Nanometer antibody of programmed death receptor 1 as well as preparation method and application of nanometer antibody

Abstract

The invention relates to a nano antibody of a programmed death receptor 1 as well as a preparation method and application of the nano antibody. The nano antibody comprises a heavy chain variable region, an amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence as shown in SEQ ID NO.1, an amino acid sequence of CDR2 of the heavy chain variable region comprises a sequence as shown in SEQ ID NO.2, and an amino acid sequence of CDR3 of the heavy chain variable region comprises a sequence as shown in SEQ ID NO.3. The PD-1 protein is used for immunizing alpaca, an antibody display library is constructed, an antibody combined with the PD-1 protein is screened, the nano antibody of the programmed death receptor 1 with high specificity and affinity is obtained, and the nano antibody has the capacity of blocking combination of PD-1 and PD-L1 and the function of enhancing immune cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies and relates to a nano antibody of programmed death receptor 1 and a preparation method and application thereof. Background Art

[0002] Programmed death 1 (PD-1) is an important immunosuppressive molecule that belongs to the immunoglobulin superfamily. PD-1 is expressed in activated T cells and B cells, and its main function is to inhibit cell activation, which is a normal self-stabilizing mechanism of the immune system, because excessive T / B cell activation can cause autoimmune diseases. After PD-1 binds to its ligands PD-L1 and PD-L2, it can initiate the programmed death of T cells, allowing tumor cells to escape immune defense. In the tumor microenvironment, tumor cells may highly express PD-L1 and PD-L2, resulting in continuous activation of the PD-1 pathway in the tumor microenvironment, inhibition of T cell function, and inability to kill tumor cells. PD-1 antibodies can block this pathway and partially restore the function of T cells, allowing these cells to continue to kill tumor cells. Therefore, anti-programmed death 1 (PD-1) antibodies have the potential to develop drugs to prevent tumor immune escape.

[0003] Anti-PD-1 antibodies are a type of immune checkpoint inhibitor that targets the PD-1 / PD-L1 signaling pathway. They block the binding of PD-1 to its ligand PD-L1, restore T cell activity, and enhance the body's immune response to tumor cells. Currently, a variety of PD-1 / PD-L1 antibody drugs have been approved for marketing worldwide for the treatment of various types of cancer, such as non-small cell lung cancer and melanoma. The clinical application of PD-1 antibodies has achieved significant therapeutic effects, but there are also some problems and challenges, such as: individual differences in efficacy: different patients have significant differences in their response to PD-1 antibody treatment, and only some patients can achieve long-term efficacy; drug resistance: some patients may develop drug resistance after receiving PD-1 antibody treatment, resulting in treatment failure.

[0004] In summary, the development of new anti-PD-1 antibodies and the discovery of effective immune checkpoint inhibitors are of great significance in the field of tumor treatment. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a nano antibody against programmed death receptor 1 and a preparation method and application thereof, develops a new nano antibody against programmed death receptor 1, and provides a new therapeutic strategy for blocking the PD-1 / PD-L1 pathway.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a nanobody against programmed death receptor 1, comprising a heavy chain variable region, wherein the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO.3.

[0008] The present invention utilizes PD-1 protein to immunize alpacas, constructs an antibody display library, screens antibodies that bind to PD-1 protein, obtains nano antibodies to programmed death receptor 1 with high specificity and affinity, and has the ability to block the binding of PD-1 and PD-L1, thereby enhancing the function of immune cells.

[0009] Preferably, the amino acid sequence of the nanobody against programmed death receptor 1 includes the sequence shown in SEQ ID NO.4.

[0010] SEQ ID NO.1: ANAFSKYD.

[0011] SEQ ID NO. 2: LVARITSGLST.

[0012] SEQ ID NO. 3: NREIRGSGIWYPLDY.

[0013] SEQ ID NO.4:

[0014] MAAVQLVDSGGGLVQPGGSLRLSCAASANAFSKYDVGWYRQAPGKQR ELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNRE IRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGHEG.

[0015] In a second aspect, the present invention provides a recombinant programmed death receptor 1 nanobody, wherein the recombinant programmed death receptor 1 nanobody contains a human Fc region and the programmed death receptor 1 nanobody described in the first aspect.

[0016] Preferably, the human Fc region comprises Ig Fc.

[0017] Preferably, the Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

[0018] Preferably, the amino acid sequence of the Fc segment of IgG1 includes the sequence shown in SEQ ID NO.5.

[0019] SEQ ID NO.5:

[0020] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0021] Preferably, the amino acid sequence of the recombinant programmed death receptor 1 nanobody includes the sequence shown in SEQ ID NO.6.

[0022] SEQ ID NO.6:

[0023] MAAVQLVDSGGGLVQPGGSLRLSCAASANAFSKYDVGWYRQAPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGHEGAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0024] In a third aspect, the present invention provides a nucleic acid molecule encoding the Nanobody against programmed death receptor 1 described in the first aspect or the Nanobody against recombinant programmed death receptor 1 described in the second aspect.

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

[0026] In a fifth aspect, the present invention provides a recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in the third aspect.

[0027] In a sixth aspect, the present invention provides a method for preparing the Nanobody of programmed death receptor 1 described in the first aspect or the Nanobody of recombinant programmed death receptor 1 described in the second aspect, the preparation method comprising:

[0028] Inserting the encoding nucleic acid of the Nanobody to the programmed death receptor 1 described in the first aspect or the recombinant Nanobody to the programmed death receptor 1 described in the second aspect into an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a host cell, or inserting the encoding nucleic acid of the Nanobody to the programmed death receptor 1 described in the first aspect or the recombinant Nanobody to the programmed death receptor 1 described in the second aspect into the host cell genome;

[0029] Obtain recombinant cells, culture the recombinant cells, and purify antibodies.

[0030] Preferably, the host cell comprises a mammalian cell.

[0031] In the seventh aspect, the present invention provides the use of the nano antibody against programmed death receptor 1 described in the first aspect, the recombinant nano antibody against programmed death receptor 1 described in the second aspect, the nucleic acid molecule described in the third aspect, the recombinant expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect in the preparation of a preparation binding to programmed death receptor 1.

[0032] The present invention obtains a nano-antibody with high specificity and affinity for programmed death receptor 1, and has the ability to block the binding of PD-1 and PD-L1, enhance the function of immune cells, and can be used as an immune checkpoint inhibitor in tumor treatment, etc.

[0033] Preferably, the preparation that binds to programmed death receptor 1 includes a drug for treating tumors.

[0034] Preferably, the tumor includes at least one of lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer, thyroid cancer, endometrial cancer, brain cancer, pancreatic cancer, nasopharyngeal cancer, cervical cancer, lymphoma, ovarian cancer, bone tumor, melanoma, bladder cancer, prostate cancer or testicular cancer.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] The present invention utilizes PD-1 protein to immunize alpacas, constructs an antibody display library, screens antibodies that bind to PD-1 protein, and obtains nano antibodies with high specificity and affinity for programmed death receptor 1. The nano antibodies have the ability to block the binding of PD-1 and PD-L1, and can be applied to the development of tumor therapeutic drugs. At the same time, nano antibodies are easier to transform, such as humanization, multivalent construction, etc., and have low production costs, and are suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the electrophoresis result of the heavy chain antibody fragment amplified by the first round of PCR;

[0038] Figure 2 This is the electrophoresis result of the second round of PCR amplification of the VHH region fragment;

[0039] Figure 3 This is a graph showing the specificity of nanoantibodies detected by flow cytometry;

[0040] Figure 4 EC for ELISA detection of nanobodies 50 Result graph;

[0041] Figure 5 This is a graph showing the results of the nano-antibody blocking function test;

[0042] Figure 6 IC of Nanobodies for ELISA 50 Result graph. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0044] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0045] In response to the need to develop new and highly effective anti-PD-1 antibodies, the present invention immunizes alpacas to explore new anti-PD-1 nanoantibodies.

[0046] The reagents used in the specific embodiments include:

[0047] Streptavidin PE(eBioscience,CAT#12-4317-87);

[0048] PE-anti-human IgG (eBioscience, CAT#12-4998-82);

[0049] PBS (bico, CAT#14190-250);

[0050] Adjuvant immune adjuvant (GERBU, CAT#3030);

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

[0052] HRP-Streptavidin (Boster, CAT#BA1088);

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

[0054] DNA fragment recovery kit (TakaRa, CAT#9761);

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

[0056] THE TM V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0057] Nivolumab (MedChemExpress, HY-P9903) anti-PD1 flow cytometry antibody (BD Pharmingen, 560779).

[0058] Example 1

[0059] In this example, alpaca immunization and antibody titer determination were performed.

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

[0061] Alpacas were immunized with PD-1 protein (health status of alpacas before immunization: weight about 50-70kg, age about 2-3 years old; normal physical development, good nutritional status and mental state, shiny alpaca back hair; no abnormal movement and behavior, no abnormal limb posture such as lameness and incoordination, alertness and quick reaction; no external trauma). Each time, the alpaca was injected on both sides near the cervical lymph nodes, 2 points on each side, and about 500μg of emulsified antigen was injected at each point. Immunization was performed once every 3 weeks, and 4 immunizations were performed. Peripheral blood was collected before immunization, two weeks after the second immunization, the third immunization, and the fourth immunization, and the upper serum was collected by centrifugation. The binding titer of alpaca serum and PD-1 protein before and after immunization was detected based on the ELISA method.

[0062] The binding titer of serum to PD-1 protein before immunization of alpaca was less than 1000, indicating that the content of anti-PD-1 specific antibodies contained in it was extremely low. On the contrary, the binding titer of serum to PD-1 protein after the fourth immunization of alpaca was more than 32K (thousand), indicating that the content of anti-PD-1 specific antibodies in alpaca serum increased significantly after multiple immunizations, and the immunization was successful, which can be used for the construction of yeast display library.

[0063] Example 2

[0064] In this example, a nanobody yeast display library was constructed and the library was panned.

[0065] (1) Heavy chain variable region (VHH) fragment cloning

[0066] After confirming that the alpaca serum in Example 1 contained anti-PD-1 specific antibodies, peripheral blood was collected, PBMCs were separated, and RNA was extracted. TM II 1 st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, specific primers (upstream primer binds to the signal peptide of VHH antibody ORF, the primer sequence is 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO.7), downstream primer binds to the CH2 region, the primer sequence is 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO.8)) were used to PCR amplify the alpaca heavy chain antibody sequence. The results are shown in Figure 1As shown, 1% agarose was used for electrophoresis analysis of PCR products, and the target fragment with a molecular weight of about 750 bp was recovered and separated. The first round PCR product was then used as a template, and specific primers (the upstream primer was combined with the antibody FR1 region, and the primer sequence was (5'-ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGT CTGG-3' (SEQ ID NO.9); the downstream primer was combined with the antibody Hinge and FR4 regions, and the 5' end contained the SfiI restriction site GGCCACGAAGGCC (SEQ ID NO.10), and the primer sequence was 5'-GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO.11)) to amplify the heavy chain antibody VHH fragment. The results are shown in Figure 2 As shown, 1% agarose was used to perform electrophoresis analysis on the PCR product, and the target fragment with a molecular weight of about 450 bp was separated, indicating that the heavy chain antibody VHH fragment was successfully amplified. The target band was recovered using a gel recovery kit.

[0067] (2) Electroporation of library vectors and detection of library capacity and diversity

[0068] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated using 1% agarose gel, and a 5000bp vector fragment was cut for gel recovery. The yeast competent strain frozen at -80°C was streaked onto a YPD solid culture medium plate in advance and activated at 30°C for 4 days. A single colony of yeast competent strain was inoculated into 50mL YPD culture medium and shaken at 250rpm and 30°C for 2 days. After mixing the linearized vector fragment and the PCR product, add them to the electroporation cup and electroporate; the yeast competent strain after electroporation was transferred to a culture bottle and shaken at 220rpm and 30°C for 1h. Take 20μL of the resuspension, dilute it 5000 times with SDCAA, aspirate 100μL, apply it to the SDCAA plate, culture it for 2 days, calculate the reservoir capacity, and continue to culture the rest of the bacterial solution for 24h. The remaining bacterial liquid was collected into a 50 mL centrifuge tube, centrifuged at 3000 × g for 5 min, the supernatant was discarded, 10 mL SDCAA was added for resuspending, 50% glycerol: resuspending liquid was mixed at a ratio of 1:1, and stored at -80°C. The results showed that the library capacity of the nanoantibody yeast display library obtained from alpaca PBMC was 1.2 × 10 9 After Sanger sequencing, the results showed that the yeast library had large sequence differences, no repeated sequences, good diversity, and could be used for subsequent experiments.

[0069] (3) Panning of yeast display library

[0070] 1) Pretreatment of streptavidin magnetic beads and yeast cells

[0071] In order to screen alpaca antibodies with high affinity for PD-1, a yeast display library was prepared. Take 8 mL of yeast library (about 2×10 8 Yeast cells) were transferred to a 50 mL centrifuge tube, centrifuged at 3000 × g for 5 min, and the supernatant was removed; at the same time, sterile 5% PBSA (PBS + 5% BSA) was diluted to 0.5% PBSA; the yeast library was resuspended with 1 mL 0.5% PBSA, added to a 1.5 mL centrifuge tube, centrifuged at 3000 × g for 5 min, and the supernatant was removed; and 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.

[0072] 2) Yeast display library panning

[0073] In order to screen alpaca antibodies with high affinity for PD-1, the yeast library was screened.

[0074] Negative selection with empty magnetic beads: Place the centrifuge tube on the magnetic rack, add the washed yeast solution to the centrifuge tube containing the empty magnetic beads, put it in a bag, and incubate at 4°C for 60 minutes; Place the empty magnetic bead centrifuge tube with yeast on the magnetic rack for 10 minutes, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube, incubate at 4°C for 30 minutes, and after the incubation, place it on the magnetic rack for 15 minutes, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube.

[0075] Biotin-PD-1-His magnetic separation: add 100 μL of 50 μg / mL Biotin-PD-1-His protein solution (biotin-coupled, diluted with 0.5% PBSA) to a centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 60 minutes; then add 1 mL of 0.5% PBSA, let it stand for 5 minutes, keep the centrifuge tube on the magnetic stand, and use a pipette to remove the supernatant, then add 1 mL of 0.5% PBSA, remove the centrifuge tube from the magnetic stand, mix it with a pipette, place the centrifuge tube on the magnetic stand again, let it stand for 5 minutes, keep the centrifuge tube on the magnetic stand, and use a pipette to remove the supernatant; repeat the above cleaning steps 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 25°C 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 25°C for 5 minutes, and discard the supernatant; repeat the washing twice, and the whole operation is performed in accordance with the aseptic 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.

[0076] 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 selection with Biotin-PD-1-His was incubated with the PD-1 protein containing the His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, added flow antibodies with anti-His tags and anti-V5 tags, incubated at 4°C for 60 minutes, then centrifuged to remove the supernatant, resuspended the cells in 1mL PBS, then centrifuged to remove the supernatant, resuspended the cells in 500μL PBS, and analyzed by flow cytometry. The number of yeast that can bind to the PD-1 protein in the yeast antibody display library of the alpaca increased by 38% and 35% before and after sorting, respectively, and subsequent experiments can be carried out.

[0077] Monoclonal selection: After overnight culture, monoclones were picked and inoculated into the culture medium to induce expression. After 48 hours, the bacterial solution was incubated with the PD-1 protein containing the His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, and the precipitate was resuspended with a solution containing a biotin-conjugated anti-His tag flow antibody, incubated at 4°C for 60 minutes, then centrifuged to remove the supernatant, and the precipitate was resuspended with a solution containing PE-Streptavidin (phycoerythrin-labeled streptavidin), incubated at 4°C for 60 minutes, resuspended with 1mL PBS, then centrifuged to remove the supernatant, and resuspended with 500μL PBS for flow analysis. Finally, a monoclone with a high positive rate was selected and named 195-4-D3. 195-4-D3 was lysed with 0.2% SDS (incubated at 95°C for 10 minutes), centrifuged, and 0.5μL of the bacterial solution supernatant was taken as a template for PCR amplification and testing.

[0078] Further analysis of the antibody sequence information showed that the CDR1 region sequence of Nanobody 195-4-D3 was shown in SEQ ID NO.1, the CDR2 region sequence was shown in SEQ ID NO.2, the CDR3 region sequence was shown in SEQ ID NO.3, and the overall sequence was shown in SEQ ID NO.4.

[0079] SEQ ID NO.1: ANAFSKYD.

[0080] SEQ ID NO. 2: LVARITSGLST.

[0081] SEQ ID NO. 3: NREIRGSGIWYPLDY.

[0082] SEQ ID NO.4:

[0083] MAAVQLVDSGGGLVQPGGSLRLSCAASANAFSKYDVGWYRQAPGKQR ELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNRE IRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGHEG.

[0084] Example 3

[0085] This embodiment performs the expression of recombinant nanoantibodies and the detection of binding to target proteins.

[0086] Design a recombinant antibody containing nanobody 195-4-D3 and IgG1 Fc region (SEQ ID NO.6), PCR amplify 195-4-D3 VHH nucleic acid sequence, CMV promoter sequence and IgG1 Fc nucleic acid sequence, take 50 μL PCR product, add 1 / 10 volume 10× loading buffer (loading buffer), use 1% agarose for electrophoresis analysis, CMV band size is 750bp, Fc band size is 1400bp, VHH band size is 500bp. Cut the target band from the gel and purify the PCR product. The 195-4-D3 VHH sequence, CMV promoter and IgG1 Fc sequence were connected, and then 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The target band was cut out from the gel, and the PCR product was purified. After the obtained PCR product was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant nanoantibody 195-4-D3.

[0087] The binding specificity of 195-4-D3 was tested. The culture supernatant containing 195-4-D3 was mixed with 3×10 5 CHO-K1 and CHO-K1-PD-1 cells were incubated at 25°C for 1 hour. The supernatant was discarded after centrifugation, and the cells were washed 3 times with PBS. 100 μl of PE-labeled Anti-human IgG antibody (1:500 dilution) was added, incubated at 25°C in the dark for 45 minutes, centrifuged at 800×g at 25°C for 5 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. The cells were resuspended in PBS and analyzed by flow cytometry. The alpaca antibody that does not bind to PD1 was used as a negative control, and the anti-PD1 flow cytometry antibody was used as a positive control.

[0088] The results are as follows Figure 3 As shown, the expression supernatant of the negative control group did not significantly bind to CHO-K1 or CHO-K1-PD-1; the positive control antibody 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. 195-4-D3 did not bind to CHO-K1 cells, but significantly bound to CHO-K1-PD-1, and was able to detect most CHO-K1-PD-1 cells expressing PD-1 protein, indicating that the nanoantibody 195-4-D3 had good affinity and specificity.

[0089] Example 4

[0090] This study determined the purification and half effective concentration (EC) of nanobodies. 50).

[0091] To determine the EC of the nanobody 195-4-D3 50 The expression plasmid was transiently transfected into 293F cells and cultured in a shaking flask to express and purify the antibody.

[0092] 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 25℃ for 2h. Wash 5 times with PBST, add different concentrations of 195-4-D3 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 25℃ for 60min. Wash 5 times with PBST, dilute HRP-Protein A at 1:50000, add 100μL / well into ELISA plate, and incubate at 25℃ for 45min. Wash 5 times with PBST, add 100μL TMB colorimetric solution to each well, and color at 25℃ in the dark for 10min. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[0093] The results are shown in Table 1 and Figure 4 As shown, the EC value of 195-4-D3 for CHO-K1-PD-1 50 The value was 0.062 μg / mL, indicating that 195-4-D3 had a higher affinity.

[0094] Table 1

[0095]

[0096] Example 5

[0097] This example performs antibody blocking function detection.

[0098] After binding to PD-1, PD-L1 can induce T cell apoptosis, dysfunction, and exhaustion, thereby inhibiting the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+T cells, thereby achieving tumor immune escape. In order to detect the function of 195-4-D3 in blocking the binding of PD-1 and PD-L1, 100 μL of cell suspension was added to a 96-well plate, and each well contained 2×10 4 Effector cells Jurkat-PD-1-Luciferase (expressing PD-1 molecules, which can inhibit the expression of luciferase fluorescent protein after binding to PD-L1) and 8×10 4Target cells CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of Luciferase fluorescent protein in Jurkat-PD-1-Luciferase cells). Then, 100 μL of 195-4-D3 of different concentrations was added to the corresponding wells, so that the final concentrations in the corresponding wells were 120 μg / mL, 40 μg / mL, 13.3 μg / mL, 4.4 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.16 μg / mL, 0.05 μg / mL, 0.02 μg / mL and 0 μg / mL. After 18 hours of co-culture, 20 μL of Ne-Glo reagent was added to each well, and the Luciferase fluorescence value was read.

[0099] result Figure 5 As shown, the maximum induction effect value of 195-4-D3 is 1.56 (the difference between the maximum Luciferase fluorescence value and the 0 μg / mL Luciferase fluorescence value), indicating that 195-4-D3 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.

[0100] Example 6

[0101] In this example, the half inhibitory concentration (IC 50 )experiment.

[0102] IC50 refers to the concentration of the inhibitor when the "reaction" is inhibited by half. The reaction here can be an enzyme-catalyzed reaction, an antigen-antibody reaction, etc. IC50 reflects the inhibitory ability of PD-1 antibodies. In order to detect the half-inhibitory concentration of 195-4-D3 blocking the binding of PD-1 and PD-L1, the 0.2μg / well PD-1 protease plate pre-coated with PBST was washed twice, and then different concentrations (25μg / mL, 8.3μg / mL, 2.8μg / mL, 0.9μg / mL, 0.3μg / mL, 0μg / mL) of 195-4-D3 candidate antibodies and anti-PD-1 positive control antibody Nivolumab were added, and incubated at 25℃ for 20min. Biotin-PD-L1 protein (final concentration 4μg / mL) was added and incubated at 25℃ for 40min. Wash 5 times with PBST, dilute the secondary antibody (streptavidin-horseradish peroxidase, HRP-Streptavidin) at 1:10000, add 100 μL / well to the ELISA plate, and incubate at 25°C for 40 minutes. Wash 5 times with PBST, add 100 μL TMB colorimetric solution to each well and color at 25°C in the dark for 12 minutes. Add 50 μL stop solution to each well, and read the absorbance at 450 nm on the ELISA reader.

[0103] The results are shown in Table 2 and Figure 6 As shown, the IC of 195-4-D3 50 The value was 0.8 μg / mL, indicating that 195-4-D3 could effectively block the binding of PD-1 and PD-L1 proteins.

[0104] Table 2

[0105]

[0106] In summary, the present invention explores a new type of anti-PD-1 nanoantibody that can bind to PD-1 with high specificity and can effectively block the binding of PD-1 and PD-L1, and can be used in the development of tumor treatment drugs.

[0107] 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 against programmed death receptor 1, characterized in that: The nanobody comprises a heavy chain variable region, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO.

3.

2. The nanobody of programmed death receptor 1 according to claim 1, characterized in that The amino acid sequence of the nanobody against programmed death receptor 1 includes the sequence shown in SEQ ID NO.

4.

3. A recombinant programmed death receptor 1 nanobody, characterized in that: The recombinant programmed death receptor 1 nanobody contains a human Fc region and the programmed death receptor 1 nanobody according to claim 1 or 2.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody against programmed death receptor 1 according to claim 1 or 2 or the Nanobody against recombinant programmed death receptor 1 according to claim 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 4.

6. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule of claim 4.

7. A method for preparing the nanobody of programmed death receptor 1 according to claim 1 or 2 or the nanobody of recombinant programmed death receptor 1 according to claim 3, characterized in that: The preparation method comprises: Inserting the encoding nucleic acid of the Nanobody to the programmed death receptor 1 according to claim 1 or 2 or the recombinant Nanobody to the programmed death receptor 1 according to claim 3 into an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a host cell, or inserting the encoding nucleic acid of the Nanobody to the programmed death receptor 1 according to claim 1 or 2 or the recombinant Nanobody to the programmed death receptor 1 according to claim 3 into the host cell genome; Obtain recombinant cells, culture the recombinant cells, and purify antibodies.

8. The method for preparing a nanobody against programmed death receptor 1 according to claim 7, characterized in that: The host cells include mammalian cells.

9. Use of the Nanobody against programmed death receptor 1 according to claim 1 or 2, the Nanobody against recombinant programmed death receptor 1 according to claim 3, the nucleic acid molecule according to claim 4, the recombinant expression vector according to claim 5 or the recombinant cell according to claim 6 in the preparation of a preparation that binds to programmed death receptor 1.

10. The use according to claim 9, characterized in that: The preparations binding to programmed death receptor 1 include drugs for treating tumors; Preferably, the tumor includes at least one of lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer, thyroid cancer, endometrial cancer, brain cancer, pancreatic cancer, nasopharyngeal cancer, cervical cancer, lymphoma, ovarian cancer, bone tumor, melanoma, bladder cancer, prostate cancer or testicular cancer.

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