifuge tube, centrifuged at 3,000 × g for 15 min, and the supernatant was removed; and washed again with 0.5% PBSA buffer. Prepare three 1.5mL centrifuge tubes (two centrifuge tubes for negative panning of empty magnetic beads, and one centrifuge tube for positive panning), add 1mL of 0.5% PBSA buffer to each centrifuge tube; fully pipette the packaged streptavidin magnetic beads to resuspend them, and add 10μL of magnetic beads to the 1.5mL centrifuge tube; put these centrifuge tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4℃ for 15min; place the centrifuge tube on a magnetic separation stand for 15min, and remove the supernatant with a pipette; then add 1mL of 0.5% PBSA buffer again, rotate and incubate at 4℃ for another 15min, and remove the supernatant.

[0076] (2) Yeast display library screening

[0077] Negative selection of empty magnetic beads: Place the centrifuge tube on the magnetic separation rack, add the washed yeast solution to the centrifuge tube containing empty magnetic beads, put it in a bag, and rotate and incubate at 4°C for 1 hour; Place the empty magnetic bead centrifuge tube with incubated yeast on the magnetic separation rack for 20 minutes, add the yeast solution to a new empty magnetic bead centrifuge tube, rotate and incubate at 4°C for 40 minutes. After the incubation, place it on the magnetic separation rack for 25 minutes, and then add the yeast solution to a new empty magnetic bead centrifuge tube.

[0078] Biotin-PD-1-His tag magnetic separation: add 100 μL of 50 μg / mL biotin-PD-1-His tag protein solution (biotin-coupled, diluted with 0.5% PBSA buffer) to the centrifuge tube containing streptavidin magnetic beads, put the centrifuge tube in a bag, and rotate and incubate at 4°C for 1 hour; then add 1 mL of 0.5% PBSA buffer, let it stand for 15 minutes, and keep the centrifuge tube on the magnetic separation stand, and remove the supernatant with a pipette, then add 1 mL of 0.5% PBSA buffer, remove the centrifuge tube from the magnetic separation stand, mix it with a pipette, and place the centrifuge tube on the magnetic separation stand again, let it stand for 15 minutes, and keep the centrifuge tube on the magnetic separation stand, and remove the supernatant with a pipette; repeat the above washing steps once more to obtain positive selection magnetic beads coated with biotin-PD-1-His tag. Add the positive selection magnetic beads bound to the biotin-PD-1-His tag to the yeast cells that have completed the negative selection, and put them into a bag and place them on a rotating mixer, incubate them at 4°C for 1 hour, and after the incubation, place them on a magnetic separation rack and let them stand at 25°C for 25 minutes; keep the centrifuge tube on the magnetic separation rack and use a pipette to discard the unbound yeast solution; remove the 1.5mL centrifuge tube from the magnetic separation rack, and add 1mL of sterile 0.5% PBSA buffer to the centrifuge tube with a pipette, gently blow the magnetic beads, and then transfer them to a sterile 1.5mL centrifuge tube, place the centrifuge tube on the magnetic separation rack, let it stand at 25°C for 15 minutes, and discard the supernatant; repeat the washing twice, and the whole operation is performed in accordance with the sterile operation requirements. After washing, resuspend the magnetic beads and adhered yeast cells in 1 mL of SD-CAA medium, add 20 μL of the resuspended solution to 180 μL of SD-CAA medium and coat two plates, with a coating volume of 100 μL per plate, and then add 5 μL of the resuspended solution to 95 μL of SD-CAA medium and coat one plate, i.e., a total of 3 plates need to be coated.

[0079] Yeast flow cytometry after selection: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into the yeast. The yeast that has undergone negative selection with empty magnetic beads and magnetic separation of biotin-PD-1-His tag was incubated with PD-1 protein containing His tag at 4°C for 1 hour, then centrifuged to discard the supernatant, added anti-His tag and anti-V5 tag flow cytometry antibodies, incubated at 4°C for 1 hour, then centrifuged to discard the supernatant, resuspended the cells in 1mL PBS buffer, then centrifuged to discard the supernatant, resuspended the cells in 500μL PBS buffer, and analyzed by flow cytometry. The results are as follows: Figure 2 As shown, the yeasts that can bind to PD-1 protein in the yeast antibody libraries of 98# alpaca and 195# alpaca increased by 38% and 35% respectively before and after sorting, and subsequent experiments can be carried out.

[0080] Monoclonal selection: After overnight culture, monoclones were picked and inoculated into culture medium to induce expression. After 48 hours, the bacterial solution was incubated with PD-1 protein containing His tag at 4°C for 1 hour, then centrifuged to discard the supernatant, and the precipitate was resuspended with a solution containing biotin-conjugated anti-His tag flow antibody, incubated at 4°C for 1 hour, then centrifuged to discard the supernatant, and the precipitate was resuspended with a solution containing PE-streptavidin, incubated at 4°C for 1 hour, resuspended with 1mL PBS buffer, then centrifuged to discard the supernatant, and resuspended with 500μL PBS buffer for flow analysis. Finally, the monoclonal 195-5-H1 with a high positive rate was selected. 195-5-H1 was lysed with 0.2% SDS buffer (95°C for 10 minutes), centrifuged, and 0.5μL of the bacterial supernatant was taken as a template for PCR amplification and testing.

[0081] Example 4 Expression of recombinant single domain antibodies and detection of binding to target proteins

[0082] In order to express the recombinant single-domain antibody 195-5-H1, the candidate 195-5-H1 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were PCR amplified. 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and electrophoresis was performed. The band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band size of VHH was 500 bp. The PCR product was purified. The 195-5-H1 VHH sequence, CMV promoter and IgG1 Fc sequence were connected by PCR, and then 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, electrophoresis was performed, the target band was cut from the gel, and the PCR product was purified. After the obtained PCR product was transiently transfected into HEK293 cells, the cell culture supernatant contained the recombinant single-domain antibody 195-5-H1.

[0083] The supernatant containing 195-5-H1 was mixed with 1×10 6 CHO-K1 or CHO-K1-PD-1 cells were incubated at 25°C for 2 hours. After centrifugation at 1000×g for 15 minutes at 25°C, the supernatant was discarded and the cells were washed three times with PBS buffer. 100μL PE-labeled anti-human IgG antibody (1:500 dilution) was added and incubated at 25°C in the dark for 55 minutes. After centrifugation at 800×g for 15 minutes at 25°C, the supernatant was discarded and the cells were washed five times with PBS buffer. The cells were resuspended in 500μL PBS buffer and flow cytometry was performed to detect the binding specificity of 195-5-H1.

[0084] The results are as follows Figure 3As shown, the expression supernatant of the negative control group did not significantly bind to CHO-K1 or CHO-K1-PD-1; the positive control antibody significantly bound to CHO-K1-PD-1 but not to CHO-K1 cells, indicating that CHO-K1 cells do not express PD-1 protein, while CHO-K1-PD-1 cells express PD-1 protein and can be used as detection cells for alpaca antibodies. The 195-5-H1 antibody did not bind to CHO-K1 cells, but significantly bound to CHO-K1-PD-1, and was able to detect the expression of PD-1 protein in most CHO-K1-PD-1 cells, indicating that the 195-5-H1 antibody had good affinity and specificity.

[0085] Example 5 Purification of recombinant antibodies and half effective concentration (EC 50 ) determination

[0086] To determine the EC of 195-5-H1 antibody 50 , transiently transfect the relevant expression plasmid into HEK293 cells, and shake culture to express and purify the antibody. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30mL PBS buffer, 30mL 0.1M sodium hydroxide, and 30mL PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20mgIgG / mL Protein A magnetic beads) to the HEK293 cell shaker according to the required amount of the sample. Incubate in a shaking incubator at 150rpm and 25℃ for 2h. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. After washing twice with 30mL PBS buffer and ultrapure water, resuspend them with 1mL elution buffer. After standing at 25℃ for 15min, collect the magnetic beads with a magnetic separation rack and transfer them to a 15mL centrifuge tube. After eluting the Protein A magnetic beads twice, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with PBS buffer at least 100 times the volume of the sample, first at 25°C for 2 hours, then the solution was changed once and then dialyzed at 8°C for 16 hours.

[0087] Use coating solution to dilute PD-1 protein to 2μg / mL, add 100μL / well to 96-well plate, and coat overnight at 4℃. Wash 3 times with PBST buffer, add 200μL / well blocking solution, and block at 25℃ for 3h. Wash 3 times with PBST buffer, add different concentrations of 195-5-H1 candidate antibody (10μg / mL, 3.3μg / mL, 1.1μg / mL, 0.37μg / mL, 0.12μg / mL, 0.04μg / mL, 0.014μg / mL, 0μg / mL), and let stand at 25℃ for 1h. Wash 5 times with PBST buffer, dilute HRP-Protein A at 1:50,000, add 100μL / well to the ELISA plate, and let stand at 25℃ for 55min. Wash 3 times with PBST buffer, add 100μLTMB colorimetric solution to each well and color for 10min at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[0088] The results are shown in Table 3 and Figure 4 As shown, the EC value of 195-5-H1 for CHO-K1-PD-1 50 The value was 0.669 μg / mL, indicating that 195-5-H1 had a higher affinity.

[0089] Table 3

[0090]

[0091] Example 6 Detection of blocking function of recombinant antibodies

[0092] In a 96-well culture plate, add 100 μL of cell suspension, including 2×10 4 Effector cells Jurkat-PD-1-Luc (expressing luciferase and 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 luciferase in Jurkat-PD-1-Luc cells). 100 μL of 195-5-H1 of different concentrations was added to the corresponding wells, so that the final concentrations in the corresponding wells were 120 μg / mL, 40 μg / mL, 13.3 μg / mL, 4.4 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.16 μg / mL, 0.05 μg / mL, 0.02 μg / mL, and 0 μg / mL. After 16 hours of co-culture, 20 μL of One-Glo reagent was added to each well and the fluorescence value was read.

[0093] result Figure 5As shown, the maximum induction effect value of 195-5-H1 is 2.03 (the difference between the maximum fluorescence value and the fluorescence value of 0 μg / mL), indicating that 195-5-H1 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-Luc to express luciferase, and can be used to develop immune-enhancing drugs.

[0094] Example 7 Recombinant Antibody Half Inhibitory Concentration (IC 50 )experiment

[0095] To detect the IC of 195-5-H1 blocking the binding of PD-1 to PD-L1 50 , dilute PD-1 protein to 2μg / mL with coating solution. Add 100μL / well to 96-well plate and coat overnight at 4℃. Wash 3 times with PBST buffer, add blocking solution at 200μL / well, and block at 25℃ for 3h. Wash 3 times with PBST buffer, add 195-5-H1 candidate antibody at different concentrations (25μg / mL, 8.3μg / mL, 2.8μg / mL, 0.9μg / mL, 0.3μg / mL, 0μg / mL), and let stand at 25℃ for 25min. Add biotin-PD-L1 protein (final concentration 4μg / mL) and let stand at 25℃ for 55min. Wash 5 times with PBST buffer, dilute the secondary antibody (streptavidin-HRP) at 1:10,000, add 100μL / well to 96-well ELISA plate, and let stand at 25℃ for 55min. Wash with PBST buffer 5 times, add 100 μL TMB colorimetric solution to each well and color for 15 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.

[0096] The results are shown in Table 4 and Figure 6 As shown, IC 195-5-H1 50 The value was 0.61 μg / mL, indicating that 195-5-H1 could block the binding of PD-1 and PD-L1 proteins.

[0097] Table 4

[0098]

[0099] In summary, the anti-programmed death receptor 1 antibody H1 of the present invention is small in size and can penetrate into tissues and cells, especially the blood-brain barrier; it has strong stability and can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90°C; it has a simple structure, can be modified, and has high specificity.

[0100] 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 antibody H1, characterized in that The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the anti-programmed death receptor 1 antibody H1 include the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The anti-programmed death receptor 1 antibody H1 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-programmed death receptor 1 antibody H1 includes the sequence shown in SEQ ID NO.

4.

3. A heavy chain antibody, characterized in that The heavy chain antibody comprises the anti-programmed death receptor 1 antibody H1 according to claim 1 or 2 and the crystallizable segment Ig Fc of human immunoglobulin.

4. The heavy chain antibody according to claim 3, 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.

5.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-programmed death receptor 1 antibody H1 according to claim 1 or 2.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.

7.

7. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 5 or 6.

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 antibody H1 according to claim 1 or 2 and immune cells.

10. Use of any one of the anti-programmed death receptor 1 antibody H1 according to claim 1 or 2, the heavy chain antibody according to claim 3 or 4, the nucleic acid molecule 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 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.

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