ERalpha36-targeted shark single-domain antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a shark single-domain antibody targeting ERalpha36 as well as a preparation method and application of the shark single-domain antibody, the shark single-domain antibodies FR6 and TH4 provided by the invention are high in stability and affinity, have good binding force and specificity with ERalpha36, have important scientific significance in research of the shark single-domain antibody targeting ERalpha36 and development of antibody drugs, and have broad application prospects. And particularly, a new research direction and application prospects are provided for personalized precise medical treatment of tumors such as breast cancer and the like.

Description

Technical Field

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

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

[0003] At present, the classic 66 kDa estrogen receptor (ERα66) needs to rely on the traditional hormone binding mechanism for functional regulation. The nuclear localization of traditional ERα will also affect the response speed to external signal stimulation. ERα36, as a splicing variant of ERα, is often located in the cell membrane or cytoplasm. This feature enables it to respond quickly to external signal stimulation and activate non-genomic signaling pathways. It is an ideal candidate target. How to develop antibodies targeting ERα36 to fill the gap in existing targeted treatments for breast cancer and other tumors and improve the specificity and effectiveness of targeted therapy is of great research value. Summary of the invention

[0004] In order to solve the problems raised by the background technology, the present invention provides a shark single-domain antibody targeting ERα36 and a preparation method and application thereof.

[0005] The technical solution of the present invention is as follows: The present invention provides a shark single-domain antibody targeting ERα36, and the amino acid sequence thereof is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0006] The nucleotide sequence encoding the antibody gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0007] The antibody comprises a framework region and a complementarity determining region; The framework region includes FR1, FR2 and FR3; Wherein, the amino acid sequence of FR1 is shown in SEQ ID NO.5; The amino acid sequence of FR2 is shown in SEQ ID NO.6 or SEQ ID NO.7; The amino acid sequence of FR3 is shown in SEQ ID NO.8 or SEQ ID NO.9; The complementary determining region includes CDR1 and CDR3; Wherein, the amino acid sequence of CDR1 is shown in SEQ ID NO.10 or SEQ ID NO.11; The amino acid sequence of CDR3 is shown in SEQ ID NO.12 or SEQ ID NO.13.

[0008] The present invention also provides a method for preparing the shark single-domain antibody targeting ERα36, which comprises extracting RNA from sharks immunized with ERα36, reversely transcribing the RNA into cDNA, amplifying the RNA, enzymatically digesting the RNA, and connecting the RNA to a phagemid vector to construct a shark phage antibody library that specifically recognizes ERα36, and selecting the shark phage antibodies to obtain the shark single-domain antibody targeting ERα36.

[0009] The present invention also provides a use of the shark single-domain antibody targeting ERα36 in the preparation of an ERα36 targeting agent.

[0010] The present invention also provides a use of the shark single-domain antibody targeting ERα36 in the preparation of a drug targeting ERα36-positive tumor cells.

[0011] The tumor is positive breast cancer or triple-negative breast cancer.

[0012] Beneficial effects: The shark single-domain antibodies FR6 and TH4 provided by the present invention have high stability, strong affinity, and good binding ability and specificity to ERα36, which are of great scientific significance for the research on shark single-domain antibodies targeting ERα36 and the development of antibody drugs, especially for the personalized precision medicine of tumors such as breast cancer, providing new research directions and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the results of serum titer determination of sharks immunized with ERα36 antigen polypeptide.

[0014] Figure 2 Results were validated by plating for three rounds of biopanning.

[0015] Figure 3 The results are from specific phage polyclonal ELISA.

[0016] Figure 4 This is the result of specific phage monoclonal ELISA identification.

[0017] Figure 5 This is the protein expression result of shark nanoantibodies FR6 and TH4 after purification.

[0018] Figure 6 The affinity test results of ERα36-specific shark single-domain antibody, where (a) is FR6 and (b) is TH4.

[0019] Figure 7This is the ELISA test result of ERα36 binding to shark single domain antibodies FR6 and TH4.

[0020] Figure 8 The results of flow cytometry analysis show that shark single domain antibodies FR6 and TH4 bind to MCF-7, BT474 and MDA-MB-468 cells with different ERα36 expressions.

[0021] Fig. 9 The results of immunofluorescence experiments on MDA-MB-468 cells using shark single-domain antibodies targeting ERα36. DETAILED DESCRIPTION

[0022] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0023] The present invention provides a shark single-domain antibody targeting ERα36, and the amino acid sequence thereof is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0024] Also provided is a gene encoding the shark single-domain antibody targeting ERα36, and the nucleotide sequence encoding the antibody gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0025] The antibody comprises a framework region and a complementarity determining region; The framework region includes FR1, FR2 and FR3; Wherein, the amino acid sequence of FR1 is shown in SEQ ID NO.5; The amino acid sequence of FR2 is shown in SEQ ID NO.6 or SEQ ID NO.7; The amino acid sequence of FR3 is shown in SEQ ID NO.8 or SEQ ID NO.9.

[0026] The complementary determining region includes CDR1 and CDR3; Wherein, the amino acid sequence of CDR1 is shown in SEQ ID NO.10 or SEQ ID NO.11; The amino acid sequence of CDR3 is shown in SEQ ID NO.12 or SEQ ID NO.13.

[0027] The present invention also provides a method for preparing the shark single domain antibody targeting ERα36, wherein RNA of sharks immunized with ERα36 is extracted, reverse transcribed into cDNA, amplified, enzyme-cut and connected to a phagemid vector in sequence, a shark phage antibody library specifically recognizing ERα36 is constructed, and shark phage antibodies are selected to obtain shark single domain antibodies targeting ERα36. The specific operation is as follows.

[0028] Example 1 Construction of a shark phage antibody library specifically recognizing ERα36

[0029] 1. Striped Bamboo Shark Immunity During the first immunization, 300 μg of a peptide consisting of a short peptide of 27 amino acids at the tail of ERα36 and a partial fragment of the E / F domain coupled to KLH protein (purchased from GenScript Biotech Co., Ltd.) was dissolved in PBS and mixed with complete Freund's adjuvant at a ratio of 1:1 to immunize the striped bamboo shark 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 the striped bamboo shark for the second time; subsequently, this immunization was performed for a total of 6 times to determine the titer of the anti-ERα36 antibody produced in the shark, such as Figure 1 The negative control was PBS. As the immunization rounds increased, the serum titer increased, indicating that anti-ERα36 antibodies were produced in sharks.

[0030] 2. Construction of variable domain (VNAR) library (1) Obtaining cDNA After the sixth immunization of sharks, peripheral blood lymphocytes (PBMCs) or spleen tissues were collected, RNA was extracted using the TRIZOL method, and then reverse transcribed into cDNA; (2) Cloning of target gene The cDNA was amplified by PCR using the primer sequences (Table 1), and the VNAR region coding for the striped bamboo shark was amplified by PCR.

[0031] Table 1 Primer sequences for library construction

[0032] The reaction system for PCR amplification was as follows: 0.5 μL bacterial solution, 0.2 μL upstream primer, 0.2 μL downstream primer, 5 μL 2×Hieff® Robust PCR Master Mix, ddH 2 Add 2% HO to 10 μL.

[0033] 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, repeated for 30 cycles; final extension at 72°C for 5 min; and storage at 4°C.

[0034] The obtained PCR product was digested with SfiI and ligated to the phagemid vector pComb3xss.

[0035] 3. Library construction The ligation product was electrotransformed into competent E. coli TG1 to form the original phage library. 2 , 10 3 , 10 4 , 10 5 100 μL of each gradient dilution was applied to freshly prepared 2×YT / A100 (1.65% tryptone, 1% yeast extract, 0.5% NaCl solution, fixed to 1 L), dissolved in double distilled water, and cultured at 37°C overnight. 5 The library capacity of the constructed shark phage antibody library that specifically recognizes ERα36 was calculated by multiplying the number of colonies on the plate. The calculated library capacity is 4.27×10 8 indivual.

[0036] Example 2 Panning of shark single domain antibody phage targeting ERα36 The original phage library of Example 1 was used to prepare 1×10 12 pfu / mL of helper phage were used to infect TG1 competent cells for panning.

[0037] 1. The first round of selection: 1. Coating: Coat the ELISA plate in the following manner, set up duplicate wells, and incubate at 4°C overnight.

[0038] ① Streptavidin coating, 10 μg / mL, 200 μL / well; ② Coated with 5% skim milk powder (MPBS), 300 μL / well; ③ Coated with 5% bovine serum albumin (BSA), 300 μL / well.

[0039] 2. Collect phages: centrifuge the bacterial solution shaken overnight at 3800 rpm for 60 min, filter the supernatant with a 0.22 μm filter membrane, add PEG / NaCl with 1 / 4 volume of the bacterial solution, and place on ice for 30 min; centrifuge at 3800 rpm and 4 ℃ for 60 min, discard the supernatant, invert on absorbent paper to dry, resuspend with PBS, and measure OD280 to calculate the titer.

[0040] 3. Wash the plate three times: wash the plate with PBST (PBS containing 0.1% Tween-20), fill the entire well (about 300 μL), and tap the plate.

[0041] 4. Biotinylated ERα36 Peptide Coating and BSA Negative Panning In step 1, add biotinylated ERα36 peptide, 100 μg / mL, 200 μL / well, and incubate for 2 h; ③ Add the library capacity to 1×10 11 phage, 200 μL / well, incubate for 2 h; 5. Blocking with MPBS Negative Elution After washing each well three times, add MPBS (300 μL / well) to ① and block for 2 h; remove phage from ③ and add to ② and incubate for 2 h 6. Activate TG1: Take 10 μL of TG1 strain shaken overnight and add it to 2 mL of fresh 2x YT medium. Cultivate at 37℃ 200rpm for 3 hours to reach the logarithmic growth phase (OD600=0.6).

[0042] 7. Incubation: Take out the phage in well ② and put it into an EP tube. Wash the plate 3 times. Add the above phage to well ① and incubate at 37℃ for 2h.

[0043] 8. Wash the plate 10 times.

[0044] 9. Infection: Add activated TG1 to well ①, 200 μL / well, incubate at 37℃ for 30 min, collect infected TG1, replace with fresh TG1, and repeat this process 3 times.

[0045] 10. Coating verification The bacterial solution was diluted to 100, 1000, 10000, and 100000 times by limiting dilution and plated (Amp + ), culture at 37℃ overnight, and observe the number of clones that grow, which is the TG1 that is successfully infected. When the input TG1 is the same, if the number of clones that grow increases significantly, it means that it is enriched, and the following experiment can be carried out.

[0046] 11. Auxiliary phage superinfection: Add 20 times the amount of M13K07 to the infected TG1 and let it stand at 37℃ for 1 hour.

[0047] 12. Progeny phage amplification The bacterial solution was centrifuged at 3800 rpm and 25°C for 30 min, the supernatant was discarded, and the culture was resuspended in 10 mL of fresh 2xYT. Kana (50 μg / mL), Amp (1:100 μg / mL), and IPTG (0.1 mM) were added and cultured at 30°C and 200 rpm overnight.

[0048] 2. Second round of selection The second round of panning steps were the same as the first round, except that the concentration of the coated antigen in step 1 was reduced to 4 μg / mL; in step 8, the plate was washed 20 times with PBST, and the rest of the experimental operation steps were the same as the first round of panning.

[0049] 3. The third round of selection The third round of panning steps were the same as the first round, except that the concentration of the coated antigen in step 1 was reduced to 1 μg / mL; in step 8, the plate was washed 25 times with PBST, and the rest of the experimental operation steps were the same as the first round of panning.

[0050] The titer of the selected phages increased significantly in the second round, and the polyclonal ELISA test showed that specific phages were also enriched (see Table 2 and Figure 2 , 3 ).

[0051] Table 2 The titer results of the original and three rounds of panning phage single domain antibody library

[0052] In order to obtain a single phage with high affinity, 300 single colonies were picked from the plate obtained from the third round of panning for identification. Among the 300 single clones picked, 22 were positive (OD 450 The positive rate was 7.3%. To avoid cross contamination of bacterial culture and improper experimental operation, these 22 monoclones were selected for secondary identification. Figure 4 Seven of the clones were identified as expected.

[0053] Furthermore, the OD 450 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 sequenced were consistent with the expected size. Through sequence alignment, two groups of sequences were screened out according to the difference in CDR3 domain sequence, and their amino acid sequences were shown in SEQ ID NO.1 or SEQ ID NO.2, respectively recorded as FR6 and TH4.

[0054] The nucleotide sequences encoding FR6 and TH4 are shown in SEQ ID NO.3 or SEQ ID NO.4, respectively.

[0055] The FR6 and TH4 sequences were compared with the NCBI database, and the results showed that these sequences were all single-domain antibody gene sequences derived from sharks.

[0056] Shark single domain antibodies FR6 and TH4 include framework regions FR and complementary determining regions CDR, wherein the framework region FR includes the amino acid sequences of FR1, FR2, and FR3, and the complementary determining region CDR includes the amino acid sequences of CDR1 and CDR3, as shown in Table 3 and Table 4, respectively: Table 3 Amino acid sequences of FR1, FR2, and FR3

[0057] Table 4 Amino acid sequences of CDR1 and CDR3

[0058] Example 3 In vitro recombinant expression and purification of shark single domain antibodies The shark single domain antibody gene VNAR fragments FR6 and TH4 in Example 2 were connected to the pET28a vector respectively. The connection system is shown in Table 5: Table 5 Connection system

[0059] Prepare the reaction system according to Table 5, mix gently, and connect at 37℃ for 30 min. The ligation product is directly used for the next transformation. E. coli DH10β (DE3) competent cells, and then the successfully constructed recombinant expression vector was transformed into E. coli In BL21 (DE3) competent cells, His tag was used for purification using nickel ion chelating filler, and each purified protein was packaged and stored at -80℃. Figure 5 FR6 and TH4 are shown as a single band.

[0060] SPR was used to identify the binding force between ERα36 and 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) in a ratio of 1:1. The protein was placed on the Graft-to-then-from-COOH surface of the biochip with 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 11.1-500μM, and it was diluted with PBST buffer at a certain ratio. The prepared compounds of different concentrations were injected for detection. The data obtained were analyzed and fitted according to the PLEXERA SPR Date Analysis Module (DAM) analysis software to obtain the binding kinetic constants. The test results are shown in Table 6. Figure 6 The affinity constants of FR6 and TH4 binding to ERα36 were 1.07×10 -8 and 5.73×10 -8 .

[0061] Table 6 Affinity constants of ERα36 and shark single domain antibodies FR6 and TH4 detected by SPR

[0062] Example 4 ELISA detection of ERα36 binding to shark single domain antibody (1) Dilute streptavidin to 10 μg / mL with PBS, add 100 μL to each well for coating, and incubate at 4°C overnight; (2) Discard the supernatant coating solution and wash each well with 200 μL PBST for 3 times; (3) Biotinylated ERα36 peptide was diluted to 10 μg / mL with PBS, 100 μL was added to each well for coating, and incubated at 37°C for 2 h. (4) Block the antigen wells with PBST containing 5% skim milk, 200 μL per well, at 37°C for 2 h; (5) Discard the blocking solution in the wells and wash each well with 200 μL PBST for 3 times; (6) Add PBS, shark single-domain antibodies FR6 and TH4, add 100 µL to each well, and incubate at 37°C for 1 h.

[0063] Set the concentration gradient to 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM (6) Discard the liquid and wash each well three times with 200 μL PBST; (7) Add HRP-labeled HA tag antibody, dilute 1:2000, add 100 μL to each well, and incubate at 37°C for 1 h; (8) Discard the liquid and wash each well three times with 200 μL PBST; (9) Add 100 μL TMB colorimetric solution to each well, react at room temperature for 15 min, and then add 100 μL 1M H 2 SO 4 Stop the reaction and read the OD value at 450 nm.

[0064] Depend on Figure 7 It can be seen that both FR6 and TH4 shark nanobodies can bind to ERα36 peptide, EC 50 2.203 and 2.277 nM respectively.

[0065] Example 5 Flow cytometry detection of the binding of shark single domain antibodies to breast cancer cells 1) Cell preparation: Culture cells and collect tumor cells (MCF-7, BT-474, MDA-MB-468) in the logarithmic growth phase; 2) Cell grouping: PBS was used as the negative control group, commercial ERα36 antibody (Abmart, M000803) was used as the positive control group, and shark single-domain antibodies FR6 and TH4 were used as experimental groups; 3) Resuspend the cells of each group with PBS buffer, place at 4°C, centrifuge at 1000 rpm for 3 min, and carefully remove the supernatant with a pipette; 4) Incubate with primary antibody; add 100 μL PBS to the negative control, and add 100 μL shark single domain antibody FR6, TH4 (1 μg / mL) and commercial ERα36 antibody (diluted to 1 μg / mL with PBS) to the experimental group, respectively, to allow them to fully contact with the primary antibody, incubate on ice for 30 minutes, and gently flick to mix every 10 minutes; 5) Washing: After the primary antibody incubation is completed, centrifuge at 4°C, 1000 rpm for 3 minutes, and carefully remove the supernatant with a pipette tip; 6) Add 500 μL 1× PBS buffer, pipette to resuspend, and wash 1-2 times; 7) Incubation with secondary antibody: For the negative control group, PBS buffer was used to dilute the goat anti-rabbit and anti-HA secondary antibodies labeled with Alex488; for the positive control group (commercial ERα36 antibody), PBS buffer was used to dilute the goat anti-rabbit secondary antibody labeled with Alex488; for the experimental group (shark antibody FR6, TH4), PBS buffer was used to dilute the His secondary antibody labeled with Alex488, resuspend and mix, and incubate on ice for 30 minutes. The whole process was carried out in the dark; 8) Washing: After the secondary antibody incubation is completed, the washing steps are the same as 5); 9) Add PBS buffer to each group of cells and resuspend them, 500 μL / group. After resuspending, transfer them into flow cytometry tubes and wait for detection on the machine. Be careful to avoid light.

[0066] Test results such as Figure 8 As shown, shark single domain antibodies FR6 and TH4 targeting ERα36 do not bind to breast cancer cells MCF-7 that lowly express ERα36, but have high levels of binding to breast cancer cells BT-474 and MDA-MB-468 that highly express ERα36. The results show that shark single domain antibodies FR6 and TH4 can specifically bind to breast cancer cell lines BT-474 and MDA-MB-468 that highly express ERα36.

[0067] Example 6 Immunofluorescence experiment of shark single domain antibody targeting ERα36 In this experiment, MDA-MB-468 breast cancer cells were selected for detection (1) Inoculate cells onto a 12-well plate slide and culture overnight. When the cell confluence reaches 60-70%, discard the culture medium. (2) After washing once with PBS, add 4% PFA paraformaldehyde to fix for 30 min, and then wash three times with PBS.

[0068] (3) Add 4% BSA to each well, block for 1 h at room temperature, and then wash three times with PBS.

[0069] (4) Add PBS, shark single-domain antibodies FR6, TH4, and commercial ERα36 antibodies at a concentration of 1-2 μg / mL, and incubate in a 4°C refrigerator overnight.

[0070] (5) The next day, the cells were washed three times with PBS, HA-tagged fluorescence was added to the wells incubated with shark single domain antibody, and goat anti-fluorescent antibody was added to the wells incubated with commercial ERα36 antibody. The cells were incubated at room temperature for 2 h, and then washed three times with PBS.

[0071] (6) Add anti-fading agent containing DAPI, seal the slides, and take photos using a fluorescence microscope.

[0072] The results of immunofluorescence experiments were Fig. 9 As shown, the results indicate that shark single domain antibodies FR6 and TH4 can be localized on the ERα36 protein on the MDA-MB-468 cell membrane and have good targeting to ERα36.

[0073] The present invention comprises the recombinant plasmid of the gene encoding the shark single-domain antibody targeting ERα36.

[0074] The present invention comprises the recombinant strain of the gene encoding the shark single-domain antibody targeting ERα36.

[0075] The present invention comprises the use of the shark single-domain antibody in the preparation of a targeting agent targeting ERα36.

[0076] The present invention also provides a use of the shark single-domain antibody targeting ERα36 in the preparation of an ERα36 targeting agent.

[0077] The present invention also provides a use of the shark single-domain antibody targeting ERα36 in the preparation of a drug targeting ERα36-positive tumor cells.

[0078] Furthermore, the tumor is ER-positive 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 shark single domain antibody targeting ERα36 according to claim 1, characterized in that The nucleotide sequence encoding the antibody gene is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The shark single domain antibody targeting ERα36 according to claim 1, characterized in that The antibody comprises a framework region and a complementarity determining region; The framework region includes FR1, FR2 and FR3; Wherein, the amino acid sequence of FR1 is shown in SEQ ID NO.5; The amino acid sequence of FR2 is shown in SEQ ID NO.6 or SEQ ID NO.7; The amino acid sequence of FR3 is shown in SEQ ID NO.8 or SEQ ID NO.9; The complementary determining region includes CDR1 and CDR3; Wherein, the amino acid sequence of CDR1 is shown in SEQ ID NO.10 or SEQ ID NO.11; The amino acid sequence of CDR3 is shown in SEQ ID NO.12 or SEQ ID NO.

13.

4. A method for preparing a shark single domain antibody targeting ERα36 as claimed in claim 1, characterized in that: RNA from sharks immunized against ERα36 was extracted, reverse transcribed into cDNA, and then amplified, enzyme-digested and ligated to a phagemid vector to construct a shark phage antibody library that specifically recognizes ERα36. After panning the shark phage antibodies, a shark single-domain antibody targeting ERα36 was obtained.

5. Use of the shark single-domain antibody targeting ERα36 as claimed in claim 1 in the preparation of an ERα36 targeting agent.

6. Use of the shark single-domain antibody targeting ERα36 as claimed in claim 1 in the preparation of a drug targeting ERα36-positive tumor cells.

7. The use according to claim 6, characterized in that: The tumor is positive breast cancer or triple-negative breast cancer.

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