Anti-ceacam1 single-domain antibody and application thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to an anti-CEACAM1 single-domain antibody and application thereof, and a nucleotide sequence thereof is any one of SEQ ID NO. 1 to SEQ ID NO. 3. The application uses phage display technology to perform multiple rounds of screening on a llama CEACAM1 nanobody M13 phage display library, to enrich phages capable of specifically binding to a CEACAM1 receptor, to construct a recombinant plasmid after gene amplification and sequencing identification of positive clones, to express and purify by using an E. coli expression system, and finally to obtain an anti-CEACAM1 single-domain antibody with high purity and good affinity. The anti-CEACAM1 single-domain antibody can specifically target a tumor surface CEACAM1 antigen, and can enhance NK cell activity to exert a tumor killing function, and has important practical significance and application prospect in immunotherapy of tumors and related basic research.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-CEACAM1 single-domain antibody and its application. Background Technology

[0002] Cancer, with its high incidence and mortality rates, is a major public health problem worldwide. Tumor immunotherapy is one of the effective treatments for cancer. Nanobodies, derived from camel serum lacking the light chain, are a class of single-domain antibodies containing only the heavy chain variable region (VHH). With a molecular weight of only about 15 kDa, they are easily and stably expressed, exhibiting low immunogenicity. Phage display technology can be used to screen for more diverse single-domain antibody fragments with higher affinity for antigens. Due to their small molecular weight, good water solubility, and strong tissue penetration, single-domain antibody fragments are widely used in the construction of novel antibodies.

[0003] Single-domain antibodies, due to their unique physicochemical properties and ease of modification and expression, hold great potential in tumor immunotherapy. Currently marketed single-domain antibody drugs include Caplacizumab, Envita® (KN035), and CARVYKTI®. Caplacizumab is used to treat adult patients with acquired thrombotic thrombocytopenic purpura (aTTP). Envita® (KN035) is currently mainly used to treat microsatellite instability-high advanced colorectal cancer, MSI-H advanced gastric cancer, and advanced solid tumors with DNA mismatch repair deficiency, and is undergoing clinical trials for other advanced solid tumors such as advanced endometrial cancer, advanced colorectal cancer, and metastatic biliary tract cancer. CARVYKTI® is mainly used to treat relapsed or refractory multiple myeloma (R / RMM). Many more single-domain antibody drugs are currently in the initial stages of research or clinical trials.

[0004] Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein that is poorly expressed in normal epithelial tissues but highly expressed in cancer cells such as liver cancer, melanoma, pancreatic cancer, metastatic colorectal cancer, and non-small cell lung cancer. It promotes tumor cell proliferation and metastasis by mediating tumor cell adhesion through homotropism and heterotropism. CEACAM1 is also expressed on CD16. - CD56 +High expression of CEACAM1 on NK cells leads to its isotropic interaction with CEACAM1 on the surface of tumor cells, inhibiting NK cell cytolysis and facilitating tumor cell immune escape. Studies have found that liver cancer patients with high CEACAM1 expression have a worse prognosis. Regulated CEACAM1 on the surface of liver cancer cells weakens NK cell-mediated cytotoxicity, and the use of anti-CEACAM1 antibodies can enhance NK cell-mediated cell killing and reduce tumor size. Therefore, developing highly active, high-affinity anti-CEACAM1 single-domain antibodies is of great significance for the preparation of drugs to treat or prevent CEACAM1-related diseases. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the purpose of this invention is to provide an anti-CEACAM1 single-domain antibody and its applications. This invention provides a highly active, high-affinity anti-CEACAM1 single-domain antibody that can not only specifically target tumor surface antigens but also enhance NK cell activity to exert its tumor-killing function, which is of great significance for cancer treatment.

[0006] The first aspect of the present invention provides an anti-CEACAM1 single-domain antibody, wherein the nucleotide sequence of the anti-CEACAM1 single-domain antibody is any one of those shown in SEQ ID NO.1 to SEQ ID NO.3.

[0007] A second aspect of the present invention provides a recombinant plasmid containing the anti-CEACAM1 single-domain antibody described above.

[0008] Furthermore, the recombinant plasmid uses the expression vector pSmart I as its backbone vector.

[0009] Further, the recombinant plasmid is prepared by the following steps: Using the DNA of the anti-CEACAM1 single-domain antibody described above as a template, PCR amplification was performed using primers with nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 to obtain PCR products; Using restriction endonucleases BamH I and Hind III. The expression vector pSmart I was double-digested to obtain the linearized pSmartI vector. The PCR product was ligated with the pSmart I linearized vector to obtain the recombinant plasmid.

[0010] Furthermore, each 50µL PCR reaction system contains: 5µL of 1 ng / µL template, 25µL of 2×PCR Master Mix, 1µL each of primers with the sequences shown in SEQ ID NO.4 and SEQ ID NO.5 (10µM), and sterile ultrapure water to make up the difference.

[0011] Furthermore, the PCR reaction procedure is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 59℃~61℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 35 cycles; 72℃ final extension for 5 min.

[0012] A third aspect of the present invention provides a recombinant cell containing the nucleotide sequence of the anti-CEACAM1 single-domain antibody integrated into the recombinant plasmid or host cell genome described above.

[0013] Furthermore, the recombinant cells use Escherichia coli expression strain BL21(DE3) as the host cell.

[0014] A fourth aspect of this invention provides a method for preparing an anti-CEACAM1 single-domain antibody, comprising the following steps: The recombinant cells described above were induced and cultured with isopropyl-β-D-thiogalactoside for 20 h. The recombinant cells were lysed, and the culture medium was centrifuged at 4 °C and 12,000 rpm for 15 min. The supernatant was collected and purified using a Ni-NTA affinity chromatography column to obtain the anti-CEACAM1 single-domain antibody.

[0015] The fifth aspect of this invention provides the application of the above-described anti-CEACAM1 single-domain antibody in the preparation of drugs, reagents, detection plates or kits, wherein the drug is a drug for treating or preventing diseases related to CEACAM1; the diseases include liver cancer, melanoma, pancreatic cancer, metastatic colorectal cancer and non-small cell lung cancer.

[0016] Furthermore, the reagents, detection plates, or kits are used to detect CEACAM1 protein in samples.

[0017] In summary, compared with the prior art, the present invention has the following advantages and effects: This invention provides an anti-CEACAM1 single-domain antibody and its applications. The nucleotide sequence of this anti-CEACAM1 single-domain antibody is any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.3. Experiments show that the binding ability of the anti-CEACAM1 single-domain antibody to the CEACAM1 antigen at a concentration of 10 μg / mL is concentration-dependent, and it can bind to the surface antigen of CEACAM1-positive tumor cells, exhibiting advantages such as high purity and good affinity. This invention also provides recombinant plasmids and recombinant cells containing the anti-CEACAM1 single-domain antibody sequence. The anti-CEACAM1 single-domain antibody, recombinant plasmid, and recombinant cells can not only be used to detect CEACAM1 protein, but also to prepare drugs for treating or preventing CEACAM1-related diseases, which has important practical significance in tumor immunotherapy and research. Attached Figure Description

[0018] Figure 1 This is a graph showing the phage ELISA detection results in Example 1; Figure 1 In the figure, A represents the phage ELISA result coated with 10 ng CEACAM1 antigen; Figure 1 B in the figure represents the phage ELISA result coated with 1 ng CEACAM1 antigen; Figure 1 In the figure, C represents the ELISA result of soluble supernatant coated with 10 ng CEACAM1 antigen.

[0019] Figure 2 The image shows the construction of the recombinant plasmid for the anti-CEACAM1 single-domain antibody and the results of vector enzyme digestion. Figure 2 In the image, A represents the electrophoresis diagram of the PCR product of the anti-CEACAM1 single-domain antibody; Figure 2 In the image, B represents the result of double enzyme digestion of the pSmart I expression vector.

[0020] Figure 3 The image shows the purification and identification results of the anti-CEACAM1 single-domain antibody. Figure 3 In the image, A is the SDS-PAGE electrophoresis result of the nickel column purified product; Figure 3 B in the image represents the SDS-PAGE electrophoresis image of the SUMO tag-removed product. Figure 3 C in the figure represents the Western blot identification of the product after the SUMO tag was removed.

[0021] Figure 4 This is a graph showing the ELISA results of the binding of anti-CEACAM1 single-domain antibody to CEACAM1 antigen.

[0022] Figure 5 The image shows the flow cytometry results of the binding of anti-CEACAM1 single-domain antibody to human hepatocellular carcinoma cells HepG2. Figure 5 A in the diagram represents the binding of D5 to HepG2 human liver cancer cells; Figure 5 B in the diagram represents the binding of D16 to HepG2 human liver cancer cells; Figure 5 In the diagram, C represents the binding of D24 to HepG2 human liver cancer cells.

[0023] Figure 6 The image shows the flow cytometry results of the binding of anti-CEACAM1 single-domain antibody to human colorectal cancer cells HCT116. Figure 6 A in the diagram represents the binding of D5 cells to human colorectal cancer cells HCT116. Figure 6 B in the diagram represents the binding of D16 to human colorectal cancer cells HCT116. Figure 6 C in the figure represents the binding of D24 to human colorectal cancer cells HCT116. Detailed Implementation

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0025] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0026] Carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein that is poorly expressed in normal epithelial tissues but highly expressed in cancer cells such as melanoma, pancreatic cancer, metastatic colorectal cancer, and non-small cell lung cancer. CEACAM1 mediates tumor cell adhesion through homotropism and heterotropism, thereby promoting tumor cell proliferation and metastasis. Anti-CEACAM1 antibodies can enhance NK cell activity and exert their tumor-killing function. Single-domain antibodies, due to their unique physicochemical properties and ease of modification and expression, have broad application potential in tumor immunotherapy. Therefore, developing highly active, high-affinity anti-CEACAM1 single-domain antibodies is of great significance for tumor treatment.

[0027] This invention provides an anti-CEACAM1 single-domain antibody and its application. First, anti-CEACAM1 single-domain antibodies were screened using phage display technology, and three recombinant plasmids with correct sequences that could be expressed in *E. coli* were constructed. The recombinant plasmids were transformed, expressed, and purified to obtain the target protein with a purity of over 95%. After further removal of the SUMO tag, Western blotting confirmed that the three single-domain antibodies provided by this invention had the correct molecular weight and high purity. Subsequently, ELISA and flow cytometry were used to assess their binding to the CEACAM1 antigen at the molecular and cellular levels, respectively. The results showed that all three single-domain antibodies provided by this invention could specifically bind to the CEACAM1 antigen, and the binding ability was concentration-dependent.

[0028] In a specific embodiment of the present invention, the 2×A8 Fast HiFi PCR Master Mix and the One StepSeamless Cloning Kit (homological recombinase kit) were purchased from Beijing Adley Biotechnology Co., Ltd.; the restriction endonuclease QuickCut™ B... amH I and QuickCut™ HindIII cells were purchased from NEB Corporation, USA; pSmart I expression vector and SUMOProtease were purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.; DH5α and BL21 competent cells were purchased from Tiangen Biotech Co., Ltd.; CoraLite488-labeled goat anti-mouse IgG (H+L), anti-DYKDDDDK tag (FLAG® tag) monoclonal antibody and HRP-labeled goat anti-mouse IgG (H+L) antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.; human hepatocellular carcinoma cells HepG2 and human colorectal carcinoma cells HCT-116 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; and ultrasensitive luminescent reagents were purchased from Nanjing Pronoun Biotechnology Co., Ltd.

[0029] Example 1: Phage library screening for anti-CEACAM1 single-domain antibodies (1) Using human CEACAM1 as the antigen, phage display technology was applied to extract the alpaca CEACAM1 nanobody M13 phage display library (library size 2.2 × 10⁻⁶). 9 The specific steps for screening single-domain antibodies against CEACAM1 are as follows: First, a selection process was performed: 100 μL of 5 μg / mL CEACAM1 antigen was coated into each well of an ELISA plate and incubated overnight at 4°C. The coating solution was discarded, and the plate was washed three times with PBST. 100 μL of blocking buffer (PBST containing 2% skim milk powder) was added to each well, and the plate was blocked at 37°C for 2 hours. After washing three times with PBST, a phage display library (1.2 × 10⁻⁶) was added. 12 PFU was incubated at 37°C for 1 h. Unbound phages were aspirated and washed 15 times with PBST. 100 μL of 0.1 M glycine-hydrochloric acid solution (pH 2.5) was added to each well, and the reaction was carried out at 37°C for 7 min. The liquid in the wells was repeatedly pipetted to elute the adsorbed phages, and then neutralized to neutral with 1 M Tris-HCl solution (pH 8.8). The eluted phages were used to infect TG1 cells in logarithmic growth phase, and the phages were amplified and recovered for the next round of panning. The same panning process was repeated twice, and positive clones were enriched. Finally, specific anti-CEACAM1 nanobodies were obtained from the CEACAM1 phage immune library using phage display technology. The phage enrichment results of the two rounds of panning are shown in Table 1. As can be seen from Table 1, after two rounds of enrichment, the number of phage particles specifically binding to CEACAM1 antibody increased significantly, with an enrichment level of 920-fold.

[0030] Table 1. Phage screening enrichment results (2) After two rounds of screening, 480 single-clone colonies were randomly selected and further screened using phage enzyme-linked immunosorbent assay (Phage-ELISA). The specific steps are as follows: 480 single colonies were randomly selected and added to 1 mL of 2×YT medium. The culture was incubated overnight at 37°C and 200 rpm. The next day, the culture was centrifuged at 5000 rpm for 10 min, and 600 μL of the supernatant was carefully transferred to a 1.5 mL centrifuge tube. This was the Phage-ELISA sample. CEACAM1 antigen was diluted to 0.1 μg / mL with PBS and coated with 100 μL / well of the ELISA plate. The plate was incubated overnight at 4°C. The plate was washed three times with PBST, and 200 μL of blocking buffer (PBS containing 2% skim milk powder) was added to each well. The plate was blocked at 37°C for 1 h. After three washes, 100 μL of the Phage-ELISA sample was added to each well and incubated at 37°C for 1 h. After three washes, 100 μL of anti-M13-HRP antibody was added to each well (dilution ratio as recommended in the manufacturer's instructions) and incubated at 37°C for 1 h. Wash three times, add 100 μL of TMB substrate per well, and incubate at 37°C in the dark for 15 min. Finally, stop the reaction by adding 100 μL of 2 M H₂SO₄ per well and read the OD values ​​using a microplate reader. 450 Value. The amount of CEACAM1 antigen coated per well in this step is 10 ng. Negative control (PBS) OD 450 The value is below 0.1, therefore OD is used. 450 >2.0 was determined to be a positive clone. A total of 91 positive clones were obtained, as shown in the results below. Figure 1 As shown in A in the diagram.

[0031] Reduce the CEACAM1 antigen coating amount to 1 ng / well, and repeat the 91 positive clones using the Phage-ELISA method described above. (Diagram showing OD values) 450 The OD values ​​were sorted from highest to lowest, and the 30 positive clones with the highest OD values ​​were selected for PCR sequencing, yielding a total of 11 single sequences. The results are as follows: Figure 1 As shown in B in the diagram.

[0032] (3) Eleven single-sequence clones were induced to express: single clones were inoculated into 2×YT medium, IPTG was added to a final concentration of 0.2 mmol / L, and expression was induced at 37℃ for 6 h. The bacterial cells were collected, and the periplasmic cavity soluble protein was extracted using the osmotic shock method. The periplasmic cavity soluble protein supernatant was diluted 1250 times, and its binding titer with CEACAM1 antigen was determined by ELISA.

[0033] The results are as follows Figure 1 As shown in C, PBS was used as a negative control, based on OD... 450 Based on the numerical values, three anti-CEACAM1 nanobodies with different DNA and protein sequences were obtained and named D5, D16, and D24, respectively. The nucleotide sequences of D5, D16, and D24 are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively.

[0034] SEQ ID NO.1: GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCACCTATTTTATGAAGTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAATTATTAATCGCGACGGTAGTAGTACAACCTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGACTCTGCAAATGAACAATCTGAAACCTGAGGACACGGCCATGTATTACTGTGCAACAGCCGATAGGGATGAAAGCCGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC; SEQ ID NO.2: GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTAAGACTCTCCTGTGCAGCCTCTGGAATCTCCTTCAGTGGTGTTCTCACGGGGTGGTACCGCCAGGCTCCAGGGAAACGGCGTGAGATGGTCGCAGTTATTATGAGTGATGGTACCACAAGTTATGCAGAATCCGCGAAGGGCCGATTCGCCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACTGGAGGACACGGCCGTCTATTACTGTAGTATGGGCTCTGGGCGGCTCTCCACCTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC; SEQ ID NO.3: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCCGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCGATAGCTATGCCATGGCCTGGCACCGCCAGGCTCCTGGGAAGGAGCGTGAGTTTGTCGCATCTATTAGTTGGAGTGGTGGTGTCACATACTA TGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAACACACGGTGTATCTGCAAATGAACAGCCTGAAAGTTGAGGACACGGCCGTATATTACTGTAATACTAGAGACTCGCGGGGAGGATCTGAGCATTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGC.

[0035] Example 2: Construction and identification of recombinant plasmids containing anti-CEACAM1 single-domain antibodies (1) Using the plasmid DNA of anti-CEACAM1 single-domain antibodies D5, D16, and D24 obtained in Example 1 as templates, specific primers CEA-3' and CEA-5' were designed for PCR reaction. The nucleotide sequences of primers CEA-3' and CEA-5' are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0036] SEQ ID NO.4: 5'-GCTCGAGTGCGGCCGCAAGCTTTTCACTTATCGTCGTCATCCTTGTAATCGCTGGAGACGGTGACCTGG-3'; SEQ ID NO.5: 5'-ACAGATTGGTGCCCAAGGATCCGATGTGCAGCTGCAGGAGTCT-3'; Each 50µL PCR reaction system contains: 5µL of 1 ng / µL template, 25µL of 2×PCR Master Mix, 1µL of 10µM primer CEA-3', 1µL of 10µM primer CEA-5', and sterile ultrapure water to make up the difference.

[0037] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 35 cycles; 72℃ final extension for 5 min.

[0038] After PCR amplification, the products were analyzed by 1.5% agarose gel electrophoresis. The results are as follows: Figure 2 As shown in Figure A, the PCR product is a single, clear band with a molecular weight of approximately 360 bp, which is consistent with the theoretically expected size.

[0039] (2) Use of restriction endonucleases BamH I and Hind III. The pSmart I expression vector was subjected to double enzyme digestion, and the digestion products were identified by agarose gel electrophoresis. The results are as follows: Figure 2 As shown in Figure B, the pSmart I expression vector was completely cleaved, and the pSmart I linearized vector fragment was recovered. The anti-CEACAM1 single-domain antibody gene fragment obtained from the above PCR amplification was ligated to the pSmart I linearized vector using homologous recombinase. The ligation product was transformed into... E. coli DH5α competent cells were cultured at 37°C for 2 hours, and single clones were selected for PCR identification. Positive clones were sent to a sequencing company for DNA sequencing to confirm the correctness of the inserted sequence, and recombinant plasmids were obtained.

[0040] Example 3: Expression, purification, and identification of anti-CEACAM1 single-domain antibody 1. Expression and purification of anti-CEACAM1 single-domain antibody The recombinant plasmid obtained in Example 2 was transformed into the *E. coli* expression strain BL21(DE3). Single colonies were picked for expansion culture, and the expression conditions were optimized. The optimal expression conditions were: during expansion culture, isopropyl-β-D-thiogalactoside (IPTG) was added to the bacterial culture to a final concentration of 0.2 mmol / L, and induction was performed for 20 h. After induction under these conditions, the bacterial culture was collected, the bacterial cells were lysed, and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and loaded onto a Ni-NTA affinity chromatography column. The column was washed with wash buffer containing 10 mmol / L, 20 mmol / L, and 30 mmol / L imidazole, respectively, followed by gradient elution with eluents containing 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L imidazole. The eluents of each concentration were collected, placed in dialysis bags, and dialyzed overnight in PBS at 4 °C.

[0041] The purification effect was detected by 12% SDS-PAGE electrophoresis. The purification results of anti-CEACAM1 single-domain antibodies D5, D16, and D24 are as follows: Figure 3 As shown in A: After Ni-NTA affinity purification, the purity of the target protein is greater than 95%, and a distinct protein band appears at a molecular weight of approximately 36 kDa, which is consistent with the expected molecular weight (Sumo tag approximately 20 kDa + single-domain antibody approximately 16-18 kDa).

[0042] Because the pSmart I vector carries a Sumo tag (molecular weight approximately 20 kDa), which may affect subsequent activity assays of the single-domain antibody, the tag was removed using the SUMO protease. The SUMO protease was mixed with the anti-CEACAM1 single-domain antibody at a mass ratio of 1:200 and digested in a 30°C water bath for 1 hour. After digestion, the reaction solution was passed through a Ni-NTA affinity chromatography column again. The Sumo tag containing the 6×His tag bound to the nickel column, while the tag-removed single-domain antibody did not bind to the nickel column and remained in the permeate (FT). The permeate was collected, concentrated, and quantified to a concentration of approximately 0.5 mg / mL. It was then aliquoted and stored at -80°C, avoiding repeated freeze-thaw cycles.

[0043] The enzyme digestion and purification efficiency was detected by 12% SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown in B in the figure. The molecular weight of the single-domain antibody after enzyme digestion is approximately 18 kDa. The permeation band is single, clear, and of high purity, which is in line with expectations.

[0044] 2. Identification of anti-CEACAM1 single-domain antibodies Quantification of the anti-CEACAM1 single-domain antibody sample obtained in step 1 with the SUMO tag removed: Take 40 µL of protein solution and mix with 10 µL of 5× protein loading buffer, heat in a boiling water bath for 5 min, then briefly centrifuge and perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0045] After electrophoresis, the separated protein bands were transferred to a PVDF membrane. Following transfer, the PVDF membrane was placed in blocking buffer containing 5% skim milk and blocked at room temperature for 2 hours. After blocking, anti-DYKDDDDK tag (FLAG® tag) monoclonal antibody diluted 1:5000 with TBST buffer was added and incubated at room temperature for 2 hours. The membrane was then washed three times with TBST buffer for 5 minutes each time. Goat anti-mouse IgG (H+L)-HRP antibody (diluted 1:10000) was then added and incubated at room temperature for 1 hour. The membrane was washed three more times with TBST buffer for 5 minutes each time. Finally, a hypersensitive chemiluminescent substrate (hypersensitive luminescent reagent) was applied to the membrane, and after an appropriate reaction time in the dark, the membrane was exposed and developed using a gel imaging system.

[0046] Western blot results are as follows Figure 3 As shown in C. The results showed that the detected antibody band was single, indicating that the purified product was a single-domain antibody against CEACAM1.

[0047] Example 4: Affinity determination of anti-CEACAM1 single-domain antibody 1. Affinity analysis of anti-CEACAM1 single-domain antibody The binding activity of the purified anti-CEACAM1 single-domain antibody to CEACAM1 antigen was detected by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: CEACAM1 antigen was diluted to 0.1 μg / mL with phosphate-buffered saline (PBS), and 100 μL was added to each well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. The coating solution was discarded, and 200 μL of PBS solution containing 2% skim milk powder (PBS-2% skim milk powder) was added to each well. The plate was blocked at 37°C for 2 h. The blocking solution was discarded, and 300 μL of PBST washing buffer (PBS containing 0.05% Tween-20) was added to each well. The plate was washed three times, with shaking for 1 min each time, and the washing buffer was discarded afterward. Three purified anti-CEACAM1 single-domain antibodies were serially diluted with PBS (concentrations of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL), 100 μL per well, and incubated at 37°C for 1.5 h. Wash three times with PBST. Add 100 μL of anti-DYKDDDDK tag (FLAG® tag) monoclonal antibody (dilution 1:5000) per well and incubate at 37°C for 1 h. Wash three times. Add 100 μL of goat anti-mouse IgG (H+L)-HRP antibody (dilution 1:10000) per well and incubate at 37°C for 1 h. Wash three times. Add 100 μL of TMB substrate chromogenic solution to each well and incubate at 37°C in the dark for 30 min. Add 100 μL of 2 M sulfuric acid stop solution to each well and immediately measure the absorbance (OD) at 450 nm using a microplate reader. 450 ).

[0048] The results are as follows Figure 4 As shown, all three anti-CEACAM1 single-domain antibodies bound to the CEACAM1 antigen to varying degrees at a concentration of 10 μg / mL, and the binding ability was concentration-dependent; as the antibody concentration decreased, the OD... 450 The value gradually decreases.

[0049] 2. Flow cytometry analysis of tumor cell surface antigen binding activity Flow cytometry was used to further detect the binding ability of anti-CEACAM1 single-domain antibody to CEACAM1-positive tumor cell surface antigen. The specific steps were as follows: Human hepatocellular carcinoma cells HepG2 and human colorectal carcinoma cells HCT-116 were washed twice with PBS containing 0.2% bovine serum albumin (BSA), and then... 6Cells / tubes were aliquoted into flow cytometry tubes. Three anti-CEACAM1 single-domain antibodies (final concentration 10 μg / mL) were added separately and incubated on ice for 1 h. Cells were washed three times with PBS containing 0.2% BSA. Anti-DYKDDDDK-tagged (FLAG®-tagged) monoclonal antibody (1:500 dilution) was added again and incubated on ice for 1 h. Cells were washed three times with PBS containing 0.2% BSA. CoraLite488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added and incubated on ice in the dark for 1 h. Cells were washed three times with PBS containing 0.2% BSA. Cells were resuspended in 500 μL of PBS in each tube, filtered through a 70 μm cell sieve, and the fluorescence intensity was detected by flow cytometry. Cells without single-domain antibodies were used as a negative control.

[0050] The results are as follows Figure 5 and Figure 6 As shown, where Figure 5 In the diagram, A, B, and C represent the binding of D5, D16, and D24 cells to HepG2 human liver cancer cells, respectively. Figure 6 In the diagram, A, B, and C represent the association of D5, D16, and D24 with human colorectal cancer cells HCT-116, respectively. Figure 5 and Figure 6 The results show that the fluorescence intensity of the sample peak shifted significantly to the right compared to the negative control. All three antibodies could bind to HepG2 and HCT116 cells, with D16 showing a stronger binding signal, indicating that the anti-CEACAM1 single-domain antibody prepared in this invention can effectively bind to the CEACAM1 antigen expressed on the surface of tumor cells.

[0051] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A single-domain antibody against CEACAM1, characterized in that, The nucleotide sequence of the anti-CEACAM1 single-domain antibody is any one of those shown in SEQ ID NO.1 to SEQ ID NO.

3.

2. A recombinant plasmid, characterized in that, The recombinant plasmid contains the anti-CEACAM1 single-domain antibody as described in claim 1.

3. The recombinant plasmid according to claim 2, characterized in that, The recombinant plasmid was prepared by the following steps: Using the DNA of the anti-CEACAM1 single-domain antibody as described in claim 1 as a template, PCR amplification was performed using primers with nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 to obtain PCR products; The PCR product was ligated with the expression vector pSmart I to obtain the recombinant plasmid.

4. The recombinant plasmid according to claim 3, characterized in that, Each 50µL PCR reaction system contains: 5µL of 1 ng / µL template, 25µL of 2×PCR Master Mix, 1µL each of primers with the sequences shown in SEQ ID NO.4 and SEQ ID NO.5 (10µM), and sterile ultrapure water to make up the volume.

5. The recombinant plasmid according to claim 3, characterized in that, The PCR reaction procedure was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 59℃~61℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 35 cycles; 72℃ final extension for 5 min.

6. A recombinant cell, characterized in that, The recombinant cell contains the recombinant plasmid of claim 2 or the genome of the recombinant cell is integrated with the nucleotide sequence of claim 1.

7. A method for preparing an anti-CEACAM1 single-domain antibody, characterized in that, Includes the following steps: The recombinant cells of claim 6 were induced and cultured, the recombinant cells were lysed, and the culture supernatant was separated and purified to obtain the anti-CEACAM1 single-domain antibody.

8. The use of the anti-CEACAM1 single-domain antibody according to claim 1 in the preparation of drugs, reagents, detection plates or kits, characterized in that, The drug is a medicine for the treatment or prevention of diseases related to CEACAM1; the diseases include liver cancer, melanoma, pancreatic cancer, metastatic colorectal cancer, and non-small cell lung cancer.

9. The application according to claim 8, characterized in that, The reagents, detection plates, or kits are used to detect CEACAM1 protein in samples.

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