TWI447125B - Kit and method of use of targeting peptide for diagnosis and therapy of cancer - Google Patents

Description

Kits and methods for targeting peptides for diagnosis and treatment of cancer

The present invention relates to a method and kit for transporting a reagent to a cancer cell for the diagnosis or treatment of cancer.

Glycosaminoglycans (GAGs) are non-branched polysaccharides composed of repeating disaccharide units with different post-translational modifications along the length and structure. GAGs such as heparin/heparan sulfate (HS) and chondroitin sulfate (CS) are synthesized along the serine-glycine concensus motif. The core protein (such as proteoglycan) is linked and secreted on the cell membrane and in the extracellular matrix (ECM). HS is not a free GAG chain in tissue and is linked to the core proteins of six major classes of HS proteoglycans (HSPGs). These include the phospholipid myosin family (glypicans 1-6) and the transmembrane multi-ligand proteoglycan family (syndecans 1-4) that are attached to the cell surface glycosylphosphatidylinositol (GPI). ), perlecan, type XVIII collagen, human agrin (agrin), and HS co-receptor proteoglycan beta glycan, neuropilin-1, neuropilin-2, and CD -44 variant (epican). As one of the most complex biopolymers, HSPGs provide a structural framework by regulating growth factor, gradient formation and signaling activity of interleukins and morphogens, and extracellular matrix excision enzymes such as matrix metalloproteinases ( The location and activity of matrix metalloproteinases (MMPs) mediate cell-cell communication. HSPGs will also end Growth factor receptors regulate biological processes in many cell growth, development, differentiation, morphogenesis, tissue organization, media remodeling, and metastasis. Therefore, HSPGs play a key role in driving molecular networks such as developmental, inflammatory, immune responses and cancer.

The eosinophils are granules that develop in the bone marrow before they migrate to the blood. They are part of the white blood cells and the immune system and are responsible for combating multicellular parasites and infection sources in invading vertebrates. The eosinophilic cycle is eosinophilic degranulation and lysis in situ after migration to tissue. Activation of eosinophils in the tissue with degranulation produces new in situ interleukin-5 (IL-5) and extracellular release of basic eosinophilic globule proteins, including eosinophil cationic (eosinophil cationic). Protein (ECP) and eosinophil derived neurotoxin (EDN). Detection of eosinophil infiltration in tissue preparations from various tumor tissues including the chest, cervix, colon and lung. The combination of eosinophil granule protein and interleukin confirmed its anticancer activity and may mediate tumor cell apoptosis. . Eosinophils and mechanisms associated with mast cells to control allergies and asthma. ECP and EDN are also referred to as human ribonuclease 3 (RNase 3) and human ribonuclease 2 (RNase 2), respectively. ECP is mainly expressed in tissues including liver, spleen and placenta, and has multifunctional properties including ribonuclease decomposition, cytotoxicity, antibacterial, antiviral, antiparasitic and heparin binding activities. In addition, ECP is extracellularly accumulated in inflamed tissues in tissue damage caused by bronchial asthma or Crohn's disease. ECP binds to cell surface GAGs, particularly HS on bronchial epithelial cells, and enters cells by lipid-rafted dependent macropinocytosis. The structure of the ECP has been demonstrated and can be refined to a resolution of up to 1.75 Å with a folding topology of three alpha helices and five beta strands. The most interesting feature is the up to 19 surface-oriented arginine residues. The basic characteristics of the ECP very high isoelectric point (pI = 10.8), which promotes the interaction of ECP with negatively charged molecules on the cell surface.

Structurally related to heparin-related HS, a wide range of different growth factors participate in a variety of physiological and pathological processes, including nutrient metabolism, wound healing, cell signaling, morphogenesis, and cellular crosstalk. ECP contains a major heparin-binding motif, RWRCK, located in the loop 3 region between the α2 helix and the β1 strand (Fan, TC, Chang, HT, Chen, IW, Wang, HY, and Chang, MD (2007) A heparan sulfate -facilitated and raft-dependent macropinocytosis of eosinophil cationic protein, Traffic 8 , 1778-1795). Based on this core heparin-binding motif, a GAG-binding peptide (GBP) with 10 amino acids (residues 32-41 across ECP) has been shown to possess GAG recognition activity.

Epithelial cell carcinoma is a malignant tumor that originates in epithelial cells of the ectoderm and endoderm. When the epithelial cells transform from the original features and enter a process called epithelial-to-mesenchymal transition (EMT), the GAGs on the cell surface begin to be abnormal when they are transferred to the second position due to loss of cell attachment. Performance has altered the binding activity of various growth factor receptors. Currently, there is cumulative evidence that proteoglycans and GAGs alter performance in many cancer cells (Table 1). For example, syndecan-1 is overexpressed in patients with myelocarcinoma and is associated with poor prognosis. Syndecan-2 is usually overexpressed in colon cancer. Syndecan-4 is upregulated in hepatocellular carcinoma and malignant mesothelioma with concomitant tumor cell proliferation. Glypican-1 is overexpressed in breast cancer and brain cancer (glioma), while glypican-3 is overexpressed in liver cancer, lung squamous cell carcinoma, metastatic melanoma, Merck cell carcinoma, and ovarian cancer.

In the present invention, various normal and cancer cell lines were examined, and it was confirmed that the preference of GBP was combined with high metastatic adenocarcinoma, which was presumed to be caused by the difference in the degree of expression of sulfated GAG on normal and cancer cells. The present invention also demonstrates by tissue microarrays that GBP can detect lung tumor types, particularly adenocarcinoma and squamous cell carcinoma. In addition, GBP shows in vivo tumor targeting activity, implying its feasibility of targeting epithelial cell carcinoma.

Unless otherwise defined, the terms used herein have their meaning as commonly understood by those of ordinary skill in the art. As used throughout this application, the following terms shall have the following meaning: the term "GBP" means a glycosaminoglycan (GAG) combination with 10 amino acids. The peptide has an amino acid sequence of NYRWRCKNQN (SEQ ID NO: 1).

Accordingly, the present invention provides a method of transporting a reagent to a cancer cell, comprising: (a) obtaining a peptide that binds to a cancer cell, wherein the peptide comprises a cancer target having the amino acid sequence of SEQ ID NO: To a motif in which the peptide is linked or fused to an agent to be targeted to a cancer cell; and (b) to expose the peptide to a population of cells suspected of having cancer cells. In a preferred embodiment, the population of cells is in a mammalian individual. Preferably, the mammalian system is a human individual. In a preferred embodiment, the population of cells is selected from the group consisting of thin sections of tissue, thick sections of tissue, blood, and circulating tumor cells. In a preferred embodiment, the cancer is epithelial cell carcinoma. Preferably, the epithelial cell carcinoma is an adenocarcinoma or squamous cell carcinoma. More preferably, the adenocarcinoma is a highly metastatic adenocarcinoma. More preferably, the adenocarcinoma is selected from the group consisting of lung adenocarcinoma and colon adenocarcinoma. In a preferred embodiment, the method further comprises detecting cancer cells in the population of cells. In a preferred embodiment, the method further comprises diagnosing cancer. In a preferred embodiment, the agent is a therapeutic or contrast agent. Preferably, the agent and peptide are administered to an individual with or suspected of having cancer for treatment or visualization. In a preferred embodiment, the therapeutic agent is a drug, a chemotherapeutic agent, a radioisotope, an apoptotic agent, an anti-angiogenic agent, a survival factor, an anti-apoptotic agent, an enzyme, a hormone, a hormone antagonist, an interleukin, Cytotoxic agents, cytotoxic agents, cytostatic agents, growth factors, peptides, proteins, antibiotics, antibodies, Fc fragments of antibodies, nucleic acids, antigens, viruses, bacteriophages, bacteria, liposomes, microparticles, magnetic beads, microelements , yeast cells, mammalian cells, cells or expression vectors. Preferably, the agent is an anti-angiogenic agent selected from the group consisting of thrombospondin, angiostatin 5, pigment epithelium-derived factor, angiotensin, laminin peptides, Fibronectin wins Fibronectin peptides, plasminogen activator inhibitors, tissue inhibitors of metalloproteinases, interferon, interleukin 12, platelet factor 4, IP-10, Gro-B, thrombus Thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM 101, Marimastat, pentose polysulfate Pentosan polysuiphate), angiopoietin 2 (Regeneron), alpha-interferon, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, Thalidomide, Pentoxifylline, genistein (genistein), TNP-470, endostatin, paclitaxel, docetaxel, polyamines, proteasome inhibitors, kinase inhibitors, message peptides, Accutin, cidofovir, vincristine, A group consisting of bleomycin, AGM-1470, platelet factor 4, and minocycline. In another embodiment, the reagent is an interleukin selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18. , a group consisting of interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-γ) or granule macrophage colony-stimulating factor (GM-CSF). In a preferred embodiment, the contrast agent is a tracking substance selected from the group consisting of fluorescent labels, chemiluminescent proteins, radioisotopes, and magnetic nanoparticles. Preferably, the contrast agent is a radioisotope selected from the group consisting of 砈-211, carbon-14, chrome-51, chloro-36, cobalt-57, cobalt-58, copper-67, Eu-152, gallium-67, Hydrogen-3, iodine-123, iodine-125, iodine-131, indium-111, iron-59, phosphorus-32, 铼-186, 铼-188, selenium-75, sulfur-35, 鍀-99m and 钇- A group of 90. In a more preferred embodiment, the peptide which binds to cancer cells is SEQ ID NO: 1.

The invention also provides a kit for performing the above method, comprising a combination of cancer A peptide of a diseased cell, wherein the peptide comprises a cancer targeting motif having the amino acid sequence of SEQ ID NO: 1. Preferably, the peptide is SEQ ID NO: 1.

The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the various aspects and features of the present invention.

Example 1: Materials and methods Fluorescent assisted sugar electrophoresis (FACE)

Based on previous studies (Calabro, A., Benavides, M., Tammi, M., Hascall, VC, and Midura, RJ (2000) Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE ), Glycobiology 10 , 273-281), the sugar was labeled with 2-aminoacridone (AMAC) (Invitrogen, Cat. No. A6289). Briefly, fifty micrograms of GAGs were dried in a freeze dryer followed by incubation with 40 μl of 1.25 M 2-aminoacridone (AMAC) / 85% DMSO / 15% acetic acid for 15 minutes at 25 °C. Forty microliters of 1.25 M sodium cyanoborohydride (NaBH ₃ CN) was added and incubated at 37 ° C for 16 hours. Thereafter, 720 μl of 99% ice-cold ethanol was added at -20 ° C over a period of 15 minutes, followed by centrifugation at 11,000 x g for 5 minutes at 4 °C. The supernatant was carefully removed with a centrifugal vacuum concentrator (Spin Vacuum) and a freezer (coolsafeTM) and lyophilized. The dried precipitate is re-dissolved in an appropriate volume (often 20 μL or 50 μL) in sterile deionized water depending on the strength of the labeled probe. Store prepared AMAC labeled probes at -80 °C and avoid light.

The AMAC labeled probe and protein were mixed and incubated for 15 minutes at 25 °C. The complex was then loaded into a 1% acacia gel and electrophoresed in pH 8.0 buffer containing 40 mM triacetic acid, 1 mM EDTA for 20 to 30 minutes. This test was performed in dark or red light to avoid light. The AMAC labeled probe was observed under UV light and scanned with a transilluminator (ONLY Science Co., Ltd.).

Cell culture

Table 2 is a list of cell lines. The cells were cultured in medium supplemented with heat-inactivated 10% (v/v) fetal calf serum (FBS) (Gibco, Invitrogen, USA) and 1% (v/v) glutamate-penicillin-biosera. (Gibco, Invitrogen, USA). The cells were grown on a 100-mm disk and incubated at 37 ° C in 5% CO ₂ .

Flow cytometry

The cells were plated in a 6-well plate (2.0 x 10 ⁵ cells/well) and cultured in medium. After 24 hours, the cells were incubated with the fluorescently labeled peptide dissolved in the medium for 1 hour. The cells were collected, washed and suspended in PBS. The treated cells were subjected to fluorescence analysis on a FACScalibur flow cytometer (FACS, BD Biosciences, Franklin Lakes, NJ) with an excitation wavelength of 488 nm and collected emission waves between 515 and 545 nm.

Immunohistochemical staining

A 5 μm slide was dried in a 60 ° C oven for 1 hour. Briefly, sections were deparaffinized with xylene (J.T. Baker Phillipsburg, N) for 15 minutes and rehydrated in a gradient ethanol solution. The sections were cultured in a solution of 3% hydrogen peroxide in distilled water for 10 minutes to block the activity of endogenous peroxidase. The sections were then washed and placed in phosphate buffer for 10 minutes. Antigen retrieval was achieved by heat treatment with 10 mM pH 6.0 citrate buffer solution or 0.1% trypsin treatment for 5 minutes (for 10E4). The slides were blocked in 3% BSA solution for 1 hour and at 2 μM After the eGFP-GBP fusion protein was cultured for 2 hours (for eGFP), the sections were cultured overnight at 10 °C with 10E4 antibody (diluted 1:200) and eGFP antibody (diluted 1:200). These sections were incubated in super enhancer buffer for 20 minutes and incubated in a second antibody broad HRP conjugate broad spectrum for 30 minutes at room temperature, followed by phosphate buffer. Wash three times. The cells were stained with 3,3'-diaminobenzidine (DAB, 0.2 mg/ml, Pierce, Rockford, IL, USA) for 3 minutes, followed by contrast staining with Mayer's hematoxylin for 1 minute. Each slide was then dehydrated by soaking in 95% alcohol and 100% alcohol, then soaked in xylene for 15 minutes and covered with a coverslip.

Immunohistochemical staining score

According to the intensity of nuclear or cytoplasmic staining (no staining = 0; weak staining = 1; moderate staining = 2; strong staining = 3) and staining cell range (0-10% = 0; 11-50% = 1; 51-80) %=2; 81-100%=3) Give each tumor a score. The positive fraction of the stained cell intensity and range is multiplied to obtain the final immune response score, with a minimum score of 0 and a maximum score of 9.

Animal mode

Adult female Balb/c mice were purchased from the National Institute of Animals in Taiwan and raised in the same place. Mouse colon cancer CT-26 cell line was purchased from ATCC (Rockville, Maryland). The cells were cultured in a medium containing 10% heat-inactivated fetal bovine serum at 37 ° C in a humidified atmosphere containing 5% Co ₂ . Briefly, four to six week old Balb/c mice were injected subcutaneously with 5 x 10 ⁵ CT-26 cells (100 μl in volume). When the tumor volume grew to approximately (8-9) ± 1 mm ³ , the mice were divided into two groups and 5 nmol enhanced green fluorescent protein (eGFP) or eGFP-GBP was injected intravenously through the tail. One hour after the injection, all animals were killed by CO ₂ asphyxiation. The lung, trachea, kidney, small intestine and tumor tissue were removed and immediately fixed in 10% neutral buffered formaldehyde. Tissue samples were processed by standard methods to prepare paraffin-embedded bulk samples (Fan, TC, Fang, SL, Hwang, CS, Hsu, CY, Lu, XA, Hung, SC, Lin, SC, and Chang, MD (2008) Characterization of molecular interactions between eosinophil cationic protein and heparin, The Journal of biological chemistry 283 , 25468-25474). The sample block was sliced into 6 μm slices and examined with a hypersensitive non-Biotin HRP detection system (BioGenex Laboratories, San Ramon, CA) as previously described (Fan, TC, Fang, SL, Hwang, CS, Hsu, CY, Lu, XA, Hung, SC, Lin, SC, and Chang, MD (2008) Characterization of molecular interactions between eosinophil cationic protein and heparin, The Journal of biological chemistry 283 , 25468-25474). At the same time, the sheets were also visualized with an optical microscope (Zeiss-Axioplan, Germany).

MRI analysis

When the tumor volume grew to approximately (8-9) ± 1 mm ³ , each mouse was intravenously injected (iv) 150 μl MNP-GBP (0.06 emu/g), followed by MRI reconnaissance scan to accurately obtain the mouse internal tumor. s position. The magnetic nanoparticles were Fe ₃ O ₄ nanoparticles (GABC Co.) coated with dextran, and had an average diameter of 52 nm. The magnetic fluid containing magnetic nanoparticles has a saturation magnetization of 0.06 eum/g. The peptide-magnetic nanoparticles uniformly dispersed in PBS were intravenously injected into the mouse from the tail. The volume of magnetic fluid injected into each mouse was 150 μl. After the injection, the position of the MNP was detected by magnetic resonance imaging (MRI). MRI was performed on the 7-Tesla system before and 1 day after injection. Mice were anesthetized with 0.5 ml ketamine and 0.5 ml rompun for MR scanning. Multiple transverse images were obtained using a fat-saturated 3D gradient echo pulse sequence (Turbo FLASH), TR=5.17 ms; TE=2.49 ms; flip angle=10°; FOV = 256 mm; sheet thickness = 2 mm; matrix = 256 x 256; pixel size = 1 x 1 x 2 mm; average = 10. In vivo MR images were cross-sections of the control group rats and the injected group across the abdominal aorta.

Statistical Analysis

All data are shown as mean ± standard deviation (SD) and n is the number of experiments performed. Statistical analysis was performed using GraphPad Prism v 4.02 (GraphPad Software, USA) with an unpaired Student's t test to compare the two means. One-way variation analysis (ANOVA) was used to test for differences between multiple treatments, followed by Dunnett's test. A P value of less than 0.05 is considered to be statistically significant.

result GBP tumor cell binding activity

GBP detects general glycosaminoglycans, including low molecular weight heparin, chondroitin sulfate, and dermatan sulfate (Figure 1A). In addition, GBP will recognize sulfated HS, especially O -sulfated heparin (Figure 1B). The most prevalent structure in heparin is the trisulfated disaccharide at the 2- O position of IdoA and the 2- N , 6- O position of GlcNAc. Monoclonal anti-acetyl heparin sulfate (10E4) antibody detects the N HS - glucosamine sulfate (N -sulfated glucosamine) epitope (epitope). Therefore, signals of 10E4 and FITC-GBP were detected by flow cytometry in various normal and cancer cell lines to quantitatively determine the correlation between GBP binding and HSPGs expression (Table 2). As shown in Table 3, GBP showed high binding amount to cancer cell lines, such as human lung adenocarcinoma H460, lung adenocarcinoma CL1-3, lung large cell carcinoma PC9, human AGS gastric adenocarcinoma, human HCT-116 colon rectum Cancer cells, human HepG2 hepatocyte cancer cells, and mouse CT-26 colon adenocarcinoma cells. In addition, GBP will also bind to CL3, A549, H157, CL1-5, Caco-2 and HT-29. Compared with the amount of binding of GBP in Beas-2B cells, GBP binds much more to cancer cell lines (Table 3). In addition, GBP showed a higher degree of binding in highly metastatic tumors of lung adenocarcinoma and colon adenocarcinoma than lower metastatic tumors, indicating that GBP prefers to connect to selective cancer cell surfaces and abnormally increases when tumors expand. The performance of GAGs. Although the expression of N -sulfate glucosamine on HS in normal lung Beas-2B cells and MRC-5 cells was higher than that in lung cancer cell lines, GBP recognized that all lung cancer cell lines were more prominent than the general lung cell lines. Combining the above, these observations indicate that GBP binds significantly to highly metastatic epithelial cancer.

Immunohistochemical screening of arrays of various cancerous tissues

Use of 37 cases of cancer from 16 different tissues (esophagus, stomach, colon, rectum, liver, lung, kidney, breast, cervix, ovary, bladder, lymph nodes, skin, brain, prostate, pancreas) Multiple organ cancer microarray specimens (BCN9636, US Biomax, Rockville, USA) were screened by immunohistochemistry (IHC). Single-antibody HS antibodies and eGFP-GBP were used to detect signals in multiple cancer tissue arrays. Table 4 summarizes the clinicopathological features of these cases.

Table 4 Tissue diagnosis of human cancer tissues

In adenocarcinomas, HS molecules are highly expressed in lung tissue (Fig. 2A) and in the colon (Fig. 2B), stomach (Fig. 2C), pancreas (Fig. 2D), prostate (Fig. 2E), and rectum (Fig. 2F). There are fewer performances. Similarly, the eGFP-GBP signal was also significantly observed in lung tissue (Fig. 3A), but in the colon (Fig. 3B), stomach (Fig. 3C), pancreas (Fig. 3D), prostate (Fig. 3E), and rectum. (Figure 3F) is rare. As for the negative control group, the anti-eGFP antibody showed no signal (Fig. 3G). In addition, HS molecules are highly expressed in lung squamous cell carcinoma tissues (Fig. 4A), as in adenocarcinoma lung tissues (Fig. 2A), but squamous from tissues of the esophagus (Fig. 4B) and skin (Fig. 4C). Cellular cancer shows only a small amount of HS. Similarly, the eGFP-GBP signal was clearly detected in lung cells (Fig. 5A), but the immunolocalization of tissues in the esophagus (Fig. 5B) and skin (Fig. 5C) was weak. Among other cancers, only hepatocytes Cancer showed weak HS expression (Fig. 6A) and eGFP-GBP signal (Fig. 6B), but stellate cell tumor, B cell lymphoma, clear cell carcinoma, invasive ductal carcinoma, transitional cell carcinoma, and serous adenocarcinoma None of the signals are shown. Pathological proliferation in tissues or organs is accompanied by an increase in the number of cells, which may be an indication of an abnormal or precancerous lesion. Surprisingly, both HS performance (Figures 7A-7C) and eGFP-GBP binding strength (Figures 7D-7F) increased significantly with cancer progression, such as normal tissue (Figures 7A and 7D), proliferating normal tissues. (Figures 7B and 7E) and IHC images of adenocarcinoma (Figures 7C and 7F) are shown. In summary, HS performance and GBP-targeted staining intensity are clearly stronger in adenocarcinoma and squamous cell carcinoma of lung tissue than in other cancers, which provides an important clue that GBP can be used as A new detection tool to identify early to mid-stage lung cancer.

Performance pattern of glycosaminoglycans in lung cancer

IHC data were quantified using the specimen scores of 61 lung cancers in the tissue array. In these 61 cases, the samples included normal lung tissue, adenocarcinoma, squamous cell carcinoma, bronchioloalveolar carcinoma, and large cell carcinoma (Table 5). In tumor or non-tumor areas, membrane-associated glycosaminoglycans such as HS and CS have varying degrees of performance. HS and CS did not appear on the surface epithelium of normal lung tissue, as did the fibroblasts in the lamina propria (Figs. 8A and 9A).

Table 5 Histological diagnosis of human lung tissue from 61 tests

Examples of adenocarcinoma (Figs. 8B & 8C and 9B & 9C) and squamous cell carcinoma (Figs. 8D & 8E and 9D & 9E) show strong staining of HS and CS expression in the extracellular membrane. In addition, compared to early cancer, GBP confirmed a higher degree of discrimination for more aggressive tumor progression (Figures 8C & 8E and 9C & 9E). Bronchioloalveolar carcinoma showed slight staining of HS and CS on the membrane (Figures 8F and 9F). Large cell carcinoma showed high HS expression on the membrane (Fig. 8G), but CS was weak (Fig. 9G). The staining score of lung cancer confirmed the IHC results of HS and CS in different types of lung cancer performance patterns (Table 6): (1) normal lung tissue staining was 100% negative (4/4); (2) adenocarcinoma was 27.3% negative ( 6/22), 59.1% weakly or moderately positive (13/22) and 13.6% strong positive (3/22); (3) Squamous cell carcinoma staining is 40% weakly positive or moderately positive (6/ 15) and 60% strong positive (9/15); (4) 20% negative (2/10), 80% weak or moderate positive (8/10); (5) large Cellulose staining was 90% weakly positive or moderately positive (9/10) and 10% strongly positive (1/10). From these results, it can be inferred that HS and CS have relatively high expression in adenocarcinoma and squamous cell carcinoma.

GBP targeting lung cancer

GBP has previously shown a high degree of binding to lung cancer in vitro. The molecular probe capability of eGFP-GBP was also assessed by an array of lung cancer tissues. Vascular endothelial cells and fibroblasts in normal lung tissue can detect weak eGFP-GBP signals (Fig. 10A). Similarly, the targeting signal of eGFP-GBP in adenocarcinoma (Fig. 10B) and squamous cell carcinoma (Fig. 10C) showed very strong staining intensity. Furthermore, the targeting intensity of eGFP-GBP was also shown to be strong in bronchioloalveolar carcinoma (Fig. 10D) and large cell carcinoma (Fig. 10E). The IHC results for eGFP-GBP targeting intensity in different types of lung cancer are summarized in Table 6: (1) Normal lung tissue staining is 50% negative (2/4) and 50% weak positive (2/4); (2) The staining of adenocarcinoma was 13.6% negative (3/22), 54.5% weakly positive or moderately positive (12/22) and 31.9% strong positive (7/22); (3) staining of squamous cell carcinoma was 20% Negative (3/15), 46.7% weakly positive or moderately positive (7/15) and 33.3% strong positive (5/15); (4) Bronchoalveolar alveolar carcinoma staining was 40% negative (4/10), 40% weak positive or moderate positive (4/10) and 20% Strong positive (2/10); (5) Staining of large cell carcinoma is 90% weakly positive or moderately positive (9/10) and 10% strong positive (1/10). Analysis of these tissue microarrays indicated that the targeting intensity of eGFP-GBP was again confirmed to be similar to that of HS and CS in all lung cancer cases, suggesting that eGFP-GBP preferentially targets adenocarcinoma and squamous Cellular cancer.

Probe activity of GBP in tumor animal model

The mouse colon adenocarcinoma cell line CT-26 showed high HSPG expression and high GBP binding activity (Table 3). Therefore, CT-26 tumor-bearing mice were selected as the in vivo GBP targeting mode. One hour after injection of recombinant eGFP-GBP and eGFP protein into CT-26 tumor-bearing mice, the mice were sacrificed and dissected for tissue dissection. The target activity of eGFP-GBP in vivo was verified by staining tissue sections with anti-eGFP antibody as IHC. The eGFP-GBP signal was only weakly detected in the bronchial epithelium (Fig. 11A), small intestine villi (Fig. 11C), kidney (Fig. 11E), and liver (Fig. 11G), but was strong in cancerous tissues (Fig. 11I). . Both the kidney and the liver are considered to be the main excretory organs of mammals, which excrete most of the waste and toxins. Therefore, most of the foreign protein is detected in the kidney (Fig. 11E) and the liver (Fig. 11G). Surprisingly, a significant high amount of eGFP-GBP signal was detected in CT-26 cancer tissue (Fig. 11I). For the negative control group (injected eGFP), the eGFP signal detected by the eGFP antibody was not observed in the bronchial epithelium (Fig. 11B), the small intestine (Fig. 11D) or the CT-26 tumor tissue (Fig. 11J). However, a clear eGFP signal can be detected in the excretory organ kidney (Fig. 11F) and the liver (Fig. 11H) due to the rapid transport of the circulatory system. These data indicate that GBP can also highly discriminate adenocarcinoma cells in mouse mode.

Tumor targeting of MNP-GBP

Magnetic Nanoparticles (MNP) are used in nuclear magnetic resonance (MR) imaging analysis, which provides a non-invasive, in-vivo monitor in animal models. T2-weighted contrast MR images (also known as relaxation-enhanced proton-density-weighted MR images) show proton signals, especially proton signals on water. Several organs such as kidney, liver and tumor tissues are rich in water and are easily imaged by a T2-weighted MRI system. When MNP (Fe ₃ O ₄ ) coated with dextran (52 nm in diameter) was bound to the above-mentioned tissues, a decrease in signal intensity was observed.

FITC-GBP (Table 3) and eGFP-GBP fusion protein (Fig. 11I) recognize CT-26, indicating that MNP-GBP may also recognize cancer cells in vivo. To test the targeting ability of MNP in the CT26 mouse model, 12-week-old female Balb/c mice implanted with CT-26 tumors were injected intravenously with 150 μl MNP (0.06 emu/g). Tumor mice were imaged at kidney and tumor location at 0, 0.5, and 21 hours after MNP injection, and ddH ₂ O was used as positive control to normalize light density (Figures 12A, 12B, and 12C, circles indicated by yellow arrows) Shape signal). The MRI contrast intensity of the excretory organ kidneys was enhanced 0.5 h after the injection of MNP (Fig. 12B) but returned to the original condition 21 hours after the injection (Fig. 12C). However, the MRI contrast intensity of the tumors did not increase at 0.5 and 21 hours after injection of MNP (Fig. 12E and Fig. 12F), indicating that MNP alone was not targeted to tumor tissues. The MRI contrast intensities of kidneys and tumors were also quantified in Figures 12G and 12H, respectively.

The use of GBP conjugated with MNP is used as a targeting agent for tumor imaging detection on MRI systems. To monitor MNP-GBP targeting in colon cancer-bearing mice, 12-week-old female Balb/c mice implanted with CT-26 tumors were injected intravenously with 150 μl MNP-GBP (0.06 emu/g). And collect MRI comparison data within 24 hours. First detect before injection The MRI signal intensity of excretory organ kidney and CT-26 solid tumor location was individually set at 100% (Figures 13A and 13D). After MNP-GBP injection, the MRI comparison of the kidneys lost 55% in 0.5 hours (Fig. 13B) and returned to 100% at 24 hours (Fig. 13C). No difference was observed in the signal intensity at 0.5 hour after MNP-GBP injection (Fig. 13E). However, it was apparent that MNP-GBP was bound to the tumor 24 hours after the injection, and the MRI contrast intensity of the CT-26 tumor tissue was about 30% lower than that before the injection (Fig. 13F). The MRI contrast intensities of kidneys and tumors were quantified in Figures 13G and 13H, respectively. Tumor histological sections were stained with Prussian blue to detect iron nanoparticles in tumors 24 hours after injection. As expected, the MNP-GBP signal was not detected in normal tissues, but the Prussian blue signal showed that MNP-GBP recognized the edge of tumor tissue 24 hours after injection (Fig. 11I). Importantly, tumor histological sections showed that MNP-GBP was selectively targeted to CT-26 tumor tissue at high magnification. These results indicate that GBP will direct MNP localization to tumor cells, strongly supporting the hypothesis that GBP not only binds to cancer cells in vitro but also targets tumor cells in vivo.

A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. The kits of the present invention and their manufacturing procedures and methods are representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

The present invention has been described in detail with reference to the preferred embodiments of the invention, It should be understood that the spirit and scope of the invention are not to be

All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described, but Various changes are possible within the scope of the patent application of the invention. Therefore, it is to be understood that the present invention has been disclosed and described herein in accordance with the preferred embodiments and the features of the present invention. Within the scope.

Figure 1 shows an electropherogram of the indicated concentration of GBP bound to various glycosaminoglycans (GAGs). (A) AMAC-labeled LMWH, chondroitin sulfate (CS) and dermatan sulfate (DS) and (B) AMAC-labeled LMWH, de -2- O -sulfated heparin, de -6- O -sulfated heparin And N -acetyl- de - O -sulfated heparin (0.33 nmol) was pre-mixed with the indicated multiple molar excess of GBP, followed by incubation with the peptide (or no peptide) in PBS at 25 °C for 15 minutes. Perform fluorescent assisted sugar electrophoresis (FACE). The reaction product was separated on a 1% acacia gel. The reaction probe and protein are displayed above the gel. The relative intensity (%) of the free probe to the unlabeled competitor is shown below the gel.

Figure 2 shows the performance of heparin sulfate (HS) in various cancer tissues of adenocarcinoma. Immunohistochemistry (IHC) localization of HS was performed with an anti-HS antibody by Supersensitive non-biotin HRP detection system. A representative IHC staining pattern (brown) of HS was tested in lung (A), colon (B), stomach (C), pancreas (D), prostate (E), and rectum (F). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E and F are both 400×)

Figure 3 shows the targeted results of eGFP-GBP in various cancer tissues of adenocarcinoma. IHC localization of eGFP-GBP was performed with an anti-eGFP antibody by an ultrasensitive non-biotin HRP detection system. Representative IHC staining patterns (brown) of eGFP-GBP were detected in lung (A), colon (B), stomach (C), pancreas (D), prostate (E), and rectum (F). The anti-eGFP anti-system that was not cultured in eGFP-GBP was detected in lung cancer tissues as a negative reaction (G). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E, F, and G are both 400×)

Figure 4 shows the performance of HS in various cancerous tissues of squamous cell carcinoma. IHC localization of HS was performed with an anti-HS antibody by an ultrasensitive non-biotin HRP detection system. A representative IHC staining profile (brown) of HS was tested in lung (A), esophagus (B) and skin (C). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B and C are both 400×)

Figure 5 shows the targeting results of eGFP-GBP in various cancer tissues of squamous cell carcinoma. IHC of eGFP-GBP with anti-eGFP antibody by ultrasensitive non-Biotin HRP detection system Bit. A representative IHC staining map (brown) of eGFP-GBP was detected in lung (A), esophagus (B) and skin (C). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B and C are both 400×)

Figure 6 shows the performance of HS in liver cancer tissues of hepatocellular carcinoma and the targeted results of eGFP-GBP. IHC localization of HS and eGFP-GBP was performed by an ultrasensitive non-biotin HRP detection system. A representative IHC staining map (brown) of HS (A) and eGFP-GBP (B) was detected in the liver. The nuclei were stained blue with hematoxylin contrast staining. (magnification: A and B are both 400×)

Figure 7 shows the immune response of HS expression and eGFP-GBP binding in cancer progression. Detection of HS produced by anti-HS antibody and anti-eGFP antibody in normal lung tissues (A and D), adjacent normal lung tissues (stromal hyperplasia) (B and E), and lung adenocarcinoma (C and F) IHC staining map (dark brown) (A to C) and GBP IHC staining map (dark brown) (D to F). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E and F are both 400×)

Figure 8 shows the performance of HS in tumor lung tissue. IHC localization of HS was performed with an anti-HS antibody by an ultrasensitive non-biotin HRP detection system. In normal lung tissue (A), low-graded adenocarcinoma (B), high-grade adenocarcinoma (C), low-grade squamous cell carcinoma (D), high malignancy HS-represented IHC staining (brown) was detected in squamous cell carcinoma (E), bronchioloalveolar carcinoma (F), and large cell carcinoma (G). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E, F, and G For 400×)

Figure 9 shows the performance of CS in tumor lung tissue. IHC localization of CS was performed with an anti-CS antibody by an ultrasensitive non-biotin HRP detection system. In normal lung tissue (A), low-grade adenocarcinoma (B), high-grade adenocarcinoma (C), low-grade squamous cell carcinoma (D), high-grade squamous cell carcinoma (E), bronchiole A representative IHC staining map (brown) of CS was detected in alveolar carcinoma (F) and large cell carcinoma (G). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E, F, and G are both 400×)

Figure 10 shows the targeted results of eGFP-GBP in tumor lung tissue. IHC localization of eGFP-GBP was performed with an anti-eGFP antibody by an ultrasensitive non-biotin HRP detection system. In normal lung tissue (A), low-grade adenocarcinoma (B), high-grade adenocarcinoma (C), low-grade squamous cell carcinoma (D), high-grade squamous cell carcinoma (E), bronchiole A representative IHC staining map (brown) of eGFP-GBP was detected in alveolar carcinoma (F) and large cell carcinoma (G). The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E, F, and G are both 400×)

Figure 11 shows the targeted results of eGFP-GBP in a mouse cancer model. IHC localization of GBP was performed with an anti-eGFP antibody by an ultrasensitive non-biotin HRP detection system. One hour after intravenous (iv) injection, a representative IHC staining pattern of eGFP-GBP was detected in bronchial epithelial (A), small intestine villi (C), liver (E), kidney (G), and cancer (I) tissues ( brown). Simultaneous treatment of lung (B), small intestine (D), liver (F), kidney (H) and cancer (J) tissue injection A slice of eGFP was used as a control group. The nuclei were stained blue with hematoxylin contrast staining. (magnification: A, B, C, D, E, F, G, H, I, J are 400 ×)

Figure 12 shows the distribution of magnetic nanoparticles (MNP) in CT-26 tumor mice. Magnetic resonance imaging (MRI) axis images (yellow arrows) of kidney (AC) and CT-26 tumors (DF) were obtained after intravenous injection of 150 μl MNP (0.06 emu/g) for 30 minutes and 21 hours. ddH ₂ O is used as a positive control group (white sphere) that normalizes the light density. MRI signal quantification represented by MNPs targeted to kidney (G) and CT-26 tumor (H) after 30 minutes and 21 hours of intravenous injection.

Figure 13 shows the results of targeting MNP-GBP to CT-26 tumors in vivo. MRI images of mouse kidney (A, yellow arrow) and tumor (D, yellow arrow) were monitored before intravenous injection of 150 μl MNP-GBP (0.06 emu/g). MRI images of mouse kidney (B, white arrow) and tumor (E, white arrow) were obtained 0.5 hours after injection. Similarly, mouse kidney (C, white arrow) and tumor (F, white arrow) images were obtained 24 hours after injection. ddH ₂ O is used as a positive control group (white sphere) that normalizes the light density. MRI comparison of renal tissue (G) and tumor (H) at 24 hours after MNP-GBP injection. (I) Photomicrograph of tumor tissue at 24 hours after MNP-GBP injection, the magnification was 400×, and Pristine blue staining was used to detect the accumulation of divalent iron (black dots) by MNP-GBP.

<110> National Tsinghua University

<120> Sets and methods for targeting peptides for the diagnosis and treatment of cancer

<130> 1767-NTHU-TW

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Human (Homo sapiens)

<220>

<221> PEPTIDE

<222> (1)..(10)

<400> 1

Claims (5)

A use of a peptide for the preparation of a composition for the specific transport of an agent to an adenocarcinoma or squamous cell carcinoma cell, wherein the peptide comprises a cancer targeting motif having an amino acid sequence of sequence 1 and The agent to be targeted to the cancer cell is ligated or fused. The use of claim 1, wherein the agent is a therapeutic agent or a contrast agent. The use according to claim 2, wherein the therapeutic agent is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptotic agent, an anti-angiogenic agent, a survival factor, an anti-apoptotic agent, an enzyme, a hormone, a hormone antagonist. , interleukins, cytotoxic agents, cytocidal agents, cytostatic agents, growth factors, peptides, proteins, antibiotics, antibodies, Fab fragments of antibodies, nucleic acids, antigens, viruses, bacteriophages, bacteria, liposomes, microparticles, Magnetic beads, microelements, yeast cells, mammalian cells, cells or expression vectors. The use of claim 2, wherein the contrast agent is a tracking substance selected from the group consisting of fluorescent labels, chemiluminescent proteins, radioisotopes, and magnetic nanoparticles. The use of claim 1, wherein the peptide is sequence 1.