TW201627011A - Lung cancer specific peptides for targeted drug delivery and molecular imaging - Google Patents

Abstract

A conjugate is disclosed. The conjugate comprises (a) an isolated or a synthetic targeting peptide of less than 15 amino acid residues in length, comprising an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-8; and (b) a component conjugated to the targeting peptide, the component being selected from the group consisting of a drug delivery vehicle, an anti-cancer drug, a micelle, a nanoparticle, a liposome, a polymer, a lipid, an oligonucleotide, a peptide, a polypeptide, a protein, a cell, an imaging agent, and a labeling agent. Methods of treating lung cancer and detecting lung cancer cells are also disclosed.

Description

Lung cancer-specific peptide for targeted drug delivery and molecular imaging

The present invention relates generally to a drug delivery system and, more particularly, to a lung cancer target drug delivery system.

Lung cancer is the leading cause of cancer-related mortality in men and women. It is estimated that 159,480 people died of lung cancer in the United States in 2013, accounting for about 27% of all cancer deaths. For therapeutic purposes, lung cancer is histopathologically classified as small cells (15%) or non-small cells (84%), the latter consisting of large cell carcinoma (LCC), adenocarcinoma, and squamous Composition of squamous cell carcinoma (SCC). Although surgery, radiation therapy, chemotherapy, and even EGFR target therapy such as cetuximab (Erbitux), erlotinib (Tarceva), and gefitinib (Iressa) It has been used to treat lung cancer of different stages or types, but the five-year survival rate of small cell carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) is still low, 6% respectively. And 18%.

One of the main reasons for this disappointing result is the lack of selectivity in conventional chemotherapy for cancer treatment, which leads to severe damage to the narrow therapeutic window and normal tissues. Another reason is the high interstitial fluid pressure of solid tumors (interstitial fluid) Pressure; IFP) makes it difficult for anticancer agents or even small molecule tyrosine kinase inhibitors commonly used for target treatment to enter tumor sites. It has been shown that the accumulation of drugs in normal viscera is about 10 to 20 times higher than that of the same weight tumor site, and many anticancer drugs cannot penetrate the vasculature with a thickness of 40-50 μm or more (equivalent to the combined diameter of 3-5 cells). ). These shortcomings often lead to limited therapeutic functions and multiple drug resistance, which affect clinical outcomes.

In one aspect, the invention relates to a conjugate comprising: (a) an isolated or synthetic target peptide having a length of less than 15 amino acid residues, comprising one selected from the group consisting of SEQ ID NOs: 1- a sequence of 8 constituent groups having at least 90% identity amino acid sequence; and (b) a component conjugated to the target peptide, the component being selected from a drug delivery vehicle, resistant A group consisting of a cancer drug, a microcell, a nanoparticle, a liposome, a polymer, a lipid, an oligonucleotide, a peptide, a peptide, a protein, a cell, a contrast agent, and a labeling agent.

The contrast agent can be iron oxide. The iron oxide can be coated in the liposome.

The target peptide can comprise at least one motif selected from the group consisting of MHLXW, NPWXE, and WXEMM motifs, wherein X is any amino acid residue.

The aforementioned conjugate may exhibit at least one of the following characteristics: (a) increased lung cancer cell binding compared to the control group of liposomes; (b) increased lung cancer cell endocytosis compared to the control group of liposomes; (c) reduction of cytotoxic cancer cells at half maximum inhibitory concentration (IC _50); (d) enhance the efficacy of anticancer drugs in vivo; (e) reducing the size of a lung tumor in vivo; and (f) to extend the overall survival of the individual having lung cancer rate.

In another aspect, the invention relates to an isolated or synthetic target peptide having a length of less than 15 amino acid residues, comprising a sequence selected from the group consisting of SEQ ID NOs: 1-8 An amino acid sequence having at least 90% identity, wherein the isolated or synthesized target peptide has the activity of binding to human lung cancer cells rather than normal cells. The lung cancer cell may be at least one selected from the group consisting of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The lung cancer may be at least one selected from the group consisting of adenocarcinoma, papillary adenocarcinoma, bronchioloalveolar carcinoma, squamous cell carcinoma, large cell carcinoma, and small cell carcinoma. .

In a specific embodiment of the invention, the isolated or synthesized target peptide comprises at least one substituent modification relative to a sequence selected from the group consisting of SEQ ID NOs: 1-8.

In another embodiment of the present invention, the isolated or synthesized target peptide is conjugated to a component selected from the group consisting of a drug delivery vehicle, a liposome, a polymer, a lipid, A group of cells, contrast agents, and labeling agents.

The isolated or synthesized target peptide can be conjugated to a PEG-phospholipid derivative, a liposome, or a PEGylated liposome. The PEG-phospholipid derivative may be selected from the group consisting of NHS-PEG-DSPE, PEG-DSPE.

The isolated or synthesized target peptide may further comprise an anticancer drug or a fluorescent dye which is coated in the liposome or PEGylated liposome.

In still another aspect, the invention relates to a composition comprising: (a) a liposome or a pegylated liposome: and (b) at least one of the aforementioned isolated or synthesized target peptides, conjugated thereof Binds to the surface of the liposome or PEGylated microlipid.

In one embodiment of the invention, the composition may comprise at least two isolated or synthesized target peptides conjugated to the surface of the liposomes or PEGylated microlipids. Each liposome or PEGylated liposome can have a different target peptide conjugated thereto.

The composition may further comprise at least one anti-cancer drug which is coated in a liposome or a PEGylated liposome. The anticancer drug may be at least one selected from the group consisting of doxorubicin and vinorelbine.

The composition may comprise one or more of the aforementioned isolated or synthesized peptides.

In another embodiment of the invention, the composition comprises: (a) an isolated or synthesized peptide as set forth in SEQ ID NO: 3; (b) isolated as shown in SEQ ID NO: 1 or Synthetic peptide; or (c) an isolated or synthesized peptide as set forth in SEQ ID NO: 1 and SEQ ID NO: 3.

In still another aspect, the invention relates to a method of treating lung cancer comprising administering a composition of the foregoing to an individual in need thereof. Also provided is the use of a composition described herein for the manufacture of a medicament for the treatment of lung cancer.

In still another aspect, the invention relates to a method for detecting a lung cancer cell, comprising: (1) exposing the cancer cell to a conjugate, comprising: at least one target that is separated or synthesized as in the second item of the patent application; a peptide; and a contrast agent or labeling agent, conjugated to at least one isolated or synthesized target Peptide; (2) removing a conjugate that is not bound to a cancer cell, and detecting a conjugated contrast agent or labeling agent that binds to the isolated or synthesized target peptide of the cancer cell; or (i) exposure The cancer cell is in a conjugate comprising: at least one isolated peptide synthesized or synthesized as in claim 2; and at least one phage displaying at least one peptide on its surface, which is displayed on the phage At least one peptide has an amino acid sequence equivalent to the isolated or synthesized target peptide as in claim 2; (ii) removing the conjugate that is not bound to the lung cancer cell, and detecting binding to the lung cancer At least one phage of the cell.

The cancer cells can be present in tissue specimens, such as surgical tissue specimens. One or more of the aforementioned isolated or synthesized peptides can react and bind to lung cancer tissue specimens.

These and other aspects of the present invention will be apparent from the following description of the preferred embodiments of the invention.

The drawings illustrate one or more embodiments of the invention, and in the written description Wherever possible, the same reference numerals refer to the

Figure 1A shows immunofluorescence staining of H460 large cell carcinoma and H1993 adenocarcinoma cell lines by FITC-calibrated HSP1, HSP2, and HSP4 peptides. The nucleus was stained with DAPI. Scale bar, 50μm.

Figure 1B is a list of the percentage of IFA positive staining cells of HSP1, HSP2, and HSP4-FITC in H460 and H1993 cell lines.

Figure 2 shows in vivo tumor homing ability verification of H460 target phage. (A) SCID mice with H460 xenografts were injected intravenously with selected phage colonies. After 8 minutes, the free phage was washed by PBS perfusion, followed by removal of allograft masses and organs to determine phage valence (n=3). Among the four phage strains of similar sequence of group 1, HPC2, 3, and 4 showed better tumor homing ability, and HPC1 was the best of group 2. (B) HILYTE ^TM Fluor 750-tagged HPC1,2,4, and contrast control phage body. These measured 24 hours HILYTE ^TM Fluor 750 labeled phage tissue distribution after the injection, and to measure the signal strength IVIS200 software in organs and tumors. *, P <0.05; * * *, P < 0.001 (n = 3). (C) Fluorescence images of anatomical organs of HPC1-HL750-injected mice were obtained and compared to control phage.

Figure 3 shows that HSP1, HSP2, and HSP4 peptides enhance the liposome SRB internalization and the liposome doxorubicin cytotoxicity in human lung cancer cell line H460. (A) H460 cells were incubated at 37 ° C and the kinetics of HSP1-LSRB, HSP2-LSRB, HSP4-LSRB, and LSRB were taken up. After washing with acid glycine buffer, the surface-bound microlipid dye was removed and the internalized SRB (n=4) was quantified. (B) H460 cell line was treated with increasing doses of target or non-targeted liposome, doxorubicin (LD), followed by cell viability (n=6) by MTT assay. The IC _{50 of} HSP1-LD, HSP2-LD, HSP4-LD, and LD were 3.512 μM, 3.388 μM, 4.646 μM, and 43.865 μM, respectively. The number of suitable peptides of the inserted HSPs 1, 2, and 4 of each liposome was tested.

Figure 4 shows the efficacy of HSP1-LD, HSP2-LD, and HSP4-LD in human lung large cell carcinoma xenografts. (A) H460-derived lung cancer xenograft mice with an average tumor size of ~75 mm ³ , with FD, LD, HSP1-LD, HSP2-LD, HSP4-LD (1 mg/kg/injection, once a week) ), or intravenous injection of an equal volume of PBS. Each group has n = 8. At each point , the average tumor volume. (B) L460-derived lung cancer mice with a mean tumor size of ~500 mm ³ administered intravenously to FD, LD, HSP1-LD, HSP2-LD, HSP4-LD (2 mg/kg/injection) , twice a week), or an equal volume of PBS. Each group has n = 7. Detailed statistics on the efficacy of HSP1-LD, HSP2-LD, and HSP4-LD are shown separately in (CE). Error bar , SE. *, P <0.05; **, P <0.01; ***, P < 0.001. (F) The Kaplan-Meier survival graph shows that the target drug has a longer life span than the non-targeted drug for large tumor treatment.

Figure 5 shows the efficacy of HSP1-LD, HSP2-LD, and HSP4-LD in human lung adenocarcinoma allografts. (A) H1993-derived lung cancer xenograft mice with an average tumor size of ~300 mm ³ administered intravenously to FD, LD, HSP1-LD, HSP2-LD, HSP4-LD (1 mg/kg/injection) , twice a week), or an equal volume of PBS. Each group has n = 7. At each point , the average tumor volume. Detailed statistics on the efficacy of HSP1-LD, HSP2-LD, and HSP4-LD are shown separately in (BD). Error bar , SE. *, P <0.05; **, P <0.01; ***, P < 0.001. (E) Changes in body weight during the course of treatment. NS, no significant. (F) The Caber-Mer survival curve shows that the life cycle of mice treated with HSP1 and HSP2 mediated liposomes is significantly longer than the other groups of the H1993 model.

Figure 6 shows the combination of HSP4-LD and HSP4-LV in human lung large cell carcinoma xenografts. (A) H460-derived lung cancer xenograft mice with an average tumor size of ~200 mm ³ , which were administered intravenously with a combination of FD/FV, LD/LV, or HSP4-LD/HSP4-LV. 1/2 mpk, or an equal volume of PBS, which is twice a week for a total of four weeks. Each group n = 8. At each point , the average tumor volume. Error bar , SE. *, P <0.05; **, P <0.01. (B) Changes in body weight during the course of treatment. NS, no significant. (C) The Caber-Mer survival curve shows that the life cycle of mice treated with HSP4-mediated lipophilic drugs is significantly longer than the other groups of the H460 model. (D) The median survival days of LD and LV of HSP4 target were significantly prolonged

Figure 7 shows the combination treatment of HSP4-LD and HSP4-LV to treat the orthotopic model of H460 large cell carcinoma. (A) Pharmacokinetic angiography of intravenously combined therapy of H460 cells of luciferase-expressing H460 cells, which were administered intravenously with FD/FV, LD/LV, or HSP4-LD/HSP4-LV, respectively. The combined dose is 1/2 mpk, or an equal volume of PBS. A total of 5 x 10 ⁵ cell lines were transplanted with MATRIGEL®, and the treatment started 4 days after cancer xenografts (every 2 days, 8 times). Each group n = 8. (B) Quantification of tumor luminescence signals with IVIS200 software. *, P <0.05. (C) Changes in body weight during the course of treatment. (D) Camembert-Mer survival curve, and (E) median survival time of the drug-treated mice.

Figure 8 shows the combination of HSP4-LD and HSP4-LV treatment to treat the orthotopic pattern of A549 adenocarcinoma. (A) Pharmacokinetic angiography of intravenously combined therapy of allogeneic transplanted mice of A549 cells expressed by luciferase, which were administered intravenously to FD/FV, LD/LV, HSP4-LD/HSP4-LV, respectively. Binding dose 1/2 mpk, or an equal volume of PBS. The total number of 5 × 10 ⁵ cell lines to MATRIGEL® xenografts and treatment began five days after cancer cell xenograft (once every two days, a total of eight times). Each group has n = 7. (B) Quantification of tumor luminescence signals with IVIS200 software. **, P <0.01; ***, P <0.001. (C) Changes in body weight during the course of treatment. The overall survival rate (D) and median survival days (E) of LD and LV of HSP4 target were significantly prolonged.

Figure 9 shows IHC staining of HPC1, 2, and 4 in NSCLC and SCLC clinical specimens. Representative micrographs of paraffin sections of several human lung cancer surgical specimens were tested with HPC1, HpC2, and HPC4 phage strains (2~5×10 ⁸ pfu/μl). In contrast, these target phage were unable to detect normal bronchioles. A helper phage was used as a negative control group. Scale bar, 100μm.

The invention is more particularly described in the following examples, which are intended to be illustrative only, and many modifications and variations will be apparent to those skilled in the art. Specific embodiments of the invention are now described in detail. Referring to the drawings, the same numerals represent the same components. The meaning of "a", "an" and "the" are used in the meaning of the description and the scope of the claims. In the meantime, the meaning of "inside" and "inside" are used in the context of the description and the scope of the entire patent application, unless the context clearly dictates otherwise. In addition, the description may use a title or sub-headings to facilitate the reader, which should not affect the scope of the invention. Moreover, some of the terms used in this specification are more specifically defined as follows.

definition

The terms used in the specification generally have their ordinary meaning in the specific context of the art, the present invention, and the use of each term. The specific terms used to describe the invention are discussed below or elsewhere in the specification to provide additional guidance to the practitioner of the invention. For convenience, specific terms such as italics and/or quotation marks may be used. The use of highlighting does not affect the scope and meaning of the term; whether or not it is highlighted, the terms and meanings of terms in the same context are the same. It should be understood that the same thing can be described in more than one way. Thus, any and a plurality of terms referred to herein may use alternative language and synonyms, and are not substituted in any particular sense, regardless of the terms set forth or discussed herein. This article provides synonyms for specific terms. The statement of one or more synonyms does not preclude the use of other synonyms. The examples used in the specification are used for illustration only, and the examples include examples of any terms referred to herein, and are not intended to limit the scope and meaning of the invention or any exemplary terms. As such, the invention is not limited to the specific embodiments provided herein.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. In the event of a conflict, this document, including definitions, will be used as a benchmark.

The words "about", "about", or "about" as used herein shall generally mean within 20% of a given value or range, preferably within 10%, and more preferably between %Inside. The numerical quantities given in this paper are approximate, which means that if there is no express requirement, the words "about", "about" or "about" can be inferred.

The term "drug delivery vehicle" refers to a vehicle that delivers a drug to a patient in a manner that increases the concentration of the drug in a portion of the body relative to other sites. Drug delivery vehicles include, but are not limited to, polymeric micelles, liposomes, lipoprotein drug carriers, nanoparticle drug carriers, dendrimers, cells, polypeptides, and the like. An ideal drug delivery vehicle must be non-toxic, biocompatible, non-immunogenic, biodegradable, and must be protected from host defense mechanisms. The terms "treatment" or "treatment" refer to the administration of an effective amount of a compound to a subject in need thereof, which has the symptoms or predisposition to cancer or evolves into a disease for the purpose of healing, relieving, soothing, and remedying. , to improve, or prevent the disease, its symptoms, or the tendency to evolve into the disease. The individual can be identified by a health care professional based on the results of any applicable diagnostic method (see U.S. Patent Application Serial No. 14/499,201, the disclosure of which is incorporated herein by reference).

The terms "treatment" or "treatment" refer to the administration of an effective amount of a compound to a subject in need of cancer, or the symptoms or predisposition of the disease, for the purpose of curing, relieving, soothing, treating, ameliorating, or Prevent the disease, its symptoms, or its propensity. This individual can be defined by a health care professional based on the results of any applicable diagnostic method.

The term "an effective amount" refers to the amount of active compound required to impart a medical benefit to a treated individual. The effective dosage will vary depending on the route of administration, the use of excipients, and the possibility of being used in conjunction with other medical treatments, as understood by those skilled in the art.

Guidance for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in the US Department of Health and Human Services, Food and Drug Administration, "Guidelines for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers) "discloses a" therapeutically effective amount "can be calculated from the formula: HED = animal dose (mg / kg) × (animal weight (kg) / human weight (kg)) ^0.33.

In this study, the present invention isolates lung cancer-specific peptides using phage display peptide libraries and biopanning techniques. The present invention recognizes three novel peptides, HSP1, HSP2, and HSP4, which bind to several NSCLCs (including LCC, adenocarcinoma, and SCC) and SCLC in cell lines and clinical surgical specimens rather than in general lung tissue. In vivo studies have further confirmed that these HSP 1, 2, or 4 peptide-mediated drug delivery systems enhance efficacy and bioavailability. These data confirm the prospects for these three novel peptides for therapeutic applications.

Iron oxide binding peptides are disclosed in U.S. Patent Publication Nos. 20100158837 and US20090208420. Superparamagnetic iron oxide (USPIO) is a liposome which has been revealed by Frascione D et al. (Int J Nanomedicine. 2012; 7: 2349-59).

The term "marker" includes but is not limited to "fluorescent marker".

The contrast agent is designed to provide more information about internal organs, cellular processes, and tumors, as well as normal tissues. It can be used to diagnose diseases and monitor treatment effects.

Example

It is not intended to limit the scope of the invention, and the exemplary apparatus, apparatus, methods, and related results of the embodiments of the invention are described below. It should be noted that the title or sub-headings may be used in the examples for the convenience of the reader and should not be construed as limiting the scope of the invention in any way. In addition, the specific theory is proposed and disclosed herein; however, it should not be construed as limiting the scope of the invention in any way, as long as the invention is implemented in accordance with the invention and does not contemplate any particular theory or mechanism of action.

Materials and methods

Cell line and culture

NCI-H460, NCI-H661, NCI-H1993, NCI-H441, NCI-H520, NCI-H1688, A549 human lung cancer cell lines, and NL20 human bronchial epithelial cell lines were purchased from the American Type Culture Collection; ATCC®), and is certified by the ATCC, based on DNA profiling, cytogenetic analysis, and isomerism. These cell lines were cultured according to the ATCC recommended method and passaged for less than 6 months after resuscitation. Create CL1-5 cells and periodically verify their growth, morphology, and no mold. The human normal nasal mucosal epithelial (NNM) cells are primary cultured cells derived from nasal polyps and cultured in DMEM.

Phage display biopanning program

The human lung large cell carcinoma cell line H460 cell line was cultured in a UV-treated inactivated control group of helper phage (no insertion phage). Subsequently, a library of peptides displayed by phage was initially added, which initially contained 5 x 10 ¹⁰ plaque-forming units (pfu). After washing, the combined phage system was eluted with lysate (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4) on ice. . The extracted phage pool was expanded and evaluated for strength in E. coli ER2738 medium. The recovered phage system is used as an input to the second round of panning. In the fourth and fifth rounds of biopanning, phage colonies were randomly selected for ELISA screening (draft), which is incorporated herein by reference.

Determination of phage colonies by cytokine-binding immunosorbent assay (ELISA)

Cancer or control NNM cells were seeded in 96-well ELISA plates. Adding individual phage colonies to the culture plate of the coated cells, and culturing the mouse anti-M13 monoclonal antibody conjugated with horseradish peroxidase (HRP), followed by culturing the peroxidase substrate O- phenylenediamine dihydrochloride. The reaction was terminated and the luminescence value was measured at 490 nm using an ELISA plate reader. The selected phage lineage was further analyzed by DNA sequencing, wherein the primer 5'- CCCTCATAGTTAGCGT AACG-3 ' (SEQ ID NO: 12) corresponds to the pIII gene sequence.

Peptide synthesis and labeling

Synthetic target peptides HSP1 (GAMHLPWHMGTL; SEQ ID NO: 1), HSP2 (NPWEEQGYRYSM; SEQ ID NO: 2), HSP4 (NNPWREMMYIEI; SEQ ID NO: 3), and control peptide (derived from BSA protein) The sequence of 12 amino acids, KATEEQLKTVME; SEQ ID NO: 13) was prepared in Fmoc SPPS using a CEM Liberty automated microwave peptide synthesizer and purified to 95% purity by reverse high performance liquid chromatography. These peptides were conjugated to a fluorescent isothiocyanate (FITC) line and added to the C-terminus of the peptide with FITC. Peptide synthesis, conjugate binding, and purification were performed by the Academia Sinica Cell and Individual Biology Institute Peptide Synthesis Core Facility (Taipei, Taiwan).

Flow cytometry analysis

Collecting lung cancer cell lines or control cells in an enzyme-free cell separation buffer, Thereafter, the cells were cultured at 20 μg/mL FITC conjugated HSP1, 2, 4, or control peptide for 1 hour at 4 °C. After washing, it was analyzed by flow cytometry.

Immunofluorescence staining of lung cancer cells by synthetic peptides

The H460 and H1993 cell lines were grown and grown to approximately 50% full on coverslips. After the cells were fixed with 2% polyoxymethylene, they were cultured at 10 μg/mL FITC-labeled HSP1, 2, 4, or a control peptide. Subsequently, the slide was counterstained with 4',6-diamidino-2-phenylindole (DAPI), mounted, and under a Leica universal fluorescent microscope. Observation. The software merged images with MetaMorph image analysis.

Immunohistochemical staining of human surgical specimens

Eleven cases of lung adenocarcinoma and ten cases of lung squamous cell carcinoma paraffin tissue sections were taken from the National Taiwan University Hospital (NTUH) organization library, which was approved by the NTUH Institutional Review Board. In order to increase the number of cases of lung cancer specimens and histopathological subtypes, the present invention also obtains commercial tissue microarray sections, which are composed of a total of 120 cases of lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell carcinoma, and the like. And agreed by AS-IRB03-102103. To localize the phage to the lung cancer tissue, the tissue was cultured in HPC1, HPC2, HPC4, or control phage (2-5×10 ⁸ pfu/μl). After washing with PBS, sections were treated with anti-M13 mouse mAb (GE Healthcare) for 1 hour at room temperature. After the washing step, immunoreactivity was detected using a biotin-free hypersensitive polymer-HRP detection system. The slides were slightly counterstained with hematoxylin, mounted with AQUATEX® (Merck), and observed by light microscopy.

In vivo tumor homing test and angiography

5×10 ⁶ H460 cells were injected subcutaneously into the dorsolateral ventral of SCID mice. The lung cancer allograft size matched (about 300 mm ³ ) mice were injected intravenously with 2 x ¹⁰⁹ pfu of the target phage or control phage. After 8 minutes of phage cycling, the mice were sacrificed and perfused with 50 ml PBS to wash away unbound phage. Subsequently, allograft tumors and mouse organs were diced and homogenized. The phage bound to each tissue was recovered by adding ER2738 bacteria, and the titer was evaluated on an IPTG/X-Gal agar plate. In vivo systemic arteriongraphy, HPC1,2,4, and the control group phage-based fluorescent dye to acid HILYTE ^TM Fluor 750 NHS ester (HL750) tag, by NHS-based functional group. The same H460 allograft model was injected intravenously with 5 x ¹⁰⁹ pfu of HL750-labeled target phage or control phage. Fluorescence of the mice and tissues was taken at a given time point with a Xenogen IVIS200 imaging system (excitation wavelength: 710/760 nm; emission wavelength: 810/875 nm). The fluorescence intensity of the tissue was calculated by subtracting the background value using the Living Image software (Xenogen).

Preparation of synthetic peptide conjugated liposome doxorubicin, venosamine, and SRB

The peptide is coupled to NHS-PEG-DSPE at a molar ratio of 1.1:1 [N-hydroxy amber succinimide-carboxy-polyethylene glycol (MW, 3400) derived distearyl decyl phosphonium decylethanolamine ]. The PEGylated peptide-PEG-DSPE conjugate was purified by SEPHADEX® G-15 (GE healthcare) gel filtration chromatography followed by lyophilization. Quantitative HPLC analysis of the conjugate and drawn up in a MALDI-TOF-MS (BRUKER MICROFLEX TM) qualitative analysis.

Lipid film hydration method is used to prepare PEGylated liposome composed of distearyl phosphatidylcholine choline, cholesterol, and PEG-DSPE, and then used for coating Sorbice, vonolpin, or incorporated sulforhodamine B-DSPE, having a particle size ranging from 65 to 75 nm. The HSP 1, 2, or 4-PEG-DSPE was then co-cultured by shaking at 60 ° C for 1 hour to incorporate preformed liposomes at a transition temperature of the lipid bilayer. After incubation, each of the liposomes showed approximately 500 peptide molecules on the surface. SEPHADEX ^TM G-25 gel filtration chromatography system for removal of free drug release, the non-conjugated peptides, and do not incorporate the conjugate. The concentration of doxorubicin and von vonpine for the fractionation of the extract was determined by measuring the fluorescence excitation/emission wavelengths of 485/590 and 520/570 nm, respectively, using a spectrophotometer (Spectra Max M5, Molecular). Devices).

Targeted peptide conjugated LSRB or LD uptake in human lung cancer cells

The H460 and H1993 cell lines were grown to 90% full in 24-well plates and 20, 10, 5, 2.5, 1.25, 0.625 μM HSP1, 2, 4-lipid sulfonium rhodamine B (1) was added to the complete medium. , 2,4-liposomal sulforhodamine B, LSRB) or LSRB. The cell line was cultured at 37 ° C for the following periods: 10, 30 minutes, 1, 2, 4, 8, 16, and 24 hours. At the indicated time points, the cells were washed with PBS, and the LSRB which was not internalized on the cell surface was removed by adding 0.1 M glycine (pH 2.8) for 10 minutes. Subsequently, the cells were lysed with 200 μl of 1% Triton X-100. The low concentration uptake of LD in H1993 cells was performed in the same procedure. For SRB or doxorubicin extraction, 300 μl of IPA (isopropanol containing 0.75 N HCl) was added to the lysate and shaken for 30 minutes. The lysate was centrifuged at 12,000 rpm for 5 minutes and the uptake was measured by measuring the fluorescence excitation/emission wavelength of 520/570 nm (for SRB) and 485/590 nm (for doxorubicin) using fluorescence luminosity. Measure (SPECTRAMAX® M5, Molecular Devices). The concentration of SRB and doxorubicin was calculated by interpolation using a standard curve.

Endocytosis test

The H460 cell line was cultured in HSP1, HSP2, HSP4-LSRB, or LSRB for 10 minutes at 4 °C and 37 °C. After washing with PBS, the cells were fixed with 4% polyoxymethylene, blocked with 1% BSA, and then cell membranes with WGA-ALEXA FLUOR® 467 and DAPI. And nuclear staining. All fluorescence images were taken from a conjugated focus microscope.

In vitro cytotoxicity assay of LD with target peptide conjugated

The H460 cell line was seeded in 96-well culture plates (5000 cells/well) containing complete medium and cultured overnight. On alternate days, cells were treated with different concentrations (0-100 μM) of HSP1-LD, HSP2-LD, HSP4-LD, or LD for 24 hours at 37 °C. After removing excess drug, the cells were washed once with PBS and continued to culture in fresh medium for 48 hours at 37 °C. Cell viability was determined by adding 50 μl of MTT (thiazole blue tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide; Sigma-Aldrich) to each well of the culture plate. After 3.5 hours of MTT incubation, 150 μl of dimethyl hydrazine (DMSO; Mallinckrodt Baker) was added to each well for 10 minutes, and the optical value (SPECTRAMAX® M5, Molecular Devices) was measured at 540 nm using a microdisc.

Animal model for ligand target treatment research

Human NSCLC cells were injected subcutaneously into the dorsolateral ventral of 4-6 week old female SCID mice. The tumor size was consistent (small tumors were about 75 mm ³ ; large tumors were about 300 or 500 mm ³ ). The mice were then randomly assigned to different treatment groups, and intravenously injected with liposome, doxorubicin (LD), and liposome. (LV), target peptide (HSP1, HSP2, or HSP4) conjugated to LD or LV, free doxorubicin (FD), free vonopine (LV), or an equal volume of saline. The dose and duration of administration are described in the schematic, depending on the experimental design. The body weight and tumor size of the mice were measured twice a week. The tumor volume was calculated as follows: length x (width) ² x 0.52. The Passport Protection Department is conducted in accordance with the guidelines of the Academia Sinica (Taipei, Taiwan). The program was approved by the Academia Sinica Animal Experimental Ethics Committee.

Orthotopic lung cancer model

SCID mice (6 weeks old) were anesthetized with isoflurane mixed oxygen and placed in the right lateral position. The skin covering the midline of the left chest wall was prepared with alcohol, and the lower chest wall and the intercostal space were exposed. 50 μl of serum-free medium containing H460 or A549 cells (5 × 10 ⁵ cells) overexpressing luciferase was added to MATRIGEL® Matrix (2:1) and injected into the left lateral thoracic midline. After tumor injection, the mice were turned to the left lateral position and observed for 45 to 60 minutes until completely awakened.

After medication before each administration, intraperitoneal injection LUCIFERIN ^TM (Caliper Life Sciences) 10 minutes to IVIS200 system (Xenogen Corporation, Alameda, CA) and the imaging performance of quantization cancer luciferase.

Pharmacokinetics and biodistribution studies

Single dose (2mg/kg) of free drug doxorubicin (FD), liposome doxorubicin (LD), or target (HSP1, HSP2, or HSP4) LD injection into H460 lung cancer Graft (~300 mm ³ ) tail vein of SCID mice. Blood was collected from the mice under anesthesia and sacrifice (three mice per group) at 1 hour and 24 hours after the injection. Subsequently, the mice were perfused through the heart with 50 ml of PBS, and the allograft tumors and organs (brain, lung, heart, liver, and kidney) were weighed, weighed, and homogenized to calculate the dormitories of the tissues. The total doxorubicin was quantified by measuring the fluorescence of λ _Ex/Em = 485/590 nm using a fluorophotometer (SPECTRAMAX® M5, Molecular Devices).

Statistical Analysis

P values were calculated on a double-sided unpaired Student's t test. For all analyses, P < 0.05 was considered to be significantly different.

result

Identify three novel peptides that bind to several human lung cancer cells

In this study, the present invention isolated phage that bind to H460 non-small cell lung cancer (NSCLC) cells as a random peptide library displayed by phage. After five rounds of affinity screening (biopanning), the combined phage valence increased by a factor of 9 (Figure S1). 94 phage colonies were randomly selected from the fourth and fifth rounds of biopanning, and ELISA screening of the cells was performed. 47 of these selected phages have a higher affinity for H460 cells. Subsequently, the present invention further tests the binding activity of these H460-binding strains to other cell lines, including human lung adenocarcinoma H1993, CL1-5, A549, murine Lewis lung carcinoma 3LL, or human normal nasal mucosal epithelial NNM cells. By phagocytizing the phage strain with the highest lung cancer binding but the weakest normal cell reactivity, the present invention identified 13 phage strains which showed two sets of consensus sequences (Table 1). Interestingly, HPC1, 5, and 13 show the same sequence "GAMHLPWHMGTL" (SEQ ID NO: 1). Table 1 shows that the phage selected from H460 cells showed a sequence alignment of the peptides. Of the 47 phage strains randomly selected after 15 rounds of biopanning, 13 phage strains with higher H460 binding affinity were identified and the peptide sequences shown were aligned. * The phage display of the consensus amino acid series is in the box.

To explore whether these similar peptides show similar binding properties of phage to different lung cancers, the present invention compares the binding strength of the two groups of phage to H460, H1993, CL1-5, A549, and 3LL, which is determined by cell ELSA. (The manuscript is paid, and is incorporated herein by reference). This data shows that although HPC2, 3, and 4 display similar sequences containing the NPW-E (SEQ ID NO: 14) motif, HPC 3, 4, and 6 exhibit more similar binding properties in various lung cancers. This shows that W-EMM (SEQ ID NO: 15) mimics the phantom to play a more important role in binding to lung cancer than the NPW-E motif, which is composed of HPC3 and 4 and thus two dimorphs, but behaves similarly HPC6, which contains only W-EMM simulation phantoms. The other groups of the phage display the MHL-W (SEQ ID NO: 16) consensus sequence with similar lung cancer binding properties. Based on these findings, the present invention was selected for HPC1, HPC2, and HPC4 for further study because of their typical MHL-W motif, NPW-E motif, and W-EMM motif.

To determine whether HPC1, HPC2, and HPC4, which are shown in the peptide sequence, have lung cancer binding function, the present invention synthesizes HSP1, HSP2, and HSP4 peptides, respectively having the amino acid sequence GAMHLPWHMGTL (SEQ ID NO: 1), NPWEEQGYRYSM (SEQ ID NO: 2), and NNPWREMMYIEI (SEQ ID NO: 3). The "SP" of "HSP" is represented by The HPC phage displays the "Synthetic Peptide". The HSP1, HSP2, or HSP4 synthetic peptide or its fluorescein isothiocyanate (FITC) conjugate will be used in subsequent experiments. To determine whether HSP1, 2, and 4 peptides can bind to target molecules expressed on the surface of lung cancer cells, the surface binding activity of each FITC conjugated peptide is determined by flow cytometry and immunofluorescence staining. (Figure 1A). Based on FACS data, all of these three FITC-labeled peptides are clearly associated with pathological subtypes of several human lung cancer cell lines, including large cell carcinoma (H460 and H661), adenocarcinoma (H1993, H441, CL1-5, and A549). ), squamous cell carcinoma (H520), and small cell carcinoma (H1688), but not to human normal bronchial epithelial cells (NL20). In addition, HSP1, 2, and 4 showed different fluorescence intensity binding characteristics in each lung cancer cell, indicating that these peptides can target different molecules on the cell surface.

In the cellular IFA assay (Fig. 1A), FITC-labeled HSP 1, 2, or 4 binds to a major group of H460 large cell carcinoma cells and H1993 adenocarcinoma cells, whereas the FITC-labeled control peptide does not bind. The FITC positive cells represent the peptide target to the cell expressing the molecule. Thus, the present invention calculates the percentage of H460 and H1993 cells positive for such tripeptide staining (Fig. 1B). Based on this statistical table and fluorescence map, the present inventors have found that the target exhibits a ratio of cells to a complete group and a receptor density on the cell surface. It is noteworthy that HSP4 shows a higher cell surface receptor density due to its stronger fluorescence intensity, although it has a lower positive ratio in H1993 cells. In addition, the HSP4 peptide showed optimal reactivity to H460 cells, regardless of positive cell or receptor density. These results show that HSP1, 2, and 4 bind to NSCLC and SCLC cells in vitro and have different binding properties.

In vivo tumor homing and angiography of HPC1, 2, and 4

In order to study the in vivo targeting ability of the selected phage colony, the present invention will treat each strain Intravenous injection into H460-derived tumor xenograft mice. After perfusion, the present invention measures phage valence of tumors and normal organs. The tumor homing ability was determined by the ratio of the tumor phage valence to the normal organ phage valence and compared to the control phage. In the first set of phage (HPC2, 3, 4, 6) with consensus sequences, HPC2, 3, and 4 showed significant tumor homing ability, while HPC6 only specific recessive tumor localization ability (Fig. 2A). This is another reason for the selection of HPC2 and HPC4 rather than HPC6 in the present invention to illustrate the NPW motif and the W-EMM motif for further investigation. Phages of MHL-W, HPC1, but not HPC9, which share a consensus sequence in other groups, exhibited remarkable tumor homing ability (Fig. 2A).

Further, the present invention is to HILYTE ^TM Fluor 750 (HL750) Fluorescent dye labeled phage, which can be excited with radiation for a particular wavelength range (excitation wavelength: 710 / 760nm; radiation wavelength: 810 / 875nm) of the contrast body. H460 heteroenzyme SCID mice with different sizes were injected with HPC1-HL750, HPC2-HL750, HPC4-HL750, or control phage HL750, and monitored by IVIS200 series. The HL750-labeled phage can be imaged in the IVIS200 imaging system during systemic cycling in mice. At 6 hours post injection, the target phage, which accumulates in the tumor tissue, becomes quite distinct and clearly visible. Fluorescence images of mice and anatomical tissues were captured 24 hours after injection (Fig. 2C). The tumor fluorescence intensity in the target phage group was about 3 to 4 times higher than that in the control group phage (Fig. 2B). These results show that all HPC1, HPC2, and HPC4 have significant tumor homing ability.

HSP1, 2, and 4 synthetic peptides improve liposome drug binding, intracellular delivery, and cytotoxicity

Since "receptor-mediated endocytosis" is important for targeted drug delivery due to enhanced drug penetration, release, and effects, the present invention then tests for HSP1, HSP2. Or HSP4 can promote the internalization of liposome drugs to human lung cancer cells. For material preparation, HSP1, HSP2, and HSP4 are conjugated to NHS-PEG-DSPE before the phospholipid DSPE is inserted into the outer surface of the liposome nanoparticles. These nanoparticles contain sulforhodamine B (SRB; fluorescent reagent) or doxorubicin. Unlike chemotherapeutic drugs, the fluorescent dye SRB does not cause cell death even at high concentrations, making it an ideal tool for measuring the absorption of living cells. During the course of the experiment, the present inventors found that the target peptide (HSP1, 2, or 4) conjugated to the liposome SRB (LSRB) promotes the liposome internalization to H460 compared to the non-target LSRB (Fig. 3A) ) and H1993 cells. Interestingly, in H460 cells, the present invention also observed significant intracellular trafficking of HSP2 at low LSRB concentrations, but at high concentrations it was no different from non-target LSRB (Fig. 3A). Conversely, HSP1 and HSP4 showed better absorption at high doses. One of the possible explanations for this phenomenon is that the receptor for HSP2 on H460 cells is saturated at high concentrations. This phenomenon indicates that HSP1, 2, or 4 can target different receptors on the surface of the cell, depending on the receptor density.

In terms of visual contrast, the present invention also uses a conjugated focal microscope to examine the LSRB of the target peptide conjugated binding of lung cancer cells. In the cytoplasm of H460 cells cultured at 37 °C of HSP1-LSRB, HSP2-LSRB, or HSP4-LSRB, a large amount of LSRB was observed, whereas cells cultured with non-target LSRB detected only a small amount of SRB fluorescence. At 4 ° C, LSRB conjugated to the three target peptides binds to the outer membrane of H460 cells. It is worth noting that HSP1 peptides showed stronger binding ability than HSP2 and HSP4, but not internalization ability. The evidence is strong binding strength at 4 °C, but the SRB fluorescence of cytoplasm at 37 °C is better. weak.

In addition, the present invention tests whether HSP 1, 2, and 4 mediated microliposome drugs enhance drug efficacy due to their enhanced targeting and endocytosis capabilities. The present invention is an in vitro toxicity test of HSP1, 2, or 4 conjugated liposome doxorubicin such as doxorubicin (LD) in H460 cells (Fig. 3B). Compared with LD, these three target peptides LD significantly enhance the cytotoxicity of drugs against cancer cells. HSP1, HSP2, and HSP4, at their optimal peptide ratio, reduced the half-maximal inhibitory concentration ( IC ₅₀ ) of H460 cells by 12.5, 13, and 9.4-fold (Fig. 3B).

In short, HSP1, 2, and 4 target peptides not only bind to lung cancer cells with high specificity, but also drive the internalization of liposome drugs and enhance the in vitro efficacy.

Efficacy of HSP1, HSP2, and HSP4-mediated drug delivery systems in human large cell carcinoma and adenocarcinoma xenograft models

In addition, in order to determine whether HSP1, 2, and 4 can enhance the in vivo chemotherapy effect of the anticancer drug, the present invention couples the peptide to the PEGylated liposome, doxorubicin (LD), and is formulated into a standard. Target drug delivery system. In the first experiment, SCID mice with H460 human lung large cell carcinoma xenografts were administered intravenously with HSP1-LD, HSP2-LD, HSP4-LD, non-target LD, free doxorubicin (FD), or an equal volume of PBS (Figure 4). These LDs inspection target in the present invention are small tumors (average tumor size ~ 75mm ³⁾ (FIG. 4A) large tumors (average tumor size ~ 500mm ³⁾ The efficacy and (FIG. 4B-D). Anticancer effects were assessed by measuring the mean tumor volume, and side effects were predicted by measuring changes in body weight during treatment. The small tumor mouse was treated with 1 mg/kg doxorubicin, four times a week. This tumor volume was significantly reduced in the target LD group (Fig. 4A). In the treatment of large tumor mice, compared with the LD group, the target peptides HSP1, 2, and 4 significantly enhanced LD efficacy in H460 large tumors, especially HSP2 and HSP4-LD, which showed a doubling of tumor volume ( Figure 4B-E). In vivo biodistribution and pharmacokinetic studies support this finding, HSP2 and HSP4 have better LD delivery to H460 tumor tissue drug delivery. During the treatment period, overall survival was observed to be prolonged (Fig. 4F) and the body weight was unchanged.

The present invention also tests HSP1, 2, and 4-LD in H1993 human lung adenocarcinoma allograft The efficacy of the plant model (Figure 5). H1993 large tumor mice of different sizes were injected with 1 mg/kg of HSP1-LD, HSP2-LD, HSP4-LD, LD, FD, or an equal volume of PBS twice a week for three weeks. Figure 5C shows that administration of HSP 1, 2, and 4-LD did not cause significant weight loss compared to the LD group. By measuring tumor volume, HSP4-LD showed the best efficacy, starting from the 10.5th day after treatment (after 3 injections) due to a significant reduction in tumor volume (Fig. 5B). However, in terms of overall survival, patients treated with HSP1 and HSP2-LD survived for an additional 50-60 days compared to LD-treated mice (Fig. 5D). These data show that tumor volume reduction does not translate into longer survival rates. One of the phenomena may be explained by the fact that although HSP4 has the highest number of receptors on the surface of H1993 cells (Fig. 1A), only 55.93% of H1993 cells exhibit HSP4 receptors, which are smaller than HSP1 and 2, compared to other dipeptides. (Figure 1B). This may provide more opportunities for HSP4-negative cells, which may become anti-drug cells during HSP4-LD treatment in H1993 mode.

HSP1, 2, and 4 target peptides in biodistribution assays to enhance tumor drug delivery in vivo

To detect the anti-cancer mechanism of action of HSP1, 2, or 4 conjugated liposomes in vivo, the present invention performs pharmacokinetic and biodistribution tests to measure the accumulation of tumor tissue. H460 xenograft tumor mice were injected intravenously with a single dose of 2 mg/kg FD, LD, HSP1-LD, HSP2-LD, or HSP4-LD. After 1 hour and 24 hours of systemic circulation, the concentration of doxorubicin, such as serum, tumor, and normal organs, was assessed by measuring a single polysoline such as bisin fluorescence after the purification step. The average intratumoral doxorubicin concentration in the HSP1, HSP2, and HSP4-LD groups was approximately 1.5, 2, and 2 times higher than that in the LD group, respectively. This data provides evidence of better tumor inhibition of HSP2 and 4 compared to the H460 large tumor treatment group using the target-LD and explains the better tumor inhibition of previous experiments (Fig. 4B-D). Because Doxorubicin acts by embedding DNA, it also measures A drug that accumulates the accumulation of cancer cells. The results of each tumor are usually similar. The liposome formulas (LD, HSP1-LD, HSP2-LD, and HSP4-LD) show a similar biodistribution of plasma to normal organs, with the free form of doxorubicin such as bismuth in plasma having a shorter half-life. The results of this experiment show that HSP1, 2, and 4 enhance the penetration of anticancer drugs into tumors and lead to higher accumulation of drugs at target sites in the cells, thereby enhancing the efficacy of doxorubicin. In addition, such target peptides do not increase the accumulation of doxorubicin in animal models such as the brain, heart, lungs, liver, and kidneys.

Combined improvement based on targeted microlipids to improve overall survival

Due to the genetic instability and genetic heterogeneity of cancer biology, monotherapy of single drugs often enhances the redundant signaling pathway, which accelerates chemotherapy-resistant mutations and relapses. The use of multiple chemotherapeutic drugs combined with different mechanisms of action has become the primary strategy for the treatment of drug-resistant cancer. Thus, the present invention co-delivers HSP4-LD and HSP4 conjugated liposome vinorepine (LV) with a binding ratio of 1:2 as a microtubule inhibitor to treat the H460 xenograft mode (Fig. 6). The prescription of the two drugs has been tested and optimized for synergy in the body (data not shown). Figures 6C-D show that the HSP4-mediated binding target liposome treatment group has a longer overall survival compared to the non-targeted liposome or free drug treated group. Compared with the non-targeted liposome treatment group, the combined microliposome treatment group prolonged the median survival rate by up to 11 days (74 days control 63 days).

The present invention also investigates the efficacy of this 1:2 combination of LD and LV in the orthotopic model of H460 large cell carcinoma (Fig. 7) and A549 adenocarcinoma (Fig. 8), which successfully summarizes the interaction of the tumor microenvironment. In the H460 orthotopic mode, the HSP4-mediated combination of the liposome-treated luciferase showed a significant reduction in tumor weight compared to the free drug-treated group (Fig. 7A-B), whereas the non-targeted liposome-treated group There was no significant difference between the free drug treatment groups. Because the H460 positive mode is extremely intense, all Both mice developed severe weight loss due to cancer cachexia syndrome (Fig. 7C). However, the median survival time of the target liposome group was observed to be extended by 6.5 days compared to the non-targeted liposome group (77.5 days versus 71 days). In the A549 in situ mode, the HSP4-mediated combination of the liposome-treated group significantly prolonged the overall survival and increased the median survival time to 47 days compared to the non-targeted liposome-treated group (131 days of control versus 84 days). ) (Fig. 8D-E). The present invention demonstrates that the HSP4 target peptide not only improves the efficacy of the nanomedicine (Fig. 8A-B), but also reduces adverse effects by reducing body weight loss (Fig. 8C).

Binding activity of HPC1, 2, and 4 to clinical surgical specimens of human lung cancer

The response of target drugs to sections or surgical specimens of cancer patients is one of the most difficult challenges in clinical drug development. Here, the present invention tests whether HSP 1, 2, or 4 can react with several different types of human lung cancer specimens, including adenocarcinoma, papillary adenocarcinoma, bronchioloalveolar carcinoma (BAC), squamous cell carcinoma ( Squamous cell carcinoma (SCC), large cell carcinoma, and small cell carcinoma. Since the M13 phage particle is composed of many coat proteins, this signal is amplified at the immunostaining step and is more visible than using the peptide itself. For this reason, the present invention performs human tissue staining with HPC1, 2, and 4 phage. Table 2A lists the percentage of positive rates obtained by detecting several different types of lung cancer with HPC 1, 2, and 4. In general, HPC4 showed the best reactivity (>80%) in almost all types of lung cancer, followed by HPC1 (>50%). In addition, HPC 1, 2, and 4 also recognized lung metastatic adenocarcinoma or SCC (Table 2B), but did not respond to normal lung tissue or normal lung tissue surrounding the cancer (Table 2C). Figure 9 shows an example of immunohistochemical staining performed on successive portions of individual tumors. These data show that HPC1, 2, and 4 can not only identify NSCLC, but also identify SCLC surgical specimens, but will not cross-react with normal lung tissue. Table 2. Human lung cancer surgical specimens were detected by immunohistochemical staining with HPC 1, 2, and 4. (A) Several clinical human lung cancer biopsy histopathology subtypes, which are HPC1, 2, or 4 Immunostaining was compared to control phage. Calculate and catalog the percentage of positive reactions. (B) IHC data for adenocarcinoma metastasized from lung and SCC. (C) Normal lung tissue and normal lung tissue surrounding the tumor confirmed tumor specificity with HPC1, HPC2, and HPC4. Reaction area: +++, >50%; ++, 50~20%; +, <20%; -, 0%.

Compared with monoclonal antibodies, it exhibits large size, poor tumor penetration, and high immunogenicity. When used as a target ligand (Cheng and Allen, 2010), peptide ligands are better for payload transport. The choice is due to its smaller size, lower immunogenicity, and higher tumor penetration, and its synthesis and production are more cost effective. In the present study, the present invention identified three novel peptides, HSP 1, 2, and 4, which selectively bind to several human lung cancers, rather than normal lung tissue, whether in vitro, in vivo, or in clinical samples. Thirteen phage colonies (HPC1-13) with higher in vitro lung cancer binding were divided into two categories according to different consensus sequences. The first group showed "MHL-W" motif (HPC1), while the other group The "NPW-E or W-EMM" motif (HPC2 and 4) is displayed. Although HPC2 and 4 show more similar sequences, their binding patterns are different and have their own functions in a series of experiments, such as cell ELISA binding assay, FACS analysis, cellular IFA staining (Fig. 1A-B), dose dependence and time. The procedure LSRB absorption test (Fig. 3A), even human surgical specimen detection (Fig. 9, Table 2). These results indicate that HSPs 1, 2, and 4 may target different protein molecules on the surface of lung cancer cells due to their different positive staining rates, reaction intensities, or receptor densities.

HSP1, 2, and 4-mediated DDS can specifically bind to lung cancer cells, which in turn triggers "receptor-mediated endocytosis" to discharge payloads to their intracellular target sites (eg, Doxorubicin) the target site for the DNA), leading to in vivo by approximately 10-fold IC ₅₀ (FIG. 3). Similarly, HSP1, 2, and 4 mediated microliposome drugs significantly improved in vivo drug bioavailability (Figure S8), resulting in an increased therapeutic index (Figures 4-8). It should be noted that in the H460 allograft mode, HSP2 and 4 performed better in LD delivery, which was a 2-fold increase in tumor accumulation and efficacy (Fig. 4B-C). Unlike the members of the consensus sequence, HPC2, HPC4, which positively stained almost all types of NSCLC and SCLC surgical specimens, showed the best clinical reactivity (Table 2). In addition, preclinical data indicate that HSP-mediated binding of liposomes has improved overall survival (Figure 6-8). This strategy provides a novel and better tailored combination of prescriptions to overcome clinical chemical resistance. And delay the recurrence of cancer.

The IHC data (Figure 9, Table 2) also shows that the W-EMM motif of HSP4 may contribute more to this effect than the NPW-E motif and is therefore critical for clinical applications. More advanced research is needed to understand the function of each motif in order to select the appropriate motif for endocytosis and other clinical tests. Therefore, the present invention can modify the peptide sequence to be perfect and multifunctional.

Further research is needed to understand the receptor proteins expressed on the surface of lung cancer cells targeted by HSP1, HSP2, and HSP4, and to investigate their individual downstream intracellular signals, which are important for the transfer of released substances. Identification of the target protein also provides information on safety and toxicity characteristics that are critical to the clinical development of the targeted drug. According to the present invention, HSP1, 2, and 4 lung cancer target peptides have a significant potential to develop into "treatment nanoparticles", which have a wide range of clinical applications, including target treatment, concomitant diagnosis, and non-invasive imaging.

The foregoing description of the preferred embodiments of the present invention is intended to In view of the above implementation, There may be many improvements and changes.

The specific embodiments and examples are intended to be illustrative of the invention and the embodiments of the invention, use. Other embodiments are apparent to those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims, rather than the foregoing description and exemplary embodiments.

Some references, which may include patents, patent applications, and various publications, are hereby incorporated by reference. The citation and/or discussion of the reference materials is provided for the purpose of clarifying the content of the present invention and is not to be construed as an admission that any of this reference is a prior art. All of the references cited and discussed in this specification are hereby incorporated by reference in its entirety in its entirety herein in its entirety in its entirety in its entirety herein in its entirety in

<110> Academia Sinica

<120> Lung cancer-specific peptides for targeted drug delivery and molecular imaging

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Claims (20)

A conjugate comprising: (a) an isolated or synthesized target peptide having a length of less than 15 amino acid residues, comprising a group selected from the group consisting of SEQ ID NOs: 1-8 a sequence having at least 90% identity amino acid sequence; and (b) a component conjugated to the target peptide, the component being selected from the group consisting of drug delivery vehicles, anticancer drugs, micelles, A group consisting of nanoparticles, liposomes, polymers, lipids, oligonucleotides, peptides, peptides, proteins, cells, contrast agents, and labeling agents. An isolated or synthetic target peptide having a length of less than 15 amino acid residues, comprising an amino acid having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-8 A sequence wherein the isolated or synthesized target peptide peptide has activity for binding to human lung cancer cells but not normal cells. An isolated peptide synthesized or synthesized according to claim 2, which is conjugated to a component selected from the group consisting of a drug delivery vehicle, a liposome, a polymer, a lipid, a cell, A group of contrast agents and marker agents. The isolated peptide synthesized or synthesized as in claim 2 of the patent application is conjugated to a PEG-phospholipid derivative, a liposome, or a PEGylated liposome. The isolated peptide synthesized or synthesized according to claim 4, wherein the PEG-phospholipid derivative is selected from the group consisting of NHS-PEG-DSPE, PEG-DSPE. The target peptide isolated or synthesized according to claim 4 of the patent application, further comprising an anticancer drug or a fluorescent dye, which is coated in the liposome or PEGylated liposome. A composition comprising: (a) a liposome or a pegylated liposome; and (b) at least one of the isolated peptides isolated or synthesized as in claim 2 of the patent application, conjugated to the liposome or Pegylated microlipid surface. The composition of claim 7, comprising at least two isolated or synthesized target peptides conjugated to the surface of the liposome or the PEGylated microlipid. The composition of claim 7, wherein each of the liposomes or the PEGylated liposomes has a different target peptide conjugated thereto. The composition of claim 7, further comprising at least one anti-cancer drug coated in the liposome or the PEGylated liposome. The composition of claim 2, wherein the lung cancer cell line is selected from the group consisting of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The composition of claim 1, wherein the contrast agent is iron oxide. The composition of claim 12, wherein the iron oxide is coated in the liposome. The composition of claim 1, wherein the target peptide comprises at least one motif selected from the group consisting of MHLXW, NPWXE, and WXEMM motifs, wherein X is any Amino acid residue. A composition comprising one or more peptides isolated or synthesized as in claim 2 of the scope of the patent application. An isolated peptide synthesized or synthesized as in claim 2, which comprises at least one substituent modification relative to a sequence selected from the group consisting of SEQ ID NOS: 1-8. The composition of claim 15 wherein the composition comprises: (a) an isolated or synthesized peptide as set forth in SEQ ID NO: 3; (b) an isolated or synthesized peptide as shown in SEQ ID NO: 1; or (c) an isolated or synthesized peptide as set forth in SEQ ID NO: 1 and SEQ ID NO: 3. A use of a composition according to claim 10 of the patent application for the manufacture of a medicament for the treatment of lung cancer. A method for detecting a lung cancer cell, comprising: (1) exposing the cancer cell to a conjugate, comprising: at least one isolated peptide synthesized or synthesized as in claim 2; and a contrast agent or a labeling agent Conjugating to at least one isolated or synthesized target peptide; (2) removing the conjugate that is not bound to the cancer cell, and detecting the isolated or synthesized target peptide that binds to the cancer cell a conjugated contrast agent or labeling agent; or (i) exposing the cancer cell to a conjugate comprising: at least one isolated peptide synthesized or synthesized as in claim 2; and at least one a phage displaying at least one peptide on its surface, the at least one peptide displayed on the phage having an amino acid sequence equivalent to the isolated or synthesized target peptide as in claim 2; Removing the conjugate that is not bound to the cancer cell and detecting the at least one phage that binds to the cancer cell. The method of claim 19, wherein the cancer cell is present in a tissue sample.