Lung cancer targeting peptides and uses thereof

Abstract

The application discloses a lung cancer targeting peptide and application thereof. In particular, the amino acid sequence of the lung cancer targeting peptide is selected from SEQ ID NO: 1-3. In addition, the application also provides the lung cancer targeting peptide, a polynucleotide thereof, a vector containing the polynucleotide and cells, and application of the lung cancer targeting peptide in preparation of a medicament or a kit for treating and diagnosing cancer (in particular, non-small cell lung cancer). The lung cancer targeting peptide has good targeting specificity for cancer cells in vitro and in vivo, and provides a basis for in-depth research of in-vivo and in-vitro drug tests and research and development of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a lung cancer targeting peptide and use thereof. BACKGROUND

[0002] Lung cancer is one of the highest incidence and mortality rates of malignant tumors in the world, and non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. The A549 cell line is a human lung adenocarcinoma cell line commonly used for the study of NSCLC, derived from the lung cancer tissue of a 58-year-old white male. A549 cells have strong invasion and metastasis ability, which is closely related to the high recurrence rate and low survival rate of lung cancer patients. It seriously affects the quality of life and survival of patients. Traditional treatment methods usually use chemotherapy and radiotherapy to inhibit the progression of A549 tumors. However, A549 cells have poor response to traditional chemotherapeutic drugs (such as cisplatin and paclitaxel), are prone to drug resistance, and chemotherapy and radiotherapy have significant systemic side effects (such as bone marrow suppression, gastrointestinal reactions, and nephrotoxicity), which also limit their long-term application, especially for patients with poor physical condition. Therefore, the development of anti-lung cancer drugs with good targeting and high efficiency has become a pressing problem to be solved.

[0003] Phage display biopanning technology is a highly efficient molecular screening technology, widely used in the fields of molecular-molecular interaction, antibody development, and target discovery. This technology is based on phage display library, which integrates foreign polypeptide or protein genes into phage coat protein genes and makes them express, thereby obtaining phage with coat protein displaying polypeptide or protein. Through multiple rounds of "adsorption-elution-amplification" selection process, phage clones with high affinity to specific target molecules (such as antibodies, receptors, and cell surface markers) can be screened from a phage library containing billions of clones. Thus, from a large phage library, a targeting peptide with high affinity to specific tumor tissues or organs can be selected. SUMMARY

[0004] The present application aims to provide a lung cancer targeting peptide. In particular, the lung cancer targeting peptide of the present application can specifically bind to non-small cell lung cancer cells. Specifically, the present application uses M13 filamentous phage polypeptide library to perform in vivo and in vitro combined screening on A549 tumor-bearing mice and A549 tumors, and obtains a polypeptide sequence that can effectively target lung cancer cells and lung cancer solid tumors. It has the advantages of simplicity, high efficiency, safety, etc., and provides a new method for the development of targeted therapy drugs for lung cancer (particularly, non-small cell lung cancer) and cancer detection. On this basis, the present application also provides the use of the lung cancer targeting peptide in biomedical materials / drugs / kit for the diagnosis, targeting and / or treatment of cancer (particularly, non-small cell lung cancer).

[0005] In this regard, the present application includes, but is not limited to, the following:

[0006] In one aspect, the present application provides a lung cancer targeting peptide, the amino acid sequence of which is selected from the group consisting of SEQ ID NO: 1-3. Preferably, the lung cancer targeting peptide of the present application is SEQ ID NO: 1 (FSAPWPT). Preferably, the lung cancer targeting peptide of the present application is SEQ ID NO: 2 (GTFCNLV). Preferably, the lung cancer targeting peptide of the present application is SEQ ID NO: 3 (YFGQNNP).

[0007] In yet another aspect, the present application provides a lung cancer targeting peptide, the amino acid sequence of which is set forth in SEQ ID NO: 1.

[0008] In yet another aspect, the present application provides an isolated polynucleotide encoding the lung cancer targeting peptide of the present application.

[0009] In yet another aspect, the present application provides a recombinant vector comprising the isolated polynucleotide of the present application.

[0010] In yet another aspect, the present application provides a cell comprising the recombinant vector of the present application, or having integrated into its genome the isolated polynucleotide of the present application.

[0011] In yet another aspect, the present application provides a bioactive substance, characterized in that it comprises the lung cancer targeting peptide of the present application, which is selected from the group consisting of:

[0012] (1) an engineered phage;

[0013] (2) a fusion protein; and

[0014] (3) a drug conjugate.

[0015] In yet another aspect, the present application provides the use of the lung cancer targeting peptide, the isolated polynucleotide, the recombinant vector, the host cell and / or the bioactive substance of the present application in the preparation or screening of a biomedical material and / or a drug for treating cancer, which is non-small cell lung cancer.

[0016] In yet another aspect, the present application provides the use of the lung cancer targeting peptide, the isolated polynucleotide, the recombinant vector, the host cell and / or the bioactive substance of the present application in the preparation or screening of a kit for diagnosing cancer, which is non-small cell lung cancer.

[0017] In one aspect, the lung cancer targeting peptide, the cell or the bioactive substance of the present application binds to the cancer cell membrane surface of non-small cell lung cancer.

[0018] In one aspect, the cancer cell of non-small cell lung cancer according to the present application is A549 cell.

[0019] In yet another aspect, the present application provides a pharmaceutical complex comprising the lung cancer targeting peptide, the isolated polynucleotide, the recombinant vector, the cell and / or the bioactive substance according to the present application, and a preparation or a drug for cancer treatment, and a pharmaceutical carrier, wherein the cancer is non-small cell lung cancer.

[0020] The preparation or drug for tumor treatment includes a biological preparation, a chemical drug, a radiotherapy drug, a photothermal therapy drug, a photodynamic therapy drug, and a cryotherapy drug. In particular, a protein, a peptide, a nucleic acid, an antibiotic, an anti-inflammatory drug, an anti-tumor drug, a neuroprotective agent, a chemotherapeutic agent, a cytotoxin, a radioisotope, a fluorescent marker, a luminescent substance, a chromogenic substance, or an enzyme.

[0021] The pharmaceutical carrier for tumor treatment includes any one of a nano-pharmaceutical carrier and a micro-pharmaceutical carrier, such as a liposome, an inorganic particle, an organic particle, a polymeric micelle, a polymeric vesicle, a carbon material, a microorganism, and the like.

[0022] Compared with the prior art, the screening method in vivo and in vitro adopted by the present application obtains a specific lung cancer targeting peptide, which has specific targeting effect on A549 cell line and also has specific targeting effect on A549 tumor in vivo, thereby providing a basis for in-depth research of in vivo and in vitro drug testing and development of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 After analyzing the sequencing results in Example 4, three polypeptide sequences with higher frequency and their corresponding frequencies appeared.

[0024] Figure 2 In Example 5, the phage titer experiment was used to verify the targeting binding ability of the polypeptide to lung cancer. DETAILED DESCRIPTION

[0025] The present application will be further described in detail below with reference to the examples and drawings. The following examples are preferred embodiments of the present application, and the present application is not limited to the following examples. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

[0026] Example 1: Construction of A549 tumor-bearing mouse biological model

[0027] (1) Environmental adaptation: 6-8 week-old male Blab / nude mice (purchased from Hangzhou Medical Science Institute) were selected and adapted for 7 days in a specific pathogen-free (SPF) environment;

[0028] (2) Tumor inoculation: about 1 x 106A549 cells were inoculated in the armpit of mice, and waited for about 21 days until the tumor size was about 50-100 mm 6 3 when the tumor size was about 50-100 mm

[0029] Example 2 In vivo screening of lung cancer targeting peptides

[0030] (1) In vivo circulation of M13 phage library: Ph.D.-7 TM phage polypeptide library (manufacturer: New England Biolabs, catalog number: E8211S) was injected into tumor-bearing mice through the tail vein, and the phage library was circulated in the tumor-bearing mice for 1 h;

[0031] (2) Heart perfusion: the mice were anesthetized with gas isoflurane, and the heart was perfused with sterilized normal saline to wash the phages that were not specifically bound to A549 tumor;

[0032] (3) Tissue grinding: the intact tumor tissue was peeled off, and the tissue was transferred to a grinding rod containing tissue cell lysis solution (Solebo, R0020), and the tumor tissue was ground thoroughly;

[0033] (4) Centrifugation: the ground tissue homogenate was centrifuged (1000 rpm, 20 min, 4°C), and the supernatant was reserved;

[0034] (5) Phage amplification: the tissue homogenate supernatant was transferred to E. coli ER2738 bacteria that were shaken to the logarithmic phase in advance, and phage amplification was performed at 37°C with vigorous shaking for 4 h;

[0035] (6) Phage purification: the amplified phage-bacteria mixture was centrifuged (12000g, 20 min at room temperature), and the supernatant was added with one-sixth volume of PEG / NaCl reagent (20% PEG, 2.5M NaCl) and mixed evenly. After overnight sedimentation at 4°C, the supernatant was removed by centrifugation (9000 rpm for 30 min), and the precipitate was resuspended with PBS to obtain purified phage, which was used as the sub-library for the next round of screening.

[0036] Example 3 In vitro screening of lung cancer targeting peptides

[0037] (1) Cell culture: A549 cells were subcultured in a T25 culture flask, and the next morning when the confluence reached 90%, the screening was started;

[0038] (2) Cell starvation: the cell culture medium in the culture flask was aspirated, and the cells were gently washed once with PBS, then 5 ml of serum-free cell culture medium was added, and the cells were incubated at 37°C for 1 h;

[0039] ​(3) Phage library affinity: Aspirate the serum-free medium from the cells, dilute the phage sub-library obtained in Example 2 to a concentration of 1 x 10 12 pfu / ml, add to the culture flask, and incubate at room temperature for 1 h;

[0040] (4) Washing: After incubation, aspirate the phage library and wash the cells 10 times with washing buffer (0.1% Tween 20 and 0.5% BSA);

[0041] (5) Elution: Remove the washing solution from the culture flask, add glycine elution solution (0.2M glycine-hydrochloric acid, pH 2.2, 1 mg / mL BSA), and incubate on ice for 10 min to elute the phage specifically bound to A549;

[0042] (6) Neutralization: Add 1M Tris-HCl (pH 9.1) neutralization solution to the eluted phage, mix, and then adjust the pH to neutral;

[0043] (7) Phage amplification: Add the collected affinity phage to ER2738 that has been shaken to the logarithmic phase in advance, and perform phage amplification at 37°C with vigorous shaking for 4 h;

[0044] (8) Phage purification: Centrifuge the amplified phage-bacteria mixture (12000g, 20 min at room temperature), add one-sixth the volume of PEG / NaCl reagent (20% PEG, 3.5M NaCl), mix well, and centrifuge (9000rpm, 30 min) after overnight sedimentation at 4°C to remove the supernatant. Resuspend the precipitate with PBS to obtain purified phage, which is used as a sub-library for the next round of screening.

[0045] Example 4: Phage plating and sequencing

[0046] (1) In vivo screening plating: Dilute the tissue homogenate collected in step (3) of Example 2 to a certain multiple, incubate 10μl with 200μl of ER2738 E. coli solution for 15 min, and then evenly spread on IPTG / Xgal solid LB plates (IPTG 0.1mM, Xgal 40μg / mL, tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, and agar powder 15g / L, pH 7.4). Place in a 37°C incubator and incubate overnight. The next day, count the blue plaques on the plate;

[0047] (2) In vitro screening coating plate: the bacteriophage collected in step (6) of Example 3 is diluted to a certain multiple, 10 μl of which is incubated with 200 μl of ER2738 E. coli liquid for 15 min, and then evenly coated on an IPTG / Xgal solid LB plate, which is placed in a 37°C incubator and cultured overnight. The next day, the blue plaques on the plate are counted;

[0048] (3) Gene sequencing to obtain lung cancer targeting peptide sequence: the bacteriophage obtained after the fifth round of screening (in which the first and third rounds are in vivo screening, and the second, fourth and fifth rounds are in vitro screening) is picked after coating and expanded, and the plasmid is extracted for gene sequencing to obtain the targeting peptide sequence displayed on the surface of the bacteriophage. After multiple rounds of in vivo and in vitro screening, three polypeptides with higher frequency are selected, and the polypeptide sequence and frequency statistics are shown in Table 1. Figure 1

[0049] Example 5: Verification of the lung cancer targeting ability of the polypeptide sequence obtained by screening through in vivo experiments

[0050] (1) Amplification and purification of the bacteriophage specifically targeting A549 tumor cells obtained by screening: the bacteriophage displaying lung cancer cell specific polypeptide sequences obtained by screening, as well as random polypeptide sequence bacteriophage (RS) and wild type bacteriophage (WT), are taken 200 μl and added to 20 ml of ER2738 E. coli liquid activated to the logarithmic growth phase, incubated for 30 min, and then shaken vigorously at 37°C overnight. The bacteriophage-bacteria mixture after shaking is centrifuged (12000g at room temperature for 20 min), the supernatant is taken, one-sixth volume of PEG / NaCl reagent (20% PEG, 3.5M NaCl) is added and mixed evenly, and after overnight precipitation at 4°C, the supernatant is removed by centrifugation (9000rpm for 30 min), and the precipitate is resuspended with PBS to obtain purified bacteriophage;

[0051] (2) Construction of A549 tumor-bearing mouse biological model: 6-8 week old male Blab / nude mice are selected, and after 7 days of adaptation in a specific pathogen-free (SPF) environment, tumor inoculation is performed. About 1×10 6 A549 cells are inoculated into the armpit of the mouse, and after about 21 days, when the tumor size is about 50-100mm 3 , the tumor-bearing mouse model is completed;

[0052] (3) In vivo circulation of bacteriophage: the purified bacteriophage (1×10 10 pfu) is injected into the tumor-bearing mouse body through the tail vein, and circulates for 1 h;

[0053] ​(4) Tissue sampling: After 1 h of in vivo phage circulation, the mice were anesthetized with gas and heart perfused with sterile saline to remove the phage not bound to the tumor tissue. After perfusion, the mouse skin tissue was carefully cut and the tumor was completely peeled off;

[0054] (5) Tissue grinding: After the peeled tumor tissue was weighed, it was placed in a grinding rod containing a tissue lysis solution and ground thoroughly;

[0055] (6) Centrifugation: The ground tissue homogenate was centrifuged (1000 rpm, 20 min, 4°C), and the precipitate was discarded to obtain the tissue homogenate supernatant containing phage;

[0056] (7) Plate counting: The collected tissue homogenate supernatant was diluted to a certain multiple, 10 μl of which was incubated with 200 μl of ER2738 E. coli liquid for 15 min, and then evenly spread on an IPTG / Xgal solid LB plate and placed in a 37°C incubator for overnight culture. The next day, the blue plaques on the plate were counted;

[0057] (8) Targeting ability quantification: The number of phage blue plaques counted in step (7) was divided by the mass of the tumor tissue to obtain the number of phage that could be bound per unit mass of tumor tissue.

[0058] As shown in Figure 2 Compared with wild-type phage (WT) and random polypeptide sequence phage (RS), the phage displaying polypeptides FSAPWPT (FT), GTFCNLV (GV) and YFGQNNP (YP) have significantly higher affinity for tumor cells, and the phage displaying polypeptide FSAPWPT (FT) exhibits the strongest affinity.

[0059] The genes and protein sequences involved in the present application are as follows:

[0060] SEQ ID NO: 1

[0061] Name: Amino acid sequence of lung cancer targeting peptide

[0062] Source: Artificial sequence (Artificial Sequence)

[0063] FSAPWPT.

[0064] SEQ ID NO: 2

[0065] Name: Amino acid sequence of lung cancer targeting peptide

[0066] Source: Artificial sequence (Artificial Sequence)

[0067] GTFCNLV.

[0068] SEQ ID NO: 3

[0069] Name: Amino acid sequence of lung cancer targeting peptide

[0070] Source: Artificial Sequence

[0071] YFGQNNP.

Claims

1. A lung cancer targeting peptide, characterized by, The amino acid sequence of the lung cancer targeting peptide is selected from SEQ ID NOs: 1-3.

2. A lung cancer targeting peptide, characterized by, The amino acid sequence of the lung cancer targeting peptide is as shown in SEQ ID NO:

1.

3. An isolated polynucleotide, comprising, The lung cancer targeting peptide according to claim 1 or 2.

4. A recombinant vector, characterized in that, The isolated polynucleotide according to claim 3.

5. A cell, comprising: The recombinant vector according to claim 4, or the genome of which is integrated with the isolated polynucleotide according to claim 3.

Images