Anti-PD-1 single-domain antibody and application thereof

Abstract

The invention relates to an anti-PD-1 single-domain antibody and application thereof. The complementarity determining region of the VHH chain of the anti-PD-1 single-domain antibody comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2 and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3. The invention further discloses a preparation method of the anti-PD-1 single-domain antibody. 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 single-domain antibody with high specificity and affinity is obtained, and the single-domain 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 field of biotechnology and relates to an anti-PD-1 single domain antibody and its application. Background Art

[0002] Programmed death 1 (PD-1) is an immune checkpoint molecule that is mainly expressed on the surface of T cells and plays a key role in regulating the immune system. The main function of PD-1 is to inhibit the activation of T cells, thereby preventing the immune system from overreacting and maintaining immune homeostasis. When PD-1 binds to its ligands PD-L1 or PD-L2, it transmits inhibitory signals, resulting in decreased or inactivated T cell function. In some diseases, such as cancer, the expression of PD-1 may be abnormally elevated, resulting in a weakened immune response of T cells to tumor cells. Therefore, PD-1 has become an important target for cancer immunotherapy. Immune checkpoint inhibitors targeting PD-1 have been widely used in the treatment of a variety of cancers, which can improve the therapeutic effect by blocking the binding of PD-1 to its ligands and restoring the anti-tumor activity of T cells.

[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 anti-PD-1 antibodies has achieved significant therapeutic effects, but there are also some problems and challenges, such as: Individual differences in efficacy: There are significant differences in the treatment response of different patients to PD-1 antibodies, 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 an anti-PD-1 single domain antibody and its application, and develops a new anti-PD-1 single domain antibody, in order to bring a more effective and safer treatment plan for tumor treatment.

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

[0007] In a first aspect, the present invention provides an anti-PD-1 single domain antibody, wherein the complementarity determining region of the VHH chain of the anti-PD-1 single domain antibody comprises: a CDR1 having an amino acid sequence as shown in SEQ ID NO.1, a CDR2 having an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO.3.

[0008] The present invention uses PD-1 protein to immunize alpacas, constructs an antibody display library, and screens antibodies that can bind to PD-1 protein. The anti-PD-1 single domain antibody obtained by this method has high specificity and affinity, and can effectively block the binding between PD-1 and PD-L1, thereby enhancing the function of immune cells and combating the immune escape mechanism of tumors. This anti-PD-1 single domain antibody, also known as nanobody, has a small molecular weight and a simple structure. It consists of a domain of the heavy chain variable region, has high affinity and strong specificity, strong tissue penetration, weak immunogenicity to the human body, and can be relatively easily transformed and amplified, so it has broad application prospects in the fields of diagnosis and treatment.

[0009] Preferably, the amino acid sequence of the anti-PD-1 single domain antibody includes the sequence shown in SEQ ID NO.4.

[0010] SEQ ID NO.1: GNIFSLNDMT.

[0011] SEQ ID NO.2: ITSGLSTNYAD.

[0012] SEQ ID NO. 3: NREIRGSSGSWYPLHY.

[0013] SEQ ID NO.4:

[0014] MAQLQLVESGGGLVQPGGSLRLSCAASGNIFSLNDMTWYRQTPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSSEPKTPKPQPAAAS.

[0015] Preferably, the anti-PD-1 single domain antibody further comprises a human Fc region (which may be referred to as a recombinant anti-PD-1 single domain antibody).

[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 anti-PD-1 single domain antibody includes the sequence shown in SEQ ID NO.6.

[0022] SEQ ID NO.6:

[0023] MAQLQLVESGGGLVQPGGSLRLSCAASGNIFSLNDMTWYRQTPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSSEPKTPKPQPAAASAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0024] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-PD-1 single domain antibody described in the first aspect.

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

[0027] In a fifth aspect, the present invention provides the use of the anti-PD-1 single domain antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant expression vector described in the third aspect, or the engineered cell described in the fourth aspect in the preparation of a preparation that binds to programmed death receptor 1.

[0028] The present invention obtains an anti-PD-1 single domain antibody with high specificity and affinity, which can be used to develop reagents for immune detection of PD1. It 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.

[0029] In a sixth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the anti-PD-1 single domain antibody described in the first aspect and a conjugated substance conjugated thereto.

[0030] Preferably, the coupling substance includes at least one of a cytotoxin, a radioactive isotope, a luminescent substance, a colorimetric substance or an enzyme.

[0031] In a seventh aspect, the present invention provides a pharmaceutical composition, comprising the anti-PD-1 single domain antibody described in the first aspect.

[0032] Preferably, the pharmaceutical composition is a drug for treating tumors.

[0033] 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.

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

[0035] 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 anti-PD-1 single-domain antibodies with high specificity and affinity, and has the ability to block the binding of PD-1 and PD-L1, which can be applied to the development of tumor therapeutic drugs. At the same time, single-domain 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

[0036] Figure 1 This is the electrophoresis result of the first round of PCR amplification of heavy chain antibody fragments;

[0037] Figure 2 This is the electrophoresis result of the second round of PCR amplification of the VHH region fragment;

[0038] Figure 3 This is a graph showing the specificity results of single domain antibodies detected by flow cytometry;

[0039] Figure 4 EC detection of single domain antibodies for ELISA 50 Result graph;

[0040] Figure 5 This is a diagram showing the results of the single domain antibody blocking function test;

[0041] Fig. 6A The blocking curve of single domain antibody detected by ELISA;

[0042] Figure 6B The blocking curve of negative control antibody for ELISA test;

[0043] Figure 6C This is the blocking curve of the positive control antibody in ELISA test. DETAILED DESCRIPTION

[0044] 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 simple 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.

[0045] 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.

[0046] Unless otherwise defined, the scientific and technical terms and abbreviations used in conjunction with the present invention shall have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs. Some of the terms and abbreviations used in the present invention are listed below.

[0047] The term "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogs that may be present at a specific defined position. The three-letter abbreviations of amino acids and the single-letter abbreviations of nucleotides mentioned in the present invention are the forms generally accepted in the art, and the single-letter abbreviations of amino acids are the forms recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0048] The terms "single domain antibody", "VHH", "nanobody" and "single domain antibody" (sdAb) have the same meaning and are used interchangeably, referring to cloning the variable region of an antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0049] Fc region: fragment crystallizable region, Fc region.

[0050] Complementarity determining region: complementarity determining region, CDR, refers to the antigen complementary binding region of an antibody.

[0051] The term "host cell" refers to cells into which exogenous nucleic acid has been introduced and their progeny, which can be transformed or transfected with nucleotides encoding polypeptides to express exogenous polypeptides. The host cells described in the present invention include, but are not limited to, 293F cells (human embryonic kidney cells), CHO cells (Chinese hamster ovary cells), BHK cells (BabyHamster Kidney cells), myeloma cells, yeast, insect cells or prokaryotic cells such as Escherichia coli, etc. It should be noted that the "host cell" described in the present invention refers not only to cells into which exogenous nucleic acid has been introduced, but also to the progeny of the cell. Since the progeny cells may mutate during cell division, they still fall within the scope of the term described in the present invention.

[0052] The reagents used in the specific embodiments include:

[0053] PBS (bico, CAT# 14190-250);

[0054] Streptavidin PE (eBioscience, CAT# 12-4317-87);

[0055] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82);

[0056] Adjuvant immune adjuvant (GERBU, CAT# 3030);

[0057] HRP-Streptavidin(Boster, CAT# BA1088);

[0058] HRP-ProteinA(Boster, CAT# BA1080);

[0059] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0060] DNA fragment recovery kit (TakaRa, CAT# 9761);

[0061] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (Novus, AT#NB7242);

[0062] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0063] Anti-PD1 flow cytometry antibody (BD Pharmingen, 560779);

[0064] Nivolumab (MedChemExpress, HY-P9903).

[0065] Example 1

[0066] In this example, alpaca immunization and antibody titer determination were performed.

[0067] Alpacas were immunized with PD-1 protein (health status before immunization: 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, alert and quick reaction; no external trauma). Each time, the alpaca was injected on both sides near the cervical lymph nodes, with 2 injections on each side, and about 500 μg 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 ELISA.

[0068] The antibody titer is expressed in the form of "1:X" (X represents the maximum dilution multiple at which the antibody can be detected). The results are shown in Table 1. The binding titer of the serum of the alpaca before immunization with the PD-1 protein was lower than 1:1000 (no significant difference from the PBS group, P > 0.05), indicating that the content of anti-PD-1 specific antibodies contained therein was extremely low. On the contrary, the binding titer of the serum of the alpaca after the fourth immunization with the PD-1 protein exceeded 1:64000 (significantly different from the PBS group, P < 0.05), indicating that the content of anti-PD-1 specific antibodies in the serum of the alpaca increased significantly after multiple immunizations, and the immunization was successful, which can be used for the construction of yeast display libraries.

[0069] Table 1

[0070]

[0071] Example 2

[0072] In this example, a single domain antibody yeast display library was constructed and library screening was performed.

[0073] (1) Heavy chain variable region (VHH) fragment cloning

[0074] After confirming that the alpaca serum in Example 1 contained anti-PD-1 specific antibodies, peripheral blood was collected, PBMC was separated, and RNA was extracted. st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. The heavy chain antibody sequence was amplified by PCR using primers (the upstream primer binds to the signal peptide of the VHH antibody ORF, the primer sequence is 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO.7), and the downstream primer binds to the CH2 region, the primer sequence is 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO.8)).

[0075] The results are as follows Figure 1 As shown, 1% agarose was used to recover the target fragment of about 750 bp. The first round PCR product was then used as a template to amplify the heavy chain antibody VHH fragment using primers (the upstream primer binds to the FR1 region, the primer sequence is (5'-ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO.9); the downstream primer binds to the Hinge and FR4 regions and contains the SfiI site GGCCACGAAGGCC (SEQ ID NO.10), the primer sequence is 5'-GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO.11)). The results are shown in Figure 2 As shown, 1% agarose was used for electrophoresis and the target fragment of about 450 bp was recovered to obtain the heavy chain antibody VHH fragment library.

[0076] (2) Electroporation of library vectors and detection of library capacity and diversity

[0077] The yeast display vector plasmid pYDisplay and the obtained VHH fragment library were digested with SfiI endonuclease and the linearized vector and target fragment were recovered. After mixing the two, add them to the electroporation cup, electroporate the yeast competent cells, and shake at 220 rpm and 30℃ for 1 h. Take 20 μL of the resuspension, dilute 5000 times with SD-CAA medium, take 100 μL, apply to SD-CAA plate, culture for 2 days, calculate the library capacity, and continue to culture the rest of the bacterial solution for 24 h. Collect the remaining bacterial solution into a 50 mL centrifuge tube, centrifuge at 3000×g for 5 min, discard the supernatant, add 10 mL SD-CAA to resuspend, add glycerol with a final concentration of 20%~25%, and freeze at -80℃. The results showed that the library capacity of the single-domain antibody yeast display library obtained from alpaca PBMC was 1.2×10 9 Sanger sequencing showed that the yeast library sequences were highly different, with no repetitive sequences and good diversity.

[0078] (3) Screening of yeast display libraries

[0079] (3-1) Pretreatment of Streptavidin Magnetic Beads and Yeast Cells

[0080] Take 8 mL of yeast library (about 2×10 8Yeast cells) into a centrifuge tube, centrifuge at 3000×g for 5 min, and discard the supernatant; at the same time, dilute 5% PBSA (PBS+5% BSA) to 0.5% PBSA; resuspend the yeast library with 1 mL 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 3 centrifuge tubes (2 centrifuge tubes for negative screening of empty magnetic beads, 1 centrifuge tube for positive screening), add 1 mL 0.5% PBSA to each centrifuge tube; resuspend the aliquoted streptavidin magnetic beads with a pipette, take 10 μL of magnetic beads and add them to a 1.5 mL centrifuge tube; put these centrifuge tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4℃ for 5 min; place the centrifuge tube on a magnetic stand for 5 min, and remove the supernatant with a pipette; then add 1 mL 0.5% PBSA again, rotate and incubate at 4℃ for another 5 min, and remove the supernatant.

[0081] (3-2) Screening of yeast display libraries

[0082] In order to screen alpaca antibodies with high affinity for PD-1, the yeast library was screened.

[0083] Empty magnetic bead negative screening: Place the centrifuge tube on the magnetic rack, add the cleaned yeast solution to the centrifuge tube containing the empty magnetic beads, and put it in a bag, and rotate and incubate at 4℃ for 1 hour. Place the centrifuge tube on the magnetic rack for 10 minutes, pipette the yeast solution into a new empty magnetic bead centrifuge tube, and rotate and incubate at 4℃ for 30 minutes. After the incubation, place it on the magnetic rack for 15 minutes, pipette the yeast solution and transfer it to a new empty magnetic bead centrifuge tube.

[0084] 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 the centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 1 h. Then add 1 mL of 0.5% PBSA, let it stand for 5 min, keep the centrifuge tube on the magnetic stand, and remove the supernatant with a pipette. 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 min, keep the centrifuge tube on the magnetic stand, and remove the supernatant with a pipette; Repeat the above washing steps once more to obtain positive screening magnetic beads coated with Biotin-PD-1-His. Add the positive screening magnetic beads bound to Biotin-PD-1-His to the yeast cells that have completed the negative screening, and put them into a bag and place them on a rotating mixer, and rotate and incubate at 4℃ for 1 hour. After the incubation, place it on a magnetic stand and let it stand at 25℃ for 15 minutes; use a pipette to absorb the unbound yeast solution. Remove the 1.5 mL centrifuge tube from the magnetic stand, and add 1 mL 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 1.5 mL centrifuge tube, place the centrifuge tube on a magnetic stand, let it stand at 25℃ for 5 minutes, and discard the supernatant; repeat the wash twice. After washing, resuspend the magnetic beads and adhered yeast cells in 1 mL SD-CAA medium, pipette 20 μL of the resuspended solution into 180 μL SD-CAA medium and coat the plate. The volume of each plate is 100 μL. Pipet 5 μL of the resuspended solution into 95 μL SD-CAA medium and coat one plate. That is, a total of 3 plates need to be coated.

[0085] Yeast flow cytometry after screening: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into yeast. The yeast that has undergone empty magnetic bead negative screening and Biotin-PD-1-His magnetic sorting was incubated with the PD-1 protein containing the His tag at 4°C for 1 h, centrifuged to remove the supernatant, added anti-His tag and anti-V5 tag flow antibodies, and incubated at 4°C for 1 h. Centrifuged to remove the supernatant, and resuspended the cells in 1 mL PBS. Centrifuged to remove the supernatant, resuspended the cells in 500 μL PBS, and performed flow analysis. The yeast that can bind to the PD-1 protein in the yeast antibody display library of the alpaca before and after sorting increased by 35%, respectively, and can be used for subsequent experiments.

[0086] Monoclonal selection: After overnight culture, monoclones were picked and inoculated into culture medium to induce expression. After 48 hours, the bacterial solution was mixed with PD-1 protein containing His tag and incubated at 4℃ for 1 hour. The supernatant was removed by centrifugation, and the precipitate was resuspended in a solution containing biotin-coupled anti-His tag flow cytometry antibody, and incubated at 4℃ for 1 hour. The supernatant was removed by centrifugation, and the precipitate was resuspended in a buffer containing PE-Streptavidin, and incubated at 4℃ for 1 hour. The cells were resuspended in 1 mL PBS, the supernatant was removed by centrifugation, and the cells were resuspended in 500 μL PBS for flow cytometry analysis. Finally, a monoclone with a high positive rate was selected and named 195-4-E3. 195-4-E3 was lysed with 0.2% SDS (incubated at 95℃ for 10 min) and centrifuged. 0.5 μL of the bacterial supernatant was taken as a template for PCR amplification and sequencing.

[0087] Further analysis of the antibody sequence information showed that the CDR1 region sequence of the single-domain antibody 195-4-E3 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 single-domain antibody sequence was shown in SEQ ID NO.4.

[0088] Example 3

[0089] This embodiment performs the expression of recombinant single domain antibodies and the detection of binding to target proteins.

[0090] A recombinant antibody containing single-domain antibody 195-4-E3 and IgG1 Fc region (SEQ ID NO.6) was designed. 195-4-E3 VHH nucleic acid sequence, CMV promoter sequence and IgG1 Fc nucleic acid sequence were amplified by PCR. 50 μL of PCR product was added with 1 / 10 volume of 10× loading buffer and analyzed by electrophoresis 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-E3 VHH sequence, CMV promoter and IgG1 Fc sequence were connected to the expression vector and transiently transfected into HEK293 cells. The harvested cell culture supernatant contained the recombinant single-domain antibody 195-4-E3.

[0091] The culture supernatant containing 195-4-E3 was mixed with 3×10 5CHO-K1 and CHO-K1-PD-1 cells (CHO-K1 cells overexpressing PD-1) were incubated at 25℃ for 1 h. 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 and incubated at 25℃ in the dark for 45 min. After centrifugation at 800×g, 25℃ for 5 min, 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 anti-PD1 flow cytometry antibody was used as a positive control.

[0092] The results are as follows Figure 3 As shown, the expression supernatant of the negative control group did not bind significantly to CHO-K1 or CHO-K1-PD-1; the positive control antibody only significantly bound to CHO-K1-PD-1 cells, indicating that both CHO-K1 cells and CHO-K1-PD-1 cells can be used for PD-1 antibody binding detection. 195-4-E3 did not bind to CHO-K1 cells, but had significant binding to CHO-K1-PD-1, and most CHO-K1-PD-1 cells expressing PD-1 protein could be detected, indicating that the single-domain antibody 195-4-E3 had good affinity and specificity.

[0093] Example 4

[0094] This study determined the purification and half effective concentration (EC) of single domain antibodies. 50 ).

[0095] The recombinant single-domain antibody expression plasmid was transiently transfected into 293F cells and cultured in a shaking flask to express and purify the antibody. Since the target antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification.

[0096] PD-1 protein was diluted to 2 μg / mL using coating solution, and added to 96-well ELISA plate at 100 μL / well, and coated overnight at 4°C. Washed 5 times with PBST, added blocking solution at 200 μL / well, and blocked at 25°C for 2 h. Washed 5 times with PBST, added gradient dilutions of 195-4-E3 antibody (10 μg / mL, 3.3 μg / mL, 1.1 μg / mL, 0.37 μg / mL, 0.12 μg / mL, 0.04μg / mL, 0.014 μg / mL, 0 μg / mL), and incubated at 25°C for 1 h. After washing 5 times with PBST, HRP-Protein A was diluted 1:50000 and added to the ELISA plate at 100 μL / well, and incubated at 25°C for 45 min. After washing with PBST for 5 times, 100 μL of TMB colorimetric solution was added to each well, and color was developed at 25°C in the dark for 10 min. 50 μL of stop solution was added to each well, and the absorbance was read at 450 nm on an ELISA reader.

[0097] The results are shown in Table 2 and Figure 4 As shown, the EC value of 195-4-E3 for CHO-K1-PD-1 50 The value was 0.062 μg / mL, indicating that 195-4-E3 had a higher affinity.

[0098] Table 2

[0099]

[0100] Example 5

[0101] This example performs antibody blocking function detection.

[0102] 100 μL / well of cell suspension was added to a 96-well plate, with each well containing 2×10 4 Effector cells Jurkat-PD-1-Luciferase (expressing PD-1 molecules, PD-L1 binding to PD-1 on the cells can inhibit the expression of Luciferase protein) and 8×10 4Target cells CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 on Jurkat-PD-1-Luciferase cells and inhibit the expression of Luciferase). 100 μL of 195-4-E3 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 h of co-culture, 20 μL of One-Glo reagent was added to each well, and the fluorescence value of Luciferase was read by a microplate reader.

[0103] result Figure 5 As shown, the maximum induction effect value of 195-4-E3 is 1.67 (the difference between the maximum Luciferase fluorescence value and the 0 μg / mL Luciferase fluorescence value), indicating that 195-4-E3 has a strong ability to block the binding of PD-1 and PD-L1, can effectively induce Jurkat-PD-1-Luciferase to express Luciferase, and can be used to develop immune-enhancing drugs.

[0104] Example 6

[0105] In this example, the half inhibitory concentration (IC 50 )experiment.

[0106] PD-1 protein was diluted to 2 μg / mL with coating solution, and 100 μL / well was pipetted into a 96-well ELISA plate, and coated overnight at 4°C. Washed 5 times with PBST buffer, 200 μL / well was added with blocking solution, and blocked at 25°C for 2 h. Washed 5 times with PBST buffer, and different concentrations of 195-4-E3 antibody (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0 μg / mL) and anti-PD-1 positive control antibody Nivolumab were added, and incubated at 25°C for 15 min. Biotin-labeled PD-L1 protein (final concentration 4 μg / mL) was added, and incubated at 25°C for 45 min. After washing 5 times with PBST Biotin-PD-L1, the secondary antibody (Streptavidin-HRP) was diluted 1: 10000, added to the ELISA plate at 100 μL / well, and incubated at 25°C for 45 min. Wash 5 times with PBST buffer, add 100 μL TMB colorimetric solution to each well, and color at 25°C in the dark for 10 min. Add 50 μL stop solution to each well, and read the absorbance at 450 nm on the ELISA reader. The PD-1 antibody 195-3-F11-3 without blocking function screened from the same batch of antibodies was used as a negative control, and the Nivolumab antibody was used as a positive control.

[0107] The results are shown in Table 3 and Figure 6A-6C As shown, Fig. 6A is the blocking curve of 195-4-E3, Figure 6B is the blocking curve of negative control antibody, Figure 6C This is the blocking curve of the positive control antibody. The IC of 195-4-E3 is 50 The value is 0.31 μg / mL, and the IC 50 The IC value of 195-4-E3 is 0.3 μg / mL. 50 The value was not significantly different from that of the positive control antibody Nivolumab, indicating that the blocking ability of 195-4-E3 was equivalent to that of the positive control antibody Nivolumab. In addition, in the antibody concentration range of 2.778 μg / mL to 25 μg / mL, the OD450 value of 195-4-E3 was significantly lower than that of the positive control antibody (P < 0.05), indicating that the inhibitory effect of 195-4-E3 on PD-1 has greater room for improvement with increasing concentration, and has better application prospects.

[0108] Table 3

[0109]

[0110] In summary, the present invention immunizes alpacas with PD-1 protein, constructs an antibody display library, and screens antibodies that bind to PD-1 protein, successfully obtaining the anti-PD-1 single-domain antibody 195-4-E3 with high specificity and affinity. This single-domain antibody can not only effectively block the interaction between PD-1 and PD-L1, but also has a significantly stronger blocking effect than the existing Nivolumab at high concentrations, and can also enhance the function of immune cells, which has important application prospects in the field of tumor immunotherapy.

[0111] The applicant declares that the above is only a specific implementation 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 are within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-PD-1 single domain antibody, characterized in that , the complementary determining region of the VHH chain of the anti-PD-1 single domain antibody comprises: The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

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

4.

3. The anti-PD-1 single domain antibody according to claim 1, characterized in that , the anti-PD-1 single domain antibody further comprises a human Fc region; The human Fc region 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.

5.

4. A nucleic acid molecule, characterized in that , the nucleic acid molecule encodes the anti-PD-1 single domain antibody according to any one of claims 1-3.

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

6. An engineered cell, characterized in that , the engineered cell contains the nucleic acid molecule described in claim 4.

7. Use of the anti-PD-1 single domain antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant expression vector according to claim 5 or the engineered cell according to claim 6 in the preparation of a preparation that binds to programmed death receptor 1. 8.An antibody conjugate, characterized in that , the antibody conjugate includes the anti-PD-1 single domain antibody according to any one of claims 1-3 and a conjugated substance conjugated thereto.

9. The antibody conjugate according to claim 8, characterized in that , the coupling substance includes at least one of a cytotoxin, a radioactive isotope, a luminescent substance, a color-developing substance or an enzyme.

10. A pharmaceutical composition, characterized in that , the pharmaceutical composition comprises the anti-PD-1 single domain antibody according to any one of claims 1-3.

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