WO2011126482A1 - Immunoassay for the diagnosis of prostate cancer - Google Patents

Abstract

The invention provides methods and kits for diagnosing prostate disease states in a patient in general. Improved methods and kits for the detection of prostate cancer-specific biomarkers, such as the PSA-ACT complex, in a biological sample taken from a patient, are discussed.

Description

IMMUNOASSAY FOR THE DIAGNOSIS OF PROSTATE CANCER

FIELD OF THE INVENTION

[0001] The invention relates to the field of medical diagnostics. BACKGROUND OF THE APPLICATION

[0002] Prostate cancer is the most common cancer, excluding skin cancer, and the second leading cause of cancer-related death, in men in the United States. According to the National Cancer Institute, in 2008 alone, 186,320 new cases of prostate cancer were diagnosed and 28,660 men died from the disease. Each year, an estimated $8 billion is spent in the U.S on prostate cancer treatment and diagnosis.

[0003] In the absence of preventative screening, prostate cancer typically progresses insidiously for many years before overt symptoms become apparent, at which point the disease is already at an advanced stage and subsequent mortality is high. Early diagnosis of prostate cancer in asymptomatic patients has improved with the widespread use of sensitive blood serum assays for the detection of the prostate- specific antigen or PSA. PSA (also known as kallikrein III, seminin, semenogelase, γ-seminoprotein and P-30 antigen) is a 34 kD serine protease and glycoprotein that is synthesized almost exclusively by the epithelial cells of the prostate gland. Present in small quantities in the serum of healthy men (0-4ng/mL), it is often elevated

(>4ng/mL) in men with either localized or metastatic prostate cancer (CaP). This is an arbitrary threshold chosen to minimize the number of false positive samples, while capturing just over half of the true positives in a screening population. Elevated PSA levels are not a reliable biomarker for prostate cancer because other prostate disorders such as prostatitis (inflammation of the prostate) and benign prostatic hyperplasia (BPH) (enlargement of the prostate) also generate elevated levels of PSA. Moreover, the occurrence of BPH increases as men age, concurrent with the typical onset of prostatic adenocarcinoma. Perhaps a fourth of men have some degree of hyperplasia by the fifth decade of life and by the eighth decade, over 90% of males will have prostatic hyperplasia. Thus, benign conditions can contribute to elevated PSA levels even though no cancer is actually present.

[0004] The inability of conventional PSA diagnostic tests to distinguish between elevated serum PSA levels caused by benign prostate conditions and elevated serum PSA levels caused by prostate adenocarcinoma results in high number of false positive results. This lack of specificity results in misdiagnoses and ultimately leads to medical procedures, such as biopsies, that have potential risks, significant financial costs and contributes to unnecessary anxiety for the patient and his family.

[0005] In an effort to improve the specificity of PSA diagnostic tests, researchers have focused on ways to distinguish between cancerous and benign conditions, and between slow- growing cancers and fast-growing, potentially lethal cancers. A major breakthrough came with the realization that the proteolytic activity of PSA in serum is inhibited by the formation of complexes with a serum protease inhibitor, -alpha-l-antichymotrypsin (al-ACT) as well as other serum proteins such as alpha-2-macroglobulin (a2-M), and other acute phase proteins. Complexed PSA is the predominant form detected in the serum of prostate cancer patients and levels of complexed PSA have been shown to increase with disease progression.

[0006] Despite this progress, current assays remain unreliable because of the significant number of false negative and false positive results. For example, the Immuno 1 PSA Assay, commercialized by Bayer Diagnostics (now part of Siemens), computes indirectly the amount of PSA-ACT complex present within peripheral blood samples by subtracting the molar amount of, fPSA (free PSA) from the molar amount of tPSA (total PSA), i.e. both bound and free PSA (see U.S. Patents 6,107,049; 5,928, 878 and 5,840,501). As shown in Figure 7 of U.S. Patent 6,107,049, the improvement of the assay specificity is only 5-7% compared with the Bayer total PSA assay, which has a specificity of 25% on average.

[0007] In its 2008 recommendation for men aged 75 or less, the U.S. Preventive Services Task Force (USPSTF) found inadequate evidence to determine whether treatment for prostate cancer detected by screening using current PSA assays improves health outcomes compared with treatment after clinical detection.

[0008] For the forgoing reasons, there is an unmet and vital need for an improved, reliable PSA-ACT assay with more acceptable specificity for use in the routine screening for prostate cancer.

SUMMARY OF THE INVENTION

[0009] The application discloses an immunoassay for the improved detection of prostate cancer-specific biomarkers in biological samples taken from a patient. Methods are described for the analysis of patient samples using a PS A- ACT complex-specific monoclonal antibody. The application further pertains to kits containing reagents for the detection of prostate cancer- specific biomarkers for use in a clinical or non-clinical setting.

[0010] In one embodiment, the application discloses a diagnostic assay for the detection of PSA-ACT having both a high specificity and a high sensitivity that is clinically relevant and statistically significant for the reliable diagnosis of a prostate disease state in a patient.

[0011] In one embodiment, a kit is described for the diagnosis of a prostate disease state in a patient. In one aspect, the kit contains reagents for a diagnostic assay having both a high specificity and a high sensitivity, e.g., a specificity of at least 65% and a sensitivity of at least 70%. In another aspect, the kit contains reagents for a diagnostic assay having a specificity of at least 65% and a sensitivity of at least 80% or at least 90%. In another aspect, the kit contains reagents for a diagnostic assay having a sensitivity of at least 70% and a specificity of at least 70% or at least 75% or at least 80% or at least 85% or at least 90%.

[0012] In one aspect, the prostate disease states that the disclosed methods are capable of diagnosing include benign prostatic hyperplasia, prostatic intraepithelial neoplasia, metastatic prostatic adenocarcinoma or primary prostatic adenocarcinoma.

[0013] In other aspects, the diagnostic assay permits the differential diagnosis between any of the prostate disease states, for example, between benign prostatic hyperplasia and prostatic intraepithelial neoplasia; between prostatic intraepithelial neoplasia and metastatic prostatic adenocarcinoma; between prostatic intraepithelial neoplasia and primary prostatic adenocarcinoma, or between benign prostatic hyperplasia and primary prostatic adenocarcinoma.

[0014] In one aspect, the reagents of the application comprise PS A- ACT binding molecules.

[0015] In one feature, the PS A- ACT binding molecule is an antibody, a scFv, a single domain antibody, Fv, a diabody, a tandem diabody, Fab, Fab', F(ab)₂, a polyclonal antibody or a monoclonal antibody.

[0016] In one embodiment, the PSA-ACT binding molecule does not bind to non- complexed PSA or non-complexed ACT.

[0017] In one embodiment, the PSA-ACT binding molecule is a nucleic acid, e.g. a R A or DNA aptamers. [0018] In one feature, the PS A- ACT binding molecule is conjugated to a labeling compound such as a radioactive agent, an enzyme, a fluorescent compound or an electron transfer agent.

[0019] In another aspect, the diagnostic assay comprises an immunoassay or a sandwich immunoassay.

[0020] In a further aspect, the diagnostic assay also includes a PSA binding molecule.

[0021] In one aspect, the specificity and the sensitivity of the assay are determined at a cut-level no more than 4ng/mL PSA-ACT complex.

[0022] In one embodiment, a kit is described for the diagnosis of prostate cancer in a patient. The kit contains reagents for collecting a biological sample from a patient, PSA binding molecules for capturing free PSA and complexed PSA within the sample, reagents for isolating proteins bound to the PSA binding molecules, PS A- ACT complex-specific binding molecules for contacting the proteins bound to the PSA binding molecules, reagents for measuring the amount of PSA-ACT complexes in the sample. The kit may further include instructions for determining the prostate disease state in the patient. The determination of a patient's prostate disease state has a specificity of at least 65% and a sensitivity of at least 70% at a pre-defined concentration of PSA-ACT complex within the patient's sample.

[0023] In one aspect, the determination of a patient's prostate disease state has a specificity of at least 65% and a sensitivity of at least 80% or at least 90%. In another aspect, the determination of a patient's prostate disease state has a sensitivity of at least 70% and a specificity of at least 70% or at least 75% or at least 80% or at least 85% or at least 90%.

[0024] The biological sample can be blood serum, semen or any biological sample containing prostatic fluid.

[0025] In another aspect, a method is described for diagnosing a prostate disease state in a patient comprising a diagnostic assay having both a high specificity and a high sensitivity, e.g., a specificity of at least 65% and a sensitivity of at least 70%.

[0026] In one embodiment, a method of diagnosing prostate cancer in a patient is described that comprises the steps of providing a biological sample from a patient, contacting the sample with a PSA binding molecule, isolating proteins bound to the PSA binding molecule, contacting the proteins bound to the PSA binding molecule with a PS A- ACT complex- specific binding molecule, measuring the amount of PSA-ACT complexes in the sample, and determining the prostate disease state in the patient. The determination of a patient's prostate disease state has a specificity of at least 65% and a sensitivity of at least 70% at a pre-defined concentration of PSA-ACT complex within said sample.

[0027] In one embodiment, the determination of a patient's prostate disease state has a specificity of at least 65% and a sensitivity of at least 80%.

[0028] In yet another embodiment, the determination of a patient's disease state has a specificity of at least 65% and a sensitivity of at least 90%.

[0029] In another embodiment, the determination of a patient's disease state has a specificity of at least 70% and a sensitivity of at least 70%.

[0030] In another embodiment, the determination of a patient's disease state has a specificity of at least 75% and a sensitivity of at least 70%.

[0031] In another embodiment, the determination of a patient's disease state has a specificity of at least 80% and a sensitivity of at least 70%.

[0032] In another embodiment, the determination of a patient's disease state has a specificity of at least 85% and a sensitivity of at least 70%.

[0033] In another embodiment, the determination of a patient's disease state has a specificity of at least 90% and a sensitivity of at least 70%.

[0034] In one feature, the pre-defined concentration of PSA-ACT complex is no more than 4ng/mL of PSA-ACT complex.

[0035] The PS A- ACT complex-specific binding molecule can be a monoclonal antibody, a scFv, a single domain antibody, Fv, a diabody, a tandem diabody, Fab, Fab', F(ab)₂ or a polyclonal antibody.

[0036] In another embodiment, a method of diagnosing prostate cancer in a patient is described which comprises the steps of providing a semen sample from a patient, contacting the sample with a PSA binding molecule, isolating proteins bound to the PSA binding molecule, contacting the proteins bound to the PSA binding molecule with a PSA-ACT complex-specific binding molecule, measuring the amount of PSA- ACT complexes in the sample, and determining the prostate disease state in the patient. The determination of the prostate disease state in the patient has a specificity of at least 65% and a sensitivity of at least 70% at a pre-defined concentration of PS A- ACT complex within the sample. [0037] Alternatively, the determination of a patient's prostate disease state has a specificity of at least 65% and a sensitivity of at least 80% or a specificity of at least 65% and a sensitivity of at least 90%, a specificity of at least 70% and a sensitivity of at least 70%, a specificity of at least 75% and a sensitivity of at least 70% or a specificity of at least 80% and a sensitivity of at least 70% or a specificity of at least 85% and a sensitivity of at least 70% or a specificity of at least 90% and a sensitivity of at least 70%.

[0038] It should be understood that this application is not limited to the embodiments disclosed in this Summary, and it is intended to cover modifications and variations that are within the scope of those of sufficient skill in the field, and as defined by the claims.

[0039] The previously described embodiments have many advantages, including the ability to screen for prostate cancer biomarkers, such as the PSA-ACT, in a patient and predict the incidence of prostate cancer with high specificity and high sensitivity, e.g., a specificity of least 75% and a sensitivity of at least 70%. Convenient, user-friendly and reliable diagnostic kits are also described that may be used in a clinical setting or as "over-the-counter" home diagnostic kits.

[0040] The methods and materials disclosed herein give results that cannot be predicted based on the prior art known to the inventor(s). Previously reported PSA-ACT immunoassays (for example, as described in U.S. Patents Nos. 6,107,049; 5,928, 878 and 5,840,501) all fail to achieve the specificity and the sensitivity of the diagnostic assays disclosed herein despite a long-felt need for such improvements.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows a calibration curve of the PSA-ACT antigen ( 1 ^st data set).

[0042] FIG. 2A and FIG. 2B depict the frequency distribution of healthy male serum samples (1^st data set).

[0043] FIG. 3 shows the calibration curve of the PSA- ACT antigen (2^nd data set).

[0044] FIG. 4A shows a ROC Curve Analysis of 59 PSA Serum Samples (2^nd data set).

[0045] FIG. 4B shows a ROC Curve Analysis of 92 PSA Serum Samples (2^nd data set).

[0046] FIG. 4C shows a ROC Curve Analysis of 98 PSA Serum Samples (2^nd data set).

[0047] FIG. 4D shows a ROC Curve Analysis of 140 PSA Serum Samples (2^nd data set). [0048] FIG. 5A depicts Distribution of Cancer and BPH amongst the 59 PSA serum samples (2^nd data set).

[0049] FIG. 5B depicts the distribution of Cancer and BPH amongst the 92 PSA serum samples (2^nd data set).

DETAILED DESCRIPTION

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art. The following definitions are provided to help interpret the disclosure and claims of this application. In the event a definition in this section is not consistent with definitions elsewhere, the definition set forth in this section will control.

[0051] As used herein, "PS A- ACT" refers to the prostate serum antigen complexed with the serine protease inhibitor, a-chymotrypsin.

[0052] As used herein, "sensitivity" refers to the diagnostic sensitivity of a biomarker assay which is the assay's ability to detect a disease state in a population or group and is expressed as a proportion or percentage: the number of persons who have both the disease state and who are correctly identified as such by the assay divided by the number of all the persons who are in fact at said disease state including those identified as such by the assay and missed by the assay, i.e., true positives/ true positives + false negatives. Strong diagnostic sensitivity improves negative predictive values. Negative predictive value is the probability that a person whose test result is negative does not have prostate cancer (that is, of every 100 patients who have negative test results, the number of patients who do not have prostate disease state, e.g. prostate cancer).

[0053] In one embodiment, the PSA-ACT diagnostic assays described in this application have a sensitivity of at least 90% at a cut-off level of 3.5 ng mL of PSA-ACT.

[0054] As used herein, "specificity" refers to the diagnostic specificity of a biomarker assay, which is the assay's ability to detect healthy persons in a population or group correctly and is expressed as a proportion or percentage: the number of persons who are healthy and who are correctly identified as such by the assay divided by the number of all persons who are in fact healthy including those identified as such by the assay and misidentified by the assay as positive, i.e. true negatives/true negatives + false positives. Strong diagnostic specificity improves positive predictive values. Positive predictive value is the probability that a person whose test result is positive truly has the prostate cancer (that is, of every 100 patients who have positive test results, the number of patients who have prostate disease state, e.g. prostate cancer).

[0055] In one embodiment, the herein described PS A- ACT diagnostic assay has a specificity of at least 75% at a cut-off level of 3.5 ng/mL of PSA-ACT.

[0056] Sensitivity and specificity define the operating characteristics of an assay, but it is the predictive value (positive or negative) of the assay that is generally of diagnostic importance to clinician and patient. For example, if an assay has perfect diagnostic sensitivity and perfect diagnostic specificity, then all persons who have positive test results have prostate cancer and all persons who have negative test results do not. A common means of depicting the accuracy of a diagnostic assay is the Receiver Operating Characteristic curve (or ROC curve), which plots the true positive rate (sensitivity) against the false positive rate (1 -specificity) for the different possible cutoff points of a diagnostic test. An exemplary ROC curve for the herein described PS A- ACT diagnostic assay is shown in FIG. 3 at a cut-off of 3.5 ng/mL of PSA- ACT.

[0057] In certain embodiments, the PSA-ACT diagnostic assays described in this application have a high sensitivity, e.g., a sensitivity of at least 70% and a specificity of at least 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 95% or at least 99% or at least 99.9% at a cut-off of 3.5 ng/mL of PSA-ACT.

[0058] In certain embodiments, the PSA-ACT diagnostic assays described in this application can have a high specificity, e.g., a specificity of at least 90% and a sensitivity of at least 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 95% or at least 99% or at least 99.9% at a cut-off of 3.5 ng/mL of PSA-ACT.

[0059] As used herein, a "prostate disease state" refers to any disease state of the prostate that can be diagnosed by a change in the expression levels of a biomarker in a biological sample taken from a patient. Diagnosis of a "prostate disease state" may be determined in conjunction with other means of prostate cancer detection including, but not limited to, digital examination, ultrasonography (transrectal ultrasound) or trans-rectal biopsy.

[0060] In one embodiment, a "prostate disease state" refers to those diseases

characterized by an abnormal cellular proliferation of the prostatic tissue including noncancerous conditions, including benign prostatic hyperplasia (BPH) (enlargement of the prostate), and pre-cancerous prostate lesions, including prostatic intraepithelial neoplasia (ΡΓΝ), particularly high-grade PIN (HGPIN), atypical small acinar proliferation (ASAP). PIN refers to the precancerous end of a morphologic spectrum involving cellular proliferation within prostatic ducts, ductules, and acini.

[0061] In other embodiments, a "prostate disease state" refers to pre-malignant and malignant prostate adenocarcinoma also referred to as primary prostatic adenocarcinoma and metastatic adenocarcinoma respectively. Prostatic adenocarcinomas are usually graded clinically according to the Gleason grading system (grades 1-5) based on the pattern of growth. The stage is determined by the size and location of the cancer, whether it has invaded the prostatic capsule or seminal vesicle, and whether it has metastasized. The prognosis of prostatic adenocarcinoma varies widely with tumor stage and grade. Cancers with low Gleason scores are generally low grade and not aggressive. Advanced prostatic adenocarcinomas have a high Gleason grade and typically cause urinary obstruction, metastasize to regional (pelvic) lymph nodes and to the bones, causing blastic metastases in most cases.

[0062] As used herein the phrase "prostate cancer" refers to cancers of the prostate tissue and/or other tissues of the male genitalia, or reproductive or urinary tracts.

[0063] As used herein, a "prostate cancer biomarker" refers to one or more molecules whose expression changes as a consequence of the onset of prostate cancer. A "prostate cancer biomarker" may be a DNA, RNA, protein or lipid. The expression of a biomarker may increase or decrease as a result of prostate cancer. In one embodiment, changes in the expression of a "prostate cancer biomarker" occurs in asymptomatic patients. In another embodiment, the absence of expression of the "prostate cancer biomarker" is indicative of prostate cancer. In yet another embodiment, a "prostate cancer biomarker" refers to certain proteins or peptides synthesized by the prostate gland including, but not limited to, prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostatic inhibin (PIP). In other embodiment, a "prostate cancer biomarker includes, but is not limited to, kallikrein-related peptidase 2 (hK2), early prostate cancer antigen (EPCA), PCA3, hepsin, prostate stem cell antigen, or {alpha}- methylacyl-CoA racemase (AMACR).

[0064] In one embodiment, the diagnostic assay detects one or more antibodies to a "prostate cancer biomarker." [0065] In another embodiment, the PSA- ACT diagnostic assay can diagnose a prostate disease state characteristic of a Gleason grade of 1 or less, or a Gleason grade of 2 or less, or a Gleason grade of 3 or less, or a Gleason grade of 4 or less, or a Gleason grade of 5 or less.

[0066] As used herein, a pre-defined concentration of PSA- ACT complex is less than 0.5 ng/mL, less than Ing/mL, less than 2 ng/mL, less than 3 ng/mL, less than 4 ng/mL. In one embodiment, the pre-defined concentration of PSA- ACT complex is between 0 - 4 ng/mL or between 4 - 10 ng/mL or between 10-50 ng/mL PSA-ACT or 5 ng/mL or greater. In one embodiment, the pre-defined concentration of PS A- ACT complex is greater than 1 ng/mL. In one embodiment, the pre-defined concentration of PS A- ACT complex is 3.5 ng/mL.

[0067] As used herein, a "binding molecule" refers to a member of a binding pair, i.e., two different molecules wherein one of the molecules specifically binds to the second molecule through chemical or physical means. In a preferred embodiment, binding molecules refer to antigen and antibody binding pair members. In another embodiment, binding molecules refer to one or more RNA or DNA aptamers, e.g., SELEX generated aptamers or non-SELEX generated aptamers. Other binding pairs include, as examples without limitation, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S-protein), and the like.

[0068] The term "antibody," as used herein, refers to an immunoglobulin molecule, which is able to specifically bind to one or more epitopes on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or produced, e.g., by immunization, synthesis or genetic engineering (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y). Antibodies can be monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies (such as, but not limited to, a bird (for example, a duck or goose), a shark or whale, a mammal, including a non-primate (for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, mouse, etc) or a non-human primate (for example, a monkey, such as a cynomologous monkey, a chimpanzee, etc), recombinant antibodies, chimeric antibodies, single- chain Fvs ("scFv"), single chain antibodies, single domain antibodies, diabodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFv"), and anti-idiotypic ("anti-Id") antibodies (including, for example, anti-Id antibodies to antibodies of the present application), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAland IgA2) or subclass.

[0069] A "recombinant antibody" as used herein, is an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody or parts thereof and which DNA molecule expresses an antibody protein or parts thereof, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. Recombinant antibodies may be selected for increased or improved affinity via the screening of a combinatory antibody library under stringent binding conditions. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0 125 023 Bl; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0 120 694 Bl; Neuberger et al., International Publication No. WO86/01533; Neuberger et al., European Patent No. 0 194 276 Bl; issued to Winter et al, U.S. Pat. No. 5,225,539; issued to Winter et al., European Patent No. 0 239 400 Bl; Queen et al., European Patent No. 0 451 216 Bl ; and Padlan et al., EP 0 519 596 Al. See also, Newman et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird et al., Science, 242:423-426 (1988)) regarding single chain antibodies. The contents of each of these patent documents and references are hereby incorporated by reference herein in their entirety.

[0070] As used herein, a "nucleic acid" may be DNA or RNA. Nucleic acids may also include modified nucleotides. The terms "nucleic acid" and "oligonucleotide" are used interchangeably to refer to a molecule comprising multiple nucleotides. As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base containing polymer.

[0071] An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody, which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen can have one or more than one epitope.

[0072] The term "epitope" is meant to refer to that portion of the antigen capable of being recognized by and bound by an epitope-specific binding molecule such as an antibody at one or more of the antibody's antigen binding region. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three- dimensional structural characteristics as well as specific charge characteristics.

[0073] As used herein, the term "sample" refers to a material suspected of containing at least one prostate cancer biomarker. In one embodiment, the prostate cancer biomarker is PSA- ACT. The sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample. The sample can be derived from any biological source suspected of containing a prostate cancer biomarker, including, but not limited to, whole blood, blood plasma, blood serum, lymphatic fluid, any sub- fraction of whole blood, urine, semen or other biological sample containing prostatic seminal fluid. The sample can be pretreated prior to use, such as preparing plasma or serum from blood, diluting viscous fluids, or the like; methods of treatment can involve filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents. Besides physiological fluids, other liquid samples can be used. In addition, a solid material suspected of containing a prostate cancer biomarker can be used as the sample. In some instances it may be beneficial to modify a solid sample to form a liquid medium or to release the prostate cancer biomarker.

[0074] As used herein, an "immunoassay" is defined as any method using a preferential binding of an antigen with a second material, a binding molecule, usually an antibody or another substance having an antigen binding site, which binds preferentially with an epitope of the antigen. Preferential binding, as used herein, refers to binding between binding partners that is selective and generally specific, and demonstrates less than 10%, preferably less than 5%, cross- reactive non-specific binding. Immunoassays are used when an unknown concentration of an analyte within a sample needs to be quantified. The immunoassay methods provided herein include any known to those of skill in the art, including, but not limited to, sandwich immunoassays, competition inhibition immunoassays, ELISAs (Enzyme-Linked Immunosorbent Assays), agglutination, precipitation, or radioimmunoassays or antigen-down immunoassays or immunometric assays for example. A number of different types of immunoassays are well known using a variety of protocols and labels. Immunoassays may be homogeneous, i.e.

performed in a single phase, or heterogeneous, where antigen or antibody is linked to an insoluble solid support upon which the assay is performed. The reaction steps may be performed simultaneously or sequentially. Assay formats include, but are not limited to, for example, assays performed in test tubes, wells or on immunochromatographic test strips, as well as dipstick or lateral flow immunoassays.

[0075] As used herein, a "sandwich immunoassay" is a method using two antibodies, which bind to different sites on an antigen. The primary antibody or capture antibody, which is highly specific for the antigen, is typically attached to a solid surface. The sample containing the antigen is then added. After an appropriate incubation that permits the binding of the antigen to the capture antibody, non-binding material is washed away. A second and labeled antibody or detection antibody is then added which binds to a different epitope on the antigen than the primary antibody. As a result the antigen is "sandwiched" between the two antibodies.

[0076] As used herein, "diagnose" refers to detecting and identifying a prostate disease state in a subject. The term also encompasses assessing or evaluating the disease status

(progression, regression, stabilization, response to treatment, etc.) in a patient known to have a prostate disease state. In one embodiment, the diagnosis includes a patient's medical history, familial history and results of digital examinations, ultrasonography (transrectal ultrasound) and/or trans-rectal needle biopsy.

[0077] As used herein, a "PSA-ACT binding molecule" is any molecule with a binding affinity with a dissociation constant of at least 10^"5 or 10^"6 or 10^"7 or 10^"8 or 10^~9M. In one embodiment, a "PSA-ACT binding molecule" is an antibody or a nucleic acid, e.g., an RNA or DNA aptamer.

[0078] Features and advantages of the present application will become apparent from the following description. Applicants are providing this description, which includes drawings and examples of specific embodiments, to give a broad representation of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description and by practice of the invention. The scope of the invention is not intended to be limited to the particular forms disclosed and the application covers all modifications, equivalents, and alternatives falling within the spirit and scope of the application as defined by the claims.

[0079] The application relates to diagnostic assays for the detection of the PSA-ACT prostate cancer-specific biomarker in biological samples taken from a patient. A person of ordinary skill in the art will recognize many different immunoassay formats may be employed for the detection of PSA-ACT in a biological sample.

[0080] For example, in a radioimmunoassay (RIA), the amount of test antigen present in a sample is measured indirectly employing a limited amount of antibody (or antigen-binding molecule) to compete for labeled antigen. An IRM A (immunoradiometric assay) is a form of radioimmunoassay in which excess specific labeled antibody is added directly to the test antigen being measured. In one class of IRMA assays, the antigen is insolubilized and reacted with labeled antibody (or antigen-binding molecule). When the antigen is insolubilized by reaction with a solid-phase antibody (or antigen-binding molecule), the assay is termed a "two-site IRMA", "junction test" or "sandwich assay." Sandwich assays are further classified according to their methodology as forward, reverse or simultaneous sandwich assays.

[0081] In a forward sandwich immunoassay, a sample containing the antigen can be first incubated with a solid-phase immunoadsorbent containing immobilized antibody. Incubation is continued for a sufficient period of time to allow antigen in the sample to bind to the

immobilized antibody on the solid-phase immunoadsorbent. The solid-phase immunoadsorbent can then be separated from the incubation mixture and washed to remove excess antigen and other substances which may be present in the sample. The solid-phase immunoadsorbent containing antigen bound to the immobilized antibody is subsequently incubated with labeled antibody capable of binding to the antigen. After the second incubation, another wash is performed to remove unbound labeled antibody from the solid-phase immunoadsorbent thereby removing non-specifically bound labeled antibody. Labeled antibody bound to the solid-phase immunoadsorbent is then detected and the amount of labeled antibody detected can serve as a direct measure of the amount of antigen present in the sample. Such forward sandwich assays are described in the patent literature, and in particular, in U.S. Pat. Nos. 3,867,517 and 4,012,294, issued to Chung-Mei Ling, each of which is incorporated by reference herein in its entirety. [0082] In a reverse sandwich assay, a sample is incubated with labeled antibody after which the solid-phase immunoadsorbent containing immobilized antibody is added and incubated. A washing step is then performed after the second incubation period. A reverse sandwich assay has been described in the patent literature in U.S. Pat. No. 4,098,876, issued to Roger N. Piasio et al., the contents of which are hereby incorporated by reference herein in its entirety.

[0083] In a simultaneous sandwich assay, a sample is incubated simultaneously in one step with both an immunoadsorbent containing immobilized antibody for the antigen and labeled antibody specific for the antigen. The amount of antigen present in the sample can be computed by determining the amount of labeled antibody bound to the immunoadsorbent. A simultaneous sandwich assay has been described in the patent literature in U.S. Pat. No. 4,837,167, issued to Hubert J. P. Schoemaker et al., the contents of which are hereby incorporated by reference herein in its entirety.

[0084] Many different types of solid-phase immunoadsorbents can be employed including solid supports such as a plate, a stick, a tube or a well formed from or coated with appropriate materials. In another embodiment, the immunoabsorbents are particles made of latex, although other types of particles to which a antigen-binding molecules may be coupled are also included within the scope of this application. Inert particles may be comprised of any suitable material, such as glass or ceramics or carbon, and/or one or more polymers, such as, for example, nylon, polytetrafluoroethylene (TEFLON™), polystyrene, polyacrylamide, styrene- divinylbenzene polymers such as Sephadex, Sepharose or Sephacryl (sold by Pharmacia AB, Uppsala, Sweden), agarose, cellulose, cellulose derivatives, or dextran, and/or can comprise metals. Porous glass or silica gel particles may also be suitable. Further examples of particles include, but are not limited to, plastic particles, ceramic particles, carbon particles, polystyrene microbeads, glass beads, magnetic beads, hollow glass spheres, metal particles, particles of complex compositions, microfabricated or micromachined particles. The particle size may be from 0.1 micron to 1000 microns. Preferably, the particle size is from 1 to 200 microns. These particles are generally in the form of beads, beaded gels or microspheres, although they may have any shape. In principle, any ligand may be covalently bound to a solid-phase matrix such as agarose beads (e.g., Sepharose Pharmacia) using known techniques, for example as described by Heam et al., Methods in Enzymology Vol. 35:102-117 (1987). Generally, the beads are first activated by a chemical agent, such as glutaraldehyde, carbonyldiimidizole, cyanogen bromide hydroxysuccinimide, tosyl chloride or the like. The chosen ligand is then covalently attached to the beads, resulting in an extremely stable linkage of the ligand to the support.

[0085] A competitive binding assay is based upon the competition of labeled and unlabeled ligand for a limited number of antibody binding sites. Competitive inhibition assays are often used to measure small analytes. These assays are also used when a matched pair of antibodies to the analyte does not exist. Only one antibody is used in a competitive binding ELISA. This is due to the steric hindrance that occurs if two antibodies would attempt to bind to a very small molecule. A fixed amount of labeled ligand (tracer) and a variable amount of unlabeled ligand are incubated with the antibody. According to law of mass action the amount of labeled ligand is a function of the total concentration of labeled and unlabeled ligand. As the concentration of unlabeled ligand is increased, less labeled ligand can bind to the antibody and the measured response decreases. Thus the lower the signal, the more unlabeled analyte there is in the sample. The standard curve of a competitive binding assay has a negative slope. Typically, either the inhibitor or the antibody is bound to a solid support (as described above), while the other component of the pair is labeled in some fashion to render it detectable. Methods that are used to detect and quantitate the presence of antigen in a sample are also referred to as serologic diagnostic methods.

[0086] Labels are well known in the art and include, e.g., radionuclides (e.g., Iodine-125, Iodine-131, Indium- 111, Bismuth-210), enzymes which produce an absorptive or fluorescent detector group when reacted with a specific substrate (e.g., horseradish peroxidase, N- methylumbelliferone-o- -D-galactosidase), dyes (chromophores), fluorescent compounds (e.g., fluorescein, rhodamine, phycoerythrin, cyanine dyes, other compound emitting fluorescence energy), electron dense compounds (e.g., gold and ferric chloride compounds). Biotin/avidin labeling systems or binding partners as described herein can also be used. Coupled assays can also be used for detecting labels.

[0087] The label may be directly linked to the component (the inhibitor or antibody) or may be bound to it indirectly, e.g., by attaching the label to another molecule capable of recognizing a component of the antigen/antibody pair. For example, an antibody (or antigen- binding fragment) can be indirectly labeled by attaching an enzyme, fluorescent marker or radionuclide to an isotype- specific antibody which recognizes the non- ariable region of the antigen-specific antibody (or antigen-binding fragment). In another embodiment, the label can be attached to an antibody (or antigen-binding fragment) which recognizes an available epitope of the antigen after it has been bound to the specific antibody (or antigen-binding fragment). Many other variants of this broad concept are possible and known in the art.

[0088] In one preferred embodiment, the label is a dye (such as, nitrophenyl) attached to the unbound component or reagent (unbound inhibitor or antibody) via a phosphate linker. After incubation of the labeled component with the immobilized binding partner, the presence of binding can then be determined by subjecting the solid support to a phosphatase enzyme, causing hydrolysis of the dye. The presence (and amount) of the dye can then be measured by absorbance, indicating the amount of binding of the two components.

[0089] In yet another embodiment, a biomarker binding molecule can be a nucleic acid, e.g., an R A or DNA aptamer selected using, for example, the SELEX process (Systematic Evolution of Ligands by Exponential enrichment). This method provides for the in vitro selection of nucleic acid molecules that are able to bind with high specificity to target molecules and is described in U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands", U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled "Nucleic Acid Ligands," and more recently "Method For Generating Aptamers With Improved Off-Rates," U.S. Patent Application No. 2009/0004667, each of which is specifically incorporated by reference herein in its entirety. In another embodiment, nucleic acid aptamers may be selected by screening libraries of structurally defined RNA motifs, as described in "Methods for identifying ligands that target nucleic acid molecules and nucleic acid structural motifs," U.S. Patent Application Publication No. 2008/0188377, the contents of which are hereby incorporated by reference herein in their entirety.

[0090] In each assay, the sample, antibody and, optionally, an inhibitor, are incubated under conditions and for a period of time sufficient to allow antigen or the inhibitor to bind to the antibody, i.e., under conditions suitable for the formation of a complex between the antigen and antibody. In general, it is usually desirable to provide incubation conditions sufficient to bind as much antigen as possible because this maximizes the binding of labeled antibody or antigen- binding fragment to the antigen thereby increasing the signal. Suitable temperatures are generally below the temperature at which denaturation can occur.

[0091] A person of ordinary skill in the art will recognize assays based on the detection of prostate cancer biomarkers, such as PS A- ACT, provide a reliable, non-invasive means of diagnosing the earliest stages of prostate cancer and monitoring disease progression as well as the efficacy of disease treatment, including surgery, chemotherapy and/or radiation therapy. The presence of PS A- ACT may be correlated to the presence of various stages of prostate cancer. If patients are undergoing successful treatment and the cancer is in remission, the level of PSA- ACT is reduced. PSA-ACT immunoreactivity can be used to monitor the course of cancer therapy and will be invaluable for the determination of the effectiveness of the various treatment regimens over time. A decrease in the level over time indicates a reduced tumor burden in the patient. In contrast, no change or an increase indicates ineffectiveness of therapy or the continued growth of the tumor.

[0092] The PSA- ACT diagnostic assay described herein may be combined with immunoassays detecting one or more other biomarkers characteristic of a prostate disease state, e.g. prostate cancer, including but not limited to, prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostatic inhibin (PIP). In other embodiment, a "prostate cancer biomarker includes, but is not limited to, kallikrein-related peptidase 2 (hK2), early prostate cancer antigen (EPCA), PCA3, hepsin, prostate stem cell antigen, or {alpha} -methylacyl-CoA racemase (AMACR). By combining the PSA-ACT assay with immunoassays for the detection of other prostate cancer biomarkers, the overall sensitivity and/or specificity of the assay may be further improved.

[0093] In another embodiment, the PSA-ACT immunoassay may be combined with the immunodetection of one or more prostate disease state biomarkers identified by expression profiling, as described in U.S. Patent No. 6,949,342 (entitled "Prostate cancer diagnosis and outcome prediction by expression analysis"), the contents of which is hereby incorporated by reference herein in its entirety.

[0094] In other embodiments, the application discloses kits for detecting the presence of the prostate cancer biomarker, PSA-ACT, in a biological sample taken from a patient. Such kits can include an antibody or antigen-binding fragment which binds PSA and PSA containing complexes, as well as one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or antibody fragment and PSA- ACT. The antibody or antigen binding fragment compositions can be provided in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes. The antibodies or antigen- binding fragments, which can be labeled or unlabeled, can be included in the kits with adjunct ingredients (e.g., buffers, such as Tris, phosphate and carbonate, stabilizers, excipients, biocides, inert proteins, e.g., bovine serum albumin and/or non-ionic detergents). The components (e.g., antibody, ancillary reagents) of the kit can be packaged separately or together within suitable containment means (e.g., bottle, box, envelope, tube). When the kit comprises a plurality of individually packaged components, the individual packages can be contained within a single larger containment means (e.g., bottle, box, envelope, tube). The kits contain detailed

instructions for use in either a clinical or a non-clinical setting as part of an "over the counter" home diagnostic kit.

[0095] In a particular embodiment, the kit comprises a primary PSA-specific "capture" antibody in a separate vial or container. In one aspect, the kit provides a microtiter place in which the primary "capture" antibody is bound to the bottom of each well of the microtiter plate. In another aspect, the primary "capture" antibody may be covalently bound to inert particles such beads. In another embodiment, the primary capture antibody is bound to magnetic beads. In one aspect the kit includes a custom-made magnetic for separating the magnetic beads from reaction mixtures.

[0096] In a further embodiment, the kit also comprises a secondary 'detection' antibody with high affinity to PSA-ACT that can be detected using a detection method (e.g., radiation, colorimetric, enzymatic, chemiluminescence, etc.), either directly or indirectly using well established methods known in the art. In one aspect, the kit comprises a Horseradish peroxidase (HRP) conjugated PSA- ACT detection antibody together with HRP substrate and reaction buffers.

[0097] The kit may include a series of calibration materials (calibrators) comprised of materials that emulate PSA-ACT in patient samples that can be used to establish an appropriate response curve to map detection signal into concentration of PS A- ACT.; and any required blocking agents and buffers that inhibit nonspecific binding or any other signal generating reactions that are unrelated to PSA- ACT concentration. The calibrators are stable over the useful lifetime of the kit.

[0098] In another embodiment, the kit may include reagents such as binding molecules for the detection of other prostate disease state biomarkers, including, but not limited to, alpha-2- macroglobulin (ot2-M), prostatic acid phosphatase (PAP), prostatic inhibin (PIP), kallikrein- related peptidase 2 (hK2), early prostate cancer antigen (EPCA), PC A3, hepsin, prostate stem cell antigen, or {alpha} -methylacyl-CoA racemase (AMACR).

[0099] The present invention will now be illustrated by the following examples, which are not to be considered limiting in any way.

EXAMPLE 1 : PS A- ACT standard curve using a sandwich immunoassay (1^st data set)

[00100] Sandwich immunoassays use a matched pair of antibodies, one for analyte capture on a solid surface and one for detection that binds to the antigen/hapten/analyte. Antibodies need to be affinity-purified for optimal results. In a PSA-ACT sandwich immunoassay, the capture antibody is an anti-PSA monoclonal antibody (1.0 ug/mL) which binds to epitopes on both free PSA and PSA molecules of complexed PSA, and the capture antibody is an anti-PSA- ACT monoclonal antibody (1.5 ug/mL) which detects only PSA- ACT complex antigen in the clinical samples. The assays also tested Egenix CI, an anti-PSA capture monoclonal antibody coated onto ELISA plates, and Egenix Dl, an anti- PS A- ACT detection monoclonal antibody which was conjugated to horseradish peroxidase (HRP). The antibodies were stored in a buffer containing 100 mrnol/L PBS phosphate buffer, pH 7.4, and 150 mmol/L NaCl. The purity of each of the antibody preparations was at least 98%, as tested by sodium dodecyl sulfate- polyacrylamide gel electrophoresis under reducing conditions, using the Pharmacia Phastgel Electrophoresis System followed by quantitative densitometric scanning. PSA-ACT complex antigen was purchased from BioSpecific and stored in PBS buffer, and 3% BSA blocking agent dissolved in the PBS buffer is used for blocking non-specific antigens binding to PSA antibodies.

[00101] The capture monoclonal antibody was purchased from RA Biosources in

Belmont, CA and the PSA- ACT antigen (CODE: 1-029- ACT) from Biospecific in Emeryville, CA.

[00102] The detection PSA-ACT antibody was purchased from Biospecific in Emeryville, CA. The detection antibody was conjugated to HRP at 1 : 1 ratio. It was prepared by RA

Biosources in Belmont, CA.

[00103] In determining a standard curve for PSA-ACT detection, both the primary capture antibody and the secondary detection antibody are titrated across a plate using a high, low and zero level of PS A- ACT antigen. The desired working range of the PSA-ACT was determined to be 0-325ng mL. [00104] Both antibodies were then diluted in coating buffer (50 mM sodium carbonate pH 9.6) at 0.5, 1 and 2 mg/niL and 100 μΐ was added to each well of a 96-well microtiter plate (Greiner). The plate containing the primary capture antibody was incubated overnight at 4°C and used the next day. The primary capture antibody solution was removed from the microtiter plate by aspirating or dumping the plate. 200 μΐ of blocking buffer (% BSA, TBS, 0.1% Tween-20) is then added to each well of the 96-well microtiter plate and incubated for one hour at room temperature. The blocking buffer was removed from the plate by aspirating or dumping the plate. A PSA-ACT standard was diluted in dilution buffer (1% BSA, PBS or TBS, 0.1 % Tween-20) to 0-325ng/mL. 100 μΐ of the standard was added to each well in the microtiter plate and incubated for 2.5 hours at room temperature. The plates were then washed 3 times with wash buffer (PBS or TBS, 0.1% Tween-20). A biotinylated secondary antibody was diluted serially at 1 :200, 1 :1000, 1:5000 and 1:25,000 in antibody diluent (1% BSA, PBS or TBS, 0.1 % Tween-20). 100 μΐ of detection antibody was added to each well of the microtiter plate and incubated for 1 1/2 hours at room temperature. Again the plates were washed 3 times with wash buffer. Streptavidin- HRP was diluted according to manufacture instructions in antibody diluent and 100 μΐ was added to each well in the microtiter plate and incubated for 1 hr at room temperature. HRP substrate (TMB: 3,3', 5,5'-tetramethyl benzidine) was added and incubated an additional 10-20 minutes at room temperature to allow for color development. The enzymatic reaction was stopped by adding 2M H2SO₄ solution (at a 1 :1 volume with the HRP/TMB substrate/enzyme solution). The absorbance of each well was then read at 450nm using a spectrophotometric/ colorimetric plate reader. The results of the titration were shown as the standard curve in FIG. 1.

EXAMPLE 2: Clinical performance of PS A- ACT sandwich immunoassay (1^st data set)

[00105] A total of 249 serum samples including from 148 BPH patients, 24 prostate cancer patients and 77 healthy males were used for a clinical performance evaluation of the assay. All subjects were of Asian origin and 50 years of age or older. The results show that the PSA- ACT immunoassay has a sensitivity of 91.6% and a specificity of 76.4% at a cut-off level of 3.5 ng mL PS A- ACT. By contrast, CanAg tPSA sandwich immunoassay conducted on the same sample pool demonstrated 45.95% specificity at a cut-off level of 2.5 ng/mL of total PSA although achieving a similar sensitivity of 91% (see data in Tables 1, 2 and 3). EXAMPLE 3: Distribution of healthy male serum samples (1^st data set)

[00106] A total of 56 healthy male serum samples were freshly collected from hospitals in August of 2008 according to standard protocol. All subjects were of Asian origins and at least 50 years of age. 56 samples were tested for the distribution of PS A- ACT concentrations by means of PSA- ACT sandwich immunoassay. The results demonstrate an acceptable frequency distribution of PS A- ACT concentrations has been achieved under a robust standard PS A- ACT quantitative measurement curve in healthy male serum samples as shown in Table 3, FIG. 1, FIG. 2A, FIG. 2B, and FIG. 3. 98% of the samples fall in the range from 1 to 5 ng/mL PSA- ACT (1 fPSA (32 kD) = 3.125 PSA-ACT 110 kD) molecule (see FIG. 2A and FIG. 2B).

EXAMPLE 4: Detection of PS A- ACT in combination with one or more additional prostate cancer biomarkers using a bead-based multiplex sandwich fluorescent immunoassay.

[00107] Capture and detection antibodies for prostatic acid phosphatase, prostatic inhibin (also called PSP94), kallikrein-related peptidase 2, early prostate cancer antigen (EPCA), PC A3, hepsin, prostate stem cell antigen, or {alpha}-methylacyl-CoA racemase (AMACR) are obtained commercially. These biomarkers are described in greater detail in the patent documents: U.S. Patent No. 7,094,533 (entitled "Therapeutic and diagnostic applications of prostatic acid phosphatase in prostate cancer"), PCT application WO/2004/078123 (entitled "Early Prostate Cancer Antigens (Epca), Polynucleotide Sequences Encoding Them, And Their Use"), U.S. Patent Application Nos. 2004/0009164 and 2006/0029984 (entitled "PSP94 diagnostic reagents and assays"), U.S. Patent No. 6,093,796 (entitled "Recombinant hK2 polypeptide"), U.S. Patent No. 7,008,765 (entitled "PC A3, PCA3 genes, and methods of use"), WO02064839 (entitled "Methods For The Diagnosis And Treatment Of Tumors Employing The Hepsin Gene"), U.S. Patent No. 6,960,443 (entitled "Methods of detecting prostate stem cell antigen in cancer") and U.S. Patent No. US20050136493 (entitled "AMACR cancer markers"). The contents of all the cited patent documents are hereby incorporated by reference herein in their entirety.

[00108] Multiplex Bead-Based Luminex® Assays (Luminex Corp.) permit the

simultaneous detection of picogram quantities of prostate disease state biomarkers present in a biological sample. The use of the suspension bead-based technology enables the multiplexing capabilities of the Luminex® assays. The xMAP® technology uses 5.6 micron polystyrene microspheres, which are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number, or bead region, allowing differentiation of one bead from another. Beads covalently bound to different antibodies can be mixed in the same assay utilizing a 96-well microplate format. At the completion of the sandwich immunoassay, beads are read, using the Luminex® 100™ or 200™ detection system, in single- file by dual lasers for classification and quantification of each analyte.

[00109] An example of the multiplex sandwich immunoassay is described in U.S. Patent Application No. 2007/0207508 (entitled "Method And Assay Kit For Simultaneously Detecting Multiple Tumor Markers"), the contents of which are hereby incorporated by reference herein in its entirety.

EXAMPLE 5: PS A- ACT standard curve using a sandwich immunoassay (2^nd data set)

[00110] PSA-ACT complex antigen was used in the calibration for the quantitative measurement of PS A- ACT complex in serum samples as described in Example 1. A dynamic calibration range from 0 to 325 ng mL of PSA- ACT was used to generate a standard curve shown in FIG. 4.

EXAMPLE 6: Clinical performance of PS A- ACT sandwich immunoassay (2^nd data set)

[00111] The clinical performance of the PS A- ACT immunoassay was tested using 59 serum samples, which include 34 biopsy-confirmed prostate cancer patients and 24 biopsy- confirmed BPH patients. These samples were drawn before the biopsy procedure and obtained from Oncologists and Medical Schools. The clinical results demonstrated the PSA- ACT immunoassay achieves 88.2% sensitivity and 68% specificity as shown in FIG. 4 A of the ROC analysis. By comparison with currently used tPSA immunoassay performance (an averaged 90% sensitivity and an averaged 25% specificity), the PS A- ACT assay showed a comparable sensitivity and an approximate 2 fold improvement in the specificity of the assay.

[00112] ROC curves for cohorts of an additional 92, 98 and 140 serum samples are shown in FIG. 4B and FIG. 4C. FIG. 5B depicts the distribution of Cancer and BPH amongst the 92 serum samples. The PS A- ACT immunoassays were performed according to the protocol of Example 1. The amount of PS A- ACT within each sample was then determined using the calibration curve shown in FIG. 4. [00113] Currently used total PSA (tPSA) and free PSA (fPSA) assays and the ratios of tPSA to fPSA have not showed significant clinical utility in discrimination between prostate cancer and BPH patients because of their low specificity. Those tests have demonstrated a low specificity of about 25% leading to high levels of false negatives. But PSA is still the best serum marker for prostate cancer although many other markers have been reported. Complexed PSA, such as PS A- ACT has been demonstrated to be a dominant PSA form detected in serum of prostate cancer patients and represents an important biomarker. Over 85% of the PSA-ACT can be detected in prostate cancer patient serum samples by the equal molar total PSA assays.

Advanced prostate cancer patients contain even higher PS A- ACT. PS A- ACT complex protein is considered as a prostate cancer associated biomarker. PSA-ACT immunoassay can be developed in two years for the significant improvement on prostate cancer diagnosis.

[00114] A large quantity of PSA-ACT can be isolated from high PSA serum samples using affinity chromatography. These purified PS A- ACT can be used an immunogen for novel anti- PSA-ACT monoclonal antibody (mAb) generation. New PSA- ACT mAbs can be utilized in the immunoassay development for the quantitation of native PSA-ACT from prostate patient serum samples. Such a PSA- ACT immunoassay is believed to have very high sensitivity (-90%) and specificity (up to 75%) over total PSA assays (90% sensitivity and 10-31% specificity) and the currently used "complexed PSA assay" (tPSA minus fPSA with 85% sensitivity and 37% specificity) in distinguishing prostate cancer from BPH patients in the gray zone of 4-10 ng PSA/mL.

THEORETICAL DISCUSSION

[00115] Although the theoretical description given herein is thought to be correct, the operation of the systems and methods described and claimed herein does not depend upon the accuracy or validity of the theoretical description. That is, later theoretical developments that may explain the observed results on a basis different from the theory presented herein will not detract from the inventions described herein.

[00116] Any patent, patent application, or publication identified in the specification is hereby incorporated by reference herein in its entirety. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

[00117] While the present invention has been particularly shown and described with reference to the preferred mode as illustrated in the drawing, it will be understood by one skilled in the art that various changes in detail may be affected therein without departing from the spirit and scope of the invention as defined by the claims.

Table 1. Specificity of 148 BPH Serum Samples

265 0.140 0.63 1.09 BPH

270 0.140 0.63 0.49 BPH

166 0.142 0.63 1.32 BPH

194 0.167 0.64 0.92 BPH

266 0.143 0.64 1.52 BPH

324 0.171 0.66 1.56 BPH

274 0.150 0.67 0.82 BPH

227 0.153 0.68 0.19 BPH

246 0.156 0.70 0.66 BPH

327 0.176 0.70 2.28 BPH

191 0.178 0.71 0.38 BPH

206 0.160 0.71 0.25 BPH

205 0.164 0.74 1.39 BPH

278 0.170 0.77 0.24 BPH

351 0.186 0.77 1.45 BPH

250 0.174 0.78 0.26 BPH

323 0.188 0.79 2.94 BPH

214 0.344 0.80 1.17 BPH

20 0.170 0.83 3.32 BPH

332 0.197 0.87 6.69 BPH

68 0.179 0.89 9.82 BPH

263 0.203 0.95 0.36 BPH

269 0.203 0.95 3.22 BPH

33 0.146 1.06 0.30 BPH

202 0.220 1.07 2.21 BPH

64 0.202 1.07 6.94 BPH

97 0.206 1.10 3.89 BPH

211 0.377 1.13 5.36 BPH

123 0.166 1.14 0.36 BPH

350 0.224 1.14 0.93 BPH

272 0.235 1.18 1.09 BPH

131 0.215 1.18 9.34 BPH

156 0.215 1.19 5.52 BPH

16 0.234 1.37 3.38 BPH

85 0.226 1.40 0.41 BPH

93 0.229 1.41 0.51 BPH

3 0.246 1.51 25.01 BPH

252 0.273 1.51 6.03 BPH

249 0.275 1.53 2.89 BPH

87 0.253 1.54 2.76 BPH

115 0.257 1.56 1.85 BPH

136 0.274 1.66 0.76 BPH

141 0.258 1.66 14.82 BPH

86 0.276 1.67 1.18 BPH

229 0.290 1.69 2.59 BPH 84 39 0.282 1.70 0.50 BPH

85 173 0.264 1.71 2.74 BPH

86 54 0.287 1.73 1.89 BPH

87 349 0.268 1.79 5.05 BPH

88 167 0.269 1.81 4.51 BPH

89 262 0.301 1.81 2.86 BPH

90 185 0.304 1.84 1.6 BPH

91 117 0.273 1.86 9.55 BPH

92 114 0.315 1.91 5.58 BPH

93 142 0.316 1.92 2.00 BPH

94 153 0.316 1.92 2.36 BPH

95 121 0.328 2.00 26.30 BPH

96 79 0.331 2.02 10.81 BPH

97 152 0.333 2.03 2.99 BPH

98 128 0.285 2.05 11.95 BPH

99 148 0.336 2.06 3.65 BPH

100 328 0.283 2.08 1.38 BPH

101 101 0.348 2.14 0.64 BPH

102 52 0.291 2.15 9.39 BPH

103 170 0.288 2.19 2.58 BPH

104 134 0.354 2.19 3.40 BPH

105 140 0.358 2.22 3.79 BPH

106 82 0.367 2.29 5.12 BPH

107 132 0.389 2.47 6.77 BPH

108 84 0.400 2.57 2.41 BPH

109 20# 0.110 2.61 3.32 BPH

110 129 0.427 2.83 5.13 BPH

111 359 0.332 2.96 0.88 BPH

112 230 0.470 3.05 7.42 BPH

113 97# 0.117 3.06 3.89 BPH

114 2 0.355 3.56 24.20 BPH

115 156# 0.130 3.86 5.52 BPH

116 277 0.424 4.09 5.71 BPH

117 16# 0.135 4.21 3.38 BPH

118 352 0.392 4.74 24.99 BPH

119 117# 0.150 5.14 9.55 BPH

120 184 0.601 5.18 2.85 BPH

121 330 0.377 5.42 20.73 BPH

122 171 0.412 5.55 2.45 BPH

123 52# 0.162 5.94 9.39 BPH

124 64# 0.162 5.94 6.94 BPH

125 6# 0.172 6.58 6.72 BPH

126 193# 0.177 6.87 3.14 BPH

127 6 0.440 6.92 6.72 BPH

128 264 0.509 7.17 7.32 BPH 129 158 0.456 7.90 9.21 BPH

130 141# 0.196 8.09 14.82 BPH

131 3# 0.202 8.51 25.01 BPH

132 131# 0.206 8.76 9.34 BPH

133 157# 0.218 9.50 13.77 BPH

134 200 0.578 9.64 10.89 BPH

135 68# 0.221 9.69 9.82 BPH

136 239 0.591 11.07 11.28 BPH

137 167# 0.265 12.54 4.51 BPH

138 161 0.517 12.67 11.14 BPH

139 158# 0.288 14.02 9.21 BPH

140 157 0.530 14.10 13.77 BPH

141 307 0.513 21.64 6.17 BPH

142 168# 0.523 29.08 28.56 BPH

143 128# 0.530 29.50 11.95 BPH

144 161# 0.813 47.64 11.14 BPH

145 2# 0.943 56.01 24.20 BPH

146 H10 1.279 161.84 15.39 BPH

147 189# 2.595 161.87 24.07 BPH

148 189 1.455 222.60 24.07 BPH

Table 2. Sensitivity of 24 Prostate Cancer Serum Samples

Table 3. Specificity of Serum Samples of Healthy Men Specificity = 92.2%

(77-6)777* 100 = 92.2%

A-ACT tPSA Status Serial No.

2.012 Normal 39

2.012 Normal 40

2.012 Normal 41

2.012 Normal 42

2.074 Normal 43

2.198 Normal 44

2.198 Normal 45

2.198 Normal 46

2.198 Normal 47

2.259 Normal 48

2.259 Normal 49

2.444 Normal 50

2.506 Normal 51

2.506 Normal 52

2.568 Normal 53

2.568 Normal 54

2.568 Normal 55

2.568 Normal 56

2.630 Normal 57

2.630 Normal 58

2.630 Normal 59

2.691 Normal 60

2.753 Normal 61

2.753 Normal 62

2.815 Normal 63

2.877 Normal 64

2.877 Normal 65

2.938 Normal 66

3.000 Normal 67

3.185 Normal 68

3.185 Normal 69

3.309 Normal 70

3.370 Normal 71

3.432 Normal 72

3.679 Normal 73

3.679 Normal 74

4.358 Normal 75

4.790 Normal 76

46.457 Normal Outlier 77

Claims

What is claimed is:

1. A kit for diagnosing a prostate disease state in a patient comprising reagents for a diagnostic assay having a specificity of at least 65% and a sensitivity of at least 70%.

2. The kit of claim 1 , wherein said diagnostic assay has a sensitivity of at least 80%.

3. The kit of claim 1 , wherein said diagnostic assay has a sensitivity of at least 90%.

4. The kit of claim 1 , wherein said diagnostic assay has a specificity of at least 70%.

5. The kit of claim 1, wherein said diagnostic assay has a specificity of at least 75%.

6. The kit of claim 1, wherein said diagnostic assay has a specificity of at least 80%.

7. The kit of claim 1, wherein said diagnostic assay permits the differential diagnosis between benign prostatic hyperplasia and prostatic intraepithelial neoplasia

8. The kit of claim 1, wherein said diagnostic assay permits the differential diagnosis between prostatic intraepithelial neoplasia and metastatic prostatic adenocarcinoma.

9. The kit of claim 1 , wherein said diagnostic assay permits the differential diagnosis between prostatic intraepithelial neoplasia and primary prostatic adenocarcinoma.

10. The kit of claim 1 , wherein said diagnostic assay permits the differential diagnosis between benign prostatic hyperplasia and primary prostatic adenocarcinoma.

11. The kit of claim 1, wherein said reagents comprise a PS A- ACT binding molecule.

12. The kit of claim 11 , wherein said PSA-ACT binding molecule is an antibody.

13. The kit of claim 12, wherein said antibody is a monoclonal antibody.

14. The kit of claim 11 , wherein said PS A- ACT binding molecule is the Egenix D 1

monoclonal antibody.

15. The kit of claim 11 , wherein said PSA-ACT binding molecule is selected from the group consisting of scFv, a single domain antibody, Fv, a diabody, a tandem diabody, Fab, Fab', F ab)2 and a polyclonal antibody.

16. The kit of claim 11 , wherein said PS A- ACT binding molecule does not bind to non- complexed PSA or non-complexed ACT.

17. The kit of claim 11 , wherein said PSA-ACT binding molecule is conjugated to a labeling compound selected from the group consisting of a radioactive agent, an enzyme, a fluorescent compound, a luminescent compound and an electron transfer agent.

18. The kit of claim 1, wherein said diagnostic assay comprises an immunoassay.

19. The kit of claim 18, wherein said immunoassay comprises a sandwich

immunoassay.

20. The kit of claim 11, further comprising a PSA binding molecule.

21. The kit of claim 1, wherein said specificity and said sensitivity are determined at a cut-off of less than 4ng/mL PSA-ACT complex.

22. A kit for diagnosing prostate cancer in a patient comprising:

a) reagents for collecting a biological sample from a patient;

b) PSA binding molecules for capturing free PSA and complexed PSA within said sample ;

c) reagents for isolating proteins bound to said PSA binding molecule; d) PSA-ACT complex-specific binding molecules for contacting the proteins bound to said PSA binding molecules;

e) reagents for measuring the amount of PSA-ACT complexes in said sample, and f) instructions for determining the prostate disease state in said patient;

wherein said determination has a specificity of at least 65% and a sensitivity of at least 70% at a pre-defined concentration of PSA- ACT complex within said sample.

23. The kit of claim 22, wherein said biological sample is a blood sample.

24. The kit of claim 22, wherein said biological sample is semen.

25. The kit of claim 22, wherein said biological sample comprises prostatic fluid.

26. The kit of claim 22, wherein said pre-defined concentration of PSA-ACT complex is less than 4ng/mL of PSA-ACT complex.

27. A method for diagnosing a prostate disease state in a patient comprising a diagnostic assay having a specificity of at least 65% and a sensitivity of at least 70%.

28. The method of claim 27, wherein said diagnostic assay has a sensitivity of at least 80%.

29. The method of claim 27, wherein said diagnostic assay has a sensitivity of at least 90%.

30. The method of claim 27, wherein said diagnostic assay has a specificity of at least 70%.

31. The method of claim 27, wherein said diagnostic assay has a specificity of at least 75%.

32. The method of claim 27, wherein said diagnostic assay has a specificity of at least 80%.

33. The method of claim 27, wherein said diagnostic assay comprises an immunoassay.

34. The method of claim 27, wherein said diagnostic assay comprises a PSA-ACT binding molecule.

35. The method of claim 34, wherein said PSA- ACT binding molecule is an antibody.

36. The method of claim 35, wherein said antibody is a monoclonal antibody.

37. The method of claim 34, wherein said PSA-ACT binding molecule is the Egenix Dl monoclonal antibody.

38. A method of diagnosing prostate cancer in a patient comprising the steps of:

a) providing a biological sample from a patient;

b) contacting said sample with a PSA binding molecule;

c) isolating proteins bound to said PSA binding molecule;

d) contacting the proteins bound to said PSA binding molecule with a PS A- ACT complex-specific binding molecule;

e) measuring the amount of PS A- ACT complexes in said sample, and

f) determining the prostate disease state in said patient;

wherein said determination has a specificity of at least 65% and a sensitivity of at least 70% at a pre-defined concentration of PSA-ACT complex within said sample.

39. The method of claim 38, wherein said biological sample is a blood sample.

40. The method of claim 38, wherein said biological sample is semen.

41. The method of claim 38, wherein said biological sample comprises prostatic fluid.

42. The method of claim 38, wherein said pre-defined concentration of PSA-ACT complex is less than 4ng mL of PS A- ACT complex.

The method of claim 38, wherein said prostate disease state is prostatic intraepithelial neoplasia.

The method of claim 38, wherein said PSA-ACT complex-specific binding molecule is a monoclonal antibody.

The method of claim 38, wherein said PSA-ACT complex-specific binding molecule is selected from the group consisting of scFv, a single domain antibody, Fv, a diabody, a tandem diabody, Fab, Fab', F(ab)₂ and a polyclonal antibody.

The method of claim 38, wherein said PS A- ACT complex-specific binding molecule is the Egenix Dl monoclonal antibody.

The method of claim 38, wherein said PS A- ACT complex-specific binding molecule is a nucleic acid.

The method of claim 38, wherein said PSA-ACT complex-specific binding molecule is conjugated to a labeling compound selected from the group consisting of a radioactive agent, an enzyme, a fluorescent compound and an electron transfer agent.

A method of diagnosing prostate cancer in a patient comprising the steps of:

a) providing a semen sample from a patient;

b) contacting said sample with a PSA binding molecule;

c) isolating proteins bound to said PSA binding molecule;

d) contacting the proteins bound to said PSA binding molecule with a PSA-ACT complex-specific binding molecule;

e) measuring the amount of PSA-ACT complexes in said sample, and

f) determining the prostate disease state in said patient;

wherein said determination has a specificity of at least 65% and a sensitivity of at least 70% at a pre-defined concentration of PSA-ACT complex within said sample.

50. The method of claim 49, wherein said pre-defined concentration of PSA-ACT complex is less than 4ng/mL of PSA-ACT complex.

51. The method of claim 49, wherein said PSA- ACT complex-specific binding molecule is a monoclonal antibody.

52. The method of claim 49, wherein said PSA-ACT complex-specific binding molecule is the Egenix Dl monoclonal antibody.