Shark single-domain antibody targeting ERα36, its preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to shark single-domain antibodies targeting ERα36, their preparation methods and applications. The provided shark single-domain antibodies FR6 and TH4 have high stability, strong affinity, and good binding ability and specificity with ERα36, which have important scientific significance for the research of shark single-domain antibodies targeting ERα36 and the development of antibody drugs. In particular, it provides a new research direction and application prospect for the personalized precision medicine of tumors such as breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a shark single-domain antibody targeting ERα36, its preparation method, and application. Background Art

[0002] Breast cancer is one of the most common malignant tumors worldwide. Among them, triple-negative breast cancer (TNBC) has limited existing targeted treatment means due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2).

[0003] Currently, the classical 66 kDa estrogen receptor (ERα66) requires a traditional hormone-binding mechanism for functional regulation, and the nuclear localization of traditional ERα also affects the response speed to external signal stimuli. ERα36, as a splicing variant of ERα, is often located in the cell membrane or cytoplasm, which enables it to rapidly respond to external signal stimuli and activate the non-genomic signaling pathway, making it an ideal candidate target. How to develop antibodies targeting ERα36 to fill the gap in existing tumor-targeted treatment methods such as breast cancer and improve the specificity and effectiveness of targeted treatment has important research value. Summary of the Invention

[0004] To solve the problems raised in the background art, the present invention provides a shark single-domain antibody targeting ERα36, its preparation method, and application.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention provides a shark single-domain antibody targeting ERα36, and its amino acid sequence is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0007] The nucleotide sequence encoding the antibody gene is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0008] The antibody includes a framework region and a complementarity-determining region;

[0009] The framework region includes FR1, FR2, and FR3;

[0010] Among them, the amino acid sequence of FR1 is shown as SEQ ID NO.5;

[0011] The amino acid sequence of FR2 is shown as SEQ ID NO.6 or SEQ ID NO.7;

[0012] The amino acid sequence of FR3 is shown as SEQ ID NO.8 or SEQ ID NO.9;

[0013] The complementary determining regions include CDR1 and CDR3;

[0014] Among them, the amino acid sequence of CDR1 is shown in SEQ ID NO.10 or SEQ ID NO.11;

[0015] The amino acid sequence of CDR3 is shown in SEQ ID NO.12 or SEQ ID NO.13.

[0016] The present invention also provides a method for preparing the shark single-domain antibody targeting ERα36. RNA of sharks immunized with ERα36 is extracted, reverse-transcribed into cDNA, and then amplified and ligated to a phagemid vector by enzymatic digestion in sequence to construct a shark phage antibody library specifically recognizing ERα36. After panning the shark phage antibody, a shark single-domain antibody targeting ERα36 is obtained.

[0017] The present invention also provides an application of the shark single-domain antibody targeting ERα36 in preparing an ERα36 targeting agent.

[0018] The present invention also provides an application of the shark single-domain antibody targeting ERα36 in preparing a drug targeting ERα36-positive tumor cells.

[0019] The tumors are positive breast cancer and triple-negative breast cancer.

[0020] Beneficial effects: The shark single-domain antibodies FR6 and TH4 provided by the present invention have high stability, strong affinity, and good binding force and specificity with ERα36, which have important scientific significance for the research of shark single-domain antibodies targeting ERα36 and the development of antibody drugs, and especially provide a new research direction and application prospect for the personalized precision medicine of tumors such as breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the determination result of the serum titer of sharks immunized with ERα36 antigen polypeptide.

[0022] Figure 2 It is the verification result of the coating plate for three rounds of biopanning.

[0023] Figure 3 It is the identification result of specific phage polyclonal ELISA.

[0024] Figure 4 It is the identification result of specific phage monoclonal ELISA.

[0025] Figure 5 It is the protein expression result after purification of shark nanobodies FR6 and TH4.

[0026] Figure 6Results of affinity detection of ERα36-specific shark single-domain antibody, where (a) is FR6 and (b) is TH4.

[0027] Figure 7 Results of ELISA assay for the binding of ERα36 to shark single-domain antibodies FR6 and TH4.

[0028] Figure 8 Results of flow cytometry assay for the binding of shark single-domain antibodies FR6 and TH4 to MCF-7, BT474, and MDA-MB-468 cells with different ERα36 expressions.

[0029] Figure 9 Results of immunofluorescence assay of shark single-domain antibody targeting ERα36 on MDA-MB-468 cells. Detailed implementation manners

[0030] The following examples are intended to illustrate the present invention rather than further limit the present invention.

[0031] The present invention provides a shark single-domain antibody targeting ERα36, and its amino acid sequence is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0032] It also provides a gene encoding the shark single-domain antibody targeting ERα36, and the nucleotide sequence encoding the antibody gene is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0033] The antibody includes a framework region and a complementary determining region;

[0034] The framework region includes FR1, FR2, and FR3;

[0035] Among them, the amino acid sequence of FR1 is shown as SEQ ID NO.5;

[0036] The amino acid sequence of FR2 is shown as SEQ ID NO.6 or SEQ ID NO.7;

[0037] The amino acid sequence of FR3 is shown as SEQ ID NO.8 or SEQ ID NO.9.

[0038] The complementary determining region includes CDR1 and CDR3;

[0039] Among them, the amino acid sequence of CDR1 is shown as SEQ ID NO.10 or SEQ ID NO.11;

[0040] The amino acid sequence of CDR3 is shown as SEQ ID NO.12 or SEQ ID NO.13.

[0041] The present invention also provides a method for preparing the shark single-domain antibody targeting ERα36. RNA is extracted from sharks immunized with ERα36, reverse transcribed into cDNA, and then amplified and ligated to a phagemid vector by enzymatic digestion in sequence to construct a shark phage antibody library specifically recognizing ERα36. After panning the shark phage antibodies, a shark single-domain antibody targeting ERα36 is obtained. The specific operations are as follows in the examples.

[0042] Example 1 Construction of a shark phage antibody library specifically recognizing ERα36

[0043] 1. Immunization of Chiloscyllium plagiosum

[0044] For the first immunization, 300 μg of a polypeptide composed of the C-terminal 27 amino acid short peptide of ERα36 conjugated with KLH protein and a partial fragment of the E / F domain (purchased from GenScript Biotech Corporation) was dissolved in PBS and mixed with complete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the first time; 3 weeks later, 100 μg of the antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the second time; subsequently, a total of 6 immunizations were carried out in sequence. The antibody titer of anti-ERα36 produced in the shark was measured. As Figure 1 The negative control was PBS. As the number of immunization rounds increased, the serum titer was detected to increase, and antibodies against ERα36 were produced in the shark.

[0045] 2. Construction of the variable domain (VNAR) library

[0046] (1) Obtaining cDNA

[0047] After the sixth immunization of the shark, peripheral blood lymphocytes PBMC or spleen tissue were collected, and RNA was extracted using the TRIZOL method and then reverse transcribed into cDNA;

[0048] (2) Cloning of the target gene

[0049] The cDNA was amplified by PCR using the primer sequences (Table 1), and the VNAR region encoding of Chiloscyllium plagiosum was obtained by PCR amplification.

[0050] Table 1 Primer sequences for library construction

[0051]

[0052] Among them, the reaction system for PCR amplification was: 0.5 μL of bacterial solution, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 5 μL of 2×Hieff® Robust PCR Master Mix, and ddH 2 O was supplemented to 10 μL.

[0053] The amplification conditions of PCR were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 10 s, repeating 30 cycles; final extension at 72°C for 5 min; stored at 4°C.

[0054] The obtained PCR product was digested with SfiI and ligated to the phagemid vector pComb3xss.

[0055] 3. Library construction

[0056] The ligation product was electrotransformed into competent Escherichia coli TG1 to form the original phage library. Subsequently, the bacterial solution was diluted 10 2 , 10 3 , 10 4 , 10 5 times. 100 μL of the serially diluted bacterial solution was taken and spread on freshly prepared 2×YT / A100 (a solution prepared with 1.65% tryptone, 1% yeast extract, 0.5% NaCl, made up to 1 L), dissolved in double-distilled water, and cultured overnight at 37°C. According to the colony growth, the number of colonies on the plate diluted 10 5 times was counted, and the library capacity of the shark phage antibody library specifically recognizing ERα36 was calculated. The calculated library capacity was 4.27×10 8 colonies.

[0057] Example 2 Panning of shark single-domain antibody phages targeting ERα36

[0058] Helper phages at 1×10 12 pfu / mL were prepared from the original phage library in Example 1 and used to infect competent TG1 cells for panning.

[0059] I. First-round panning:

[0060] 1. Coating: The ELISA plates were coated as follows, with duplicate wells, overnight at 4°C.

[0061] ① Coating streptavidin, 10 μg / mL, 200 μL / well;

[0062] ② Coating 5% skim milk powder (MPBS), 300 μL / well;

[0063] ③ Coating 5% bovine serum albumin (BSA), 300 μL / well.

[0064] 2. Phage collection: Centrifuge the overnight-shaken bacterial solution at 3800 rpm for 60 min. Filter the supernatant through a 0.22 μm filter membrane. Add PEG / NaCl at 1 / 4 of the bacterial solution volume and incubate on ice for 30 min. Centrifuge at 3800 rpm and 4 °C for 60 min. Discard the supernatant, invert and air-dry on absorbent paper, resuspend with PBS, and measure the titer by calculating OD280.

[0065] 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.

[0066] 4. Coating with biotinylated ERα36 polypeptide and negative panning with BSA

[0067] In step one, ① Add biotinylated ERα36 polypeptide at 100 μg / mL, 200 μL / well, and incubate for 2 h;

[0068] ③ Add phage with a library capacity of 1×10 11 , 200 μL / well, and incubate for 2 h;

[0069] 5. Blocking and negative panning with MPBS

[0070] After washing each well three times, ① Add MPBS, 300 μL / well, and block for 2 h; Take out the phage in ③ and add it to ②, and incubate for 2 h

[0071] 6. Activate TG1: Take 10 μL of the overnight-shaken TG1 strain and add it to 2 mL of fresh 2x YT medium. Culture at 37 °C and 200 rpm for 3 h until the logarithmic growth phase (OD600 = 0.6).

[0072] 7. 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 2 h.

[0073] 8. Wash the plate 10 times.

[0074] 9. Infection: Add the activated TG1 to well ①, 200 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.

[0075] 10. Plate coating for verification

[0076] Dilute the bacterial solution to 100, 1000, 10000, and 100000 times respectively by the method of limited dilution and coat the plates (Amp +), Incubate overnight at 37°C, 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 experiments can be carried out.

[0077] 11. Helper phage superinfection: Add M13K07 at 20 times the bacterial cell volume to the infected TG1 and let it stand at 37°C for 1 h.

[0078] 12. Amplification of progeny phages

[0079] Centrifuge the bacterial solution at 3800 rpm at 25°C for 30 min, discard the supernatant, resuspend in 10 mL of fresh 2xYT, add Kana (50 μg / mL), Amp (1:100 μg / mL), IPTG (0.1 mM), and incubate overnight at 30°C with shaking at 200 rpm.

[0080] II. Second round of panning

[0081] The steps of the second round of panning are the same as those of the first round, except that the concentration of the coated antigen in step 1 is decreased to 4 μg / mL; in step 8, wash the well plate 20 times with PBST, and the rest of the experimental operation steps are the same as those of the first round of panning.

[0082] III. Third round of panning

[0083] The steps of the third round of panning are the same as those of the first round, except that the concentration of the coated antigen in step 1 is decreased to 1 μg / mL; in step 8, wash the well plate 25 times with PBST, and the rest of the experimental operation steps are the same as those of the first round of panning.

[0084] The titer of the panned phages increased significantly in the second round, and specific phages were also enriched as detected by polyclonal ELISA (see Table 2 and Figure 2 、 3 ).

[0085] Table 2 Titers of the original and panned phage single-domain antibody libraries

[0086]

[0087] To obtain highly affinity single phages, 300 single colonies were picked from the plates obtained in the third round of panning for identification. Among the 300 picked monoclonal colonies, 22 were positive (OD 450 value > 0.5), with a positive rate of 7.3%. To avoid cross-contamination of the bacterial solution during culture and improper experimental operations, these 22 monoclonal colonies were picked out for secondary identification. As Figure 4 shown, 7 of them met the expectations.

[0088] Furthermore, for the OD values in the secondary identification results450 The bacterial solution with a value greater than 1 was sent to a sequencing company for sequencing. The sequencing results were analyzed, and all the sequences obtained by sequencing were consistent with the expected size. Through sequence alignment, two groups of sequences were screened out based on the sequence differences in the CDR3 domain, and their amino acid sequences are shown in SEQ ID NO.1 or SEQ ID NO.2, denoted as FR6 and TH4 respectively.

[0089] The nucleotide sequences encoding FR6 and TH4 are shown in SEQ ID NO.3 or SEQ ID NO.4 respectively.

[0090] The sequences of FR6 and TH4 were aligned with the NCBI database, and the results showed that these sequences were all single-domain antibody gene sequences derived from sharks.

[0091] The shark single-domain antibodies FR6 and TH4 include a framework region FR and complementarity-determining regions CDR. Among them, the framework region FR includes the amino acid sequences of FR1, FR2, and FR3, and the complementarity-determining regions CDR include the amino acid sequences of CDR1 and CDR3, as shown in Table 3 and Table 4 respectively:

[0092] Table 3 Amino acid sequences of FR1, FR2, and FR3

[0093]

[0094] Table 4 Amino acid sequences of CDR1 and CDR3

[0095]

[0096] Example 3 In vitro recombinant expression and purification of shark single-domain antibodies

[0097] The VNAR fragments FR6 and TH4 of the shark single-domain antibody genes in Example 2 were respectively ligated to the pET28a vector. The ligation system is shown in Table 5:

[0098] Table 5 Ligation system

[0099]

[0100] According to Table 5, the reaction system was prepared, gently mixed, and then ligated at 37°C for 30 min. The ligation product was directly used for the next transformation. That is, it was transformed into E. coli DH10β(DE3) competent cells, and then the successfully constructed recombinant expression vector was transformed into E. coli BL21(DE3) competent cells. Purification was carried out using nickel ion chelating filler with His tag, and each purified protein was aliquoted and stored at -80°C. The results showed that Figure 5 FR6 and TH4 presented single bands.

[0101] Use SPR to identify the binding affinity of ERα36 with shark single-domain antibodies FR6 and TH4. The chip surface was activated by mixing 0.4M EDC (N-ethyl-N’-dimethylaminopropylcarbodiimide) and 0.1M NHS (N-hydroxysuccinimide) at a ratio of 1:1. The protein was immobilized on the Graft-to-then-from-COOH surface of the biochip with the help of PDMS, and 1M ethanolamine (pH = 8.5) was injected for 30 minutes to block the activated chip surface. The initial concentration of the compound was 11.1 - 500 μM, and it was diluted with PBST buffer in a certain proportion. The prepared compounds with different concentrations were injected for detection. The obtained data were analyzed and fitted using the PLEXERA SPR Date Analysis Module (DAM) analysis software to obtain the binding kinetic constants. The detection results are shown in Table 6, Figure 6 The measured affinity constants of FR6 and TH4 binding to ERα36 are 1.07×10 -8 and 5.73×10 -8 .

[0102] Table 6 Affinity constants of ERα36 binding to shark single-domain antibodies FR6 and TH4 detected by SPR

[0103]

[0104] Example 4 Detection of the binding of ERα36 to shark single-domain antibodies by ELISA

[0105] (1) Dilute streptavidin to 10 μg / mL with PBS, add 100 μL to each well for coating, and incubate overnight at 4°C;

[0106] (2) Discard the supernatant coating solution, wash each well with 200 μL of PBST, and wash 3 times;

[0107] (3) Dilute biotinylated ERα36 polypeptide to 10 μg / mL with PBS, add 100 μL to each well for coating, and incubate at 37°C for 2 h

[0108] (4) Block the antigen wells with PBST containing 5% skim milk, 200 μL per well, and incubate at 37°C for 2 h;

[0109] (5) Discard the blocking solution in the wells, wash each well with 200 μL of PBST, and wash 3 times;

[0110] (6) Add PBS, shark single-domain antibodies FR6 and TH4, 100 µL to each well, and incubate at 37°C for 1 h.

[0111] Set the concentration gradient to 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM

[0112] (6) Discard the liquid, and wash 3 times with 200 μL of PBST per well;

[0113] (7) Add the HRP-labeled HA-tag antibody, diluted 1:2000, add 100 μL per well, and incubate at 37 °C for 1 h;

[0114] (8) Discard the liquid, and wash 3 times with 200 μL of PBST per well;

[0115] (9) Add 100 μL of TMB chromogenic solution per well, react at room temperature for 15 min, and add 100 μL of 1 M H 2 SO 4 to terminate the reaction. Read the OD value at 450 nm.

[0116] It can be seen from Figure 7 that both FR6 and TH4 shark nanobodies can bind to the ERα36 polypeptide, and the EC 50 are 2.203 and 2.277 nM respectively.

[0117] Example 5 Detection of the binding of shark single-domain antibody to breast cancer cells by flow cytometry

[0118] 1) Cell preparation: Culture cells and collect tumor cells (MCF-7, BT-474, MDA-MB-468) in the logarithmic growth phase;

[0119] 2) Cell grouping: PBS is used as the negative control group, the commercial ERα36 antibody (Abmart, M000803) is used as the positive control group, and the shark single-domain antibodies FR6 and TH4 are used as the experimental groups;

[0120] 3) Resuspend the cells in each group with PBS buffer, place them at 4 °C, centrifuge at 1000 rmp for 3 min, and carefully aspirate the supernatant with a pipette;

[0121] 4) Incubate the primary antibody; add 100 μL of PBS to the negative control, and add 100 μL of the shark single-domain antibodies FR6 and TH4 (1 μg / mL) and the commercial ERα36 antibody (diluted to 1 μg / mL with PBS) to the experimental groups respectively, so that they are fully contacted with the primary antibody, incubate on ice for 30 min, and gently flick and mix every 10 min;

[0122] 5) Washing: After the incubation of the primary antibody, centrifuge at 4 °C and 1000 rpm for 3 min, and carefully aspirate the supernatant with a pipette tip;

[0123] 6) Add 500 μL of 1×PBS buffer, pipette to resuspend; wash 1 - 2 times;

[0124] 7) Incubate with secondary antibody: In the negative control group, dilute the goat anti - rabbit and anti - HA secondary antibodies labeled with Alex488 with PBS buffer. In the positive control group (commercial ERα36 antibody), dilute the goat anti - rabbit secondary antibody labeled with Alex488 with PBS buffer. In the experimental group (shark antibodies FR6, TH4), dilute the His secondary antibody labeled with Alex488 with PBS buffer, resuspend and mix well, incubate on ice for 30 min, and keep the whole process in the dark.

[0125] 8) Wash: After the incubation with secondary antibody, the washing steps are the same as in 5).

[0126] 9) Resuspend the cells in each group with PBS buffer, 500 μL per group, transfer the resuspended cells into flow cytometry tubes, and wait for detection by flow cytometry, paying attention to keep it in the dark.

[0127] The detection results are as Figure 8 shown. The shark single - domain antibodies FR6 and TH4 targeting ERα36 do not bind to breast cancer cells MCF - 7 with low expression of ERα36; while they have a high level of binding to breast cancer cells BT - 474 and MDA - MB - 468 with high expression of ERα36. The results prove that the shark single - domain antibodies FR6 and TH4 can specifically bind to breast cancer cell lines BT - 474 and MDA - MB - 468 with high expression of ERα36.

[0128] Example 6 Immunofluorescence Experiment of Shark Single - Domain Antibody Targeting ERα36

[0129] In this experiment, MDA - MB - 468 breast cancer cells were selected for detection

[0130] (1) Seed the cells onto the coverslips in 12 - well plates. After overnight culture, when the cell confluence reaches 60 - 70%, discard the culture medium.

[0131] (2) After washing once with PBS, add 4% paraformaldehyde (PFA) to fix for 30 min, and then wash three times with PBS.

[0132] (3) Add 4% BSA to each well, block at room temperature for 1 h, and then wash three times with PBS.

[0133] (4) Add PBS, shark single - domain antibodies FR6, TH4, and commercial ERα36 antibody respectively, with a concentration of 1 - 2 μg / mL, and incubate overnight in a 4℃ refrigerator.

[0134] (5)Wash three times with PBS the next day. Add the fluorescence of the HA tag to the wells incubated with the shark single-domain antibody, and add the goat anti-fluorescence antibody to the wells incubated with the commercial ERα36 antibody. Incubate at room temperature for 2 h, and then wash three times with PBS.

[0135] (6)Add the quenching agent containing DAPI. After sealing the slides, take pictures using a fluorescence microscope.

[0136] The results of the immunofluorescence assay are as Figure 9 shown. The results indicate that the shark single-domain antibodies FR6 and TH4 can localize to the ERα36 protein on the cell membrane of MDA-MB-468, and have good targeting ability for ERα36.

[0137] The present invention encompasses a recombinant plasmid containing the encoding gene of the shark single-domain antibody targeting ERα36 as described above.

[0138] The present invention encompasses a recombinant strain containing the encoding gene of the shark single-domain antibody targeting ERα36 as described above.

[0139] The present invention encompasses the application of the shark single-domain antibody as described above in the preparation of an ERα36 targeting agent.

[0140] The present invention also provides the application of the shark single-domain antibody targeting ERα36 as described above in the preparation of an ERα36 targeting agent.

[0141] The present invention also provides the application of the shark single-domain antibody targeting ERα36 as described above in the preparation of a drug targeting ERα36-positive tumor cells.

[0142] Furthermore, the tumor is ER-positive drug-resistant breast cancer or triple-negative breast cancer.

Claims

1. A shark single-domain antibody targeting ERα36, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The gene encoding the shark single-domain antibody targeting ERα36 according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. Use of the shark single-domain antibody targeting ERα36 as claimed in claim 1 in the preparation of breast cancer drugs.

4. The use according to claim 3, characterized in that: The breast cancer is ER-positive drug-resistant breast cancer or triple-negative breast cancer.

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