WO2016140860A2

WO2016140860A2 - Peptide linker for attachment of thiol-reactive molecules to surfaces

Info

Publication number: WO2016140860A2
Application number: PCT/US2016/019550
Authority: WO
Inventors: Kenneth P. OLIVE; Sam HOLMSTROM
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2015-02-25
Filing date: 2016-02-25
Publication date: 2016-09-09
Also published as: WO2016140860A3

Abstract

The current invention is in the field of assays, specifically an improvement in binding thiol-reactive molecules to high-binding plastic, such as that used in enzyme-linked immunosorbent assay or ELISA, using a peptide that has novel linking properties. The use of the peptide has particular utility in the field of cancer screening and diagnostics, and the testing of efficacy of cancer therapeutic agents.

Description

PEPTIDE LINKER FOR ATTACHMENT OF THIOL-REACTIVE MOLECULES TO

SURFACES

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. patent application serial No.

62/120,571 filed February 25, 2015, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The current invention is in the field of assays, specifically an improvement in binding thiol-reactive molecules to high-binding plastic, such as that used in enzyme-linked immunosorbent assay or ELISA, using a peptide that has novel linking properties. The use of the peptide has particular utility in the field of cancer screening and diagnostics.

The peptide linker of the current invention can also be used to bind thiol-reactive molecules to other materials and surfaces for other purposes such as purification.

BACKGROUND OF THE INVENTION

The enzyme-linked immunosorbent assay or ELISA is an immunoassaying technique which tests for specific antigens in a sample. ELISA is a powerful tool for testing over a variety of applications due to its high sensitivity. However, some assays for specific molecules and compounds can be time-consuming and expensive. One example of such molecules is those that react with thiol groups.

While there are commercially available plates for ELISA assays involving thiol- reactive molecules, they are either expensive, proprietary, and/or require dangerous instrumentation such as x-rays. One such commercial product is a glutathione-coated plate marketed by Pierce Biotechnology. However, the process to make these plates is proprietary and the plates are expensive to purchase.

Thus, there is a need for an inexpensive and easy method to utilize ELISA and other assay formats for thiol-reactive molecules and compounds, as well as for other molecules that are difficult to attach to plastic plates and other assay formats. SUMMARY OF THE INVENTION

The current invention is a composition comprising peptides that link, anchor, and/or immobilize thiol-reactive molecules and compounds to surfaces including assay format surfaces, such as plastic plates and wells used in ELISA assays, beads, resins, affinity columns and tissue culture plates (hereinafter "peptide linker").

One embodiment of the present invention is a peptide linker comprising lysine amino acids or arginine amino acids, and cysteine amino acids that bind to thiol-reactive molecules and compounds, to surfaces including assay platforms. In a preferred embodiment, the peptide linker comprises lysine and cysteine residues. In a more preferred embodiment, the peptide linker comprises an equal number of lysine and cysteine residues. In a more preferred embodiment, the peptide linker comprises six lysine amino acids and six cysteine amino acids. In a more preferred embodiment, the lysine amino acids and cysteine amino acids are alternating in groups in the peptide linker.

In more preferred embodiments, the peptide linker consists essentially of lysine amino acids or arginine amino acids, and cysteine amino acids that bind to thiol-reactive molecules and compounds, to surfaces including assay platforms. In a more preferred embodiment, the peptide linker consists essentially of lysine and cysteine residues. In a more preferred embodiment, the peptide linker consists essentially of an equal number of lysine and cysteine residues. In a more preferred embodiment, the peptide linker consists essentially of six lysine amino acids and six cysteine amino acids. In a more preferred embodiment, the lysine amino acids and cysteine amino acids are alternating in groups in the peptide linker.

In a most preferred embodiment, the peptide linker has the amino acid sequence: cys- cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1). In another preferred embodiment the peptide linker with this sequence comprises no other reagents that would materially change the peptide linker.

In a further embodiment, the peptide linker binds to thiol-reactive compounds, including but not limited to nitro- aromatic compounds. A preferred nitro-aromatic compound to which the peptide linker binds is pimonidazole or PIMO, which is a 2-nitro-imidazole.

The preferred assay format in which the peptide linker is used is a plastic plate or well used in an ELISA assay. The peptide linker can also be used with a variety of other formats, surfaces and materials, including but not limited to, resins, beads, columns and culture plates. The peptide linker of the present invention can be used in any method that now uses glutathione, including many commercial applications that are proprietary and expensive, such as glutathione agarose resins, beads and columns.

A further embodiment of the present invention is a method of preparing assay format platforms that are capable of binding thiol-reactive molecules and compounds using the peptide linker, and an ELISA assay including the peptide linker.

A further embodiment of the present invention is a method of detecting the presence of a thiol-reactive compound or molecule in a sample, utilizing an enzyme-linked immunosorbent assay and the peptide linker to bind thiol-reactive compounds and molecules. This method could comprise the steps of: contacting or incubating the sample which may have a thiol-reactive compound or molecule, to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, i.e, the peptide linker; allowing a thiol-reactive compound or molecule in the sample to bind to the composition; contacting or incubating the sample bound to the surface, via the peptide linker, with an antibody that recognizes or binds to the thiol-reactive compound or molecule, wherein the antibody generates a detectable signal when it recognizes or binds to the thiol-reactive molecule or compound; detecting and/or measuring the signal; and determining the presence of the thiol- reactive compound or molecule in the sample. In this embodiment, an increase in signal or a greater signal via a control indicates the presence of the thiol-reactive compound or molecule in the sample.

This method could also comprise the steps of: labeling a purified thiol-reactive antigen, which when bound to an antibody, generates a detectable signal; contacting or incubating the sample which may have the thiol-reactive antigen to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, i.e, the peptide linker; allowing the thiol-reactive antigen in the sample to bind to the composition; contacting or incubating the sample bound to the surface, via the peptide linker, with the labeled antigen- antibody complex; and detecting and/or measuring the signal.

This method could also comprise the steps of: contacting a sample which may have a thiol-reactive compound or molecule with a first antibody that recognizes or binds to the thiol-reactive compound or molecule; contacting or incubating the sample which may have a thiol-reactive compound or molecule, to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, i.e, the peptide linker, said composition having linked, anchored, and/or immobilized a thiol-reactive compound or molecule to the surface; allowing any first antibody not bound to the thiol-reactive compound or molecule in the sample to bind to the thiol-reactive compound or molecule bound to the surface via the composition; contacting or incubating the first antibody bound to the surface with a second antibody that recognizes or binds to the first antibody, wherein the second antibody generates a detectable signal when it binds to the first antibody; detecting and/or measuring the signal; and determining the presence of the thiol-reactive compound or molecule in the sample. In this embodiment, a decrease in signal indicates the presence of the thiol-reactive compound or molecule in the sample.

A further embodiment of the present invention is a method of detecting hypoxic tissue, including cancerous tissue and tumors, utilizing an enzyme-linked immunosorbent assay and the peptide linker to detect nitro-aromatic compounds. In a preferred embodiment, the nitro-aromatic compound is pimonidazole or PIMO. As will be described below, traditional immunochemical methods for detecting PIMO in hypoxic tumors can be unreliable. The method of the invention, an ELISA with the peptide linker, allows for the accurate diagnosis of hypoxic cancers in a non-invasive fashion, but also can be used to determine if a particular tumor or cancerous tissue or portions thereof are hypoxic in order to better target treatment. In other words, the method is a non-invasive accurate way to determine if the cancer could be treated by hypoxia-targeted anti-cancer drugs. The method can also be used to assay particular regions of tissue and tumors for hypoxia, and to monitor hypoxia-targeted anti-cancer treatment in a subject.

In some embodiments, this method would comprise: administering a nitro-aromatic compound or molecule to the subject; isolating protein from a sample from the subject; contacting or incubating the sample of protein on a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, i. e., the peptide linker; allowing the nitro-aromatic compound or molecule to bind to the composition; contacting or incubating the sample of protein bound to the surface, via the peptide linker, with an antibody that recognizes or binds to the nitro-aromatic compound or molecule, wherein the antibody generates a detectable signal when it recognizes or binds to the nitro-aromatic compound; and detecting and/or measuring the signal. The presence of the nitro-aromatic compound in the protein is an indication that the tissue is hypoxic. In some embodiments, if the tissue is hypoxic, it is considered cancerous. In some embodiments, if the tissue was known to be cancerous and determined to be hypoxic, it would be considered to be a candidate for hypoxia-targeted anti-cancer treatments. In these embodiments, an increase in signal or a greater signal via a control indicates the presence of the nitro-aromatic compound or molecule in the protein.

In other embodiments, this method would also comprise: administering a nitro- aromatic compound or molecule to the subject; isolating protein from a sample from the subject; contacting or incubating the sample of protein with a first antibody that recognizes or binds to the nitro-aromatic compound or molecule; contacting or incubating the sample of protein-antibody to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, i.e., the peptide linker, that has linked, anchored, and/or immobilized a nitro-aromatic compound or molecule to the surface; allowing any first antibody not bound to the nitro-aromatic compound or molecule in the sample to bind to the nitro-aromatic molecule or compound bound to the surface via the peptide linker; contacting or incubating the first antibody bound to the surface with a second antibody that recognizes or binds to the first antibody and when bound generates a detectable signal; and detecting and/or measuring the signal. The presence of the nitro-aromatic compound in the protein is an indication that the tissue is hypoxic. In some embodiments, if the tissue is hypoxic, it is considered cancerous. In some embodiments, if the tissue was known to be cancerous and determined to be hypoxic, it would be considered to be a candidate for hypoxia-targeted anti-cancer treatments. In these embodiments, a decrease in signal or a lesser signal via a control indicates the presence of the nitro-aromatic compound or molecule in the protein.

The peptide linker in any embodiment can be used in any of the methods of the invention to detect hypoxic and/or cancerous tissue.

The current invention also includes kits that can be used to perform the methods and assays of the invention, in particular, in a high throughput screening format.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, certain embodiments of the invention are depicted in drawings. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 depicts an image of immunohistochemistry (IHC) for pimonidazole performed on a pancreatic tumor from the KPC mouse model showing small areas of intense staining making up a minority of the total tumor area (200X magnification).

Figure 2 shows the schematic for a competitive ELISA using the peptide linker to measure pimonidazole adducts in tissues.

Figure 3 depicts a standard curve generated with known amounts of free pimonidazole as a competitor. Pimonidazole adducts levels were extrapolated based on a previously established conversion of luM free pimonidazole competitor equals 40nM adduct competitor.

Figure 4 is a graph showing pimonidazole adduct levels from wild type kidneys and KPC tumors from mice subjected to normoxic (white bars) or hypoxic (black bars) conditions. Data is represented as average pimonidazole levels relative to normoxic kidney ± SEM. Control data are shown for a KPC tumor from a mouse not injected with pimonidazole.

Figure 5A is a graph depicting the quantification of 3D photoacoustic measurements of percent (%) total oxygen saturation through the depth of each of four pancreatic tumors (white bars), compared to a surrounding area of normal pancreas tissue (black bars).

Figure 5B is a graph depicting the quantification of total hemoglobin measurements through the depth of four pancreatic tumors (white bars), relative to normal pancreas tissue (black bars). Total hemoglobin is expressed relative to the average levels found within surrounding pancreas.

Figure 6 is a frequency plot of 45 readings from 9 tumors using the Oxylite™ method, showing the distribution of p02 readings within the binned ranges indicated under each bar. The percent of tumor measurements falling within the ranges of severe hypoxia (0 - 10 mmHg), mild hypoxia (11 - 30 mmHg), or normoxia (31 - 150 mmHg) are also indicated.

Figure 7 is a plot of partial oxygen pressure measurements for the kidney, spleen and pancreas of four wild type mice using the Oxylite method.

Figure 8 is a graph of results of a comparison of various peptide linkers with regard to the detection of PIMO. DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

The term "peptide" includes any sequence of two or more amino acids. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the left.

The term "amino acid," includes the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, l ,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, alpha-methylalanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (Ci-C₆) alkyl, phenyl or benzyl ester or amide).

The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

The term "subject" as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors. Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications.

The term "agent" as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

The term "detect", "detecting", "detection" and the like as used herein means to discover or identify the presence or existence of.

The term "link" as used herein means to form a connection.

The term "anchor" as used herein means to secure into place.

The term "immobilize" as used herein means to fix in place.

The term "surface" means the outer face, outside, or exterior boundary.

The term "peptide linker" as used herein means the composition of the invention, made up of specific amino acids, and capable of linking, anchoring and/or immobilizing thiol-reactive compounds and molecules to assay surfaces.

The Peptide Linker and Use in ELISAs

Enzyme-linked immuosorbent assays, otherwise known as ELISAs, are powerful tools for detecting and quantifying specific substances in a sample. Substances that can be detected include, but are not limited to, peptides, proteins, antibodies, and antigens.

The first molecule in an ELISA is immobilized on a solid surface, which is often made of a plastic material, such as polystyrene. This material passively binds molecules. It is this binding and immobilization of the molecules that makes an ELISA easy to design and perform. A preferred format for ELISA is a microtiter plate, such as a 96- or 384-well plate, made of polystyrene.

It is also preferred that a bicarbonate buffer be used in the ELISA procedure. The bicarbonate buffer has a high pH which helps to bind hydrophilic compounds.

A detection enzyme or other tag is linked directly to the final molecule, e.g., antibody, or introduced through a secondary antibody. The detection enzyme or tag can also be linked to a protein such as streptavidin if the final molecule is biotin labeled. Horseradish peroxidase and alkaline phosphatase can also be used, as well as β-galactosidase, acetylcholinesterase, and catalase.

The key step in the design and development of an ELISA to detect a specific molecule of interest is to optimize the plate-coating or immobilization conditions of the molecule. This is achieved directly by passive adsorption of the molecule to the plastic solid surface, which occurs through hydrophobic interactions between the plastic and the non-polar protein residues, or indirectly, e.g, via a capture antibody attached to the plate.

The antigen is then detected directly by a labeled primary antibody or indirectly by a labeled secondary antibody. One format is a sandwich assay where the molecule of interest is "sandwiched" between two primary antibodies- one that captures it to the plate and one that detects it.

Another format is a competitive assay. One variation of this format is to label purified antigen rather than an antibody. Unlabeled antigen from the sample and labeled antigen compete for binding to the capture antibody. A decrease in signal from the purified antigen indicates the presence of the antigen in the samples when compared to the wells with labeled antigen alone.

A further variation of this format is to incubate a sample with a first antibody which recognizes an antigen bound to plate or well. When the sample is contacted with the plate or well, any first antibody not bound to the antigen in the sample will bind to the antigen on the plate or well. A second antibody which binds to the first antibody and generates a signal is then added to the plate or well. A decrease in signal indicates the first antibody has bound to the antigen in the sample and is not available to bind to the antigen bound to the plate or well.

However, not all molecules are easily amenable to absorption to plastic and other surfaces. These include molecules that react with thiols, which are functional groups that contain a carbon-bonded sulfhydryl group. These molecules include a variety of nitro- aromatic compounds, including but not limited to, nitro-benzenes, nitro-furans, nitro- thiazoles, nitro-pyrroles, nitro-diazoles, and nitro-triazoles. One such example is pimonidazole or PIMO which is a 2-nitro-imidazole. These nitro-aromatic compounds, and PIMO in particular, are useful as markers for cancerous tissue that is hypoxic (U.S. Patent No. 5,086,068).

Other groups of compounds that react with thiols include but are not limited to, haloacetyls, maleimides, aziridines, acrylolys, arylating agents, vinylsulfones, pyridyl disulfides, TNB-thiols, and disulfide reducing agents. Additionally, reactions such as alkylation, oxidation, reduction, and ion-complexion are easily performed on thiols, making them useful and efficient in binding many other compounds and molecules.

The current invention solves this problem and simplifies the key step of immobilization of these thiol-reacting compounds. The current invention, which is a peptide linker, can be used in ELISA to immobilize the thiol-reacting compound to the plastic solid surface or in other assay formats or surfaces or purification surfaces such as affinity columns, or the binding of thiol-reactive molecules to tissue culture plates.

The novel peptide linker comprises three types of amino acids, arginine, cysteine and lysine.

Cysteine contains two thiol groups that bind to the thiol-reactive molecules. While one cysteine can be used in the peptide linker of the invention, the more cysteines in the peptide, increases the detection of the signal in the assay (Example 5). Thus, it is preferred that the peptide linker of the present invention comprise at least two cysteines, and more preferably at least three cysteines, and more preferably at least six cysteines.

Lysine polymers and arginine assist the peptide in binding to the solid surface of an assay, an affinity column or a tissue culture plate. While there are other positively charged amino acids such as histadine, lysine outperformed histadine in comparison experiments (Example 5).

In one embodiment, the peptide linker comprises an equal number of lysine and cysteine residues. In another embodiment, the peptide linker comprises an unequal number of lysine and cysteine residues. In a preferred embodiment, the peptide linker comprises six lysine amino acids and six cysteine amino acids. In a more preferred embodiment, the lysine amino acids and cysteine amino acids are alternating in groups in the peptide linker.

The preferred peptide, shown below comprises six cysteine residues and six lysine residues, in alternating groups of three:

cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

As shown in Examples, the peptide linker set forth above was reacted with chemically reduced pimonidazole to create pimonidazole-peptide adducts. The cysteine residues reacted with the activated PIMO and the lysine residues in the peptide facilitated coating of an ELISA plate. As shown in Example 1 with the use of an anti-PIMO antibody, the peptide linker bound the PIMO to the ELISA plates. As shown in Example 5, this particular configuration of peptide linker allowed an increased detection of PIMO over a peptide linker using histadine, meaning it facilitated the coating of the ELISA surface with the PIMO. Shorter and longer peptides, as well as peptides of different configurations, can also be used as a linker.

Examples of peptide linkers contemplated by the invention are:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO: 2)

arg-arg-arg-arg-arg-arg-cys (SEQ ID NO: 3)

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4)

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5)

lys-lys-lys-cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys-cys-cys-cys (SEQ ID NO:

6)

The peptide linker of the current invention can be prepared by various means, including but not limited to, recombinant expression, purification from cell culture, and chemical synthesis. The peptide linker of the invention are preferably provided in purified or substantially purified form, i.e., substantially free from other polypeptides, such as free from naturally-occurring polypeptides, particularly from other E. coli or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure.

ELISA Assay To Test for Hypoxia in Tumors Utilizing the Linker Peptide

Many cancers are hypoxic including pancreatic ductal adenocarcinoma (PDA), a particularly aggressive malignancy with a median overall survival of just 6 months (Siegel et al. (2014). The poor perfusion of pancreatic tumors suggests, by extension, that these tumors should be significantly hypoxic. Indeed, direct measurements of intratumoral oxygen tension in a small number of human PDA patients using an oxygen microelectrode showed severe hypoxia in the range of 0 - 5.3 mmHg (Koong et al. (2000))

One such use of PIMO or other nitro-aromatic compounds is as a marker for tumor hypoxia. The administration of a nitro-aromatic compound to a subject results in the selective binding of the compound to any hypoxic tissue if present in the subject. Using a labeled antibody, hypoxic tissue can be visualized using conventional immunochemical techniques (U.S. Patent No. 5,086,068).

A genetically engineered mouse model, designated KPC (K-ras^{LSL G12D/+};

LSL R172H/+

2^ ; Pdxl-Cre mouse model (Hingorani et al. (2005)) recapitulates biological and histological characteristics of human PDA and has been widely utilized for preclinical studies in pancreatic cancer. However, two reports have subsequently presented evidence, based on immunostaining for pimonidazole, that tumors arising in KPC mice, and a closely related model, are not widely hypoxic (Breast Cancer Linkage Consortium (1997); Cook et al. (2012)).

In order to help resolve these discordant observations, the ELISA assay using the peptide linker was used to directly examine the oxygenation of pancreatic ductal adenocarcinomas arising in KPC mice. The results from this assay demonstrated that pimonidazole is clearly activated in KPC tumor tissues (Example 3). This was in clear agreement with the results using the fluorescence quenching Oxylite™ system, which is particularly sensitive to the lowest levels of oxygen saturation, and showed that the pancreatic tumor tissues arising in the KPC model experience widespread, severe hypoxia in the range of 0 - 10 mmHg (Example 5). These results were also consistent with findings using photoacoustic imaging, an in vivo platform capable of direct quantitative measurements of blood oxygenation (Example 4). However, traditional immunohistochemical detection of hypoxia using staining of PIMO had varying results with a lack of pimonidazole staining in the majority of tumor tissue (Example 2).

The finding of severe hypoxia in KPC pancreatic tumors using two different in vivo methodologies calls into question the accuracy of traditional immunohistochemical detection of hypoxia by pimonidazole. However, the ELISA assay using the peptide linker accurately and efficiently found the hypoxia in the tumor tissue using PIMO making it an improved method for detecting, screening for and/or identifying tumor tissue in a subject.

Thus, one purpose of this invention is to solve the problems in using PIMO to detect hypoxic tissue and cancer. This invention also aims to present a simple, accurate, noninvasive, and high-throughput method for cancer detection and diagnosis.

Such a method would detect PIMO binding to hypoxic tissue in a non-invasive accurate manner. PIMO would be administered to the subject following standard protocol. One such protocol is to inject PIMO intraperitoneal at a dose of about 120 mg/kg. Samples of biological tissue and/or bodily fluid are taken from the subject. Examples of tissue that would be of interest are those that are suspected of being cancerous, including but not limited to, lung, liver, stomach, breast, epithelial, and intestinal. A preferred tissue would be from the pancreas. Bodily fluid would include blood, sputum, urine, and cerebrospinal fluid. Protein is then isolated from the tissue or bodily fluid and assayed using an ELISA with the peptide linker.

The potential presence of physiological hypoxia in tumors forms the primary rationale for the development of hypoxia-targeted anti-cancer drugs such as TH-302, a hypoxia- activated chemotherapy developed for pancreatic ductal adenocarcinoma (NCT01746979) (Duan et al. (2008); Jung et al. (2012)). This agent is an analog of pimonidazole that forms adducts under hypoxic conditions and then fragments to release a DNA alkylating agent, providing preferential cytotoxic targeting of tumor cells. In clinical trials of TH-302, some patients did not benefit from the drug. One interpretation of the outcome of these results is that some patients had more hypoxia than others, and therefore a subset benefitted, while others did not. To that point, an accurate and efficient assay to determine which patients are likely to benefit from such targeted treatment might produce better results. Thus, ELISA assays using the peptide linker could also be used to determine which patients and which tumors might benefit from hypoxia-activated chemotherapy, such as TH-302, by administering and subsequently measuring PIMO in the cancerous tissue in a non-invasive, less expensive, and perhaps most importantly, accurate fashion, as shown by the examples. Indeed the data in the examples support the basic rationale for targeting hypoxia in pancreatic cancer and show that an ELISA assay with the peptide linker is accurate and useful for determining which cancers and patients would benefit from treatment based upon hypoxia.

The peptide linker of the present invention would be useful in ELISA and other assays that would measure the concentration of hypoxia-activated chemotherapy and anti-cancer drugs in various tissues, both healthy and cancerous, after administration of the drug. This would indicate the patient's responses to the drug in a non-invasive fashion.

The finding of micro-regional diversity in both Oxylite™ readings also suggests a possible mechanism of escape for tumors treated with hypoxia-activated chemotherapy such as TH-302. Pools of cells located close to blood vessels likely experience higher local p(¾ levels and therefore would fail to activate the agent. Such islands of normoxia have been described in many experimental systems and imply that substantial heterogeneity in microenvironmental conditions likely exists within any given tumor. This heterogeneity in tumor oxygen environment can also be determined using an ELISA assay with the peptide linker by taking samples of the tumor from many regions and testing for hypoxia by the detection of PIMO.

Other Uses of the Peptide Linker

The peptide linker of the current invention can also be used to bind thiol-reactive molecules and compounds to many types of materials and surfaces, including but not limited to, sepharose, magnetic particles, latex beads, nanoparticles, macrobeads, tissue culture plates, and dipsticks. ELISAs and other assays using the peptide linker can be used as the basis of pharmacological assays to detect the presence of specific thiol-reactive compounds for which a specific antibody is available. In this method, the peptide linker could be used to immobilize a thiol-reactive compound of interest to an ELISA plate or related material, which would then be used as the basis for a competitive ELISA. This method could be used to screen for hypoxia-targeted anti-cancer drugs.

The peptide linker can also be used in affinity chromatography columns to bind and purify thiol-reactive molecules and compounds. In these applications, the lysine would bind to any material used for affinity chromatography including sepharose. Another version of this application would involve immobilizing a thiol-reactive compound via the peptide linker, to a material used for affinity chromatography, such as sepharose. The material treated in this way could then be used to isolate molecules or compounds that bind to the immobilized compound from complex mixtures (including biological lysates) through affinity chromatography or other purification techniques.

Kits

The current invention includes kits comprising the novel peptide linker for binding thiol-reactive molecules.

One embodiment of the kit is a 96- or 384- well ELISA plate with the attached peptide linker, other reagents for performing the assay such as an antibody that recognizes the molecule of interest, and instructions for use.

A further embodiment is a kit for identifying hypoxic tissue including cancerous tissue comprising a 96- or 384- well ELISA plate with the attached peptide linker, an antibody that recognizes pimonidazole, or another nitro-aromatic compound, other reagents for performing the assay, and instructions for use.

A further embodiment is a kit for identifying hypoxic tissue including cancerous tissue comprising a 96- or 384- well ELISA plate with the attached PIMO-peptide linker conjugate, an antibody that recognizes pimonidazole or another nitro-aromatic compound, other reagents for performing the assay, and instructions for use.

A further embodiment is a kit for identifying hypoxia-targeted anti-cancer drugs, e.g.,

TH-302, in tissues including cancerous tissue comprising a 96- or 384- well ELISA plate with the attached peptide linker, an antibody that recognizes the hypoxia-targeted anti-cancer drug, pimonidazole or another nitro-aromatic compound, other reagents for performing the assay, and instructions for use. A further embodiment is a kit for identifying hypoxia-targeted anti-cancer drugs, e.g., TH-302, in tissues including cancerous tissue comprising a 96- or 384- well ELISA plate with the attached hypoxia-targeted anti-cancer drugs or PIMO-peptide linker conjugate, an antibody that recognizes the hypoxia-targeted anti-cancer drug, pimonidazole, other reagents for performing the assay, and instructions for use.

In these embodiments of the kit, other reagents for performing the assay may include but are not limited to, pimonidazole, and reagents for purifying protein from a sample from the subject. The methods and assays of the present invention can be automated for convenient high-throughput screening to, for example, test a large number of tissue samples for hypoxia and cancerous tissue. Automated methods can be used to detect binding of the labeled assay components. Both qualitative and quantitative measurements can be made using the methods and assays of the invention using automated techniques known in the art.

Computer programs can be utilized to process samples, record output and/or process data. Such programs are known in the art.

Examples

The present invention may be better understood by reference to the following non- limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention.

Example 1- Preparation of ELISA Plates using the Peptide Linker

A peptide linker (hereafter referred to as lysteine or peptide linker) containing alternating groups of three cysteine residues (to react with activated pimonidazole) and three lysine residues (to bind to plastic in the manner of a poly-lysine coating) was developed and synthesized to 95% purity by Lifetein, Inc. Lysteine comprises the following amino acid sequence: cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

To generate a solid phase antigen, pimonidazole was first activated by reduction using

250 grams zinc metal dust (Sigma) added to 1 milliliter of an aqueous solution of 40 mM ammonium chloride and 5 milligrams of pimonidazole HC1 (Hypoxyprobe Inc.) under acidic conditions (Raleigh et al. (1992)). Complete activation of the nitro-imidizole group was noted at 10 minutes of reaction time at 60°C, as characterized by a loss of absorbance above 260nm present at the beginning of the reaction.

After filtration to remove the zinc dust, 0.5 milligrams of lysteine was reacted with 1.5 milligrams of activated pimonidazole in a 1.5 ml volume of 10 mM Tris Buffer pH 7.5, for 3 hours at 37° C to make pimo-lysteine. During this step, the activated 2-nitro-imidizole groups irreversibly react with the thiols present on the cysteine.

In order to attach the pimonidazole-linked peptide to high binding ELISA plates, 1 μg/well of pimo-lysteine (calculated from initial pure peptide concentrations) was incubated in 96-well high-binding ELISA plates with bicarbonate buffer (pH 9.6) for 30 minutes, followed by blocking with 0.5% porcine gelatin for 30 min. After washing with Tris-buffered saline 0.05% Tween-20, plates were ready for use.

The binding of the PIMO to the ELISA plate surface was verified by detection using a high titer rabbit anti-pimonidazole antibody (clone PAb27, kindly provided by Hypoxyprobe, Inc.). 10 μΐ of antibody in a total of 100 μΐ was added to the ELISA plates at concentrations of 1 : 1000, 1 :2000, and 1 :4000 along with negative controls.

Example 2- Mice Model of Pancreatic Cancer and In Vivo Pimonidazole Treatment and Immunohistochemistry

KPC mice

Kras^{LSL G12D/+}; _E53^{LSL R172H/+}; Pdxl-Cre (KPC) mice were bred by crossing the

Kras^LSL'^G12D, ¹⁸ _E53LSL.RI72H 19 ^ Pdxl-Cre²⁰ strains (Hingorani et al. (2005)). Triple mutant mice were palpated twice weekly for evidence of early tumors beginning at 8 weeks of each, followed by subsequent B-mode ultrasound screening using a VisualSonics 2100 Vevo High Resolution System, as previously described (Sastra and Olive (2013)). Following detection, tumors were monitored twice weekly until reaching a mean diameter of 6 mm.

In vivo pimonidazole treatment under normoxia and hypoxia and immunochemistry Tumor-bearing KPC mice were injected i.p. with 120 mg/kg of pimonidazole HC1 under either normoxic (n=5) or hypoxic (n=5) conditions. For hypoxia treatment, KPC mice were preconditioned for two hours in 10% (¾ in a Biospherix hypoxia chamber supplemented with nitrogen, followed by one hour at 6% (¾. After preconditioning, mice were quickly removed from the hypoxia chamber and injected with pimonidazole, and immediately returned to the chamber. They were then kept for one more hour at 6% (¾. After treatment, all mice were euthanized by isoflurane overdose, tumors were rapidly harvested, and samples were preserved in formalin for 24 hours followed by transfer to 70% ethanol. Other samples were rapidly frozen in Optimum Cutting Temperature compound (O.C.T., Tissue-Tek). As a control, pancreas and kidney tissues were harvested from wild type mice subjected to either normoxic (n=4) or hypoxic (n=4) conditions, as described above.

Tumor-bearing KPC mice (identified by high resolution ultrasound) were injected with pimonidazole, a nitroimidizole compound that is activated to form adducts with cellular thiols under hypoxic conditions (Varghese et al. (1976); Varia et al. (1998)). Pimonidazole adduct formation was assessed in tumor tissues using a rabbit anti-pimonidazole antibody (Hypoxyprobe, Inc.) and traditional immunohistochemical techniques. Consistent with previous reports (Cook et al. (2012); Guillaumond et al. (2013)), KPC pancreatic tumors were largely free of staining except for occasional intensely-stained patches immediately adjacent to regions of microscopic necrosis, which are often observed in this model (Figure 1). These areas made up approximately 5-10% of the tumor area, suggesting that the bulk of tumor tissue in this model is not strongly hypoxic. Example 3- Use of ELISA Assay with Peptide Linker to Detect Hypoxic Tissue

Materials and Methods

A previously described competitive ELISA (Arteel et al. (1995)), protocol to measure levels of pimonidazole adducts in tissues was modified using the plates as described in Example 1, i.e., using the peptide linker or lysteine, to attach the PIMO, and the mice in Example 2.

To prepare samples, fresh frozen OCT-embedded tissue cryosections were cut at 10 μπι and homogenized in Tris-buffered saline solution 0.5% tween-20. For tumors, an adjacent section was stained with hematoxylin and eosin to confirm that the samples were contained only tumor tissue. In cases where PDA tumor sections contained large necrotic areas or non- tumor tissue (e.g. normal/diseased pancreas), these regions were marked on the slide and markings were used to guide their removal from the block face by macrodissection prior to collecting subsequent sections for homogenization (5-10 sections at 10 microns/tumor). Following BCA protein concentration measurements of homogenized lysate (Pierce), 5 μg of tissue protein was digested with proteinase K (Sigma) at 1 μg/μL in a total of 125

overnight at 50° C. Proteinase K was inactivated with 20 mM PMSF for 5 minutes at room temperature and boiled for 10 minutes. Samples were centrifuged to remove precipitate formed by proteinase K inactivation and transferred to new tube. All subsequent steps were carried out at room temperature. To set up competition reaction on a low-binding 96-well plate (Figure 2), a starting amount of 1.6 ug of digested lysate was added in a total of 200 ul with buffer conditions adjusted to Tris buffered saline with 0.05% Tween-20. Free pimonidazole was used as a standard starting at 2 μΜ. Samples and standard were diluted in a 2-fold series in final volumes of 100 μί. Next, 25 μL· of a high titer rabbit anti-pimonidazole antibody (clone PAb27, kindly provided by Hypoxyprobe, Inc.) diluted at 1 :4000 was added to each well. Competition was performed with 600 rpm shaking for 1 hour. 100 μL· of competition reaction was transferred to antigen-coated ELISA plate and incubated with shaking for 1 hour. After washing plate 3 times, 100 μL· of alkaline phosphatase conjugated secondary antibody (Sigma) was added to ELISA plate wells with shaking for 1 hour. Following 3 washes, 100 μΕ of 1 mg/ml alkaline phosphatase surface (Sigma) in 10% diethanolamine buffer (pH 9.8) was added to each well and the maximal slope of colorimetric development at 405 nm was kinetically determined over 40 minutes at 5 minutes read intervals

A standard curve was used to convert maximal slopes to approximate levels of pimonidazole adducts in tissue samples (Figure 3). To calculate the conversion, a previously reported approximation was used where it was observed that 1 μΜ free pimonidazole corresponds to 400 nM tissue adducts (Raleigh et al. (1992)). During testing of the ELISA method, it was ensured that antigen-free control wells incubated directly with primary antibody followed by secondary antibody did not show any alkaline phosphatase activity. Additionally, no competition for pimonidazole primary antibody was detected in PDA tumor lysate from untreated KPC mice (Figure 4).

Results

To test the performance of this assay using tissue samples, wild type mice were exposed to hypoxia as described in Example 1, and pimonidazole concentrations in kidney tissues were measured. As expected, exposure to environmental hypoxia resulted in increased pimonidazole levels in normal kidneys (Figure 4).

Next the ELISA was used to measure pimonidazole concentrations in tumors from KPC mice, under ambient oxygen conditions. Consistent with in vivo data, KPC pancreatic tumors had high levels of pimonidazole, exceeding even that of kidney tissues from hypoxia- exposed mice (Figure 4). A trend towards even greater concentrations of pimonidazole was noted in hypoxia-exposed KPC mice.

It was concluded that the ELISA using the peptide linker detected the pimonidazole adducts formed in the hypoxic environment of KPC pancreatic tumors, that were not adequately detected using the traditional immunohistochemical protocols. Example 4- Photoacoustic Imaging in Murine PDA Confirms the Results of the ELISA Materials and Methods

Photoacoustic imaging is a technology that uses laser absorption to induce an acoustic signal in tissues that can be detected, at depth, by an ultrasound transducer. By tuning the wavelength of light, the concentration of different light-absorbing molecules (chromophores) can be quantified. By stimulating with 750nm and 805nm lasers, one can distinguish the concentrations of oxy- and deoxy-hemoglobin, respectively, producing measures of total hemoglobin concentration, percent oxygenated saturation, and total oxygenated hemoglobin. For comparison to the ELISA method used in Example 3, photoacoustic imaging on four tumor-bearing KPC mice was performed as previously described (Gerling et al. (2014)).

Results

Substantially lower levels of oxygen were found in the blood pool of the pancreatic tumors compared to surrounding pancreatic tissue. Interestingly, total hemoglobin levels were only approximately 1.5-fold lower in tumors compared to pancreas. See Figures 5A and 5B. All four tumors were verified as PDA by histopathology (data not shown). This data provided independent evidence that the ductal adenocarcinomas arising in KPC mice were severely hypoxic.

Example 5-Oxylite™ Analysis of Murine PDA Confirms the Results of the ELISA

Materials and Methods

Intratumoral partial oxygen pressures in KPC mice (n=8) were measured using the Oxylite™ fluorescence quenching-based system (Oxford Optronics). Tumor-bearing KPC mice were anesthetized under 98% O2 and 2% isoflurane while immobilized in a supine position on a heated stage. Hair was removed with depilatory cream around the abdomen and the tumor was visualized by ultrasound as above. A syringe with a 21G needle was attached to a stereotactic mount and inserted through the skin and abdominal wall. Real-time ultrasound imaging was used to visually guide the needle in-plane with the image through the center of the tumor until reaching the far edge. With the needle in place, the syringe was carefully removed and the bare-fibre oxygen-sensing Oxylite™ probe was then attached to the stereotactic mount and threaded through the needle bore until the probe tip was localized at the far edge of the tumor. The needle was fully retracted over the fibre and an initial p02 measurement was taken at the far site. Prior to each measurement, the probe was allowed to equilibrate for at 3-5 minutes until readings stabilized. After the initial reading, the fibre was retracted incrementally through the needle track, with readings taken every 1 - 2 mm, through the full depth of the tumor. Measurements within 1 mm of the edge of the tumor were excluded from the analysis since the needle frequently punctured the far wall of the tumor, allowing oxygen from the abdominal cavity into the wound (as made apparent by a sharp spike in readings).

To compare tumor to normal tissue, partial pressures were also measured in pancreas and kidney of WT mice (n=4).

After completing measurements, mice were euthanized by isoflurane overdose and tissue was harvested for formalin fixation for 24 hours prior to paraffin embedding. All tumors were verified as pancreatic ductal adenocarcinoma by a blinded observer experienced in mouse tumor pathology (KPO).

Results

It was found that 69% of the readings were in the severely hypoxic range (0 - 10 mmHg), while only a minority were in the mild hypoxic or normoxic ranges (10 - 30 mmHg and 30 - 150 mmHg respectively) (Figure 6). Precise examination of ultrasound images showing the location of the Oxylite™ probe tip at each measurement point demonstrated that normoxic readings were acquired in the area of cysts; moving the probe into solid tumor tissue brought p(¾ levels into a severely hypoxic range of 2 mmHg.

As a control, oxygen tension in normal tissues of wild type mice were measured, and found that kidney, pancreas and spleen tissues were within the normoxic range (>50 mmHg) (Figure 7). One kidney showed measurements in the range of 27 mmHg, consistent with prior reports that the outer medulla/inner cortex of the kidney can be mildly hypoxic (Aukland and

Krog (I960)).

Thus direct measurement of oxygen tension in pancreatic tumors using a fluorescence-based oxygen sensor demonstrated that pancreatic tumors in KPC mice were severely hypoxic verifying the results using the ELISA and the peptide linker.

Example 6- Comparison of Peptide Linker

Materials and Methods

The peptide linker or lysteine, with the amino acid sequence cys-cys-cys-lys-lys-lys- cys-cys-cys-lys-lys-lys (SEQ ID NO: 1), was compared to two other peptides with the amino acids sequences of: lys-lys-lys-lys-lys-lys-cys ("K6C") (SEQ ID NO 2); and his-his-his-his- his-his-cys ("H6C") (SEQ ID NO: 7), using the ELISA assay as described in Example 1. Results

As shown in Figure 8, in terms of relative detection of PIMO, the lysines in the lysteine outperforms histadines for binding to plate, and lysteine performed slightly better even though the six lysines in the K6C peptide were grouped as a consecutive sequence.

Controls with peptide alone with no conjugation reaction with PIMO showed no detection of signal as expected.

REFERENCES

Arteel et al. (1995) Br. /. Cancer 72:889-95

Aukland and Krog ( 1960) Nature 188 : 671

Breast Cancer Linkage Consortium (1997) Lancet 349: 1505-10

Cook et al. (2012) /. Exp. Med. 209:437-44

Duan et al. (2008) Journal of Medicinal Chemistry 51 :2412-20

Gerling et al. (2014) Theranostics 4:604- 13

Guillaumond et al. (2013) Proc Natl Acad Sci 110:3919-24

Hingorani et al. (2005) Cancer Cell 7:469-83

Jung et al. (2012) Xenobiotica 42:372-88

Koong et al. (2000) International Journal of Radiation Oncology, Biology, Physics 48:919- 22

Raleigh et al. (1992) International Journal of Radiation Oncology, Biology, Physics 22:403-5 Sastra and Olive (2013) Methods in Molecular Biology, 980:249-66

Siegel ei aZ. (2014) Cancer J. Clin. 64:9-29

Varghese et al. (1976) Cancer Research 36:3761-5

Varia et al. (1998) Gynecologic Oncology 71 :270-7

Claims

I. A composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition capable of linking thiol-reactive molecules and compounds to surfaces.

2. The composition of claim 1, wherein the composition comprises an equal number of cysteine residues and lysine or arginine residues.

3. The composition of claim 1, wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

4. The composition of claim 1 , comprising the amino acid sequence of cys-cys-cys-lys-lys- lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

5. The composition of claim 1, wherein the thiol-reactive compound is chosen from the group consisting of nitro-benzenes, nitro-furans, nitro-thiazoles, nitro-pyrroles, nitro- diazoles, and nitro-triazoles.

6. The composition of claim 1, wherein the thiol-reactive compound is pimonidazole.

7. The composition of claim 1, wherein the thiol-reactive compound is a hypoxia-tissue targeted anti-cancer agent.

8. The composition of claim 1, wherein the surface is a well or plate used in an enzyme- linked immunosorbent assay, sepharose, magnetic particles, latex beads, nanoparticles, macrobeads, tissue culture plates, or dipsticks.

9. A composition consisting essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition capable of linking thiol-reactive molecules and compounds to surfaces.

10. The composition of claim 9, wherein the composition consists essentially of an equal number of cysteine residues and lysine or arginine residues.

I I. The composition of claim 9, consisting essentially of the amino acid sequence of cys-cys- cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

12. An enzyme-linked immunosorbent assay for the detection of thiol-reactive compounds comprising the composition of claim 1.

13. An enzyme-linked immunosorbent assay for the detection of thiol-reactive compounds comprising the composition of claim 9.

14. The assay of claims 12 or 13, wherein the thiol-reactive compound is chosen from the group consisting of nitro-benzenes, nitro-furans, nitro-thiazoles, nitro-pyrroles, nitro- diazoles, and nitro-triazoles.

15. The assay of claims 12 or 13, wherein the thiol-reactive compound is pimonidazole.

16. The assay of claim 12 or 13, wherein the thiol-reactive compound is hypoxia-tissue targeted anti-cancer agent.

17. A method of detecting hypoxic tissue in a subject, comprising:

a. administering a nitro-aromatic compound to the subject;

b. isolating protein from a sample from the subject;

c. contacting or incubating the protein to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine;

d. allowing the nitro-aromatic compound in the protein to bind to the composition;

e. contacting or incubating the protein bound to the composition with an antibody that recognizes or binds to the nitro-aromatic compound, wherein the antibody generates a detectable signal when it recognizes or binds to the nitro- aromatic compound;

f. detecting and/or measuring the signal; and

g. determining the presence of the nitro-aromatic compound in the protein, wherein the presence of the nitro-aromatic compound is an indication that the tissue from the subject is hypoxic.

18. The method of claim 17, wherein the hypoxic tissue is cancerous.

19. The method of claim 17, wherein the hypoxic tissue is pancreatic.

20. The method of claim 18, wherein the indication that the tissue is hypoxic further indicates that the cancer can be treated by a hypoxia-targeted anti-cancer drug.

21. The method of claim 17, wherein the nitro-aromatic compound is pimonidazole.

22. The method of claim 17, wherein the sample is biological tissue or bodily fluid.

23. The method of claim 17, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

24. The method of claim 17, wherein the composition has the amino acid sequence chosen from the group consisting of: lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

25. The method of claim 17, wherein the composition has the amino acid sequence comprising: cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

26. The method of claim 17, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

27. A method of detecting cancer in a subject, comprising:

a. administering a nitro-aromatic compound to the subject;

b. isolating protein from a sample from the subject;

c. contacting or incubating the protein on a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine;

d. allowing the nitro-aromatic compound in the protein to bind to the composition; e. contacting or incubating the sample of protein bound to the composition with an antibody that recognizes or binds to the nitro-aromatic compound, wherein the antibody generates a detectable signal when it recognizes or binds to the nitro-aromatic compound;

f. detecting and/or measuring the signal; and

g. determining the presence of the nitro-aromatic compound in the protein, wherein the presence of the nitro-aromatic compound is an indication the subject is afflicted with cancer.

28. The method of claim 27, wherein the nitro-aromatic compound is pimonidazole.

29. The method of claim 27, wherein the sample is biological tissue or bodily fluid.

30. The method of claim 27, wherein the sample is pancreatic tissue.

31. The method of claim 27, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

32. The method of claim 27, wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2); arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

33. The method of claim 27, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

34. The method of claim 27, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

35. A method for the detection of a hypoxia-tissue targeted anti-cancer agent, in a subject, who has had the hypoxia-tissue targeted anti-cancer agent administered for the treatment of cancer, comprising:

a. isolating protein from a sample from the subject;

b. contacting or incubating the protein on a solid surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine;

c. allowing the hypoxia-tissue targeted anti-cancer agent in the protein to bind to the composition;

d. contacting or incubating the protein bound to the composition with an antibody that recognizes or binds to the hypoxia-tissue targeted anti-cancer agent, wherein the antibody generates a detectable signal when it recognizes or binds to the hypoxia-tissue targeted anti-cancer agent;

e. detecting and/or measuring the signal; and

f. detecting the presence of the hypoxia-tissue targeted anti-cancer agent in the protein, wherein the hypoxia-tissue targeted anti-cancer agent comprises a nitro- aromatic compound.

36. The method of claim 35, wherein the sample is biological tissue or bodily fluid.

37. The method of claim 35, wherein the sample is cancerous tissue.

38. The method of claim 35, wherein the sample is healthy tissue.

39. The method of claim 35, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

40. The method of claim 35, wherein the composition has the amino acid sequence chosen from the group consisting of: lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

41. The method of claim 35, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

42. The method of claim 35, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

43. A method of detecting hypoxic tissue in a subject, comprising:

a. administering a nitro-aromatic compound to the subject;

b. isolating protein from a sample from the subject;

c. contacting or incubating the protein with a first antibody that recognizes or binds to the nitro-aromatic compound;

d. contacting or incubating the protein that has been contacted or incubated with the first antibody to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition having linked, anchored, and/or immobilized a nitro-aromatic compound to the surface;

e. allowing any first antibody not bound to the nitro-aromatic compound in the protein to bind to the nitro-aromatic compound bound to the surface via the composition;

f. contacting or incubating the first antibody bound to the surface with a second antibody that recognizes or binds to the first antibody and when bound generates a detectable signal;

g. detecting and/or measuring the signal; and

h. determining the presence of the nitro-aromatic compound in the protein, wherein the presence of the nitro-aromatic compound is an indication that the tissue from the subject is hypoxic.

44. The method of claim 43, wherein the hypoxic tissue is cancerous.

45. The method of claim 43, wherein the hypoxic tissue is pancreatic.

46. The method of claim 44, wherein the indication that the tissue is hypoxic further indicates that the cancer can be treated by a hypoxia-targeted anti-cancer drug.

47. The method of claim 43, wherein the sample is biological tissue or bodily fluid.

48. The method of claim 43, wherein the nitro-aromatic compound is pimonidazole.

49. The method of claim 43, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

50. The method of claim 43, wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

51. The method of claim 43, wherein the composition has the amino acid sequence comprising: cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

52. The method of claim 43, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

53. A method of detecting cancer in a subject, comprising:

a. administering a nitro-aromatic compound to the subject;

b. isolating protein from a sample from the subject;

e. allowing any first antibody not bound to the nitro-aromatic compound in the protein to bind to the nitro-aromatic compound bound to the surface via the composition; f. contacting or incubating the first antibody bound to the surface with a second antibody that recognizes or binds to the first antibody and when bound generates a detectable signal;

g. detecting and/or measuring the signal; and

h. determining the presence of the nitro-aromatic compound in the protein, wherein the presence of the nitro-aromatic compound is an indication that the subject is afflicted with cancer.

54. The method of claim 53, wherein the nitro-aromatic compound is pimonidazole.

55. The method of claim 53, wherein the sample is biological tissue or bodily fluid.

56. The method of claim 53, wherein the sample is cancerous tissue.

57. The method of claim 53, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

58. The method of claim 53, wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

lys-lys-lys-cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys-cys-cys-cys (SEQ ID NO: 6).

59. The method of claim 53, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

60. The method of claim 53, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

61. The method of claim 53, wherein the cancer is pancreatic.

62. A method for the detection and measurement of a hypoxia-tissue targeted anti-cancer agent, in a subject, who has had the hypoxia-tissue targeted anti-cancer agent administered for the treatment of cancer comprising:

a. isolating protein from a sample from the subject;

b. contacting or incubating the protein with a first antibody that recognizes or binds to the hypoxia-tissue targeted anti-cancer agent;

c. contacting or incubating the protein that has been contacted or incubated with the first antibody to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition having linked, anchored, and/or immobilized a nitro-aromatic compound to the surface;

d. allowing any first antibody not bound to the hypoxia-tissue targeted anti-cancer agent in the sample to bind to the nitro-aromatic molecule or compound bound to the surface via the peptide linker;

e. contacting or incubating the first antibody bound to the surface with a second antibody that recognizes or binds to the first antibody and when bound generates a detectable signal;

f. detecting and/or measuring the signal; and

g. determining the presence of the hypoxia-tissue targeted anti-cancer agent in the protein, wherein the hypoxia-tissue targeted anti-cancer agent comprises a nitro- aromatic compound.

63. The method of claim 62, wherein the sample is biological tissue or bodily fluid.

64. The method of claim 62, wherein the sample is cancerous tissue.

65. The method of claim 62, wherein the sample is healthy tissue.

66. The method of claim 62, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

67. The method of claim 62, wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

68. The method of claim 62, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

69. The method of claim 62, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

70. The method of claim 61, wherein the cancer is pancreatic.

71. A kit comprising: a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition capable of linking thiol-reactive molecules and compounds to solid surfaces; and a 94 or 384-well microtiter plate.

72. The kit of claim 71, further comprising an antibody that recognizes or binds a thiol- reactive molecule or compound.

73. The kit of claim 71, further comprising a nitro-aromatic compound.

74. The kit of claim 73, wherein the nitro-aromatic compound is pimonidazole.

75. The kit of claim 71 , wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

76. The kit of claim 71, wherein the composition has the amino acid sequence of cys-cys-cys- lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

77. The kit of claim 71 , wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition capable of linking thiol-reactive molecules.

78. A method of detecting the presence of a thiol-reactive compound in a sample, comprising: a. contacting the sample to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine;

b. allowing a thiol-reactive compound in the sample to bind to the composition; c. contacting or incubating the sample bound to the composition with an antibody that recognizes or binds to the thiol-reactive compound, wherein the antibody generates a detectable signal when it recognizes or binds to the thiol-reactive compound;

d. detecting and/or measuring the signal; and

e. determining the presence of the thiol-reactive compound in a sample.

79. The method of claim 78, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

80. The method of claim 78, wherein the composition has the amino acid sequence chosen from the group consisting of: lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

81. The method of claim 78, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

82. The method of claim 78, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

83. The method of claim 78, wherein the sample is a test agent and the determination of the presence of the thiol-reactive compound indicates the test agent could be a hypoxia- targeted anti-cancer drug.

84. A method of detecting the presence of a thiol-reactive compound in a sample, comprising: a. contacting the sample with a first antibody that recognizes or binds to the thiol- reactive compound;

b. contacting or incubating the sample that has been contacted or incubated with the first antibody to a surface comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition having linked, anchored, and/or immobilized a thiol-reactive compound to the surface;

c. allowing any first antibody not bound to the thiol-reactive compound in the sample to bind to the thiol-reactive compound bound to the surface via the composition;

d. contacting or incubating the first antibody bound to the surface with a second antibody that recognizes or binds to the first antibody and when bound generates a detectable signal;

e. detecting and/or measuring the signal; and

f. determining the presence of the thiol-reactive compound in the sample.

85. The method of claim 84, wherein the surface is a well or plate used in an enzyme-linked immunosorbent assay.

86. The method of claim 84, wherein the composition has the amino acid sequence chosen from the group consisting of: lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

87. The method of claim 84, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

88. The method of claim 84, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.

89. The method of claim 84, wherein the sample is a test agent and the determination of the presence of the thiol-reactive compound indicates the test agent could be a hypoxia- targeted anti-cancer drug.

90. A method of purifying a compound that binds to thiol-reactive compounds or molecules from a mixture, comprising:

a. contacting the mixture with a material comprising a composition comprising at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine, said composition having linked, anchored, and/or immobilized a thiol-reactive compound to the material; b. allowing the compound to bind to the thiol-reactive compound linked, anchored, and/or immobilized to the material;

c. washing the unbound mixture from the material; and

d. eluting the compound from the material.

91. The method of claim 90, wherein the composition has the amino acid sequence chosen from the group consisting of:

lys-lys-lys-lys-lys-lys-cys (SEQ ID NO:2);

arg-arg-arg-arg-arg-arg-cys; (SEQ ID NO: 3);

lys-lys-lys-lys-lys-lys-cys-cys-cys-cys-cys-cys (SEQ ID NO: 4);

lys-cys-lys-cys-lys-cys-lys-cys-lys-cys-lys-cys (SEQ ID NO: 5); and

6).

92. The method of claim 90, wherein the composition comprises the amino acid sequence of cys-cys-cys-lys-lys-lys-cys-cys-cys-lys-lys-lys (SEQ ID NO: 1).

93. The method of claim 90, wherein the composition consists essentially of at least one cysteine residue and at least one residue of a second amino acid chosen from the group consisting of lysine and arginine.