FAP-targeted shark single-domain antibody 4A1 and application thereof

Abstract

The invention discloses a shark single-domain antibody 4A1 targeting FAP and an application of the shark single-domain antibody 4A1. The amino acid sequence of the shark single-domain antibody 4A1 is as shown in SEQ ID No. 1, and the nucleotide sequence of the encoding gene of the shark single-domain antibody 4A1 is as shown in SEQ ID No. 2. The molecular weight of the shark single-domain antibody 4A1 prepared by the invention is about 15 kDa and is smaller than that of a traditional monoclonal antibody, the stability is high, the tissue permeability is good, and the 4A1 can be expressed by utilizing an escherichia coli prokaryotic expression system. The antibody can be used for constructing immunotoxin fusion protein, the immunotoxin has good killing activity on FAP-positive tumor cells or cancer-related fibroblasts, subsequent effective treatment can be carried out on tumors independently or in combination with other anti-tumor drugs, and a new thought is provided for tumor treatment and antibody drug research and development.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a shark single-domain antibody 4A1 targeting FAP and its application. Background Art

[0002] Fibroblast activation protein (FAP) is a cell surface antigen first reported by Wolf Rettig in 1986. It is a type II transmembrane serine protease and also a member of the dipeptidyl peptidase (DPP) family. The DPP family can hydrolyze the proline bond of the two amino acids at the N-terminus of proteins. And different from other members of the DPP family, FAP also has endopeptidase activity, that is, it can cleave the peptide after proline in the Gly-Pro sequence. As a specific tumor marker, FAP has special biological characteristics (including enzymatic and non-enzymatic effects) and plays an important role in the growth, invasion, metastasis and immunosuppression of tumor cells. FAP is mostly expressed on stromal fibroblasts of epithelial carcinomas, most soft tissue sarcomas, granulation tissue of wound healing and some fetal mesenchymal fibroblasts. In contrast, FAP is not expressed in normal fibroblasts and the stroma of benign epithelial tumors.

[0003] Given the special structure and biological functions of FAP, as well as its important role in the occurrence and development of tumor treatment and the characteristics of selective expression in different tissues. In recent years, the application of FAP in tumor treatment has attracted more and more researchers' attention. Currently, a variety of strategies have been explored, including small molecule inhibitors, prodrugs, tumor vaccines, and monoclonal antibodies. Among them, monoclonal antibodies have been widely used in scientific research and clinical treatment due to their excellent specificity. In fact, monoclonal antibodies have dominated the biopharmaceutical market, and many successful drugs produced in recent years are based on immunoglobulins. However, despite many successful cases, immunoglobulins also have several limitations; for example, their size may limit tissue penetration, the production cost of recombinant monoclonal antibodies is relatively high, and they often show limited stability under non-physiological conditions. Protein engineering has achieved certain success in providing highly stable and functional antibody fragments, but at the same time, the magical nature has provided another possible alternative. In 1993, antibodies with only heavy chains were discovered in camelids (camels, llamas, and alpacas). In 1995, a novel immunoglobulin IgNAR different from traditional antibodies was discovered in nurse sharks. When it was first discovered, it had a relatively high homology with the T cell receptor (TCR), so it was named the new antigen receptor (New antigen receptor, NAR). However, subsequent studies found that this molecule has many similar structural and functional characteristics to immunoglobulins, so it was renamed IgNAR. Different from traditional antibodies, IgNAR is a homodimer composed of two heavy chains and lacks light chains. Scientists found that after cloning and expressing its heavy chain variable region alone, it still retains the ability to bind to antigens, and this part was subsequently named VNAR (Variable new antigen receptor). Compared with traditional antibodies, VNAR has the following characteristics: small size, low immunogenicity, better barrier penetration ability, recognition ability of hidden epitopes, simple structure, and easy coupling with other molecules; easy to produce, and the simple structure can use prokaryotic expression systems; single-domain antibodies also have excellent thermal stability and chemical stability and can maintain activity in harsh environments. These characteristics of single-domain antibodies have laid a good foundation for the development of therapeutic drugs for various administration routes (such as oral agents). Due to its excellent physiological characteristics and structural properties, the research on single-domain antibodies in industries such as medicine and food processing is becoming increasingly popular. Therefore, researching and developing shark single-domain antibodies targeting FAP has important research value and significance for the treatment and diagnosis of tumors. Summary of the Invention

[0004] The object of the present invention is to provide a shark single-domain antibody targeting FAP, its preparation method and application. The present invention screened a novel shark single-domain antibody 4A1, which has high affinity and high specificity for FAP. The immunotoxin prepared with 4A1 as the ligand has good in vitro killing activity against fibroblasts expressing FAP.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions: The present invention provides a shark single-domain antibody 4A1 targeting FAP, and its amino acid sequence is shown in SEQ ID No.1.

[0006] Further, the shark single-domain antibody 4A1 comprises fixed amino acid sequences FR1, FR2, FR3a, FR3b and FR4, hypervariable regions HV2 and HV4, and complementarity-determining regions CDR1 and CDR3.

[0007] The present invention also provides the coding gene of the above-mentioned shark single-domain antibody, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a shark single-domain antibody immunotoxin targeting FAP, and the toxin is specifically Pseudomonas aeruginosa exotoxin PE38.

[0009] The present invention also provides a shark single-domain antibody immunotoxin targeting FAP, and the amino acid sequence of the shark single-domain antibody immunotoxin is shown in SEQ ID NO.3.

[0010] The present invention also provides the coding gene of the above-mentioned shark single-domain antibody immunotoxin, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.4.

[0011] The present invention also provides a recombinant plasmid containing the above-mentioned coding gene.

[0012] The present invention also provides a recombinant strain containing the above-mentioned coding gene.

[0013] The present invention also provides the application of the above-mentioned shark single-domain antibody in the preparation of FAP protein-targeted drugs.

[0014] The present invention also provides the application of the above-mentioned shark single-domain antibody in the preparation of detection agents or drugs for diagnosing or treating tumors.

[0015] The single-domain antibody of the present invention has the following advantages: 1. The shark single-domain antibody 4A1 prepared by the present invention has a small molecular weight, only about 15 kDa, which is smaller than traditional monoclonal antibodies; 2. The shark single-domain antibody 4A1 has high stability, good tissue permeability, is easier to store and transport than conventional antibodies, and has good affinity and specificity for FAP; 3. It is easier to express and genetically engineered. 4A1 is expressed using the Escherichia coli prokaryotic expression system, and a large amount of shark single-domain antibody is finally obtained; 4. The shark single-domain antibody immunotoxin can express an antibody fusion toxin protein with good solubility and high tumor suppression activity. This immunotoxin provides a new strategy for the development of anti-tumor drugs targeting FAP; Its sequence consists of FR1, FR2, FR3a, FR3b and FR4, HV2, HV4, CDR1 and CDR3. Among them, FR1 and FR4 are fixed amino acid sequences, HV2 and HV4 are hypervariable regions, which affect the antigen-binding site, and CDR1 and CDR3 are the complementarity-determining regions of the antibody, which determine the binding to different antigens. In this case, it has the ability to recognize fibroblast activation protein (FAP). VNAR is the antibody structure with the smallest molecular weight. Therefore, VNAR overcomes the disadvantages and deficiencies of traditional antibodies in application. In addition, the present invention selects the barred bamboo shark (Chiloscyllium plagiosum) as the model animal for antibody preparation to prepare VNAR. It does not belong to endangered shark species, has a small body size, is easy to breed artificially, and is suitable for antibody development.

[0016] The shark nanobody 4A1 can be applied to drugs for diagnosing and treating a wide range of cancers. The gene encoding 4A1, recombinant plasmid or recombinant cell containing the gene can be applied to the preparation of immunoassay diagnostic kits, flow cytometry, cellular immunofluorescence detection, and the treatment and diagnosis of a wide range of cancers. Description of the Drawings

[0017] Figure 1 It is the result of the determination of the serum titer after shark immunization; Figure 2 It is for PCR amplification of the shark nanobody gene. Lanes 1 and 5: Primer combination one; Lanes 2 and 6: Primer combination two; Lanes 3 and 7: Primer combination three; Figure 3 It is the result of the determination of the positive rate of 22 monoclonal clones randomly selected from the single-domain antibody phage library. M is the Marker; Figure 4a It is the result of panning phage polyclonal ELISA; Figure 4b It is the result of phage monoclonal ELISA; Figure 5 It is the result of the expression and purification of the single-domain antibody. M is the Marker, and the size of the 4A1 antibody is around 15 kDa.

[0018] Figure 6a To detect the binding of 4A1 to FAP by ELISA experiment; Figure 6b To detect the binding of 4A1 to NIH-3T3 cells expressing FAP by flow cytometry. NIH-0 is the blank control, and the cells are untreated; NIH-1 is the negative control, and the cells are only incubated with the fluorescent secondary antibody; NIH-2 and NIH-3 are the experimental groups. The concentration of the primary antibody in NIH-2 is 10 μg / ml, and the concentration of the primary antibody in NIH-3 is 50 μg / ml; Figure 6c To detect the binding of 4A1 to HEK-293 cells expressing FAP by immunofluorescence experiment. The concentration of the primary antibody in the experimental group is 50 μg / ml, the primary antibody in the negative control group is an irrelevant shark nanobody that does not target FAP, and the primary antibody in the positive control group is a commercial rabbit monoclonal antibody against FAP. The secondary antibody is a goat anti-rabbit A488 fluorescent antibody; Figure 7 For the internalization experiment results of 4A1 antibody, detected by flow cytometry experiment. NIH-3T3 0 is the blank control, and the cells are untreated; NIH-3T3 1 is the positive control, and the experimental steps are the same as those for the flow cytometry detection of binding; for NIH-3T3 15+, after incubating the primary antibody at 4°C for 30 min, incubate at 37°C for 15 min to activate internalization, and the remaining steps are the same; the remaining groups of 30+, 60+, and 120+ are treated at 37°C for 30 min, 60 min, and 120 min respectively; Figure 8 For the docking results of 4A1 antibody and the FAP antigen mimetic molecule, and it is the abstract drawing; Figure 9 For the purification results of the single-domain antibody 4A1 fused with Pseudomonas exotoxin A (PE38) protein. M is the Marker, and the size of the 4A1-PE38 fusion protein is around 55 kDa; Figure 10 To detect the killing results of the 4A1-PE38 immunotoxin on NIH-3T3 cells expressing FAP and wild type by CCK8 experiment. Detailed implementation manners

[0019] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners. However, the scope required to be protected by the present invention is not limited to the scope described in the examples. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the kit biological materials can be obtained from commercial channels unless otherwise specified.

[0020] Example 1: Construction of a shark phage antibody library targeting recombinant human GPC3 protein 1. Immunization of Chiloscyllium plagiosum (1)Primary immunization: Dissolve 400 μg of human FAP recombinant protein in PBS and mix it with complete Freund's adjuvant at a ratio of 1:1 to immunize the bamboo shark for the first time; (2)Second immunization: Two weeks after the primary immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize the bamboo shark for the second time; (3)Third immunization: Two weeks after the second immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize the bamboo shark for the third time; (4)Fourth immunization: Two weeks after the third immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize the bamboo shark for the fourth time; (5)Fifth immunization: Two weeks after the fourth immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize the bamboo shark for the fifth time; (6)Sixth immunization: Two weeks after the fifth immunization, take 300 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize the bamboo shark for the sixth time.

[0021] As Figure 1 shown, after six immunizations, the shark had a good immune response to the antigen FAP, and the titer reached about 1:500.

[0022] 2. Construction of VNAR library (1)Obtaining cDNA Immunize the shark with human FAP recombinant protein, collect peripheral blood lymphocytes PBMC and spleen tissue, extract RNA by the TRIZOL method, and then reverse transcribe it into cDNA.

[0023] (2)Cloning of target gene Use the primer sequences (SEQ ID NO.5 - 12) to perform PCR amplification on the cDNA (see Figure 2 ), and the obtained PCR product is digested with Sfi I and ligated to the phagemid vector pComb3xss.

[0024] Combination 1 (upstream primers NewVF1 + NewVF2, downstream primers NewVR1 + NewVR2) and combination 2 (upstream primers NewVF2 + BamVF3, downstream primers BamVR3 + BamVR4) use the following reaction systems (Tables 1 and 2), combination 3 (upstream primer NewVF4, downstream primer BamVR4) uses the following reaction system (Tables 1 and 2). The sequence of primer NewVF1 is shown in SEQ ID NO.5, the sequence of primer NewVF2 is shown in SEQ ID NO.6, the sequence of primer BamVF3 is shown in SEQ ID NO.7, the sequence of primer BamVF4 is shown in SEQ ID NO.8, the sequence of primer NewVR1 is shown in SEQ ID NO.9, the sequence of primer NewVR2 is shown in SEQ ID NO.10, the sequence of primer BamVR3 is shown in SEQ ID NO.11, and the sequence of primer BamVR4 is shown in SEQ ID NO.12: Table 1 PCR reaction system Reagent Dosage 2×Phanta Max Buffer 25μl dNTP Mix (10 mM each) 1μl Forward Primer (10μM) 2μl each Reverse Primer (10μM) 2μl each Phanta Max Super-Fidelity DNA Polymerase 1μl cDNA 1μl <![CDATA[ddH2O]]> up to 50μl Table 2 PCR reaction program Cycling Steps Temperature Time Number of Cycles Pre-denaturation 95℃ 3 min 1 cycles Denaturation 95℃ 15 s 30 cycles Annealing 60℃ 15 s 30 cycles Extension 72℃ 30 s 30 cycles Final Extension 72℃ 5 min 1 cycles The nucleic acid electrophoresis result of amplifying the VNAR region of Chiloscyllium plagiosum by PCR method is as Figure 2 .

[0025] (3) Construction of phagemid The enzyme digestion system of the PCR product is as follows: Table 3 Enzyme digestion system of PCR product Reagent Dosage rCutSmart™ Buffer 5μl Ⅰ 1μl PCR Product 1μg <![CDATA[ddH2O]]> up to 50μl After thorough mixing, digest at 50 °C for 3 h. After verifying complete digestion by agarose gel electrophoresis, perform gel recovery to recover the digested PCR product, obtaining the shark nanobody gene fragment for subsequent construction of the pComb3XSS recombinant plasmid.

[0026] The enzyme digestion system of the pComb3XSS plasmid is as follows: Table 4 Enzyme digestion system of pComb3XSS plasmid Reagent Dosage rCutSmart™ Buffer 5μl Ⅰ 1μl pComb3XSS Plasmid 1μg <![CDATA[ddH2O]]> up to 50μl After thorough mixing, digest at 50 °C for 3 h. After verifying complete digestion by agarose gel electrophoresis, perform gel recovery to recover the digested pComb3XSS plasmid, obtaining the linearized pComb3XSS plasmid for subsequent construction of the pComb3XSS recombinant plasmid.

[0027] The ligation system of T4 DNA ligase is as follows: Table 5 Ligation system of T4 DNA ligase Reagent Dosage T4 DNA Ligase 1μl 10×T4 DNA Ligase Buffer 2μl Linearized pComb3XSS Plasmid 50ng Shark Nanobody Gene Fragment 30ng <![CDATA[ddH2O]]> up to 20μl Incubate at 16 °C for 12 h, inactivate T4 DNA Ligase at 65 °C for 10 min, and then recover the ligation product, namely the pComb3XSS recombinant plasmid.

[0028] 3. Library construction Electrotransform the ligation product into competent Escherichia coli TG1 to form the original phage library. Subsequently, dilute the bacterial solution by 10 6 -, 10 7 -, 10 8 times. Take 100 μl of the serially diluted bacterial solution and spread it on freshly prepared 2×YT / A100 (16 g tryptone, 10 g yeast extract, 5 g NaCl, dissolve in 900 ml double-distilled water and incubate overnight at 37 °C). Count the number of colonies on the plate diluted 10 7 times according to the colony growth, and calculate the library capacity of the constructed shark single-domain antibody library. The quality evaluation of the VNAR library is shown in Table 6, and the gene insertion rate is as Figure 3 shown.

[0029] Table 6: Quality evaluation of the phage library Library Capacity Insertion Rate Diversity <![CDATA[2×10 8 > 100% 20 / 20 Example 2: Panning of FAP shark single-domain antibody phages Use the original phage library in Example 1, amplify it with helper phage, and then prepare phages for panning.

[0030] I. First-round panning: 1. Coating: Two wells of a 96-well plate, 100 μl / well, overnight at 4 °C.

[0031] ① Coat with human FAP recombinant protein, 10 μg / ml; ② Coat with 5% MPBS.

[0032] 2. Harvest phages Centrifuge the bacterial solution shaken overnight in the previous round at 12000g for 5 min, filter the supernatant through a 0.22 μm filter membrane, add PEG at 1 / 4 of the bacterial solution volume, and incubate on ice for 15 - 30 min; centrifuge at 10000g at 4 °C for 15 min, discard the supernatant, invert and air-dry on absorbent paper, resuspend with PBS, and measure OD280 (OD280 = 1 is equivalent to 2.5x10 12 pfu / ml).

[0033] 3. Wash the plate three times Wash the plate with PBST (PBS containing 0.1% Tween-20), fill the whole well (about 300 μl), and tap the plate.

[0034] 4. Incubate at 37 °C for 1 h to seal.

[0035] ① Add 3% BSA or 5% skim milk powder, the same as the coating, 300 μl / well; ② Add the diluted phage, 100 μl / well; Calculation method: Make the 400 μl of phage added contain 500 times the library capacity to ensure complete panning.

[0036] 5. Activate TG1: Take 10 μl of the TG1 strain that has been shaken overnight and add it to 2 ml of fresh 2x YT medium. Incubate at 37 °C with shaking at 200 rpm for 3 h until the logarithmic growth phase is reached (OD600 = 0.5 - 0.6).

[0037] 6. Incubation: Take out the phage in well ② into an EP tube, wash the plate 3 times, add the above phage to well ①, and incubate at 37 °C for 1 - 1.5 h.

[0038] 7. Wash the plate 6 times.

[0039] 8. Infection: Add the activated TG1 to well ①, 100 μl / well, let it stand at 37 °C for 30 min, collect the infected TG1, replace it with fresh TG1, and repeat this 3 times.

[0040] 9. Verify by plating Dilute the bacterial solution to 1000, 10000, 100000, 1000000 times (1 ml volume) by the method of limited dilution, and then take 100 μl from each and plate them (Amp + ), incubate at 37 °C overnight, observe the number of colonies grown, which is the successfully infected TG1. When the same amount of TG1 is inoculated, a significant increase in the number of colonies grown indicates enrichment, and the following experiment can be carried out.

[0041] 10. Superinfect with helper phage: Add M13K07 with 20 times the bacterial cell amount to the infected TG1, let it stand at 37 °C for 1 h (shake once at 30 min).

[0042] 11. Amplify progeny phage Centrifuge the bacterial solution at 3000 g at 25 °C for 5 min, discard the supernatant, resuspend it in 10 ml of fresh 2x YT, add Kana (50 μg / ml), Amp (100 μg / ml), and incubate at 30 °C with shaking at 200 rpm overnight.

[0043] II. Second-round panning The second round of panning steps is the same as the first round, but the concentration of the coated antigen in step 1 needs to be decreased to 4 μg / ml; when blocking in step 4, choose PBS containing 3% BSA to block the microtiter plate and incubate overnight at 4°C; in step 7, wash the microtiter plate 8 times with PBST, and the rest of the experimental operation steps are the same as those in the first round of panning.

[0044] III. The third round of panning The third round of panning steps is the same as the first round, but the concentration of the coated antigen in step 1 needs to be decreased to 2 μg / ml; when blocking in step 4, choose PBS containing 5% skim milk powder to block the microtiter plate and incubate overnight at 4°C; in step 7, wash the microtiter plate 10 times with PBST, and the rest of the experimental operation steps are the same as those in the first round of panning.

[0045] The titer of the panned phages increased significantly in the second and third rounds. Specific phages were also enriched by polyclonal ELISA detection (see Table 7 and Figure 4a ).

[0046] Table 7: Titre results of the original and three-round panned phage single-domain antibody libraries Library Original Phage Antibody Library First Round Phage Antibody Library Second Round Phage Antibody Library Third Round Phage Antibody Library Titer / each <![CDATA[2×10 13 > <![CDATA[8×10 6 > <![CDATA[3.2×10 6 > <![CDATA[2.6×10 7 > To obtain high-affinity single phages, 95 single colonies were picked from the plates obtained in the third round of panning for identification. Among the 95 picked monoclonal clones, 30 were positive monoclonal clones (see Figure 4b ), and the positive rate was 31.5%. The bacterial solutions of these 30 monoclonal colonies were sent to a sequencing company for sequencing. After translation and sequence alignment of the sequencing results of the positive monoclonal clones, shark nanobody sequences were obtained and named 4A1.

[0047] The amino acid sequence of 4A1 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; when the 4A1 sequence was aligned with the NCBI database, the results showed that these sequences were all single-domain antibody gene sequences from sharks.

[0048] The shark single-domain antibody 4A1 includes a framework region FR and complementarity-determining regions CDR. The framework region FR includes the amino acid sequences of FR1, CDR1, FR2, FR3a, HV2, HV4, FR3b, CDR3, and FR4, which are as follows: FR1: ARLEQTPTTTTKEAGESLTINCVLR; FR2: TNWHFTKKGATKKE; HV2: SLSNG; FR3a: GRYAETV; HV4: NKASK; FR3b: SFSLRISDLRVEDSGTYHC; FR4:EGGGTILTVKP; The complementarity-determining regions CDR include the amino acid sequences of CDR1 and CDR3, corresponding to the above respective FRs. The CDR amino acid sequences of the shark single-domain antibody VNAR-F10 are respectively: Number CDR1 CDR3 4A1 DSSCALAS KALAFGCQFQGYY Example 3: In vitro recombinant expression and purification of single-domain antibody 4A1 Using the positive clone phagemid as a template, according to the sequence information of the sequencing results, the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.14 were designed, and the 4A1 sequence was amplified by PCR. The pET28a vector was digested with Not I、 Nco I by double digestion. The shark single-domain antibody gene 4A1 fragment was ligated to the pET28a vector by homologous recombination, and the above ligation product was transformed into E. coli BL21(DE3) competent cells. Purification was carried out using a nickel ion chelating filler with the his tag. Each purified protein was aliquoted and stored at -80 °C. The impurity removal condition during the purification process was 50 mM imidazole (PBS solution), and the elution condition was 200 mM imidazole concentration (PBS solution). The results were as Figure 5 shown, and the position of the target band was consistent with the theoretical molecular weight of 4A1.

[0049] Example 4: ELISA detection of the binding of shark nanobody to recombinant human FAP protein Add 100 μl / well of 2 mg / ml recombinant human GPC3 protein to the enzyme-linked immunosorbent assay (ELISA) plate and incubate overnight at 4 °C; discard the supernatant, and wash 3 times with 300 μl / well of phosphate buffered saline with 0.1% Tween 20 (PBST); add 200 μl / well of PBS containing 5% skim milk powder and incubate at 37 °C for 1 h, then wash 3 times with 300 μl / well of PBST; add 100 μl / well of shark nanobodies at different concentrations (0.1, 1, 5, 10, 50, 100, 200 μg / ml) and incubate at 37 °C for 1 h, then wash 3 times with 300 μl / well of PBST; add 100 μl / well of HRP-anti-HA tag antibody (diluted 1:10000 in PBS containing 5% skim milk powder) and incubate at 37 °C for 1 h, then wash 3 times with 300 μl / well of PBST; add 100 μl of TMB substrate to each well and incubate in the dark at 37 °C for 10 - 15 min. After color development, add 50 μl of 1 mol / l H2SO4 to each well to terminate the reaction, and detect the OD450 value. According to the ELISA resultsFigure 6a 4A1 can specifically bind to recombinant human FAP protein.

[0050] Example 5: Detection of the binding of shark nanobody to cells expressing FAP by immunofluorescence and flow cytometry Flow cytometry: Culture the modified NIH-3T3 cells with high expression of human FAP, and digest the cells; Resuspend the cells with PBS buffer, centrifuge at 1000 rpm at 4 °C for 5 min horizontally; Resuspend the cells with PBS buffer, after counting, aliquot into 1,000,000 cells / 200 μl per tube, centrifuge at 1000 rpm at 4 °C for 5 min; Resuspend the cells in the experimental group with 4A1 antibody diluted with FACS buffer (concentrations of 10 μg / ml and 50 μg / ml), incubate at 4 °C for 30 min, then centrifuge at 1000 rpm at 4 °C for 5 min horizontally; Discard the supernatant, wash 3 times with PBS, add anti-His-A488 antibody diluted with FACS buffer (dilution ratio of 1:300), incubate at 4 °C for 30 min, then centrifuge at 1000 rpm at 4 °C for 5 min; Discard the supernatant, wash 3 times with PBS, and detect by machine.

[0051] The detection results are as Figure 6b shown. NIH-0 is the blank control, NIH-1 is the control group incubated only with the fluorescent secondary antibody, and the 4A1 concentrations in the experimental groups NIH-2 and NIH-3 are 10 μg / ml and 50 μg / ml. The results prove that the shark single-domain antibody 4A1 targeting FAP has a high level of binding to the mouse fibroblast NIH-3T3 with high expression of FAP.

[0052] Immunofluorescence experiment: Place glass slides in a 12-well plate in advance, inoculate the modified HEK-293 cells with high expression of human FAP, and the inoculation density is 5×10 5 cells / well; After culturing overnight, discard the medium, wash 3 times with PBS, add 4% paraformaldehyde to fix for 10 min, and wash 3 times with PBS; 3% BSA dissolved in PBS is used as the blocking solution, 1 ml / well, incubate at room temperature for 1 h, and wash 3 times with PBS; Dilute the shark nanobody with the blocking solution, and the final concentration is about 50 μg / ml, 1 ml / well, incubate at 4 °C for 1 h, and wash 3 times with PBS; Dilute the anti-His-A488 antibody with the blocking solution (diluted 1:300), incubate at 4 °C for 1 h, and wash 3 times with PBS; Add DAPI to stain for 5 min, and wash 3 times with PBS; After adding the anti-fluorescence quenching agent and mounting the coverslip, take pictures with a fluorescence microscope. The primary antibody in the negative control group is an irrelevant shark nanobody that does not target FAP, the primary antibody in the positive control group is a commercial rabbit monoclonal antibody against FAP, and the secondary antibody is a goat anti-rabbit A488 fluorescent antibody.

[0053] The immunofluorescence results are asFigure 6c As shown, 4A1 can specifically bind to HEK-293 cells highly expressing FAP, further demonstrating the specific binding of 4A1 to cells highly expressing FAP.

[0054] Example 6: Detection of internalization of shark nanobody by flow cytometry The flow cytometry experiment was generally the same as the flow experiment in Example 5. The difference was that after the primary antibody in the experimental group was incubated at 4°C for 30 min, it was incubated at 37°C for 15 min, 30 min, 60 min, and 120 min to activate internalization. The remaining steps were the same as above. Whether internalization occurred was judged by detecting whether the fluorescence decreased. The results are as Figure 7 shown, where NIH-3T3 0 was the blank control, NIH-3T3 1 was the positive control group, and the rest were the experimental groups. The antibody had internalized after incubation at 37°C for 15 min.

[0055] Example 7: Expression of shark nanobody immunotoxin fusion protein The relevant genes were obtained by synthesis. The protein expression steps were the same as those in Example 3. The results are as Figure 9 shown, and its size was about 55 kDa.

[0056] Example 8: Detection of killing of FAP-positive cells by immunotoxin fusion protein by CCK8 assay The modified NIH-3T3 cells highly expressing human FAP and wild-type NIH-3T3 cells not expressing FAP were cultured. After digestion, they were resuspended with medium and added to a 96-well plate at 8000 cells / well. After overnight culture, different concentrations of 4A1-PE38 antibody (volume 100 μl) were added according to each column as a group. After treatment for 48 h, 20 μl of CCK8 reagent was added to each well. After incubation at 37°C for 2 h, the OD 450 value was measured. The results are as Figure 10 shown. 4A1-PE38 can specifically and efficiently kill only NIH3T3 cells highly expressing FAP, and its IC 50 was about 500 pM.

[0057] The above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A shark single-domain antibody 4A1 targeting FAP and its application, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The shark single-domain antibody 4A1 according to claim 1, wherein The shark single-domain antibody 4A1 comprises the fixed amino acid sequences FR1, FR2, FR3a, FR3b and FR4, the hypervariable regions HV2 and HV4, and the complementarity-determining regions CDR1 and CDR3.

3. The shark single-domain antibody 4A1 according to claim 1, wherein The amino acid sequence of the CDR3 is KALAFGCQFQGYY.

4. The coding gene of the shark single-domain antibody according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

5. The encoding gene of the shark single-domain antibody according to claim 4, characterized in that, The sequence of the forward primer for cloning the encoding gene is shown in SEQ ID NO. 13, and the sequence of the reverse primer is shown in SEQ ID NO.

14.

6. A shark single-domain antibody immunotoxin targeting FAP, characterized in that, The amino acid sequence of the shark single-domain antibody immunotoxin is shown in SEQ ID NO.

3.

7. The coding gene of the shark single-domain antibody immunotoxin according to claim 6, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

8. Use of the shark single-domain antibody 4A1 according to claim 1 in the preparation of an FAP protein-targeted drug.

9. Use of the shark single-domain antibody 4A1 according to claim 1 in the preparation of a tumor detection reagent and a drug for treating tumors.

10. Use of the immunotoxin according to claim 6 in the preparation of a detection agent or a drug for diagnosing or treating tumors.

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