Single-domain antibody for resisting programmed death molecule 1 and application of single-domain antibody

Abstract

The invention relates to a single-domain antibody for resisting a programmed death molecule 1 and application of the single-domain antibody. An amino acid sequence of a CDR1 of the single-domain antibody comprises a sequence as shown in SEQ ID NO.1, an amino acid sequence of a CDR2 of the single-domain antibody comprises a sequence as shown in SEQ ID NO.2, and an amino acid sequence of a CDR3 of the single-domain antibody comprises a sequence as shown in SEQ ID NO.3. The invention further relates to a preparation method of the single-domain antibody. According to the present invention, by using the PD-1 protein to immunize the alpaca, the single-domain antibody with characteristics of strong specificity and high affinity with PD-1 is obtained, such that the combination of PD-1 and PD-L1 can be blocked, the immune cell function can be enhanced, and the tumor killing ability of the T cell can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to a single-domain antibody against programmed death molecule 1 and an application thereof. Background Art

[0002] In the immune system, T cell-mediated cellular immunity is mainly used to eliminate tumors, and T cells need to be activated to exert anti-tumor effects. The activation amplitude and quality of T cells depend on the balance between activation signals and inhibitory signals. Immune checkpoints can be considered as the brake system of immune cells, which refers to a series of molecules expressed on immune cells that can regulate the degree of immune activation, so that the activation level of T cells remains within the normal range and prevents damage to healthy tissues of the body.

[0003] 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 PD-L2. 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. Currently, there are many monoclonal antibodies targeting PD-1 on the market, including Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Cindilimab, etc. These PD-1 monoclonal antibodies have been successfully used in clinical practice.

[0004] CN111808196A discloses an anti-PD-1 antibody or an antigen-binding fragment thereof, as well as a preparation method and use. The anti-PD-1 antibody or its antigen-binding fragment can effectively block the binding of PD-1 to the ligand, and has strong specificity and high affinity for binding to human PD-1. In vivo experiments also show that it is well tolerated by non-human animals, non-toxic, and significantly inhibits tumor growth.

[0005] The research carriers of ordinary antibodies are mainly mice and rabbits. There is a special antibody lacking light chains in the blood of camelids and cartilaginous fish, 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 nano antibody) 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. Compared with ordinary antibodies, alpaca antibodies have many advantages: First, they are small in size, one-tenth of ordinary antibodies, which enables them to penetrate into tissues and cells, especially the blood-brain barrier. Second, they are highly stable. Nano antibodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90°C. Third, they are simple in structure. Nano antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs and are suitable for large-scale production. Fourth, it is highly specific. The alpaca’s immune system can produce antibodies that are highly specific and have affinity for specific antigens, and have weak immunogenicity to humans, so the risk of causing an immune response is low.

[0006] In summary, how to use PD-1 antibodies to block the PD-1 / PD-L1 pathway to restore T cell tumor activity, activate T cells, kill tumor cells, and apply relevant technologies to prepare anti-PD-1 alpaca antibodies to make antibody drugs or cell drugs has become one of the urgent problems to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a single-domain antibody against programmed death molecule 1 and its application. Alpacas are immunized with PD-1 protein to obtain a single-domain antibody with strong specificity and high affinity for PD-1. At the same time, it can block the binding of PD-1 and PD-L1 and enhance the function of immune cells.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a single-domain antibody against programmed death molecule 1, wherein the amino acid sequence of CDR1 of the single-domain antibody includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the single-domain antibody includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 of the single-domain antibody includes the sequence shown in SEQ ID NO.3.

[0010] SEQ ID NO. 1: ASGSIFSIHDMG.

[0011] SEQ ID NO. 2: ARITSGLSTNYAD.

[0012] SEQ ID NO. 3: NREIRGSGIWYPLDY.

[0013] Preferably, the amino acid sequence of the heavy chain variable region of the single-domain antibody includes the sequence shown in SEQ ID NO.4.

[0014] SEQ ID NO.4: MAQVKLEESGGGLVQPGGSLRLSCAASGSIFSIHDMGWYRQAPGKQR ELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAAS.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the single domain antibody described in the first aspect.

[0016] Preferably, the nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.5.

[0017] SEQ ID NO.5: ATGGCTCAGGTAAAACTAGAGGAGAGTGGAGGAGGCTTGGTGCAA CCCGGCGGTTCACTTCGGTTAAGCTGTGCCGCTTCCGGGTCAATCTTTAGCATTCATGACATGGGTTGGTATAGACAAGCACCAGGGAAGCAACGTGAACTTGTAGCTAGAATAACAAGTGGCTTATCTACCAACTATGCCGATAGCGTTAAGGGTAGATTTACGATCTCACGTGACAACGCC AAAAATACTGTATATCTTCAAATGGACTCATTAAAACCGGAGGATACCGCAGTATATTACTGTAATCGCGAAATCCGTGGGTCAAGTGGGATATGGTATCCGCTGGATTATTGGGGCCAAGGAACTCAGGTTACAGTCTCTTCCGCTCATCACAGTGAAGACCCCAGTTCCGCAGCGGCCAGC.

[0018] In a third aspect, the present invention provides a heavy chain antibody, wherein the heavy chain antibody comprises the single domain antibody described in the first aspect.

[0019] Preferably, the heavy chain antibody further comprises Ig Fc.

[0020] Preferably, the Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

[0021] Preferably, the amino acid sequence of the Fc segment of IgG1 includes the sequence shown in SEQ ID NO.6.

[0022] SEQ ID NO.6:EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] Preferably, the amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.7.

[0024] SEQ ID NO.7: MAQVKLEESGGGLVQPGGSLRLSCAASGSIFSIHDMGWYRQAPGKQR ELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGIWYPLDYWGQGTQVTVSSAHHSEDPSSAAASAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

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

[0026] Preferably, the expression vector contains the gene encoding the heavy chain antibody described in the third aspect.

[0027] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains the expression vector described in the fourth aspect.

[0028] In a sixth aspect, the present invention provides a pharmaceutical composition comprising any one or a combination of at least two of the single domain antibody described in the first aspect, the heavy chain antibody described in the third aspect, or the host cell described in the fifth aspect.

[0029] Preferably, the pharmaceutical composition further comprises immune cells.

[0030] Preferably, the immune cells include T cells

[0031] In the seventh aspect, the present invention provides the use of any one or a combination of at least two of the single domain antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the heavy chain antibody described in the third aspect, the expression vector described in the fourth aspect, the host cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of tumor treatment drugs.

[0032] Preferably, the tumor includes any one of colorectal cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, bile duct cancer, lung cancer or nasopharyngeal cancer, or a combination of at least two of them.

[0033] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

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

[0035] The present invention uses PD-1 protein to immunize alpacas, and after determining the titer of PD-1 specific antibodies in alpacas serum by ELISA, separates alpacas peripheral blood mononuclear cells, extracts RNA and reverse transcribes to obtain cDNA. Alpaca single-domain antibody specific primers are used to amplify the VHH sequence and clone it into a yeast expression plasmid to construct a yeast 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. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to PD-1 is detected by ELISA. After the positive antibodies are expressed and purified, their affinity for binding to PD-1 protein is detected. Antibody clones with strong affinity to PD-1 are selected to detect the ability to block the binding of PD-1 and PD-L1. The results show that alpaca antibodies have high specificity and can block the binding of PD-1 and PD-L1 at the same time, thereby enhancing the function of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a graph showing the specificity results of recombinant single domain antibodies detected by flow cytometry.

[0037] Figure 2 ELISA for detecting EC of recombinant single domain antibodies 50 Result graph.

[0038] Figure 3 This is a graph showing the results of the recombinant antibody blocking function test.

[0039] Figure 4 IC of recombinant single domain antibodies for ELISA 50 Result graph. DETAILED DESCRIPTION

[0040] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0041] 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.

[0042] Reagents used in the following examples:

[0043] PE-anti-Human IgG antibody (eBioscience, CAT#12-4998-82);

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

[0045] PBS Buffer(bico, CAT#14190-250);

[0046] Adjuvant immune adjuvant (GERBU, CAT#3030);

[0047] HRP-ProteinA (Boster, CAT#BA1080);

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

[0049] cDNA Synthesis Kit (TaKaRa, CAT#6210B);

[0050] DNA fragment recovery kit (TakaRa, CAT#9761);

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

[0052] THE TM V5 Tag Antibody [iFluor 647] (GenScript, CAT# A01805);

[0053] Nivolumab (MedChemExpress, HY-P9903).

[0054] Example 1 Alpaca immunization and antibody titer determination

[0055] Two alpacas (98# and 195#) were immunized once every three weeks with recombinant PD-1 protein for a total of four immunizations. Peripheral blood was collected before immunization, two weeks after the second immunization, the third immunization, and the fourth immunization, and centrifuged at 800×g for 10 minutes to collect the upper serum.

[0056] In a 96-well ELISA plate, add 1 μg / mL of PD-1-His recombinant protein at 100 μL / well and coat overnight at 4°C. After washing 5 times with PBST, add 200 μL / well of blocking solution and block at 37°C for 2 hours, and then wash the plate 5 times with PBST. Use PBS to gradient dilute the above-collected serum, add 100 μL of gradient diluted serum to the 96-well ELISA plate, and incubate at room temperature for 1 hour. After washing 5 times with PBST, add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour. After washing the plate 5 times with PBST, add 100 μL / well TMB colorimetric solution and incubate at room temperature in the dark for 15 minutes. Add 50 μL / well stop solution and use an ELISA reader to read the OD in the well. 450 The titer test results of the serum from the 98# alpaca immunization are shown in Table 1, and the titer test results of the serum from the 195# alpaca immunization are shown in Table 2.

[0057] Table 1

[0058]

[0059] Table 2

[0060]

[0061]

[0062] As shown in Tables 1 and 2, the binding titers of the serum of the two alpacas before immunization to the PD-1 protein were both lower than 1:2000, indicating that the content of anti-PD-1 specific antibodies contained therein was extremely low. On the contrary, the binding titers of the serum of the 98# alpaca and the 195# alpaca after the fourth immunization to the PD-1 protein were both higher than 1:64K, indicating that the content of anti-PD-1 specific antibodies in the alpaca serum was significantly increased.

[0063] Example 2 Construction of single domain antibody yeast display library

[0064] (1) VHH antibody fragment cloning

[0065] 100 mL of peripheral blood was collected from each of the two alpacas, peripheral blood mononuclear cells (PBMC) were separated, RNA was extracted and reverse transcribed to obtain cDNA. Primers were used to amplify the alpaca heavy chain antibody sequence by PCR. The upstream primer SEQ ID NO.8 had a sequence of 5'-GTCCTGGCTGCTCTTCTACAAGG-3', and the downstream primer SEQ ID NO.9 had a sequence of 5'-GGTACGTGCTGTTGAACTGTTCC-3'. The PCR product was analyzed by agarose electrophoresis, and the target fragment of about 750 bp was recovered.

[0066] The first-round PCR product was used as a template, and specific primers were used to amplify the heavy chain antibody VHH fragment. The PCR product was analyzed by agarose electrophoresis, and the target fragment of about 450 bp was recovered to obtain the VHH fragment library.

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

[0068] Streak the competent yeast strain onto a YPD solid medium plate and activate at 30°C for 3-5 days. Inoculate a single colony of competent yeast into 50mL YPD medium and shake at 250rpm and 30°C for 1-2 days. Mix the yeast surface display vector pYDisplay and the VHH fragment library after SfiI digestion, add them to the electroporation cup, and electroporate; transfer the competent yeast after electroporation to the culture bottle and shake at 220rpm and 30°C for 1h. Take 20μL of the resuspension, dilute it 5000 times with SDCAA, take 100μL, apply it to the SDCAA plate, culture 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 at 3000×g for 5min, discard the supernatant, add 10mL SDCAA to resuspend, mix with 50% glycerol: resuspension = 1:1, and freeze at -80°C.

[0069] The results showed that the single domain antibody yeast display library obtained from 98# alpaca PBMC had a library capacity of 2.4×10 9The single domain antibody yeast display library obtained from 195# alpaca PBMC has a capacity of 1.2×10 9 .

[0070] Example 3 Panning of yeast display library

[0071] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0072] Take 2×10 8 Yeast library, centrifuge at 3000×g for 5min, remove the supernatant, resuspend in 1mL 0.5% PBSA in a 1.5mL centrifuge tube, centrifuge at 3000×g for 5min, and discard the supernatant. Add 1mL 0.5% PBSA and 10μL magnetic beads to 3 centrifuge tubes, fix them on a rotating mixer, and rotate and incubate at 4℃ for 5min. Place the centrifuge tubes on a magnetic stand for 5min and remove the supernatant. Add 1mL 0.5% PBSA again, rotate and incubate at 4℃ for another 5min, and remove the supernatant.

[0073] (2) Yeast display library screening

[0074] Negative panning: Add the yeast solution to the centrifuge tube containing empty magnetic beads and incubate at 4°C for 60 minutes. Place the empty magnetic bead centrifuge tube containing yeast on a magnetic rack for 10 minutes, transfer the yeast solution to a new empty magnetic bead centrifuge tube, and incubate at 4°C for 30 minutes. After the incubation, place it on a magnetic rack, pipette the yeast solution and add it to a new empty magnetic bead centrifuge tube.

[0075] Biotin-PD-1-His magnetic separation: Add 100μL of 50μg / mL biotin-PD-1-His protein solution to the centrifuge tube containing streptavidin magnetic beads, and incubate at 4℃ for 60min; then add 1mL of 0.5% PBSA, let stand for 5min, and keep the centrifuge tube on the magnetic rack to remove the supernatant. Then add 1mL of 0.5% PBSA, mix by pipetting, and place the centrifuge tube on the magnetic rack again, let stand for 5min, and remove the supernatant; repeat the above washing steps once again 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, and incubate at 4℃ for 60min. After the incubation, place it on the magnetic rack, let stand at room temperature for 15min, and discard the unbound yeast solution. Add 1mL of sterile 0.5% PBSA buffer, gently pipette, place on a magnetic stand, let stand at room temperature for 5min, discard the supernatant, and repeat washing twice. The whole process is performed in accordance with the requirements of aseptic operation. After washing, resuspend the magnetic beads and adhered yeast cells with 1mL SDCAA medium, and pipette 20μL of the resuspension into 180μL SDCAA medium to coat two plates. The volume of each plate is 100μL, and then 5μL of the resuspension is pipetted into 95μL SDCAA medium to coat one plate.

[0076] 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 selected yeast was incubated with the PD-1 protein containing the His tag at 4°C for 60 minutes and the supernatant was removed. Anti-His tag and anti-V5 tag flow cytometry antibodies were added, incubated at 4°C for 60 minutes and the supernatant was removed by centrifugation. The cells were resuspended in 1mL PBS and centrifuged to remove the supernatant. The cells were resuspended in 500μL PBS and analyzed by flow cytometry. Before and after sorting, the number of yeasts that could bind to the PD-1 protein in the yeast antibody display library of 98# alpaca and 195# alpaca increased by 38% and 35%, respectively.

[0077] Selection of monoclonal clones: Inoculate the monoclonal clones into the culture medium to induce expression. After 48 hours, incubate the bacterial solution with the PD-1 protein containing the His tag at 4°C for 60 minutes and centrifuge to remove the supernatant. Resuspend the precipitate with a solution containing a biotin-conjugated anti-His tag flow antibody, incubate at 4°C for 60 minutes, and centrifuge to remove the supernatant. Resuspend the precipitate with a solution containing PE-streptavidin, incubate at 4°C for 60 minutes, resuspend the cells with 1mL PBS, and centrifuge to remove the supernatant. Resuspend the cells with 500μL PBS and perform flow analysis. Finally, the monoclonal clone 195-4-G5 with a high positive rate was selected and sent for PCR amplification.

[0078] Example 4 Expression of recombinant single domain antibodies and detection of binding to target proteins

[0079] After the 195-4-G5 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were connected and transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant single domain antibody 195-4-G5.

[0080] The culture supernatant containing 195-4-G5 was incubated with CHO-K1 or CHO-K1-PD-1 cells at room temperature for 1 hour, and the cells were washed 3 times with PBS. 100 μL PE-labeled anti-human IgG antibody (1:500 dilution) was added, incubated at room temperature in the dark for 45 minutes, and the cells were washed 3 times with PBS. The cells were resuspended in 500 μL PBS and analyzed by flow cytometry to detect the binding specificity of 195-4-G5.

[0081] The results are as follows Figure 1 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. The 195-4-G5 antibody 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 195-4-G5 has good affinity and specificity.

[0082] Example 5 Purification of recombinant antibodies and half effective concentration (EC 50 ) determination

[0083] Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash Protein A magnetic beads twice with 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20mg IgG / mL Protein A magnetic beads) to the 293 cell expression supernatant according to the required amount of the sample. Incubate at room temperature for 1-4h or at 4℃ overnight at 120rpm. Collect Protein A magnetic beads with a magnetic separation rack. Wash twice with 30mL PBS buffer and deionized water, resuspend with 1mL elution buffer. After incubation at room temperature for 5min, collect magnetic beads with a magnetic separation rack. After eluting Protein A magnetic beads twice, combine the eluates, and add neutralization buffer to adjust the pH of the solution. The eluted sample is dialyzed with 100 times the volume of PBS, first dialyzed at 18-25℃ for 2h, and then dialyzed at 2-8℃ for 14-16h after changing the solution once, and the protein concentration is determined.

[0084] 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 2h. Wash 5 times with PBST, add different concentrations of 195-4-G5 candidate antibodies (Table 3), and incubate at room temperature for 60min. Wash 5 times with PBST, dilute HRP-Protein A at 1:50000, add 100μL / well to the ELISA plate, and incubate at room temperature for 45min. Wash 5 times with PBST, add 100μL TMB colorimetric solution to each well and color for 10min at room temperature in the dark. Add 50μL stop solution to each well, and read the absorbance at 450nm wavelength on the ELISA reader.

[0085] The results are shown in Table 3 and Figure 2 As shown, the EC of 195-4-G5 antibody against CHO-K1-PD-1 50 The value was 0.06 μg / mL, indicating that 195-4-G5 had a higher affinity.

[0086] Table 3

[0087]

[0088] Example 6 Detection of blocking function of recombinant antibodies

[0089] In a 96-well plate, 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). Different concentrations of 195-4-G5 were added to the corresponding wells, so that the final concentrations in the corresponding wells were 120μg / mL, 40μg / mL, 13.3μg / mL, 4.4μg / mL, 1.6μg / mL, 0.5μg / mL, 0.16μg / mL, 0.05μg / mL, 0.02μg / mL, and 0μg / mL, respectively. After 18 hours of co-culture, 20μL of Ne-Glo reagent was added to each well and the fluorescence value was read.

[0090] result Figure 3 As shown, the maximum induction effect value of 195-4-G5 is 1.66 (the difference between the maximum fluorescence value and the fluorescence value of 0 μg / mL), indicating that 195-4-G5 has a strong ability to block the binding of PD-1 and PD-L1, and can effectively promote the expression of luciferase in Jurkat-PD-1-luciferase cells.

[0091] Example 7 Recombinant Antibody Half Inhibitory Concentration (IC 50 )experiment

[0092] In order to detect the half-inhibitory concentration of 195-4-G5 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 the 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 195-4-G5 candidate antibodies (Table 4) respectively, 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 with PBST 5 times, add 100 μL TMB color development solution to each well and color for 10 minutes at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[0093] The results are shown in Table 4 and Figure 4 As shown, the IC of 195-4-G5 50 The value was 0.42 μg / mL, indicating that 195-4-G5 could block the binding of PD-1 and PD-L1 proteins.

[0094] Table 4

[0095]

[0096] In summary, the present invention uses PD-1 protein to immunize alpacas, uses alpaca single domain antibody specific primers to amplify VHH sequences, and clones them into yeast expression plasmids to construct yeast display libraries, perform positive and negative screening, pick monoclones from the enriched products, and detect the specific binding of candidate antibodies to PD-1 by ELISA. After the positive antibodies are expressed and purified, their affinity for binding to PD-1 protein is detected, and antibody clones with strong affinity to PD-1 are selected to detect the ability to block the binding of PD-1 and PD-L1. The results show that the single domain antibodies provided by the present invention have high specificity, can block the binding of PD-1 and PD-L1, and enhance the function of immune cells.

[0097] 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 single domain antibody against programmed death molecule 1, characterized in that: The amino acid sequence of CDR1 of the single-domain antibody includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the single-domain antibody includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 of the single-domain antibody includes the sequence shown in SEQ ID NO.

3.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the single-domain antibody includes the sequence shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single domain antibody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.

5.

5. A heavy chain antibody, characterized in that The heavy chain antibody comprises the single domain antibody of claim 1 or 2.

6. The heavy chain antibody according to claim 5, characterized in that The heavy chain antibody also includes Ig Fc; The Ig Fc includes any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4; The amino acid sequence of the Fc segment of IgG1 includes the sequence shown in SEQ ID NO.6; The amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.

7.

7. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 3 or 4.

8. A host cell, characterized in that The host cell contains the expression vector according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises any one or a combination of at least two of the single domain antibody of claim 1 or 2, the heavy chain antibody of claim 5 or 6, or the host cell of claim 8; The pharmaceutical composition also includes immune cells.

10. Use of any one or a combination of at least two of the single domain antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the heavy chain antibody according to claim 5 or 6, the expression vector according to claim 7, the host cell according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of tumor therapeutic drugs.

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