Targeted TROP2 shark single-domain antibody and application thereof

Abstract

The invention discloses a shark single-domain antibody targeting to TROP2 and an application of the shark single-domain antibody. The shark single-domain antibody targeting the TROP2 and the immunotoxin of the shark single-domain antibody are 4C6 with the amino acid sequence shown as SEQ ID NO.1. The invention further discloses a nucleotide sequence of the coding gene of the shark single-domain antibody and the immunotoxin of the shark single-domain antibody. The nucleotide sequence of the coding gene is shown as SEQ ID NO.2. The shark single-domain antibody prepared by the invention is small in molecular weight, high in stability and good in tissue permeability, has good affinity and specificity with TROP2, and has good selectivity for TROP2 positive tumor cells. The antibody can be used for constructing immunotoxin, an antibody-coupled drug (ADC), a nuclide-coupled drug (RDC), antibody fusion protein, CAR-T, CAR-NK cells and the like, and can also be used for developing in-vitro diagnostic reagents and scientific research reagents. The invention lays a foundation for tumor treatment and development of antibody drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a shark single-domain antibody targeting TROP2 and its application. Background Art

[0002] Human trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein belonging to the tumor-associated calcium signal transduction protein (TACSTD) family. The expression level of TROP2 is low in normal cells, but it is significantly elevated in a variety of malignant tumors, including breast cancer, pancreatic cancer, colorectal cancer, and lung cancer, etc. This difference in expression makes TROP2 an important tumor target and has received extensive attention in recent years. Research shows that TROP2 not only promotes the proliferation, invasion, and metastasis of tumor cells, but is also closely related to the poor prognosis of patients. Therefore, developing precise targeted therapeutic molecules against TROP2 has important clinical significance.

[0003] Traditional antibody drugs are mainly based on IgG-type antibodies. Although these molecules have high specificity, they have a large molecular weight, poor tissue permeability, and may exhibit off-target toxicity in the complex tumor microenvironment. In addition, the engineering modification of IgG antibodies is relatively complex, and for some highly hydrophilic or inaccessible tumor targets, their therapeutic effects may be limited.

[0004] Shark single-domain antibodies (IgNAR) are a class of antibodies unique to cartilaginous fish. Different from traditional IgG antibodies, they consist only of heavy chains, with a compact and stable structure. The variable region (VNAR) of shark single-domain antibodies is the smallest known natural antibody binding domain, with a molecular weight of only 12 - 15 kDa, significantly smaller than conventional antibodies. VNAR has unique characteristics, including a small molecular weight, good solubility, strong tissue permeability, flexible engineering modification, and low immunogenicity. These advantages make it an ideal candidate molecule for the development of next-generation antibody drugs. In addition, due to its unique binding mode, VNAR can target cryptic epitopes that traditional antibodies cannot effectively recognize, thus expanding its application scope in disease treatment.

[0005] Therefore, the development of novel targeted shark single-domain antibodies provides a new solution for the development of tumor targeted diagnostic reagents, protein drugs, and cell therapy drugs such as CaR-T. Its design not only overcomes the deficiencies of traditional antibody drugs in tumor diagnosis and treatment, but also expands the new direction of antibody drug development, with broad application prospects. Summary of the Invention

[0006] The present invention provides a preparation method and application of a shark single-domain antibody 4C6 targeting TROP2. The present invention screens and obtains a novel shark single-domain antibody VNAR-4C68, which has high affinity and high specificity for TROP2 and good targeting ability for triple-negative breast cancer.

[0007] To achieve the above invention object, the present invention is implemented by the following technical solutions: The present invention provides a shark single-domain antibody targeting TROP2, and its amino acid sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] Furthermore, the shark single-domain antibody comprises fixed amino acid sequences FR1 and FR4, hypervariable regions HV2 and HV4, and complementary determining regions CDR1 and CDR3.

[0009] The present invention also provides a coding gene of the shark single-domain antibody, and its nucleotide sequences are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] The present invention also provides a recombinant plasmid and a recombinant protein containing the coding gene.

[0011] The present invention also provides a recombinant strain containing the coding gene.

[0012] The present invention also provides a preparation method of the shark single-domain antibody, which comprises the following steps: (1) Immunize sharks with human TROP2 recombinant protein to obtain immunized sharks; (2) Collect blood or tissues of the immunized sharks, extract RNA and reverse transcribe it into cDNA, perform PCR amplification, and ligate the PCR product to a phagemid vector to obtain a ligation product; (3) Electrotransform the ligation product into Escherichia coli, culture it overnight, and then perform three rounds of shark single-domain antibody phage panning using human TROP2 recombinant protein; (4) Identify the single colonies of Escherichia coli obtained by panning, select single colonies with an OD 450 value greater than 0.8 for sequencing, and compare and analyze the sequences conforming to the shark origin to obtain the shark single-domain antibody.

[0013] Furthermore, in the step (2), the upstream primer sequence of the PCR amplification primer is as shown in SEQ ID NO.5~SEQ ID NO.9; the downstream primer sequence is as shown in SEQ ID NO.10~SEQ ID NO.14.

[0014] The present invention also provides the use of the shark single-domain antibody or the recombinant immunotoxin protein in the preparation of a TROP2 targeting agent.

[0015] The present invention also provides the use of the shark single-domain antibody or the recombinant immunotoxin protein in the preparation of a detection agent or drug for diagnosing or treating tumors.

[0016] Further, the tumors are triple-negative breast cancer and pancreatic cancer.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The shark single-domain antibody 4C6 prepared by the present invention has a small molecular weight, only about 16 kDa, which is smaller than traditional monoclonal antibodies. 2. The shark single-domain antibody 4C6 has high stability, good tissue permeability, and good affinity and specificity for TROP2. Therefore, it has important scientific significance for the research on shark single-domain antibodies targeting TROP2 and the development of antibody drugs. 3. The shark single-domain antibody immunotoxin can express an antibody fusion toxin protein with good solubility and high tumor inhibitory activity, which provides a new strategy for the development of anti-tumor drugs targeting TROP2. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the SDS-PAGE result after purification of the shark single-domain antibody 4C6; M: Protein Marker; 1: Whole protein; 2: Precipitate; 3: Supernatant; 4: Penetrate; 5 - 15: 20 mM, 25 mM, 50 mM, 100 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 500 mM imidazole elution buffer. Figure 2 It is the result of detecting the binding force between the single-domain antibody 4C6 and TROP2 protein by indirect ELISA method. Figure 3 shows the binding of the shark single-domain antibody 4C6 to HUVEC and MDA-MB-468 cells detected by flow cytometry. A. Binding of 4C6 to HUVEC cells; B. Binding of the shark single-domain antibody 4C6 to MDA-MB-468 cells. Figure 4 shows the detection results of the affinity of the shark nanobody 4C6 for HUVEC and MDA-MB-468 cells. A is the binding to MDA-MB-468 cells; B is the binding to HUVEC cells. Figure 5 It is the immunofluorescence result of the binding of the shark single-domain antibody 4C6 to MDA-MB-468 cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection required 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 Human Recombinant TROP2 Protein 1. Immunization of Chiloscyllium plagiosum Select a healthy Chiloscyllium plagiosum and immunize it with human recombinant TROP2 protein (provided by Novus Biologicals Co., Ltd.) as the antigen. At the first immunization, dissolve 150 μg of the antigen in PBS and mix it with complete Freund's adjuvant at a ratio of 1:1, and complete the injection through multiple subcutaneous injections on the back. Subsequent immunizations are carried out every 3 weeks after the first immunization. For the second to fourth immunizations, 100 μg of the antigen is mixed with incomplete Freund's adjuvant at a ratio of 1:1 and injected in the same way. The fifth immunization is carried out 3 weeks after the fourth immunization. Take 2 μg of the antigen dissolved in PBS and complete the injection through intravenous injection. Collect the peripheral blood of Chiloscyllium plagiosum after immunization for constructing the phage display library.

[0021] 2. Construction of the VNAR Library Immunize sharks with human recombinant TROP2 protein, collect peripheral blood lymphocytes PBMC or spleen tissue, extract RNA using the TRIZOL method, and then reverse transcribe it into cDNA; Use the primer sequences (SEQ ID NO.5 - 14) to perform PCR amplification on the cDNA, and the obtained PCR product is Sfi digested with I enzyme and ligated to the phagemid vector pComb3xss.

[0022] The primers used are: BamVF1: CAATTTGATTGGGCCCAGGCGGCCgccsmacggsttgaacaaac ac (SEQ ID NO.3); BamVR1: CAATTTGATTGGGCCGGCCTGGCCagggttcacagtcasarkg gtsc (SEQ ID NO.4); Amplify the VNAR region of Chiloscyllium plagiosum by PCR 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 2 、10 3 、10 4 、10 5times, 100 μL of each gradient-diluted bacterial solution was spread onto freshly prepared 2×YT / A100 (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, dissolved in 900 mL of double-distilled water and cultured overnight at 37°C. According to the colony growth situation, the number of colonies on the plate diluted 10 5 times was counted, and the library capacity of the constructed shark single-domain antibody library could be calculated. The quality evaluation of the VNAR library is shown in Table 1, including library capacity, gene insertion rate, and gene diversity After amplifying the original phage library, biopanning was carried out. The enrichment of specific phages was verified by polyclonal and monoclonal ELISA, and high-affinity single phages were obtained. The single-domain antibody 4C6 was screened out by sequence alignment. The amino acid sequence of 4C6 is shown in SEQ ID NO.1, and the encoded nucleotide sequence is shown in SEQ ID NO.2; The 4C6 sequence was aligned with the NCBI database, and the results showed that these sequences were all single-domain antibody gene sequences derived from sharks.

[0023] The shark single-domain antibody 4C6 includes the framework region FR and the complementarity-determining region CDR. Among them, the framework region FR and the hypervariable region HV include the amino acid sequences of FR1, FR2, FR3a, HV2, HV4, FR3b, and FR4, which are as follows: FR1: VDQTPTTTTKEAGESLTINCVLR; FR2: TYWYFTKKGAT; HV2: KKESLSNG; FR3a: GRYAETV; HV4: NKASK FR3b: SFSLRISDLRVEDSGTYHC.

[0024] FR4: EGGGTILTVK The complementarity-determining region CDR includes the amino acid sequences of CDR1 and CDR3. Corresponding to the above-mentioned each FR, the CDR amino acid sequences of the shark single-domain antibody 5G8 are respectively:

[0025] Example 2: Recombinant expression and purification of single-domain antibody The VNAR fragment 4C6 of the shark single-domain antibody in Example 1 was ligated with the pET28a vector by homologous recombination. The ligation product was directly used for the next transformation. The above ligation product was directly transformed into E. coli BL21(DE3) competent cells, and then bacterial liquid PCR was carried out to verify whether the recombinant expression vector was successfully constructed.

[0026] Purification was carried out using Ni ion affinity filler with his tag, and the purified protein ( Figure 1 ) was stored at -80 °C after being aliquoted.

[0027] Example 3: Affinity of single-domain antibody 4C6 to TROP2 protein ELISA detection: ① Dilute TROP2 in PBS to a final concentration of 1 μg / mL, and coat 100 μL / well on the ELISA plate overnight at 4 °C.

[0028] ② Discard the liquid in the plate, add 200 μL / well of PBST, and wash 3 times.

[0029] ③ Add 200 μL / well of MPBS and incubate at 37 °C for 1 h.

[0030] ④ Discard the liquid in the plate, add 200 μL / well of PBST, and wash 3 times.

[0031] ⑤ Dilute the antibody in MPBS, with the highest concentration of 1000 nM, and perform 2-fold dilution. A total of 7 concentration gradients are set, and the diluted antibody is added correspondingly, 100 μL / well, and incubated at 37 °C for 1 h.

[0032] ⑥ Discard the liquid in the plate, add 200 μL / well of PBST, and wash 3 times.

[0033] ⑦ Add HRP-anti-His (diluted 1:10000 in MPBS), 100 μL / well, and incubate at 37 °C for 1 h.

[0034] ⑧ Discard the liquid in the plate, add 200 μL / well of PBST, and wash 3 times.

[0035] ⑨ Add 100 μL / well of the combined solution, react at room temperature in the dark for 10 min, and determine the actual reaction time by observing the color specifically.

[0036] ⑩ Add 50 μL / well of 1 M H2SO4 to terminate the reaction, gently shake the plate to mix the liquid in the wells, and immediately measure the OD with an ELISA reader 450 .

[0037] As Figure 2 shown, the EC50 value of 4C6 is 3.093 nM, indicating good binding ability to TROP2.

[0038] Example 4: Detection of the binding of single-domain antibody to cells by flow cytometry 1) Cell preparation: Culture cells and collect tumor cells in the logarithmic growth phase; 2) Cell grouping: PBS is used as the negative control group, and shark antibody 4C6 is used as the experimental group; 3) Resuspend each group of cells with PBS buffer, place at 4 °C, centrifuge at 1000 rmp for 3 min, and carefully aspirate the supernatant with a pipette. 4) Incubate with the primary antibody; add 100 μL of PBS to the negative control, and add 100 μL of shark antibody 4C6 (10 μg / mL) (diluted to 1 μg / mL with PBS) to the experimental groups respectively, allowing them to fully contact with the primary antibody, incubate on ice for 30 min, and gently flick and mix every 10 min. 5) Washing: After the incubation with the primary antibody, centrifuge at 4 °C, 1000 rpm for 3 min, and carefully aspirate the supernatant with the tip of the pipette. 6) Add 500 μL of 1×PBS buffer, pipette and resuspend; wash 1 - 2 times. 7) Incubate with the secondary antibody: Dilute the anti-His secondary antibody labeled with Alex488 with PBS buffer at 1:200 for the negative control group, and dilute the anti-His secondary antibody labeled with Alex488 with PBS buffer at 1:200 for the shark antibody 4C6, 100 μL per group, resuspend and mix well, incubate on ice for 30 min, and keep the whole process in the dark. 8) Washing: After the incubation with the secondary antibody, the washing steps are the same as in 5). 9) Resuspend the cells in each group with PBS buffer, 350 μL per group, transfer the resuspended cells into flow cytometry tubes, and wait for detection on the machine, paying attention to keeping in the dark.

[0039] The detection results are shown in Figure 3. The shark single-domain antibody 4C6 targeting TROP2 does not bind to human umbilical vein endothelial cells HUVEC that do not express TROP2; while the shark single-domain antibody 4C6 has a high level of binding to breast cancer cells MDA-MB-468 cells with high expression of TROP2.

[0040] Example 5: Detection of the affinity of anti-TROP2 shark nanobody 4C6 with HUVEC and MDA-MB-468 cells and The affinity identification of the purified shark nanobody 4C6 at the HUVEC and MDA-MB-468 cell levels was completed according to the method described in Example 4. The results are shown in Figure 4 and Table 1, indicating that the shark single-domain antibody has strong binding activity to TROP2-high-expressing cells, and the EC50 value is 4.519 nmol / L.

[0041] Example 6: Immunofluorescence experiment of shark single-domain antibody 4C6. MDA-MB-468 cells were directly detected with biotinylated single-domain antibody.

[0042] (1) Seed MDA-MB-468 cells onto the coverslips in 12-well plates. After overnight culture, when the cell confluence reaches 60 - 70%, discard the culture medium.

[0043] After washing once with PBS, add 4% paraformaldehyde (PFA) and fix for 30 min, then wash three times with PBS.

[0044] Add 4% BSA (dissolved in PBS) and incubate at room temperature for 1 h, then wash three times with PBS.

[0045] Add single-domain antibody 4C6 (diluted with 4% BSA to 2 μg / mL of single-domain antibody) and incubate overnight in a 4°C refrigerator.

[0046] The next day, wash three times with PBS, add the corresponding fluorescence-labeled anti-His-tag antibody, incubate at room temperature for 2 h, and then wash three times with PBS.

[0047] Add DAPI (0.1 - 1 μg / mL) and stain for 5 min, then wash three times with PBS. After washing once with sterile water, add an anti-quenching agent to mount the slides and take confocal microscopy images.

[0048] The results of the immunofluorescence assay are shown in Figure 5 , and the single-domain antibody 4C6 can localize the TROP2 protein on the cell membrane of MDA-MB-468.

Claims

1. A shark single-domain antibody 4C6 targeting TROP2, characterized in that , The CDR3 amino acid sequence of the antibody is: KAYSLVGIGCRTVGIFGH.

2. Comprising the shark single-domain antibody described in claim 1, characterized in that , The shark single-domain antibody comprises fixed amino acid sequences FR1 and FR4, hypervariable regions HV2 and HV4, and complementary determining regions CDR1 and CDR3.

3. A shark single-domain antibody 4C6 targeting TROP2, characterized in that , The amino acid sequence of the immune antibody is as shown in SEQ ID No.

1.

4. The coding gene of the shark single-domain antibody according to claim 3, characterized in that , The nucleotide sequence of the encoding gene is as shown in SEQ ID NO.

2.

5. The antibody can be used to construct immunotoxins, antibody-drug conjugates (ADCs), radionuclide-drug conjugates (RDCs), antibody fusion proteins, CAR-T and CAR-NK cells, etc., characterized in that , The use is formed by coupling or fusing with an antibody containing the sequence recited in claim 1.

6. A recombinant plasmid comprising the encoding gene recited in claim 4.

7. A recombinant strain comprising the encoding gene recited in claim 4.

8. Use of the shark single-domain antibody recited in claim 2 or 3 in the preparation of a TROP2 targeting agent.

9. Use of the targeting agent recited in any one of claims 5-7 in the preparation of a TROP2 targeting agent.

10. Use of the shark single-domain antibody recited in claim 2 or 3 in the preparation of a detection agent or drug for diagnosing or treating tumors.

11. Use of the targeting preparation recited in any one of claims 5-7 in the preparation of a detection agent or drug for diagnosing or treating tumors.

12. The application according to claim 11, characterized in that , The tumor is a tumor with positive TROP2 expression.

13. The application according to claim 12, characterized in that , The tumor is triple-negative breast cancer.

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