Single-chain antibody for resisting human secretory FGL1 protein as well as preparation method and application of single-chain antibody

Abstract

The invention provides a single-chain antibody for resisting human secretory FGL1 protein as well as a preparation method and application of the single-chain antibody. The amino acid sequence of the single-chain antibody for resisting the human secretory FGL1 protein is selected from one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6. The anti-human secretory FGL1 protein single-chain antibody provided by the invention has the characteristics of high specificity and high affinity to the human secretory FGL1 protein, can specifically recognize the human secretory FGL1 protein and improve the binding force of the human secretory FGL1 protein and the human secretory FGL1 protein, has a good treatment effect on malignant tumors, and is of great significance in clinical treatment of the malignant tumors. Moreover, the single-chain antibody for resisting the human secretory FGL1 protein is purely humanized, so that the immunogenicity is relatively low, and the immunological rejection is reduced. Besides, the single-chain antibody for resisting the human secretory FGL1 protein also has the advantages of small molecular weight, low production cost and the like, in the clinical aspect, the inspection and treatment cost is reduced, the diagnosis sensitivity and specificity are improved, and the immunotherapy effect is expected to be improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a single-chain antibody against human secretory FGL1 protein, and a preparation method and application thereof. Background Art

[0002] Antibodies, as functional proteins, have a typical "Y"-shaped structure consisting of four polypeptide chains: two identical light chains and one heavy chain. Sequence analysis reveals that the four polypeptide chains are composed of a series of similar unit domains. The C-terminal sequences of the heavy and light chains of antibodies are highly conserved, known as the conserved region. The corresponding N-termini of the protein have rich amino acid sequence diversity, known as the variable region. Within the variable region, there are even more complex and diverse amino acid sequences, known as the high-frequency mutation region, which is closely related to specific antigen recognition.

[0003] Single-chain antibodies, also known as single-chain variable region fragments (scFv), are small molecule proteins composed of variable region fragments of an antibody's heavy and light chains, connected by a short peptide chain. Compared to traditional monoclonal antibodies, scFvs offer numerous advantages, including a smaller molecular weight, improved target cell adhesion, a shorter in vivo half-life, lower immunogenicity, and a lower likelihood of host rejection. Furthermore, scFvs' simple structure facilitates genetic engineering, reducing the cost of mass production.

[0004] Currently, scFv can be prepared by a variety of methods, including Escherichia coli expression system, mammalian expression system, yeast surface display technology, and phage surface display technology, and has important applications in targeted therapy, imaging diagnosis, intracellular immunity, biological detection, etc.

[0005] Human secretory FGL1 (fibrinogen-like protein 1, FGL1) is a protein highly expressed in the liver. Also known as hepatocyte-derived fibrinogen-related protein 1 (HFREP1) or hepassocin (HPS), it is encoded by the FGL1 gene and belongs to the fibrinogen superfamily. The full-length FGL1 protein consists of 312 amino acids, contains a hydrophobic leader peptide, and shares high homology with the β- and γ-subunits of fibrinogen and other fibrinogen-related proteins.

[0006] The LAG-3 protein is encoded by the lymphocyte-activation gene-3 (LAG-3). Abnormal expression of LAG-3 in malignant tumors can cause tumor cells to escape the immune system. FGL1 protein is a functional ligand of LAG-3. Binding between the two inhibits T cell proliferation and immune activity, promoting immune escape. Therefore, the LAG-3-FGL1 pathway may represent a novel immune escape mechanism in malignancies such as liver cancer, non-small cell lung cancer, oral squamous cell carcinoma, and esophageal cancer.

[0007] The existing phage single-chain variable region fragment libraries have the following problems: First, the phage libraries commonly used on the market are mostly derived from immune animals. The target antigen protein is prepared to immunize mice, rabbits, alpacas and other experimental animals, and the spleen or peripheral blood mononuclear cells are collected and isolated to construct the single-chain antibody library. The single-chain antibodies obtained by this method are highly immunogenic and may induce severe immune rejection reactions in the host; second, there are currently no therapeutic FGL1 monoclonal antibodies on the market, and previous studies have found that FGL1, as a secretory protein, plays an important role in the activation of the FGL1 / LAG3 immune checkpoint pathway. Summary of the Invention

[0008] In order to solve the problems of high immunogenicity of existing single-chain antibodies against human secretory FGL1 protein and improve the specific recognition effect of single-chain antibodies on human secretory FGL1 protein, the present invention provides a single-chain antibody against human secretory FGL1 protein and its preparation method and application.

[0009] According to the first aspect of the present invention, a single-chain antibody against human secretory FGL1 protein is provided, wherein the amino acid sequence of the single-chain antibody is selected from one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6.

[0010] The inventors of the present application isolated peripheral blood mononuclear cells (PBMCs) from the blood of healthy volunteers and extracted RNA from the PBMCs. The extracted RNA was reverse transcribed and amplified using RT-PCR technology to obtain the heavy chain encoding gene and the light chain encoding gene of the single-chain antibody, and the two were randomly recombined and connected using universal linker primers to obtain the single-chain antibody encoding gene (i.e., the single-chain variable region fragment encoding gene, referred to as scFv fragment). The single-chain antibody encoding gene was then connected to an expression vector by electroporation to construct a phage library. The phage library was then subjected to five rounds of panning using human FGL1 protein as an antigen, and finally five phage antibodies with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6 were screened, namely, the single-chain antibody against human secretory FGL1 protein (FGL1) provided by the present invention. This single-chain antibody (scFv) has high specificity and affinity for the human secretory FGL1 protein. It can specifically recognize the human secretory FGL1 protein and enhance the binding affinity between the two, demonstrating excellent therapeutic effects against malignant tumors and possessing extremely important clinical significance. Furthermore, this single-chain antibody against the human secretory FGL1 protein is fully humanized, significantly reducing its immunogenicity and lowering its immune rejection after infusion in actual clinical applications. Furthermore, this single-chain antibody against the human secretory FGL1 protein has the advantages of a small molecular weight and low production cost. In clinical practice, this reduces testing and treatment costs, improves diagnostic sensitivity and specificity, and is expected to enhance immunotherapeutic efficacy.

[0011] According to a second aspect of the present invention, a gene encoding the above-mentioned single-chain antibody against human secretory FGL1 protein is provided.

[0012] Preferably, the nucleotide sequence of the gene encoding the single-chain antibody against human FGL1 protein is selected from one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.

[0013] According to the third aspect of the present invention, a nucleic acid molecule is provided, which comprises a gene encoding the above-mentioned single-chain antibody against human secretory FGL1 protein.

[0014] According to a fourth aspect of the present invention, an expression vector is provided, which comprises the above-mentioned nucleic acid molecule.

[0015] According to a fifth aspect of the present invention, a cell is provided, which contains the above-mentioned expression vector.

[0016] According to a sixth aspect of the present invention, a human secretory FGL1 protein inhibitor is provided. The human secretory FGL1 protein inhibitor comprises the above-mentioned single-chain antibody against human secretory FGL1 protein.

[0017] According to a seventh aspect of the present invention, there is provided use of the above-mentioned single-chain antibody against human secretory FGL1 protein in the preparation of a product for detecting human secretory FGL1 protein.

[0018] The single-chain antibody against human secretory FGL1 protein provided by the present invention is used in the preparation of a product for detecting human FGL1 protein. The prepared product can specifically recognize human secretory FGL1 protein, thereby achieving the purpose of detecting human secretory FGL1 protein.

[0019] Preferably, the above-mentioned product includes at least one of a reagent, a kit, and a chip.

[0020] According to an eighth aspect of the present invention, a kit for detecting human secretory FGL1 protein is provided, wherein the kit comprises the above-mentioned single-chain antibody against human secretory FGL1 protein.

[0021] According to a ninth aspect of the present invention, there is provided use of the above-mentioned single-chain antibody against human secretory FGL1 protein in the preparation of a drug for treating malignant tumors.

[0022] Preferably, the above-mentioned malignant tumor includes at least one of liver cancer, non-small cell lung cancer, oral squamous cell carcinoma, and esophageal cancer.

[0023] Previous studies have found that FGL1 is highly expressed in malignant tumors such as liver cancer, non-small cell lung cancer, oral squamous cell carcinoma, and esophageal cancer, and plays an important role in the FGL1 / LAG3 immunosuppressive pathway and T cell anti-tumor immunity. The single-chain antibody against human secretory FGL1 protein provided by the present invention is used in the preparation of a drug for treating malignant tumors. The drug can specifically recognize human secretory FGL1 protein and has a good therapeutic effect on malignant tumors such as liver cancer, non-small cell lung cancer, oral squamous cell carcinoma, and esophageal cancer. At the same time, it reduces the immune rejection reaction of the drug in actual clinical application, which is of great significance for the clinical treatment of malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the electrophoresis identification result of RNA extracted from peripheral blood mononuclear cells of healthy volunteers in Example 1.

[0025] Figure 2 This is the electrophoresis identification result of the heavy chain encoding gene and light chain encoding gene of the single-chain antibody amplified by RT-PCR technology in Example 1.

[0026] Figure 3This is the electrophoresis identification result of Example 1, in which the heavy chain encoding gene and the light chain encoding gene of the amplified single-chain antibody were spliced into scFv.

[0027] Figure 4 This is a graph showing the results of Example 3 using a competitive ELISA experiment to detect the blocking activity of a single-chain antibody (FGL1scFv) against human secretory FGL1 protein. DETAILED DESCRIPTION

[0028] The following is a further clear and complete description of the technical features of the technical solution provided by the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present invention.

[0029] Example 1 Establishment of a Pure Human Phage Library

[0030] This example aims to establish a purely human phage display library for subsequent screening and preparation of pure humanized single-chain antibodies against human secretory FGL1 protein. The purely human phage display library was constructed according to the following steps:

[0031] 1. Recruit healthy volunteers, draw blood from the volunteers, and use density gradient centrifugation to separate peripheral blood mononuclear cells (PBMCs) from the blood.

[0032] 2. Place the PBMC obtained above in a test tube, then add chloroform to the test tube and shake it vigorously for 1 minute. Let it stand for 5 minutes, centrifuge it at 4°C and 11000 rpm for 10 minutes, take the supernatant and add an equal volume of isopropanol, mix well, let it stand at room temperature for 20-30 minutes, centrifuge it at 4°C and 11000 rpm for 10 minutes, discard the supernatant, add 1 mL of 75% ethanol solution, wash the precipitate twice, centrifuge it at 4°C and 7000 rpm for 5 minutes, discard the supernatant, and the resulting precipitate is the extracted RNA. After drying at room temperature, add an appropriate amount of DEPC water to dissolve it and identify the extracted RNA by electrophoresis. The results are as follows: Figure 1 As shown, lane 1 represents marker 5000, and lanes 2 to 24 represent the extracted RNA.

[0033] Table 1 PCR amplification system

[0034] Components volume 2×KOD PCR Master mix 10 μL Upstream primer (2 μM) 0.8μL Downstream primer (2 μM) 0.8μL template 1 μL <![CDATA[ddH2O]]> 7.4μL

[0035] Table 2 PCR amplification program

[0036]

[0037] 3. Primers for amplifying the heavy and light chains of the single-chain antibody were designed using Primer 5.0 software. Referring to the PCR amplification system shown in Table 1 and the PCR amplification program shown in Table 2, the heavy chain encoding gene and the light chain encoding gene of the single-chain antibody were amplified from the RNA extracted above using RT-PCR technology. The heavy chain encoding gene and the light chain encoding gene of the single-chain antibody were identified by electrophoresis. The results are as follows: Figure 2 As shown, lanes 1-30 are electrophoretic patterns of VH heavy chain PCR amplification, lanes 31-69 are electrophoretic patterns of VK light chain PCR amplification, lanes 70-94 are electrophoretic patterns of VL light chain PCR amplification, and lanes 13, 26, 43, 68, and 82 are all markers; then, the heavy chain encoding gene and the light chain encoding gene were randomly recombined using a universal linker primer (G4S Linker) to obtain a single-chain antibody encoding gene (i.e., a single-chain variable region fragment encoding gene, referred to as scFv fragment), and the obtained single-chain antibody encoding gene was amplified using PCR technology and referring to the scFV PCR amplification system shown in Table 3 and the scFV PCR amplification procedure shown in Table 4. The amplified single-chain antibody encoding gene was then identified by electrophoresis, and the results are shown in FIG. Figure 3 As shown, lane 1 represents Marker 5000, lane 2 represents VK light chain + VH heavy chain spliced into scFv, lane 3 represents VL light chain + VH heavy chain spliced into scFv, and IgG light chains are divided into two types, namely lambda chain and kappa chain, namely VK light chain and VL light chain.

[0038] Table 3 scFv PCR amplification system

[0039] Components volume 2×KOD PCR Master mix 25 μL Upstream primer (2 μM) 2μL Downstream primer (2 μM) 2μL VH (300ng) -μL VK / VL (300ng) -μL <![CDATA[ddH2O]]> -μL

[0040] Table 4 PCR amplification procedure of scFv

[0041]

[0042] 4. The recovered scFv fragment and the E. coli pcomb3X expression vector were digested with sfiI enzyme, and then the digested scFv fragment was mixed with the E. coli pcomb3X expression vector and connected overnight at 4°C using T4 ligase to construct a phagemid vector (i.e., a phage library). The ligated phagemid vector products were mixed with the XL1-Blue competent state in an ice bath, and 80 μL / cup were dispensed into an electric transfer cup and electroporated at 1800V. After electroporation, the mixture was transferred to 2YT-ATG medium containing ampicillin and tetracycline, and the volume was fixed to 200 mL using 2YT-ATG. The library after fixed volume was shaken and cultured at 37°C and 250 rpm for 1 hour to recover. At this time, it was the antibody bacterial library. 100 μL of bacterial solution was taken from the library and diluted 10 times using 2YT medium (100 μL bacterial solution + 900 μL After diluting the 2YT (2YT) 1000-fold, 100 μL of the diluted bacterial solution was aspirated and applied to a 2YT-ATG semi-solid medium plate. The plate was incubated at 37°C overnight. The next day, the monoclonal colonies were counted and the number of transformants was calculated. The monoclonal colony count was as follows: monoclonal colony count * dilution factor per unit volume * total library volume = 488 * 10000 * 200 = 9.76 * 10 8 ; 50 monoclonal colonies were picked from the coated plates for sequencing. The sequencing results are shown in Table 5.

[0043] Table 5 Phage library sequencing results

[0044] Correct 43 Mutation / no signal (number) 7 No load (units) 1 Repeated sequences none Accuracy 86％

[0045] Example 2 Screening of single-chain antibodies against human secretory FGL1 protein

[0046] This embodiment provides a single-chain antibody against human secretory FGL1 protein, wherein the amino acid sequence of the single-chain antibody is selected from one of SEQ ID NOs: 1 to 8, and the single-chain antibody is prepared by the following steps:

[0047] 1. Prepare human secretory FGL1 protein and mix it with coating solution (carbonate buffer (CBS, pH = 9.0)) in a 96-well ELISA plate at 4°C overnight. After overnight, discard the coating solution, wash, and block the 96-well plate with blocking solution (PBS buffer containing 2% BSA) for 2 hours, then wash and pat dry.

[0048] 2. The diluted phage library (i.e., the phage vector constructed in Example 1) was added to a 96-well plate and incubated at 4°C for 2 h. The plate was then washed 15 times with a washing solution (PBST, phosphate-buffered saline (PBST)). The plate was then eluted with an acidic elution solution (glycine-hydrochloric acid buffer Gly-HCl, pH = 2.2). The plate was immediately neutralized with an alkaline buffer (Tris-HCl, pH = 9.0). Finally, an XL1-Blue bacterial solution (Tris-HCl, pH = 9.0) in the logarithmic growth phase was added for neutralization. The XL1-Blue bacterial solution in the logarithmic growth phase (the genotype of the Escherichia coli strain XL1-Blue is recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac[F′proAB, lac1 q Z△M15Tn10(Tet γ )], purchased from Stratagene. Tn10 is tetracycline-resistant and recombination-deficient, allowing superinfection with M13 phage. The cells were cultured at 37°C and eluted twice. The two elutions were combined, and the phages were amplified and titered. This constituted the first round of panning. Following the above steps, a total of five rounds of phage panning were performed. The titer results of the input and output phages during the panning process are shown in Table 6.

[0049] Table 6 Titers of input phage and output phage during phage panning

[0050]

[0051]

[0052] 3. The phage libraries obtained by the second, third, fourth, and fifth rounds of panning using the human secretory FGL1 protein were used for ELISA detection. The ELISA detection system and the OD value detection results at 450 nm are shown in Table 7. Based on the test results, the phage libraries obtained from FGL1-4th and FGL1-5th were used for subsequent monoclonal screening.

[0053] Table 7 Elisa detection system and OD value detection results at 450nm

[0054]

[0055] 4. FGL1 Monoclonal Screening

[0056] (1) 96 monoclonal clones were selected from the phage libraries obtained from FGL1-4th and FGL1-5th. After phage expression, the supernatant was obtained for ELISA detection to evaluate the affinity of phage antibodies to the target antigen. First, a 96-well plate was coated with antigen (FGL1 protein) at a concentration of 0.5 μg / mL at 4°C overnight; the wells were blocked with 3% milk powder for 1 hour; the phage expression supernatant was diluted 10 times and added to the 96-well plate at a volume of 100 μL per well and incubated for 1 hour; the secondary antibody M13-HRP (0.3 μg / mL) was added at a volume of 100 μL per well and incubated for 1 hour; then TMB color development solution was added at a volume of 200 μL per well for color development for 20 minutes; the color stop solution was added at a volume of 50 μL per well; the absorbance OD value was detected at a wavelength of 450 nm. The test results are shown in Tables 8 and 9.

[0057] Table 8 Final detection results of antigen FGL1 from phage libraries obtained from FGL1-4th and FGL1-5th

[0058] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.099 0.0546 0.0548 0.0727 0.19 0.0643 0.0736 0.0495 0.2444 0.0896 0.0631 0.1022 B 0.1127 0.053 0.0900 0.0733 0.0721 0.0519 0.0695 0.0839 0.0401 0.0731 0.0932 0.0757 C 0.1026 0.0459 0.0438 0.0838 0.087 0.0529 0.1026 0.0909 0.2098 0.0417 0.0573 0.059 D 0.0613 0.0775 0.0873 0.1113 0.0611 0.0674 0.0598 0.0711 0.0803 0.0494 0.0468 0.099 E 0.0585 0.076 0.0607 0.049 0.0323 0.0839 0.0368 0.0565 0.0513 0.0646 0.0582 0.0734 F 0.1474 0.1165 0.0826 0.0581 0.0454 0.0743 0.0876 0.0398 0.0482 0.0366 0.1048 0.0594 G 0.0667 0.0828 0.0524 0.0403 0.0409 0.0463 0.04 0.0452 0.0539 0.0522 0.0427 0.0632 H 0.1253 0.1253 0.0690 0.1174 0.0757 0.0769 0.0653 0.0661 0.0629 0.06 0.0669 0.1093

[0059] Table 9 Negative test results of antigen FGL1 of phage library obtained from FGL1-4th and FGL1-5th after blocking with 3% milk powder

[0060] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.0962 0.0601 0.0355 0.0664 0.1608 0.074 0.0999 0.0665 0.3268 0.1104 0.0579 0.0748 B 0.0589 0.0273 0.0513 0.0588 0.072 0.0567 0.0602 0.0428 0.0434 0.0656 0.0539 0.0479 C 0.0767 0.0393 0.0346 0.067 0.0642 0.0428 0.0573 0.0585 0.2921 0.0345 0.0451 0.061 D 0.0634 0.0648 0.0677 0.122 0.0967 0.0593 0.0567 0.0548 0.0666 0.064 0.0603 0.15 E 0.0689 0.0527 0.0338 0.0294 0.0329 0.0512 0.033 0.0358 0.0508 0.1072 0.0512 0.0912 F 0.0784 0.0577 0.0267 0.0376 0.0378 0.0881 0.0511 0.0292 0.0294 0.0389 0.0548 0.0602 G 0.062 0.0741 0.0353 0.0384 0.0325 0.0445 0.0344 0.029 0.0277 0.0352 0.0565 0.0453 H 0.2034 0.0568 0.0306 0.1713 0.042 0.0578 0.0338 0.0354 0.0273 0.038 0.0441 0.0592

[0061] (2) 144 monoclonal clones were selected from the phage libraries obtained from FGL1-4th and FGL1-5th, and the supernatant was obtained after phage expression for Elisa detection. The plate was coated with antigen FGL1 protein, the antigen sample concentration was 2μg / mL, and the plate was incubated at 4℃ overnight; the whole well was blocked with 3% milk powder for 1 hour; the phage expression supernatant was diluted 2 times and added to the 48-well plate at a volume of 100μL per well and incubated for 1 hour; the secondary antibody M13-HRP (0.3μg / mL) was added at a volume of 100μL per well and incubated for 1 hour; then TMB color development solution was added at a volume of 200μL per well for color development for 20 minutes; the color stop solution was added at a volume of 50μL per well; the plate was stained at a wavelength of 450nm. The absorbance OD value was detected. The detection results of the first plate antigen FGL1 are shown in Tables 10 and 11, and the detection results of the second plate antigen FGL1 are shown in Tables 12 and 13. Among them, 144 monoclonal clones were selected from the FGL1-4th and FGL1-5th libraries, and the supernatant after phage expression was used for ELISA detection. The first plate + the second plate had a total of 144 monoclonal clones. The detection results of the first plate and the second plate mentioned in Tables 10, 11, 12, and 13 are the detection results of 144 monoclonal clones.

[0062] Table 10 Final detection results of the first plate antigen FGL1 of the phage library obtained from FGL1-4th and FGL1-5th

[0063] 1 2 3 4 5 6 A 1.3631 0.7443 0.7078 0.9842 0.944 0.8687 B 0.9787 0.8093 1.2924 0.5967 1.0455 0.1535 C 0.7718 0.6463 0.7958 0.9052 0.8018 0.4648 D 0.536 0.4177 0.7485 0.8027 0.8419 0.1744 E 0.7243 0.7643 0.7482 1.0386 0.6839 0.2188 F 0.8468 0.8504 0.8320 0.9949 0.8565 0.7184 G 0.8576 0.6998 0.9768 1.0337 0.854 0.761 H 1.0147 0.7566 0.1518 1 0.219 0.5235

[0064] Table 11 Negative test results of the first plate antigen FGL1 of the phage library obtained from FGL1-4th and FGL1-5th after blocking with 3% milk powder

[0065]

[0066]

[0067] Table 12 Final test results of the second plate antigen FGL1 of the phage library obtained from FGL1-4th and FGL1-5th

[0068] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.1968 0.1654 0.1872 0.222 0.5008 0.1428 0.1752 0.2029 0.2865 0.2674 0.7034 0.2123 B 0.1863 0.237 0.2132 0.1699 0.4203 0.2234 0.2809 0.1402 0.3812 0.1703 0.1692 0.1794 C 0.1372 0.1587 0.1774 0.1218 0.1216 0.0901 0.3824 0.1194 0.2636 0.1037 0.1013 0.18 D 0.3189 0.115 0.1914 0.1403 0.2556 0.1045 0.1845 0.0723 0.1807 0.1132 0.1409 0.1734 E 0.2773 0.2203 0.1469 0.1791 0.3208 0.1455 0.23 0.2774 0.1375 0.2059 0.1598 0.2385 F 0.1506 0.1763 0.2595 0.1552 0.1418 0.3766 0.2695 0.1507 0.2017 0.1716 0.1567 0.1576 G 0.1781 0.2615 0.1503 0.1936 0.163 0.1646 0.4939 0.104 0.2234 0.2564 0.2303 0.2879 H 0.2757 0.4512 0.3049 0.2173 0.1134 0.1103 0.177 0.1004 0.284 0.1512 0.1747 2.6522

[0069] Table 13 Negative test results of the second plate antigen FGL1 of the phage library obtained from FGL1-4th and FGL1-5th after blocking with 3% milk powder

[0070] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.4539 0.3135 0.3329 0.4176 0.4618 0.2704 0.3996 0.3187 0.3988 0.3924 0.4709 0.3722 B 0.3686 0.3495 0.2952 0.4326 0.429 0.2925 0.3733 0.3379 0.5168 0.3298 0.3245 0.4 C 0.4303 0.3629 0.2903 0.3373 0.2932 0.3208 0.2891 0.2239 0.3898 0.2453 0.3778 0.2785 D 0.4655 0.3661 0.3607 0.4431 0.3707 0.3287 0.2682 0.1606 0.3234 0.2578 0.3249 0.3624 E 0.5743 0.3692 0.3421 0.4149 0.4423 0.3533 0.3651 0.4311 0.2582 0.2783 0.3939 0.3509 F 0.4816 0.3094 0.3921 0.3521 0.3197 0.3798 0.3724 0.3154 0.1971 0.1057 0.1327 0.1351 G 0.4622 0.3835 0.3692 0.3235 0.3721 0.3525 0.2975 0.1511 0.1683 0.1143 0.1092 0.1525 H 0.339 0.3156 0.3346 0.3918 0.1236 0.3644 0.3282 0.1703 0.3152 0.1629 0.0794 0.073

[0071] 5. Based on the above monoclonal detection results, 8 monoclonal clones were selected from the first plates of FGL1-4th and FGL1-5th. The negative test results after 3% milk powder blocking are shown in Table 14. These 8 clones were then sequenced. The amino acid sequences of the 8 monoclonal clones are shown in SEQ ID NOs: 1 to 8 (i.e., the single-chain antibodies against human secretory FGL1 protein provided by the present invention), and the nucleotide sequences of the encoding genes are shown in SEQ ID NOs: 9 to 16, as specifically shown in Table 15.

[0072] Table 14 Negative test results of 8 selected monoclonals after 3% milk powder blocking

[0073]

[0074] Table 15 Amino acid sequences and encoding genes of single-chain antibodies against human secretory FGL1 protein

[0075]

[0076] It should be noted that among the eight monoclonal antibodies mentioned above: the first six bases of the nucleotide sequence of clone H004 were mutated based on the primers and lack a light chain; the amino acid sequence of clone H007 contains two stop codons, indicating a frameshift and is not a complete antibody sequence; and the sequencing of clone H008 was incorrectly tailed, possibly due to excessive length or a problem with the sequence itself. Therefore, the single-chain antibodies against human secretory FGL1 protein corresponding to clones H004, H007, and H008 may not be considered for subsequent use in practical applications.

[0077] Example 3 Blocking Activity Detection of Single-chain Antibody Against Human Secretory FGL1 Protein (FGL1 scFv)

[0078] This example aims to select 12 positive phage clones from Example 2 and use competitive ELISA to test single-chain antibodies (FGL1) against human secretory FGL1 protein. The blocking activity of the scFv was detected. The specific experimental steps were as follows: different concentrations of FGL1 protein were fixed on the surface of a polystyrene microplate by physical adsorption, and irrelevant proteins were added thereto to block nonspecific binding sites. Subsequently, an Anti-FGL1 antibody standard or a test sample (the single-chain antibody against human secretory FGL1 protein obtained in Example 2) was added, followed by the competitive ligand Biotin-labeled LAG-3 protein, and then avidin-coupled peroxidase (HRP) was added. Finally, a colorimetric substrate was added, and the absorbance OD value of the system was detected to reflect the binding ability of the FGL1 protein (antigen) and the competitive ligand LAG-3. The inhibition rate of the single-chain antibody against human secretory FGL1 protein on FGL1 / LAG-3 binding in the test sample was further calculated. Finally, three phage clones with significant blocking activity were screened, and the results are shown in FIG. Figure 4 As shown, Figure 4 The horizontal axis represents the logarithm of the concentration (nM) of the single-chain antibody (FGL1 scFv) against human secretory FGL1 protein (Log2), and the vertical axis represents the Mean OD 450 The average absorbance at a wavelength of 450 nm (OD 450 ).

[0079] Depend on Figure 4 It can be seen that the single-chain antibody against human secretory FGL1 protein (FGL1 scFv) provided by the present invention can target human FGL1 protein, thereby blocking the binding activity of the Anti-FGL1 antibody to FGL1 / LAG-3.

[0080] In summary, the inventors of the present application isolated peripheral blood mononuclear cells (PBMCs) from the blood of healthy volunteers and extracted RNA from the PBMCs. The extracted RNA was reverse transcribed and amplified using RT-PCR technology to obtain the heavy chain encoding gene and the light chain encoding gene of the single-chain antibody, and the two were randomly recombined and linked using universal linker primers to obtain the single-chain antibody encoding gene (i.e., the single-chain variable region fragment encoding gene, referred to as scFv fragment). The single-chain antibody encoding gene was then linked to an expression vector by electroporation to construct a phage library. The phage library was then subjected to five rounds of panning using human secretory FGL1 protein to screen and obtain eight phage antibodies with amino acid sequences as shown in SEQ ID NOs: 1 to 8. The amino acid sequences of the antibodies and the nucleotide sequences of the corresponding encoding genes were further examined, and three monoclonal clones with problems were excluded. Finally, five phage antibodies with amino acid sequences as shown in SEQ ID NOs: 1, 2, 3, 5, and 6 were obtained, namely, the single-chain antibody against human secretory FGL1 protein (FGL1) provided by the present invention. The single-chain antibody (scFv) has high specificity and affinity for human FGL1 protein and low immunogenicity.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims

1. A single-chain antibody against human secretory FGL1 protein, characterized in that: The amino acid sequence of the single-chain antibody is selected from one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:

6.

2. A gene encoding the single-chain antibody against human secretory FGL1 protein according to claim 1.

3. The gene encoding the single-chain antibody against human secretory FGL1 protein according to claim 2, characterized in that: The nucleotide sequence of the gene is selected from one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO:

14.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule comprises a gene encoding the single-chain antibody against human secretory FGL1 protein according to claim 1 .

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 4.

6. A cell, characterized in that: The cell contains the expression vector according to claim 5.

7. A human secretory FGL1 protein inhibitor, characterized by: The human secretory FGL1 protein inhibitor comprises the single-chain antibody against human secretory FGL1 protein as claimed in claim 1.

8. Use of the single-chain antibody against human secretory FGL1 protein according to claim 1 in the preparation of a product for detecting human secretory FGL1 protein.

9. A kit for detecting human secretory FGL1 protein, characterized in that: The kit comprises the single-chain antibody against human secretory FGL1 protein as claimed in claim 1.

10. Use of the single-chain antibody against human secretory FGL1 protein according to claim 1 in the preparation of a drug for treating malignant tumors.

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