An anti-PD-1 single domain antibody and its application

Abstract

The present invention relates to an anti-PD-1 single-domain antibody and its application. The complementarity-determining regions of the VHH chain of the anti-PD-1 single-domain antibody include: CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3. In the present invention, alpacas are immunized with PD-1 protein to construct an antibody display library, antibodies that bind to the PD-1 protein are screened, and single-domain antibodies with high specificity and affinity are obtained, and they have the ability to block the binding of PD-1 and PD-L1, enhancing the function of 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 mainly expressed on the surface of T cells and plays a key role in the regulation of the immune system. The main function of PD-1 is to inhibit the activation of T cells, thereby preventing the overreaction of the immune system and maintaining immune homeostasis. When PD-1 binds to its ligands PD-L1 or PD-L2, inhibitory signals are transmitted, resulting in a decrease or inactivation of T cell function. In some diseases, such as cancer, the expression of PD-1 may be abnormally elevated, leading to 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 various cancers. By blocking the binding of PD-1 to its ligands, the anti-tumor activity of T cells is restored, thereby improving the therapeutic effect.

[0003] Anti-PD-1 antibodies are a class of immune checkpoint inhibitors targeting the PD-1 / PD-L1 signaling pathway. By blocking the binding of PD-1 to its ligand PD-L1, the activity of T cells is restored, and the immune response of the body to tumor cells is enhanced. Currently, a variety of PD-1 / PD-L1 antibody drugs have been approved for marketing globally and are used to treat various types of cancers, such as non-small cell lung cancer, melanoma, etc. 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 responses of different patients to PD-1 antibodies, and only some patients can obtain long-term efficacy; drug resistance problems: some patients may develop drug resistance after receiving PD-1 antibody treatment, resulting in treatment failure.

[0004] In summary, developing novel anti-PD-1 antibodies and exploring effective immune checkpoint inhibitors are of great significance for the field of tumor treatment. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art and the actual needs, the present invention provides an anti-PD-1 single-domain antibody and its application, and develops a novel 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, and the complementarity-determining regions of the VHH chain of the anti-PD-1 single-domain antibody include: CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3.

[0008] The present invention immunizes alpacas with PD-1 protein, constructs an antibody display library, and screens out antibodies that can bind to PD-1 protein. The anti-PD-1 single-domain antibody obtained by this method has high specificity and affinity, can effectively block the binding between PD-1 and PD-L1, thereby enhancing the function of immune cells and counteracting the immune escape mechanism of tumors. This anti-PD-1 single-domain antibody, also known as a nanobody, has a small molecular weight, a simple structure, consists of a single domain of the heavy-chain variable region, has high affinity and strong specificity, strong tissue penetration, and weak immunogenicity to the human body, and can be relatively easily modified and amplified. Therefore, 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 includes a human Fc region (which can be called a recombinant anti-PD-1 single-domain antibody).

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

[0017] Preferably, the Ig Fc includes 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] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[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] MAQLQLVESGGGLVQPGGSLRLSCAASGNIFSLNDMTWYRQTPGKQRELVARITSGLSTNYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSSEPKTPKPQPAAASAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0024] In a second aspect, the present invention provides a nucleic acid molecule that encodes 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, and 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, and 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 for binding to programmed death receptor 1.

[0028] The present invention has discovered an anti-PD-1 single-domain antibody with high specificity and affinity, which can be developed into a reagent for immunologically detecting PD1, 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 applications such as tumor treatment.

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

[0030] Preferably, the conjugate substance includes at least one of a cytotoxin, a radioisotope, a luminescent substance, a chromogenic substance, or an enzyme.

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

[0032] Preferably, the pharmaceutical composition is a tumor treatment drug.

[0033] Preferably, the tumor includes at least one of lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer, thyroid cancer, endometrial cancer, brain cancer, pancreatic cancer, nasopharyngeal cancer, cervical cancer, lymphoma, ovarian cancer, bone tumor, melanoma, bladder cancer, prostate cancer, or testicular cancer.

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

[0035] The present invention immunizes alpacas with PD-1 protein, constructs an antibody display library, screens for antibodies that bind to the PD-1 protein, obtains an anti-PD-1 single-domain antibody with high specificity and affinity, and has the ability to block the binding of PD-1 and PD-L1. It can be used for the development of tumor treatment drugs. At the same time, single-domain antibodies are easier to modify, such as humanization, multivalency construction, etc., and have low production costs and are suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the electrophoresis result diagram of the heavy chain antibody fragment amplified by the first round of PCR;

[0037] Figure 2 It is the electrophoresis result diagram of the VHH region fragment amplified by the second round of PCR;

[0038] Figure 3 It is the result diagram of detecting the specificity of the single-domain antibody by flow cytometry;

[0039] Figure 4 It is for detecting the EC of the single-domain antibody by ELISA 50 result diagram;

[0040] Figure 5 It is the result diagram of detecting the blocking function of the single-domain antibody;

[0041] Figure 6A It is the blocking curve diagram of detecting the single-domain antibody by ELISA;

[0042] Figure 6B It is the blocking curve diagram of detecting the negative control antibody by ELISA;

[0043] Figure 6C It is the blocking curve diagram of detecting the positive control antibody by ELISA. Detailed implementation manners

[0044] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0045] For those not specifying specific technologies or conditions in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field, or in accordance with the product specifications. For those reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0046] Unless otherwise defined, the scientific and technical terms and their abbreviations used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The following lists some of the terms and abbreviations used in the present invention.

[0047] The term "amino acid" refers to one of the 20 naturally occurring amino acids or any unnatural analog 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 in the forms generally accepted in the art. The single-letter abbreviations of amino acids are in 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 the variable region of the heavy chain of a cloned antibody, and constructing 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: CDR, refers to the antigen complementary binding region of an antibody.

[0051] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced and its progeny, which can be transformed or transfected with nucleotides encoding a polypeptide, thereby expressing the exogenous polypeptide. 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 (Baby Hamster Kidney cells), myeloma cells, yeast, insect cells, or prokaryotic cells such as Escherichia coli, etc. It should be noted that the "host cells" described in the present invention not only refer to cells into which exogenous nucleic acid has been introduced, but also include the progeny of such cells. Although progeny cells may undergo mutations during cell division, they still fall within the scope of the terms described in the present invention.

[0052] The reagents used in the specific examples 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 Antibody (BD Pharmingen, 560779);

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

[0065] Example 1

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

[0067] Alpacas were immunized with PD-1 protein (Health status of alpacas before immunization: Normal physical development, good nutritional status and mental state, shiny alpaca wool; No abnormalities in movement and behavior, no abnormal limb postures such as lameness and incoordination, alert and responsive; No external injuries were observed externally). Each time, injections were made on the left and right sides near the cervical lymph nodes of the alpacas, with 2 injection points on each side, and approximately 500 μg of emulsified antigen was injected at each point. Immunization was carried out once every 3 weeks for 4 times. Peripheral blood was collected before immunization, 2 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 to PD-1 protein before and after immunization was detected based on the ELISA method.

[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). As shown in Table 1, the binding titers of the sera of alpacas before immunization to PD-1 protein were all lower than 1:1000 (no significant difference from the PBS group, P > 0.05), indicating that the content of anti-PD-1 specific antibodies in them was extremely low. On the contrary, the binding titers of the sera of alpacas after the fourth immunization to PD-1 protein all exceeded 1:64000 (significant difference from the PBS group, P < 0.05), indicating that the content of anti-PD-1 specific antibodies in alpaca serum increased significantly after multiple immunizations, and the immunization was successful and could be used for the construction of a yeast display library.

[0069] Table 1

[0070]

[0071] Example 2

[0072] In this example, a single-domain antibody yeast display library was constructed and the library was screened.

[0073] (1) Cloning of the heavy chain variable region (VHH) fragment

[0074] After confirming that the alpaca serum in Example 1 contained anti-PD-1 specific antibodies, peripheral blood was collected, PBMC was isolated, and RNA was extracted. Reverse transcription was carried out using the PrimeScript™ II 1 st Strand cDNA Synthesis Kit 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 Figure 1 shown. The target fragment of about 750 bp was subjected to electrophoresis using 1% agarose and recovered. Then, using the first-round PCR product as a template, primers (the upstream primer binds to the FR1 region, and 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), and the primer sequence is 5’-GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG-3’ (SEQ ID NO.11)) were used to amplify the VHH fragment of the heavy-chain antibody. The results are as Figure 2 shown. The target fragment of about 450 bp was subjected to electrophoresis using 1% agarose and recovered to obtain a VHH fragment library of the heavy-chain antibody.

[0076] (2) Electroporation of the library vector 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 for single-enzyme digestion, and the linearized vector and target fragment were recovered. After mixing the two, they were added to an electroporation cuvette. After electroporating the yeast competent cells, they were cultured with shaking at 220 rpm and 30 °C for 1 h. Take 20 μL of the resuspension, dilute it 5000-fold with SD-CAA medium, pipette 100 μL, spread it on an SD-CAA plate, culture for 2 days, calculate the library capacity, and continue to culture the remaining 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, resuspend with 10 mL of SD-CAA, add glycerol with a final concentration of 20% - 25%, and store at -80 °C. 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 had large differences, no repeated sequences, and good diversity.

[0078] (3) Screening of the yeast display library

[0079] (3-1) Pretreatment of streptavidin magnetic beads and yeast cells

[0080] Take 8 mL of the yeast library (about 2×10 8Yeast cells) were transferred to a centrifuge tube and centrifuged at 3000×g for 5 min. The supernatant was discarded. At the same time, 5% PBSA (PBS + 5% BSA) was diluted to 0.5% PBSA. The yeast library was resuspended in 1 mL of 0.5% PBSA, transferred to a 1.5 mL centrifuge tube, and centrifuged at 3000×g for 5 min. The supernatant was discarded. It was washed again with 0.5% PBSA. Three centrifuge tubes were prepared (2 for negative selection with empty magnetic beads and 1 for positive selection). 1 mL of 0.5% PBSA was added to each centrifuge tube. The aliquoted streptavidin magnetic beads were resuspended with a pipette, and 10 μL of magnetic beads was added to a 1.5 mL centrifuge tube. These centrifuge tubes were placed in a bag and fixed to a rotary mixer, and incubated with rotation at 4°C for 5 min. The centrifuge tubes were placed on a magnetic stand for 5 min, and the supernatant was aspirated with a pipette. Subsequently, 1 mL of 0.5% PBSA was added again, and it was incubated with rotation at 4°C for another 5 min, and the supernatant was discarded.

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

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

[0083] Negative selection with empty magnetic beads: The centrifuge tube was placed on a magnetic stand, and the washed yeast cell suspension was added to the centrifuge tube containing empty magnetic beads. It was placed in a bag and incubated with rotation at 4°C for 1 h. The centrifuge tube was placed on the magnetic stand for 10 min, and the yeast cell suspension was aspirated and added to a new centrifuge tube with empty magnetic beads, and incubated with rotation at 4°C for 30 min. After the incubation, it was placed on the magnetic stand for 15 min, and the yeast cell suspension was aspirated and transferred to a new centrifuge tube with empty magnetic beads.

[0084] Biotin-PD-1-His Magnetic Sorting: Add 100 μL of 50 μg / mL Biotin-PD-1-His protein solution (biotin-conjugated, diluted with 0.5% PBSA) into a centrifuge tube containing streptavidin magnetic beads. Place the centrifuge tube in a bag and incubate it with rotation at 4°C for 1 h. Then add 1 mL of 0.5% PBSA, let it stand for 5 min while keeping the centrifuge tube on a magnetic stand, and aspirate the supernatant with a pipette. Then add 1 mL of 0.5% PBSA, remove the centrifuge tube from the magnetic stand, mix well by pipetting, place the centrifuge tube back on the magnetic stand again, let it stand for 5 min while keeping the centrifuge tube on the magnetic stand, and aspirate the supernatant; repeat the above washing steps one more time to obtain the positively selected magnetic beads coated with Biotin-PD-1-His. Add the positively selected magnetic beads conjugated with Biotin-PD-1-His to the yeast cells that have completed negative selection, place them in a bag on a rotary mixer, and incubate with rotation at 4°C for 1 h. After incubation, place it on the magnetic stand and let it stand at 25°C for 15 min; aspirate the unbound yeast cell suspension with a pipette. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 1 mL of sterile 0.5% PBSA buffer to the centrifuge tube with a pipette, gently pipette the magnetic beads, then transfer them to a sterile 1.5 mL centrifuge tube, place the centrifuge tube on the magnetic stand, let it stand at 25°C for 5 min, and discard the supernatant; repeat the washing two more times. After washing, resuspend the magnetic beads and the adhered yeast cells in 1 mL of SD-CAA medium, aspirate 20 μL of the resuspended solution into 180 μL of SD-CAA medium for plating, with each plate having a volume of 100 μL. Then aspirate 5 μL of the resuspended solution into 95 μL of SD-CAA medium for plating one plate, so a total of 3 plates need to be plated.

[0085] Flow Cytometry Detection of Yeast after Screening: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into yeast cells. Incubate the yeast cells that have undergone negative selection with empty magnetic beads and Biotin-PD-1-His magnetic sorting with the PD-1 protein containing a His tag at 4°C with rotation for 1 h, centrifuge to remove the supernatant, add flow antibodies against the His tag and the V5 tag, and incubate at 4°C with rotation for 1 h. Centrifuge to remove the supernatant, resuspend the cells in 1 mL of PBS. Centrifuge to remove the supernatant, resuspend the cells in 500 μL of PBS, and perform flow cytometry analysis. The yeast cells that can bind to the PD-1 protein in the alpaca yeast antibody display library before and after sorting increased by 35% respectively, which can be used for subsequent experiments.

[0086] Monoclonal selection: After overnight culture, pick monoclonal colonies and inoculate them into the medium for induced expression. After 48 h, mix the bacterial solution with the His-tagged PD-1 protein and incubate at 4 °C with rotation for 1 h. Centrifuge to remove the supernatant, resuspend the precipitate with a solution containing biotin-conjugated anti-His-tag flow antibody, and incubate at 4 °C with rotation for 1 h. Centrifuge to remove the supernatant, resuspend the precipitate with a buffer containing PE-Streptavidin, and incubate at 4 °C with rotation for 1 h. Resuspend the cells with 1 mL PBS, centrifuge to remove the supernatant, and resuspend the cells with 500 μL PBS for flow cytometry analysis. Finally, select the monoclonal colonies with a high positive rate and name them 195-4-E3. Lyse 195-4-E3 with 0.2% SDS (incubate at 95 °C for 10 min) and centrifuge. Take 0.5 μL of the supernatant of the bacterial solution as a template for PCR amplification and send it for sequencing.

[0087] Further analyze the sequence information of the antibody. The CDR1 region sequence of the single-domain antibody 195-4-E3 is shown in SEQ ID NO.1, the CDR2 region sequence is shown in SEQ ID NO.2, the CDR3 region sequence is shown in SEQ ID NO.3, and the single-domain antibody sequence is shown in SEQ ID NO.4.

[0088] Example 3

[0089] In this example, the expression of the recombinant single-domain antibody and the detection of its binding to the target protein were carried out.

[0090] Design a recombinant antibody containing the single-domain antibody 195-4-E3 and the IgG1 Fc region (SEQ ID NO.6). PCR amplify the 195-4-E3 VHH nucleic acid sequence, the CMV promoter sequence, and the IgG1 Fc nucleic acid sequence respectively. Take 50 μL of the PCR product, add 1 / 10 volume of 10× loading buffer, and perform electrophoresis analysis using 1% agarose. The band size of CMV is 750 bp, the band size of Fc is 1400 bp, and the band size of VHH is 500 bp. Cut the target band from the gel and purify the PCR product. Connect the 195-4-E3 VHH sequence, the CMV promoter, and the IgG1 Fc sequence into the expression vector and transiently transfect it into HEK293 cells. The harvested cell culture supernatant contains the recombinant single-domain antibody 195-4-E3.

[0091] Use the culture supernatant containing 195-4-E3 to react with 3×10 5CHO-K1 and CHO-K1-PD-1 cells (CHO-K1 cells overexpressing PD-1) were incubated at 25 °C for 1 h. The supernatant was discarded by centrifugation, and the cells were washed 3 times with PBS. 100 μl of PE-labeled Anti-human IgG antibody (diluted 1:500) was added, and the cells were incubated at 25 °C in the dark for 45 min. After centrifugation at 800×g at 25 °C for 5 min, the supernatant was discarded, and the cells were washed 3 times with PBS. The cells were resuspended with PBS for flow cytometry analysis. The flow antibody against PD1 was used as a positive control.

[0092] The results are as Figure 3 shown. The expression supernatant of the negative control group did not show significant binding to either 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 the binding detection of PD-1 antibodies. 195-4-E3 did not bind to CHO-K1 cells but showed significant binding to CHO-K1-PD-1, and most of the CHO-K1-PD-1 cells expressing the PD-1 protein could be detected, indicating that the single-domain antibody 195-4-E3 had good affinity and specificity.

[0093] Example 4

[0094] In this example, the purification of the single-domain antibody and the half-maximal effective concentration (EC 50 ) were determined.

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

[0096] Dilute the PD-1 protein with the coating solution to 2 μg / mL, add 100 μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash 5 times with PBST, add 200 μL per well of the blocking solution, and block at 25°C for 2 h. Wash 5 times with PBST, add the serially diluted 195-4-E3 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), and incubate at 25°C for 1 h. After washing 5 times with PBST, dilute HRP-Protein A at 1:50000 and add 100 μL per well to the ELISA plate, and incubate at 25°C for 45 min. After washing 5 times with PBST, add 100 μL of the TMB chromogenic solution to each well, and develop color at 25°C in the dark for 10 min. Add 50 μL of the stop solution to each well, and read the absorbance at a wavelength of 450 nm on an ELISA reader.

[0097] The results are shown in Table 2 and Figure 4 as follows. The EC 50 value of 195-4-E3 for CHO-K1-PD-1 is 0.062 μg / mL, indicating that 195-4-E3 has a high affinity.

[0098] Table 2

[0099]

[0100] Example 5

[0101] In this example, the antibody blocking function was detected.

[0102] Add 100 μL per well of the cell suspension to a 96-well plate. Each well contains 2×10 4 effector cells Jurkat-PD-1-Luciferase (expressing the PD-1 molecule, and the binding of PD-L1 to PD-1 on this cell can inhibit the expression of its Luciferase protein) and 8×10 4One target cell, CHO-K1-PD-L1 (expressing the PD-L1 molecule, which can bind to PD-1 on Jurkat-PD-1-Luciferase cells and inhibit the expression of its Luciferase), was used. 100 μL of 195-4-E3 at different concentrations was added to the corresponding wells to make the final concentrations in the corresponding wells 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 co-culturing for 18 h, 20 μL of One-Glo reagent was added to each well, and the Luciferase fluorescence value was read using a microplate reader.

[0103] Results Figure 5 As shown, the maximum induction effect value of 195-4-E3 was 1.67 (the difference between the maximum Luciferase fluorescence value and the Luciferase fluorescence value at 0 μg / mL), 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 for the research and development of immune-enhancing drugs.

[0104] Example 6

[0105] In this example, the half-inhibitory concentration (IC 50 ) experiment of the recombinant antibody was carried out.

[0106] Dilute the PD-1 protein with coating buffer to 2 μg / mL, aspirate 100 μL per well into a 96-well ELISA plate, and coat overnight at 4°C. Wash 5 times with PBST buffer, add 200 μL per well of blocking solution, and block at 25°C for 2 h. Wash 5 times with PBST buffer, and add 195-4-E3 antibodies at different concentrations (25 μg / mL, 8.3 μg / mL, 2.8 μg / mL, 0.9 μg / mL, 0.3 μg / mL, 0 μg / mL) and the anti-PD-1 positive control antibody Nivolumab respectively, and incubate at 25°C for 15 min. Add biotin-labeled PD-L1 protein (final concentration 4 μg / mL), and incubate at 25°C for 45 min. After washing 5 times with PBST Biotin-PD-L1, dilute the secondary antibody (Streptavidin-HRP) at 1:10000, add 100 μL per well to the ELISA plate, and incubate at 25°C for 45 min. Wash 5 times with PBST buffer, add 100 μL of TMB chromogenic solution to each well, and develop color at 25°C in the dark for 10 min. Add 50 μL of stop solution to each well, and read the absorbance at a wavelength of 450 nm on an ELISA reader. Use the PD-1 antibody 195-3-F11-3, which has no blocking function, screened from the same batch of antibodies, as the negative control, and the Nivolumab antibody as the positive control.

[0107] The results are shown in Table 3 and Figures 6A - 6C as follows, where Figure 6A is the blocking curve of 195-4-E3, Figure 6B is the blocking curve of the negative control antibody, Figure 6C is the blocking curve of the positive control antibody. The IC 50 value of 195-4-E3 is 0.31 μg / mL, and the IC 50 value of the positive control antibody is 0.3 μg / mL. The IC 50 value of 195-4-E3 has no significant difference from that of the positive control antibody Nivolumab, indicating that the blocking ability of 195-4-E3 is comparable to that of the positive control antibody Nivolumab. In addition, in the range of antibody concentration from 2.778 μg / mL to 25 μg / mL, the OD450 value of 195-4-E3 is 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 a greater room for improvement with the increase of 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 out 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 Nivolumab of the prior art at high concentrations, and can also enhance the function of immune cells, showing important application prospects in the field of tumor immunotherapy.

[0111] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the 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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