Nanobody of programmed death receptor 1 and preparation method and application thereof

Abstract

The present application relates to a kind of nanobody of programmed death receptor 1 and its preparation method and application.The nanobody includes heavy chain variable region, the amino acid sequence of CDR1 of the heavy chain variable region includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID NO.2, the amino acid sequence of CDR3 includes the sequence shown in SEQ ID NO.3.The present application utilizes PD-1 protein immunization alpaca, constructs antibody display library, screens the antibody binding PD-1 protein, obtains the nanobody of programmed death receptor 1 with high specificity and affinity, and has the ability to block the combination of PD-1 and PD-L1, enhances the function of immune cell.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibodies, and relates to a nanobody of a programmed death receptor 1 and a preparation method and application thereof. BACKGROUND

[0002] Programmed death molecule 1 (PD-1) is an important immunosuppressive molecule belonging to the immunoglobulin superfamily. PD-1 is expressed in activated T cells and B cells, and mainly functions to inhibit the activation of cells, 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, the programmed death of T cells can be initiated, and tumor cells can obtain immune escape. In the tumor microenvironment, tumor cells can highly express PD-L1 and PD-L2, leading to continuous activation of the PD-1 pathway in the tumor microenvironment, and the function of T cells is inhibited and cannot kill tumor cells. The antibody of PD-1 can block this pathway, partially restore the function of T cells, so that these cells can continue to kill tumor cells, and therefore, the anti-programmed death molecule 1 (PD-1) antibody has the potential to develop drugs to prevent tumor immune escape.

[0003] Anti-PD-1 antibody is a kind of immune checkpoint inhibitor targeting PD-1 / PD-L1 signal pathway, which restores the activity of T cells by blocking the binding of PD-1 and its ligand PD-L1, and enhances the immune response of the body to tumor cells. At present, a variety of PD-1 / PD-L1 antibody drugs have been approved for marketing worldwide, which are used for treating various types of cancer, such as non-small cell lung cancer, melanoma, etc. The application of PD-1 antibody in clinic has achieved remarkable therapeutic effect, but at the same time, there are some problems and challenges, for example: individual difference of curative effect: there is a significant difference in the treatment response of different patients to PD-1 antibody, only part of the patients can obtain long-term curative effect; drug resistance problem: some patients may develop drug resistance after receiving PD-1 antibody treatment, leading to treatment failure.

[0004] In summary, developing new anti-PD-1 antibodies and exploring effective immune checkpoint inhibitors are of great significance to the field of tumor treatment. SUMMARY

[0005] In view of the deficiencies of the prior art and actual needs, the application provides a nanobody of a programmed death receptor 1 and a preparation method and application thereof, and develops a new nanobody of a programmed death receptor 1, thereby providing a new treatment strategy for blocking the PD-1 / PD-L1 pathway.

[0006] To achieve the above purpose, the application adopts the following technical solutions:

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

[0008] The present application uses PD-1 protein to immunize a llama, constructs an antibody display library, screens an antibody binding to PD-1 protein, and obtains a nanobody of programmed death receptor 1 with high specificity and affinity, and the ability to block the combination of PD-1 and PD-L1, and enhance the function of immune cells.

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

[0010] SEQ ID NO. 1: ANAFSKYD.

[0011] SEQ ID NO. 2: LVARITSGLST.

[0012] SEQ ID NO. 3: NREIRGSSGIWYPLDY.

[0013] SEQ ID NO. 4:

[0014] MAAVQLVDSGGGLVQPGGSLRLSCAASANAFSKYDVGWYRQAPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGHEG.

[0015] In a second aspect, the present application provides a recombinant nanobody of programmed death receptor 1, which comprises a human Fc region and the nanobody of programmed death receptor 1 in the first aspect.

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

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

[0018] Preferably, the Fc segment of IgG1 comprises a sequence shown in SEQ ID NO. 5.

[0019] SEQ ID NO. 5:

[0020] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0021] Preferably, the amino acid sequence of the recombinant Nanobody of Programmed Death Receptor 1 comprises the sequence set forth in SEQ ID NO. 6.

[0022] SEQ ID NO. 6:

[0023] MAAVQLVDSGGGLVQPGGSLRLSCAASANAFSKYDVGWYRQAPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGHEGAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0024] In a third aspect, the present application provides a nucleic acid molecule encoding the Nanobody of Programmed Death Receptor 1 of the first aspect or the recombinant Nanobody of Programmed Death Receptor 1 of the second aspect.

[0025] In a fourth aspect, the present application provides a recombinant expression vector comprising the nucleic acid molecule of the second aspect.

[0026] In a fifth aspect, the present application provides a recombinant cell comprising the nucleic acid molecule of the third aspect.

[0027] In a sixth aspect, the present application provides a method for preparing the Nanobody of Programmed Death Receptor 1 of the first aspect or the recombinant Nanobody of Programmed Death Receptor 1 of the second aspect, the method comprising:

[0028] inserting the nucleic acid encoding the Nanobody of Programmed Death Receptor 1 of the first aspect or the recombinant Nanobody of Programmed Death Receptor 1 of the second aspect into an expression vector to obtain a recombinant vector, introducing the recombinant vector into a host cell, or inserting the nucleic acid encoding the Nanobody of Programmed Death Receptor 1 of the first aspect or the recombinant Nanobody of Programmed Death Receptor 1 of the second aspect into the genome of a host cell;

[0029] obtaining a recombinant cell, culturing the recombinant cell, and purifying the antibody.

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

[0031] In a seventh aspect, the present application provides use of the Nanobody of Programmed Death Receptor 1 of the first aspect, the recombinant Nanobody of Programmed Death Receptor 1 of the second aspect, the nucleic acid molecule of the third aspect, the recombinant expression vector of the fourth aspect, or the recombinant cell of the fifth aspect in the preparation of a preparation that binds to Programmed Death Receptor 1.

[0032] The present application mines the Nanobody of Programmed Death Receptor 1 with high specificity and affinity, 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 for tumor treatment, etc.

[0033] Preferably, the preparation that binds to Programmed Death Receptor 1 comprises a drug for treating tumors.

[0034] Preferably, the tumor comprises 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 application has at least the following beneficial effects:

[0036] The application utilizes the PD-1 protein to immunize the llama, constructs an antibody display library, screens the antibody combined with the PD-1 protein, obtains the nanobody of the programmed death receptor 1 with high specificity and affinity, and has the ability to block the combination of the PD-1 and the PD-L1, can be applied to the development of tumor treatment drugs, meanwhile, the nanobody is easier to be reformed, such as humanization, multivalent construction, etc., and has low production cost, is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

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

[0038] Figure 2 It is a result graph of electrophoresis of the VHH region fragment amplified by the second round of PCR;

[0039] Figure 3 It is a result graph of flow cytometry detection of the specificity of the nanobody;

[0040] Figure 4 It is a result graph of ELISA detection of the EC of the nanobody; 50 Result graph;

[0041] Figure 5 It is a result graph of nanobody blocking function detection;

[0042] Figure 6 It is a result graph of ELISA detection of the IC of the nanobody. 50 Result graph. DETAILED DESCRIPTION

[0043] The technical solutions of the application will be further described below by combining the drawings and through specific embodiments. However, the following examples are only simple examples of the application, and do not represent or limit the protection scope of the application, and the protection scope of the application is subject to the claims.

[0044] If the specific technology or condition is not indicated in the examples, the technology or condition described in the literature in the field or the product instruction is used. If the used reagent or instrument is not indicated by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0045] The application aims at the demand of developing new high-efficiency anti-PD-1 antibody, and immunizes the llama to mine new anti-PD-1 nanobody.

[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 immunoadjuvant (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 (KPL, CAT#A01805);

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

[0058] Example 1

[0059] This example carries out the immunization of Llama and the determination of antibody titer.

[0060] (1) Llama immunization and serum titer detection

[0061] The llama (the health condition of the llama before immunization: the weight is about 50-70 kg, the age is about 2-3 years old; the physical development is normal, the nutritional status and the mental state are good, the llama back hair is bright; the movement and behavior are normal, there is no abnormality such as lameness, uncoordination and the like, there is alertness and rapid response; there is no external observation of external trauma) is immunized by using the PD-1 protein. Each time, the llama is injected in the left and right sides near the cervical lymph nodes, each side is divided into 2 points, each point is injected about 500 μg of the emulsified antigen, the immunization is performed once every 3 weeks, and the immunization is performed 4 times. The peripheral blood is collected before immunization, after the second immunization, the third immunization and the fourth immunization for two weeks, the upper serum is collected by centrifugation. The binding titer of the serum of the llama before and after immunization with the PD-1 protein is detected based on the ELISA method.

[0062] The binding titer of the serum of the llama before immunization with the PD-1 protein is less than 1000, which indicates that the content of the anti-PD-1 specific antibody contained therein is extremely low. On the contrary, the binding titer of the serum of the llama after the fourth immunization with the PD-1 protein is more than 32K (thousand), which indicates that the content of the anti-PD-1 specific antibody in the serum of the llama is significantly improved after multiple immunizations, the immunization is successful, and the llama can be used for the construction of the yeast display library.

[0063] Example 2

[0064] In this embodiment, the nanobody yeast display library is constructed and the library is screened.

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

[0066] After confirming that the serum of the llama in Example 1 contains the anti-PD-1 specific antibody, the peripheral blood is collected, the PBMC is separated, and the RNA is extracted. The PrimeScript TM II 1 st Strand cDNA Synthesis Kit is used for reverse transcription to obtain the cDNA. The llama heavy chain antibody sequence is amplified by PCR with the PBMC cDNA as the template and specific primers (the upstream primer is combined with the signal peptide of the VHH antibody ORF, the primer sequence is 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO. 7), and the downstream primer is combined with the CH2 region, the primer sequence is 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO. 8)). The results are as follows: Figure 1The PCR products were analyzed by electrophoresis using 1% agarose gel, and the target fragment with a molecular weight of about 750 bp was recovered. The first round of PCR products were used as templates to amplify the heavy chain antibody VHH fragment using specific primers (the upstream primer was bound to the antibody FR1 region, and the primer sequence was (5'- ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGT CTGG-3' (SEQ ID NO. 9); the downstream primer was bound to the antibody Hinge and FR4 region, and the 5' end contained the Sfil enzyme cutting site GGCCACGAAGGCC (SEQ ID NO. 10), and the primer sequence was 5'-GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO. 11)). The results are shown in Figure 2. Figure 2 As shown in Figure 2, the PCR products were analyzed by electrophoresis using 1% agarose gel, 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 subjected to single enzyme digestion using Sfil endonuclease. The pYDisplay vector fragment was separated using 1% agarose gel, and the 5000 bp vector fragment was cut and recovered. The yeast competent strain stored at -80°C was streaked onto YPD solid medium plates and activated at 30°C for 4 days. Single colony yeast competent was inoculated into 50 mL YPD medium, and incubated at 250 rpm and 30°C for 2 days. After mixing the linearized vector fragment and the PCR product, the mixture was added to an electroporation cup and subjected to electroporation. The yeast competent after electroporation was transferred to a culture bottle and incubated at 220 rpm and 30°C for 1 h. 20 μL of the resuspended solution was diluted 5000-fold with SDCAA, 100 μL of the diluted solution was taken and plated on SDCAA plates, and incubated for 2 days. The library capacity was calculated, and the remaining bacterial solution was further incubated for 24 h. The remaining bacterial solution was collected into a 50 mL centrifuge tube, centrifuged at 3000 x g for 5 min, and the supernatant was discarded. 10 mL of SDCAA was added for resuspension, and 50% glycerol was added to the resuspension solution at a ratio of 1:1, and the mixture was stored at -80°C. The results showed that the library capacity of the nanobody yeast display library obtained from the PBMC of a llama was 1.2 x 10 9 Sanger sequencing showed that the sequences of the yeast library were different, there were no repeated sequences, and the diversity was good, which could be used for subsequent experiments.

[0069] (3) Screening of the yeast display library

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

[0071] To screen for alpaca antibodies with high affinity for PD-1, a yeast display library was prepared. 8 mL of the yeast library (approximately 2 × 10⁻⁶ cells / mL) was used. 8 Add yeast cells to a 50 mL centrifuge tube, centrifuge at 3000×g for 5 min, 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 1.5 mL centrifuge tube, centrifuge at 3000×g for 5 min, and discard the supernatant; wash again with 0.5% PBSA. Prepare three 1.5 mL centrifuge tubes (two for negative panning of 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 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.

[0072] 2) Yeast display library selection

[0073] To screen for alpaca antibodies with high affinity for PD-1, a yeast library was screened.

[0074] Negative selection with empty magnetic beads: 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 minutes. Place the centrifuge tube containing empty magnetic beads with yeast culture on a magnetic rack for 10 minutes, then add the yeast culture to a new centrifuge tube containing empty magnetic beads. Incubate at 4°C for 30 minutes. After incubation, place the tube on a magnetic rack for 15 minutes, then add the yeast culture to a new centrifuge tube containing empty magnetic beads.

[0075] Biotin-PD-1-His magnetic sorting: 100 μL of 50 μg / mL Biotin-PD-1-His protein solution (biotin coupling, 0.5% PBSA dilution) was added to a centrifuge tube containing streptavidin magnetic beads, the centrifuge tube was placed in a bag, and 4°C rotation incubation was performed for 60 min; then 1 mL of 0.5% PBSA was added, and the centrifuge tube was allowed to stand for 5 min, and the supernatant was removed with a pipette, and then 1 mL of 0.5% PBSA was added, the centrifuge tube was removed from the magnetic stand, and the mixture was mixed by pipetting, and then the centrifuge tube was placed on the magnetic stand again, and the supernatant was removed with a pipette; the above washing step was repeated once again, and the Biotin-PD-1-His coated positive selection magnetic beads were obtained. The Biotin-PD-1-His combined positive selection magnetic beads were added to the yeast cells after negative selection, and the mixture was placed in a bag and placed on a rotation mixer at 4°C for 60 min. After incubation, the mixture was placed on a magnetic stand and allowed to stand at 25°C for 15 min. The centrifuge tube was kept on the magnetic stand, and the unbound yeast solution was removed with a pipette. The 1.5 mL centrifuge tube was removed from the magnetic stand, and 1 mL of sterile 0.5% PBSA buffer was added to the centrifuge tube with a pipette. The magnetic beads were gently blown and then transferred to a sterile 1.5 mL centrifuge tube. The centrifuge tube was placed on the magnetic stand and allowed to stand at 25°C for 5 min. The supernatant was discarded. The washing step was repeated twice, and the whole process was performed according to the sterile operation requirements. After washing, the magnetic beads and adhered yeast cells were resuspended in 1 mL of SDCAA medium, and 20 μL of the resuspension was taken to 180 μL of SDCAA medium for plating on two plates, each with a volume of 100 μL. Five μL of the resuspension was taken to 95 μL of SDCAA medium for plating on one plate, for a total of three plates.

[0076] Flow cytometry detection of yeast 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 subjected to negative selection with empty magnetic beads and Biotin-PD-1-His magnetic sorting was incubated with PD-1 protein containing His tag at 4°C for 60 min, followed by centrifugation to remove the supernatant. Anti-His tag and anti-V5 tag flow cytometry antibodies were added, and the mixture was incubated at 4°C for 60 min. The supernatant was removed by centrifugation, and the cells were resuspended in 1 mL of PBS. The supernatant was removed by centrifugation, and the cells were resuspended in 500 μL of PBS. Flow cytometry analysis was performed. The number of yeast in the llama's yeast antibody display library that could bind to PD-1 protein increased by 38% and 35% before and after sorting, respectively, and subsequent experiments could be performed.

[0077] Monoclonal picking: After overnight culture, single clone was picked and inoculated into culture medium to induce expression. After 48h, the bacteria solution was incubated with His-tagged PD-1 protein at 4℃ for 60min. Then the supernatant was removed by centrifugation, the precipitate was resuspended with solution containing biotin-conjugated anti-His tag flow antibody, incubated at 4℃ for 60min, then the supernatant was removed by centrifugation, the precipitate was resuspended with solution containing PE-Streptavidin, incubated at 4℃ for 60min, 1mL PBS was used to resuspend the cells, then the supernatant was removed by centrifugation, 500μL PBS was used to resuspend the cells, and flow cytometry analysis was performed. Finally, a single clone with high positive rate was selected, named 195-4-D3. 195-4-D3 was lysed using 0.2% SDS (incubated at 95℃ for 10min), centrifuged, and 0.5μL of the bacterial solution supernatant was used as a template for PCR amplification and sequencing.

[0078] Further analysis of the sequence information of the antibody, the CDR1 region sequence of nanobody 195-4-D3 is shown as SEQ ID NO. 1, the CDR2 region sequence is shown as SEQ ID NO. 2, the CDR3 region sequence is shown as SEQ ID NO. 3, and the overall sequence is shown as SEQ ID NO. 4.

[0079] SEQ ID NO. 1: ANAFSKYD.

[0080] SEQ ID NO. 2: LVARITSGLST.

[0081] SEQ ID NO. 3: NREIRGSSGIWYPLDY.

[0082] SEQ ID NO. 4:

[0083] MAAVQLVDSGGGLVQPGGSLRLSCAASANAFSKYDVGWYRQAPGKQR ELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNRE IRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASGHEG.

[0084] Example 3

[0085] This embodiment carries out expression of recombinant nanobody and detection of binding with target protein.

[0086] The recombinant antibody containing the nanobody 195-4-D3 and IgG1 Fc region (SEQ ID NO. 6) was designed, the 195-4-D3 VHH nucleic acid sequence, CMV promoter sequence and IgG1 Fc nucleic acid sequence were PCR amplified, 50 μL of the PCR product was added with 1 / 10 volume of 10x loading buffer, and electrophoresis analysis was performed using 1% agarose, the band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band size of VHH was 500 bp. The target band was cut from the gel, and the PCR product was purified. The 195-4-D3 VHH sequence, CMV promoter and IgG1 Fc sequence were ligated, and then 50 μL of the PCR product was added with 1 / 10 volume of 10x loading buffer, and electrophoresis analysis was performed 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 harvested cell culture supernatant contained the recombinant nanobody 195-4-D3.

[0087] The binding specificity of 195-4-D3 was detected. The culture supernatant containing 195-4-D3 was incubated with 3x10 5 CHO-K1 and CHO-K1-PD-1 cells for 1 h at 25°C, respectively. The supernatant was discarded by centrifugation, and the cells were washed with PBS for 3 times. 100 μl of PE-labeled Anti-human IgG antibody (1:500 dilution) was added, and the cells were incubated at 25°C for 45 min in the dark. After centrifugation at 800xg for 5 min at 25°C, the supernatant was discarded, and the cells were washed with PBS for 3 times. The cells were resuspended with PBS, and flow cytometry analysis was performed. A llama antibody not binding to PD1 was used as a negative control, and a flow cytometry antibody against PD1 was used as a positive control.

[0088] The results are shown in Figure 3 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 the CHO-K1 cells did not express PD-1 protein, and the CHO-K1-PD-1 cells expressed PD-1 protein, which could be used as a detection cell for the llama antibody. 195-4-D3 did not bind to CHO-K1 cells, but significantly bound to CHO-K1-PD-1, and most of the CHO-K1-PD-1 cells expressed PD-1 protein, indicating that the nanobody 195-4-D3 had good affinity and specificity.

[0089] Example 4

[0090] In this embodiment, the purification and half maximal effective concentration (EC 50).

[0091] To determine the EC50 of the Nanobody 195-4-D3 antibody, the following experiment was performed. 50 The expression plasmid was transiently transfected into 293F cells, which were cultured in a shaker flask, to express and purify the antibody.

[0092] PD-1 protein was diluted to 2 μg / mL with coating solution, 100 μL / well was taken to a 96-well enzyme-labeled plate, and coated at 4°C overnight. PBST was washed 5 times, 200 μL / well of blocking solution was added, and blocked at 25°C for 2 h. PBST was washed 5 times, and different concentrations of 195-4-D3 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) were added, and incubated at 25°C for 60 min. PBST was washed 5 times, HRP-Protein A was diluted 1:50000, 100 μL / well was added to the enzyme-labeled plate, and incubated at 25°C for 45 min. PBST was washed 5 times, 100 μL of TMB color developing solution was added to each well, and developed at 25°C for 10 min in the dark. 50 μL of stop solution was added to each well, and the absorbance was read on an enzyme-labeled instrument at a wavelength of 450 nm.

[0093] The results are shown in Table 1 and Figure 4 The EC50 of 195-4-D3 against CHO-K1-PD-1 was 0.062 μg / mL, indicating that 195-4-D3 has high affinity. 50

[0094] Table 1

[0095]

[0096] Example 5

[0097] This example detects the blocking function of the antibody.

[0098] PD-L1 can induce T cell apoptosis, dysfunction, and exhaustion after binding to PD-1, thereby inhibiting the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+ T cells, and achieving tumor immune escape. To detect the function of 195-4-D3 in blocking the binding of PD-1 to PD-L1, 100 μL of cell suspension was added to a 96-well plate, including 2×10 4 effector cells Jurkat-PD-1-Luciferase (expressing PD-1 molecules, which can inhibit the expression of luciferase (Luciferase) fluorescent protein after binding to PD-L1) and 8×10 4 ​Target cell CHO-K1-PD-L1 (express PD-L1 molecule, can bind with PD-1, inhibit the expression of Jurkat-PD-1-Luciferase Luciferase fluorescent protein). Then add 100 μL of different concentrations of 195-4-D3 to the corresponding wells, so that the final concentration in the corresponding wells is 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 18 h of co-culture, add 20 μL One-Glo reagent to each well and read the Luciferase fluorescence value.

[0099] Results 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 Luciferase fluorescence value at 0 μg / mL), indicating that 195-4-D3 has 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 immune-enhancing drugs.

[0100] Example 6

[0101] This example carries out a semi-inhibitory concentration (IC50) experiment of recombinant antibody. 50 ) experiment.

[0102] IC50 refers to the concentration of an inhibitor when a "reaction" is inhibited by half, where the reaction can be an enzyme catalytic reaction, an antigen-antibody reaction, etc. IC50 reflects the inhibitory ability of PD-1 antibody. In order to detect the semi-inhibitory concentration of 195-4-D3 in blocking the binding of PD-1 and PD-L1, the 0.2 μg / well PD-1 protein-coated plate was washed with PBST twice in advance, and then different concentrations (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0 μg / mL) of 195-4-D3 candidate antibodies and anti-PD-1 positive control antibody Nivolumab were added, and incubated at 25°C for 20 min. Biotin-PD-L1 protein (final concentration 4 μg / mL) was added and incubated at 25°C for 40 min. PBST was washed 5 times, the secondary antibody (streptavidin-horseradish peroxidase, HRP-Streptavidin) was diluted at 1:10000, 100 μL / well was added to the enzyme-labeled plate, and incubated at 25°C for 40 min. PBST was washed 5 times, 100 μL TMB color developing solution was added to each well, and color development was carried out at 25°C for 12 min in the dark. 50 μL of stop solution was added to each well, and the absorbance was read on an enzyme-labeled instrument at a wavelength of 450 nm.

[0103] Results are shown in Table 2 and Figure 6 The IC50 value of 195-4-D3 is 0.8 μg / mL, indicating that 195-4-D3 can efficiently block the binding of PD-1 and PD-L1 proteins. 50 Results are shown in Table 2 and

[0104] Table 2

[0105]

[0106] In summary, the present application mines a new anti-PD-1 nanobody, which can specifically bind to PD-1 and efficiently block the binding of PD-1 and PD-L1, and can be applied to the development of tumor treatment drugs.

[0107] The applicant states that the above description is merely a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily conceived by those skilled in the art, and all such changes or replacements fall within the protection scope and disclosure scope of the present application.

Claims

1. A nanobody of programmed death receptor 1, characterized in that, The amino acid sequence of the programmed death receptor 1 nanobody is shown in SEQ ID NO.

4.

2. A nanobody of recombinant programmed death receptor 1, characterized in that, The recombinant programmed death receptor 1 nanobody contains a human Fc region and the programmed death receptor 1 nanobody as described in claim 1.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody of programmed death receptor 1 as described in claim 1 or the recombinant programmed death receptor 1 nanobody as described in claim 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecules as described in claim 3.

6. A method for preparing a nanobody of programmed death receptor 1 as described in claim 1 or a recombinant nanobody of programmed death receptor 1 as described in claim 2, characterized in that, The preparation method includes: The encoding nucleic acid of the programmed death receptor 1 nanobody of claim 1 or the recombinant programmed death receptor 1 nanobody of claim 2 is inserted into the expression vector to obtain a recombinant vector, and the recombinant vector is introduced into the host cell; or, the encoding nucleic acid of the programmed death receptor 1 nanobody of claim 1 or the recombinant programmed death receptor 1 nanobody of claim 2 is inserted into the genome of the host cell. Recombinant cells were obtained, cultured, and then the antibodies were purified.

7. The method for preparing the nanobody of programmed death receptor 1 according to claim 6, characterized in that, The host cells include mammalian cells.

8. The use of the nanobody of programmed death receptor 1 according to claim 1, the recombinant nanobody of programmed death receptor 1 according to claim 2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the recombinant cell according to claim 6 in the preparation of tumor therapeutic drugs, characterized in that, The tumor is at least one of the following: lung cancer, stomach 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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