Anti-BTLA nano antibody, preparation method and application

Abstract

The invention relates to an anti-BTLA nano antibody in the technical field of biological medicines. The amino acid sequence of the nano antibody is selected from one of SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3. The preparation method comprises three steps of BTLA antigen purification, phage display library panning and nano antibody expression purification, a high-purity antigen is obtained through secondary purification of Ni resin and an anion column, high-affinity clone is screened through four rounds of panning and ELISA verification, and a final product is high in purity and stable in yield. The nano antibody has the advantages of high specificity, high stability, excellent tissue penetrability and low-cost production, can efficiently block a BTLA-HVEM channel and activate T cell immune response, has a remarkable effect in treatment of tumors such as melanoma and lymphoma and rheumatoid arthritis, can be used as an ELISA or immunohistochemical reagent to detect the BTLA expression level, and has a wide application prospect. Important clinical application values and commercial prospects are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an anti-BTLA nanobody, a preparation method and application, and especially to a preparation method of a nanobody against B and T lymphocyte attenuator (BTLA) and application thereof in tumor immunotherapy. BACKGROUND

[0002] Precise regulation of immune system homeostasis is the key to maintaining the health of the body. Immune checkpoint molecules play a core role in the establishment of immune tolerance and disease prevention and control by regulating the intensity of immune response. B and T lymphocyte attenuator (BTLA) is an important immune checkpoint molecule, mainly expressed on the surface of lymphocytes, which negatively regulates the activation of T cells and B cells by binding to the ligand HVEM, and plays a key role in immune tolerance and autoimmune diseases. In recent years, antibodies targeting BTLA have shown broad application prospects in tumor immunotherapy, autoimmune diseases and inflammatory diseases.

[0003] As mentioned in the anti-BTLA antibody drug composition and its use disclosed in CN 113134081 A and the anti-BTLA antibody disclosed in CN 112638943 A, however, the existing antibodies (such as IgG) all have the problems of large molecular weight, poor tissue penetration, high production cost, and may cause immunogenicity. Nanobodies (VHH) as a new type of therapeutic antibody fragment have unique advantages such as small molecular weight (about 15 kDa), strong stability, excellent penetration, easy recombinant expression and humanization modification, which can effectively overcome the shortcomings of traditional antibodies. However, the research on specific nanobodies against BTLA is still in its infancy, and there is a lack of candidate molecules with high affinity, strong stability and low cost production characteristics. Therefore, the development of an anti-BTLA nanobody that can efficiently block the BTLA-HVEM pathway and activate immune response has important clinical value and commercial prospects for improving the effect of immunotherapy and reducing treatment costs, and can fill the technical gap in this field. SUMMARY

[0004] The present application aims to provide a specific nanobody against BTLA, which has solved the defects of the prior art.

[0005] To achieve the above-mentioned purpose, one of the purposes of the present application is to provide an anti-BTLA nanobody, wherein the amino acid sequence of the nanobody is selected from one of SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3.

[0006] Specifically, the nanobody comprises a framework region and a complementarity determining region containing a key binding site; the N-terminus or C-terminus of the nanobody can be connected to a His tag or a Strep tag, and is expressed in 293F cells, CHO cells or E. coli through a pATX1 or pTXs1 vector system.

[0007] The second object of the present application is to provide a nucleic acid encoding the anti-BTLA nanobody.

[0008] The third object of the present application is to provide an expression vector comprising an expression frame of the nucleic acid.

[0009] The fourth object of the present application is to provide a host cell comprising the expression vector.

[0010] The fifth object of the present application is to provide a kit of anti-BTLA nanobody.

[0011] The sixth object of the present application is to provide an application of the anti-BTLA nanobody in preparing a drug for preventing and / or treating tumors or autoimmune diseases.

[0012] Further, the tumor is selected from melanoma and lymphoma.

[0013] The seventh object of the present application is to provide a preparation method of the anti-BTLA nanobody, characterized in that the expression vector of claim 4 is constructed, the expression vector is transferred into a host cell, expression is induced, and purification is performed to obtain the product.

[0014] Further, the method comprises the following steps: (1) BTLA antigen purification: the nucleotide sequence of BTLA is obtained and cloned into a suitable expression vector, transformed into a host cell for expression, and after the expression product is collected, at least one purification treatment is performed to obtain a BTLA antigen with a purity of ≥90%; (2) phage display library panning: the BTLA antigen is used as a target to coat a solid phase carrier, a VHH phage display library is added, and specific binding phages are enriched through multiple rounds of negative screening and positive screening, and high-affinity positive clones are screened through immunological detection methods; (3) nanobody expression and purification: the coding sequence of the positive clone is cloned into an expression vector, transformed into a suitable host cell for expression, and after the expression supernatant is collected, affinity purification treatment is performed to obtain an anti-BTLA nanobody pure product.

[0015] Further, the preparation method comprises: Step 1: BTLA antigen purification The nucleotide sequence of BTLA is obtained through sequencing, and the obtained nucleotide sequence is cloned into a pATX2 vector.

[0016] The obtained pATX2-BTLA plasmid is transformed into 293F cell strain, a single colony is selected in LB, and activated at 37℃ overnight. 1.0ml sample is taken for expression test on the 5th day after transfection, and after rapid purification, SDS-PAGE and WB detection are performed respectively.

[0017] On the 6th day after transfection, the culture supernatant is collected, and Ni resin purification is performed multiple times, gradient elution is performed through buffer A and buffer B, the sample is collected for SDS-PAGE analysis, and the protein purity is determined to be >90%.

[0018] Step 2: Phage display library panning (1) The immunotube is coated with BTLA protein (40 μg / mL), and the displayed library is combined with the antigen, and after 4 rounds of negative screening (containing NC-his control) and positive screening, specific phage is eluted and enriched; (2) The positive clones are verified by polyclonal ELISA and monoclonal ELISA, and high-affinity nanobodies (such as clone 24000333F-2-R4P1-F8) are screened.

[0019] Step 3: Expression and purification of nanobodies (1) The positive clones are subcloned into the vector pATX1, and 293F or CHO cells (30-400 mL system) are transfected; (2) The culture supernatant is collected and purified by Strep-tactin resin or Ni column (buffer: PBS pH 7.5, eluent containing 300 mM imidazole or 50 mM Biotin); (3) The purity is verified by SDS-PAGE, the final product concentration is 0.14-0.57 mg / mL, and the yield is 0.28-0.71 mg.

[0020] The application provides an anti-BTLA nanobody and a preparation method thereof. The nanobody can efficiently bind to BTLA, block the interaction with HVEM, and activate T cell immune response. Compared with the prior art, the application has the following advantages: 1. High specificity: the nanobody screened by phage display technology has high specificity for BTLA. 2. Strong stability: the nanobody remains active under high temperature and extreme pH conditions, is suitable for long-term storage and in vivo application, retains >95% activity after being stored in PBS at 4℃ for 30 days, and is resistant to repeated freeze-thawing. 3. Excellent penetration: the small molecular weight (about 15 kDa) makes it easy to penetrate tumor tissues or inflammatory sites. 4. Low-cost production: can be efficiently expressed by prokaryotic or mammalian systems, the purification process is simple, the expression amount in a mammalian system is 5-10 mg / L, and the purification yield is >80%.

[0021] The nanobodies of this invention can be used to treat tumors (melanoma, lymphoma), autoimmune diseases (rheumatoid arthritis), or as ELISA or immunohistochemical reagents to detect BTLA expression levels. Attached Figure Description

[0022] Figure 1 Here is a flowchart of the BTLA nanobody screening process; A: Schematic diagram of BTLA nanobody construction; B: First round of PCR; C: Second round of PCR; D: Library insertion rate in colony PCR analysis.

[0023] Figure 2 This is a structural diagram of a BTLA nanobody.

[0024] Figure 3 SDS-PAGE image of BTLA nanobody; Coomassie Brilliant Blue staining.

[0025] Figure 4 This is a schematic diagram of an ELISA experiment in which BTLA nanobodies are bound to antigens for screening.

[0026] Figure 5 A schematic diagram illustrating how BTLA nanobodies enhance the expression of CD25 activation molecules in CD8+ T cells.

[0027] Figure 6 A schematic diagram illustrating the anti-tumor effect of BTLA nanobodies on CD8+ T cells. Detailed Implementation

[0028] The following detailed description illustrates the specific implementation method: Step 1: BTLA antigen purification (1) Nucleotide sequence of BTLA CCGCCACCATGAAGACCCTGCCTGCCATGCTGGGCACAGGCAAGCTGTTCTGGGTGTTCTTCCTGATCCCCTACCTGGATATCTGGAATATCCACGGCAAGGAGAGCTGCGACGTGCAGCTGTACATCAAGAGACAGAGCGAGCACAGCATCCTGGCCGGCGACCCTTTCGAGCTGG; AGTGTCCTGTGAAGTACTGCGCCAATAGGCCTCACGTGACATGGTGCAAGCTGAATGGCACCACCTGTGTGAAGCTGGAGGATAGGCAGACCAGCTGGAAGGAGGAGAAGAATATCTCCTTCTTCATCCTGCACTTCGAGCCTGTGCTGCCCAACGACAACGGCAGCTACAGGTGCAG; The sequence CGCCAACTTTCAGAGCAATCTGATCGAGTCCCACTCCACCACCCTGTACGTGACCGACGTGAAGAGCGCCAGCGAGAGACCCTCCAAGGACGAGATGGCCTCCGGCAGCCACCACCACCACCACCATCACTGAGCGGCCGC was obtained by sequencing by Pujian Biotechnology (Wuhan) Co., Ltd., and the obtained nucleotide sequence was cloned into the pATX2 vector.

[0029] (2) The obtained pATX2-BTLA plasmid was transformed into the pATX2 strain, and single colonies were selected and activated overnight at 37°C. On the 5th day after transfection, 1.0 ml of sample was taken for expression testing. After rapid purification, SDS-PAGE and WB detection were performed respectively.

[0030] (3) On day 6 post-transfection, the culture supernatant was collected and purified using His-tagged Ni resin. The Ni resin column was equilibrated with PBS pH 7.5. The culture supernatant was then loaded onto the equilibrated column and washed sequentially with PBS pH 7.5 buffer containing 30 mM and 50 mM imidazole. Elution was then performed with PBS pH 7.5 buffer containing 300 mM imidazole, and the eluent was collected. Subsequently, the protein collected by Ni resin was subjected to secondary purification using an anion exchange column. A HiTrap Q HP 5 ml resin column was used. The column was first equilibrated with buffer A containing 20 mM Tris pH 7.5. The protein collected by Ni resin purification was then loaded onto the equilibrated anion exchange column and eluted using a gradient of buffer A (20 mM Tris pH 7.5) and buffer B (20 mM Tris pH 7.5, 1000 mM NaCl). The elution products were collected. Finally, a second amplification purification was performed, repeating the above His-tagged Ni resin purification process. The procedure for Ni resin purification and anion exchange column secondary purification is as follows: First, equilibrate the Ni resin column with PBS pH 7.5, load the sample, wash with PBS pH 7.5 buffer containing 30mM and 50mM imidazole, elute with PBS pH 7.5 buffer containing 300mM imidazole and collect the elution product, then use Ni resin to collect the protein for secondary purification with anion exchange column. Equilibrate a 5 ml HiTrap QHP resin column with buffer A, load the sample, elute with a gradient of buffer A and buffer B, collect the sample and perform SDS-PAGE analysis to determine protein purity >90%.

[0031] Step 2: Alpaca Immunization (1) Emulsify the purified BTLA antigen and adjuvant at a 1:1 ratio to form a homogeneous mixture and store at 4°C. Take a blank alpaca and inject it into the lymph nodes near the alpaca's neck on both sides, with two injection points on each side. Inject approximately 0.4 mL of emulsified antigen at each point. Observe the alpaca for half an hour after immunization to confirm that it is in good condition and has no adverse symptoms. Immunize once every 2 weeks, for at least 4 immunizations. Before each antigen immunization, collect 5 mL of blood for immune evaluation. On the same day, centrifuge the blood at 400 xg for 30 minutes using a pre-cooled 25°C centrifuge, separate and preserve the supernatant serum for subsequent antibody titer detection.

[0032] (2) Lymphocyte isolation: On day 7 after the fourth immunization, 50 ml of blood was collected from the jugular vein of the alpaca. 15 ml of cell separation medium was added to a 50 ml centrifuge tube, followed by 15 ml of blood. The tube was centrifuged at 400 xg for 30 minutes at a pre-cooled temperature of 25°C. The supernatant serum was collected and stored in a new centrifuge tube at -80°C. The middle layer of immune cells (resembling cotton) was pipetted into a 50 ml centrifuge tube, 10 ml of PBS buffer was added, and the tube was centrifuged at 400 xg for 20 minutes at 25°C. The supernatant was discarded, 5 ml of PBS buffer was added, and the cell count was determined using a hemocytometer. The cell count was then centrifuged at 400 xg for 20 minutes at 25°C. The supernatant was discarded, and the isolated lymphocytes were lysed using RNAiso Plus to obtain a 10⁷ / mL cell lysate, which was stored at -80°C.

[0033] Step 3: Phage Library Construction (1) RNA extraction: Lymphocyte samples preserved with TRIzol reagent were thawed on ice and transferred to 1.5 mL centrifuge tubes. 1 / 5 volume of chloroform was added, and the mixture was vigorously vortexed and allowed to stand at room temperature for 5 minutes. Then, the mixture was centrifuged at 12,000 × g for 15 minutes at 4°C. The supernatant was carefully transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was gently inverted and allowed to stand at room temperature for 10 minutes. Next, the mixture was centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was discarded. The precipitate was washed once with pre-chilled 75% ethanol (centrifuged at 7,500 × g for 5 minutes at 4°C), the ethanol was discarded, and the precipitate was air-dried at room temperature. The RNA precipitate was dissolved in an appropriate amount of RNase-free water. Following the reverse transcription kit instructions, the total RNA was divided into two aliquots, and reverse transcribed using Oligo(dT)18 primers and RandomHexamers primers, respectively, to synthesize cDNA.

[0034] (2) Antibody fragment amplification: Using the synthesized cDNA as a template, the first round of PCR amplification was performed using Alpa001F / R primers and Taq DNA Polymerase (Hot Start Version). The 50 μL reaction system contained: 2 μL cDNA template, 2 μL each of Alpa001F / R primers, 5 μL 10× Taq Buffer, 4 μL dNTP mixture, 0.25 μL Taq (HS) enzyme, and ddH2O to make up the volume. The reaction program was: 98℃ pre-denaturation for 3 minutes; followed by 22 cycles (each cycle: 95℃ for 30 seconds, 57℃ for 30 seconds, 72℃ for 40 seconds, with the extension time increasing by 2 seconds per cycle); and a final extension at 72℃ for 5 minutes. A portion of the product was subjected to 1% agarose gel electrophoresis, and two specific bands were visible at approximately 1.0 kb and 0.7 kb. The 0.7 kb band was excised and purified using a DNA gel extraction kit. Using the purified and recovered DNA fragment as a template, a second round of PCR amplification was performed using Alpa002F / R primers and TaqDNA Polymerase (Hot Start Version). The 50 μL reaction volume was the same as above, only the primers were changed. The reaction program was: 98℃ pre-denaturation for 3 minutes; followed by 12 cycles (each cycle: 95℃ for 50 seconds, 55℃ for 30 seconds, 72℃ for 40 seconds); and a final extension at 72℃ for 10 minutes. The PCR products were recovered using a DNA purification and recovery kit, and the concentration was determined before use.

[0035] (3) Cloning to phage plasmids: The antibody gene fragment purified in the previous step and the phage display vector were double-digested with Sfi I and Not I restriction endonucleases, respectively. After purification of the digestion products, they were ligated overnight at 16°C using T4 DNA ligase at an appropriate molar ratio. The ligation products were recovered using a DNA purification and recovery kit according to the instructions and dissolved in ultrapure water. 100 ng of the ligation product was gently mixed with 100 μL of TG1 electroporation competent cells thawed on ice and transferred to a pre-chilled electroporation cuvette. Electroporation was performed using the preset bacterial transformation program of the electroporator. Immediately after electroporation, 1 mL of SOC medium was added to the cuvette, and the bacterial culture was transferred to a sterile tube and incubated at 37°C and 220 rpm for 60 minutes with shaking. To obtain a high-capacity library, this transformation process should be repeated at least 20 times. All the revived bacterial cultures were mixed and evenly spread on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C overnight. After incubation, add an appropriate amount of 2×YT medium to the plate, gently scrape off all colonies with a sterile spreader, and collect the bacterial suspension. Add sterile glycerol to a final concentration of 20%, mix thoroughly, aliquot, label as a primary antibody library, and store at -80℃.

[0036] (4) Amplification and purification of the phage library: A portion of the bacterial culture was taken from the primary library stored at -80℃ and inoculated into 100 mL of 2×YT medium containing 100 μg / mL ampicillin at an inoculation rate of approximately 10^9 cells. The culture was incubated at 37℃ with shaking at 220 rpm until OD600 ≈ 0.5. Helper phage (M13K07) was added at a ratio of helper phage to bacteria of approximately 20:1, and the culture was incubated at 37℃ for 30 minutes. Then, kanamycin was added to a final concentration of 50 μg / mL, and the culture was transferred to a shaker at 30℃ and incubated overnight with shaking at 220 rpm. The overnight culture was centrifuged at 4℃, 13,000 × g for 15 minutes, and the supernatant was collected. 1 / 4 volume of pre-chilled PEG / NaCl solution (e.g., 5× PEG8000 / NaCl) was added to the supernatant, and the culture was incubated on ice for 30 minutes to precipitate the phage. The sample was then centrifuged at 13,000 × g for 10 minutes at 4°C, and the supernatant was discarded. The precipitate was resuspended in 1 mL PBS, and 250 μL of PEG / NaCl solution was added again. The sample was incubated on ice for 10 minutes. Finally, the sample was centrifuged at 16,000 × g for 15 minutes at 4°C, and the supernatant was discarded. The precipitate was thoroughly resuspended in 1 mL PBS to obtain the purified anti-human BTLA phage display antibody library. After titer determination, the sample was aliquoted and stored at 4°C for a short period.

[0037] Step 4: Antibody Screening (1) Selection: 1 mL of BTLA (40 μg / mL, CBS buffer) was coated onto each immunotube and incubated overnight at 4°C. The immunotubes were then washed three times with 5 mL PBST, and 5 mL of 5% skim milk / PBST was added. After blocking at 30°C for 1 h, the tubes were washed once with 5 mL PBS. Negative screening of the phage library was then performed. NC-his (50 μg) and 1% skim milk / PBST (or 0.2% casein / PBST) were added to a phage library of 1.0 x 10¹² pfu. PBS was added to bring the total volume to 1 mL, and the tubes were incubated at room temperature for 0.5 h. The negatively screened phages were added to the immunotubes and incubated at 30°C for 2 h. The tubes were then washed 4-6 times with 5 mL PBST. Gly-HCl (pH = 2.2) was added to the immunotubes to elute the phages, and the tubes were incubated with shaking at room temperature for about 6-8 min. Tris-HCl (pH = 2.2) was then added to the immunotubes. 9.6) Neutralize the solution to pH = 7.0-8.0; finally, dilute the eluted phage and infect logarithmic-phase Escherichia coli TG1, then plate and determine the titer.

[0038] (2) Amplification of eluted phages: The eluted phages were aspirated and added to the logarithmic-phase Escherichia coli TG1 bacterial culture. After standing at 37°C for 30 min, the culture was incubated at 220 rpm for 30 min-1 h. Then, the antibiotic Amp was added to the culture medium and the culture was incubated at 37°C and 220 rpm until the OD of the bacterial culture reached approximately 0.4-0.6. Subsequently, helper phages were added to the bacterial culture, and the culture was stood at 37°C for 30 min, then incubated at 220 rpm for 45 min-1 h. Afterward, the bacterial culture was centrifuged at 3000-5000 rpm, the supernatant was discarded, and the bacterial culture was resuspended in an equal volume of 2YT-Amp-Kan medium and incubated overnight at 30°C and 220 rpm. The next day, the culture was centrifuged at 8000 rpm and 4°C for 20 min, the supernatant was transferred to a new centrifuge tube, 1 / 4 volume of 5*PEG / NaCl solution was added, and the mixture was thoroughly mixed and placed on ice or at 4°C for 1-2 h. The culture was then centrifuged at 8000 rpm and 4°C for 30 min. After 10 min, discard the supernatant, resuspend the precipitate in about 1 mL of PBS, centrifuge at 8000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube; finally, dilute the amplified phage, infect TG1 in the logarithmic phase, plate it, and determine the titer.

[0039] (3) Polyclonal phage ELISA detection: Prepare Ag (4 μg / mL BTLA), NC1 (4 μg / mL NC-his), and NC2 (PBS) using CBS buffer. Add 100 μL to each well and incubate overnight at 4°C. Then wash the immunoassay plate three times with 300 μL PBST. Add 300 μL 5% skim milk / PBST, block at 30°C for 1 hour, and wash 2-3 times with 300 μL PBST. Perform 3-fold serial dilutions of the phage after each round of amplification using PBS (initial concentration 1×10⁻⁶). 12 Add 100 μL of diluted amplified phage (pfu / mL) to each well and incubate at 30°C for 1 hour. Wash 4-6 times with 300 μL PBST. Then add 100 μL of secondary antibody (anti-phage M13) dilution buffer and incubate at 30°C for 1 hour. Wash 4-6 times with 300 μL PBST again. Finally, add 100 μL of TMB chromogenic buffer and incubate in the dark for 3-8 minutes. Stop the reaction with 100 μL of 2M HCl and read the values ​​using a microplate reader at 450 nm-620 nm. Based on the polyclonal results, we decided to plate the fourth round of elution products for single-clonal selection.

[0040] (4) Single-clone phage ELISA screening: The elution product of R4 was selected and diluted to an appropriate concentration for infecting TG1 bacterial suspension in the logarithmic phase, followed by plate culture. The next day, 96 single clones were picked from the plate and inoculated into 96-well plates, and cultured with shaking at 37℃ and 250 rpm until the OD value of the bacterial suspension reached 0.4-0.6. Then, helper phages were added to the deep well plates, and the plates were incubated at 37℃ for 30 minutes and then cultured with shaking at 250 rpm for 45 minutes to 1 hour. After infection, the deep well plates were centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were resuspended in 2YT-Amp-Kan medium in each well and cultured with shaking at 30℃ and 250 rpm overnight. The deep well plates were centrifuged again the next day (4000 rpm, 10-15 minutes), and the supernatant was collected for subsequent ELISA experiments. In the ELISA experiment, the immunoassay plate needs to be coated first: Add 4 μg / mL BTLA antigen (Ag) to the positive control wells and 4 μg / mL NC-his protein to the negative control wells (NC), 100 μL per well, and incubate overnight at 4°C. The next day, wash three times with 300 μL PBST, then add 300 μL of 5% skim milk / PBST solution, and block at 30°C for 1 hour. After blocking, wash 2-3 times with PBST. Add 100 μL of phage supernatant to each well, incubate at 30°C for 1 hour, and then wash 4-6 times with PBST to remove unbound phage. Next, 100 μL of anti-M13 secondary antibody dilution buffer was added, and the mixture was incubated at 30°C for 1 hour. After washing 4-6 times, 100 μL of TMB chromogenic buffer was added to each well for incubation in the dark for 3-8 minutes. Finally, 100 μL of 2M HCl was added to terminate the reaction. The absorbance values ​​were measured at 450 nm and 620 nm using a microplate reader to analyze the experimental results. The Ag group polyclonal results from the fourth round of screening were sent for sequencing, and 14 sequences were obtained. We performed secondary validation on these sequences.

[0041] (5) Secondary verification of positive clones: After sequencing the positive clones obtained from single-clone screening, the repetitive sequences were removed to obtain the final positive clones, which were then verified by a secondary ELISA test to ensure the authenticity of the positive results. First, the immunoassay plate was coated. 4 μg / mL BTLA antigen was added to the positive well (Ag), 4 μg / mL NC-his protein was added to the negative control 1 (NC1), and PBS was added to the negative control 2 (NC2), 100 μL per well, and incubated overnight at 4°C. The next day, the plate was washed 3 times with 300 μL PBST, followed by the addition of 300 μL 5% skim milk / PBST solution, and blocked at 30°C for 1 hour. After blocking, the plate was washed 2-3 times with PBST. Then, 100 μL of the phage supernatant to be tested was added to each well, and the plate was incubated at 30°C for 1 hour. The plate was then washed 4-6 times with PBST to remove unbound phages. Subsequently, 100 μL of anti-M13 secondary antibody dilution buffer was added, and the mixture was incubated at 30°C for 1 hour. After washing 4-6 times, 100 μL of TMB chromogenic buffer was added to each well for incubation in the dark for 3-8 minutes. Finally, 100 μL of 2M HCl was added to terminate the reaction. The absorbance values ​​were measured at 450 nm and 620 nm using a microplate reader. After two rounds of validation, 13 different sequences were obtained, and three nucleotide sequences were selected.

[0042] 333F-2-R4P1-B5: CAGGTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGGAGCCTCTGGATTCACCTTCAGTATGTATAACATGAGCTGGTACCGCCAGGCTCCAGGGAAGGGGCGCGAGTTGGTCGCACGAATCACTCGTGATGACGGTAGTAGCACAAACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTTCTGTGTTACGGGTTGGGGCGTCTCCGGTGCGCGGGGCCAGGGGACCCAGGTCACTGTCTCCACG, translated into the amino acid sequence SEQID No: 1 is QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMSWVRQAPGKGLEWVSYINSGSDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYHCGLGTEVRGRVVRYDFGSWGPGTRVTVSP.

[0043] 333F-2-R4P1-D10: CAGGTGCAGCTCGTGGAGTCAGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACAGCCTCTGGACGCACCTTCAGTGGCTATGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTGTTGGGTGGAGTGGTGGACGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATGTGCAAATGAACAGCCTGAAACCTGGGGACACGGCCGTTTATTACTGTGCGCGTGCACCTAAAACTAGTTACTATACCAACTATGACGCCCGCCGCTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA, translated to amino acid sequence SEQ ID No: 2 is QVQLVESGGGLVQPGGSLRLSCGASGFTFSMYNMSWYRQAPGKGRELVARITRDDGSSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYFCVTGWGVSGARGQGTQVTVST.

[0044] 333F-2-R4P1-F8: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTGACATTCAGTAGCTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCATATATTAATAGTGGTAGTGATAGCACATATTATGCGGACTCCGTG AAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATCACTGTGGATTGGGGACAGAAGTACGTGGTAGAGTGGTCCGCTATGACTTTGGTTCCTGGGGCCCGGGGACCCGGGTTACCGTCTCCCCA, translated into the amino acid sequence SEQ ID No: 3 is QVQLVESGGGLVQAGGSLRLSCTASGRTFSGYVMGWFRQAPGKEREFVAAVGWSGGRTYYADSVKGRFTISRDDAKNTVYVQMNSLKPGDTAVYYCARAPKTSYYTNYDARRYDYWGQGTQVTVSS.

[0045] Step 5: Screening of nanobodies Using the quality-tested pTXs1-G59404-2 plasmid as a template, which contains a 485bp VHH gene fragment verified by sequencing, the target gene (333F-2-R4P1-F8) was amplified by PCR. The amplified product was digested with EcoRI and NotI, and then ligated to an expression vector that had undergone the same digestion treatment to construct a recombinant expression plasmid. The recombinant plasmid was transformed into TOP10 competent cells, plated on LB agar plates containing ampicillin, and positive clones were screened. Single clones were picked and inoculated into LB medium for culture, and the plasmid was extracted for restriction enzyme digestion and sequencing verification. The verified recombinant plasmid was transformed into Bacillus subtilis, inoculated into MT medium and cultured at 37°C for 4 hours, then transferred to MTN medium at a 1:200 ratio and cultured at 30°C with shaking for 72 hours. The supernatant was collected by centrifugation and analyzed using Ni... Purification was performed using a Sepharose affinity chromatography column, followed by washing and elution with Tris buffer containing 20 mM and 250 mM imidazole, respectively. The eluent was concentrated by ultrafiltration and replaced with 0.1 M NaHCO3 buffer (pH 8.3). Finally, SDS-PAGE analysis confirmed the successful expression of the nanobody.

[0046] Step 6: Verification of the binding affinity between the anti-BTLA nanobody and the BTLA protein. (1) Expression and purification of nanobodies in mammalian cells The VHH gene fragment (333F-2-R4P1-F8) was cloned into the pATX1 vector and transformed into XtenCHO cells for expression. After 5 days of culture following transfection, the supernatant was collected for preliminary analysis. On day 10, the culture supernatant was collected on a large scale and purified using Strep-tactin resin. The purification steps were: equilibration (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA), washing (same equilibration buffer), and elution (equilibration buffer containing 50 mM biotin). The purified protein was analyzed by SDS-PAGE. Figure 4 The protein samples were analyzed by reducing SDS-PAGE (MW protein marker: 1.333F-2-R4P1-F8). The final protein samples were stored in PBS (pH 7.5), aliquoted, and stored at -80°C for later use.

[0047] (2) ELISA detection of nanobody binding to target protein The target protein (BTLA) was coated onto an ELISA plate at a concentration of 4 μg / mL and incubated overnight at 4°C. After blocking, serially diluted nanobody samples were added and incubated at 37°C for 1 hour. After washing, HRP-labeled secondary antibody (anti-Strep-tagged antibody) was added and incubated at 37°C for 30 minutes. After washing again, TMB chromogenic substrate was added, and the reaction was stopped after 10 minutes. The OD450 value was then measured. Figure 5 By comparing the signal intensity of nanobodies at different concentrations, the anti-BTLA nanobody (24000333F-3-R4P1-F8) exhibited high affinity and high specificity for BTLA target proteins.

[0048] Step 7: In vivo comparison of anti-BTLA nanobody and Blank ctr antibody (1) To construct an A549 lung cancer mouse model, 30 five-week-old male mice were used. After one week of feeding, the lateral ventral skin was anesthetized and disinfected. A suspension of approximately 1×10^6 tumor cells was slowly injected using a syringe, which formed a transient vacuole. The colonization of lung cancer cells and the growth of metastatic tumors were monitored regularly. The model was considered successful when the subcutaneous tumor volume reached 200 mm³.

[0049] (2) Grouping and Drug Administration: Thirty lung cancer model mice were randomly divided into three groups: PBS group, Blank ctr group, and anti-BTLA Nb group, with 10 mice in each group. Each group of mice was injected via tail vein with PBS, Blank ctr + DC / A549 fusion cells (DC / A549-FC) to induce CD8+. + T cells, anti-BTLA Nb+DC / A549 fusion cell-induced CD8 + T cells. The mice were weighed daily to observe their condition, and the size of the tumors in each group was measured every 4 days.

[0050] (3) Results show that the measured tumor volume data were plotted as a line graph ( Figure 6 It was evident that the tumor volume growth in the Blank ctr group was almost identical to that in the PBS-treated control group, indicating no significant therapeutic effect. In contrast, the tumor volume in the anti-BTLANb group was significantly lower than the other two groups, demonstrating excellent tumor growth inhibition ability. This may be due to the activation of CD8 after BTLA blockade. + It is related to the anti-tumor activity of T cells.

[0051] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An anti-BTLA nanobody, characterized in that, The amino acid sequence of the nanobody is selected from one of SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No:

3.

2. The anti-BTLA nanobody according to claim 1, characterized in that: It includes a framework region and a complementation-determining region, the complementation-determining region containing a key binding site; the N-terminus or C-terminus of the nanobody can be linked to a His tag or a Strep tag, and it is expressed in 293F cells, CHO cells or E. coli via the pATX1 or pTXs1 vector system.

3. A nucleic acid, characterized in that, The nucleic acid encodes the anti-BTLA nanobody according to any one of claims 1 or 2.

4. An expression carrier, characterized in that, An expression frame comprising the nucleic acid of claim 3.

5. A host cell, characterized in that, It includes the expression vector as described in claim 4.

6. A kit containing the anti-BTLA nanobody as described in claim 1 or 2.

7. The use of the anti-BTLA nanobody as described in any one of claims 1 or 2 in the preparation of drugs for the prevention and / or treatment of tumors or autoimmune diseases.

8. The application according to claim 7, characterized in that, The tumor was selected from melanoma and lymphoma.

9. A method for preparing an anti-BTLA nanobody as described in claim 1 or 2, characterized in that, The expression vector of claim 4 is constructed, and then transferred into a host cell, induced to express, and purified to obtain the final product.

10. The preparation method according to claim 9, characterized in that, Specifically, the following steps are included: (1) BTLA antigen purification: Obtain the nucleotide sequence of BTLA and clone it into a suitable expression vector, transform it into host cells for expression, collect the expression product and purify it at least once to obtain BTLA antigen with a purity ≥90%; (2) Phage display library screening: The BTLA antigen is used as the target to coat the solid-phase carrier, and the VHH phage display library is added. The phages that specifically bind are enriched through multiple rounds of negative and positive screening. High-affinity positive clones are obtained by screening through immunological detection methods. (3) Expression and purification of nanobodies: The coding sequence of the positive clone is cloned into the expression vector, transformed into a suitable host cell for expression, and the expression supernatant is collected and purified by affinity treatment to obtain pure anti-BTLA nanobodies.

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