A nanoantibody specifically binding to PD-1 and its application

Abstract

This invention provides a nanobody that specifically binds to PD-1 and its applications. The heavy chain variable region of the nanobody includes CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 is shown in SEQ ID NO:1; the amino acid sequence of CDR2 is shown in SEQ ID NO:2; and the amino acid sequence of CDR3 is shown in SEQ ID NO:3. The nanobody prepared by this invention has high specificity and can block the binding of PD-1 and PD-L1, enhancing the function of immune cells, and has important application value in cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a nanobody that specifically binds to PD-1 and its applications. Background Technology

[0002] A special type of antibody, the heavy chain antibody, exists in the blood of camels and cartilaginous fishes, lacking a light chain. Compared to ordinary antibodies, it contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Cloning its variable region yields single-domain antibodies (also known as nanobodies) composed solely of the heavy chain variable region, which have advantages such as small molecular weight, strong tissue penetration, high stability, and low immunogenicity.

[0003] Compared with ordinary antibodies (the research vectors are mainly mice and rabbits), alpaca antibodies have many advantages, including: (1) small size, only one-tenth the size of ordinary antibodies, which allows them to penetrate into tissues and cells, especially the blood-brain barrier. (2) strong stability, nanobodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90 degrees Celsius. (3) simple structure, nanobodies are easier to modify, such as humanization and multivalent construction, and have low production costs, making them suitable for large-scale production. (4) high specificity, the alpaca's immune system can produce antibodies with high specificity and affinity for specific antigens, and have weak immunogenicity to humans. The risk of nanobodies causing immune responses is low.

[0004] Programmed death molecule 1 (PD-1), also known as CD279, is widely expressed on the surface of immune cells and is an important immunosuppressive molecule. PD-1 belongs to the immunoglobulin superfamily CD28 / B7 and is a type I transmembrane glycoprotein composed of 288 amino acids, serving as an immunosuppressive receptor. It has at least two ligands, PD-L1 and PDL2. Under normal circumstances, PD-1 on the surface of T cells can inhibit the function of T lymphocytes, thereby suppressing autoimmune responses and preventing the occurrence of autoimmune diseases. However, in tumors, PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, leading to downregulation of T cell proliferation and even inducing T cell apoptosis, thus promoting tumor immune escape. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate the immune system and kill tumor cells.

[0005] Currently, utilizing 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. Several PD-1-targeting monoclonal antibodies are available on the market, and these PD-1 monoclonal antibodies have been successfully used in clinical practice; however, most of these antibodies are conventional antibodies. Compared to conventional antibodies, alpaca antibodies have many advantages. Developing an alpaca antibody that specifically binds to PD-1, and preparing antibody drugs or cell-based drugs based on it, has significant application value in cancer treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a nanobody that specifically binds to PD-1 and its applications. The nanobody prepared by the present invention exhibits high specificity and can simultaneously block the binding of PD-1 and PD-L1, thereby enhancing the function of immune cells.

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

[0008] In a first aspect, the present invention provides a nanobody that specifically binds to PD-1, wherein the heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3;

[0009] The amino acid sequence of CDR1 includes that shown in SEQ ID NO:1;

[0010] The amino acid sequence of CDR2 includes that shown in SEQ ID NO:2;

[0011] The amino acid sequence of CDR3 includes that shown in SEQ ID NO:3.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the nanobody includes that shown in SEQ ID NO:4.

[0013] In a second aspect, the present invention provides a heavy chain antibody that specifically binds to PD-1, the heavy chain antibody comprising the PD-1-specific nanobody described in the first aspect and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin.

[0014] Preferably, the Ig Fc includes the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

[0015] Preferably, the IgG1 Fc amino acid sequence includes that shown in SEQ ID NO:5.

[0016] Preferably, the amino acid sequence of the heavy chain antibody includes that shown in SEQ ID NO:6.

[0017] This invention uses PD-1 protein to immunize alpacas. After each immunization, the titer of PD-1-specific antibodies in alpaca serum was determined by ELISA. A sufficiently high titer indicated the presence of B cells in the alpaca capable of secreting high-affinity anti-PD-1 protein. Subsequently, alpaca peripheral blood mononuclear cells (PBMCs) were isolated, RNA was extracted, and cDNA was obtained through reverse transcription. The VHH sequence was amplified using alpaca single-domain antibody-specific primers and cloned into a yeast expression plasmid to construct a yeast display library. The yeast display library was then subjected to positive and negative selection using biotin-PD-1 protein-streptomycin magnetic beads or streptomycin magnetic beads to enrich yeast strains that bind to PD-1 protein. A single clone was selected from the enriched product and named 195-1-A07. The specific binding of 195-1-A07 to PD-1 was detected by ELISA. After expression and purification, the affinity of 195-1-A07 for binding to PD-1 protein was assessed. The ability of 195-1-A07 to block the binding of PD-1 and PD-L1 was investigated. The results showed that 195-1-A07 has high specificity and can block the binding of PD-1 and PD-L1, thereby enhancing the function of immune cells.

[0018] The amino acid sequence of CDR1, SEQ ID NO:1: VSGSIFGIHAIS.

[0019] The amino acid sequence of CDR2, SEQ ID NO:2: TRITSTDYGE.

[0020] The amino acid sequence of CDR3, SEQ ID NO:3: NREIRGSSGIWYPLDY.

[0021] The amino acid sequence of the heavy chain variable region of the nanobody, SEQ ID NO:4:

[0022] DVQLVESGGGLVQPGGSLTLSCAVSGSIFGIHAISWYRQAPGNQRELVTRITSGTSTDYGESVKGRFIVSRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSS.

[0023] The IgG1 Fc amino acid sequence, SEQ ID NO:5:

[0024] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0025] The amino acid sequence of the heavy chain antibody, SEQ ID NO:6:

[0026] DVQLVESGGGLVQPGGSLTLSCAVSGSIFGIHAISWYRQAPGNQRELVTRITSGTSTDYGESVKGRFIVSRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0027] Thirdly, the present invention provides a nucleic acid molecule that encodes either the nanobody that specifically binds to PD-1 as described in the first aspect or the heavy chain antibody that specifically binds to PD-1 as described in the second aspect.

[0028] Fourthly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the third aspect.

[0029] Fifthly, the present invention provides a cell expressing a nanobody that specifically binds to PD-1 as described in the first aspect or a heavy chain antibody that specifically binds to PD-1 as described in the second aspect, wherein the cell contains at least one copy of the recombinant vector as described in the fourth aspect, or the nucleic acid molecule as described in the third aspect is integrated into its genome.

[0030] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the nanobody that specifically binds to PD-1 as described in the first aspect or the heavy chain antibody that specifically binds to PD-1 as described in the second aspect.

[0031] In a seventh aspect, the present invention provides a kit for detecting PD-1, the kit comprising the nanobody that specifically binds to PD-1 as described in the first aspect or the heavy chain antibody that specifically binds to PD-1 as described in the second aspect.

[0032] Eighthly, the present invention provides the use of the nanobody that specifically binds PD-1 as described in the first aspect, the heavy chain antibody that specifically binds PD-1 as described in the second aspect, the nucleic acid molecule as described in the third aspect, the recombinant vector as described in the fourth aspect, the cell as described in the fifth aspect, the pharmaceutical composition as described in the sixth aspect, or the kit for detecting PD-1 as described in the seventh aspect in the preparation of a drug for detecting and / or treating tumors.

[0033] The alpaca antibody prepared in this invention has high specificity, can block the binding of PD-1 and PD-L1, and enhance the function of immune cells. The anti-PD1 alpaca antibody can be formulated into an antibody drug using relevant technologies and can be used for cancer treatment.

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

[0035] The anti-PD-1 protein antibody prepared in this invention exhibits good affinity and specificity. 195-1-A07 does not bind to CHO-K1 cells but significantly binds to CHO-K1-PD-1, and most CHO-K1-PD-1 cells express PD-1 protein, indicating that 195-1-A07 has good affinity and specificity for PD-1 protein. Further measurements of the EC50 of 195-1-A07 against CHO-K1-PD-1 were performed. 50 The IC50 value of 195-1-A07 was 0.2 μg / mL, indicating that 195-1-A07 has high affinity. The blocking function assay of the recombinant antibody showed that 195-1-A07 has a strong ability to block the binding of PD-1 and PD-L1, and can effectively promote the expression of luciferase by Jurkat-PD-1-Luciferase, which can be used for the development of immunomodulatory drugs. Further measurement of the IC50 value of 195-1-A07 showed a result of 1.25 μg / mL, indicating that 195-1-A07 has a strong ability to block the binding of PD-1 and PD-L1 proteins. In summary, the antibody prepared in this invention has high specificity, can block the binding of PD-1 and PD-L1, and can enhance the function of immune cells, showing significant application prospects in drug preparation. Attached Figure Description

[0036] Figure 1To detect the enrichment of yeast libraries before and after sorting using flow cytometry.

[0037] Figure 2 The results of flow cytometry detection of the specificity of recombinant single-domain antibodies.

[0038] Figure 3 This is the detection curve for the blocking function of the recombinant antibody.

[0039] Figure 4 EC for ELISA detection of recombinant single-domain antibodies 50 .

[0040] Figure 5 IC50 for ELISA detection of recombinant single-domain antibodies 50 . Detailed Implementation

[0041] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] The reagents used in the following examples:

[0044] Streptavidin (PE) (eBioscience, CAT#12-4317-87);

[0045] PE-anti-human IgG (eBioscience, CAT#12-4998-82);

[0046] PBS (bico, CAT#14190-250);

[0047] HRP-Protein A (HRP-tagged protein A, Boster, CAT#BA1080);

[0048] HRP-Streptavidin (Horseradish peroxidase-streptavidin, Boster, CAT#BA1088);

[0049] PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#6210B);

[0050] DNA Fragment Recovery Kit (TakaRa, CAT#9761);

[0051] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (HRP-labeled goat anti-lama IgG secondary antibody, Novus, AT#NB7242);

[0052] THE TM V5-tagged antibody [iFluor 647], mAb (Genscript, CAT#A01805).

[0053] Example 1

[0054] In this embodiment, alpacas were immunized multiple times with PD-1 protein, and peripheral blood was collected to monitor the content of anti-PD-1 specific antibodies in the serum.

[0055] The amino acid sequence of the PD-1 protein is shown below, SEQ ID NO:7:

[0056] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL.

[0057] Two alpacas (number 98 and number 195) were immunized with PD-1 protein. The immunization experiment began after recording the ear tags of the blank alpacas. The immunization steps are as follows:

[0058] Before immunization, collect 5mL of peripheral blood; each time, inject the alpaca into the lymph nodes near the neck of the alpaca, on both sides, with 2 injection points on each side; observe the alpaca for half an hour after immunization to confirm that the alpaca is in good condition and has no discomfort symptoms; immunize once every 3 weeks, for a total of 4 immunizations.

[0059] For the first immunization, approximately 500 μg of emulsified antigen is injected.

[0060] For the second immunization, approximately 500 μg of emulsified antigen was injected; 5 mL of peripheral blood was collected two weeks after the immunization.

[0061] The third immunization involves injecting approximately 500 μg of emulsified antigen; 5 mL of peripheral blood is collected two weeks after immunization.

[0062] The fourth immunization involves injecting approximately 500 μg of emulsified antigen; 5 mL of peripheral blood is collected two weeks after immunization.

[0063] Centrifuge tubes containing blood samples at 800g for 10 min and collect the supernatant serum. Dilute PD-1-His recombinant protein to a final concentration of 1 μg / mL with sterile PBS. Add 100 μL / well of PD-1-His recombinant protein to a new 96-well microplate and incubate overnight at 4°C. Discard the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20). Add 200 μL / well of blocking buffer and incubate at 37°C for 2 hours. Discard the blocking buffer and wash the plate 5 times with PBST. Serially dilute the collected serum with PBS, adding 100 μL of each dilution to the 96-well microplate and incubating at room temperature for 1 hour. Use PBS for the control wells. Discard the liquid from the wells and wash 5 times with PBST. Add 100 μL of HRP anti-Llama. IgG (H+L) antibody (1:50000 dilution), incubated at room temperature for 1 hour; after discarding the liquid in the wells, wash the plate 5 times with PBST; add 100 μL / well TMB chromogenic buffer; incubate at room temperature in the dark for 10-15 min; add 50 μL / well stop solution; read the OD in the wells using a microplate reader. 450 value.

[0064] The test results showed that the binding titers of PD1 protein in the serum of both alpacas after four immunizations were significantly higher than those in the serum before immunization, indicating that the content of PD1-specific antibodies in the serum of the two alpacas was significantly increased after multiple rounds of immunization, and can be used for the construction of yeast display libraries.

[0065] Example 2

[0066] In this embodiment, peripheral blood mononuclear cells (PBMCs) were isolated from alpaca peripheral blood, RNA was extracted, and a single-domain antibody yeast display library was constructed.

[0067] (1) Carefully collect 100 mL of peripheral blood from an alpaca using a syringe and place it in an anticoagulant tube. Separate PBMCs using lymphocyte separation medium according to the operating procedure. The steps for separating PBMCs include: taking fresh anticoagulant whole blood, adding 1× dilution wash buffer at a ratio of 1:1 to dilute the blood (to reduce blood viscosity), and gently mixing. Carefully add an appropriate amount of mononuclear cell separation medium Ficoll to a sterile centrifuge tube, and carefully spread the diluted blood sample on top of the separation medium (separation medium: diluted whole blood = 1:2), being careful not to let the blood sample mix into the separation medium Ficoll to maintain a clear interface between the two liquids. Centrifuge at 800g, room temperature, for 20-30 min. After centrifugation, discard the plasma layer, carefully aspirate the PBMC layer (i.e., the white membrane layer) and transfer it to a 15 mL centrifuge tube. Add 10 mL of 1× dilution wash buffer to the centrifuge tube to resuspend the cells, centrifuge at 250g, room temperature, for 10 min, and discard the supernatant. Repeat this step 1-2 times for subsequent experiments.

[0068] (2) The steps for extracting RNA include:

[0069] RNA was extracted from PBCMs after PBMC isolation and then processed using PrimeScript. TM II 1 st The Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA; using PBMC cDNA as a template, the alpaca heavy chain antibody sequence was amplified by PCR with specific primers.

[0070] The upstream primer binds to the signal peptide of the VHH antibody ORF, and the primer sequence is SEQ ID NO:8: 5'GTCCTGGCTGCTCTTCTACAAGG 3'.

[0071] The downstream primer binds to the CH2 region of the VHH antibody, and the primer sequence is SEQ ID NO:9: 5'GGTACGTGCTGTTGAACTGTTCC 3'.

[0072] Using the first-round PCR product as a template, the VHH fragment of the heavy chain antibody was amplified with specific primers.

[0073] After amplification, the PCR products were analyzed by electrophoresis using 1% agarose gel. The target band (the target fragment with a molecular weight of about 450 bp) was recovered and frozen using a gel recovery kit.

[0074] (3) Electroconversion of the library carrier

[0075] The obtained VHH fragment library was ligated onto the yeast surface display vector pYDisplay.

[0076] Specifically, the yeast surface display vector pYDisplay and the obtained VHH fragment library were digested with SfiI restriction enzyme. The pYDisplay vector fragment was separated using a 1% agarose gel, and a 5000bp fragment was extracted for gel recovery.

[0077] The linearized vector fragment and the recovered PCR product were mixed and added to an electroporation cuvette for electroporation. The electroporated yeast competent cells were then transferred to culture flasks and incubated at 220 rpm and 30°C for 1 hour. 20 μL of the resuspended culture was diluted 5000 times with SDCAA, and 100 μL was plated on an SDCAA plate and incubated for 2-3 days. The library volume was calculated, and the remaining culture was incubated for another 24 hours. The remaining culture was collected in a 50 mL centrifuge tube, centrifuged at 3000 g for 5 minutes, the supernatant was discarded, and 10 mL of SDCAA was added for resuspending. The mixture was then mixed with 50% glycerol at a 1:1 ratio and stored at -80°C.

[0078] Example 3

[0079] To screen for alpaca antibodies with high affinity for PD-1, this embodiment performs panning on the constructed yeast display library. The panning steps include:

[0080] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0081] To quickly screen for alpaca antibodies with high affinity for PD-1, we prepared a yeast display library and screened the library. We took 10 mL of the yeast library (approximately 2 × 10⁻⁶ cells / mL). 8 Add yeast cells to a centrifuge tube, centrifuge at 3000g for 5 min at room temperature, and discard the supernatant; at the same time, dilute sterile 5% PBSA (PBS + 5% BSA) to 0.5% PBSA; resuspend the yeast library in 1 mL of 0.5% PBSA, add it to a centrifuge tube, centrifuge at 3000g for 5 min at room temperature, and discard the supernatant; wash again with 0.5% PBSA. 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 to each centrifuge tube. Resuspend the aliquoted streptavidin magnetic beads thoroughly by pipetting, and add 10 μL of the magnetic beads to each 1.5 mL centrifuge tube. Place these centrifuge tubes in a bag, fix them on a rotary mixer, and incubate at 4°C for 5 min. Place the centrifuge tubes on a magnetic rack for 5 min, and remove the supernatant with a pipette. Then add 1 mL of 0.5% PBSA again, incubate at 4°C for another 5 min, and remove the supernatant.

[0082] (2) Yeast Display Library Selection

[0083] (A) Negative selection of empty magnetic beads

[0084] Because yeast display libraries have certain non-specific adsorption, it is necessary to perform negative screening of the yeast display library with empty magnetic beads first. Place the centrifuge tube on a magnetic rack, add the washed yeast culture to the centrifuge tube containing empty magnetic beads, and put it in a bag. Incubate at 4°C for 60 min. Place the centrifuge tube with empty magnetic beads containing yeast on a magnetic rack for 10 min, then aspirate the yeast culture and add it to a new centrifuge tube with empty magnetic beads, being careful not to aspirate the old magnetic beads. Incubate at 4°C for 30 min. After incubation, place the tube on a magnetic rack for 15 min, and carefully aspirate the yeast culture and add it to a new centrifuge tube with empty magnetic beads.

[0085] (B) Biotin-PD-1-His magnetic separation

[0086] Add 100 μL of 50 μg / mL biotin-PD-1-His protein solution (biotin-conjugated, diluted with 0.5% PBSA) to a centrifuge tube containing streptavidin magnetic beads. Place the centrifuge tube in a bag and incubate at 4°C for 60 min. Then add 1 mL of 0.5% PBSA to the bag and let stand at room temperature for 5 min. Place the centrifuge tube on a magnetic rack throughout the process. Carefully aspirate the supernatant with a pipette and add 1 mL of 0.5% PBSA. Remove the centrifuge tube from the magnetic rack, mix by pipetting, and place the centrifuge tube back on the magnetic rack. Let stand at room temperature for 5 min. Carefully aspirate the supernatant with a pipette. Repeat the above washing steps once more to obtain biotin-PD-1-His coated positively selected magnetic beads. Add biotin-PD-1-His-bound positively panning magnetic beads to the negatively panned yeast cells, place them in a bag, and incubate on a rotary mixer at 4°C for 60 min. After incubation, place the mixture on a magnetic rack and let it stand at room temperature for 15 min. Keep the centrifuge tube on the magnetic rack and use a pipette to remove the unbound yeast culture. Remove the 1.5 mL centrifuge tube from the magnetic rack and add 1 mL of sterile 0.5% PBSA buffer to the centrifuge tube. Gently pipette the magnetic beads and then transfer them to a sterile 1.5 mL centrifuge tube. Place the centrifuge tube on a magnetic rack and let it stand at room temperature for 5 min. Discard the supernatant. Repeat the washing process twice. Perform the entire procedure according to aseptic technique. After washing, the magnetic beads and the adhered yeast cells were resuspended in 1 mL of SDCAA medium. 20 μL of the resuspension was transferred to 180 μL of SDCAA medium and plated onto two plates, each plate having a volume of 100 μL. Then, 5 μL of the resuspension was transferred to 95 μL of SDCAA medium and plated onto one plate. In total, three plates were required.

[0087] (C) Flow cytometry detection of yeast cells after selection

[0088] After panning, yeast cells (containing the V5 tag, indicating successful transfection of the expression vector into yeast) were enriched. Yeast cells that underwent negative panning with empty magnetic beads and biotin-PD-1-His magnetic sorting were incubated with His-tagged PD-1 protein at 4°C for 60 min. The supernatant was removed by centrifugation, and flow cytometry antibodies against both the anti-His and anti-V5 tags were added. The cells were incubated at 4°C for 60 min, then the supernatant was removed by centrifugation, and the cells were resuspended in 1 mL of PBS. Flow cytometry analysis was then performed.

[0089] Figure 1 To detect the enrichment of yeast libraries before and after sorting using flow cytometry, from Figure 1 It can be seen that the number of yeast cells that can bind to PD-1 protein in the yeast antibody display libraries of alpacas #98 and #195 increased by 38% and 35% respectively before and after sorting, which can be used for subsequent experiments.

[0090] (3) Single clone selection

[0091] After overnight culture, single clones were picked and inoculated into culture medium for 48 hours. Flow cytometry staining and flow cytometry analysis were performed after 48 hours.

[0092] Flow cytometry analysis showed that the monoclonal clone with a high positive rate was numbered 195-1-A07; the monoclonal clone 195-1-A07 was amplified by PCR and sequenced.

[0093] Example 4

[0094] In this embodiment, the antibody expressed by yeast with monoclonal number 195-1-A07 was recombinantly expressed and its binding specificity was detected.

[0095] (1) Recombinant expression

[0096] The candidate 195-1-A07 VHH sequence, CMV promoter sequence, and IgG1 Fc sequence were amplified using a PCR instrument. After amplification, the PCR products were analyzed by 1% agarose gel electrophoresis. The band size of the CMV promoter sequence was 750 bp, the band size of the IgG1 Fc sequence was 1400 bp, and the band size of the 195-1-A07 VHH sequence was 500 bp.

[0097] The amino acid sequence of the VHH sequence is shown in SEQ ID NO:4.

[0098] DVQLVESGGGLVQPGGSLTLSCAVSGSIFGIHAISWYRQAPGNQRELVTRITSGTSTDYGESVKGRFIVSRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSS.

[0099] The amino acid sequence of the IgG1 Fc sequence is shown in SEQ ID NO:5.

[0100] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0101] The nucleotide sequence of the CMV promoter is shown in SEQ ID NO:10.

[0102] .

[0103] The target band of the above size was recovered and purified. The 195-1-A07 VHH sequence, CMV promoter sequence, and IgG1 Fc sequence were ligated using a PCR instrument. The PCR product was then analyzed by electrophoresis with 1% agarose gel. The target band was excised from the gel, and the PCR product was purified. The obtained PCR product was transiently transfected into HEK293 cells. The culture supernatant of the harvested cells contained the recombinant single-domain antibody 195-1-A07.

[0104] (2) Combining specific detection

[0105] The binding specificity of recombinant single-domain antibody 195-1-A07 was detected.

[0106] The culture supernatant containing 195-1-A07 was then mixed with CHO-K1 (3×10⁻⁶) and 10⁻⁶ mol / L. 5 (each) or CHO-K1-PD-1 (3×10) 5(Number) cells were incubated at room temperature with shaking for 1 hour. After 1 hour, the cells were centrifuged at 800g for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in PBS. Then, the cells were centrifuged at 800g for 5 minutes, the supernatant was discarded, and the process was repeated 3 times.

[0107] Add 100 μL of PE-labeled anti-human IgG antibody (1:500 dilution) and incubate at room temperature in the dark for 45 min. After centrifugation at 800g for 5 min at room temperature, discard the supernatant and wash the cells three times with PBS. Resuspend the cells in 500 μL of PBS and perform flow cytometry analysis.

[0108] Figure 2 To obtain specific results for flow cytometry detection of recombinant single-domain antibodies. From Figure 2 It was found that 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 express PD-1 protein and can be used as detection cells for alpaca antibodies. 195-1-A07 did not bind to CHO-K1 cells but significantly bound to CHO-K1-PD-1, and most CHO-K1-PD-1 cells expressed PD-1 protein, indicating that 195-1-A07 has good affinity and specificity.

[0109] Example 5

[0110] This embodiment describes the purification of recombinant single-domain antibody 195-1-A07.

[0111] (1) The 195-1-A07 sequence obtained by amplification was synthesized with human IgG1 FC segment and cloned into expression plasmid. The expression plasmid was transiently transfected into 293F cells and cultured in shake flasks for 48 hours. Then, the antibody was expressed and purified.

[0112] (2) Since the target recombinant antibody contains human IgG fragments, affinity purification can be performed using Protein A magnetic beads. Wash the Protein A magnetic beads twice sequentially with 30 mL PBS buffer, 0.1 M sodium hydroxide, and PBS buffer, respectively. Add the appropriate volume of Protein A magnetic beads to a 293F cell shake flask according to the required sample volume (calculated at 20 mg IgG / mL Protein A magnetic beads). Incubate at 120 rpm at room temperature for 1-4 hours or overnight at 4°C. Collect the Protein A magnetic beads using a magnetic separator and transfer them to 50 mL centrifuge tubes. Wash twice with 30 mL PBS buffer and deionized water, then resuspend in 1 mL elution buffer. Incubate at room temperature for 5 min, collect the magnetic beads using a magnetic separator, and transfer them to 15 mL centrifuge tubes. Repeat the elution process twice, combine the eluents, and adjust the pH of the solution with neutralization buffer. The eluted sample was dialyzed with at least 100 times its volume of PBS, initially at 18-25°C for 2 hours, followed by one dialysis with the buffer changed, and then dialyzed again at 2-8°C for 14-16 hours. Finally, the protein concentration was determined, and the sample was filtered through a 0.22 μm sterile filter membrane, aliquoted, and stored at -80°C for later use.

[0113] Example 6

[0114] This embodiment tests the blocking function of recombinant single-domain antibody 195-1-A07. The specific steps include:

[0115] Add 100 μL of cell suspension containing 2 × 10⁶ cells to each well of a 96-well plate. 4 One effector cell, Jurkat-PD-1-Luciferase (expressing PD-1 molecules, which can inhibit luciferase expression after binding to PD-L1), and 8 × 10 4 Target cells were CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of Jurkat-PD-1-Luciferase cytoluciferase). Subsequently, 100 μL of different concentrations of 195-1-A07 were added to the corresponding wells, resulting in final antibody 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 co-culturing for 20 h, 20 μL of One-Glo reagent was added to each well, and the luciferase fluorescence value was read.

[0116] Figure 3 This is the detection curve for the blocking function of the recombinant antibody. From... Figure 3It can be seen that the maximum induction effect value of 195-1-A07 is 2.1 (the difference between the maximum luciferase fluorescence value and the luciferase fluorescence value at 0 μg / mL), indicating that 195-1-A07 has a strong ability to block the binding of PD-1 and PD-L1, and can effectively promote the expression of luciferase in effector cells, which can be used to develop immune-enhancing drugs.

[0117] Example 7

[0118] This embodiment describes the half-maximal effect concentration (EC50) of recombinant single-domain antibody 195-1-A07. 50 The determination process includes the following steps:

[0119] Dilute PD-1 protein to 2 μg / mL using coating buffer, and add 100 μL / well to a 96-well microplate. Coat overnight at 4°C. Wash 5 times with PBST, add 200 μL / well of blocking buffer, and block at room temperature for 2 hours. Wash 5 times with PBST, add different concentrations of 195-1-A07 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, 0 μg / mL), and incubate at room temperature for 60 min. Wash 5 times with PBST, dilute HRP-Protein A 1:50000, add 100 μL / well to the microplate, and incubate at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB chromogenic buffer to each well, and develop at room temperature in the dark for 10 min. Add 50 μL of stop solution to each well and read the absorbance at 450 nm wavelength on a microplate reader.

[0120] Figure 4 EC for ELISA detection of recombinant single-domain antibodies 50 Table 1 shows the EC50 detection results for antibody 195-1-A07. From Table 1 and... Figure 4 It can be seen that 195-1-A07 corresponds to the EC of CHO-K1-PD-1. 50 The value was 0.2013 μg / mL, indicating that 195-1-A07 has a high affinity.

[0121] Table 1

[0122]

[0123] Example 8

[0124] This embodiment describes the half-inhibitory concentration (IC50) of recombinant single-domain antibody 195-1-A07. 50 The detection process includes the following steps:

[0125] To determine the half-maximal inhibitory concentration (IC50) of 195-1-A07 in blocking the binding of PD-1 and PD-L1, PD-1 protein was diluted to 2 μg / mL using coating buffer, and 100 μL / well was added to each well of a 96-well microplate. The plates were incubated overnight at 4°C. After washing five times with PBST, 200 μL / well of blocking buffer was added, and the plates were blocked at room temperature for 2 hours. After washing five times with PBST, different concentrations (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, and 0 μg / mL) of the 195-1-A07 candidate antibody were added, and the plates were incubated at room temperature for 15 min. Biotin-PD-L1 protein (final concentration 4 μg / mL) was added, and the plates were incubated at room temperature for 45 min. After washing five times with PBST, the secondary antibody (Streptavidin-HRP) was diluted 1:10000, and 100 μL / well was added to each well of the microplate. The plates were incubated at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 10 min. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm using a microplate reader.

[0126] Figure 5 IC50 for ELISA detection of recombinant single-domain antibodies 50 Table 2 shows the blocking curve of 195-1-A07 antibody; Table 2 shows the IC50 of 195-1-A07 antibody. 50 Detection; from Figure 5 As shown in Table 2, IC 195-1-A07 50 The value was 1.25 μg / mL, indicating that 195-1-A07 has a strong ability to block the binding of PD-1 and PD-L1 proteins.

[0127] Table 2

[0128]

[0129]

[0130] In summary, this invention provides an alpaca antibody with high specificity that can block the binding of PD-1 and PD-L1 and enhance the function of immune cells, which has important application prospects in cancer treatment.

[0131] 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 nanobody that specifically binds to PD-1, characterized in that, The heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 is shown in SEQ ID NO:1; The amino acid sequence of CDR2 is shown in SEQ ID NO:2; The amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The nanobody that specifically binds to PD-1 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody includes that shown in SEQ ID NO:

4.

3. A heavy chain antibody that specifically binds to PD-1, characterized in that, The heavy chain antibody comprises the nanobody that specifically binds to PD-1 as described in claim 1 or 2 and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin.

4. The heavy chain antibody that specifically binds to PD-1 according to claim 3, characterized in that, The Ig Fc includes the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof; The IgG1 Fc amino acid sequence includes that shown in SEQ ID NO:5; The amino acid sequence of the heavy chain antibody includes that shown in SEQ ID NO:

6.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody that specifically binds to PD-1 as described in claim 1 or 2, or the heavy chain antibody that specifically binds to PD-1 as described in claim 3 or 4.

6. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 5.

7. A cell expressing a nanobody specifically binding to PD-1 as described in claim 1 or 2, or a heavy chain antibody specifically binding to PD-1 as described in claim 3 or 4, characterized in that, The cell contains at least one copy of the recombinant vector of claim 6, or the nucleic acid molecule of claim 5 is integrated into its genome.

8. A pharmaceutical composition, characterized in that, The composition comprises a nanobody that specifically binds to PD-1 as described in claim 1 or 2, or a heavy chain antibody that specifically binds to PD-1 as described in claim 3 or 4.

9. A kit for detecting PD-1, characterized in that, The kit comprises a nanobody that specifically binds to PD-1 as described in claim 1 or 2, or a heavy chain antibody that specifically binds to PD-1 as described in claim 3 or 4.

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