In Vivo Detection of Colon Tumorigenesis Using a Near-Infrared Peptide Targeting Overexpressed cMET

Abstract

The present disclosure provides peptides that selectively bind to the cMet protein, including labeled peptides. The present disclosure also provides methods for detecting dysplastic cells and tissues, such as in the colon, and for providing early identification of pre-cancerous and cancerous tissues.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application 62 / 808,637, filed on February 21, 2019, the entire content of which is incorporated herein by reference.

[0003] Statement of Government Interests

[0004] This invention was made with government support under Grant No. CA193377 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] Incorporation by Reference of Electronically Submitted Materials

[0006] The sequence listing, which is part of the present disclosure, is submitted simultaneously with the specification as a text file. The name of the text file containing the sequence listing is "53791A_Seqlisting.txt", which was created on February 13, 2020 and is 828 bytes in size. The subject matter of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0007] The present disclosure relates to peptide reagents, methods of using the peptide reagents to detect pre - cancer (dysplasia) or cancer of the colon, and methods of using the peptide reagents to target dysplastic or cancerous colon cells. Background Art

[0008] Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer - related death globally. It accounts for approximately 8% of all cancer deaths, with an estimated 693,900 deaths in 2012. 1-4 . Most patients present with advanced CRC when they exhibit obvious syndromic obstruction or intestinal bleeding, which is consistent with the high mortality and poor survival rates of CRC patients. 5,6 . Early detection of CRC, when it is still small and has not spread, is crucial for reducing mortality. Currently, standard white - light endoscopy, which is widely used for CRC screening, often misses pre - cancerous dysplasia, which is small, flat, and patchy. 7,8 . The miss rates for polyps and adenomas are as high as 28% and 20% respectively. 9 . In addition, some invisible flat lesions may be more invasive than visible polyps and are more likely to contain cancer than polyps. 10 . The miss rate for small, flat lesions is as high as 25%. 11。Effectively detecting invisible polyps and flat lesions is a current issue in early cancer detection. In addition, interval cancers occur when CRC develops within 5 years after colonoscopy, and the incidence is increasing (Farrar et al., Clin. Gastroenterol. Hepatol. 4:1259-1264 (2006), Sanduleanu et al., Nat. Rev. Gastroenterol. Hepatol. 9:550-554 (2012), Robertson, Gut 63:949-956 (2014)).

[0009] Advanced imaging methods are being developed to improve the performance of early CRC detection. Pigmented endoscopy uses locally applied vital dyes, and narrow-band imaging (NBI) uses filtered light in different spectral bands to highlight mucosal changes suspicious for disease (Stoffel, Cancer Prev Res 1:507-513 (2008), Kaminski, Endoscopy 46:435-449 (2014)). In these methods, contrast is generated by non-specific mechanisms that are not related to the biological processes driving CRC progression, and they have shown limited effectiveness in clinical studies.

[0010] Preclinical mouse models of disease provide important tools for studying disease development mechanisms. It has been determined that mutations in the adenomatous polyposis coli (APC) gene may be a key event in the development of most adenomas and CRCs. Previously reported genetically engineered mouse models that mimic human APC gene mutations mainly form adenomas in the small intestine (e.g., the APCMin model 56 , rather than in the distal colon, and it is difficult to image polyps and their in vivo progression using currently available small animal endoscopy tools. Hinoi et al. 57 described a genetically engineered mouse (called the CPC:Apc mouse) in which somatic mutations in the Apc allele result in a truncated Apc protein and lead to the formation of adenomas in the distal colon as early as 10 weeks. Others have developed mouse models that grow tumors in the distal colon using cancer cell implantation 58 or adenovirus activation of mutations 59 and reported the binding of cathepsin B smart probes, but surgical intervention is required to generate polyps, and the subsequent response to the injury may lead to a change in the target.

[0011] For decades, colonoscopy has been widely accepted by doctors as a procedure that helps detect and remove polyps. However, traditional white light (WL) endoscopy has difficulty visualizing small and flat colorectal lesions. To improve the ability of endoscopy to detect and characterize colorectal lesions, various endoscopic imaging techniques have been developed. The combination of molecular-targeted fluorescent reagents and wide-field techniques has shown encouraging results in detecting pre-cancerous lesions of CRC. Studies have identified several clinical and pathological biomarkers associated with CRC prognosis. As a tyrosine kinase receptor, cMet (mesenchymal-epithelial transition factor) is activated by hepatocyte growth factor and regulates multiple biological processes such as cell proliferation, diffusion, and survival.

[0012] As a member of the proto-oncogenic transmembrane tyrosine kinases, cMet is widely expressed in epithelial and endothelial cells 14 . cMet is expressed on the surface of normal epithelial cells in the digestive tract and is highly overexpressed in CRC. The 190kD cMet heterodimer consists of two subunits linked by disulfide bonds, including an extracellular 50kD α-chain and a transmembrane 145kD β-chain. The binding of cMet to its ligand, namely hepatocyte growth factor (HGF), triggers cMet dimerization and autophosphorylation, which in turn phosphorylates and activates downstream mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and signal transducer and activator of transcription (STAT) signaling pathways 15 . This pathway plays an important role in tumor growth, invasion, angiogenesis, and metastasis 16 . cMet is crucial for cell proliferation, mitosis, morphogenesis, and angiogenesis 16 . Elevated cMet levels have been detected in many cancers, such as colorectal cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, and breast cancer, as well as many sarcomas 17-19 , making cMet an important target for anti-tumor therapy.

[0013] Endoscopic imaging using exogenous fluorescent-labeled probes can produce imaging results that provide precise localization of tumor lesions, and the fluorescence provides improved contrast. To date, diagnostic molecules that have been used as targeting agents include antibodies and antibody fragments to detect pre-cancerous and malignant lesions of various types of cancer. However, the use of antibodies and antibody fragments is limited by immunogenicity, production cost, and long plasma half-life. Small molecules, RNA aptamers, and activatable probes have also been used. Peptides represent a newer class of imaging agents that are compatible with clinical applications in the digestive tract, especially for local administration. Some monoclonal antibodies have been developed as fluorescent imaging agents targeting cMet 22,23 . As mentioned above, such agents have a relatively high molecular weight and large size, which limit their ability to penetrate tissues, increase their immunogenicity, and shorten their circulating half-life.

[0014] Phage display is a powerful combinatorial technique for peptide discovery that uses recombinant DNA technology methods to generate complex peptide libraries, typically expressing up to 10 7 -10 9 unique sequences that can bind to cell surface antigens. The DNA of candidate phages can be recovered and sequenced to elucidate the positive binding peptides that can then be synthesized and manufactured. Phage display using the commercially available NEB M13 phage system has identified highly dysplastic peptide conjugates in Barrett's esophagus 60 and human colorectal dysplasia 61 . The T7 system has proven effective in in vivo screening experiments to identify peptides unique to the islet vascular system 62 , the mammary vascular system 63 , bladder tumor cells 64 and liver tissue 65 . Screening using intact tissues provides additional relevant cellular targets while taking into account subtle features in the tissue microenvironment that may affect binding.

[0015] Recent advances in in vitro organoid culture techniques have opened a new window for the development of new models for human cancer research. Lgr5+ stem cell-derived'mini-gut' organoids phenocopy the epithelium of intestinal crypts and villi 30 , indicating that organoids can exhibit self-organization capabilities and phenocopy the basic aspects of the organ from which they are derived. Organoids can be derived from healthy and tumor tissues, and advances in efficient organoid orthotopic xenotransplantation methods 31 have made it possible to develop more physiologically relevant preclinical cancer models for early cancer detection.

[0016] Accordingly, there remains a need in the art for improved CRC animal models and new products and methods for the early detection of dysplasia. New products and methods for early detection will have important clinical applications to improve CRC survival rates and reduce healthcare costs. Summary of the Invention

[0017] Transformed cells and tissues express molecular changes before gross morphological changes, thus providing opportunities for early detection of cancer. Peptides conjugated to pre-cancerous colorectal lesions have the potential to guide tissue biopsies of "invisible" lesions endoscopically and combinatorial phage display screening can be used to identify and isolate such peptides. Peptides have in vivo advantages in the gastrointestinal tract as they can be delivered locally to identify early molecular changes on the surface of epithelial cells located in the outermost layer of the mucosa where cancer originates. In addition, they can exhibit rapid binding kinetics and diffuse into diseased mucosa. Further, smaller peptides reduce the opportunity for non-specific interactions and thus reduce background issues found in larger targeting molecules such as proteins (e.g., antibodies), antibody fragments, or even peptides on the order of about 25 or more amino acids. Peptides that are relatively small, i.e., no greater than 20 amino acids, are smaller in size and lower in molecular weight compared to these other targeting molecules. These properties help overcome many challenges of probe delivery, including irregular microvascular systems, heterogeneous uptake, and transport barriers. Improved diffusion and extravasation through leaky blood vessels relative to these other targeting molecules can also result in higher concentrations and deeper penetration of the relatively small peptides of the present disclosure. In addition, the peptides of the present disclosure have a relatively low immunogenic potential. The peptides of the present disclosure are also suitable for administration to humans by a variety of routes, including intrarectal delivery, preferably intravenous delivery.

[0018] The data disclosed herein establish the ability of cMet-targeting peptides, such as the cMet-targeting peptide QQTNWSL (SEQ ID NO:1), to detect pre-cancerous colon lesions in vivo in a patient-derived organoid mouse model. In some exemplary embodiments, the cMet-targeting peptide QQTNWSL (SEQ ID NO:1) is linked to a linker such as the GGGSK linker of SEQ ID NO:2, which is in turn linked to the fluorophore Cy5.5 and is thus labeled with the fluorophore (hereinafter referred to as QQT*-Cy5.5). The data support the disclosure of such peptides, including the QQT*-Cy5.5 peptide, as reagents capable of detecting pre-cancerous dysplasia in an effective and convenient manner.

[0019] The combination of molecular imaging tools with a fluorescent peptide targeting cMet (overexpressed in pre-cancerous CRC dysplasia) holds promise for improving diagnostic efficacy. Disclosed herein is a 7-amino acid peptide conjugated to a near-infrared (NIR) fluorescent cyanine dye that specifically binds to cMet. In vitro cMet high / low expressing cell lines demonstrated binding to cMet, and the specificity of the peptide for cMet was verified in knockdown and competition studies. The disclosed peptide exhibited a high binding affinity of 57 nM with a time constant of 1.6 minutes, supporting rapid binding upon topical application. The peptide also showed binding to human adenoma / SSA organoids and spontaneous colonic adenomas expressing high levels of cMet in vivo. Specific uptake in human adenoma / SSA organoids and spontaneous adenomas demonstrated the feasibility of using the peptide for real-time in vivo imaging. Immunofluorescence studies of human proximal colon specimens further demonstrated that the peptide could be used endoscopically to detect pre-cancerous lesions and guide tissue biopsies.

[0020] As an exemplary colon dysplasia targeting peptide according to the present disclosure, the QQT*-Cy5.5 peptide functions as a short amino acid protein fragment that acts as a highly specific targeting ligand for cMet, a cell surface target that plays a key role in many biological processes. The peptides hold promise for clinical translation to detect upregulated imaging biomarkers in colorectal cancer and other cancers. They are inherently highly diverse and can be designed to bind to a wide range of cell surface targets with high specificity and affinity at the nanomolar level. Through topical application, the peptides can be efficiently delivered to the mucosal surface of the digestive tract at high concentrations to maximize target interaction and achieve rapid binding with minimal risk of toxicity. The probe platform can be flexibly labeled with multiple fluorophores for multiplex imaging and has low manufacturing costs on a large scale. The peptides also have a low immunogenic potential that allows for repeated use. These characteristics of the peptides are well-suited for clinical applications in high-volume procedures such as colonoscopy.

[0021] On the one hand, the present disclosure provides a peptide comprising the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the peptide is derivatized, e.g., by attachment to a linker. In some embodiments, the linker comprises the sequence listed in SEQ ID NO: 2 (GGGSK). In some embodiments, the derivatized peptide is labeled, e.g., with a fluorescent label. In some embodiments, the label is FITC, Cy 5.5, Cy 7, Li-Cor, a radioactive label, biotin, luciferase, 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid), 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), 5-(and-6)-carboxy-2',7'-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX (5-carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA, 5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6JOE, 6,8-difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-carboxyrhodamine 6G pH 7.0, 6-carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SE pH 9.0, 7-amino-4-methylcoumarin pH 7.0, 7-hydroxy-4-methylcoumarin, 7-hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa405, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 antibody conjugate pH 7.2, AlexaFluor 488 antibody conjugate pH 8.0, Alexa Fluor 488 hydrazide-water, Alexa Fluor 532 antibody conjugate pH7.2, Alexa Fluor 555 antibody conjugate pH 7.2, Alexa Fluor 568 antibody conjugate pH 7.2, AlexaFluor 610R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 647 antibody conjugate pH 7.2, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH7.2, Alexa Fluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugate pH 7.2, allophycocyanin pH 7.5, AMCA conjugate, aminocoumarin, APC (allophycocyanin), Atto 647, BCECF pH 5.5, BCECF pH9.0, BFP (Blue Fluorescent Protein), Calcein, Calcein pH 9.0, Cal-Red, Cal-Red Ca2+, Cal-Green, Cal-Green Ca2+, Cal-Orange, Cal-Orange Ca2+, Carboxynaphthofluorescein pH 10.0, Cascade Blue, Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow Antibody Conjugate pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5, CI-NERF pH6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy 5, CyQUANT GR-DNA, DansylCadaverine, Dansylcadaverine, MeOH, DAPI, DAPI-DNA, Dapoxyl (2-Aminoethyl) Sulfonamide, DDAO pH9.0, Di-8 ANEPPS, Di-8-ANEPPS-Lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed, DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (Enhanced Green Fluorescent Protein), Eosin, Eosin Antibody Conjugate pH 8.0, Erythrosin-5-Isothiocyanate pH 9.0, eYFP (Enhanced Yellow Fluorescent Protein), FDA, FITC Antibody Conjugate pH 8.0, FlAsH, Fluo-3, Fluo-3 Ca2+. + , Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1MNaOH, Fluorescein Antibody Conjugate pH 8.0, Fluorescein Dextran pH 8.0, Fluorescein pH 9.0, Fluoro-Emerald, FM1-43, FM 1-43 Lipid, FM 4-64, FM 4-64, 2% CHAPS, Fura Red Ca2+ + , Fura Red, High Ca, FuraRed, Low Ca, Fura-2 Ca2+, Fura-2, Fura-2, GFP(S65T), HcRed, Indo-1Ca2+ +, Indo-1, Ca-free, Indo-1, Ca-saturated, JC-1, JC-1 pH 8.2, Lissamine Rhodamine, Fluorescein Yellow, CH, Magnesium Green, Magnesium Green Mg2+, Magnesium Orange, Marina Blue, mBanana, mCherry, mHoneydew, mOrange, mPlum, mRFP, mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace 500 / 525, Green Fluorescent Nissl Stain RNA, Nile Blue, Nile Red, Nile Red Lipid, Nissl, Oregon Green 488, Oregon Green 488 Antibody Conjugate pH 8.0, Oregon Green 514, Oregon Green 514 Antibody Conjugate pH 8.0, Pacific Blue, Pacific Blue Antibody Conjugate pH8.0, Phycoerythrin, R-Phycoerythrin pH 7.5, ReAsH, Resorufin, Resorufin pH 9.0, Rhod-2, Rhod-2 Ca2 + , Rhodamine, Rhodamine 110, Rhodamine 110pH

[0022] 7.0, Rhodamine 123, MeOH, Rhodamine Green, Rhodamine Phalloidin pH 7.0, Rhodamine Red-X Antibody Conjugate pH 8.0, Rhodamine Green pH 7.0, Rhodol Green Antibody Conjugate pH 8.0, Sapphire, SBFI-Na + , Sodium Green Na + , Sulfonyl Rhodamine 101, Tetramethylrhodamine Antibody Conjugate pH 8.0, Tetramethylrhodamine Dextran pH 7.0, Texas Red-X Antibody Conjugate pH 7.2, 11 C, 13 N, 15 O, 18 F, 32 P, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 90 Y, 94 mTc, 94 Tc, 95 Tc, 99 mTc, 103 Pd, 105 Rh, 109 Pd, 111 Ag, 111In, 123 I, 124 I, 125 I, 131 I, 140 La, 149 Pm, 153 Sm, 154-159 Gd, 165 Dy, 166 Dy, 166 Ho, 169 Yb, 175 Yb, 175 Lu, 177 Lu, 186 Re, 188 Re, 192 Ir, 198 Au, 199 Au or 212 Bi.

[0023] In some embodiments of the peptides according to the present disclosure, the peptides are labeled with a fluorescent label that emits in the near-infrared range of the electromagnetic spectrum. In some embodiments, the fluorescent label is FITC or a cyanine dye, such as a Cy5.5 or Cy7 dye. In some embodiments, the peptide consists of the sequence set forth in SEQ ID NO: 1.

[0024] Another aspect according to the present disclosure relates to a method for detecting intestinal tumorigenesis, which includes: (a) contacting intestinal tissue with a labeled peptide comprising

[0025] the sequence shown in SEQ ID NO: 1; (b) measuring the binding of the labeled peptide to the intestinal tissue; and (c) detecting intestinal tumorigenesis based on the measurement of the binding. In some embodiments, the intestinal tissue is colorectal tissue. In some embodiments, the colorectal tissue is colon tissue. In some embodiments, intestinal tumorigenesis is a pre-cancerous lesion. In some embodiments, intestinal tumorigenesis is cancer, such as colorectal cancer. In some embodiments, the intestinal tissue that binds the labeled peptide cannot be discerned as a polyp by endoscopy. In some embodiments, the intestinal tissue is a polyp. In some embodiments, the binding occurs in vivo. Some embodiments of the method further include in vivo fluorescence imaging. In some of these embodiments, fluorescence imaging is obtained using a wide-field endoscope.

[0026] As described above, the present disclosure provides peptides that bind to cMet presented on dysplastic colon cells and / or cancerous colon cells. An example of the provided peptide is the QQT*-Cy5.5 peptide, which is the peptide of SEQ ID NO: 1 labeled with Cy5.5.

[0027] The present disclosure also provides a reagent comprising a peptide according to the present disclosure. In some embodiments, the reagent comprises a detectable label attached to the peptide according to the present disclosure. The detectable label can be detected, for example, by microscopy including fluorescence microscopy, ultrasound, PET, SPECT, or magnetic resonance imaging. In one embodiment, the label detectable by microscopy is a member of the cyanine dye family, fluorescein isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, CF-633, or 5-carboxytetramethylrhodamine. An exemplary cyanine dye is Cy5.5.

[0028] In some embodiments, the detectable label is attached to the peptide according to the present disclosure via a peptide linker. The terminal amino acid of the linker can be lysine, such as the lysine in the exemplary linker GGGSK (SEQ ID NO:2), as noted above. In certain aspects, the linker is an Ahx linker, i.e., an amino-terminal linker comprising 6-aminohexanoic acid.

[0029] In other embodiments, the reagent comprises a therapeutic moiety attached to the peptide according to the present disclosure. The therapeutic moiety can be a chemopreventive agent or a chemotherapeutic agent. In certain aspects, the therapeutic moiety is an anti-cancer agent, such as a potent cytotoxin having selective activity in rapidly dividing cells. Such cytotoxins include several groups of chemotherapeutic agents, such as auristatins, maytansines, and calicheamicins, as well as duocarymycins and pyrrolobenzodiazepine (PBD) dimers, all of which induce DNA damage in target cells such as tumor cells of the colon. Exemplary auristatins are monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The present disclosure contemplates maytansines and their derivatives, such as maytansanoids. The advantageous properties of using the disclosed peptides as targeting agents also allow for the effective use of earlier-generation cytotoxic agents as anti-cancer agents while limiting toxicity to acceptable levels. These older cytotoxic agents include, but are not limited to, vinca alkaloids, anthracyclines, Gleevec, paclitaxel, camptothecin, doxorubicin, methotrexate, 5-fluorouracil, chlorambucil, and any anti-cancer agent known in the art. In a related aspect, the present disclosure provides a therapeutic moiety that is an anti-inflammatory agent, such as an NSAID, such as celecoxib.

[0030] In yet another aspect, the present invention provides a composition comprising the reagent of the present invention and a pharmaceutically acceptable excipient.

[0031] In another aspect, the present disclosure provides a method for determining the therapeutic efficacy of colon cancer and / or cancer metastasis or cancer recurrence in a patient, the method comprising the steps of: administering a reagent comprising a peptide according to the present disclosure linked to a detectable marker to the colon of the patient, visualizing a first amount of cells labeled with the reagent, and comparing the first amount with a second amount of cells labeled with the reagent that was previously visualized, wherein a decrease in the first amount of cells relative to the previously visualized second amount of labeled cells indicates effective treatment. In some embodiments, a 5% decrease indicates effective treatment. In other embodiments, a decrease of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more indicates effective treatment. In some embodiments, the method further comprises obtaining a biopsy of the cells labeled with the reagent.

[0032] In another aspect, the present invention provides a method for delivering a therapeutic agent to dysplastic colon cells of a patient, which comprises the step of administering to the patient a reagent comprising a peptide according to the present invention linked to a therapeutic moiety.

[0033] In yet another aspect, the present disclosure provides a method for delivering a therapeutic agent to colon cells of a patient, which comprises the step of administering to the patient a reagent comprising a peptide according to the present invention linked to a therapeutic moiety.

[0034] In yet another aspect, the present invention provides a kit for administering a composition according to the present disclosure to a patient in need thereof, wherein the kit comprises a composition according to the present disclosure, instructions for using the composition, and a device for administering the composition to the patient.

[0035] Other features and advantages of the present disclosure will be better understood by reference to the following detailed description (including the drawings and examples). Description of the Drawings

[0036] Figure 1. The cMet-specific peptide shows the biochemical structures of A) the exemplary target peptide sequence QQTNWSL (SEQ ID NO: 1) and B) the scrambled control peptide sequence TLQWNQS (SEQ ID NO: 3). The GGGSK linker (SEQ ID NO: 2) separates the Cy5.5 fluorophore from the amino acid sequence of the labeled peptide to prevent steric hindrance. C and D) The three-dimensional models show the differences in the biochemical structures. E) The peak absorbance and F) the maximum fluorescence emission occur at λ abs = 675 nm and λ em = 710 nm.

[0037] Figure 2. In vitro validation using cMet knockdown. A) The QQT*-Cy5.5 peptide (red) and B) the AF488-labeled anti-cMet antibody (green) showed strong binding (arrows) to the surface of human HT29 colorectal cancer cells transfected with siCL non-targeting siRNA (control) using confocal microscopy. C) The control peptide TLQ*-Cy5.5 (red) showed minimal binding. D) The fluorescence intensities of the peptide and E) the antibody decreased with HT29 knockdown cells transfected with sicMet targeting siRNA. F) TLQ*-Cy5.5 (red) showed little binding. G) QQT*-Cy5.5 and anti-cMet-AF488 showed a significant decrease in intensity, with cMet knockdown being 2.9-fold and 4.1-fold, respectively. TLQ*-Cy5.5 showed a non-significant decrease (0.96-fold, P = 0.52). The intensity of QQT*-Cy5.5 was significantly higher compared to TLQ*-Cy5.5 (8.1-fold). For log-transformed data, a two-way ANOVA model was fitted with terms for 6 conditions and 6 replicate slides. The measurements were the mean of 5 cells randomly selected from 6 slides for each condition. H) Western blot showed cMet expression under each condition.

[0038] Figure 3 . cMet-specific peptide A) Adding unlabeled QQT* to compete with QQT*-Cy5.5 for binding to HT29 cells resulted in a significant decrease in fluorescence intensity in a concentration-dependent manner. TLQ* showed no significant change. For log-transformed data, a two-way ANOVA model was fitted with terms for the labeled peptide, the concentration of the unlabeled peptide, and their interaction. The measurements were the mean of 5 cells randomly selected from 3 slides for each condition. B) The binding of QQT*-Cy5.5 (red) and anti-cMet-AF488 (green) co-localized on the surface of HT29 cells (arrows), ρ = 0.73. C) Measuring the apparent dissociation constant k d = 57 nM, R 2 = 0.98. D) Measuring the apparent association time constant k = 0.62 min -1 (1.6 min) for the binding of QQT*-Cy5.5 to HT29 cells, R 2 = 0.97. These results represent 3 independent experiments.

[0039] Figure 4. Effects of peptides on cell signaling and growth. Peptide binding does not affect cell signaling. (A) From western blot, after incubation for 10, 30, and 120 min (i.e., minutes), HGF (25 ng / mL) induced phosphorylation of cMet and downstream AKT and Erk1 / 2 in HT29 cells. HGF (100 ng / mL) was used as a positive control and no HGF (none) was used as a negative control. Incubation with 5 or 100 μM of QQT*-Cy5.5 showed no effect on the expression of p-cMet (phosphorylation activity of cMet) or downstream AKT and Erk1 / 2 signaling. β-Tubulin was used as a loading control. This set of bands was cut from different parts of the same gel. The Alamar Blue assay showed that after 48 h, after addition of HGF, (B) the growth of HT29 cells increased, but (C) not in CCD841 cells. No change was seen with 5 or 100 μM of QQT*-Cy5.5. An ANOVA model with terms having 4 groups was fit to the log-transformed data with 3 independent experiments.

[0040] Figure 5. In vivo endoscopic imaging of human colon organoids CPC; in vivo imaging in Apc mice. (A) White light image shows no visibly obvious lesions (flat). (B) NIR fluorescence image after rectal administration of QQT*-Cy5.5 shows increased intensity of the flat lesion (arrow). (C) A co-registered reflectance image is obtained from the same lesion. (D) Fluorescence image collected using TLQ*-Cy5.5 (control) shows minimal signal. (E) White light image of the colon shows the presence of a polyp (arrow). (F) QQT*-Cy5.5 shows increased fluorescence intensity from the polyp (arrow). (G) A co-registered reflectance image of the polyp is collected. (H) TLQ*-Cy5.5 shows minimal signal. (I) The ratio of fluorescence and reflectance images of the flat lesion in (A) is shown. (J) The fluorescence (red), reflectance (green), and ratio (blue) intensities of the dotted line in (I) are shown. (K) In n = 8 mice, by paired t-test on log-transformed data, QQT*-Cy5.5 showed significantly higher mean (±SD) T / B ratios for flat lesions (n = 7) and polyps (n = 8) compared to adjacent normal mucosa, changing 1.7-fold and 2.1-fold, respectively. (L) White light image of the resected colon shows many polyps (arrows) on the exposed mucosal surface. (M) Fluorescence image collected in vitro shows increased intensity of the polyp after topical application of QQT*-Cy5.5. (N) Merged image. (O) In n = 5 mice, by paired t-test of log-transformed data, the mean fluorescence intensity from adenomas was 2.6-fold higher than that from adjacent normal mucosa with normal appearance. Immunohistochemistry (IHC) shows (P) higher expression of cMet in dysplasia compared to (Q) normal.

[0041] Figure 6. In vitro validation using human colon specimens. Merged images using confocal microscopy show co-localization of QQT*-Cy5.5 (red) and anti-cMet-AF488 (green) binding to A) adenoma, B) SSA, C) hyperplastic polyp (HP), and D) normal mucosa. Pearson correlation coefficients ρ = 0.82, 0.79, 0.86, and 0.75 were measured, respectively. E) Adenoma, F) SSA, G) HP, and H) immunohistochemistry (IHC) of normal colonic mucosa support cMet expression. An ANOVA model with terms for 4 groups was used on log-transformed data with 3 replicate intensity measurements per sample, showing representative histology (H&E) of I) adenoma, J) SSA, K) HP, and L) normal colonic mucosa; M) Adenoma (n = 21) had significantly higher mean fluorescence intensity compared to normal (n = 10) and HP (n = 7), with 3.0- and 2.4-fold changes, respectively. For SSA (n = 13), the mean fluorescence intensity was also significantly higher than normal and HP, with 2.8- and 2.2-fold changes, respectively. Measurements were the mean of 3 regions of interest (ROIs) of size 20 × 20 μm on each slide. 2 The mean of 3 regions of interest (ROIs) of size 20 × 20 μm on each slide.

[0042] Figure 7 . Quantitative immunofluorescence results in human colon specimens. A) The mean fluorescence intensity of adenoma (n = 21) was significantly higher than that of HP (n = 7) and normal cells (n = 10), with 2.4- and 3.0-fold changes, respectively, as determined by two-sample t-test. The results for SSA (n = 13) were also significantly higher than those of HP and normal cells, with 2.2- and 2.8-fold changes, respectively. Measurements were the mean of 3 regions of interest (ROIs) of size 25 × 25 pixels on each slide. The ANOVA model was fitted with terms for 4 groups, with 3 replicate intensity measurements per sample. B) The ROC curve showed 86% sensitivity and 88% specificity, with an area under the curve (AUC) of 0.94 for differentiating adenoma from normal cells and HP, and C) 92% sensitivity and 88% specificity, AUC = 0.95, for differentiating SSA from normal cells and HP.

[0043] Figure 8 . Under confocal microscopy, the QQT*-Cy5.5 peptide (red) and AF568-labeled anti-cMet antibody (yellow) bind to adenoma human colon organoids. Human-specific cytokeratin staining of the organoids shows successful transplantation. H&E staining of the organoid xenografts. Statistical results of in vivo imaging of QQT*-Cy5.5 and control TLQ*-Cy5.5 in human adenoma organoid xenografts. The fluorescence intensity of QQT* was 3.2-fold higher than that of TLQ*.

[0044] Figure 9.Mass spectrometry analysis of Cy5.5-labeled peptides. The experimental mass-to-charge (m / z) ratios of A) QQT*-Cy5.5 and B) TLQ*-Cy5.5 were found to be 1827.67, indicating consistency with the expected values.

[0045] Figure 10 .Peptide validation in human colorectal cancer cells in vitro. Under confocal microscopy, A) QQT*-Cy5.5 (red) and B) anti-cMet-AF488 (green) showed strong binding to the surface of HT29 cells (cMet+) (arrows). C) The control peptide TLQ*-Cy5.5 (red) showed minimal binding. In contrast, a decrease in signal was observed using D) the peptide and E) SW480 cell antibody (cMet-). F) TLQ*-Cy5.5 (red) showed little binding. G) The quantification results showed that the average intensities of QQT*-Cy5.5 and anti-cMet-AF488 were significantly higher in HT29 compared to SW480 cells, by 2.1-fold and 4.3-fold, respectively, with P = 4.5×10 -10 and 3.2×10 -17 . TLQ*-Cy5.5 showed a non-significant decrease (0.85-fold change, P = 0.07). The intensity of QQT*-Cy5.5 for HT29 was significantly greater than that of HT29 cells for TLQ*-Cy5.5 (7.5-fold change, P = 4.5×10 -21 ). For the log-transformed data, a two-way ANOVA model was fitted with terms for 6 conditions and 6 replicate slides. The measurements were the means of 5 cells randomly selected from 6 slides for each condition. H) Western blot showed the cMet expression levels in each cell.

[0046] Figure 11 .Peptide validation in mouse cells in vitro. Under confocal microscopy, A) QQT*-Cy5.5 (red) and B) anti-cMet-AF488 (green) showed strong binding to the surface of S114 cells (cMet+) (arrows). C) The control peptide TLQ*-Cy5.5 (red) showed minimal binding. In contrast, a decrease in signal was observed using D) the peptide and E) NIH3T3 cell antibody (cMet-). F) TLQ*-Cy5.5 (red) showed little binding. G) The quantification results showed that the average intensities of QQT*-Cy5.5 and anti-cMet-AF488 were significantly higher in S114 compared to NIH3T3 cells, by 2.4-fold and 6.7-fold, respectively, with P = 1.7×10 -16 and 4.0×10 -26 . TLQ*-Cy5.5 showed a non-significant decrease (0.72-fold change, P = 8.2×10 -7)。The intensity of QQT*-Cy5.5 for S114 was significantly greater than that of S114 cells for TLQ*-Cy5.5 (7.0-fold change, P = 2.6×10 -26 )。For the log-transformed data, a two-way ANOVA model was fit to terms for 6 conditions and 6 replicate slides. Measurements were the mean of 5 cells randomly selected on 6 slides for each condition. H) Western blot shows the cMet expression level in each cell. I) The mean intensities of QQT*-Cy5.5 and anti-cMet-AF488 with S114 were significantly higher than with NIH3T3 cells, with 2.4- and 6.7-fold changes, respectively. TLQ*-Cy5.5 showed a non-significant increase. The mean intensity of QQT*-Cy5.5 was significantly higher than that of TLQ*-Cy5.5, with a 7.0-fold change. The difference between S114 and NIH3T3 for QQT*-Cy5.5 was significantly higher than the difference for TQL*-Cy5.5 (P = 1.3×10 -8 )。An ANOVA model was fit to the log-transformed data for terms with 6 conditions. There were 6 replicate slides for each condition, and 10 cells were measured on each slide.

[0047] Figure 12. In vivo imaging in CPC; Apc mice. A) White light images show no gross lesions (polyps), consistent with the presence of flat lesions. B) NIR fluorescence image of QQT*-Cy5.5 shows the presence of flat lesions (arrows). C) Fluorescence-registered reflectance images were obtained. D) Images of the same region with TLQ*-Cy5.5 (control) show minimal signal. E) White light endoscopy images of the colon show the presence of spontaneous polyps (arrows) and normal-appearing mucosa. F) NIR fluorescence image after topical application of QQT*-Cy5.5 shows increased polyp intensity (arrows). G) Fluorescence-registered reflectance images were obtained. H) TLQ*-Cy5.5 images show minimal signal. I) The ratio of fluorescence and reflectance images from flat lesions is shown in Figure S4A. J) Fluorescence (red), reflectance (green), and ratio (blue) values along the dashed line in Figure S4I. K) In n = 8 mice, QQT*-Cy5.5 showed mean (±SD) T:B ratios from polyps (n = 8) and flat lesions (n = 7) higher than the T:B ratio from adjacent normal mucosa, 3.85 ± 1.1 and 3.76 ± 0.67, respectively, with P = 1.1×10 -3 and P = 7.8×10 -3 . L) Polyps and M) flat lesions adjacent to normal mucosa showed histological (H&E) signs of low-grade dysplasia.

[0048] Figure 13. CPC; Macroscopic validation of cMet expression in the colon of Apc mice. A) White light images show numerous dysplastic polyps (arrows) on the exposed mucosal surface of excised mouse colon. B) Fluorescence images show increased intensity of polyps after topical application of QQT*-Cy5.5. C) Merged images are shown. D) In n = 5 mice, the mean fluorescence intensity of 10 dysplastic areas was 2.7-fold higher than that of the uninvolved surrounding normal mucosa, with P = 1.6×10 in a paired t-test -5 . Immunohistochemistry (IHC) supports cMet overexpression in E) dysplasia and F) normal mucosa. (L) White light images of excised colon show numerous polyps (arrows) on the exposed mucosal surface. (M) Fluorescence images collected in vitro show increased intensity of polyps after topical application of QQT*-Cy5.5. (N) Merged images. (O) In n = 5 mice, by paired t-test on log-transformed data, the mean fluorescence intensity from adenomas was 2.6-fold higher than that from adjacent normal mucosa with normal appearance. Immunohistochemistry (IHC) shows higher expression of cMet in (P) dysplasia compared to (Q) normal.

[0049] Figure 14. CPC; In vivo imaging in Apc mice. A) White light endoscopy images of the colon show the presence of spontaneous polyps (arrows) and normal-appearing mucosa. B) NIR fluorescence images after topical application of QQT*-Cy5.5 show increased intensity of polyps (arrows). C) Images of the same area with TLQ*-Cy5.5 (control) show minimal signal. D) White light images show no grossly visible lesions (polyps). E) NIR fluorescence images of QQT*-Cy5.5 show the presence of flat lesions (arrows). F) TLQ*-Cy5.5 images show minimal signal. G) In n = 8 mice, QQT*-Cy5.5 showed that the mean (±SD) T:B ratios from polyps (n = 8) and flat lesions (n = 7) were higher than the T:B ratios from adjacent normal mucosa, at 3.85 ± 1.1 and 3.76 ± 0.67, respectively, with P = 1.1×10 -3 and P = 7.8×10 -3 . H) Histology (H&E) of polyps and I) flat lesions compared to adjacent normal mucosa shows signs of low-grade dysplasia.

[0050] Figure 15. Macroscopic validation of cMet expression in the colon of CPC; Apc mice. Under confocal microscopy, the binding of A) QQT*-Cy5.5 peptide (red) and B) AF488-labeled anti-cMet antibody (green) co-localized on the surface of dysplastic colonocytes in tubular adenomas (arrows). C) On the merged image, the Pearson correlation coefficient □ = 0.78 was measured. D-F) Reduced signals were observed in normal mucosa. G, I) The mean (±SD) fluorescence intensity of adenomas (n = 30) was significantly higher than that of normal (n = 30), being 6.2 ± 0.17 and 4.9 ± 0.25 respectively, and P = 2.4×10 for the paired t-test on log-transformed data -4 . H) The ROC curve showed 93% sensitivity and 87% specificity, AUC = 0.96, to distinguish dysplasia from normal. J, K) Histology (H&E) is shown.

[0051] Figure 16. In vivo imaging of normal and SSA human colon organoids. White light images show patient-derived normal mucosa (arrow) A) and patient-derived SSA (arrow) E) implanted into the colon of NOD / SCID mice. Fluorescence images were collected after local administration of QQT*-Cy5.5 and showed strong signals from SSA organoids (arrow) F), while minimal intensity from normal organoids (arrow) B). C, G) Registered reflectance images are shown. Three days later, imaging with TLQ*-Cy5.5 (control) from the same normal D) and SSA H) organoids showed little signal (arrow). I) For in vivo imaging, the mean fluorescence intensity of SSA organoids was found to be significantly higher than that of normal organoids. H&E staining shows the histology of transplanted normal J) and SSA M) organoids. Human-specific hcytokeratin staining shows successful transplantation of normal K) and SSA N) organoids. IF co-localization of cMet and QQT*-Cy5.5 on normal L) and adenoma O) organoids.

[0052] Figure 17 . Peptide characterization. A) Using confocal microscopy, the binding of QQT*-Cy5.5 to HT29 cells showed no change in the presence of HGF. B) The binding of QQT* to mouse cMet-ECD showed strong bands compared to TLQ* from the pull-down assay. Keywords: Total – 20 μg mouse cMet-ECD without EHS beads; None – EHS beads without peptide; QQT* – target peptide immobilized on EHS beads; TLQ* – control peptide immobilized on EHS beads.

[0053] Figure 18 . Western blot. For A) cMet knockdown by siRNA in HT29 cells ( Figure 2 H), B) cMet expression in HT29 and SW480 cells (Figure 10 H), C) cMet expression in S114 and NIH3T3 cells Figure 11 H), and D-H) Detection of downstream cMet signaling in HT29 cells treated with HGF (25 ng / mL) or QQT*-Cy5.5 (5 or 10 μM) Figure 4 ) and I) Binding of QQT* to murine cMet-ECD Figure 17 ) showing the uncropped gels. All blots are from the same gel but developed on different films.

[0054] Figure 19. In vivo endoscopic imaging of human colon organoids shows A) white light and B) reflection images of an adenoma implanted in the colon of NOD / SCID mice. Fluorescence images collected after local administration of C) QQT*-Cy5.5 show strong intensity from the adenoma, while D) TLQ*-Cy5.5 provides minimal signal. White light and F) reflection images of SSA are shown. Fluorescence images collected using G) QQT*-Cy5.5 show strong intensity from SSA, while H) TLQ*-Cy5.5 provides minimal signal. Normal I) white light and J) reflection images are shown. Fluorescence images collected using G) QQT*-Cy5.5 and H) TLQ*-Cy5.5 show minimal signal. Detailed Description

[0055] It is expected that relatively small peptide-based fluorescent imaging probes targeting cancer biomarkers will increase the visualization of pre-cancerous lesions and have lower immunogenicity and faster clearance. Some clinical studies have shown that peptides can be used as diagnostic tools to guide tissue biopsies in the gastrointestinal tract 27,28 . Compared to antibodies, the relatively small size of peptides makes them more easily penetrate deep tissues with minimal immunogenicity. Peptide-based imaging probes also exhibit high diversity and labeling flexibility at an affordable cost and with fast binding kinetics. These advantages make peptides very suitable reagents for in vivo imaging and clinical use.

[0056] The present disclosure provides a fluorescently labeled peptide that is specific for cMet to detect pre-cancerous dysplasia in CRC. The peptide has been shown to detect pre-cancerous colonic lesions in vivo that are flat in appearance and easily missed by colonoscopy with white light illumination. Using biopanning techniques with a phage display library, we identified a heptapeptide that specifically binds to the extracellular domain of cMet. After labeling with the near-infrared fluorescent dye Cy5.5, we topically applied this peptide to the surface of the distal colon in mice, which can minimize toxicity and reduce the risk of unintended tissue binding. Compared with scrambled control peptides, the peptide exhibited an excellent target-to-background (T / B) signal ratio. The function of the peptide as a probe or targeting ligand was examined in the experiments disclosed herein, and the results demonstrated that the peptide specific for cMet is expected to be useful for endoscopic detection of pre-cancerous lesions and for guiding the localization of biopsy tissues.

[0057] Phage display technology was used to biopan a linear heptapeptide library against the extracellular domain (ECD) of cMet, and the heptapeptide sequence QQTNWSL (SEQ ID NO:1) was identified. We covalently linked the C-terminus of this linear monomer (black) to the near-infrared (NIR) fluorophore Cy5.5 (red) via a GGGSK (SEQ ID NO:2) linker (blue), hereafter referred to as QQT*-Cy5.5. The peptide was separated from the fluorophore to minimize the effect of steric hindrance. Cy5.5 was chosen because it is less sensitive to hemoglobin absorption and tissue scattering, minimizes the effect of tissue autofluorescence, and provides the greatest light penetration depth. We achieved a purity of over 95% for both peptides using HPLC and measured an experimental mass-to-charge (m / z) ratio of 1827 on mass spectrometry, which was consistent with the expected value. We measured the apparent dissociation constant K D = 57 nM for the peptide binding to HT29 human colorectal adenocarcinoma cells. In addition, we measured the apparent association time constant k = 0.622 min-1 (1.61 min).

[0058] Overexpression of cMet is an early event in CRC tumorigenesis, making the detection of cMet expression levels a promising approach for identifying pre-cancerous dysplasia in CRC. 20,21 Moreover, its location on the cell membrane allows cMet to be accessible to imaging agents, such as fluorescent targeting agents. It has been reported that elevated cMet levels occur in a variety of cancer types, especially colorectal cancer. The results of various preclinical and clinical studies confirm that cMet is a promising target for molecular imaging, which allows real-time and in vivo monitoring of abnormal changes.

[0059] The present disclosure relates to an NIR-labeled cMet-targeting peptide for in vivo fluorescence imaging in a mouse model of polyps spontaneously developing in Cpc; Apc and a mouse model of human organ transplantation. QQTNWSL (SEQ ID NO:1) was selected against the extracellular domain of cMet by biopanning and phage display. Specific binding to cMet was verified in vitro and ex vivo using standard assays such as competition and cell binding assays. The peptide exhibited a high binding affinity with kd = 57 nM, and binding occurred within 2 minutes (k = 0.622 min-1), which is compatible with clinical use during colonoscopy. In a spontaneous mouse model of CRC, we demonstrated that the peptide was able to detect flat and polyploid colonic adenomas in vivo, which were pathologically diagnosed as low-grade dysplasia. To more accurately mimic real human physiology, a mouse model of human organ transplantation was developed. By orthotopic injection, this mouse model generated colonic polyps derived from human organs, truly recapitulating human CRC. One of the important goals of advanced imaging techniques is to distinguish between benign hyperplastic polyps and malignant lesions such as serrated polyps to avoid unnecessary costs and treatments 47 。Imaging results using the organoid transplantation mouse model disclosed herein showed that the QQT*-Cy5.5 fluorescently labeled peptide bound to human adenomas and SSAs with minimal binding to normal organoids. IF staining of different subtypes of human proximal colon tissue with QQT*-Cy5.5 further confirmed this observation, which showed that the fluorescently labeled peptide distinguished adenomas or SSAs from normal and HP with 88% sensitivity and 82% specificity and an area under the curve (AUC) of 0.94.

[0060] Antibodies as molecular imaging probes against cMet have been validated 48,49 。Although antibodies and antibody fragments can achieve high binding affinity, they are limited in diagnosis due to slow binding kinetics, long half-life, and increased background. Compared with antibodies, peptides are safer and less costly because of their lower molecular weight. Peptides have several advantages such as favorable pharmacokinetic and tissue distribution profiles, higher permeability, lower toxicity, lower immunogenicity, and ease of chemical modification 50 。Local administration of peptides delivers therapeutic agents directly to target tissues at high concentrations that are at risk of carrying the disease to maximize binding interactions and achieve high image contrast with little risk of toxicity. This approach avoids the unwanted biodistribution of exogenous agents characteristic of administration by, for example, intravenous injection to other tissues.

[0061] Recently, increasing evidence has shown that cMet is associated with other cell surface receptor tyrosine kinases (RTKs) and cell surface proteins related to tumor formation and progression in colorectal cancer, such as vascular endothelial growth factor receptor (VEGFR) 51,52and the epidermal growth factor receptor (EGFR) 53,54 Associated. Due to the complexity and heterogeneity of the disease in a broad patient population, a multiplex imaging method using multiple targets may prove beneficial 35,55 .

[0062] Linkers and polypeptides

[0063] As used herein, a "linker" is an amino acid sequence located at the terminus of a peptide of the present disclosure and is generally uncharged. In some embodiments, the linker sequence terminates in a lysine residue. Uncharged amino acids contemplated by the present disclosure include, but are not limited to, glycine, serine, cysteine, threonine, histidine, tyrosine, asparagine, and glutamine.

[0064] In some embodiments, the presence of a linker results in at least a 1% increase in the detectable binding of a reagent of the present disclosure to dysplastic or cancerous colon cells compared to the detectable binding of the reagent in the absence of a linker. In various aspects, the increase in detectable binding is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 100-fold or more.

[0065] The term "peptide" refers to a molecule of 2 to 50 amino acids, a molecule of 3 to 20 amino acids, and a molecule of 6 to 15 amino acids. The length of the peptides and linkers contemplated by the present invention can be 5 amino acids. In various aspects, the length of the polypeptide or linker can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids.

[0066] In various aspects, exemplary peptides are randomly generated by methods known in the art, carried in polypeptide libraries (e.g., but not limited to, phage display libraries), derived by protein digestion, or chemically synthesized. The peptides illustrated in the present disclosure can be obtained using phage display technology, which is an effective combinatorial method that uses recombinant DNA technology to generate a complex library of polypeptides for selection by preferential binding to cell surface targets [Scott et al., Science, 249:386 - 390 (1990)]. The protein coat of the phage, such as filamentous M13 or icosahedral T7, is genetically engineered to express a very large number (greater than 10 9 ) of different polypeptides with unique sequences for affinity binding [Cwirla et al., Proc. Natl. Acad. Sci. USA, 87:6378 - 6382 (1990)]. Selection is then performed by biopanning the phage library against cultured cells and tissues that overexpress the target. The DNA sequences of these candidate phages are then recovered and used to synthesize the polypeptides [Pasqualini et al., Nature, 380:364 - 366 (1996)]. Polypeptides that preferentially bind dysplastic mucosa are optionally labeled with a fluorescent dye, including but not limited to FITC, Cy5.5, Cy7, and Li - Cor.

[0067] Peptides include purified or a mixture of two forms, the D - form and the L - form. The present disclosure also contemplates peptides that compete with the peptides of the invention for binding to colon cells.

[0068] It should be understood that the peptides and linkers of the present invention are optionally incorporated with modifications known in the art, and the positions and amounts of such modifications are altered to obtain the best results.

[0069] Detectable label

[0070] As used herein, a "detectable label" is any label that can be used to identify the binding of the compositions of the present disclosure to intestinal tissues such as colon tissue. Non - limiting examples of detectable labels are fluorophores, chemical tags, or protein tags that can render the polypeptide visible. In some aspects, visualization is performed with the naked eye or with a device (e.g., but not limited to, an endoscope), and can also involve alternative light or energy sources.

[0071] Fluorophores, chemical, and protein tags contemplated for use in the present invention include, but are not limited to, FITC, Cy 5.5, Cy7, Li-Cor, radiolabels, biotin, luciferase, 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid), 1-anilinonaphthalen-8-sulfonic acid (1,8-ANS), 5-(and -6)-carboxy-2',7'-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX (5-carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA, 5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6JOE, 6,8-difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-carboxyrhodamine 6G pH 7.0, 6-carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SE pH 9.0, 7-amino-4-methylcoumarin pH7.0, 7-hydroxy-4-methylcoumarin, 7-hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 647, Alexa 660, Alexa 680, Alexa700, Alexa Fluor 430 antibody conjugate pH 7.2, Alexa Fluor 488 antibody conjugate pH 8.0, AlexaFluor 488 hydrazide-water, Alexa Fluor 532 antibody conjugate pH 7.2, Alexa Fluor 555 antibody conjugate pH7.2, Alexa Fluor 568 antibody conjugate pH 7.2, Alexa Fluor 610R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 647 antibody conjugate pH 7.2, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH 7.2, Alexa Fluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugate pH 7.2, allophycocyanin pH 7.5, AMCA conjugate, aminocoumarin, APC (allophycocyanin), Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (blue fluorescent protein), calcein, calcein pH 9.0, calred, calred Ca2+, calgreen, calgreen Ca2+, calorange, calorange Ca2+, carboxynaphthofluorescein pH 10.0, cascade blue, cascade blue BSA pH 7.0, Cascade Yellow, Cascade Yellow antibody conjugate, pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5, CI-NERF pH 6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy5, CyQUANT GR-DNA, Dansyl Cadaverine, Dansyl Cadaverine, MeOH, DAPI, DAPI-DNA, Dapoxyl (2-aminoethyl) sulfonamide, DDAO pH 9.0, Di-8 ANEPPS, Di-8-ANEPPS-lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed, DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (Enhanced Green Fluorescent Protein), Eosin, Eosin antibody conjugate pH 8.0, Erythrosin-5-isothiocyanate pH 9.0, eYFP (Enhanced Yellow Fluorescent Protein), FDA, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3, Fluo-3 Ca2. + , Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1M NaOH, Fluorescein antibody conjugate pH 8.0, Fluorescein dextran pH 8.0, Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43, FM 1-43 lipid, FM 4-64, FM 4-64, 2% CHAPS, Fura Red Ca2 + , Fura Red, high Ca, Fura Red, low Ca, Fura-2 Ca2+, Fura-2, Fura-2, GFP(S65T), HcRed, Indo-1 Ca2 +, Indo-1, Ca-free, Indo-1, Ca-saturated, JC-1, JC-1 pH 8.2, Lissamine Rhodamine, Fluorescein, CH, Magnesium Green, Magnesium Green Mg2+, Magnesium Orange, Marina Blue, mBanana, mCherry, mHoneydew, mOrange, mPlum, mRFP, mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace 500 / 525, Green Fluorescent Nissl Stain RNA, Nile Blue, Nile Red, Nile Red Lipid, Nissl, Oregon Green 488, Oregon Green 488 Antibody Conjugate pH 8.0, Oregon Green 514, Oregon Green 514 Antibody Conjugate pH 8.0, Pacific Blue, Pacific Blue Antibody Conjugate pH 8.0, Phycoerythrin, R-Phycoerythrin pH 7.5, ReAsH, Resorufin, Resorufin pH 9.0, Rhod-2, Rhod-2 Ca2 + , Rhodamine, Rhodamine 110, Rhodamine 110 pH 7.0, Rhodamine 123, MeOH, Rhodamine Green, Rhodamine Phalloidin pH 7.0, Rhodamine Red-X Antibody Conjugate pH 8.0, Rhodamine Green pH 7.0, Rhodol Green Antibody Conjugate pH 8.0, Sapphire, SBFI-Na + , Green Sodium Na + , Sulfonyl Rhodamine 101, Tetramethylrhodamine Antibody Conjugate pH 8.0, Tetramethylrhodamine Dextran pH 7.0, and Texas Red-X Antibody Conjugate pH 7.2.

[0072] Non-limiting examples of chemical tags contemplated by the present invention include radiolabels. By way of example and not limitation, radiolabels contemplated in the compositions and methods of the present disclosure include 11 C, 13 N, 15 O, 18 F, 32 P, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 90 Y, 94 mTc, 94 Tc, 95 Tc, 99 mTc, 103 Pd,105 Rh, 109 Pd, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 140 La, 149 Pm, 153 Sm, 154-159 Gd, 165 Dy, 166 Dy, 166 Ho, 169 Yb, 175 Yb, 175 Lu, 177 Lu, 186 Re, 188 Re, 192 Ir, 198 Au, 199 Au and 212 Bi.

[0073] One of ordinary skill in the art will appreciate that many such detectable labels can be used for in vitro, in vivo, or ex vivo visualization of the compositions of the present disclosure.

[0074] Therapeutic moiety

[0075] Therapeutic moieties contemplated by the present invention include, but are not limited to, polypeptides or peptides, small molecules, therapeutic agents, chemotherapeutic agents, or combinations thereof.

[0076] As used herein, the term "small molecule" refers to a compound such as a peptidomimetic or oligonucleotide, which may optionally be derivatized, or any other natural or synthetic low molecular weight organic compound.

[0077] "Low molecular weight" refers to a compound having a molecular weight of less than 1000 daltons, typically between 300 and 700 daltons. In various aspects, the low molecular weight compound is about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 1000 or more daltons.

[0078] In some aspects, the therapeutic moiety is a protein therapeutic. Protein therapeutics include, but are not limited to, cellular proteins or circulating proteins and fragments and derivatives thereof. Still other therapeutic moieties include polynucleotides, which include, but are not limited to, polynucleotides encoding proteins, polynucleotides encoding regulatory polynucleotides, and / or self-regulatory polynucleotides. Optionally, the composition comprises a combination of the compounds described herein.

[0079] In various aspects, the protein therapeutics include cytokines or hematopoietic factors, including but not limited to IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-α (IFN-α), consensus interferon, IFN-β, IFN-γ, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, thrombopoietin (TPO), angiopoietin,For example, Ang-1, Ang-2, Ang-4, Ang-Y (human angiopoietin-like polypeptide), vascular endothelial growth factor (VEGF), angiopoietin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophils, chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, β-endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-associated protein, growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor receptor, neurotrophin 3, neurotrophin 4, placental growth factor, placental growth factor 2, platelet-derived endothelial cell growth factor, platelet-derived growth factor, platelet-derived growth factor A chain, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor B chain, platelet-derived growth factor BB, platelet-derived growth factor receptor α, platelet-derived growth factor receptor β, pre-B cell growth-stimulating factor, stem cell factor receptor, TNF,Including TNF0, TNF1, TNF2, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor and its chimeric proteins and biological or immunological active fragments.

[0080] In various embodiments, the therapeutic moiety further includes a chemotherapeutic agent. Chemotherapeutic agents contemplated for use in the reagents of the present invention include, but are not limited to, alkylating agents, including: nitrogen mustards, such as methyl-ethylamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethyleneimine / methylmelamines, such as tetraethylenemelamine (TEM), triethylenethiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates, such as busulfan; triazines, such as (dacarbazine (DTIC)); antimetabolites, including folic acid analogs (e.g., methotrexate and trimethoprim), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, gemcitabine, cytarabine (AraC), 5-azacytidine, 2,2'-difluorodeoxycytidine), purine analogs (e.g., 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxyadenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA)); natural products, including antimitotic drugs (e.g., paclitaxel); vinca alkaloids, including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins, such as etoposide and teniposide; antibiotics, such as actinomycin D, rubidomycin, doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes, such as L-asparaginase; biologic response modifiers, such as interferon-α, IL-2, G-CSF, and GM-CSF; miscellaneous agents, including platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide;Hormones and antagonists, including adrenocortical steroid antagonists (such as prednisone and equivalents, dexamethasone and aminoglutethimide); progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens, such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone / equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens, such as flutamide.;

[0081] The doses of the therapeutic moieties or agents provided are administered in doses measured, for example, in mg / kg. The expected mg / kg doses of the disclosed therapeutic agents include from about 1 mg / kg to about 60 mg / kg. Specific dose ranges in mg / kg include from about 1 mg / kg to about 20 mg / kg, from about 5 mg / kg to about 20 mg / kg, from about 10 mg / kg to about 20 mg / kg, from about 25 mg / kg to about 50 mg / kg and from about 30 mg / kg to about 60 mg / kg. The precise effective amount for a subject will depend on the subject's weight, size and general health; the nature and extent of any medical condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0082] As used herein, "effective amount" means an amount of the reagent of the present invention sufficient to visualize the identified disease or medical condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect is detected, for example, by improvement of the clinical condition or alleviation of symptoms. The precise effective amount for a subject will depend on the subject's weight, size and general health; the nature and extent of the medical condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0083] Visualization of the reagent

[0084] Binding to colon cells is visualized by any method known to those of ordinary skill in the art. As discussed herein, visualization is, for example but not limited to, in vivo, ex vivo, in vitro or in situ visualization.

[0085] In one embodiment, visualization is performed by imaging and can be carried out using a wide-field endoscope (Olympus Corporation, Tokyo, Japan), which is specifically designed to collect fluorescence images with high spatial resolution over large mucosal surface areas at the macroscopic scale (millimeters to centimeters). This capability is required to rapidly screen large surface areas, such as those found in the distal esophagus during endoscopy, to localize regions of suspected disease [Wang et al., Gastrointestinal Endoscopy 1999; 49:447-55]. The technique has been adapted for fluorescence detection and is compatible with dye-labeled probes. The instrument can image in three different modes, including white light (WL), narrow-band imaging (NBI), and fluorescence imaging. Narrow-band imaging is a new technique that represents a variation of traditional white light illumination, which limits or narrows the wavelength range by altering the spectrum with a filter.

[0086] This method enhances the contrast in endoscopic images by adjusting the light to maximize the absorption of hemoglobin present in the vasculature of the intestinal metaplasia region, thereby providing more visual details of the esophageal mucosa. The WL and NBI images are collected by a central objective lens, and the fluorescence image is collected by a second objective lens located peripherally. There is a distance of approximately 3 mm between the centers of the white light and fluorescence objective lenses, which results in only a slight misregistration between the two images. In addition, there is an air / water nozzle to remove debris from the objective lens, and an instrument channel with a diameter of 2.8 mm is available for delivering biopsy forceps. The objective lens is forward-looking and has a 140-degree field of view (FOV) defined by the maximum illumination angle. The depth of field (DOF) of the WL / NBI imaging mode is defined by the range of distances between the distal end of the endoscope and the mucosal surface, which allows the image to be focused, and it is 7 to 100 mm, and for fluorescence, it is 5 to 100 mm. The lateral resolution measured at a distance of 10 mm from the mucosa is 15 μm for WL / NBI and 20 μm for fluorescence. A xenon light source provides illumination for all three modes, which is determined by a filter wheel located in the image processor. Illumination for all three imaging modes is provided through two fiber optic light guides. In the WL mode, the full visible spectrum (400 to 700 nm) is provided, while in the NBI mode, the filter wheel narrows the spectral bands in the red, green, and blue regions. In the fluorescence mode, a second filter wheel enters the illumination path and provides fluorescence excitation in the spectral band of 395 to 475 nm. In addition, illumination of 525 to 575 nm is provided to provide reflected light in the green spectral range centered at 550 nm. The fluorescence image is collected by a CCD detector located peripherally, which has a 490–625 nm band-pass filter to block the excitation light. Normal mucosa emits bright autofluorescence, so the composite color is bright green. Since the increased vasculature in neoplastic mucosa absorbs the autofluorescence, the displayed intensity is reduced.

[0087] The medical endoscope can be used to collect images after reagent application and incubation in the colon using 1) white light, 2) narrow band, and fluorescence. After entering the colon, 5-second videos are collected and digitized in white light and narrow band imaging modes. Imaging in this mode is used to evaluate the spatial extent of intestinal metaplasia for a comprehensive assessment of polypeptide binding. Then, approximately 3 ml of fluorescently labeled peptide at a concentration of 10 μM is topically applied to the colon using a spray catheter, carefully covering the entire mucosa. The dosage of the reagent of the present invention can be determined by those of ordinary skill in the art.

[0088] In some embodiments where the detectable label is a radiolabel, the radiolabel is detected by nuclear imaging. Nuclear imaging is understood in the art as a method of generating images by detecting radiation from different parts of the body after administration of a radioactive tracer material. The images are recorded on a computer and film.

[0089] Other methods according to the present disclosure involve obtaining a tissue sample from a patient. The tissue sample is selected from the group consisting of tissues or organs of the patient.

[0090] Formulation

[0091] In various aspects, the compositions of the present invention are formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the specific mode of administration and dosage form. The compositions are generally formulated to achieve a physiologically compatible pH, and the range is from about pH 3 to about pH 11, or from about pH 3 to about pH 7, depending on the formulation and the route of administration. In alternative embodiments, the pH is adjusted to a range of about pH 5.0 to about pH 8. In various aspects, the compositions include a therapeutically effective amount of at least one compound as described herein, and one or more pharmaceutically acceptable excipients. Optionally, the compositions contain combinations of the compounds described herein, or may include a second active ingredient for treating or preventing bacterial growth (e.g., but not limited to, antibacterial or antimicrobial agents), or may include combinations of reagents according to the present disclosure.

[0092] Suitable excipients include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated virus particles. Other exemplary excipients include antioxidants (e.g., but not limited to ascorbic acid), chelating agents (e.g., but not limited to EDTA), carbohydrates (e.g., but not limited to dextrin, hydroxyalkyl cellulose, and hydroxyalkyl methyl cellulose), stearic acid, liquids (e.g., but not limited to oils, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, etc.

[0093] The following examples are presented by way of illustration and are not intended to limit the scope of the subject matter disclosed herein.

[0094] Example

[0095] Example 1

[0096] Cell lines, media, and chemicals

[0097] The human CRC cell lines HT29, SW480, CCD841 and the mouse embryonic fibroblast cell line NIH 3T3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The S114 cell line contains NIH 3T3 cells transformed with human HGF / SF and Met and expresses cMet. We cultured HT29 cells using McCoy 5A medium (Gibco), and SW480, NIH3T3, and S114 cells using Dulbecco's Modified Eagle Medium (Gibco). Eagle's Minimum Essential Medium (Lonza) was used for CCD841 cells. All cells were cultured at 37 °C, 5% CO 2 2. Medium was supplemented with 10% fetal bovine serum (FBS). Cells were passaged using 0.25% trypsin containing EDTA (Mediatech, Manassas, VA). Cell numbers were quantified on a hemocytometer. Peptide synthesis reagents were obtained from Anaspec (Anaspec, Fremont, CA) or AAPPTEC (AAPPTEC, Louisville, KY), were of the highest available grade (>99% purity), and were used without further purification. Unless otherwise stated, solvents and other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO).

[0098] cMet-specific peptides

[0099] A phage display library of heptapeptides (Ph.D.-7 New England Biolabs) was used for biopanning against the extracellular domain of purified cMet protein, i.e., cMet-ECD (10692-H08H, Sino Biological Inc.) 32 . Candidate phages with the highest enrichment were selected for further evaluation. The reactivity of HT29 cells was evaluated using an enzyme-linked immunosorbent assay (ELISA). The binding interaction between the candidate peptides and cMet was evaluated using the non-crystallographic structures 1UX3 and 2UZX using Pepsite software 55 . Using the above protocol, phages containing the QQTNWSL (SEQ ID NO: 1) (QQT*) peptide were enriched after 4 rounds of biopanning. A randomized scrambled sequence, i.e., TLQWNQS (SEQ ID NO: 3) (TLQ*), was used as a control. Peptides were synthesized using standard Fmoc-mediated solid-phase chemistry 33, the C-terminus of the peptide was labeled with the NIR dye Cy5.5 (Lumiprobe, Hallandale Beach, FL) via a 5-amino acid (GGGSK; SEQ ID NO: 2) linker. The synthesis of the two peptides was carried out using a PS3 automated synthesizer (Protein Technologies Inc., Tucson, AZ). Fmoc- and Boc-protected L-amino acids were used and assembled on rink amide MBHA resin. The C-terminal lysine was incorporated in the form of Fmoc-Lys(ivDde)-OH, and the N-terminal amino acid was incorporated with Boc protection to avoid removal of the excess Fmoc during deprotection of the ivDde moiety prior to fluorophore labeling. After synthesis, the ivDde side-chain protecting group was removed with 5% hydrazine in DMF (3 × 10 min) with continuous stirring at room temperature (RT), and then the resin was transferred to a reaction vessel for manual dye labeling. The resin was washed with DMF and DCM 3 × 1 min. The protected resin-bound peptide was incubated overnight with Cy5.5-NHS ester in the presence of DIEA and stirred at room temperature for 24 - 48 h, and the completion of the reaction was monitored by qualitative ninhydrin test. Then the peptide was cleaved from the resin with cooled trifluoroacetic acid (TFA):triisopropylsilane:water (9.5:0.25:0.25, v / v / v) with stirring at room temperature in the dark for 4 h. After separating the peptide from the resin, the filtrate was evaporated to dryness under N 2 2 and then precipitated with cold diethyl ether by incubation overnight at -20 °C. The precipitate was centrifuged at 3000 rpm for 5 min and washed 3 times with diethyl ether. The crude peptide was suspended in 1:1 acetonitrile:H 2 2O (v / v) and purified by high performance liquid chromatography (Waters, Milford, MA) with a C18 column using a water (0.1% TFA)-acetonitrile (0.1% TFA) gradient. The final purity of the peptide was confirmed using an analytical C18 column. Mass spectrometry (MALDI-TOF, Bruker AutoFlex Speed) was used to measure the mass-to-charge ratio (m / z) of the product.

[0100] Spectral measurements

[0101] The absorption spectra of the peptides were measured using a UV-Vis spectrophotometer (NanoDrop 2000, Thermo Scientific), and fluorescence emission was collected using a fiber-optic coupled spectrophotometer (Ocean Optics) with a diode-pumped solid-state laser (Technica Laser Inc.) excited at λex = 671 nm. The spectral plots were drawn using Origin 6.1 software (OriginLab Corp).

[0102] Confocal fluorescence microscopy

[0103] HT29, SW480, S114, and NIH3T3 cells were seeded in 12-well cell culture plates with round glass coverslips until approximately 80% confluent. Cells were blocked with 1X PBS plus 2% BSA at 4 °C for 1 hour, then incubated with 5 μM peptide for 10 minutes at room temperature in the dark, washed three times, fixed with 4% PFA for 5 minutes, washed with 1X PBS, and then mounted on slides with ProLong Gold reagent containing DAPI (Invitrogen, Waltham, MA). As a positive control, after blocking with 2% BSA at 4 °C for 1 hour, 1:3000 diluted rabbit anti-cMet monoclonal primary antibody (Cell Signaling Technology, #8198) was incubated with the cells overnight at 4 °C. After that, the cells were washed three times with 1X PBS and further incubated with a 1:500 dilution of AF488-labeled goat anti-rabbit immunoglobulin G secondary antibody (Life Technologies, #A-11029) for 1 hour at room temperature, washed three times, and then mounted on slides with ProLong Gold reagent containing DAPI. Confocal fluorescence images were collected using a 63X oil immersion objective (Leica SP5 Inverted 2-Photon FLIM Confocal). Fluorescence intensities from five cells in two independent images were quantified using custom Matlab (Mathworks) software.

[0104] Downregulating cMet with siRNA

[0105] We decreased the cMet protein level with siRNA and then evaluated the binding of QQT*-Cy5.5 and TLQ*-Cy5.5 to the surface of si-cMet transfected HT29 cells to verify specific peptide binding. We used siRNA1 (SASI_Hs01_00133002, Sigma) for HT29, siRNA2 (SASI_WI_00000001, Sigma) for S114, and siRNA #1 universal negative control (SIC001, Sigma) for the negative control. We transfected the cells with Lipofectamine 2000 (11668027, Invitrogen) according to the manufacturer's instructions. Knockdown of cMet was confirmed by western blotting ( Figure 18) Incubate the cells with 5 μM peptide at RT (i.e., room temperature) for 5 minutes, then fix and mount on slides with ProLong Gold reagent containing DAPI as described previously. As a positive control, incubate the cells with a 1:3000 dilution of monoclonal rabbit anti-cMet primary antibody (Cell Signaling Technology, #8198), fix and mount on slides with ProLong Gold reagent containing DAPI. Collect confocal fluorescence images using a 63X oil immersion objective (Leica SP5 Inverted 2-Photon FLIM Confocal).

[0106] Competition of peptide binding

[0107] We used a competition assay between labeled QQT*-Cy5.5 and unlabeled QQT* or recombinant human hepatocyte growth factor (HGF, 194-HG-005, R&D) to verify the specific binding of QQT**-Cy5.5 to HT29 cells. In triplicate, grow approximately 10 3 HT29 cells on coverslips to approximately 70% confluence. First, add unlabeled QQT* and 0, 25, 50, 100, 200, and 400 μM of TLQ* peptide or 0, 5, 10, 25, 50, or 100 ng / mL of HGF and incubate with the cells at 4 °C for 30 min (i.e., minutes). Wash the cells three times with 1X PBS, then incubate with 5 μM QQT*-Cy5.5 at 4 °C for another 30 min. Wash the cells three times with 1X PBS, then fix with 4% PFA for 10 minutes. Wash the cells with 1X PBS and mount with ProLong Gold reagent containing DAPI (Invitrogen). Collect confocal fluorescence images for each concentration using a 63X objective (Leica SP5 Inverted 2-Photon FLIM Confocal), and quantify the intensity of five cells in three independent images using custom Matlab (Mathworks, Natick, MA) software.

[0108] Effect of peptide on cell signaling

[0109] Before incubation with hHGF (hHGF, 294-HG-005, R&D) or peptide, HT29 cells were treated with serum-free medium overnight to starve them. Recombinant human HGF protein was added to HT29 cells at a concentration of 25 ng / ml for 10, 30 or 120 minutes in different wells. QQT*-Cy5.5 and TLQ*-Cy5.5 were added at concentrations of 5 or 100 μM for 10, 30 and 120 minutes. Peptides were added at concentrations of 5 and 100 μM for 10, 30 or 120 minutes. Cells were then washed with 1X PBS and lysed with Pierce RIPA buffer containing Halt phosphatase inhibitor mixture (Thermo Fisher) and Halt protease inhibitor mixture (Thermo Fisher). Protein content was quantified by bicinchoninic acid assay (BCA). Anti-cMet antibody (Cell Signaling, #8198), phospho-cMet (Tyr1234 / 1235) antibody (Cell Signaling, #3077), anti-AKT antibody (Cell Signaling, #4691), anti-phospho-AKT antibody (Cell Signaling, #9271), anti-ERK1 / 2 antibody (Abcam,

[0110] #ab17942), anti-phospho-ERK1 / 2 antibody (Abcam, #ab50011) and anti-tubulin antibody (Invitrogen, #32-2600) were used according to the manufacturer's instructions.

[0111] Alamar Blue assays were performed using HT29 and CCD841 cells. After overnight culture in serum-free medium, approximately 3×10 3 cells were seeded in serum-free medium in each well of a 96-well plate at a final volume of 100 μL per well. Cells were incubated with HGF (25 ng / mL) or peptide (5 and 10 μM) at 37 °C for 48 hours. Alamar Blue reagent (10 μL), equivalent to 10% of the well volume, was added and incubated at 37 °C for 4 hours. Fluorescence was measured with excitation at λex = 530–560 nm and emission at λex = 590 nm.

[0112] Characterization of Peptide Binding

[0113] We evaluated the binding affinity of QQT*-Cy5.5 to HT29 cells by measuring the apparent dissociation constant. HT29 cells were blocked with 0.5% BSA and then approximately 10 5Cells were incubated with QQT*-Cy5.5 at concentrations of 0, 10, 25, 50, 75, 100, 125, 150 or 200 nmol / L at 4 °C for 1 hour. Then the cells were washed three times with 1X PBS containing 0.5% BSA to remove unbound peptides, and then analyzed by flow cytometry (FACS Canto; BD Biosciences, San Jose, CA). Nonlinear regression analysis was performed using Origin 6.1 data analysis software (OriginLab, Northampton, MA), and the equilibrium dissociation constant K was calculated using the sample mean value. D . K D = 1 / K A By performing least-squares fitting of the data to the nonlinear equation I[X] = (I 0 + I max k a [X]) / (I 0 + k a [X]). I 0 and I max are the initial and maximum fluorescence intensities corresponding to peptide-free and peptide-saturated, respectively, and [X] represents the concentration of bound peptide. 34 .

[0114] The time scale of QQT*-Cy5.5 binding to HT29 cells was evaluated by measuring the apparent binding time constant k. HT29 cells were blocked with 0.5% BSA, and then approximately 10 5 cells were incubated with 5 μM QQT*-Cy5.5 at 4 °C for time intervals from 0 to 20 minutes. Then the cells were washed three times with cold 1X PBS containing 0.5% BSA to remove unbound peptides. After centrifugation, the cells were fixed with 4% PFA at 4 °C for 30 minutes and then analyzed by flow cytometry. Using Flowjo software (LLC, Ashland, OR), the median fluorescence intensity (y) at different time points (t) was taken as the ratio to HT29 cells without added peptide. The rate constant k was calculated by fitting the data to the first-order kinetic model y(t) = I max [1 - exp(-kt)], where I max is the maximum value, using Prism 5.0 software (GraphPad, La Jolla, CA).

[0115] The interaction between the peptide and murine cMet was evaluated using a pull-down assay (Paul et al., Methods 54:387-395 (2011)). The peptide was immobilized on EHS activated beads (17-0906-01, GE) and incubated with purified murine cMet-ECD protein (50622-M08H, Sino Biological). After washing, the bound protein was detected by Western blotting( Figure 18 ).

[0116] Organoid specimens

[0117] Patient specimen information is listed in Table 1. Normal specimens (#87) in this study were from tissues of deceased donors; while adenoma organoids were from biopsied large adenomas: #245 (sessile serrated); #590 (tubular); #584 (tubular 20 mm); and #236 (FAP).

[0118] To authenticate the samples, short tandem repeat (STR) analysis was used to identify 15 tetranucleotide repeat loci of human genomic DNA (AMPFLSTR Identifier Plus Assay, Applied Biosystems; University of Michigan DNA Sequencing Core). In addition, amelogenin sex determination markers were run on a 3730XL genetic analyzer (Applied Biosystems). Mycoplasma contamination of the cultures was routinely tested using the Lonza MycoAlert kit (service of UMICH Transgenic Animal Model Core).

[0119] Table 1

[0120]

[0121] Table 1. Colonic normal and adenoma organoids from patients. A targeted colorectal cancer DNA sequencing panel was used to determine the presence of variants in 71 different oncogenes and tumor suppressor genes that are commonly mutated in colorectal cancer. Stop codon (*); frameshift (FS).

[0122] Organoid culture

[0123] Human organoid cultures were previously established from normal and adenoma tissues 36-38 , and were provided by the Translational Tissue Modeling Laboratory (TTML; University of Michigan).

[0124] The cultures were grown in Matrigel (diluted to 8 mg / mL with growth medium; Corning, #354234) in 6-well tissue culture plates (USA Scientific CytoOne, #CC7682-7506). The cultures were passaged by grinding and dissociating the Matrigel in cold Dulbecco's phosphate-buffered saline (DPBS), centrifuging at 300 x g, and plating on the first day with 2.5 μM CHIR99021 (Tocris; 4423), a highly selective GSK3 inhibitor, and 10 μM Y27632 (Tocris; TB1254-GMP / 10), a highly selective p160ROCK inhibitor.

[0125] Normal (#87) and sessile serrated (#245) organoids were cultured in LWRN complete medium, which contained 50% L-WRN conditioned medium (source of Wnt3a, R-spondin-3, and Noggin) 39 , Advanced DMEM / F-12 (Gibco, 12634028), N-2 Medium Supplement (Gibco; #17502048), B-27 Supplement minus vitamin A (Gibco; #12587010), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich, A9165), 2 mM GlutaMax (Gibco, #35050-061), 10 mM HEPES (Gibco, #15630080), 50 units / mL penicillin, 0.05 mg / mL streptomycin (Gibco, #15070063), 50 μg / ml Primocin (InvivoGen; ##ant-pm-1), 100 ng EGF / mL (R&D Systems, Inc., 236-EG), 10 μM SB202190 (Sigma-Aldrich; S7067), 500 nM A83-01 (R&D Tocris, #2939), and 10 μM Y27632 (Tocris; TB1254-GMP / 10). FAP adenomas (#236) were cultured in LWRN complete medium without SB202190.

[0126] Tubular adenoma organoids (#590) were cultured in Stemline complete medium, which contained Stemline TMKeratinocyte Medium II (Sigma S0196) supplemented with Stemline Growth Supplement (Sigma S9945), 2 mM GlutaMax, 4 mM L-glutamine, and 50 μg / ml Primocin. Prior to harvest for transplantation, the #590 culture was treated with 5 μM Y27632 for 18 hours. The tubular adenoma organoids #584 were cultured in 50% of the above Stemline complete medium and 50% of the above LWRN complete medium.

[0127] Cultures were harvested from Matrigel in cold DPBS, triturated 30 times with a 1 mL pipette tip, and centrifuged at 300 x g for 3 minutes at 4°C. Organoid pellets were resuspended in 10 mL cold DPBS and mechanically dissociated using a gentleMACS Octo Dissociator (Miltenyi Biotec; 130-096-427), using the program h_Tumor_01.01 then m_Lung-01.01. Organoid fragments were further dissociated by pipetting 20X with a 1 mL pipette tip. Large debris was removed using a 100 μm BSA-coated cell strainer (Corning, DL 352360). Cells were slowly centrifuged at 100 x g to reduce single cell content. Cell aggregates were resuspended in cold DPBS supplemented with 5% Matrigel and 10 μM Y27632. All plasticware, including the gentleMACS C tubes (Miltenyi; 130-093-237), was treated with 0.1% BSA in DPBS to reduce organoid adhesion.

[0128] Organoid transplantation

[0129] Acute colitis was induced in 8-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ mice (005557, Jackson Laboratory) by feeding them 3.0% DSS (molecular weight 40,000; Cat# AAJ6360622; Alfa Aesar) dissolved in water for 5 days (d). 40,41 . On day 6, donor organoids were released from type I collagen gels, dissociated with EDTA, and washed with PBS containing BSA as in the passage procedure. Approximately 2.5 - 5E5 cell aggregates in 200 μL were transplanted into each mouse, as described previously. 42,43。The organoids were instilled into the colonic lumen as a suspension using a syringe and a thin flexible catheter 4 cm in length and 2 mm in diameter. After infusion, anal margin glue was applied for 6 hours to prevent immediate expulsion of luminal contents, and then the glue was removed with ethanol. The mice were housed as normal after transplantation. The mice were housed under pathogen-free conditions and had free access to water under controlled humidity (50 ± 10%), light (12 / 12 hour light / dark cycle), and temperature (25 °C). Anesthesia was induced and maintained via a nose cone, with inhalation of a mixture of isoflurane and oxygen at a concentration of 2 - 4% and a flow rate of approximately 0.5 L / min. Organoid imaging was performed 3 weeks after transplantation.

[0130] CPC; Apc mouse model

[0131] The CPC; Apc mouse model, which can develop spontaneous adenomas in the distal colonic epithelium, was also used. 44 。Under the control of the Cdx2 promoter (CDX2P - 9.5NLS - Cre), Cre recombinase can sporadically delete the adenomatous polyposis coli (APC) allele, resulting in polyploid colonic adenomas or flat lesions. We collected images of mice (n = 8) aged from 7 to 10 months.

[0132] Peptide in vivo imaging

[0133] In vivo imaging was performed with the approval of the University of Michigan Committee on Use and Care of Animals. CPC; Apc mice were used for in vivo imaging. This mouse strain has been genetically engineered to sporadically delete the adenomatous polyposis (APC) allele under the control of the Cdx2 promoter (CDX2P - 9.5NLS - Cre), resulting in spontaneous formation of flat or polyploid adenomas in the distal colon (Hinoi et al., Cancer Res 67:9721 - 9730 (2007)). The mice were housed under pathogen - free conditions and had free access to water under controlled humidity (50 ± 10%), light (12 / 12 hour light / dark cycle), and temperature (25 °C). Before imaging, the mice were fasted for 4 - 6 hours. Anesthesia was induced and maintained via a nose cone, with inhalation of a mixture of isoflurane and oxygen at a concentration of 2 - 4% and a flow rate of 0.5 L / min.

[0134] A rigid small animal endoscope (Karl Sorz Veterinary Endoscopy) was inserted into the rectum (Liu et al., Gut 62:395 - 403 (2013)) and used to image the distal colon. 45Mucus and debris in the distal colon were removed by vigorously flushing three times with warm tap water. White light illumination was first applied to identify the presence of adenomas. The distance between the endoscope tip and the anus and the clockwise position of the polyp were recorded. The QQT*-Cy5.5 solution (100 μM, 1.5 mL) was locally delivered to the distal colon through the instrument channel (3Fr). After incubation for 5 minutes, unbound peptides, feces, and debris were flushed three times with warm tap water before image collection. Three days later, clearance of the QQT*-Cy5.5 signal was confirmed by endoscopy, and then the same mice were imaged using TLQ*-Cy5.5 as a control. The ratio of fluorescence and reflection images was determined to correct for differences in distance and geometry across the image field of view (FOV) (Joshi et al., Endoscopy 48:A1-A13 (2016)). A total of 3 independent regions of size 20 × 20 μm 2 were randomly identified from the location of the adenoma (target) and adjacent normal colonic mucosa (background).

[0135] The mean fluorescence intensity was used to calculate the target-to-background (T / B) ratio. Images were processed and analyzed using custom software in Matlab (Mathworks) (Joshi et al., Gastroenterology 152:1002-1013e1009 (2017)). Fluorescence intensity was quantified using Matlab software. The fluorescence intensity of the region of interest (ROI) was corrected by the ratio of fluorescence to reflectance, with the fluorescence of the ROI as the target (T), and an adjacent equal-area normal colonic region of the mouse was selected as the background (B). 46 Flows showing minimal motion artifacts and no debris (feces, mucus) were selected for image quantification. Individual frames were exported using custom Matlab software. Ex vivo validation of high cMet expression in murine colon tumorigenesis

[0136] After imaging, the mice were euthanized. The colon was excised and split longitudinally, the excised colon was rinsed with PBS, and opened longitudinally for imaging with an NIR fluorescence imaging system ( LI-COR Biosciences). Images were collected at 85-μm resolution using λex = 685 nm and λem = 720 nm. Images were analyzed using custom software (Image Studio, Li-Cor Biosciences). A normal colonic region of equal area adjacent to the polyp was used to measure the background. Prism software (v6.02, GraphPad) was used to plot the data.

[0137] cMet expression is increased in CPC;Apc murine colon adenomas and human proximal colon tumorigenesis by IHC

[0138] Prepare 10-μm thick serial formalin-fixed sections, de-wax, and perform antigen retrieval using standard methods. Briefly, incubate the sections in xylene three times for 3 minutes each, wash in 100% ethanol twice for 2 minutes each, and wash in 95% ethanol twice for 2 minutes each. Incubate the sections in dH 2 O twice for 5 minutes each for rehydration. Antigen exposure is carried out in boiling 10 mM 1X pH 6.0 citrate buffer for 10 minutes. After cooling at room temperature (RT) for 20 - 30 minutes, wash the sections in dH 2 O for 2 minutes, three times. Incubate the sections in 3% H 2 O 2 for 10 minutes to block endogenous peroxidase activity. Wash the sections in dH 2 O for 5 minutes, three times, and wash in phosphate-buffered saline containing Tween 20 (PBST) for 5 minutes. Block with 10% normal goat serum or DAKO protein blocker (X0909, DAKO) at room temperature for 45 minutes. Incubate the sections with a 1:100 dilution of monoclonal rabbit anti-cMet antibody (Abcam, EP1454Y, ab51067) containing 2.5% normal goat serum overnight at 4°C and wash in 0.1% TBST for 5 minutes, three times. Apply a 1:200 dilution of goat anti-rabbit secondary antibody (Abcam, ab150077) to each section and incubate at room temperature for 30 minutes. Prepare controls using the same method but without the anti-cMet primary antibody. Remove the secondary antibody by washing in 0.1% TBST for 5 minutes, three times. Then incubate the sections in pre-mixed Elite Vectastain ABC reagent (Vector Labs, PK-6100) at room temperature for 30 minutes. Wash the sections in 0.1% TBST for 5 minutes, three times, and develop the color with 3,3'-diaminobenzidine substrate. Monitor the reaction for 1 - 3 minutes and then immediately quench the reaction by dipping the slides into dH 2 O after the sections have developed color. Add hematoxylin as a counterstain for approximately 20 seconds and then dehydrate the sections in increasing concentrations of ethanol (70%, 80%, 95%, 95%, 100%, 100%). Mount the coverslips in Permount TM mounting medium (Fisher, Pittsburgh, PA, #SP15-100) in xylene. Process the serial sections for histology (H&E). Prepare controls using the same method but without the anti-cMet primary antibody. Process the serial sections for routine histology (H&E).

[0139] Immunofluorescent staining of cMet using QQT*-Cy5.5 / antibody in CPC; Apc mouse colon adenomas and human proximal colon tumorigenesis

[0140] Harvest mouse colon adenoma specimens, fix them in formalin, and embed them in paraffin. Specimens of tubular adenomas (n = 21), sessile serrated adenomas (n = 13), hyperplastic polyps (n = 7), and normal colon mucosa (n = 10) from the proximal human colon were obtained from the tissue bank of the Department of Pathology at the University of Michigan. Human specimens were processed in the same way as mouse colon specimens. Sections (5 mm thick) were cut and mounted on slides (Superfrost Plus; Fischer Scientific). The serial 5-μm sections were deparaffinized and antigen retrieval was performed as described above. The sections were blocked with 10% normal goat serum (Fisher Scientific, 50062Z) for 10 minutes at room temperature and then rinsed with PBS. The sections were incubated with 5 μM QQT*-Cy5.5 and 2% BSA for 10 minutes at room temperature. Then the sections were washed 3 times with 0.1% PBST and further incubated with a 1:200 dilution of anti-cMet primary antibody (Cell Signaling Technology, #8198) and 2% BSA for 2 hours at room temperature in the dark. The sections were washed 3 times with 0.1% PBST for 3 minutes each and then incubated with a 1:500 AF488-conjugated goat anti-rabbit secondary antibody (Abcam, ab150077) and 2% BSA for 1 hour at room temperature in the dark. After washing 3 times with 0.1% PBST for 3 minutes each, the sections were mounted with ProlongGold reagent containing DAPI (Invitrogen). Adjacent sections were processed for histology (H&E). We placed 3 boxes of size 20×20 μm 2 completely within the colonic epithelium in each image and measured the mean fluorescence intensity using custom Matlab software. Regions of saturated intensity were avoided.

[0141] Example 2

[0142] cMet-specific peptide

[0143] A highly diverse phage display library of linearized heptapeptides was biopanned against the extracellular domain (ECD) of cMet to identify the linear heptapeptide sequence QQTNWSL (SEQ ID NO:1). Using a structural model of cMet, this peptide showed the lowest P-value for binding interactions. The C-terminus of the peptide (black) was covalently linked to the fluorophore Cy5.5 (red) via a GGGSK linker (blue), hereafter referred to as QQT*-Cy5.5, Figure 1A. The linker separates the peptide from the fluorophore to prevent steric hindrance. The scrambled sequence TLQWNQS (SEQ ID NO:3) was also labeled with Cy5.5 to serve as a control, hereafter referred to as TLQ*-Cy5.5, Figure 1B. The three-dimensional models show the differences in the biochemical structures, Figures 1C, D. The peak absorption and emission of the Cy5.5-labeled peptide occur in the near-infrared (NIR) spectrum, Figures 1E, F, where hemoglobin absorption, tissue scattering, and tissue autofluorescence are minimal. The tissue penetration depth is maximized, and the effects of hemoglobin absorption, tissue scattering, and tissue autofluorescence are minimized in this protocol. The peptide synthesized by HPLC had a purity greater than 95%, and the experimental mass-to-charge ratio (m / z) measured by mass spectrometry was 1827.67, which was consistent with the expected value, Figure 9 。

[0144] Example 3

[0145] Verification of in vitro binding to cells

[0146] An siRNA knockdown experiment was performed using HT29 human colorectal cancer cells to verify the specific binding of QQT*-Cy5.5 to cMet. Under a confocal microscope, QQT*-Cy5.5 (red) and an AF488-labeled anti-cMet antibody (green) strongly bound to the surface of control HT29 cells transfected with siC (control) (arrows), Figure 2 A, B, while TLQ*-Cy5.5 showed minimal binding, Figure 2 C. A decrease in fluorescence intensity was observed using HT29 knockdown cells (sicMet), Figure 2 D, E, and TLQ*-Cy5.5 had little signal, Figure 2 F. The quantification results showed that this decrease was significant, Figure 2 G. Western blot showed the effect of cMet expression knockdown in the cells, Figure 2 H. In addition, significantly higher fluorescence intensities were observed for the binding of QQT*-Cy5.5 and anti-cMet-AF488 to HT29 cells (cMet+) compared to SW480 human colorectal cancer (cMet-) cells, Figure 10 。Similar results were found using mouse S114 (cMet+) and NIH3T3 (cMet-) cells, Figure 11 。

[0147] Example 4

[0148] Peptide characterization

[0149] The specific binding of QQT*-Cy5.5 to cMet was confirmed by adding unlabeled QQT* for competitive binding. A significant decrease in fluorescence intensity was observed in a concentration-dependent manner, Figure 3 A. These results indicate that the peptide rather than the linker or fluorophore mediates the binding interaction. In contrast, the fluorescence intensity of QQT*-Cy5.5 binding to HT29 cells did not change with the addition of hepatocyte growth factor (HGF), a known cMet ligand, at concentrations ranging from 0 to 100 ng / mL, Figure 17 A. By comparison with TLQ*, pull-down assays showed strong bands from the binding of QQT* to murine cMet-ECD, Figure 17 B. Co-localization of QQT*-Cy5.5 (red) and anti-cMet-AF488 (green) binding to the surface of HT29 cells (arrows) was observed on merged images, with a Pearson correlation coefficient of ρ = 0.73, Figure 3 B. The apparent dissociation constant of k d = 57 nM for the binding of QQT*-Cy5.5 to HT29 cells was measured, Figure 3 C, and the apparent binding time constant of k = 0.62 min -1 (1.6 min) for the same cells was measured, which supports rapid binding upon local administration, Figure 3 D.

[0150] Example 5

[0151] Effect of the peptide on cell signaling

[0152] Hepatocyte growth factor (HGF) is a known ligand of cMet and was incubated with HT29 cells (cMet+) as a positive control. Strong phosphorylation activities of cMet (p-cMet), downstream AKT (p-AKT), and ERK1 / 2 (p-ERK1 / 2) were observed on Western blots, Figure 4 . However, the addition of QQT* at concentrations of 5 or 100 μM did not result in any change in the phosphorylation of any of these downstream cell signaling markers.

[0153] More specifically, no competition was observed between QQT* and HGF, which supports the lack of interaction and effect on downstream signaling, Figure 17 A. These results indicate that the peptide and HGF bind to different sites on the cMet target. Western blots were performed to evaluate markers of downstream cell signaling activation, Figure 4A. Incubation of HGF (positive control) with HT29 cells showed strong phosphorylation activity of cMet (p-cMet), downstream AKT (p-AKT), and ERK1 / 2 (p-ERK1 / 2). In contrast, addition of QQT*-Cy5.5 at concentrations of 5 and 100 μM did not result in any change in phosphorylation of the substrates. Compared with HGF, the Alamar Blue assay showed that addition of QQT*-Cy5.5 at concentrations of 5 or 100 μM for 48 hours had no effect on the growth of HT29 and CCD841 cells. Figure 4 B, C. CCD841 normal colon cells were used to evaluate the effect of the peptide on the cell phenotype in non-tumor cells.

[0154] Example 6

[0155] In Vivo Imaging of Genetically Engineered CRC Mice

[0156] The results of the pull-down assay supported the specific binding of QQT*-Cy5.5 to murine cMet-ECD. Figure 17B. Rigid small animal endoscopes were used to collect in vivo images of CPC; Apc mice. The white light images collected using this small animal endoscope showed no visibly obvious polyps (arrows) in the colon of CPC; Apc mice, Figure 12A. This mouse was genetically engineered to somatically delete one Apc allele under Cre regulation and spontaneously form flat adenomas and polyploid adenomas. Then QQT*-Cy5.5 was locally applied, incubated for about 5 minutes, and the unbound peptide was rinsed off. The NIR fluorescence images collected after staining with QQT*-Cy5.5 showed the presence of flat lesions (arrows), Figure 12B. Fluorescence-registered reflection images were obtained, Figure 12C. Fluorescence images collected from the same lesion using TLQ*-Cy5.5 (control) 3 days later showed little signal, Figure 12D. Similar results were obtained from representative polyploid lesions, Figure 5E-H. The ratios of the fluorescence and reflection images from the flat lesions were determined to correct for differences in distance and geometry across the image field of view (FOV) to allow accurate quantification of image intensity, Figure 5I. The fluorescence, reflectance, and ratio values of the dashed line in Figure 5I are shown, Figure 5J. Images collected from polyps were processed similarly. White light images collected in different mice showed the presence of polyps (arrows), Figure 12E (also see Figure 14A). The fluorescence images collected showed strong intensity from pre-malignant lesions (arrows), Figure 12F. Fluorescence-registered reflection images were obtained, Figure 12G. Fluorescence images collected from the same polyp using TLQ*-Cy5.5 3 days later showed minimal signal, Figure 12H. The ratio of the fluorescence and reflection images from the flat lesion in Figure 12A was used to correct for differences in distance and geometry across the image field of view and allow accurate quantification of image intensity, Figure 12I. The fluorescence, reflectance, and ratio values of the dashed line in Figure 12I are shown, Figure 12J. It was found that for both polyps and flat lesions, the average T:B ratio of QQT*-Cy5.5 was significantly greater than that of TLQ*-Cy5.5, Figure 12K and 14G. Histology (H&E) of flat lesions and polyps showed adjacent to normal colonic mucosa and showed features of low-grade dysplasia, Figure 12L, M.

[0157] Example 7

[0158] Macroscopic verification of ex vivo mouse colon

[0159] After imaging was completed, CPC; Apc mice were euthanized, the colon was excised and split longitudinally to expose the mucosal surface for collection of macroscopic white light and fluorescence images, Figures 5L, M and 13A, B. Polyp locations were co-localized on white light and fluorescence images, Figure 13C. In n = 5 mice, significantly higher mean fluorescence intensity was found from dysplasia compared to adjacent normal colonic mucosa in the n = 10 regions, Figure 13D. Immunohistochemistry was performed with known antibodies to verify increased expression of cMet in mouse dysplasia, Figure 13E, which was compared to normal colonic mucosa, Figure 13F.

[0160] This ex vivo imaging verified the specific binding of QQT*-Cy5.5 to cMet. The colon was excised and split longitudinally to expose the mucosal surface. Co-localization of polyps was visible on the merged images, Figure 5N. The adenoma border was clearly visible. The mean fluorescence intensity of the polyps was significantly higher than that of the adjacent normal colonic mucosa, Figure 5O. Using immunohistochemistry (IHC), the expression of cMet was increased in mouse adenomas compared to normal colon, Figures 5P, Q.

[0161] Example 8

[0162] Microscopic verification of mouse colon ex vivo

[0163] Increased fluorescence staining of QQT*-Cy5.5 and anti-cMet-AF488 (arrows) was observed on the surface of dysplastic colonocytes in sections of the colon of CPC; Apc mice using confocal microscopy, Figures 15A, B. The merged images showed co-localization of peptide and antibody binding (arrows) with a correlation of ρ = 0.78, Figure 15C. Minimal staining of peptide and antibody binding to normal colonic mucosa was observed, Figures 15D - F. Quantification results showed that the mean fluorescence intensity was significantly higher in dysplasia compared to normal colonocytes, Figure 15G. The ROC curve showed 93% sensitivity and 87% specificity with an area under the curve (AUC) of 0.96 for QQT*-Cy5.5 to distinguish dysplasia from normal tissue. Histology (H&E) of mouse adenomas and normal colon is shown, Figures 15H, I.

[0164] Example 9

[0165] In vivo imaging of patient-derived colon organoids

[0166] White light images collected with a small animal endoscope showed the presence of two normal human (arrows, Figure 19A) and one tubuloadenoma organoid (arrow, Figure 19E) implanted in the colon of immunodeficient mice. Then QQT*-Cy5.5 was topically applied, incubated for about 5 minutes, and unbound peptide was rinsed off. Fluorescence images showed minimal signal from normal organoids (arrows, Figure 19B) and strong intensity from pre-malignant tubuloadenoma organoids (arrows, Figure 19F). Fluorescence-registered reflectance images were obtained (Figure 19C, G). Fluorescence images collected 3 days later using TLQ*-Cy5.5 (control) from normal and adenoma organoids showed little signal (Figure 19D, H). The statistically averaged T:B ratio of QQT*-Cy5.5 on adenomas in vivo was found to be significantly greater than that on normal organoids, Figure 19L. H&E histological staining of normal and adenoma organoids is shown in Figure 19J, M. IHC staining of human-specific hCytokeratin on the same slide demonstrated successful transplantation of human organoids (Figure 19K, N). Using these tissue sections, IF co-staining of cMet antibody and QQT*-Cy5.5 showed minimal signal for normal colon (Figure 19K), while strong intensity for adenoma organoids (Figure 19O), determining that QQT*-Cy5.5 specifically binds to human tissues with overexpressed cMet. Statistical results are shown in Figure 19P. Using tissue sections and immunofluorescence, QQT*-Cy5.5 showed strong staining for adenomas and minimal staining for normal crypts, Figure 19I.

[0167] To investigate whether QQT*-Cy5.5 also binds to SSA, we generated a mouse model transplanted with SSA using the same method. White light of normal and SSA organoids is shown in Figure 16A, E (arrows). Fluorescence images collected from normal and SSA organoids are shown in Figure 16B, F (arrows). The signal of QQT*-Cy5.5 on SSA was significantly higher than that on normal organoids. Fluorescence-registered reflectance images were obtained (Figure 16C, G). Three days later, fluorescence images collected using TLQ*-Cy5.5 (control) from these two types of organoids showed minimal signal. The statistically averaged T:B ratio of QQT*-Cy5.5 on SSA in vivo was found to be significantly greater than that on normal organoids, Figure 19L. H&E histological staining of normal and SSA organoids is shown in Figure 16I, M. IHC staining of human-specific hCytokeratin on the same slide demonstrated successful transplantation of human organoids (Figure 16J, N). Using these tissue sections, IF co-staining of cMet antibody and QQT*-Cy5.5 showed minimal signal for normal colon (Figure 19K), while strong intensity for SSA organoids (Figure 16O). In summary, these in vivo imaging results indicate that QQT*-Cy5.5 can specifically bind to adenoma and SSA human organoids with high-level expression of cMet compared to normal organoids.

[0168] Example 10

[0169] Verification of cMet Expression in Human Colon

[0170] Staining of human colon with QQT*-Cy5.5 and anti-cMet-AF488 was evaluated in n = 42 formalin-fixed, paraffin-embedded (FFPE) human colon specimens, including tubular adenomas, sessile serrated adenomas (SSAs), hyperplastic polyps (HPs), and normal mucosa. Merged fluorescence images of representative sections of adenomas, SSAs, hyperplastic polyps (HPs), and normal colon mucosa are shown, which were collected with a confocal microscope, Figures 6A-D. For each histological classification, there was strong co-localization of peptide and antibody binding. Immunofluorescence of SSA specimens showed large, dilated crypts with numerous goblet cells, indicating abnormal maturation. Immunohistochemistry was performed to verify cMet expression. Strong staining (2+ / 3+) was observed for adenomas and SSAs, while weak staining (0+ / 1+) was observed for HPs and normal cells, Figures 6E-H. Representative histology (H&E) is shown, Figures 6I-L. For adenomas or SSAs, the mean fluorescence intensity of QQT*-Cy5.5 staining was significantly higher compared to HPs or normal colon, Figure 6M. A total of n = 11 SSA lesions were described endoscopically as having a flat appearance, and n = 2 were recorded as slightly protruding from the colonoscopy report. Representative histology (H&E) is shown for each histological classification, Figures 6K-N.

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[0237] As will be apparent from the context, all publications and patents mentioned in this application are hereby incorporated by reference in their entirety or in relevant parts. Various modifications and changes to the disclosed subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described manner of making or using the disclosed subject matter are intended to be within the scope of the appended claims, where such modifications are apparent to one or more persons skilled in the relevant art. <110> The Regents of the University of Michigan <120> In Vivo Detection of Colon Tumorigenesis Using a Near - Infrared Peptide Targeting Overexpressed cMET <130> 30275 / 53791A / PC <150> US 62 / 808,637 <151> 2019 - 02 - 21 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 1 Gln Gln Thr Asn Trp Ser Leu 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic linker <400> 2 Gly Gly Gly Ser Lys 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 3 Thr Leu Gln Trp Asn Gln Ser 1 5

Claims

1. Use of a peptide consisting of the amino acid sequence QQTNWSL shown in SEQ ID NO: 1 in the preparation of a kit for detecting intestinal tumor formation in colorectal tissue, wherein the intestinal tumor formation is selected from polyps, flat lesions, and colorectal cancer.

2. The use according to claim 1, wherein the peptide is derivatized by attachment to a linker.

3. The use according to claim 2, wherein the linker consists of the sequence GGGSK listed in SEQ ID NO:

2.

4. The use according to claim 2, wherein the derivatized peptide is labeled.

5. The use according to claim 4, wherein the peptide label is a fluorescent label.

6. The use according to claim 5, wherein the label is FITC, Cy 5.5, Cy 7, 1-anilinonaphthalene-8-sulfonic acid, 5-ROX, 5-TAMRA, 7-hydroxy-4-methylcoumarin, Alexa 350, Alexa 405, Alexa 430, Alexa488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 647, Alexa 660, Alexa 680, Alexa 700, aminocoumarin, allophycocyanin, Atto 647, blue fluorescent protein, Calcein, Calcium Crimson, Calcium Green, Calcium Orange, Cascade Blue, Cascade Yellow, cyan fluorescent protein, Cy 2, Cy 3, Cy 3.5, Cy 5, dansylcadaverine, DsRed, DTAF, dTomato, enhanced green fluorescent protein, eosin, Fluo-3, Fluo-4, Fluor-Ruby, fluorescein, Fluoro-Emerald, Marina Blue, mBanana, mCherry, mHoneydew, mOrange, mPlum, mRFP, mStrawberry, mTangerine, NBD-X, NeuroTrace500 / 525, Nile blue, Nile red, Oregon Green 488, Oregon Green 514, Pacific Blue, phycoerythrin, and rhodamine.

7. The use according to claim 5, wherein the fluorescent label emits in the near-infrared range of the electromagnetic spectrum.

8. The use according to claim 5, wherein the fluorescent label is FITC or a cyanine dye.

9. The use according to claim 8, wherein the cyanine dye is Cy5.5 or Cy7.

10. The use according to claim 1, wherein the intestinal tumor formation is colorectal cancer.

11. The use according to claim 1, wherein the colorectal tissue cannot be discerned as a polyp by endoscopy.

12. The use according to claim 1, wherein the intestinal tumor formation is a polyp.

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