WO2010077921A1

WO2010077921A1 - Prostate cancer markers and uses thereof

Info

Publication number: WO2010077921A1
Application number: PCT/US2009/068204
Authority: WO
Inventors: Douglas F. Lake; Kwasi Antwi; Galen Hostetter; Michael J. Demeure
Original assignee: Arizona Board Of Regents, A Body Corporte Acting For And On Behalf Of Arizona State University
Priority date: 2008-12-17
Filing date: 2009-12-16
Publication date: 2010-07-08

Abstract

The present invention provides methods for assessing the probability of a prostate tumor in a subject, or for monitoring efficacy of treatment for prostate cancer, involving detection of QSOX1 in patient tissue samples.

Description

Prostate cancer markers and uses thereof

Cross Reference

This application claims priority to U.S. Provisional Patent Application Serial No. 61/138,361 filed December 17, 2008, incorporated by reference herein in its entirety.

Background

Prostate cancer develops in the prostate and may cause pain, erectile dysfunction, and difficulty in urinating, among other symptoms. Rates of detection of prostate cancers vary widely across the world, with South and East Asia detecting less frequently than in Europe and the United States. Prostate cancer tends to develop in men over the age of fifty and it is one of the most prevalent types of cancer in men. The presence of prostate cancer maybe indicated by symptoms, physical examination, prostate specific antigen (PSA), or biopsy. However, there is controversy about the accuracy of the PSA test and the value of screening. Thus, improved markers for prostate cancer would be of great value.

Summary of the Invention

In a first aspect, the present invention provides methods for assessing the probability of a prostate tumor in a subject, comprising

(a) contacting a prostate tissue section from a subject at risk of prostate cancer with an antibody to QSOXl, under conditions suitable to promote immunocomplex formation between the antibody and QSOXl present in the prostate tissue section;

(b) removing unbound antibody; and

(c) detecting immunocomplex formation in the prostate tissue section between the antibody and QSOXl, wherein the presence of immunocomplex formation in the prostate tissue section correlates with a probability of a prostate tumor in the subject.

In a second aspect, the present invention provides methods for monitoring efficacy of treatment of a prostate tumor in a subject, comprising (a) contacting a prostate tissue section from a subject being treated for a prostate tumor with an antibody to QSOXl, under conditions suitable to promote immunocomplex formation between the antibody and QSOXl present in the prostate tissue section, wherein the analyzing is carried out after one or more tumor treatments;

(b) removing unbound antibody; and

(c) detecting immunocomplexes the prostate tissue section, wherein a decrease in amount of immunocomplexes in the prostate tissue section compared to an amount of immunocomplexes in a prostate tissue section from the subject after diagnosis of the prostate tumor but before tumor treatment began correlates with an effective prostate tumor treatment.

Brief Description of the Figures

Figure 1 shows an exemplary immunohistochemical staining of prostate adenocarcinoma (ACA) tissue sections with anti-QSOXl antibody.

Figure 2 shows an exemplary immunohistochemical staining of prostate ACA tissue sections with anti-QSOXl antibody.

Detailed Description of the Invention

In one aspect, the present invention provides methods for assessing the probability of a prostate tumor in a subject, comprising

(b) removing unbound antibody; and

The methods of this aspect of the invention are useful, for example, in assessing the probability of a prostate tumor in a patient. The methods provide early testing for prostate cancer (both initial diagnosis and recurrence after remission), and thus provide a means to improve the prognosis and treatment of prostate cancer patients. As disclosed below, the inventors have demonstrated that immunohistochemical staining of tissue sections from patients with prostate cancer showed remarkable tumor specificity for anti-QSOXl antibodies. It appears that prostate tumor cells over-produce QSOXl, while normal adjacent prostate tissue, including normal glands, do not express QSOXl .

The prostate tumor may be of any type; in one preferred embodiment, the prostate tumor is an adenocarcinoma. In other preferred embodiments, the prostate tumor is a leiomyosarcoma or a rhabdomyosarcoma. The prostate tumor may be localized, or may include metastases to other tissue sites.

QSOXl is Quiescin Sulfhydryl Oxidase 1, also called QSCN6. The protein accession number for the long variant of QSOXl on the NCBI database is NP_002817 (SEQ ID NO:1), and the accession number for the short form is NP_001004128 (SEQ ID N0:2).

The term antibody as used herein is intended to include polyclonal antibodies, monoclonal antibodies, and antibody-like proteins (antibody-like molecules) having an antibody structure (at least partially comprising a tetrameric heavy chain-light chain, heavy chain-light chain structure) such as chimeric antibodies, humanized antibodies, and fully human antibodies.

Any suitable antibody that specifically binds QSOXl variants may be used in the methods of this aspect of the invention. Currently, a long form (747 amino acids) and a short form (604 amino acids) of QSOXl are known. Antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). hi one example, pre-immune serum is collected prior to the first immunization. A peptide as disclosed herein (with a carrier if desired), together with an appropriate adjuvant, is injected into an animal in an amount and at intervals sufficient to elicit an immune response. Animals are bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. At about 7 days after each booster immunization, or about weeldy after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20° C. Polyclonal antibodies against the polypeptides can then be purified directly by passing serum collected from the animal through a column to which non-antigen-related proteins prepared from the same expression system without the polypeptides bound.

Monoclonal antibodies can be produced by obtaining spleen cells from the animal. (See Kohler and Milstein, Nature 256, 495-497 (1975)). In one example, monoclonal antibodies (mAb) of interest are prepared by immunizing inbred mice with a polypeptide as disclosed herein, or portion thereof. The mice are immunized by the IP or SC route in an amount and at intervals sufficient to elicit an immune response. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of by the intravenous (IV) route. Lymphocytes from antibody positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions that allow formation of stable hybridomas. The antibody producing cells and fusion partner cells are fused in polyethylene glycol at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells and are screened for antibody production by an immunoassay such as solid phase immunoradioassay. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.

To generate such an antibody response, a peptide as disclosed herein is typically formulated with a pharmaceutically acceptable carrier for parenteral administration. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The formulation of such compositions, including the concentration of the polypeptide and the selection of the vehicle and other components, is within the knowledge of those of skill of the art.

Antibodies can be fragmented using conventional techniques, and the fragments screened for utility in the same manner as described herein for whole antibodies. For example, F(ab')₂ fragments can be generated by treating antibody with pepsin. The resulting F(ab')₂ fragment can be treated to reduce disulfide bridges to produce Fab¹ fragments. As used herein "specific binding" means that the antibodies recognize one or more epitope within the recited protein under suitable immunohistochemical conditions, but possess little or no detectable reactivity with other proteins under the same conditions, such as those disclosed herein. In one embodiment, antibodies against the peptides of the invention may be used The antibodies may be directed against any one or more epitopes within QSOXl or SerpinF2 so long as the antibodies retain specific binding for the protein in immunohistochemical staining. The contacting occurs under conditions suitable to promote specific binding of the antibody to QSOXl while minimizing non-specific binding.

Any suitable steps wash steps may be used to remove unbound antibody prior to detection.

Detecting immunocomplex formation can be done by any suitable method; such methods are well within the level of skill in the art, based on the present disclosure. In one embodiment, the antibody is labeled with a detectable marker, including but not limited to enzymes (such as peroxidase) that can catalyze a detectable reaction (typically based on color generation) and fluorophores (including but not limited to FITC, rhodamine, Texas Red, fluorescent proteins (GFP, EFP, etc.). In another embodiment, the antibodies are bound by secondary antibodies on which the detectable label is located.

Any suitable prostate tissue section can be used, although thin sections are preferred (ie: 4 μm to 10 μm). In one non-limiting embodiment, a prostate tissue section from a subject is placed in a fixative, and subsequently embedded in a mounting medium from which appropriate tissue sections are generated using any suitable device, such as a microtome. In one preferred embodiment, the prostate tissue section comprises seminal ducts and/or ejaculatory ducts.

The tissue section may be subjected to any desired treatments prior to contacting with the antibody, including but not limited to drying, dewaxing, rehydrating, heating, blocking, etc. One exemplary embodiment is provided in detail below.

The subject can be any mammal, preferably a human male. In one preferred embodiment of this aspect, the subject has one or more risk factors for prostate cancer selected from the group consisting of: age 50 years or more, African ancestry, family history of prostate cancer, prostatitis, exposure to Agent Orange, obesity, elevated blood levels of testosterone, elevated prostate specific antigen (PSA) count, and sexually transmitted diseases (including but not limited to Chlamydia, gonorrhea, and syphilis).

As used herein, the "probability of a prostate tumor" can comprise determining a probability that the subject will develop a prostate tumor (prognostic), or may comprise determining a probability that the subject already has a prostate tumor (diagnostic). For example, a subject in who tests positive with QSOXl antibodies can then undergo further tests to verify that a prostate tumor is present. If such further tests do not show the presence of a tumor, an attending physician can determine whether early treatment interventions (chemotherapy, radiation therapy, hormone therapy, surgery, etc.), are warranted, based on all circumstances. Alternatively, someone who tests positive with another marker of prostate cancer (such as PSA) is biopsied, and the sample is tested for QSOXl antibody staining. Positive staining would be used in assisting an attending physician to determine appropriate treatment interventions. Alternatively, an attending physician can use the results from the methods of the invention, in light of other circumstances, to determine whether the subject should begin treatment for prostate cancer. Thus, the methods of the invention lead to earlier prognosis, diagnosis, and/or treatment of prostate cancer, which will lead to improved patient prognosis.

The methods of the invention may be used in combination with other markers for prostate cancer, including but not limited to analysis of prostate specific antigen (PSA) and Gleason score evaluation. In one preferred embodiment, the methods further comprise PSA detection, where PSA is analyzed either in circulation or by immunohistochemistry. While the PSA test is widely used, it is a poor predictor of prostate cancer, since PSA levels are also generally elevated in subjects with enlargement of the prostate (benign prostatic hypertrophy (BPH)) and those with prostatitis (prostate infection). PSA levels may also be lowered in men on medication to treat BPH or baldness.

hi another aspect, the present invention provides methods for monitoring efficacy of treatment of a prostate tumor in a subject, comprising

(a) contacting &■ prostate tissue section from a subject being treated for a prostate tumor with an antibody to QSOXl, under conditions suitable to promote immunocomplex formation between the antibody and QSOXl present in the prostate tissue section, wherein the analyzing is carried out after one or more tumor treatments;

(b) removing unbound antibody; and

(c) detecting immunocomplexes in the prostate tissue section, wherein a decrease in amount of immunocomplexes in the prostate tissue seption compared to an amount of immunocomplexes in a prostate tissue section from the subject after diagnosis of the prostate tumor but before tumor treatment began correlates with an effective prostate tumor treatment. Thus, this aspect of the invention provides a valuable tool for monitoring effectiveness of treatments for prostate cancer.

All embodiments of the first aspects of the invention are suitable in this aspect as well, unless the context dictates otherwise. The subject can be any mammal, preferably a human male. Treatment options to be assessed for efficacy in this aspect of the invention include, but are not limited to, surgical removal/resection of the tumor, hormonal therapy, chemotherapy (including, but not limited to gemcitabine. 5- fluorouracil, oxaliplatin, erlotinib, abraxane or any combination of chemotherapies, and/or radiation therapy (including but not limited to brachytherapy).

Examples

Immunohistochemistty (IHC). Formalin-fixed, paraffin-embedded tissue blocks from patients who underwent surgical resection for prostate ACA were sectioned at 5μm thickness using water flotation for tissue section transfer and dried overnight at room temperature. The slides were dewaxed, rehydrated and antigen retrieved on-line on the Bond™ autostainer (Leica Microsystems, Inc. Bannockburn, IL). The Bond autostainer is intended for staining sections of formalin-fixed, paraffin-embedded tissue. All slides were cut at 5μm, baked at 6O⁰C for 60 minutes. Slides were subjected to heat induced epitope retrieval using a proprietary citrate based retrieval solution for 20 minutes. Endogenous peroxidase was blocked. For QSOXl, the tissue sections were incubated for 30 minutes with an anti-QSOXl polyclonal antibody (binds to both the long and short QSOXl variant) at 1 :75 from Proteintech Group, Inc. (Chicago, IL). The sections were visualized using the Bond™ Polymer Refine Detection kit (Leica) using diaminobenzidine chromogen as substrate. IHC optimization and staining parameters were evaluated by a board-certified pathologist, with standard scoring based on stain intensity (0 to 3) with score of 0 representing no staining and score 3 intense staining. IHC stain localization was determined in tumor cells as nuclear, cytoplasmic or membranous. The staining pattern for QSOXl was cytoplasmic.

As shown in figures 1-2, anti-QSOXl antibodies stain prostate ACA cells, but not normal adjacent glands. Anti-QSOXl antibodies did not stain (or minimally stained) tissue sections from patients with BPH.

Claims

We claim:

1. A method for assessing the probability of a prostate tumor in a subject, comprising

(b) removing unbound antibody; and

2. The method of claim 1 , wherein the subject has one or more risk factors for prostate cancer selected from the group consisting of age 50 years or more, African ancestry, family history of prostate cancer, prostatitis, exposure to Agent Orange, obesity, elevated blood levels of testosterone, and sexually transmitted diseases (including but not limited to Chlamydia, gonorrhea, and syphilis).:

3. The method of claim 1 or 2 wherein the antibody is a monoclonal antibody.

4. The method of claim 1 or 2 wherein the antibody is a polyclonal antibody.

5. The method of any one of claims 1 -4 wherein the antibody is detectably labeled.

6. The method of any one of claims 1 -5, where assessing the probability of a prostate tumor comprises determining a probability that the subject will develop a prostate tumor.

7. The method of any one of claims 1 -5, where assessing the probability of a prostate tumor comprises determining a probability that the subject has a prostate tumor.

8. A method for monitoring efficacy of treatment of a prostate tumor in a subject, comprising

(a) contacting a prostate tissue section from a subject being treated for a prostate tumor with an antibody to QSOXl, under conditions suitable to promote immunocomplex formation between the antibody and QSOXl present in the prostate tissue section, wherein the analyzing is carried out after one or more tumor treatments;

(b) removing unbound antibody; and