Anti-programmed death receptor 1 antibody H1 and its application

Abstract

The present invention discloses an anti-programmed death receptor 1 antibody H1 and its application. The amino acid sequences of the 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. The present invention uses PD-1 protein to immunize alpacas. The anti-programmed death receptor 1 antibody H1 obtained has strong affinity and 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 H1 and an application thereof. Background Art

[0002] PD-1 (programmed death 1) is a receptor protein on the surface of immune cells called T cells. It is a crucial immune checkpoint that interacts with PD-L1, a protein expressed on the surface of tumor cells. PD-L1 (programmed cell death-ligand 1) is the ligand for PD-1 and is associated with immune system suppression, transmitting inhibitory signals. Once PD-1 and PD-L1 bind, they transmit a negative regulatory signal to T cells, inducing them to enter a quiescent state. This reduces T cell proliferation in lymph nodes, preventing them from recognizing cancer cells, and leads to decreased T cell proliferation or apoptosis, effectively neutralizing the body's immune response. In tumors, PD-L1 expressed on tumor cells binds to PD-1 on the surface of T cells. This inhibitory effect on T lymphocytes leads to downregulation of T cell proliferation and even induction of T cell apoptosis, promoting tumor immune evasion. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate immune system function and kill tumor cells.

[0003] Currently, there are a variety of monoclonal antibodies targeting PD-1 on the market, including nivolumab (Nivolumab), pembrolizumab, cemiplimab, toripalimab, and cindilimab. For example, CN113227142A discloses an antibody or antigen-binding fragment thereof that binds to PD-1, which comprises a heavy chain variable region comprising CDR1, 2, and 3, and a light chain variable region comprising CDR1, 2, and 3. However, these antibodies are all ordinary 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 a focus of cancer immunotherapy. Therefore, there is an urgent need to provide a stable, structurally simple, and highly specific alpaca antibody for the preparation of antibody drugs or cell-based drugs. Summary of the Invention

[0005] In response to the deficiencies of the existing technology 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 the blood-brain barrier, has strong stability, 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.

[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 nanobody) 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 H1 of the present invention is small in size and 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.

[0012] The present invention uses PD-1 protein to immunize alpacas. After determining the titer of PD-1 specific antibodies in alpaca serum by ELISA, the alpaca peripheral blood mononuclear cells (PBMC) 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 using 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 the candidate antibodies to PD-1 is detected by ELISA. After the positive antibodies are expressed and purified, their affinity for binding to the PD-1 protein is detected. Antibody clones with strong affinity to PD-1 are selected, and the ability to block the binding of PD-1 and PD-L1 is tested. 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 the 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 comprising 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 excipients 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 or a combination of at least two 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.

[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 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 are highly stable. 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 nanoantibody is easier to modify, such as humanization and multivalent construction, 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 alpaca antibody VHH region, 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 2 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 with reference to the embodiments and drawings. It should be understood that the specific embodiments 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 experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. 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 detection

[0059] Two alpacas, 98# and 195#, were immunized with PD-1 protein. Each injection was administered bilaterally near the cervical lymph nodes, with approximately 500 μg of emulsified antigen injected at two sites on each side. Immunizations were performed every three weeks for a total of four times. Five milliliters of peripheral blood were collected before immunization, and two weeks after the second, third, and fourth immunizations. The blood was centrifuged at 1,000 × g for 20 minutes, and the supernatant serum was collected. Use sterile PBS buffer to dilute the PD-1-His tag recombinant protein to a final concentration of 1 μg / mL, add 1 μg / mL PD-1-His tag recombinant protein to a 96-well plate at 100 μL / well, and coat overnight at 4°C; then aspirate the antigen coating solution and wash three times with PBST buffer (containing 0.05% Tween-20); add 200 μL / well blocking solution and block at 37°C for 3 hours; after aspirating the blocking buffer, wash the plate three times with PBST buffer; use PBS buffer to gradiently dilute the above-collected serum, add 100 μL of the gradient diluted serum to the 96-well plate, and let it stand at 25°C for 1 hour. The control well is PBS buffer; aspirate the liquid in the well and wash three times with PBST buffer; add 100 μL HRP anti-llama IgG (H+L) antibody (1:50,000 dilution) was incubated at 25°C for 2 h. After aspirating the liquid in the wells, the plate was washed five times with PBST buffer. 100 μL / well of TMB colorimetric solution was added. The plate was incubated at 25°C in the dark for 25 min. 50 μL / well of stop solution was added. The OD value in the wells was read using a microplate 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] Antibody titers are expressed as "1:X," where X is the maximum dilution at which the antibody can be detected. The binding titers of the serum of both alpacas before immunization to the PD-1 protein were both below 1:2000, indicating that the levels of anti-PD-1-specific antibodies in them were extremely low. The binding titers of the serum after the fourth immunization to the PD-1 protein exceeded 64K, indicating that the level of anti-PD-1-specific antibodies in the alpaca serum increased significantly after multiple immunizations, indicating successful immunization.

[0066] Example 2 Construction of a 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. The pYDisplay vector fragments were separated by electrophoresis, and a 5,000-bp fragment was excised and recovered from the gel. The competent yeast strain was streaked onto a YPD solid medium plate and activated at 30°C for 7 days. A single clone of competent yeast was inoculated into 50 mL of YPD medium and cultured at 250 rpm and 30°C for 3 days. The linearized vector fragment and PCR product were mixed and added to an electroporation cuvette for transformation. The competent yeast cells were then transferred to a shake flask and incubated at 220 rpm and 30°C for 1 hour. A 20-μL aliquot of the resuspension was diluted 5000-fold with SD-CAA. 100 μL was then plated onto an SD-CAA plate and cultured for 3 days. The reservoir volume was calculated, and the remaining culture was continued overnight. The remaining bacterial suspension was collected into a 50 mL centrifuge tube and centrifuged at 3,000 × g for 15 min. The supernatant was discarded and resuspended in 10 mL of SD-CAA. The suspension was mixed with 50% glycerol and resuspension solution 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 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 and centrifuged at 5,000 × g for 15 min. The supernatant was discarded. Meanwhile, sterile 5% PBSA buffer (PBS buffer + 5% BSA) was diluted to 0.5% PBSA buffer. The yeast library was resuspended in 1 mL of 0.5% PBSA buffer and added to a 1.5 mL centrifuge tube. The library was centrifuged at 3,000 × g for 15 min. The supernatant was discarded. The cell suspension was washed again with 0.5% PBSA buffer. Prepare three 1.5 mL centrifuge tubes (two for negative panning with empty magnetic beads and one for positive panning), add 1 mL of 0.5% PBSA buffer to each centrifuge tube; thoroughly resuspend the aliquoted streptavidin magnetic beads with a pipette, and add 10 μL of magnetic beads to the 1.5 mL centrifuge tube; place the centrifuge tubes in a bag, secure them to a rotary mixer, and incubate them at 4°C for 15 minutes; place the centrifuge tubes on a magnetic separation rack for 15 minutes, and remove the supernatant with a pipette; then add 1 mL of 0.5% PBSA buffer again, incubate them at 4°C for another 15 minutes, and remove the supernatant.

[0076] (2) Yeast display library selection

[0077] Negative panning with empty magnetic beads: Place the centrifuge tube on a magnetic separation rack, add the washed yeast solution to the centrifuge tube containing empty magnetic beads, place it in a bag, and rotate and incubate at 4°C for 1 hour; place the empty magnetic bead centrifuge tube with yeast on a magnetic separation rack for 20 minutes, add the yeast solution to a new empty magnetic bead centrifuge tube, and rotate and incubate at 4°C for 40 minutes. After the incubation is completed, place it on a 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 in 0.5% PBSA buffer) to the centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and incubate with rotation 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 rack. Aspirate the supernatant with a pipette, then add 1 mL of 0.5% PBSA buffer, remove the centrifuge tube from the magnetic separation rack, pipette to mix, and place the centrifuge tube on the magnetic separation rack again. Let it stand for 15 minutes, and keep the centrifuge tube on the magnetic separation rack. Aspirate the supernatant with a pipette; repeat the above washing steps once more to obtain positive panning 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, 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 stand and let them stand at 25°C for 25 minutes; keep the centrifuge tube on the magnetic separation stand and use a pipette to discard the unbound yeast liquid; remove the 1.5mL centrifuge tube from the magnetic separation stand 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 stand, let it stand at 25°C for 15 minutes, and discard the supernatant; repeat the washing twice, and perform the entire operation 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 resuspension to 180 μL of SD-CAA medium and coat two plates, with each plate containing 100 μL. Then, add 5 μL of the resuspension to 95 μL of SD-CAA medium and coat one plate, for a total of three plates.

[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 with 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 number of yeasts capable of binding 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] Single clone selection: After overnight culture, single clones were picked and inoculated into culture medium to induce expression. 48 hours later, the cells were incubated with His-tagged PD-1 protein at 4°C for 1 hour with rotation. The supernatant was then centrifuged and discarded. The pellet was resuspended in a solution containing a biotin-conjugated anti-His tag flow cytometer antibody and incubated at 4°C for 1 hour with rotation. The supernatant was then centrifuged and discarded. The pellet was resuspended in a solution containing PE-streptavidin and incubated at 4°C for 1 hour with rotation. The cells were resuspended in 1 mL of PBS buffer and the supernatant was then centrifuged and discarded. The cells were then resuspended in 500 μL of PBS buffer and analyzed by flow cytometry. The single clone 195-5-H1, which had a high positive rate, was selected. 195-5-H1 was lysed with 0.2% SDS buffer (95°C for 10 minutes), centrifuged, and 0.5 μL of the supernatant was used as template for PCR amplification and analysis.

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

[0082] 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. A 50 μL aliquot of the PCR product was added to 1 / 10 volume of 10× loading buffer and subjected to electrophoresis. The CMV band size was 750 bp, the Fc band size was 1400 bp, and the VHH band size was 500 bp. The PCR product was then purified. The 195-5-H1 VHH sequence, CMV promoter sequence, and IgG1 Fc sequence were ligated by PCR. Subsequently, a 50 μL aliquot of the PCR product was added to 1 / 10 volume of 10× loading buffer and subjected to electrophoresis. The desired band was excised from the gel and the PCR product was purified. The resulting PCR product was transiently transfected into HEK293 cells, resulting in the cell culture supernatant containing 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. 100 μL of PE-labeled anti-human IgG antibody (1:500 dilution) was added and the cells were 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 500 μL of PBS buffer and analyzed by flow cytometry to determine 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 either CHO-K1 or CHO-K1-PD-1. The positive control antibody significantly bound to CHO-K1-PD-1 but not to CHO-K1 cells, indicating that CHO-K1 cells do not express PD-1 protein, while CHO-K1-PD-1 cells do 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 could detect PD-1 protein expression in the majority of CHO-K1-PD-1 cells, demonstrating that the 195-5-H1 antibody has 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 The relevant expression plasmids were transiently transfected into HEK293 cells and cultured with shaking for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads were used for affinity purification. The Protein A magnetic beads were washed twice with 30 mL of PBS buffer, 30 mL of 0.1 M sodium hydroxide, and 30 mL of PBS buffer, respectively. Based on the required sample volume, the appropriate volume of Protein A magnetic beads (calculated at 20 mg IgG / mL Protein A magnetic beads) was added to the HEK293 cell shaker flask. The cells were incubated in a shaking incubator at 150 rpm and 25°C for 2 hours. The Protein A magnetic beads were collected using a magnetic separation rack and transferred to a 50 mL centrifuge tube. After washing twice with 30 mL of PBS buffer and then ultrapure water, the beads were resuspended in 1 mL of elution buffer. After standing at 25°C for 15 minutes, the beads were collected using a magnetic separation rack and transferred to a 15 mL centrifuge tube. After eluting the Protein A beads twice, combine the eluates and adjust the pH of the solution with neutralization buffer. Dialyze the eluted sample against at least 100 times the volume of PBS buffer at 25°C for 2 hours, then change the buffer once and dialyze again at 8°C for 16 hours.

[0087] PD-1 protein was diluted to 2 μg / mL in coating buffer and added to a 96-well plate at 100 μL / well. Coating was allowed to proceed overnight at 4°C. The plate was washed three times with PBST buffer, and 200 μL / well of blocking buffer was added. The plate was blocked at 25°C for 3 h. The plate was washed three times with PBST buffer, and various concentrations of the 195-5-H1 candidate antibody (10 μg / mL, 3.3 μg / mL, 1.1 μg / mL, 0.37 μg / mL, 0.12 μg / mL, 0.04 μg / mL, 0.014 μg / mL, and 0 μg / mL) were added. The plate was incubated at 25°C for 1 h. The plate was washed five times with PBST buffer, and HRP-Protein A was diluted 1:50,000 and added to the plate at 100 μL / well. The plate was incubated at 25°C for 55 min. The plate was washed three times with PBST buffer, and 100 μL of TMB color development solution was added to each well. The plate was developed for 10 min at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at a wavelength of 450 nm on a microplate reader.

[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 high 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 were CHO-K1-PD-L1 (expressing PD-L1, which binds to PD-1 and inhibits luciferase expression in Jurkat-PD-1-Luc cells). 100 μL of 195-5-H1 at varying concentrations was added to the corresponding wells, resulting in final concentrations of 120 μg / mL, 40 μg / mL, 13.3 μg / mL, 4.4 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.16 μg / mL, 0.05 μg / mL, 0.02 μg / mL, and 0 μg / mL, respectively. After 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 PD-1 protein was diluted to 2 μg / mL using coating buffer. 100 μL / well was added to a 96-well plate and coated overnight at 4°C. The plate was washed three times with PBST buffer, and 200 μL / well of blocking buffer was added. The plate was blocked at 25°C for 3 h. The plate was washed three times with PBST buffer. Different concentrations of the 195-5-H1 candidate antibody (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, and 0 μg / mL) were added and incubated at 25°C for 25 min. Biotin-PD-L1 protein was added (final concentration 4 μg / mL) and incubated at 25°C for 55 min. The plate was washed five times with PBST buffer. The secondary antibody (streptavidin-HRP) was diluted 1:10,000 and added to a 96-well plate at 100 μL / well. The plate was incubated at 25°C for 55 min. Wash five times with PBST buffer, add 100 μL of TMB color development solution to each well, and develop for 15 min 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.

[0096] The results are shown in Table 4 and Figure 6 As shown, IC of 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 is highly stable 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 embodiment of the present invention, but the scope of protection 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 fall within the scope of protection and disclosure of the present invention.

Claims

1. A single-domain antibody H1 against programmed death receptor 1, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the anti-programmed death receptor 1 single-domain antibody H1 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The anti-programmed death receptor 1 single domain 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 single-domain antibody H1 is 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 single domain antibody H1 according to claim 1 or 2 and the human immunoglobulin crystallizable segment Ig Fc.

4. The heavy chain antibody according to claim 3, wherein The Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4; The Fc amino acid sequence of the 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 single-domain antibody H1 according to claim 1 or 2.

6. The nucleic acid molecule according to claim 5, wherein The nucleotide sequence of the nucleic acid molecule is 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 single-domain antibody H1 according to claim 1 or 2 and immune cells.

10. Use of any one of the anti-programmed death receptor 1 single domain 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 drug for treating tumors, characterized in that: The tumor is any one of colorectal 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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