WO2019154103A1

WO2019154103A1 - Novel polypeptide for tumor targeting and application thereof

Info

Publication number: WO2019154103A1
Application number: PCT/CN2019/073096
Authority: WO
Inventors: 魏敏杰; 赵琳; 卫倩
Original assignee: 中国医科大学
Priority date: 2018-02-09
Filing date: 2019-01-25
Publication date: 2019-08-15
Also published as: CN108341854B; CN108341854A

Abstract

The present invention relates to the field of biomedicine, and specifically provides a novel polypeptide having a targeting effect on human ovarian cancer cells, and an application thereof. The polypeptide is (1) a polypeptide having an amino acid sequence: WNPLLLTRLLPA (SEQ ID No. 1), and (2) a polypeptide derivative that is obtained by means of deletion, insertion or replacement of one or several amino acids in polypeptide molecules in (1) and has the same biological function as the polypeptide molecules in (1). The polypeptide has small relative molecular mass, high permeability, high specificity, low costs, etc., and shows unique advantages in tumor targeting diagnosis and treatment.

Description

Novel polypeptide targeted by tumor and use thereof

Technical field

The invention belongs to the field of biomedicine, and particularly relates to a novel polypeptide which has a targeting effect on human ovarian cancer cells and uses thereof.

Background technique

Ovarian cancer is one of the most common tumors in female malignant tumors. Its incidence rate is the second in gynecological tumors, and its mortality rate is the first in gynecological malignancies. Because the ovary is located in the deep pelvic cavity, the onset is more concealed, the early stage of the disease is difficult to find and the disease develops rapidly, and the lack of effective screening and early diagnosis means that more than 70% of patients are in advanced stage. At present, the 5-year survival rate of patients with advanced ovarian cancer is only 20% to 40%, while that of early patients can reach 70% to 90%. Therefore, ovarian cancer is a hot topic in early diagnosis and treatment.

Many researchers have been working for many years to find one or more combinations of detection methods to find early ovarian cancer that is easy to treat. However, an ideal method for screening for ovarian cancer and reducing its mortality has not been found so far. At present, the basic treatment method is surgery supplemented with chemotherapy. The existing anticancer drugs mainly include metal anticancer drugs, anticancer active ingredients extracted from natural products, anticancer drug targeted preparations, genetic engineering drugs, and nano controlled release anticancer drugs. There are five types of drugs, such as drugs with large toxic side effects, large dosages, and prone to acquired drug resistance, which have an impact on the treatment of tumors. With the research of tumors in the molecular field, molecular targeted therapy and other treatment methods provide new options for patients with advanced ovarian cancer. Therefore, early detection and development of ovarian cancer, sensitive diagnostic techniques, targeted therapy is the focus of research.

Phage display technology is an important technique for screening intermolecular interactions in the field of molecular biology. The main principle of phage display technology is that the gene of interest or the gene encoding the protein and polypeptide is cloned into the appropriate position of the phage surface protein gene by genetic engineering technology, and it is expressed on the surface of the phage with the amplification of phage DNA, due to the foreign gene. In contrast to the phage gene, the expression product polypeptide or protein of the foreign gene can maintain its original spatial structure and corresponding biological activity. Then, we use the target cells or target molecules to perform meiotic screening on the phage, and finally select the phage that can specifically bind to the target cell or target molecule from the phage peptide library, and sequence the DNA to obtain the corresponding polypeptide. The coding sequence. This technology enables the connection between genotypes and phenotypes of proteins or peptides, and has the characteristics of simple operation and high-throughput detection, thus becoming an efficient means for screening tumor cell-specific binding peptides, for early detection of tumors and Targeted vector research in drug therapy offers new directions.

Summary of the invention

The object of the present invention is to provide a tumor-targeting novel polypeptide and a use thereof, which can specifically bind to human ovarian cancer cells and have no effect on human ovarian epithelial cells, which is an early diagnosis and targeted drug for ovarian cancer. R&D and other aspects play an important role.

The principle of the present invention is: using human ovarian epithelial cells as a control, using human ovarian cancer cell line SK-OV-3 to perform subtraction screening on phage display peptide library, and selecting blue and white screening test to be specific to human ovarian cancer cells. Binding positive phage clones were tested and the specificity of binding of phage to human ovarian cancer cells was verified by ELISA experiments. Then, using E. coli as a vector, the purified phage is amplified and the DNA is extracted for sequencing, thereby obtaining a polypeptide coding sequence capable of specifically binding to human ovarian cancer cells, and synthesizing a fluorescently labeled positive polypeptide for fluorescent labeling-polypeptide and The verification of the binding effect of human ovarian cancer cells and tumor-bearing mice provides an experimental basis for early diagnosis and targeted therapy of ovarian cancer.

In order to achieve the above object, the present invention adopts the following technical scheme: a novel tumor-targeting polypeptide, wherein the polypeptide has the amino acid sequence of: (W) a polypeptide sequence of: WNPLLLTRLLPA (SEQ ID No. 1), (2) A polypeptide derivative of a polypeptide molecule which has one or more amino acids deleted, inserted or substituted and which has the same biological function as the polypeptide molecule of (1).

The polypeptide has a targeting effect on tumor cells and specifically binds to tumor cells.

The tumor cells are human ovarian cancer cells.

The use of a polypeptide in the preparation of a tumor diagnostic kit comprising the polypeptide or polypeptide conjugate.

The use of a polypeptide for the preparation of a medicament for the treatment of a tumor comprising the polypeptide and a pharmaceutically active ingredient, or a polypeptide and a delivery vehicle. The medicament is any pharmaceutically therapeutically acceptable dosage form, and the preferred dosage form of the medicament is an injectable preparation.

The drug is any pharmacologically acceptable dose.

Compared with the prior art, the effects of the present invention are as follows: (1) The short peptide small molecule drug has the advantages of relatively low molecular weight, weak immunogenicity, high activity and the like. (2) It can target in vivo tumors, and has the application prospects of specifically transmitting anticancer drugs, imaging agents, inorganic nanoparticles, liposomes, etc. to tumor tissues. (3) The peptides we screened can specifically bind to human ovarian cancer cells, but have no specific effect on human ovarian epithelial cells. Compared with antibody-directed drugs, which have the advantages of small molecular mass, high permeability, high specificity and low cost, they have shown unique advantages in tumor targeted diagnosis and treatment.

DRAWINGS

Figure 1 shows the binding of 40 phage clones to human ovarian epithelial cells HOSEpiC and human ovarian cancer cell line SK-OV-3.

Figure 2 shows the targeted binding of positive phage to human ovarian epithelial cells HOSEpiC and human ovarian cancer cell line SK-OV-3.

Figure 3 shows the sequencing results of positive phage DNA.

Figure 4 is a targeted binding effect of FITC-WA12 of the present invention to human ovarian epithelial cells HOSEpiC and human ovarian cancer cell line SK-OV-3.

Figure 5 is a targeted binding effect of FITC-WA12 of the present invention to tumor-bearing human ovarian cancer cell line SK-OV-3.

Detailed ways

The following description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Example 1

Experimental material

1.1 phage peptide library, cells and host bacteria.

Phage 12 peptide library, Escherichia coli E. coli ER2738, human ovarian epithelial cells HOSEpiC, human ovarian adenocarcinoma cells SK-OV-3, tumor-bearing mice.

1.2 experimental reagents

McCoy's 5A medium, RPMI-1640 medium, trypsin, FITG-labeled rabbit anti-mouse, fetal bovine serum, yeast powder, peptone, agar powder, tetracycline stock solution, Tween-20, bovine serum albumin BSA , HRP-anti-M13 filamentous phage antibody, M13 phage single-stranded DNA extraction kit, IPTG, X-gal, PEG-8000, TMB.

1.3 experimental working fluid

1×PBS, LB liquid medium, LB-Tet solid plate, top agar, IPTG/X-gal working solution, IPTG/X-gal plate, PEG-NaCl, TBS buffer, 0.1% TBST, 0.5% TBST, 4 Preparation of % paraformaldehyde fixative, TBS-NaN3 solution, 3% BSA blocking solution, sodium iodide buffer, TE buffer, TMB working solution, tetracycline stock solution.

2. Experimental methods

2.1 Cultivation of E. coli.

(1) Resuscitation of E. coli: Take the E. coli glycerol cryopreservation solution from the -80 °C refrigerator, take a small amount of scribing on the LB/Tet solid plate with the inoculating loop, and then pour the LB/Tet solid plate at 37 °C. Incubate overnight in an electrothermal incubator and pick a single colony when using.

(2) Culture of Escherichia coli: 10 ml of LB/Tet liquid medium was added to a 15 ml centrifuge tube, and a single colony of Escherichia coli was picked and added thereto. Then, the centrifuge tube was placed in a constant temperature oscillator and cultured overnight, and the relevant experiment was carried out when the OD ₆₀₀ value of the bacterial liquid was 0.5.

2.2 Subtraction screening of phage display peptide library.

(1) Preparing cells: Firstly, the 6-well culture plate was pretreated with poly-lysine, and human ovarian epithelial cells HOSEpiC and human ovarian adenocarcinoma cell line SK-OV-3 were taken and treated with trypsin and then cultured into cells. Screening was performed after successful adherence and good growth.

(2) Preparation of bacterial liquid: On the day of screening, Escherichia coli ER2738 was inoculated into 20 ml LB/Tet liquid medium, and placed in a 37 ° C constant temperature shaker for shaking culture. When the OD ₆₀₀ value of the bacterial liquid was 0.5, it was used for expansion. Increase the screening of eluted phage.

(3) Serum-free culture: The cell culture medium was aspirated, washed once with PBS, and then added to serum-free medium, and placed in a 37 ° C constant temperature cell incubator with 5% CO 2 for 1 h.

(4) Washing: The blocking solution was aspirated and washed gently with 0.1% TBST for 5 times, each time being rotated so that the bottom and edges of the micropores were washed and dried. 0.5% TBST and 1.0% TBST were used for the second and third rounds of screening, respectively.

(5) Closing the cells: Aspirate the cell culture medium, place the plate on a clean paper towel, remove the remaining medium, and seal the human ovarian epithelial cells HOSEpiC and human ovarian adenocarcinoma cells with a medium containing 1% BSA. -OV-3, placed in a 37 ° C constant temperature cell incubator with 5% CO 2 for 1 h.

(6) Adsorption: 10 μl of the original peptide library was taken, added to 990 μl of 0.5% BSA/PBS buffer, and the phage was diluted to 1.5×10 ¹¹ pfu/ml, and added to the closed human ovarian epithelial cell HOSEpiC. At 37 ° C for 1 h, the phage which can bind to human normal intestinal epithelial cells HIEC was adsorbed, and the supernatant was taken.

(7) Binding: The adsorbed phage supernatant was incubated with human ovarian adenocarcinoma cell line SK-OV-3 for 1 h.

(8) Washing: Discard the unbound phage, place the microplate on a clean paper towel and pat it vigorously to remove the remaining solution. The plate was washed 5 times with 0.1% TBST as described above.

(9) Elution: Add 1 ml of 0.2 M Glycine-HCl (pH 2.2) 1 mg/ml BSA eluate, shake slowly for 10 min on ice, then aspirate the eluate and transfer to 150 μl of the previously prepared neutralized solution ( 1M Tris-HCl, pH 9.1).

(10) Repeat the operation twice as described above.

2.3 Determination of titer of phage.

The IPTG/X-gal plate was preheated in an electrothermal incubator at 37 ° C; the appropriate amount of top agar was taken out and heated in a microwave oven, and after it was completely melted, it was taken out, and 3 ml was dispensed in each 10 ml centrifuge tube; After the phage was diluted in a ratio, 10 μl and 200 μl of E. coli were mixed and reacted for 5 min, then added to 3 ml of top agar, and then evenly spread on preheated IPTG/X-gal plate, after condensation. The titration results were observed at 37 ° C in an electrothermal incubator overnight.

2.4 Amplification and purification of phage.

(1) Amplification of phage: 20 ml of LB/Tet liquid medium was added to the Erlenmeyer flask, then E. coli bacteria solution and phage to be amplified were added at 1:100, placed at 37 ° C, and shaken vigorously in a constant temperature oscillator 4.5 h, an phage amplification solution is obtained.

(2) Purification of phage: The phage amplification solution obtained by the above procedure was centrifuged at 4 ° C, 12000 r/min for 10 min, and the supernatant was taken and precipitated by adding 1/6 volume of PEG-NaCl overnight, centrifuged at 12000 r/min for 15 min, and discarded. The supernatant was removed, the pellet was dissolved in TBS buffer, 1/6 volume of PEG-NaCl was again administered, and incubated on ice for 1 h. After centrifugation at 4 ° C, 14000 r / min for 15 min, the supernatant was discarded, and the obtained precipitate was dissolved in TBS-NaN 3 and stored in a refrigerator at 4 ° C.

2.5 enzyme-linked immunosorbent assay

(1) Preparation of 96-well plates of cells, plating rules: 16 holes of two rows of 96-well plates were added with 100 μl×PBS as a blank group; then each of the 1, 2, 3, and 4 rows was serpentinely spread. 100 μμ of human ovarian epithelial cells HOSEpiC suspension, each well of

rows

5, 6, 7, and 8 were spread in a serpentine manner with 100 μ of human ovarian adenocarcinoma cell line SK-OV-3, and then the plated cells were plated. The ELISA assay was performed overnight in a 37 ° C cell incubator with 5% CO 2 .

(2) Fixation: 96-well plates with cells were taken overnight, and the liquid in the dry wells was washed three times with PBS, and then fixed with 4% paraformaldehyde for 20 minutes.

(3) Blocking: Remove the fixed 96-well plate, take the liquid in the dry well, wash it with PBS 3 times, then add 3% hydrogen peroxide, and block it in a constant temperature incubator at 37 °C for 30 min to block Endogenous peroxidase activity.

(4) Closure: The blocked 96-well plate was taken out, and the liquid in the dry well was photographed, washed with PBS three times, and then 3% BSA/PBS was added and blocked in a 37 ° C cell incubator for 1 h.

(5) Adding phage sample: The blocked 96-well plate was taken out, and the liquid in the dry well was photographed, and the purified positive phage was added and reacted in a constant temperature incubator at 37 ° C for 1 h.

(6) Addition of primary antibody: The 96-well plate after the reaction was taken out, and the liquid in the dry well was photographed, and after washing 3 times with PBS, 1:4000 M13 antibody was added, and the mixture was allowed to stand overnight at 4 °C.

Secondary antibody: The 96-well plate after the reaction was taken out, and the liquid in the dry well was photographed, washed three times with PBS, and then added with a secondary antibody, and reacted in a 37 ° C cell incubator for 30 min.

(7) Adding substrate TMB: The 96-well plate after washing the PBS three times was added to the TMB display agent in the dark, and was placed in a 37 ° C cell incubator for 15 min.

(8) Termination: The 96-well plate after the reaction was taken out, and the reaction was terminated by adding 2 M sulfuric acid.

(9) Measurement of results: A 96-well plate in which all reactions were completed was placed in a microplate reader, and its OD value was measured at 405 nm, and the results were saved and analyzed.

Immunofluorescence experiment of 2.6 cells and positive phage

(1) Cell plating: Human ovarian epithelial cells HOSEpiC and human ovarian adenocarcinoma SK-OV-3 were plated in a six-well plate for use.

(2) Fixation: fixed with 4% paraformaldehyde for 15 min.

(3) Blocking: 4% paraformaldehyde was discarded, washed twice with PBS, and blocked with 3% BSA/PBS at 37 ° C for 30 min.

(4) Incubation of positive phage: the blocking solution was wiped and the positive phage was added at 37 ° C for 1 h.

(5) DAPI staining: washing with PBS 3 times, adding DAPI 100 μl, room temperature, 15 min

(6) Covering: After washing 3 times with PBS, the film was sealed.

Extraction and sequencing of 2.7 positive phage DNA

(1) 100 ul of iodide buffer was added to the purified phage precipitate, and 250 ul of absolute ethanol was added thereto, and the mixture was thoroughly mixed, and allowed to stand at room temperature for 20 minutes.

(2) Centrifugation: 4 ° C, 14,000 rpm, 10 min, the supernatant was discarded.

(3) Washing: The precipitate was washed with 500 ul of 70% ethanol, briefly centrifuged, and dried under vacuum.

(4) The pellet was resuspended in 30 ul TE (10 mM Tris-HCl, pH 5.0, 1 mM EDTA) buffer to prepare a DNA sequencing template solution, which was sent to Shanghai Biotech for sequencing.

As shown in Figure 1, 40 phage-expressing E. coli clones were randomly picked. The results of cell-based enzyme-linked immunosorbent assay showed that there were 31 clones with a ratio of SK-OV-3 to HOSEPIC greater than 2 in the experimental group. Is 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 22, 24, 25, 26, 27, 29, 30, 31 , 32, 33, 36, 38, 39, 40. They bind strongly to ovarian cancer cells and weakly bind to normal ovarian epithelial cells HOSEPIC. Therefore, immunofluorescence staining experiments of phage were further carried out to verify the targeted binding of phage to human ovarian cancer cells. As shown in Fig. 2, the positive phage was able to bind to human ovarian adenocarcinoma cell line SK-OV-3, and to human The binding ability of HOSEpiC in ovarian epithelial cells is weak, and there are significant differences between them, indicating that positive phage has a targeting effect on human ovarian cancer cells. Then, the phage clones showing positive binding were further sequenced, and it was confirmed that a total of 20 phage sequencing results showed the same sequence, which were 1, 5, 6, 7, 8, 9, 11, 12, 14, 18, 20, 22, 24, respectively. , 26, 27, 30, 32, 36, 38, 39. As shown in Figure 3, the polypeptide sequence was translated according to the principle of the triplet codon: WNPLLLTRLLPA (SEQ ID No. 1), WA12.

Further, immunoglobulin staining assay was used to detect the targeted binding of the polypeptide to human ovarian cancer cells. The fluorescently labeled polypeptide FITC-WA12 was used, and then the polypeptide was tested with human ovarian adenocarcinoma cell line SK-OV-3 by tumor-bearing mice. Targeting.

2.8 Immunofluorescence experiment of cells and FITC-WA12

(2) Fixation: fixed with 4% paraformaldehyde for 15 min.

(4) Incubation of FITC-WA12: Wipe the blocking solution and add FITC-WA12 at 37 ° C for 1 h.

(6) Covering: After washing 3 times with PBS, the film was sealed.

As shown in Fig. 4, the polypeptide sequence can bind to human ovarian adenocarcinoma cell line SK-OV-3, and the binding ability to human ovarian epithelial cells HOSEpiC is weak, and there is a significant difference between them, suggesting that the polypeptide has Target the role of ovarian cancer cells.

2.9 tumor-bearing mouse experiment

(1) Tumor-bearing: The nude mice were used for four weeks of age, and the human ovarian cancer cell line SK-OV-3 was inoculated into the tumor.

(2) Imaging: The polypeptide was placed in a solution of 100 μM/ml in PBS, and 0.1 ml of FITC-WA12 polypeptide solution was injected into the tail vein of the tumor-bearing mouse, and imaged on a small animal imager 10 minutes later.

The experimental results are shown in Fig. 5. After constructing human ovarian cancer-bearing mice, the peptide was fluorescently labeled and injected into the tail vein, and it was found that the fluorescence mainly accumulated in the tumor-bearing tissue, suggesting that the polypeptide may have targeted ovarian cancer tissue in vivo. The role.

Claims

A novel polypeptide targeted by a tumor, characterized in that the polypeptide is any of the following:

(1) The amino acid sequence of the polypeptide is: WNPLLLTRLLPA (SEQ ID No. 1);

(2) A polypeptide derivative which has undergone deletion, insertion or substitution of one or several amino acids in the polypeptide molecule of (1) and which has the same biological function as the polypeptide molecule of (1).
A novel tumor-targeting polypeptide according to claim 1, wherein the polypeptide has a targeting effect on tumor cells and specifically binds to tumor cells.
A novel tumor-targeting polypeptide according to claim 2, wherein the tumor cells are human ovarian cancer cells.
Use of the novel tumor-targeting polypeptide of claim 1 for the preparation of a tumor diagnostic kit, characterized in that the polypeptide or polypeptide conjugate is contained in the kit.
A novel tumor-targeting polypeptide according to claim 1 for use in the manufacture of a medicament for the treatment of tumors.
The use of the novel tumor-targeting polypeptide according to claim 5 for the preparation of a medicament for treating a tumor, characterized in that the medicament comprises the polypeptide and a pharmaceutically active ingredient, or comprises the polypeptide and a delivery carrier .
Use of a novel tumor-targeting polypeptide according to claim 5 for the preparation of a medicament for the treatment of a tumor, characterized in that the medicament is any pharmaceutically therapeutically acceptable dosage form.
The use of a novel tumor-targeting polypeptide according to claim 5 for the preparation of a medicament for the treatment of tumors, characterized in that the preferred dosage form of the medicament is an injection preparation.
Use of a novel tumor-targeting polypeptide according to claim 5 for the preparation of a medicament for the treatment of a tumor, characterized in that the medicament is any pharmacologically acceptable dose.