CN110746489A

CN110746489A - Polypeptide for specifically targeting triple-negative breast cancer and application thereof

Info

Publication number: CN110746489A
Application number: CN201911002329.0A
Authority: CN
Inventors: 马岚; 胡羽童
Original assignee: Tsinghua Berkeley Shenzhen College Preparatory Office
Current assignee: Tsinghua Berkeley Shenzhen College Preparatory Office
Priority date: 2019-10-21
Filing date: 2019-10-21
Publication date: 2020-02-04
Anticipated expiration: 2039-10-21
Also published as: CN110746489B

Abstract

The invention discloses a polypeptide for specifically targeting triple-negative breast cancer and application thereof. The polypeptide has an amino acid sequence shown as SEQ ID No. 1. The polypeptide has homology with Decorin and can be combined with a Decorin receptor on the surface of a cancer cell of the triple negative breast cancer, so that a targeting effect is achieved, meanwhile, the binding capacity of the polypeptide and other types of breast cancer cells is weak, high specificity selectivity is shown, and the polypeptide can be used for targeted therapy or imaging diagnosis of the triple negative breast cancer.

Description

Polypeptide for specifically targeting triple-negative breast cancer and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a polypeptide for specifically targeting triple-negative breast cancer and application thereof.

Background

With the improvement of living standard and the extension of average life span of people, the incidence rate of cancer in the world is rising year by year and becomes a main factor threatening the health of human beings. The data from the world cancer report shows 1400 ten thousand cases of global cancer in 2012, which will rise to 2400 ten thousand by 2035. Breast cancer is the second most prevalent cancer in the world and is the most prevalent type of cancer in women. In 2012, 167 thousands of new breast cancer cases are caused in the whole world, and account for 25% of all new cancer cases in the current year. Breast cancer can be classified according to different molecular typing: estrogen/progestin positive type (ER/PR +), HER2 high expressing type (HER2+), and triple negative type. For ER/PR + type breast cancer, endocrine therapy is currently available; for HER2+ form, treatment is currently available with HER2 mab drugs or HER2 inhibitors, such as pertuzumab, trastuzumab, lapatinib, afatinib, and the like. Triple negative breast cancer accounts for approximately 15% -20% of all breast cancers, is the most malignant breast cancer species, and has a five-year survival rate far lower than the first two types. However, no effective molecular target is found for triple-negative breast cancer, and the method mainly depends on surgical treatment, traditional chemotherapy or radiotherapy. However, the traditional cancer treatment means often has serious side effects, and damages the normal functions of the body and destroys the autoimmunity of the human body while treating the cancer. Therefore, the search for possible biological targets of triple negative breast cancer is always one of the hot spots and difficulties in the research of the field of breast cancer.

Phage display technology is an in vitro screening technique that enables polypeptides of a desired nature to be extracted from colonies of a large number of variants. Since the first proposal by Smith, phage display technology has evolved into a powerful tool for discovering new properties and for altering the properties of existing polypeptides. Phage display technology is extremely effective in finding new binding polypeptides corresponding to target molecules. The basic principle is as follows: first, a nucleotide sequence of random sequence is inserted into the capsid protein gene of the bacteriophage, so that the bacteriophage can display the polypeptide encoded by the nucleotide. Then, the target molecules are screened through the phage library, and phages capable of specifically binding to the target molecules are enriched, so that corresponding specific binding polypeptides are obtained. The phage display technology shows very powerful application potential and important role in the process of screening specific binders for various types of target molecules. Based on this, there is a need to find polypeptides capable of specifically targeting triple negative breast cancer.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a polypeptide capable of specifically targeting triple negative breast cancer and application of the polypeptide.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a polypeptide having an amino acid sequence as shown in SEQ ID No.1 (ASYSSGPHTVQY).

The embodiment of the invention has the beneficial effects that:

the polypeptide has homology with Decorin and can be combined with a Decorin receptor on the surface of a cancer cell of the triple negative breast cancer, so that a targeting effect is achieved, meanwhile, the binding capacity of the polypeptide and other types of breast cancer cells is weak, high specificity selectivity is shown, and the polypeptide can be used for targeted therapy or imaging diagnosis of the triple negative breast cancer.

In a second aspect of the invention, there is provided a conjugate comprising a polypeptide as described above and a conjugation moiety. The conjugate can be specifically combined with triple negative breast cancer cells, has high specificity selectivity, and can be used for targeted therapy or imaging diagnosis of triple negative breast cancer.

According to an embodiment of the invention, the coupling moiety is selected from at least one of an imaging agent, a therapeutic agent.

According to an embodiment of the invention, the imaging agent is at least one of a variety of imaging techniques of contrast agents, such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI), ultrasound, radionuclide scanning, Positron Emission Tomography (PET), and the like.

According to an embodiment of the invention, the imaging agent is at least one of an optical marker, such as a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, or the like.

According to an embodiment of the invention, the imaging agent is selected from at least one of a radionuclide, a radionuclide label, a molecular imaging agent, a fluorescein, a quantum dot.

According to an embodiment of the invention, the therapeutic agent is selected from at least one of a drug, a toxin, a cytokine, a carrier protein, an antibody, an enzyme, a lectin.

In a third aspect of the invention, there is provided a nucleic acid molecule encoding a polypeptide or conjugate as hereinbefore described.

In a fourth aspect of the invention, there is provided a recombinant vector comprising a nucleic acid molecule as described above. The recombinant plasmid is used for targeted therapy or imaging diagnosis of triple negative breast cancer.

According to embodiments of the present invention, the recombinant vector may be a recombinant plasmid, a phage.

According to an embodiment of the present invention, the recombinant vector is a recombinant plasmid, which inserts the above-mentioned nucleic acid molecule at the multiple cloning site of the plasmid vector.

According to an embodiment of the invention, the recombinant vector is a recombinant bacteriophage.

According to an embodiment of the present invention, the recombinant bacteriophage inserts the aforementioned nucleic acid molecule into its DNA or RNA strand, and expresses the nucleic acid molecule with the expression of coat protein, thereby efficiently expressing the aforementioned polypeptide.

According to an embodiment of the invention, the recombinant bacteriophage is an M13 bacteriophage comprising the aforementioned nucleic acid molecule.

In a fifth aspect of the invention, there is provided a recombinant cell comprising a nucleic acid molecule as described above.

According to an embodiment of the invention, the recombinant cell is obtained by transduction or transfection of the aforementioned nucleic acid molecule in a host cell.

According to an embodiment of the invention, the host cell is a gram-negative bacterium; more specifically, Escherichia coli.

In a sixth aspect of the invention, there is provided a composition comprising any one of the aforementioned polypeptides, conjugates, nucleic acid molecules, recombinant vectors, recombinant cells. The composition is a diagnostic composition or a therapeutic composition for triple negative breast cancer.

According to an embodiment of the invention, the composition is a pharmaceutical composition further comprising a drug delivery vehicle.

According to an embodiment of the present invention, the drug delivery vehicle is any one of a nano-scale vehicle system, a micro-scale vehicle system.

According to an embodiment of the present invention, the drug delivery vehicle is selected from at least one of liposomes, polymeric micelles, dendrimers, inorganic nanoparticles.

In a seventh aspect of the invention, a kit is provided, which comprises any one of the aforementioned polypeptides, conjugates. The polypeptide or the conjugate has targeting specificity on the triple-negative breast cancer cells, and the kit prepared by the polypeptide or the conjugate can be used for specifically identifying and marking the triple-negative breast cancer cells or used for diagnosis and treatment of the triple-negative breast cancer cells.

In an eighth aspect of the invention, the application of the aforementioned polypeptide, conjugate, nucleic acid molecule, recombinant vector, recombinant cell and composition in preparing a reagent for diagnosing, preventing and treating triple negative breast cancer is provided.

Drawings

FIG. 1 shows the results of an ELISA assay for the selective binding of 28 polypeptides to triple negative breast cancer cells according to one embodiment of the present invention. Wherein, the number 7 is the polypeptide claimed in this example, the experimental cell line MDA-MB-231 is triple negative breast cancer cell, and the control cell line MCF-7 is non-triple negative breast cancer cell.

FIG. 2 shows the results of immunofluorescence assay of example 2 of the present invention, in which FAM fluorophores are attached to polypeptides. Wherein, a and b represent a control cell line MCF-7 which is non-triple negative breast cancer cells; c and d represent the experimental group cell line MDA-MB-231, which is a triple negative breast cancer cell.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

1. Screening for Polypeptides

The cells are screened by using a phage peptide library by adopting a Negative-/Positive + method, and the screening is carried out for three rounds by using MCF-7 (Negative screen), MDA-MB-231 (Positive screen). Each round of screening was as follows:

1) adding 0.5% BSA into adherent MCF-7 cells to seal for 1 h;

2) phage random dodecapeptide library (Ph.D. -12 phage display peptide library kit, New England Bio-LABS, USA) at 5X 10¹⁰The titer of PFU (5. mu.L) was added to MCF-7 cell culture dishes and incubated for 1h at 37 ℃ (negative screen);

3) centrifuging at 3000rpm/min for 3min, collecting supernatant (containing bacteriophage without non-triple negative breast cancer cells), transferring into an immune tube containing MDA-MB-231 cells, and incubating for 1h (positive screening) at 37 ℃ with shaking;

4) after the incubation is finished, centrifuging at 3000rpm/min for 3 min;

5) discarding the supernatant, washing the cell precipitate with PBST solution (the concentration of Tween-20 in PBST is gradually increased from 0.1% to 0.5%) for 5 times, and then washing with PBS solution for 5 times;

6) adding 1mL of eluent, centrifuging at 12000rpm, transferring the supernatant into a new centrifuge tube, and adding 150 μ L of a neutralization solution, namely the phage obtained in the first round of panning;

7) the panned phage were amplified in e.coli ER2378 host bacteria and counted by titration.

Wherein, the titer determination and enrichment ratio in each round of phage peptide library screening are shown in Table 1

TABLE 1 enrichment ratio of different screening runs

Wherein the enrichment rate is the amount of phage eluted/added

Randomly selecting 28 independent phage blue spots from the final round of phage titration plates for amplification, sending the amplified phage blue spots to a sequencing company for sequencing, and numbering and combining phage clones with the same amino acid sequence. Sequencing results show that the amino acid sequence of the polypeptide displayed by one phage monoclonal is shown in SEQ ID No. 1.

2. Selective binding assay

And (3) testing the selective binding capacity of the 28 polypeptides obtained by the third round of screening on the triple-negative breast cancer cells by enzyme-linked immunosorbent assay (ELISA), wherein the specific steps are as follows:

1) MCF-7 and MDA-MB-231 cells are respectively inoculated in a 96-well plate, and the cells grow to proper density;

2) fixing the cells, adding 100 mu L of 4% paraformaldehyde solution into each hole, fixing for 15min at room temperature, and washing the plate with PBS solution for three times;

3) add 200. mu.L of 1% BSA-PBS-T blocking solution to each well and block for 2h at 37 ℃. Washing the plate with PBS for three times after the sealing is finished;

4) adding phage diluted by a sealing solution into each hole, incubating for 90min at room temperature, washing the plate with a PBST solution for three times after incubation is finished, and washing the plate with a PBS solution for three times;

5) adding 100 mu L of mouse M13 phage antibody marked by horseradish catalase diluted by blocking solution, incubating for 90min at room temperature, and washing the plate for three times by using PBS solution after incubation is finished;

6) adding 100 mu L of ELISA developing solution into each hole, developing for 15min in a dark place, terminating the reaction by using 2mol/L sulfuric acid, and washing the plate for three times by using PBS solution;

7) the absorbance of the solution was read at 492nm using a microplate reader.

FIG. 1 shows the results of ELISA experiments on the selective binding of 28 polypeptides of this example to triple negative breast cancer cells. Wherein, the number 7 is the polypeptide with the amino acid sequence shown in SEQ ID No.1, the MDA-MB-231 cell line in the experimental group is triple negative breast cancer cells, and the MCF-7 cell line in the control group is non-triple negative breast cancer cells. As can be seen from the figure, the polypeptide represented by the number 7 specifically binds to triple negative breast cancer cell MDA-MB-231, but poorly binds to non-triple negative breast cancer cell MCF-7, indicating that the polypeptide provided by the present invention is selectively specific for binding to triple negative breast cancer cells, and can specifically target triple negative breast cancer cells.

Example 2

Fluorescent probe for targeting triple-negative breast cancer cells

A fluorescent probe specifically targeting triple-negative breast cancer cells comprises the polypeptide shown in the number 7 in the amino acid sequence of SEQ ID No.1 in example 1 and a fluorescent group FAM (carboxyfluorescein) coupled on the peptide chain.

The method for detecting the target specificity of the fluorescent probe comprises the following specific steps:

1) synthesizing a polypeptide connected with a FAM fluorophore;

2) MCF-7 and MDA-MB-231 cells are respectively inoculated in a 24-well plate, and the cells grow to proper density;

3) removing the culture medium by suction, washing with PBS for three times, 3min each time, adding 500 μ L of 4% paraformaldehyde solution into each well, fixing at room temperature for 20min, removing the fixing solution by suction, and washing with PBS for three times, 3min each time;

4) diluting the polypeptide according to a ratio of 1:500, adding the diluted polypeptide into a corresponding pore plate, incubating for 1h at 37 ℃, and washing for three times with PBS (phosphate buffer solution) for 5min each time;

5) adding 100 μ L of DAPI dye solution into each well, incubating at room temperature for 15min, sucking off DAPI, and washing with PBS for 5min for 3 times;

6) the cover glass is clamped from the 24-well plate by using tweezers, 10 mu L of the anti-fluorescence quenching blocking tablet is dripped on the glass slide, the glass slide with the cells laid thereon is reversely buckled on the glass slide, and after the glass slide is naturally dried, the fluorescence intensity is observed by a confocal microscope and a picture is taken.

FIG. 2 shows the results of immunofluorescence assay of example 2 of the present invention, in which FAM fluorophores are attached to polypeptides. Wherein, a and b represent a control cell line MCF-7 which is non-triple negative breast cancer cells; c and d represent the experimental group cell line MDA-MB-231, which is a triple negative breast cancer cell. As shown in FIG. 2, in the control group of MCF-7 cells, no fluorescence was observed in addition to the blue fluorescence of DAPI, indicating that the polypeptide having the attached fluorophore FAM was not bound to the MCF-7 cells; in the experimental group, MDA-MB-231 cells can observe bright red fluorescence, which indicates that the polypeptide connected with the fluorescent group FAM is combined with the MDA-MB-231 cells; the results indicate that the fluorescent probe provided by the present example can specifically bind to triple negative breast cancer cells.

The above examples show that the polypeptide provided by the invention has the characteristic of targeting triple negative breast cancer cells, so that in practical application, the polypeptide can be used as a targeting polypeptide, combined with a preparation capable of killing cancer cells, used for targeting treatment of tumors, or combined with an imaging diagnostic reagent, and used for molecular imaging and diagnosis of targeted tumors.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.

SEQUENCE LISTING

<110> preparation office of the institute of Qinghua-Berkeley Shenzhen

<120> polypeptide specifically targeting triple-negative breast cancer and application thereof

<130>9

<160>1

<170>PatentIn version 3.5

<210>1

<211>12

<212>PRT

<213> Artificial sequence

<400>1

Ala Ser Tyr Ser Ser Gly Pro His Thr Val Gln Tyr

1 5 10

Claims

1. A polypeptide having an amino acid sequence as shown in SEQ ID No. 1.

2. A conjugate comprising the polypeptide of claim 1 and a conjugation moiety.

3. The conjugate of claim 2, wherein the coupling moiety is selected from at least one of a radionuclide, a radionuclide label, a molecular imaging agent, a fluorescein, a quantum dot, a drug, a toxin, a cytokine, a carrier protein, an antibody, an enzyme, a lectin.

4. A nucleic acid molecule encoding the polypeptide of claim 1, or encoding the conjugate of any one of claims 2 to 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 4.

6. A recombinant cell comprising the nucleic acid molecule of claim 4.

7. A composition comprising any one of the polypeptide of claim 1, the conjugate of claims 2 to 3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant cell of claim 6.

8. The composition of claim 7, further comprising a drug delivery vehicle;

preferably, the drug delivery vehicle is selected from at least one of liposomes, polymeric micelles, dendrimers, inorganic nanoparticles.

9. A kit comprising the polypeptide of claim 1, any one of the conjugates of claims 2 to 3.

10. Use of the polypeptide of claim 1, the conjugate of any one of claims 2 to 3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant cell of claim 6, the composition of any one of claims 7 to 8 in the preparation of an agent for the diagnosis, prevention, treatment of triple negative breast cancer.