AU2015252024B2 - Haptens, hapten conjugates, compositions thereof and method for their preparation and use - Google Patents

Abstract

HAPTENS, HAPTEN CONJUGATES, COMPOSITIONS THEREOF AND METHOD FOR THEIR PREPARATION AND USE 5OF THE DISCLOSURE A method for performing a multiplexed diagnostic assay, such as for two or more different targets in a sample, is described. One embodiment comprised contacting the sample with two or more specific binding moieties that bind specifically to two or more different targets. The two or more specific binding moieties are conjugated to 10 different haptens, and at least one of the haptens is an oxazole, a pyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, or a benzodiazepine. The sample is contacted with two or more different anti-hapten antibodies that can be detected separately. The two or more different anti-hapten 15 antibodies may be conjugated to different detectable labels. 7075696_1 (GHMafters) P80514.AU.2 SHEILAB 2/11/15 M ) Co C Cf0) -j n C / ~ ( o C14 'I04 (D

Description

The present invention provides embodiments of a method for developing more informed and effective regimes of therapy that can be administered to patients with an increased likelihood of an effective outcome (i.e., reduction or elimination of the tumor).

A diagnosis, both an initial diagnosis of disease and subsequent monitoring of the disease course (before, during, or after treatment), often is confirmed using histological examination of cell or tissue samples removed from a patient. For tumors, clinical pathologists need to be able to accurately determine whether such samples are o benign or malignant and to classify the aggressiveness of tumor samples deemed to be malignant. These determinations often form the basis for selecting a suitable course of patient treatment. Similarly, the pathologist needs to be able to detect the extent to which a cancer has grown or gone into remission, particularly as a result of or consequent to treatment, most particularly treatment with chemotherapeutic or

5 biological agents.

Histological examination involves tissue-staining procedures as disclosed herein alone or in combination with other known technologies that permit morphological features of a sample to be readily observed, such as under a light microscope. A pathologist, after examining the stained sample, typically makes a qualitative o determination of whether the tumor sample is malignant. Ascertaining a tumor’s aggressiveness merely by histological examination is difficult. A tumor’s aggressiveness often is a result of the biochemistry of the cells within the tumor, such as

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2015252024 02 Nov 2015 protein expression, suppression and protein/or phosphorylation, which may or may not be reflected by sample morphology. Assessing the biochemistry of the cells within a tumor sample using disclosed embodiments of the present invention alone, and optionally in combination with other known techniques is desirable, as is observing, and potentially quantitating, both gene expression and protein phosphorylation of tumorrelated genes or proteins, or more specifically cellular components of tumor-related signaling pathways.

Cancer therapy can be based on molecular profiling of tumors rather than simply their histology or site of the disease. Elucidating the biological effects of targeted io therapies in tumor tissue and correlating these effects with clinical response helps identify the predominant growth and survival pathways operative in tumors, thereby establishing a pattern of likely responders and conversely providing a rational for designing strategies to overcome resistance. Successful diagnostic targeting of a growth factor receptor determinea if tumor growth or survival is being driven by the targeted receptor or receptor family, by other receptors not targeted by the therapy, and whether downstream signaling suggests that another oncogenic pathway is involved. Furthermore, where more than one signaling pathway is implicated, members of those signaling pathways can be used as diagnostic targets to determine if a dual inhibitor therapy will be or is effective.

o Effective chemotherapeutic medications destroy tumor cells and not adjacent normal cells. This is accomplished using medications that affect cell activities predominantly occurring in cancer cells but not in normal cells. One difference between normal and tumorous cells is the amount of oxygen in the cells. Many tumorous cells are “hypoxic,” i.e. oxygen deficient.

5 Mammalian cells have an array of responses to balance the requirement for oxygen as an energy substrate and the inherent risk of oxidative damage to cellular macromolecules. Molecular bases for a variety of cellular and systemic mechanisms of oxygen homeostasis have been idenfied, and the mechanisms have been found to occur at every regulatory level, including gene transcription, protein translation,

0 posttranslational modification, and cellular localization (Harris, 2002, Nat Rev. 2:3847).

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Embodiments of the present invention can be used to analyze these products and/or processes. One disclosed embodiment first comprises identifying tumors. A panel of diagnostics of each tumor is used to find suitable, and preferably the best, candidate for each therapy. For example, treatment by an mTOR pathway-targeted therapy, such as rapamycin or PX-478, may not be effective unless an EGF pathway inhibitor is used in combination. Where there are high expression levels of EGF pathway components, such as pERK and pMEK, an mTOR pathway-targeted therapy is not effective. Disclosed embodiments of the present a clinician to identify a more effective combination of targeted therapies.

o Automated (computer-aided) image analysis systems known in the art can augment visual examination of tumor samples. In a representative embodiment, a cell or tissue sample is exposed to at least one disclosed hapten, hapten conjugate, such as a hapten-antibody conjugate, or composition thereof, having a detectable label, or that is recognized by an anti-hapten antibody having a detectable label. These hapten-based reagents and processes can be specific for a particular biological marker, such as those disclosed herein. An image, typically a magnified image, of the sample is then processed by a computer that receives the image, typically from a charge-coupled device (CCD) or camera such as a television camera. Such a system can be used, for example, to detect and measure expression and activation levels of desired targets, such

0 as HIF-Ια, pMEK, pERK, mTOR, pmTOR, pAKT, pTSC2, pS6, and p4EBPl in a sample, or any additional diagnostic biomarkers. Thus, disclosed embodiments of the present invention provide a more accurate cancer diagnosis and better characterization of gene expression in histologically identified cancer cells, most particularly with regard to expression of tumor marker genes or genes known to be expressed in

5 particular cancer types and subtypes (e.g., having different degrees of malignancy).

This information allows a clinician to determine a more effective therapy regimen, and to monitor the results of an implemented therapy regimen. For example, drugs with clinical efficacy for certain tumor types or subtypes can be administered to patients whose cells are so identified.

o Another drawback of conventional therapies is that the efficacy of specific therapeutic agents in treating a particular disease in an individual human patient is unpredictable. This unpredictability has to date substantially precluded determining,

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2015252024 02 Nov 2015 prior to starting therapy, whether one or more selected agents would be active or to render an accurate prognosis or course of treatment in an individual patient. This is especially important because a particular disease presents the clinician with a choice of treatment regimens, without any current way of assessing which regimen will be most efficacious for a particular individual. Disclosed embodiments of the present invention are able to better assess the expected efficacy of a proposed therapeutic agent (or combination of agents) in an individual patient. Disclosed embodiments are advantageous for the additional reasons that they are both time- and cost-effective in assessing the efficacy of chemotherapeutic regimens and are minimally traumatic to io cancer patients.

As a result, disclosed embodiments of the present method can be used to identify a disease that will respond to the proposed treatment, such as a mammalian tumor that responds to particular inhibitor, such as an mTOR pathway inhibitor, or a dual mTOR pathway inhibitor and EGF pathway inhibitor therapy. Further, disclosed embodiments of the invention can be used to select a patient for a particular treatment, or can be used to identify a disease that does not respond to directed therapies. Further, methods of this invention can be used to select a subject that will not likely be responsive to a proposed treatment.

Patterns of expression, or other cellular processes, such as phosphorylation, o cellular products, etc., are detected and optionally quantified using disclosed embodiments of the present invention. For example, expression and/or phosphorylation patterns of polypeptides can be detected using biodetection reagents specific for the polypeptides. Exemplary biodetection reagents include antibodies and nucleic acid probes, typically a collection of one or more nucleic acid fragments whose

5 hybridization to a sample can be detected. The antibody and probe may be unlabeled or labeled so that its binding to the target or sample can be detected. For example, the antibody or probe might be conjugated to at least one disclosed hapten, alone or in combination with other disclosed or known haptens. An anti-hapten antibody having a detectable label can be administered to a sample in a manner effective to allow the anti30 hapten antibody to complex with the hapten. The complex is then visualized.

Nucleic acid probes mahy be from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., one or more clones, an isolated

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2015252024 02 Nov 2015 whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The nucleic acid probe also may be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. The probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854).

One of ordinary skill in the art will recognize that the precise sequence of the o particular probes can be modified to a certain degree to produce probes that are substantially identical, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived. The term nucleic acid refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides that have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. One

0 of skill in the art would recognize how to use a nucleic acid probes for screening of cancer cells in a sample by reference, for example, to U.S. Patent 6,326,148, directed to screening of colon carcinoma cells.

Polypeptides associated with cancer can be quantified by image analysis using a suitable primary antibody against biomarkers, including but not limited HIF-Ια, pMEK,

5 pERK, mTOR, pmTOR, pAKT, pTSC2, pS6, and p4EBPl, detected directly or using an appropriate secondary antibody (such as rabbit anti-mouse IgG when using mouse primary antibodies) and/or a tertiary avidin (or Strepavidin) biotin complex (“ABC”).

Examples of reagents useful in the practice of the methods of the invention as exemplified herein include antibodies specific for HIF-Ια, including but not limited to

0 the mouse monoclonal antibody VMSI760-4285, obtained from Ventana Medical

Systems, Inc. (Tucson, AZ). Other reagents useful in the practice of the methods of this invention include, but are not limited to, rabbit polyclonal antibody Abeam 2732

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2015252024 02 Nov 2015 specific to mTOR, rabbit polyclonal antibody CST 2971 specific to pmTOR, rabbit polyclonal antibody CST 3614 specific to mTSC2, rabbit polyclonal antibody CST 2211 specific to pS6, rabbit monoclonal antibody CST 3787 specific to pAKT, rabbit polyclonal antibody CST 9121 specific to pMEK, rabbit polyclonal antibody VMSI

760-4228 specific to mERK (p44/p42), and rabbit polyclonal antibody CST 9455 specific to m4EBPl.

Further, predictive patterns or products, such as peptides or phosphorylation thereof, can be compared to a sample that has not received treatment, such as a nontumor tissue or cell sample. The non-tumor tissue or cell sample can be obtained from io a non-tumor tissue or cell sample from the same individual, or alternatively, a nontumor tissue or cell sample from a different individual. A detected pattern for a polypeptide is referred to as decreased in the mammalian tumor, tissue, or cell sample, if there is less polypeptide detected as compared to the a non-tumor tissue or cell sample. A detected pattern for a polypeptide is referred to as increased in the mammalian tumor, tissue, or cell sample, if there is more polypeptide detected as compared to the a non-tumor tissue or cell sample. A detected pattern for a polypeptide is referred to as normal in the mammalian tumor, tissue, or cell sample, if there is the same, or approximately the same, polypeptide detected as compared to a non-tumor tissue or cell sample.

o Target protein amounts may be quantified by measuring the average optical density of the stained antigens. Concomitantly, the proportion or percentage of total tissue area stained can be readily calculated, for example as the area stained above a control level (such as an antibody threshold level) in the second image. Following visualization of nuclei containing biomarkers, the percentage or amount of such cells in

5 tissue derived from patients after treatment are compared to the percentage or amount of such cells in untreated tissue. For purposes of the invention, “determining” a pattern of expression, phosphorylation, or both expression and phosphorylation of polypeptides is understood broadly to mean merely obtaining the expression level information on such polypeptide(s), either through direct examination or indirectly from, for example, a

0 contract diagnostic service.

IX. Miscellaneous Utilities

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Although the examples presented herein to exemplify the invention primarily pertain to immunohistochemical assays, the disclosed haptens, the disclosed haptenlabeled probes and the disclosed detection methods can be applied to any type of immunoassay, nucleic acid-based assay or peptide nucleic acid (PNA)-based assay.

For example, the disclosed haptens can form a component of a detection scheme for enzyme-linked immunosorbent assays (ELISA); protein, nucleic acid and PNA microarray assays; and, for flow cytometric assays. Furthermore, immunohistochemical assays such as those specifically detailed herein also can be applied for detection of target molecules in tissue arrays.

io The disclosed haptens, antibodies conjugated to haptens and detection methods can be utilized for detection of target molecules in standard (indirect), sandwich and competitive format ELISA assays. In a standard format ELISA, target molecules are non-specific ally adhered to a substrate (such as a nitrocellulose substrate) and are subsequently detected by one or more primary antibodies that specifically bind to the desired target or targets. In one embodiment, the primary antibodies are labeled with different haptens as disclosed herein, and disclosed anti-hapten antibodies conjugated to detectable labels (more specifically, enzymes in an ELISA, but other detectable labels such as quantum dots could be substituted) are subsequently added for visualization of the presence of target molecules adhered to the substrate. Alternatively, in the o sandwich format, capture antibodies specific to one or more target molecule are adhered (covalently or non-covalently) to a substrate and a sample is added, allowing any target molecules present to also become adhered to the substrate through the interaction with the capture antibodies. After washing to remove non-target molecules that are nonspecifically bound to the substrate, one or more detection antibodies that bind to the

5 desired targets at a different site from the capture antibodies are added. In one embodiment, the detection antibody or antibodies are labeled with one or more disclosed haptens and then one or more disclosed anti-hapten antibodies conjugated to the same or different detectable label are added for subsequent visualization of the targets adhered to the substrate. In any format, amplification of the visualization signal

0 is possible utilizing additional intermediate antibodies such as species-specific antiantibodies.

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In other embodiments, disclosed haptens, disclosed hapten-labeled antibodies and hapten-labeled nucleic acids, and disclosed detection methods can be employed for target detection in blot assays, wherein proteins or nucleic acids that are separated by electrophoresis are blotted non-specifically to a substrate, which substrate is then queried for the presence of particular nucleic acid sequences or proteins. For example, in a Southern Blot assay, nucleic acids are separated by agarose gel electrophoresis and blotted from the gel in relative position to one another onto a nitrocellulose filter, which filter can then be probed with hapten-labeled nucleic acid probes that can then be detected by utilizing disclosed anti-hapten antibodies conjugated to detectable labels.

io Detection of targets in Northern (RNA) and Western (Protein) blots also are possible utilizing the disclosed haptens.

The disclosed haptens, hapten-labeled probes and detection methods also can be utilized in target detection schemes using microarrays, such as for normal and reverse phase protein microarrays and for nucleic acid microarrays (including cDNA and oligonucleotide microarrays). For example, in a reverse phase protein microarray, samples are spotted individually onto a substrate where proteins in the samples become non-specifically bound to the substrate. Subsequently, multiple different antibodies (such as disclosed antibody-hapten conjugates) are used to probe the spots for the presence of particular proteins. Each spot can be probed for a plurality of different o proteins simultaneously, or alternatively each spot can be probed for a different protein, or each spot can be probed for the same protein (such as where each spot is from a different sample taken from a subject at different times, for example, following administration of a drug to the subject). In the case of nucleic acid microarrays, disclosed hapten labeled probes can be utilized in a detection scheme.

5 Tissue microarrays are advantageously used to implement disclosed embodiments of the invention, to rapidly screen multiple tissue samples under uniform staining and scoring conditions. (Hoos et al., 2001, Am J Pathol. 158: 1245-51). Scoring of the stained arrays can be accomplished manually using the standard 0 to 3+ scale, or by an automated system that accurately quantifies the staining observed. For

0 example, with disclosed drug therapy embodiments, this analysis can be used to identify biomarkers that best predict patient outcome following treatment. Patient “probability of response” ranging from 0 to 100 percent can be predicted based upon the expression,

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2015252024 02 Nov 2015 phosphorylation or both of a small set of ligands, receptors, signaling proteins or predictive combinations thereof. Additional samples from cancer patients can be analyzed, either as an alternative to or in addition to tissue microarray results. For example, analysis of samples from breast cancer patients can confirm the conclusions from the tissue arrays, if the patient’s responses correlate with a specific pattern of receptor expression and/or downstream signaling.

The disclosed haptens, hapten-labeled probes and detection methods also find utility in flow cytometry, where cells are probed for the presence of one or more target molecules (e.g. particular proteins or nucleic acid sequences) and possibly sorted io according to the presence or absence of one or more target molecules (such as in fluorescence assisted cell sorting, FACS). In one embodiment, one or more disclosed hapten-labeled antibodies or hapten-labeled nucleic acid probes are contacted to a plurality of cells, and then the cells are contacted with one or more anti-hapten antibodies that are conjugated to one or more differentially detectable labels.

X. Antigen/Antibody Recognition and Target Generally

Any antibody that specifically binds the hapten of interest, or an epitope from the antigen of interest, can be used in the methods disclosed herein. In one example the sequence of the specificity determining regions of each complementarity determining o region (CDR) of an antibody that specifically binds the antigen of interest is determined. Residues that are outside the specificity determining region (SDR, nonligand contacting sites) may be substituted. For example, at most one, two or three amino acids can be substituted. The production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well

5 known in the art. For example, humanized antibodies can be routinely produced. The antibody or antibody fragment can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor monoclonal antibody that specifically binds the antigen of interest and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light

0 chain frameworks. Generally, the humanized immunoglobulin specifically binds to

RET with an affinity constant of at least 10⁷ M’¹, such as at least 10⁸ M’¹ at least 5 X 10⁸

M’¹ or at least 10⁹ M’¹.

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Humanized monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci.

io U.S.A 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J.

Immunol. 150:2844, 1993. The antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, IgG2, IgG3 and IgG₄.

In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework.

o Human framework regions, and mutations that can be made in a humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No.

5,585,089, which is incorporated herein by reference).

Exemplary human antibodies are LEN and 21/28 CL. These are framework regions that are used in a variety of antibodies that bind tumor markers. A person of

5 ordinary skill in the art will appreciate that others could be used, and that these regions are exemplary only. The sequences of the heavy and light chain frameworks are known in the art.

Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such

0 as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region and are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor. These fragments include:

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2015252024 02 Nov 2015 (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating 5 whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab'h, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

io (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). To produce these antibodies, the V_H and the V_L can be expressed from two individual nucleic acid constructs in a host cell. If the Vh and the Vl are o expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light

5 chain variable region are chemically linked by disulfide bonds.

In an additional example, the Fv fragments comprise V_H and V_L chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the Vh and V_L domains connected by an oligonucleotide. The structural gene is inserted into

0 an expression vector, which is subsequently introduced into a host cell such as E. coli.

The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see

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Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio/Technology 11:1271, 1993; and Sandhu, supra).

Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or 5 by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2· This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl io groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al.,

Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments o bind to the antigen that is recognized by the intact antibody.

One of ordinary skill in the art will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the Vh and the Vl

5 regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the Vh and the Vl regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of

0 ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

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2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, one of skill in the art can readily review SEQ ID NOs: 1-2 and 5-10, locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.

Effector molecules, such as therapeutic, diagnostic, or detection moieties can be io linked to an antibody that specifically binds an antigen of interest, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (15 NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL.

0 The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are

5 polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, label (such as

0 enzymes or fluorescent molecules) drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

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XI. Methods of Producing Antibodies using DNA Generally

Exemplary nucleic acids encoding sequences encoding an antibody that specifically binds an antigen of interest can be prepared by cloning techniques.

Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from io manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.

(Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the

0 transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting the cDNA which encodes a variable region from an antibody that specifically binds an antigen of interest

5 into a vector.

Once the nucleic acids encoding the antibody or fragment thereof are isolated and cloned, the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. One or more DNA sequences encoding the antibody or fragment thereof can be expressed in vitro by DNA transfer

0 into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during

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2015252024 02 Nov 2015 replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

Polynucleotide sequences encoding the antibody or fragment thereof can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading io frame of that gene to permit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the antibody or fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out by o conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCh method using procedures well known in the art. Alternatively, MgCh or RbCl can be used. Transformation can also be performed after forming a protoplast of

5 the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences

0 encoding the antibody or fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.

Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or

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Isolation and purification of recombinantly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity io columns, column chromatography, and the like (see, generally, R. Scopes, Protein

Purification, Springer-Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies disclosed herein. See, Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun,

Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989 and Ward et al.,

Nature 341:544, 1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well known in the art, a

5 reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated by reference herein, and especially

0 as described by Buchner et al., supra.

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Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, the heavy and 5 light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is io completed.

In addition to recombinant methods, the antibodies disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater o length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'dicycylohexylcarbodiimide) are well known in the art.

5 XII. Antigens

Exemplary antigens of interest include those listed below:

Table 1

Exemplary antigens of interest (target antigens)

Viral Target Antigens	Exemplary Target Antigen Sequences from the Target Antigens	SEQ ID NO:
BK	TLYKKMEQDVKVAHQ	1
	GNLPLMRKAYLRKCK	22
	TFSRMKYNICMGKCI	23
JC	SITEVECFL	2
Epstein-Barr (EBV)	QPRAPIRPI	3

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cytomegalovirus (CMV)	NLVPMVATV	4
HPV	YMLDLQPET(T)	5
Influenza A	GILGFVFTL	6
Tumor Target Antigens and their derivative peptides
PRAME	LYVDSLFFL	7
WT1	RMFPNAPYL	8
Survivin	ELTLGEFLKL	9
AFP	GVALQTMKQ	10
ELF2M	ETVSEQSNV	11
proteinase 3 and its peptide PR1	VLQELNVTV	12
neutrophil elastase	VLQELNVTV	13
MAGE	EADPTGHSY	14
MART	AAGIGILTV	15
tyrosinase	RHRPLQEVYPEANAPIGHNRE	16
GP100	WNRQLYPEWTEAQRLD	17
NY-Eso-1	VLLKEFTVSG	18
Herceptin	KIFGSLAFL	19
carcino-embryonic antigen (CEA)	HLFGYSWYK	20
PSA	FLTPKKLQCV	21
Fungal Target Antigen
Blastomyces dermatitidis	CELDNSHEDYNWNLWFKWCSGHGR TGHGKHFYDCDWDPSHGDYSWYLW DPSHGDYSWYLWDYLCGNGHHPYD DYLCGNGHHPYDCELDNSHEDYSW DPYNCDWDPYHEKYDWDLWNKWCN KYDWDLWNKWCNKDPYNCDWDPYH	24 25 26 27 28 29

Table 2

Exemplary tumors and their tumor antigens

Tumor	Tumor Associated Target Antigens
Acute myelogenous leukemia	Wilms tumor 1 (WT1), preferentially expressed antigen of melanoma (PRAME), PR1, proteinase 3, elastase, cathepsin G
Chronic myelogenous leukemia	WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G
Myelodysplastic syndrome	WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G
Acute lymphoblastic leukemia	PRAME
Chronic lymphocytic leukemia	Surviving
Non-Hodgkin’s lymphoma	Surviving
Multiple myeloma	New York esophageous 1 (NY-Esol)
Malignant melanoma	MAGE, MART, Tyrosinase, PRAME GP100
Breast cancer	WT1, herceptin
Lung cancer	WT1
Prostate cancer	Prostate-specific antigen (PSA)
Colon cancer	Carcinoembryonic antigen (CEA)
Renal cell carcinoma (RCC)	Fibroblast growth factor 5 (FGF-5)

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Any antigenic peptide (such as an immunogenic fragment) from an antigen of interest can be used to generate a population of T cells specific for that antigen of interest. Numerous such antigenic peptides are known in the art, such as viral and tumor antigens. This disclosure is not limited to using specific antigen peptides.

Particular examples of antigenic peptides from antigens of interest, include, but are not limited to, those antigens that are viral, fungal, and tumor associated, such as those shown in Table 1. Additional antigenic peptides are known in the art (for example see Novellino et al., Cancer Immunol. Immunother. 54(3):187-207, 2005, and Chen et al., io Cytotherapy, 4:41-8, 2002, both herein incorporated by reference).

Although Table 1 discloses particular fragments of full-length antigens of interest, one skilled in the art will recognize that other fragments or the full-length protein can also be used in the methods disclosed herein. In one example, an antigen of interest is an “immunogenic fragment” of a full-length antigen sequence. An “immunogenic fragment” refers to a portion of a protein which can be used to induce an immune response, such as a B cell response, such as the production of antibodies. Typically, such fragments are 8 to 12 contiguous amino acids of a full length antigen, although longer fragments may of course also be used. In particular examples, the immunogenic fragment is 8-100 contiguous amino acids from a full-length target

0 antigen sequence, such as 8-50 amino acids, 8-20 amino acids, or 10, 20, 30, 40, 50,

100 or 200 contiguous amino acids from a full-length target antigen sequence.

Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required to produce antigenic molecules have been identified (see, for example, Southwood et al., J. Immunol. 160:3363, 1998; Rammensee et al.,

Immunogenetics 41:178, 1995; Rammensee et al., J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, Curr. Opin. Immunol. 6:13, 1994; Sette and Grey, Curr. Opin. Immunol. 4:79, 1992).

In view of the many possible embodiments to which the principles of the

0 disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the

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2015252024 02 Nov 2015 following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to io preclude the presence or addition of further features in various embodiments of the invention.

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Claims (28)

1. We claim:

   1. A hapten-carrier conjugate comprising a hapten coupled to a carrier where the hapten is an oxazole, the hapten-carrier conjugate having a formula (hapten)_m-(linker)_n(carrier)p where m is 1, n is 1, and p is 1 and wherein:

   the linker is a polymer comprising from 1 to about 15 ethylene glycol units;

   the carrier is selected from the group consisting of an amino acid, a polypeptide, a protein, a nucleoside, a nucleotide, a nucleotide chain, a nucleic acid, DNA, RNA, mRNA, a polymer, aminoalkyl agarose, aminopropyl glass and cross-linked dextran;

   wherein the hapten-carrier conjugate is a product of a reaction between the hapten and carrier with a homobifunctional or heretobifunctional linker having the general structure:

   A-|-CH₂C h₂-o-|-b wherein y is an integer from 1 to 15, wherein A and B are independently selected from a reactive group that reacts with a corresponding reactive group on the hapten and/or the carrier, and wherein the reactive group is selected from the group consisting of an amine-reactive group selected from the group consisting of an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acid chloride, an aldehyde, a glyoxal, an epoxide, an oxirane, a carbonate, an arylating agent, an imidoester, a carbodiimide, an anhydride, and combinations thereof; a thiol-reactive functional group selected from the group consisting of a haloacetyl, an alkyl halide, a maleimide, an aziridine, an acryloyl derivative, an arylating agent; a thiol-disulfide exchange reagent selected form the group consisting of a pyridyl disulfide, a TNBthiol, a disulfide reductant, and combinations thereof; a carboxylate reactive functional groups selected from the group consisting of a diazoalkane, a diazoacetyl compound, a carbonyldiimidazole compound, and a carbodiimide; a hydroxylreactive functional groups selected from the group consisting of an epoxide, an oxirane, a carbonyldiimidazole, a Ν,Ν'-disuccinimidyl carbonate, a Nhydroxysuccinimidyl chloroformate, an alkyl halogen, and an isocyanate; aldehyde and ketone reactive functional groups selected from the group consisting of a hydrazine, a Schiff base, and combinations thereof; an active hydrogen-reactive compound selected from a diazonium derivative, and combinations thereof; a photoreactive chemical functional group selected from the group consisting of an aryl

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   2015252024 20 Dec 2017 azide, a halogenated aryl azide, a benzophonone, a diazo compound, a diazirine derivative, and combinations thereof; and where the hapten is an oxazole having a formula .T where R1-R3 independently are selected from hydrogen, acyl, aldehyde (-C(O)H), alkoxy, alkyl having 20 or fewer carbon atoms, heteroalkyl having 20 or fewer carbon atoms, hydroxyalkyl having 20 or fewer carbon atoms, amido (-C(O)NH₂), amino (-NH₂), aryl, alkylaryl wherein the alkyl chain has 20 or fewer carbon atoms, carboxyl (-C(O)OH), carboxylate (-C(O)O'), cycloalkyl having 20 or fewer carbon atoms, cyano, alkylester wherein the alkyl chain has 20 or fewer carbons, ether, fluoro, chloro, bromo, iodo,, hydroxyl (-OH), hydroxylamine (-NHOH), alkylketone having 20 or fewer carbon atoms, nitro (-NO₂), sulfhydryl (-SH), and sulfoxide, at least one of the R1-R3 substituents is coupled to the linker, and Y is oxygen.
2. 2. The hapten-carrier conjugate according to claim 1, where the hapten is an oxazole sulfonamide having a formula where R₃-R₆ independently are selected from hydrogen, acyl, aldehyde (-C(O)H), alkoxy, alkyl having 20 or fewer carbon atoms, heteroalkyl having 20 or fewer carbon atoms, hydroxyalkyl having 20 or fewer carbon atoms, amido (-C(O)NH₂), amino (-NH₂), aryl, alkyl aryl wherein the alkyl chain has 20 or fewer carbon atoms, carboxyl (-C(O)Off), carboxylate (-C(O)O’), cycloalkyl having 20 or fewer carbon atoms, cyano, alkyl ester wherein the alkyl chain has 20 or fewer carbons, ether, fluoro, chloro, bromo, iodo, hydroxyl (-OH), hydroxylamine (-NHOH), alkyl ketone

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   Y is oxygen.

   2015252024 20 Dec 2017
3. 3. The hapten-carrier conjugate according to claim 1 or claim 2, where the carrier is a protein.
4. 4. The hapten-carrier conjugate of any one of claims 1 to 3, where the carrier is bovine thyroglobulin, keyhole limpet hemocyanin, or bovine serum albumin.
5. 5. The hapten-carrier conjugate according to any one of claims 1 to 4, where the linker has from about 2 to about 4 ethylene glycol units.
6. 6. The hapten-carrier conjugate according to any one of claims 1 to 5, where the carrier is a specific binding carrier.
7. 7. The hapten-carrier conjugate according to claim 6, where the carrier is a protein, a nucleic acid, or an antibody.
8. 8. The hapten-carrier conjugate according to any one of claims 1 to 7, where the carrier is an immunogenic carrier.
9. 9. The hapten-carrier conjugate according to claim 8, where the carrier is a polymer carrier or a protein carrier.
10. 10. An immunogenic hapten-carrier conjugate according to any one of claims 1 to 9.
11. 11. A pharmaceutical composition comprising diagnostically or therapeutically effective amounts of a hapten-carrier conjugate comprising the hapten-carrier conjugate according to any one of claims 1 to 9.
12. 12. A kit, comprising:

    a hapten-dCTP conjugate, where the hapten is an oxazole, where the hapten and dCTP are coupled by a linker which is a polymer comprising from 1 to about 15 ethylene glycol units, where the hapten is an oxazole having a formula

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    R-i

    R₂

    R3 where R1-R3 independently are selected from hydrogen, acyl, aldehyde (-C(O)H), alkoxy, alkyl having 20 or fewer carbon atoms, heteroalkyl having 20 or fewer carbon atoms, hydroxyalkyl having 20 or fewer carbon atoms, amido (-C(O)NH₂), amino (-NH₂), aryl, alkyl aryl wherein the alkyl chain has 20 or fewer carbon atoms, carboxyl (-C(O)OH), carboxylate (-C(O)O'), cycloalkyl having 20 or fewer carbon atoms, cyano, alkylester wherein the alkyl chain has 20 or fewer carbons, ether, fluoro, chloro, bromo, iodo, hydroxyl (-OH), hydroxylamine (-NHOH), alkyl ketone having 20 or fewer carbon atoms, nitro (-NO₂), sulfhydryl (-SH), and sulfoxide, at least one of the R1-R3 substituents is coupled to the linker, and Y is oxygen; and an anti-hapten antibody.
13. 13. The kit according to claim 12, where the anti-hapten antibody is conjugated to a detectable label.
14. 14. The kit according to claim 13, where the detectable label is an enzyme, a chromophore, a quantum dot, or combinations thereof.
15. 15. An immunoassay process, comprising:

    providing the hapten-carrier conjugate according to any one of claims 1 to 9, the hapten-carrier conjugate being suitable for performing the immunoassay; and using the hapten-carrier conjugate in at least one step of the immunoassay.
16. 16. The immunoassay according to claim 15, selected from enzyme-linked immunosorbent assays (ELISA); protein, PNA microarray assays; flow cytometric assays; target detection in blot assays, normal and reverse phase protein microarrays; and nucleic acid microarrays.
17. 17. A method for identifying a mammalian tumor, comprising assaying a sample obtained from the mammalian tumor to detect a pattern of expression, phosphorylation or both expression and phosphorylation using the hapten-carrier conjugate according to any one of claims 1 to 9.
18. 18. A method for assessing a response to drug therapy in an individual, comprising:

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    2015252024 20 Dec 2017 obtaining a first tissue or cell sample from the individual before exposing the individual to a drug therapy;

    obtaining a second tissue or cell sample from the individual after exposing the individual to the drug therapy;

    detecting a biochemical product and/or process affected by the therapy from the first sample and the second sample, where detecting comprises using the hapten-carrier conjugate according to any one of claims 1 to 9;

    comparing results for the first sample to the second; and determining whether the drug therapy had a positive, negative or null effect.
19. 19. A method for making the hapten-carrier conjugate according to any one of claims 1 to 9, comprising:

    providing a hapten which is an oxazole; and coupling the hapten to a linker that is coupled to a carrier.
20. 20. A method for detecting a molecule of interest in a biological sample, comprising:

    contacting the biological sample with the hapten-carrier conjugate according to any one of claims 1 to 9, wherein the hapten-carrier conjugate is a hapten-antibody conjugate or a nucleic acid hapten conjugate; and detecting a signal generated by the conjugate after treatment with an anti-hapten antibody having at least one detectable label.
21. 21. A hapten-deoxycitidinetriphosphate (dCTP) conjugate, where the hapten is an oxazole, where the hapten and dCTP coupled by a linker which is a polymer comprising from 1 to about 15 ethylene glycol units, and where the hapten is an oxazole having a formula .T where Ri-R₃ independently are selected from hydrogen, acyl, aldehyde (-C(O)H), alkoxy, alkyl having 20 or fewer carbon atoms, heteroalkyl having 20 or fewer

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    2015252024 20 Dec 2017 carbon atoms, hydroxyalkyl having 20 or fewer carbon atoms, amido (-C(O)NH₂), amino (-NH2), aryl, alkyl aryl wherein the alkyl chain has 20 or fewer carbon atoms, carboxyl (-C(O)OH), carboxylate (-C(O)O'), cycloalkyl having 20 or fewer carbon atoms, cyano, alkylester wherein the alkyl chain has 20 or fewer carbons, ether, fluoro, chloro, bromo, iodo, hydroxyl (-OH), hydroxylamine (-NHOH), alkyl ketone having 20 or fewer carbon atoms, nitro (-NO₂), sulfhydryl (-SH), and sulfoxide, at least one of the Ri-R₃ substituents is coupled to the linker, and Y is oxygen.

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