A single-domain antibody against programmed death molecule 1 and application thereof

Abstract

The present application relates to a kind of single-domain antibody of anti-programmed cell death molecule 1 and its application, 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, the amino acid sequence of CDR3 of the single-domain antibody includes the sequence shown in SEQ ID NO.3.The present application is obtained by using PD-1 protein immunizes alpaca, a kind of single-domain antibody with high specificity, high affinity with PD-1, while can block the combination of PD-1 and PD-L1, enhance the function of immune cell, improve the ability of T cell to eliminate tumor.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more particularly to a single-domain antibody against programmed death molecule 1 and its application. Background Technology

[0002] In the immune system, T-cell-mediated cellular immunity is primarily used to eliminate tumors. T cells need to be activated to exert their anti-tumor effects, and the magnitude and quality of T-cell activation depend on the balance between activating and inhibitory signals. Immune checkpoints can be considered the braking system of immune cells, referring to a series of molecules expressed on immune cells that regulate the level of immune activation, keeping T-cell activation levels within a normal range and preventing damage to healthy tissues.

[0003] Programmed death molecule 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 and is a type I transmembrane glycoprotein composed of 288 amino acids, serving as 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 suppressing autoimmune responses and preventing the occurrence of autoimmune diseases. However, in tumors, PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, leading to downregulation of T cell proliferation and even inducing T cell apoptosis, thus promoting tumor immune escape. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate the immune system and kill tumor cells. Currently, there are various PD-1 targeted monoclonal antibodies available on the market, including nivolumab, pembrolizumab, cimiplimab, toripalimab, and cindilimab. These PD-1 monoclonal antibodies have been successfully used in clinical practice.

[0004] CN111808196A discloses an anti-PD-1 antibody or its antigen-binding fragment, as well as a preparation method and uses. This anti-PD-1 antibody or its antigen-binding fragment can effectively block the binding of PD-1 to its ligand, exhibits strong specificity and high affinity for human PD-1, and in vivo experiments have shown that it is well tolerated in non-human animals, is non-toxic, and significantly inhibits tumor growth.

[0005] Research vectors for conventional antibodies primarily use mice and rabbits. However, a special type of antibody, the heavy chain antibody, exists in the blood of camels and cartilaginous fishes, lacking a light chain. Compared to conventional antibodies, it contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Cloning its variable region yields single-domain antibodies (also known as nanobodies) composed solely of the heavy chain variable region, which possess advantages such as small molecular weight, strong tissue penetration, high stability, and low immunogenicity. Compared to conventional antibodies, alpaca antibodies have several advantages: First, they are small, only one-tenth the size of conventional antibodies, allowing them to penetrate tissues and cells, particularly the blood-brain barrier. Second, they are highly stable, maintaining stability under extreme temperature and pH conditions and retaining biological activity even at temperatures as high as 90°C. Third, they have a simple structure, making them easier to modify, such as through humanization and multivalent construction, and their low production cost makes them suitable for large-scale production. Fourth, it has high specificity. The alpaca's immune system can produce antibodies that have high specificity and affinity for specific antigens, and have weak immunogenicity to humans, thus posing a low risk of immune response.

[0006] In summary, how to utilize PD-1 antibodies to block the PD-1 / PD-L1 pathway to restore T cell tumor activity, activate T cells, kill tumor cells, and prepare anti-PD-1 alpaca antibodies using related technologies 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] To address the aforementioned technical problems, this invention provides a single-domain antibody against programmed death molecule 1 and its application. By immunizing alpacas with PD-1 protein, a highly specific single-domain antibody with high affinity for PD-1 was obtained. Simultaneously, it can block the binding of PD-1 and PD-L1, thereby enhancing the function of immune cells.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a 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] Secondly, the present invention provides a nucleic acid molecule that encodes 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] Thirdly, the present invention provides a heavy chain antibody, the heavy chain antibody comprising the single-domain antibody described in the first aspect.

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

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

[0021] Preferably, the Fc segment amino acid sequence of the 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] Fourthly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

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

[0027] Fifthly, the present invention provides a host cell containing 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 includes immune cells.

[0030] Preferably, the immune cells include T cells.

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

[0032] Preferably, the tumor includes any one or a combination of at least two of the following: 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.

[0033] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

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

[0035] This invention uses PD-1 protein to immunize alpacas. After determining the titer of PD-1-specific antibodies in alpaca serum by ELISA, peripheral blood mononuclear cells of alpacas are isolated, RNA is extracted, and cDNA is obtained through reverse transcription. The VHH sequence is amplified using alpaca single-domain antibody-specific primers and cloned into a yeast expression plasmid to construct a yeast display library. The yeast display library is then subjected to positive and negative screening using biotin-PD-1 protein-streptomycin magnetic beads or streptomycin magnetic beads to enrich yeast cells that bind to PD-1 protein. Single clones are selected from the enriched products, and the specific binding of candidate antibodies to PD-1 is detected by ELISA. After expression and purification of positive antibodies, their affinity for PD-1 protein is tested. Antibody clones with strong affinity for PD-1 are selected, and their ability to block the binding of PD-1 and PD-L1 is tested. The results show that the alpaca antibody has high specificity and can block the binding of PD-1 and PD-L1, thereby enhancing the function of immune cells. Attached Figure Description

[0036] Figure 1 The figure shows the specificity results of flow cytometry detection of recombinant single-domain antibodies.

[0037] Figure 2 EC for ELISA detection of recombinant single-domain antibodies 50 Result image.

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

[0039] Figure 4 IC50 for ELISA detection of recombinant single-domain antibodies 50 Result image. Detailed Implementation

[0040] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] The 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: Determination of Alpaca Immunity and Antibody Titer

[0055] Two alpacas (98# and 195#) were immunized with recombinant PD-1 protein every 3 weeks for a total of 4 immunizations. Peripheral blood was collected before immunization, two weeks after the second immunization, and two weeks after the third and fourth immunizations. The blood was centrifuged at 800×g for 10 min, and the supernatant serum was collected.

[0056] In a 96-well microplate, add 100 μL / well of PD-1-His recombinant protein (1 μg / mL) and coat overnight at 4°C. After washing five times with PBST, add 200 μL / well of blocking buffer and block at 37°C for 2 h. Wash the plate five times with PBST again. Serially dilute the collected serum with PBS, adding 100 μL of each dilution to the 96-well microplate and incubate at room temperature for 1 h. After washing five times with PBST, add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 h. After washing five times with PBST, add 100 μL / well of TMB chromogenic buffer and incubate at room temperature in the dark for 15 min. Add 50 μL / well of stop solution and read the OD values ​​in the wells using a microplate reader. 450 The titer of serum from alpaca #98 after immunization is shown in Table 1, and the titer of serum from alpaca #195 after immunization is 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 PD-1 protein in the serum of both alpacas before immunization were below 1:2000, indicating that the content of anti-PD-1 specific antibodies in their serum was extremely low. Conversely, the binding titers of PD-1 protein in the serum of alpacas #98 and #195 after the fourth immunization were both above 1:64K, indicating that the content of anti-PD-1 specific antibodies in the alpaca serum was significantly increased.

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

[0064] (1) Cloning of VHH antibody fragments

[0065] Peripheral blood was collected from two alpacas (100 mL each). Peripheral blood mononuclear cells (PBMCs) were isolated, RNA was extracted, and cDNA was obtained by reverse transcription. The alpaca heavy chain antibody sequence was amplified by primer PCR. The upstream primer (SEQ ID NO. 8) sequence was 5'-GTCCTGGCTGCTCTTCTACAAGG-3', and the downstream primer (SEQ ID NO. 9) sequence was 5'-GGTACGTGCTGTTGAACTGTTCC-3'. The PCR products were analyzed by agarose gel electrophoresis, and the target fragment of approximately 750 bp was recovered.

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

[0067] (2) Electroconversion of library carriers and detection of library capacity and diversity

[0068] Strawberry competent cells were streaked onto YPD solid medium plates and activated at 30°C for 3-5 days. Single colonies of competent yeast were inoculated into 50 mL of YPD medium and incubated at 250 rpm and 30°C for 1-2 days. The yeast surface display vector pYDisplay digested with SfiI and the VHH fragment library were mixed and added to an electroporation cuvette for electroporation. The electroporated competent yeast cells were then transferred to culture flasks and incubated at 220 rpm and 30°C for 1 hour. 20 μL of the resuspension was diluted 5000 times with SDCAA, and 100 μL was plated on an SDCAA plate and incubated for 2-3 days. The library volume was calculated, and the remaining culture was incubated for another 24 hours. The remaining culture was collected into 50 mL centrifuge tubes, centrifuged at 3000×g for 5 min, the supernatant was discarded, and the cells were resuspended in 10 mL of SDCAA. The mixture was then mixed with 50% glycerol at a 1:1 ratio and stored at -80°C.

[0069] The results showed that the single-domain antibody yeast display library obtained from alpaca PBMC #98 had a volume of 2.4 × 10⁻⁶. 9The single-domain antibody yeast display library obtained from alpaca PBMC #195 has a volume of 1.2 × 10⁻⁶. 9 .

[0070] Example 3: Selection of yeast display library

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

[0072] Take 2×10 8 Yeast library, centrifuged at 3000×g for 5 min, supernatant discarded, resuspended in 1 mL 0.5% PBSA in 1.5 mL centrifuge tubes, centrifuged at 3000×g for 5 min, supernatant discarded. Add 1 mL 0.5% PBSA and 10 μL magnetic beads to three centrifuge tubes, fix on a rotary mixer, and incubate at 4°C for 5 min. Place the centrifuge tubes on a magnetic rack for 5 min, then discard the supernatant. Add 1 mL 0.5% PBSA again, incubate at 4°C for another 5 min, then discard the supernatant.

[0073] (2) Yeast Display Library Selection

[0074] Negative selection: Add the yeast culture to a centrifuge tube containing empty magnetic beads and incubate at 4°C with rotation for 60 min. Place the centrifuge tube containing the incubated yeast on a magnetic rack for 10 min, then transfer the yeast culture to a new centrifuge tube containing empty magnetic beads and incubate at 4°C with rotation for 30 min. After incubation, place the centrifuge tube on a magnetic rack and aspirate the yeast culture to a new centrifuge tube containing empty magnetic beads.

[0075] Biotin-PD-1-His magnetic sorting: 100 μL of 50 μg / mL biotin-PD-1-His protein solution was added to a centrifuge tube containing streptavidin magnetic beads and incubated at 4°C for 60 min. Then, 1 mL of 0.5% PBSA was added, and the mixture was allowed to stand for 5 min, while the centrifuge tube remained on a magnetic rack. The supernatant was discarded. Then, 1 mL of 0.5% PBSA was added again, and the mixture was mixed by pipetting. The centrifuge tube was again placed on a magnetic rack and allowed to stand for 5 min. The supernatant was discarded. This washing process was repeated once more to obtain biotin-PD-1-His-coated positively sorted magnetic beads. The biotin-PD-1-His-bound positively sorted magnetic beads were added to negatively sorted yeast cells and incubated at 4°C for 60 min. After incubation, the cells were placed on a magnetic rack and allowed to stand at room temperature for 15 min. The unbound yeast culture was then discarded. Add 1 mL of sterile 0.5% PBSA buffer, gently pipette, place on a magnetic rack, and incubate at room temperature for 5 min. Discard the supernatant and repeat the washing process twice, adhering to aseptic techniques throughout. After washing, resuspend the magnetic beads and adhered yeast cells in 1 mL of SDCAA medium. Transfer 20 μL of the resuspended solution to 180 μL of SDCAA medium and spread it onto two plates. Each plate should have a volume of 100 μL. Then, transfer another 5 μL of the resuspended solution to 95 μL of SDCAA medium and spread it onto one plate.

[0076] Flow cytometry analysis after sorting: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transformed into yeast. The sorted yeast cells were incubated with PD-1 protein containing the His tag at 4°C for 60 min by rotation, and the supernatant was discarded. Flow cytometry antibodies against the anti-His tag and anti-V5 tag were added, and the cells were incubated at 4°C for 60 min by rotation, and the supernatant was discarded by centrifugation. The cells were resuspended in 1 mL PBS, and the supernatant was discarded by centrifugation. The cells were resuspended in 500 μL PBS, and flow cytometry analysis was performed. Before and after sorting, the number of yeast cells capable of binding to the PD-1 protein in the yeast antibody display libraries of alpacas #98 and #195 increased by 38% and 35%, respectively.

[0077] Single-clone selection: Single clones were inoculated into culture medium to induce expression. After 48 h, the bacterial culture was incubated with PD-1 protein containing the His tag at 4 °C for 60 min by rotation, followed by centrifugation and removal of the supernatant. The pellet was resuspended in a solution containing biotin-conjugated anti-His tag flow cytometry antibody, incubated at 4 °C for 60 min by rotation, and then centrifuged to remove the supernatant. The pellet was then resuspended in a solution containing PE-streptavidin, incubated at 4 °C for 60 min by rotation, and the cells were resuspended in 1 mL PBS, followed by centrifugation and removal of the supernatant. The cells were then resuspended in 500 μL PBS for flow cytometry analysis. The single clone 195-4-G5, with a high positive rate, was finally selected and amplified by PCR for testing.

[0078] Example 4: Expression of recombinant single-domain antibody and detection of its binding to target protein

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

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

[0081] The results are as follows Figure 1 As shown, the expression supernatant of the negative control group did not significantly bind to either 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 most CHO-K1-PD-1 cells expressed PD-1 protein, indicating that 195-4-G5 has good affinity and specificity.

[0082] Example 5: Purification and half-maximal effect concentration (EC5) of recombinant antibody 50 ) Measurement

[0083] Since the target recombinant antibody contains human IgG fragments, affinity purification can be performed using Protein A magnetic beads. Wash the Protein A magnetic beads twice sequentially with 30 mL PBS buffer, 0.1 M sodium hydroxide, and PBS buffer. Add the appropriate volume of Protein A magnetic beads to the expression supernatant of 293 cells according to the required sample volume (calculated as 20 mg IgG / mL Protein A magnetic beads). Incubate at 120 rpm at room temperature for 1-4 h or overnight at 4°C. Collect the Protein A magnetic beads using a magnetic separator. Wash twice with 30 mL PBS buffer and deionized water, then resuspend in 1 mL elution buffer. Incubate at room temperature for 5 min, then collect the magnetic beads using a magnetic separator. Repeat the elution process twice with the Protein A magnetic beads, combine the eluents, and add neutralization buffer to adjust the pH. Dialyze the eluted sample with 100 times its volume of PBS, first at 18–25°C for 2 h, changing the buffer once, then at 2–8°C for 14–16 h, and determine the protein concentration.

[0084] Dilute PD-1 protein to 2 μg / mL using coating buffer, and pipette 100 μL / well into a 96-well microplate. Coat overnight at 4°C. Wash 5 times with PBST, add 200 μL / well of blocking buffer, and block at room temperature for 2 h. Wash 5 times with PBST, add different concentrations of 195-4-G5 candidate antibody (Table 3), and incubate at room temperature for 60 min. Wash 5 times with PBST, dilute HRP-Protein A 1:50000, add 100 μL / well into the microplate, and incubate at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB chromogenic solution to each well, and develop at room temperature in the dark for 10 min. Add 50 μL of stop buffer to each well, and read the absorbance at 450 nm using a microplate reader.

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

[0086] Table 3

[0087]

[0088] Example 6: Detection of Recombinant Antibody Blocking Function

[0089] In a 96-well plate, 2×10⁻⁶ ppm was added to each well. 4 One effector cell, Jurkat-PD-1-luciferase (expressing PD-1 molecules, which can inhibit luciferase expression after binding to PD-L1) and 8 × 10 4 Target cells were CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of Jurkat-PD-1-luciferase). Different concentrations of 195-4-G5 were added to the corresponding wells, resulting in final concentrations of 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 Lone-Glo reagent was added to each well, and the fluorescence values ​​were read.

[0090] result Figure 3 As shown, the maximum induction effect value of 195-4-G5 was 1.66 (the difference between the maximum fluorescence value and the fluorescence value at 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 (IC50) 50 )experiment

[0092] To determine the half-maximal inhibitory concentration (IC50) of 195-4-G5 in blocking the binding of PD-1 and PD-L1, PD-1 protein was diluted to 2 μg / mL using coating buffer, and 100 μL / well was added to each well of a 96-well microplate. The plates were incubated overnight at 4°C. After washing five times with PBST, 200 μL / well of blocking buffer was added, and the plates were blocked at room temperature for 2 h. After washing five times with PBST, different concentrations (Table 4) of the 195-4-G5 candidate antibody were added, and the plates were incubated at room temperature for 15 min. Biotin-PD-L1 protein (final concentration 4 μg / mL) was added, and the plates were incubated at room temperature for 45 min. After washing five times with PBST, 100 μL of TMB chromogenic buffer was added to each well, and the plates were incubated at room temperature in the dark for 10 min. Add 50 μL of stop solution to each well and read the absorbance at 450 nm wavelength on a microplate reader.

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

[0094] Table 4

[0095]

[0096] In summary, this invention uses PD-1 protein to immunize alpacas, amplifies the VHH sequence using alpaca single-domain antibody-specific primers, clones it into a yeast expression plasmid, constructs a yeast display library, performs positive and negative screening, selects single clones from the enriched products, and detects the specific binding of candidate antibodies to PD-1 using ELISA. After expression and purification of positive antibodies, their affinity for PD-1 protein is detected, and antibody clones with strong affinity for PD-1 are selected to test their ability to block the binding of PD-1 and PD-L1. The results show that the single-domain antibody provided by this invention has high specificity and can block the binding of PD-1 and PD-L1, thereby enhancing the function of immune cells.

[0097] 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 in the present invention fall within the protection 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 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the single-domain antibody is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 of the single-domain antibody is 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 is shown in SEQ ID NO.

4.

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

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

5.

5. A heavy chain antibody, characterized in that, The heavy chain antibody includes the single-domain antibody as described in 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 is any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4; The amino acid sequence of the Fc segment of the IgG1 is shown in SEQ ID NO.6; The amino acid sequence of the heavy chain antibody is shown in SEQ ID NO.

7.

7. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 3 or 4.

8. A host cell, characterized in that, The host cell contains the expression vector as described in 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. The use of any one or a combination of at least two of the following in the preparation of a tumor therapeutic agent: the single-domain antibody of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the heavy chain antibody of claim 5 or 6, the expression vector of claim 7, the host cell of claim 8, or the pharmaceutical composition of claim 9; wherein the tumor is any one of colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, bile duct cancer, lung cancer, or nasopharyngeal carcinoma.

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