WO2016205515A1 - GOLD NANOROD BASED DIAGNOSTIC AGENT FOR DETECTION AND QUANTIFICATION OF c-MET (HGF) RECEPTORS - Google Patents

Abstract

Provided is a nanoparticle conjugate comprising a nanoparticle linked via a linker to a target molecule with the specific affinity for c-MET receptor. Methods for diagnosing and treating patients are provided as well.

Description

GOLD NANOROD BASED DIAGNOSTIC AGENT FOR DETECTION AND QUANTIFICATION OF c-MET (HGF) RECEPTORS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims a benefit of priority from US provisional patent application 62/180,857, filed June 17, 2015, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to compositions comprising a gold nanorod conjugated with a peptide which selectively binds c-MET (HGF) receptor and cells expressing the receptor. Also provided are methods in which the compositions are used to diagnose and treat patients.

BACKGROUND

[0003] In the last several years, it has become clear that due to the heterogeneous nature of cancers, focus must shift towards treating each cancer based on its genetic makeup and expression abnormalities. In order to properly tailor treatment to each individual, evaluation of potential biomarker targets must be carefully conducted. One such subclass of biomarkers is comprised of cell-surface receptors, which are commonly overexpressed in cancerous cells.

[0004] The most widely practiced method of cell-surface receptor detection is through immunohistochemistry (IHC), which uses antibody-antigen interactions for detection. Common IHC detection is through an indirect method, where a primary antibody against the desired target antigen is then labeled with a secondary antibody directed at the lgG of the primary antibody species. This secondary antibody is usually tagged with an enzymatic reporter such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). Once the secondary antibody is bound, a substrate, usually a chromagen, is applied to the tissue to yield a colored product from the reporter that is identifiable through light microscopy. There are many commercially available kits for IHC testing of human tumor tissues, such as the FDA-approved DAKO EGFR PharmDx kit, approved for diagnosis of EGFR in colorectal tumors.

[0005] Though IHC is the preferred method of diagnosis, recent studies have shown problems with the IHC analysis of membrane protein expression due to problems inherent to the indirect IHC design such as background staining, variation in staining intensity, antibody cross-reactivity, enzyme activity, and the qualitative nature of the diagnosis method.

[0006] The rise of personalized medicine has become a promising option for cancer treatment due to the specificity of biomarker-targeted therapies. In order to properly treat a patient with targeted drugs, a testing must be performed first on biopsied samples to establish a profile of suitable biomarker targets. One such biomarker is the hepatocyte growth factor (HGF) receptor also known as c-MET receptor. Normally, c-MET receptor is only expressed in stem and progenitor cells. The c-MET receptor is overexpressed in the cell membrane of many cancers, including non-small cell lung carcinoma (NSCLC), gastric, ovarian, thyroid, breast, and colon cancers, including colorectal adenocarcinoma.

[0007] In cancers, the expression of the MET pathway affects the development of cancer through activation of oncogenic pathways such as RAS, PI3K, and STAT3, and also promotes angiogenesis in tumors. The expression of MET in tumors is also linked to resistance of anti-EGFR therapies, whereby the tumors will circumvent the EGFR pathway by internalizing the receptor and up regulating surface c-MET receptor expression to proliferate. Currently, there are only very few FDA-approved anti-cMET drugs available. These drugs include crizotinib for NSCLC and cabozantinib for thyroid cancers.

[0008] Despite the need to test for c-MET receptor expression in cancers, current methods of evaluating c-MET receptor expression have not been accurate enough to warrant a clinical diagnosis.

SUMMARY

[0009] In one aspect, the present application provides a reagent and sensitive methods using the reagent for accurate detection of c-MET receptor expression on cancer cells and cancer tissues. The reagent comprises a nanoparticle conjugate in which a nanoparticle is linked via a linker to a target molecule with the specific affinity for c-MET receptor. [0010] In some embodiments, the target molecule in the nanoparticle conjugate is selected from the group consisting of a peptide, ligand, antibody, small molecule and any combination thereof.

[0011] One of the preferred nanoparticle conjugates comprises a peptide consisting essentially of the amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1).

[0012] In some preferred embodiments, the nanoparticle is a nanorod.

[0013] Some suitable reagents comprise a nanoparticle conjugate in which the nanoparticle is a nanorod linked to a peptide consisting essentially of the amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1) by the linker comprising a thiol moiety selected from the group consisting of thioctic acid, monothioctic acid, dithioctic acid, and trithioctic acid.

[0014] In some embodiments, the linker comprises at least one or more moiety selected from the group consisting of a thiol moiety, lysine residue and ethylene glycol.

[0015] Further embodiments provide methods of treatment, including a method of treating a cancer patient, the method comprising reacting the patient's cancer tissue biopsy sample with a reagent comprising a nanoparticle conjugate comprising a nanorod linked to a peptide with specific affinity to human c-MET receptor, and wherein the reaction detects expression of c-MET receptor in the cancer tissue biopsy sample, administering to the patient an anti-cMET drug.

[0016] These methods of treatment may be particularly useful for cancer patients with non-small cell lung carcinoma, gastric cancer, ovarian cancer, thyroid cancer, breast cancer or colon cancer.

[0017] Further embodiments include methods for making the reagent comprising a nanoparticle conjugate. Such methods include a method of forming a nanoparticle conjugate comprising a nanoparticle linked to a target molecule specific for cMET receptor, with the method comprising the following steps: mixing solutions of cetyltrimethylammonium bromide and chlorauric acid, adding a solution of cetyltrimethylammonium bromide, silver nitrate, and ascorbic acid to obtain gold nanorods, purifying the gold nanorods by filtration and centrifugation; conjugating a polyethylene glycol linker modified with thiol to the gold nanorods to obtained gold nanorods with a linker; and conjugating a peptide to the gold nanorods with the linker. [0018] Various uses for the reagent comprising comprises a nanoparticle conjugate in which a nanoparticle is linked via a linker to a target molecule with the specific affinity for c-MET receptor include any of the following:

- use of the reagent as an agent for targeting cMET receptors on cancerous tissue;

- use of the reagent as an agent for targeting cMET receptors on one or more human cancers from the list comprising non-small cell lung carcinoma, gastric cancer, ovarian cancer, thyroid cancer, breast cancer, and colon cancer;

- use of the reagent as an agent for detecting cMET receptors on cancerous tissue;

- use of the reagent as an agent for detecting cMET receptors on human tissue;

- use of the reagent as a means for detecting and quantifying the number cMET receptors on cancerous tissue by utilizing polarization microscopy to image said nanoparticle conjugates bound to said cMET receptors; and

- use of said nanoparticle conjugate of claim 1 as a therapy agent for one or more human cancers selected from the group consisting of non-small cell lung carcinoma, gastric cancer, ovarian cancer, thyroid cancer, breast cancer, and colon cancer.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a scheme 1, showing a nanorod linked with peptides with specific affinity to cMET receptor.

FIG. 2 is a chemical structure of reagent GNR-1093, the portion comprising the thioctic-acid disulfide bound to the N-terminus of the 1093 peptide through a poly- lysine bridge is shown.

FIG. 3 is an HPLC chromatogram for the 1093 peptide.

FIG. 4 is a mass spectrogram for the 1093 peptide.

FIG. 5A is a UV-visible spectrum and Fig. 5B is a Zeta potential of GNR-1093.

Fig. 6 are micrographs showing detection of c-MET receptor on non-small lung cancer cells with GNR-1093. Fig. 7 is a graph for relative intensities of each sample from Fig. 6.

Figs. 8-25 are scanned images of tumor and normal tissue microarray reacted with

GNR-1093. See Table 2 for a microarray panel legend.

DETAILED DESCRIPTION

[0019] The present disclosure provides various embodiments of a reagent suitable for detection of cancer cells which express c-MET receptor. In some embodiments, the reagent can be used for identifying a cancer patient who may benefit from treatment with an anti-cMET drug which target cancer cells expressing c-MET receptor. Such cancer patients include those afflicted with non-small cell lung carcinoma (NSCLC), gastric, ovarian, thyroid, breast, or colon cancers, including colorectal adenocarcinoma.

[0020] The reagent comprises a gold nanorod conjugated through a linker with a peptide which selectively recognizes and binds human c-MET receptor expressed at the cell surface of cancer cells. Gold nanoparticles have been used as imaging labels due to their light scattering properties and ease of surface modification. While nanospheres have attracted much attention, the inventors have unexpectedly discovered that gold nanorods (GNRs) of the same volume have an optical efficiency 20 times that of the spheres. GNR also have a high affinity for thiol groups, which allows them to be stabilized with thiolated polyethylene glycol, and conjugated with a desired biomolecule such as c-MET receptor binding peptide for targeting. Gold nanorods of various length can be obtained. Preferably, the length of a gold nanorod is in the range from about 30 nm to about 60 nm. In some embodiments, the length of a gold nanorod is in the range from 30 nm to 60 nm. Suitable gold nanorods include those with the length/width ratio from 2 to about 100, from 2 to 50, from 2 to 20, from 2 to 10, and from 2 to 6.

[0021] GNRs are an attractive alternative to organic fluorophores, which can suffer from photodecomposition or sensitivity to quenching. GNRs are also biocompatible and are thus also an attractive alternative to cytotoxic quantum dots. Various detection technologies can be used with GNRs conjugated to a peptide which binds to c-MET receptor. These technologies include dark field microscopy, near-infrared (NIR) transmission imaging, photoacoustic tomography (PAT), two-photon excited luminescence (TPL) imaging, and surface enhanced Raman spectroscopy (SERS) imaging. Additional detection technologies may include polarized imaging, either in the dark field or through differential interference contrast (DIC) microscopy.

[0022] In some embodiments a nanorod-based tissue diagnostic agent specific for the c-MET receptor comprises a gold nanorod (GNR) attached through a linker to a peptide with the specific affinity for c-MET receptor. A person of skill will appreciate that suitable peptides include those obtained by screening a phage display library against human c-MET receptor or any extra-cellular portion of human c-MET receptor.

[0023] In some embodiments, an antibody specific to the c-MET receptor, a ligand specific to the c-MET receptor or a small molecule can be used instead of the peptide, or in combination with the peptide as a chimeric molecule.

[0024] The term "specific affinity" means binding of a peptide or an antibody or a ligand to the c-MET receptor with the equilibrium dissociation constant (¾) in the range from 10^"6 to 10^{"i 2}, and preferably i the range from 10^"9 to 10^"12.

[0025] In one embodiment, a gold nanorod-based c-Met tissue diagnostic agent comprises a gold nanorod conjugated through a linker to a peptide comprising the following amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1). In other embodiment, a gold nanorod-based c-Met tissue diagnostic agent comprises a gold nanorod conjugated through a linker to a peptide consisting essentially of the following amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1). In further embodiment, a gold nanorod-based c-Met tissue diagnostic agent comprises a gold nanorod conjugated through a linker to a peptide consisting of YLFSVHWPPLKA (SEQ ID NO. 1, also referred to as the 1093 peptide).

[0026] GNR- 1093 binds the c-Met receptor to give a point-specific image signal, rather than a basic membrane stain which cannot be accurately quantified. This method of c-Met detection improves on current immunohistochemical methods that involve multi-step processes and qualitative data.

[0027] One of the methods in which a gold nanorod-based c-Met tissue diagnostic agent can be used is the polarized light mode which allows to distinctly detect a GNR- peptide conjugate. Polarization microscopy is used for imaging anisotropic (directionally dependent) materials. [0028] The plasmon peaks seen in the UV absorption spectrum show that GNR has two localized plasmon resonances: the transverse and longitudinal modes. This important fact confirms that GNR is directionally dependent, given its rod shape, and is thus an anisotropic material that can be imaged using polarized tight. Using crossed polarization imaging, which can be achieved by simply adding a polarizer and analyzer to a bright field microscope, GNR can be seen as a bright gold/red color. DIC microscopy mode also uses cross polarized light, with the addition of two Wollaston prisms. These prisms bias the tight in such a way that the image seen looks more three dimensional. Since polarized tight is present, the inventors also have detected some of the GNR on samples imaged with DIC when the prisms are at the correct bias.

[0029] The 1093 peptide is comprised of the 12 amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1 and Figures 1 and 2). The 1093 peptide was identified to bind to c-MET specifically and efficiently by screening a phage display library. Due to solubility issues, the 1093 peptide as such cannot be conjugated to GNR by simply adding a thiol group. The inventors have modified the 1093 peptide to address the solubility issues, while leaving the binding sequence intact. Such modification includes attaching at least one or more moiety selected from the group consisting of ethylene glycol, lysine and any combination thereof to the N-terminus of the peptide.

[0030] As shown in Figs. 1 and 2, several lysine groups are incorporated within the structural motif of the GNR- 1093 peptide that is expected to enhance the interaction of receptors with the peptide. The thioctic acid moiety is incorporated at the far end of the binding site. The thioctic acid-disulfide interacts with gold atoms on the GNR to form a stable 6-membered ring for utilization in diagnostic testing.

[0031] A person of skill will appreciate that in some embodiments a linker comprises the thioctic acid-disulate attached to polylysine comprising several lysine residues attached to a peptide specific for c-MET receptor. Thus, the polylysine bridge connects the thioctic acid moiety to the peptide. In addition to the thioctic acid moiety or instead of the thioctic acid moiety, any moiety selected from monothioctic acid, dithioctic acid, and trithioctic acid can be used. [0032] In some embodiments, a gold nanorod-based c-MET tissue diagnostic agent comprises a gold nanorod conjugated with a peptide with the specific affinity to c- MET receptor through a linker comprising a thiol group. In some embodiments, a gold nanorod-based c-MET tissue diagnostic agent comprises a gold nanorod conjugated with a peptide with the specific affinity to c-MET receptor through a linker comprising a thiol group and ethylene glycol. In some embodiments, the thiol group is selected from at least one moiety from thioctic acid, monothioctic acid, dithioctic acid, and trithioctic acid.

[0033] Various ethylene glycols are suitable, including monoethylene glycol, diethylene glycol, and polyethylene glycol.

[0034] In some embodiments, a gold nanorod-based c-MET tissue diagnostic agent comprises a gold nanorod conjugated with the thioctic-acid disulfide bound to the N- terminus of the 1093 peptide through a poly-lysine bridge comprising several lysine residues. The number of lysine residues may vary. In some embodiments, the number of lysine residues is from 2 to 3.

[0035] In some embodiments, the polylysine bridge comprises at least two lysine residues. In other embodiments, the polylysine bridge comprises at least three lysine residues.

[0036] A peptide with the selective affinity to c-MET receptor can be synthetized and purified using high pressure liquid chromatography (HPLC). Fig. 3 reports an HPLC chromatogram for the 1093 peptide purified by HPLC. Further confirmation of the 1093 peptide purification was obtained from mass spectroscopy (MS) as shown in Fig. 4.

[0037] A further embodiment provides a method of forming a nanoparticle conjugate comprising a nanorod linked to a target molecule that is a peptide with the specific affinity for cMET receptor. In this method, gold nanorods are prepared using a seed-mediated growth method which utilizes mixing solutions of cetyltrimethylammonium bromide and chlorauric acid, followed by addition of a solution of cetyltrimethylammonium bromide, silver nitrate, and ascorbic acid, and the final solution then washed, filtered, and centrifuged to remove excess cetyltrimethylammonium bromide. In the next step, a polyethylene glycol linker is modified with thiol in a solution containing the gold nanorods. Completion of the conjugation of a peptide to the gold nanorods is then achieved by addition of a solution containing a twelve amino acid sequence YLFSVHWPPLKA peptide (SEQ ID NO. 1) or some other peptide with the selective binding to c-MET receptor to the solution containing the gold nanorods. The nanoparticle conjugates comprising the nanorod attached to the peptide through the linker are then washed. These nanoparticle conjugates retain the Zeta potential is in the range about +10 mV to about +60 mV.

[0038] Further embodiments include a method for making a nanoparticle linked to a target molecule specific for cMET receptor. This method comprises the following steps: preparing nanoparticles; conjugating a linker to the nanoparticles; and conjugating of a target molecule specific for cMET receptor to the nanoparticle. Other methods may be employed for alternative nanoparticle conjugates. At least in some embodiments, a peptide may be modified by attaching several lysine residues to its N-terminus prior being linked with a gold nanorod by a linker comprising at least a thiol moiety.

[0039] Various analytical techniques can be used to confirm conjugation of a peptide to GNR. For example, UV- visible spectrum and Zeta potential of GNR-1093 as quality control can be used as shown in Figs. 5A and 5B, respectively.

[0040] In some embodiments, a nanoparticle conjugate comprising a nanorod linked to a peptide with the affinity specific for cMET receptor is used for treating patients where patients are to be treated with anti-cMET drugs. These drugs may include crizotinib and cabozantinib, as well as drugs currently in clinical trials as shown in Table 1. In these embodiments, a sample of patient's cancer tissue is reacted with the reagent such as for example GNR-1093, and the cancer tissue positive for expression of c-MET receptor is identified. A patient with cancer tissues positive for cMET receptor expression is then treated with at least one anti-cMET drug.

TABLE 1: cMET RECEPTORS TARGETING DRUG MOLECULES

medipharma

[0041] Further embodiments provide diagnostic methods in which a cancer tissue is analyzed for expression of c-MET receptor with a reagent comprising GNR conjugated to a peptide which selectively binds c-MET receptor. Cancer cells which express c-MET receptor are then detected based on identification of cells bound to a nanoparticle conjugate comprising a nanorod linked to a peptide with the specific affinity specific for cMET receptor. Such detection methods may include any of the following: dark field microscopy, near-infrared (NIR) transmission imaging, photoacoustic tomography (PAT), two-photon excited luminescence (TPL) imaging, surface enhanced Raman spectroscopy (SERS) imaging, polarized imaging, either in the dark field or through differential interference contrast (DIC) microscopy.

[0042] The invention will be now described by the way of the following non- limiting examples.

EXAMPLE L SYNTHESIS OF A NANOROD LINKED TO A

PEPTIDE WITH SPECIFIC AFFINITY TO C-MET RECEPTOR

[0043] To synthesize a combined c-MET -targeted, GNR-based diagnostic and imaging agent GNR-1093, the 1093 peptide (SEQ ID NO. 1) was conjugated to GNRs functionalized with a polyethylene glycol. [0044] Scheme 1 shown in Fig. 1 is (PEG) surface modification synthesis. In this method, gold nanorods (GNR) of aspect ratio 3.4 were prepared using an established seed-mediated growth method. All solutions were made in fresh de-ionized water (DI H₂O). A seed solution was prepared by making a 10ml solution of 0.1M cetyltrimethylammonium bromide (CTAB). The CTAB solution was lightly heated until the CTAB had dissolved, giving a clear solution. 250 ml of a 0.01M solution of chlorauric acid (HAuCI₄) was then added to the CTAB solution while stirring. Immediately after addition of chlorauric acid, 600 ml of ice-cold 0.01M sodium borohydride (NaBH₄) was added to the solution, which changed color from gold to light brown.

[0045] The seed solution was then left to stir for 5 minutes. While the seed solution stirred, 500 ml of growth solution was then prepared. The first step was to make 250 ml of a 0.1M CTAB solution, and then heat until the CTAB had dissolved as had been done with the seed solution. 250 ml of 0.001 M chlorauric acid was then added to this CTAB solution and stirred lightly by hand. 10 ml of 0.0043 M silver nitrate (AgN0₃) was added to the solution and again stirred gently by hand. 4 ml of 0.1M ascorbic acid was then added, and the solution was stirred very gently until the solution had turned from gold-orange to clear. This last step completed the growth solution. 0.5 ml of the seed solution was then added in to the growth solution. The solution was not touched after this point due to the delicate nature of the synthesis. Minutes later the solution turned from clear to purple, indicating the formation of GNR. The GNR solution was left alone for 24 hours, and then washed of CTAB. The GNR solution was twice filtered through filter paper to remove excess CTAB. To further remove excess CTAB, the solution was then centrifuged at 16,000 RPM for 10 minutes at 25°C. The supernatant was removed and replaced with fresh DI H₂O.

[0046] The centrifuging step was then repeated. The sample of GNR was then characterized using UV-Vis spectroscopy, TEM imaging, and Zeta potential. The GNR synthesized characteristically shows a UV-Vis transverse peak near 540 nm and much larger longitudinal peak near 780 nm. The Zeta potential for GNR-CTAB is highly positive, showing stable values of +20 mV and above. In order to attach the 1093 peptide, a 750 Dalton polyethylene glycol (PEG750) linker modified with thiol was conjugated to the GNR. A solution of PEG750 was added to the GNR solution at a molar ratio of 1:2 (GNR:Peg 750). This solution was allowed to stir for 24 hours. The washing step as described above was then repeated exactly to remove unbound PEG.

[0047] After washing, GNR-PEG was then characterized through UV-Vis spectroscopy, TEM imaging, and Zeta potential. The UV-Vis spectrum of GNR-PEG showed only a slight shift in absorbance, whereas the Zeta potential became a largely negative and stable value of -15mV and below. A solution of the 1093 peptide was then added to the GNR-PEG solution at a molar ratio of 1: 1 GNR-PEG: 1093 peptide. This solution was stirred for 24 hours to ensure maximum binding of peptide to PEG. The same washing protocol as with GNR-CTAB was followed after the 24-hour period. GNR-1093 was then characterized using UV-Vis spectroscopy, TEM, and Zeta potential. The UV-Vis spectrum shifted only slightly, but the Zeta potential once again became a highly positive with a value of +20mV and above.

[0048] This example reports specific binding of the GNR-peptide reagent (GNR- 1093) to c-MET receptors in MET-overexpressing cells lines and tissues, such as NSCLC. GNR-1093 interacts with c-MET receptors and can be visualized using polarized light as described in previous sections.

[0049] To further confirm that GNR-1093 is targeting c-MET receptors, cell microarrays with varying degrees of c-MET receptors were used. If GNR-1093 is targeting c-MET receptors, then there should be a linear increase in the number of GNR present in the surface of cells with increase in c-MET receptors. As shown in Fig. 6, there was a linear increase in GNR-1093 on the surface of cells with increasing c-MET receptors.

[0050] It is important to note that cellular microarrays used in this study contained three different cell lines that have been verified by Lilly Labs (Indianapolis, IN) as being low, medium, and highly c-MET receptor expressing. For quantification of GNR on cellular microarray, we used MATLAB image processing tools.

[0051] Using MATLAB for image processing, the DAPI (4',6-diamidino-2- phenylindole, dihydrochloride) stained nuclei of the cells were isolated in order to evaluate the staining on a per cell basis. Each cell was then analyzed for expression of the intense red signals seen through polarized microscopy of gold nanorods.

[0052] The results of each image analysis were graphed on a log scale of the relative intensity of the pixels per cell. The pixel intensity surrounding each of the c- Met 'low' samples did not give much indication of GNR signals, whereas for the medium and highly MET expressing cell lines a much higher value of pixel intensity was detected.

[0053] The relative intensities of each sample were graphed according to the intensity scale, and a linear pattern was established (Figure 7). This study confirms that GNR- 1093 is selective in identifying c-MET receptors. Further, it confirms that receptors present on the surface can be quantified using GNR signals.

[0054] The following protocol was followed for imaging: paraffin was removed from the slides by immersing twice in 100% xylene for 5 minutes, and then rehydrated in graded ethanol and Dl H₂O. Once prepared for staining, slides were stained using GNR- 1093. For GNR staining, the slides were treated with 2.5% BSA solution for 10 minutes prior to addition of the nanorod solution. Once washed of BSA solution, the samples were treated with 50 ug/ml GNR- 1093 solution in a humid chamber for 2 hours. After this time, the slides were washed thoroughly with PBS and Dl H₂0. The GNR slides were mounted using fluorescent DAPI nuclear stain and imaged on a Leica DMSSOO using DAPI for nuclear identification, and either polarized or dark field light microscopy was used for gold nanorod detection. Using the scanning capabilities of Leica DMSSOO instrument, a tile-scan was constructed of one whole spot of the GNR- 1093 treated NSCLC tissue microarray.

[0055] This capability allows for a bigger picture of expression in a given tissue, and will be further explored for image processing.

EXAMPLE 3. DETECTION AND QUANTIFICIATION OF c-MET

RECEPTORS IN CANCER TISSUES

[0056] GNR- 1093 was utilized in identifying c-MET receptors present in human tissues. A tissue microarray containing 18 separate cases of non-small cell lung carcinoma (NSCLC) and normal tissue was incubated with the c-MET targeted GNR- 1093 and non-targeted GNR-PEG compounds in order to determine the presence of c- MET receptors in the tissue cases.

[0057] The tissue microarray was tested in the same manner as the cell lines from Lilly Labs, using DAPI as a nuclear counter-stain.

[0058] Pictured are the 18 tissue stained tissue sections with normal tissue, scanned at 20x and stitched using a Leica DMSSOO scanning algorithm. The stitched overview of the tissue cores shows bright areas corresponding to GNR signals in the tissue. The non-targeted GNR-PEG samples show some areas of non-specific gold signals, while the targeted GNR- 1093 samples show much brighter and higher intensity gold signals indicative of c-MET expression.

In each sample, the gold signals are localized to the areas where the cell nuclei are present, and the gold expression is confined to the membrane (Figure 8). Scanned images of all tumor and normal tissue sections analyzed are provided in Figs. 8-25. Table 2 below provides description of lung tissue sections of Figs 8-25.

TABLE 2. MICROARRAY PANEL DISPLAY

AT - Adjacent tissue 1.5 cm away from tumor

MT - Malignant tumor

NT - Normal tissue

[0059] Table 3 below describes the pathology and status of cancer in each patient.

TABLE 3. TISSUE SECTIONS SCANNED WITH GNR-1093 AND THE IMAGES ARE APPENDED ALONG WITH COMMENTS

Claims

What is claimed is:

1. A nanoparticle conjugate comprising a nanoparticle linked via a linker to a target molecule with the specific affinity for c-MET receptor.

2. The nanoparticle conjugate of claim 1, wherein said target molecule is selected from the group consisting of a peptide, ligand, antibody, small molecule and any combination thereof.

3. The nanoparticle conjugate of claim 1, wherein said target molecule is a peptide consisting essentially of the amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1).

4. The nanoparticle conjugate of claim 1, wherein the nanoparticle is a nanorod.

5. The nanoparticle conjugate of claim 1, wherein the nanoparticle is a nanorod linked to a peptide consisting essentially of the amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1) by the linker comprising a thiol moiety selected from the group consisting of thioctic acid, monothioctic acid, dithioctic acid, and trithioctic acid.

6. The nanoparticle conjugate of claim 1, wherein the linker comprises a thioctic acid moiety and from 2 to 3 lysine residues.

7. The nanoparticle conjugate of claim 1, wherein the linker comprises a thiol moiety and ethylene glycol.

8. The nanoparticle conjugate of claim 1, wherein the linker comprises at least one or more moiety selected from the group consisting of a thiol moiety, lysine residue and ethylene glycol.

9. The nanoparticle conjugate of claim 8, wherein said ethylene glycol is selected from the group consisting of monoethylene glycol, diethylene glycol, and polyethylene glycol.

10. The nanoparticle conjugate of claim 1, wherein the nanoparticle is a nanorod with the length in the range from about 30 nm to about 60 nm.

11. A method of treating a cancer patient, the method comprising:

reacting the patient's cancer tissue biopsy sample with a reagent comprising a nanoparticle conjugate comprising a nanorod linked to a peptide with the specific affinity to human c-MET receptor, and wherein the reaction detects expression of c-MET receptor in the cancer tissue biopsy sample, administering to the patient an anti-cMET drug.

12. The method of claim 11, wherein the cancer patient is afflicted with a cancer selected from the group consisting of non-small cell lung carcinoma, gastric cancer, ovarian cancer, thyroid cancer, breast cancer, and colon cancer.

13. The method of claim 11, wherein the patient's cancer tissue biopsy sample is reacted with the reagent comprising a nanorod linked to a peptide consisting essentially of the amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1) by the linker comprising a thiol moiety selected from the group consisting of thioctic acid, monothioctic acid, dithioctic acid, and trithioctic acid.

14. The method of claim 11, wherein the detection is performed by a method selected from the group consisting of dark field microscopy, near-infrared (NIR) transmission imaging, photoacoustic tomography (PAT), two-photon excited luminescence (TPL) imaging, surface enhanced Raman spectroscopy (SERS) imaging and polarized imaging.

15. A method of forming a nanoparticle conjugate comprising a nanoparticle linked to a target molecule specific for cMET receptor, with the method comprising the following steps:

mixing solutions of cetyltrimethylammonium bromide and chlorauric acid, adding a solution of cetyltrimethylammonium bromide, silver nitrate, and ascorbic acid to obtain gold nanorods,

purifying the gold nanorods by filtration and centrifugation;

conjugating a polyethylene glycol linker modified with thiol to the gold nanorods to obtained gold nanorods with a linker; and

conjugating a peptide to the gold nanorods with the linker.

16. The method of claim 15, wherein the peptide consists essentially of the amino acid sequence YLFSVHWPPLKA (SEQ ID NO. 1).

17. The method of claim 16, wherein the peptide is modified with a poly- lysine moiety at the N-terminus prior to be conjugated with a nanorod.