Nanometer antibody specifically binding to PD-1 and application thereof

Abstract

The invention provides a nano antibody capable of being specifically combined with PD-1 and application of the nano antibody. A heavy chain variable region of the nano antibody comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is as shown in SEQ ID NO: 1; the amino acid sequence of the CDR2 is as shown in SEQ ID NO: 2; the amino acid sequence of the CDR3 is as shown in SEQ ID NO: 3. The prepared nano antibody is high in specificity, can block combination of PD-1 and PD-L1 at the same time, enhances the function of immune cells, and has important application value in cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a nano antibody that specifically binds to PD-1 and an application thereof. Background Art

[0002] There is a special antibody lacking light chains in the blood of camelids and chondrichthyes, namely heavy chain antibodies. Compared with ordinary antibodies, it only contains 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 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.

[0003] Compared with ordinary antibodies (research vectors are mainly mice and rabbits), alpaca antibodies have many advantages, including: (1) small size, one-tenth of ordinary antibodies, which allows them to penetrate into tissues and cells, especially the blood-brain barrier. (2) Strong stability. Nano antibodies can maintain stability under extreme temperature and pH conditions and can still maintain biological activity at high temperatures up to 90 degrees. (3) Simple structure. Nano antibodies 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. Nano antibodies have a low risk of causing an immune response.

[0004] Programmed death 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. It is a type I transmembrane glycoprotein composed of 288 amino acids and is 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 inhibiting autoimmune responses and preventing the occurrence of autoimmune diseases. However, in tumors, after PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, it can inhibit T lymphocytes, leading to downregulation of T cell proliferation, and even inducing T cell apoptosis, promoting tumor immune escape. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate the immune system function and kill tumor cells.

[0005] Currently, the use of PD-1 antibodies to block the PD-1 / PD-L1 pathway to restore T cell tumor activity, activate T cells, and kill tumor cells has become the focus of cancer immunotherapy. There are many monoclonal antibodies targeting PD-1 on the market, which have been successfully used in clinical practice, but most of these antibodies are ordinary antibodies. Compared with ordinary antibodies, alpaca antibodies have many advantages. Developing an alpaca antibody that specifically binds to PD-1 and preparing it into antibody drugs or cell drugs based on it has important application value in the treatment of cancer. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a nano antibody that specifically binds to PD-1 and its application. The nano antibody prepared by the present invention has high specificity and can block the binding of PD-1 and PD-L1 at the same time, 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 comprises CDR1, CDR2 and CDR3;

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

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

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

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the Nanobody comprises 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, wherein the heavy chain antibody comprises the nanobody that specifically binds to PD-1 as 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 comprises the full-length or partial sequence of the Fc fragment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

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

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

[0017] The present invention uses PD-1 protein to immunize alpacas. After each immunization, the titer of PD-1 specific antibodies in alpaca serum is determined by ELISA. When the titer is high enough, it indicates that B cells that can secrete high-affinity anti-PD-1 proteins are already present in the alpaca. Subsequently, the alpaca peripheral blood mononuclear cells (PBMC) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Using alpaca single-domain antibody-specific primers, the VHH sequence is amplified and cloned into a yeast expression plasmid to construct a yeast display library. The yeast display 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. A single clone is picked from the enriched product and named 195-1-A07, and the specific binding of 195-1-A07 to PD-1 is detected by ELISA. After 195-1-A07 is expressed and purified, its affinity for binding to PD-1 protein is detected. The ability of 195-1-A07 to block the binding of PD-1 and PD-L1 was tested. The results showed that 195-1-A07 has high specificity and can block the binding of PD-1 and PD-L1 at the same time, enhancing the function of immune cells.

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

[0019] The amino acid sequence of the CDR2 is SEQ ID NO:2: TRITSGTSTDYGE.

[0020] The amino acid sequence of the CDR3 is 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] In a third aspect, the present invention provides a nucleic acid molecule encoding 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] In a fourth aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the third aspect.

[0029] In the fifth aspect, the present invention provides a cell expressing the nano antibody 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, 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] In an eighth aspect, the present invention provides the use of the nanoantibody that specifically binds to PD-1 as described in the first aspect, the heavy chain antibody that specifically binds to 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 drugs for detecting and / or treating tumors.

[0033] In the present invention, the prepared alpaca antibody has high specificity, can block the combination of PD-1 and PD-L1, and enhance the function of immune cells. The anti-PD1 alpaca antibody can be made into an antibody drug using relevant technology and can be used for the treatment of cancer.

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

[0035] The anti-PD-1 protein antibody prepared by the present invention has good affinity and specificity. 195-1-A07 does not bind to CHO-K1 cells, but has significant binding to CHO-K1-PD-1, and can detect that most CHO-K1-PD-1 cells express PD-1 protein, indicating that 195-1-A07 has good affinity and specificity for PD-1 protein. The EC value of 195-1-A07 for CHO-K1-PD-1 was further measured. 50 The result was 0.2μg / mL, indicating that 195-1-A07 has a high affinity. The blocking function test results of the recombinant antibody showed that 195-1-A07 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-Luciferase to express luciferase, and can be used to develop immune-enhancing drugs. The IC50 value of 195-1-A07 was further measured, and the result was 1.25μg / 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 by the present invention has high specificity, can block the binding of PD-1 and PD-L1, can enhance the function of immune cells, and has important application prospects in drug preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Flow cytometry was used to detect the enrichment of the yeast library before and after sorting.

[0037] Figure 2 Flow cytometry was used to detect the specificity of recombinant single domain antibodies.

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

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

[0040] Figure 5 IC of recombinant single domain antibodies for ELISA 50 . DETAILED DESCRIPTION

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

[0042] 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.

[0043] 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-labeled 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-Llama IgG secondary antibody, Novus, AT#NB7242);

[0052] THE TM V5 tag antibody [iFluor 647], mAb (GenScript, CAT#A01805).

[0053] Example 1

[0054] In this example, the alpaca was 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 (98# and 195#) were immunized with PD-1 protein, and the immunization experiment was started after recording the ear number of the blank alpaca. The immunization steps are as follows:

[0058] Before immunization, collect 5mL of peripheral blood; each time, inject the alpaca near the cervical lymph nodes on both sides, 2 points on each side for immunization; observe 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, and perform 4 immunizations;

[0059] For the first immunization, about 500 μg of emulsified antigen was injected;

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

[0061] For the third immunization, about 500 μg of emulsified antigen was injected; 5 mL of peripheral blood was collected two weeks after immunization;

[0062] For the fourth immunization, about 500 μg of emulsified antigen was injected; 5 mL of peripheral blood was collected two weeks after immunization.

[0063] Place the centrifuge tube containing the blood sample in a centrifuge, centrifuge at 800g for 10 minutes, and collect the upper serum. Use sterile PBS to dilute the PD-1-His recombinant protein to a final concentration of 1μg / mL, take a new 96-well ELISA plate, add 1μg / mL of PD-1-His recombinant protein to 100μL / well, and coat overnight at 4°C; then shake off the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20); add 200μL / well of blocking solution and block at 37°C for 2 hours; after shaking off the blocking buffer, wash the plate 5 times with PBST; use PBS to gradient dilute the above collected serum, add 100μL of gradient diluted serum to the 96-well ELISA plate, incubate at room temperature for 1 hour, and the control well is PBS; shake off the liquid in the well and wash 5 times with PBST; add 100μL HRPanti-Llama IgG (H+L) antibody (1:50000 dilution), incubate at room temperature for 1 hour; shake off the liquid in the wells, wash the plate 5 times with PBST; add 100 μL / well TMB colorimetric solution; incubate at room temperature in the dark for 10-15 minutes; add 50 μL / well stop solution; read the OD in the wells using an ELISA reader 450 value.

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

[0065] Example 2

[0066] In this example, 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 the alpaca with a syringe and place it in an anticoagulation tube. Use lymphocyte separation solution to separate PBMC according to the operating procedures. The steps of separating PBMC include: taking fresh anticoagulated whole blood, adding 1× dilution washing solution in a 1:1 ratio to dilute the blood (to reduce blood viscosity), gently mixing, and setting aside. Carefully add an appropriate amount of mononuclear cell separation solution Ficoll to a sterile centrifuge tube, and carefully spread the diluted blood sample on the surface of the separation solution (separation solution: diluted whole blood = 1:2). Be careful not to let the blood sample mix into the separation solution Ficoll to keep the interface between the two liquid surfaces clear. Centrifuge at 800g, room temperature, for 20-30 minutes. After the centrifugation is completed, the plasma layer is discarded, and the PBMC layer (i.e., the white film layer) is carefully aspirated and transferred to a 15 mL centrifuge tube. Add 10 mL of 1× dilution washing solution to the centrifuge tube to resuspend the cells, centrifuge at 250g, room temperature for 10 minutes, and discard the supernatant. Repeat this step 1-2 times and it can be used for subsequent experiments.

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

[0069] After PBMC isolation, RNA was extracted from PBCM and expressed using PrimeScript TM II 1 st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA; PBMC cDNA was used as a template and specific primers were used to PCR amplify the alpaca heavy chain antibody sequence.

[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] The first-round PCR product was then used as a template to amplify the heavy chain antibody VHH fragment using specific primers.

[0073] After amplification, 1% agarose was used for electrophoresis analysis of the PCR product, and the target band (target fragment with a molecular weight of about 450 bp) was recovered using a gel recovery kit and frozen.

[0074] (3) Electroporation of library vector

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

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

[0077] After mixing the linearized vector fragment and the recovered PCR product, add them to the electroporation cup and electroporate; transfer the yeast competent cells after electroporation to the culture bottle and shake and culture at 220rpm and 30℃ for 1h. Take 20μL of the resuspension, dilute it 5000 times with SDCAA, take 100μL, apply it to the SDCAA plate, culture it for 2-3 days, calculate the storage capacity, and continue to culture the rest of the bacterial solution for 24h. Collect the remaining bacterial solution into a 50mL centrifuge tube, centrifuge it at 3000g for 5min, discard the supernatant, add 10mL SDCAA to resuspend it, mix it with 50% glycerol: resuspension = 1:1, and freeze it at -80℃.

[0078] Example 3

[0079] In order to screen alpaca antibodies with high affinity for PD-1, the constructed yeast display library was panned in this example. The panning steps included:

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

[0081] In order to quickly screen alpaca antibodies with high affinity for PD-1, we prepared a yeast display library and screened the library. Take 10mL of yeast library (about 2×10 8 Yeast cells) were transferred to a centrifuge tube and centrifuged at room temperature at 3000 g for 5 min, and the supernatant was discarded; at the same time, sterile 5% PBSA (PBS + 5% BSA) was diluted to 0.5% PBSA; the yeast library was resuspended with 1 mL of 0.5% PBSA, added to the centrifuge tube, and centrifuged at room temperature at 3000 g for 5 min, and the supernatant was discarded; and the solution was washed again with 0.5% PBSA. Prepare three 1.5mL centrifuge tubes (two centrifuge tubes for negative panning of empty magnetic beads, and one centrifuge tube for positive panning), and add 1mL 0.5% PBSA to each centrifuge tube; use a pipette to fully pipette and resuspend the packaged streptavidin magnetic beads, and draw 10μL of magnetic beads into a 1.5mL centrifuge tube; put these centrifuge tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4℃ for 5min; place the centrifuge tube on a magnetic stand for 5min, and remove the supernatant with a pipette; then add 1mL 0.5% PBSA again, rotate and incubate at 4℃ for another 5min, and remove the supernatant.

[0082] (2) Yeast display library screening

[0083] (A) Negative panning with empty magnetic beads

[0084] Since the yeast display library has a certain amount of non-specific adsorption, it is necessary for us to first perform negative panning of the yeast display library with empty magnetic beads. Place the centrifuge tube on the magnetic rack, add the washed yeast solution to the centrifuge tube containing empty magnetic beads, and put it in a bag, and incubate at 4°C for 60 minutes; place the empty magnetic bead centrifuge tube incubated with yeast on the magnetic rack for 10 minutes, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube, being careful not to aspirate the old magnetic beads, and incubate at 4°C for 30 minutes. After the incubation, place it on the magnetic rack for 15 minutes, and carefully aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube.

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

[0086] 100 μL of 50 μg / mL biotin-PD-1-His protein solution (biotin-coupled, diluted with 0.5% PBSA) was added to the centrifuge tube containing streptavidin magnetic beads, the centrifuge tube was placed in a bag, and incubated at 4°C for 60 min; then 1 mL of 0.5% PBSA was added to the bag, and the mixture was allowed to stand at room temperature for 5 min. The centrifuge tube was placed on a magnetic rack during the whole process, and then the supernatant was carefully removed with a pipette, and 1 mL of 0.5% PBSA was added. The centrifuge tube was removed from the magnetic rack, and after mixing with a pipette, the centrifuge tube was placed on the magnetic rack again, and the mixture was allowed to stand at room temperature for 5 min. The centrifuge tube was placed on the magnetic rack during the whole process, and the supernatant was carefully removed with a pipette; the above-mentioned washing steps were repeated once more to obtain positive selection magnetic beads coated with biotin-PD-1-His. Add the positive selection magnetic beads bound to biotin-PD-1-His to the yeast cells that have completed negative selection, put them into a bag and place them on a rotating mixer, incubate them at 4°C for 60 minutes, and after the incubation, place them on a magnetic rack and let them stand at room temperature for 15 minutes; keep the centrifuge tube on the magnetic rack and use a pipette to absorb the unbound yeast solution; remove the 1.5mL centrifuge tube from the magnetic 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 rack, let it stand at room temperature for 5 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 with 1 mL of SDCAA medium, pipette 20 μL of the resuspended solution into 180 μL of SDCAA medium and coat two plates, each with a coating volume of 100 μL, then pipette 5 μL of the resuspended solution into 95 μL of SDCAA medium and coat one plate, i.e., a total of 3 plates need to be coated.

[0087] (C) Flow cytometry of yeast after panning

[0088] After panning, the yeast (the V5 tag in the vector indicates whether the expression vector has been successfully transferred into the yeast) was enriched and analyzed. The yeast that had undergone negative panning with empty magnetic beads and magnetic separation with biotin-PD-1-His was incubated with PD-1 protein containing the His tag at 4°C for 60 minutes, the supernatant was removed by centrifugation, and flow cytometry antibodies with anti-His tag and anti-V5 tag were added. The cells were incubated at 4°C for 60 minutes, the supernatant was removed by centrifugation, and the cells were resuspended in 1mL PBS. The supernatant was removed by centrifugation, and the cells were resuspended in 500μL PBS for flow cytometry analysis.

[0089] Figure 1 Flow cytometry was used to detect the enrichment of yeast library before and after sorting. Figure 1 It can be seen that the yeast capable of binding to PD-1 protein in the yeast antibody display libraries of 98# alpaca and 195# alpaca increased by 38% and 35% respectively before and after sorting, and subsequent experiments can be carried out.

[0090] (3) Monoclonal selection

[0091] After overnight culture, a single clone was picked and inoculated into culture medium and cultured for 48 hours. Flow staining and flow analysis were performed 48 hours later.

[0092] The flow cytometry analysis results showed that the monoclonal number with a high positive rate was 195-1-A07; the monoclonal number 195-1-A07 was amplified by PCR and sequenced.

[0093] Example 4

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

[0095] (1) Recombinant expression

[0096] The candidate 195-1-A07 VHH sequence, CMV promoter sequence, and IgG1Fc sequence were amplified using a PCR instrument; after amplification, the PCR products were subjected to 1% agarose electrophoresis analysis, wherein 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-A07VHH 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 sequence 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 connected using a PCR instrument, and then the PCR product was taken for electrophoresis analysis using 1% agarose, 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 culture supernatant of the harvested cells contained the recombinant single domain antibody 195-1-A07.

[0104] (2) Binding specificity detection

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

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

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

[0108] Figure 2 The results of flow cytometry testing of the specificity of recombinant single domain antibodies. Figure 2 It can be seen that the expression supernatant of the negative control group has no significant binding to CHO-K1 or CHO-K1-PD-1; the positive control antibody has significant binding to CHO-K1-PD-1 but not to CHO-K1 cells, indicating that CHO-K1 cells do not express PD-1 protein, and CHO-K1-PD-1 cells express PD-1 protein, which can be used as detection cells for alpaca antibodies. 195-1-A07 does not bind to CHO-K1 cells, but has significant binding to CHO-K1-PD-1, and can detect most CHO-K1-PD-1 cells expressing PD-1 protein, indicating that 195-1-A07 has good affinity and specificity.

[0109] Example 5

[0110] In this example, the recombinant single domain antibody 195-1-A07 was purified

[0111] (1) The amplified 195-1-A07 sequence and the human IgG1 FC segment were synthesized and cloned into an expression plasmid. The expression plasmid was transiently transfected into 293F cells and cultured in a shake flask for 48 hours, followed by antibody expression and purification.

[0112] (2) 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 30 mL PBS buffer, 0.1 M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20 mg IgG / mL Protein A magnetic beads) to the 293F cell shake flask according to the required sample volume. Incubate at room temperature for 1-4 hours or at 4°C overnight in a shaking incubator at 120 rpm. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50 mL centrifuge tube. Wash twice with 30 mL PBS buffer and deionized water, respectively, and resuspend them with 1 mL elution buffer. After incubation at room temperature for 5 minutes, collect the magnetic beads with a magnetic separation rack and transfer them to a 15 mL centrifuge tube. After eluting the Protein A magnetic beads twice, combine the eluates and add neutralization buffer to adjust the pH of the solution. The eluted sample was dialyzed with PBS at least 100 times the volume of the sample, first dialyzed at 18-25°C for 2 hours, and then dialyzed at 2-8°C for 14-16 hours after changing the solution once. Finally, the protein concentration was determined and the sample was filtered with a 0.22 μm sterile filter membrane and packaged, and stored in a -80°C refrigerator for use.

[0113] Example 6

[0114] In this example, the blocking function of the recombinant single domain antibody 195-1-A07 was tested, and the specific steps included:

[0115] In a 96-well plate, 100 μL of cell suspension containing 2×10 4 Effector cells Jurkat-PD-1-Luciferase (expressing PD-1 molecules, which can inhibit the expression of luciferase after binding to PD-L1) and 8×10 4 Target cells CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of luciferase in Jurkat-PD-1-Luciferase cells). Then, 100 μL of 195-1-A07 of different concentrations was added to the corresponding wells, so that the final concentrations of antibodies in the corresponding wells were 120 μg / mL, 40 μg / mL, 13.3 μg / mL, 4.4 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.16 μg / mL, 0.05 μg / mL, 0.02 μg / mL, and 0 μg / mL, respectively. After 20 hours of co-culture, 20 μL of One-Glo reagent was added to each well, and the luciferase fluorescence value was read.

[0116] Figure 3 This is the blocking function test curve of the recombinant antibody. 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 of 0 μg / mL), indicating that 195-1-A07 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote the expression of luciferase in effector cells, and can be used to develop immune-enhancing drugs.

[0117] Example 7

[0118] In this example, the recombinant single-domain antibody 195-1-A07 was tested for its half effective concentration (EC 50 ) determination, the specific steps include:

[0119] Use coating solution to dilute PD-1 protein to 2μg / mL, pipette 100μL / well into 96-well ELISA plate, and coat overnight at 4℃. Wash 5 times with PBST, add 200μL / well of blocking solution, and block at room temperature for 2 hours. Wash 5 times with PBST, add different concentrations of 195-1-A07 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 incubate at room temperature for 60min. Wash 5 times with PBST, dilute HRP-Protein A at 1:50000, add 100μL / well into ELISA plate, and incubate at room temperature for 45min. Wash 5 times with PBST, add 100μL TMB colorimetric solution to each well, and color at room temperature for 10min in the dark. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[0120] Figure 4 ELISA for detecting EC of recombinant single domain antibodies 50 ; Table 1 is the EC50 test results of 195-1-A07 antibody. Figure 4 It can be seen that the EC of 195-1-A07 for CHO-K1-PD-1 50 The value was 0.2013 μg / mL, indicating that 195-1-A07 had a higher affinity.

[0121] Table 1

[0122]

[0123] Example 8

[0124] In this example, the half inhibitory concentration (IC 50 ) detection, the specific steps include:

[0125] In order to detect the half-inhibitory concentration of 195-1-A07 blocking the binding of PD-1 and PD-L1, the PD-1 protein was diluted to 2 μg / mL with the coating solution, and 100 μL / well was pipetted into a 96-well ELISA plate and coated overnight at 4°C. Wash with PBST 5 times, add 200 μL / well of blocking solution, and block at room temperature for 2 hours. Wash with PBST 5 times, add different concentrations of 195-1-A07 candidate antibodies (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0 μg / mL), and incubate at room temperature for 15 minutes. Add biotin-PD-L1 protein (final concentration 4 μg / mL) and incubate at room temperature for 45 minutes. Wash with PBST 5 times, dilute the secondary antibody (Streptavidin-HRP) at 1:10000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 45 minutes. Wash 5 times with PBST, add 100 μL TMB colorimetric solution to each well and color for 10 min at room temperature in the dark, add 50 μL stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

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

[0127] Table 2

[0128]

[0129]

[0130] In summary, the present 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 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. 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 the CDR1 includes that shown in SEQ ID NO: 1; The amino acid sequence of the CDR2 includes that shown in SEQ ID NO: 2; The amino acid sequence of the 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 according to 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 comprises the full length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof; Preferably, the IgG1 Fc amino acid sequence comprises that shown in SEQ ID NO: 5; Preferably, the amino acid sequence of the heavy chain antibody comprises 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 according to claim 5.

7. A cell expressing the nanobody that specifically binds to PD-1 according to claim 1 or 2 or the heavy chain antibody that specifically binds to PD-1 according to 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 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.

9. A kit for detecting PD-1, characterized in that: The kit comprises 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.

10. Use of the nanobody that specifically binds to PD-1 according to claim 1 or 2, the heavy chain antibody that specifically binds to PD-1 according to claim 3 or 4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6, the cell according to claim 7, the pharmaceutical composition according to claim 8, or the kit for detecting PD-1 according to claim 9 in the preparation of drugs for detecting and / or treating tumors.

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