WO2011094233A1 - Procédés de classification de maladies ou de pronostic du cancer de la prostate basés sur l'expression d'antigènes testiculaires/cancéreux - Google Patents

Procédés de classification de maladies ou de pronostic du cancer de la prostate basés sur l'expression d'antigènes testiculaires/cancéreux Download PDF

Info

Publication number
WO2011094233A1
WO2011094233A1 PCT/US2011/022464 US2011022464W WO2011094233A1 WO 2011094233 A1 WO2011094233 A1 WO 2011094233A1 US 2011022464 W US2011022464 W US 2011022464W WO 2011094233 A1 WO2011094233 A1 WO 2011094233A1
Authority
WO
WIPO (PCT)
Prior art keywords
cta
pca
cancer
expression
sample
Prior art date
Application number
PCT/US2011/022464
Other languages
English (en)
Inventor
Prakash Kulkarni
Takahito Suyama
Robert H. Getzenberg
Original Assignee
The Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Johns Hopkins University filed Critical The Johns Hopkins University
Publication of WO2011094233A1 publication Critical patent/WO2011094233A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to disease classification, prognosis, diagnosis, and/or treatment of prosta te cancer (PCa), and more specifically to methods of disease classification or prognosis for PCa based on expression of cancer/iesiis antigens,
  • PCa prostate cancer
  • PSA serum prostate-specific antigen
  • CTAs cancer/testis antigens
  • This programmatic acquisition is thought to be one of the driving forces in tumori genesis.
  • Numerous studies have revealed a multitude of CTA functions such as media tors of prolifera tive signals, chromatin modifiers, and transcrip tional co-regulators.
  • the CTAs represent attracti ve immunotherapeutic targets because of their restricted expression in an immune-privileged organ. Despite these significant advances, how the CTAs are "reactivated" in malignant cells is unclear and, their role in PCa remains virtually unexplored.
  • the present in vention is based on the seminal disco very of a correlation between expression levels of cancer/testis antigens (CTAs) and disease classification of prostate cancer ( PCa).
  • CTAs cancer/testis antigens
  • PCa disease classification of prostate cancer
  • Provided herein are methods of disease classification for a subject having PCa based on expression levels of selected CTA(s) as compared with subjects not having cancer.
  • the present invention further provides kits for disease classification of PCa based on expression levels of selected CTA(s),
  • the present invention provides a method of disease
  • the method includes determining the expression level of at least one cancer/testis antigen (CTA) in a sample from the subject by analysis of CTA nucleic acid or protein, wherein a difference in the expression level of the CTA as compared to a sample from a subject not having PCa provides classification for the PCa (e.g., likelihood of recurrence; likelihood of metastases).
  • CTA cancer/testis antigen
  • the at least one CTA is PAGE4.
  • the at least one CTA further includes a CTA selected from the group consisting of CEP55, NUF2, MAGEA1 , MAGEA2, MAGEAS, MAGEA4, MAGEAS, MAGEA6, MAGEAS, MAGEA12, CSAGl , CSAG2, CTAGl, GAGE3, GAGE4, GAGE7, DDX43, FMRINB, FTHL17, SSX2, SSX3, SSX6, SSX7, XAGEl, PBK, TTK and combinations thereof.
  • the at least one CTA includes CEP55 and/or NUF2.
  • the at least one CTA includes a CT-X antigen.
  • the CT-X antigen includes a member of M AGFA subfamily or CSAG subfamily.
  • the at least one CTA is selected from the group consisting of PAGE4, CEP55, NUF2, MAGEAl, MAGEA2, MAGE A3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEAl 2, CSAG! , CSAG2, CTAGl , GAGE3, GAGE4, GAGE7, DDX43, FMR1NB, FTHL17, SSX2, SSX3, SSX6, SSX7, XAGE1, PBK, TTK and combinations thereof.
  • nucleic acid encoding the CTA is measured to determine the expression level of the CTA.
  • the expression level of the CTA is detected by quantitative real-time polymerase chain reaction (Q-PCR) or quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) and can be analyzed by methods known in the art, such as a microarray.
  • the expression level of the CTA is detected by an immunoassay, In an additional aspect, the immunoassay includes ELISA, Western blotting, or immunoprecipitation.
  • the subject is a human patient.
  • the sample contains prostate ceils and includes a tumor sample such as a biopsied tumor tissue or tumor margins.
  • the sample includes circulating tumor cells.
  • the sample includes a blood, serum or urine sample.
  • the sample includes a formalin-fixed paraffin-embedded (FFPE) sample.
  • the present invention provides a method of prognosis for PCa or PCa recurrence in a subject.
  • the method includes (a) detecting expression level of at least one cancer/testis antigen (CT A) from a sample of the subject; and (b) comparing the expression level obtained in step (a) to at least one reference sample from a subject not having cancer, [0014]
  • the expression level is measured using quantitative real-time polymerase chain reaction (Q-PCR), quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), or other methods known in the art.
  • Q-PCR quantitative real-time polymerase chain reaction
  • Q-RT-PCR quantitative reverse transcription polymerase chain reaction
  • Such nucleic acid levels can be analyzed using a microarray for example.
  • the expression level is measured using an immunoassay.
  • the at least one CTA includes CEP55 and/or NUF2.
  • the at least one CT A includes a CT-X antigen.
  • the CT-X antigen includes a member of MAGEA subfamily or CSAG subfamily.
  • the at least one CTA is selected from the group consisting of PAGE4, CEP55, NUF2, MAGEAl, MAGEA2, AGE A3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA 12, CSAG l, CSAG2, CTAG1, GAGE3, GAGE4, GAGE 7, DDX43, FMR1NB, FTHL17, SSX2, SSX3, SSX6, SSX7, XAGE1 , PBK, and TTK.
  • the subject is a human patient.
  • the sample includes a tumor sample.
  • the tumor sample includes a biopsied tumor tissue or the margins of a resected tumor, for example.
  • the sample could include circulating tumor cells.
  • the sample includes a blood, serum or urine sample.
  • the sample does not include a prostate tissue or PCa ceils.
  • the sample includes a formalin-fixed paraffin-embedded (FFPE) sample.
  • FFPE formalin-fixed paraffin-embedded
  • the method includes (a) measuring expression level of at least one cancer/testis antigen (CTA); and optionally (b) measuring at least one of the following: (i) size of the prostate; (ii) size of the PCa; (iii) blood level of prostate-specific antigen (PSA); (iv) blood level of sex hormones; (v) thymosin ⁇ 15 levels; (vi) phosphorylation level of NF-KB- p65/RelA at 254th amino acid threonine; and (vii) mR A level of PSA or PC A3; and (c) correlating measurement of (a) and (b) with the prognosis for the PCa treatment.
  • CTA cancer/testis antigen
  • the present invention provides a method for treating PCa in a subject.
  • the method includes contacting PCa cells of the subject with an agent inhibiting expression of at least one cancer/testis antigen (CTA).
  • the agent includes siR A or antisense oligonucleotide.
  • the at least one cancer/testis antigen (CTA) includes a member of MAGEA subfamily or CSAG subfamily.
  • the at least one cancer/testis antigen (CTA) includes CEP55 and/or NUF2.
  • the at least one cancer/testis antigen is selected from the group consisting of CEP55, NUF2, MAGEA 1, MAGEA2, MAGE A3, MAGEA4, MAGEA5.
  • the present invention provides a kit for disease
  • the kit includes one or more probes or primers that selectively hybridize to mRNA of at least one CTA; or one or more antibodies specifically binds to protein of at least one CTA.
  • Figure 1 shows that expression of MAGEA2 mRNA is androgen independent but is stimulated by changes in serum constituents.
  • Figure 1 A shows expression in LNCaP in response to R 1881 normalized to expression before treatment.
  • Figure IB shows expression in PC-3 ceils in response to treatment with 5AZA (3 ⁇ ), CS-FBS (10%) and R1881 (10 nM).
  • Inset shows Western blotting analysis of MAGEA2 protein in PC-3 cells grown either in control (regular) or CS-FBS -containing medium.
  • Figure 1C shows expression in CS-FBS- containing medium and in regular medium following treatment with CS-FBS for four days.
  • FIG. 2 shows that MAGEA2 is predominantly expressed in hormone- refractory/castrate-resistant prostate cancer (PCa).
  • Figure 2A shows expression in prostate samples obtained by analyzing data from Gene Expression Omnibus (accession #GDS1439).
  • Figure 2B shows expression in clinical samples from primary and from various metastatic sites. Primary PCa samples are obtained from radical prostatectomy while hormone- refractory/castrate-resistant PCa are obtained from "rapid" autopsies and propagated as xenografts. Details of these samples are also described in Tables 6 and 7.
  • RN A samples are run on a whole genome microarray. Heat map shows the predominant upregulation of MAGEA genes from subclusters II-IV indicated in parenthesis as well as the CSAG genes.
  • Figure 2C shows Q-PCR validation of a subset of samples. MAGEA2 expression is normalized to the TATA-binding protein (TBP), a house-keeping gene.
  • TBP TATA-binding protein
  • FIG. 3 shows Q-RT-PCR validation of MAGEA2 expression in PCa cell lines. PGR reactions are performed in triplicate.
  • FIG. 4 shows methylation analyses of MAGE A2 promoter D A, A fragment of the MAGEA2 promoter DNA constituting the CpG island is PCR amplified after bisulfite conversion and cloned into a plasmid vector, DNA stretches from representative clones are shown for each of the time points mdicated. The numbers of the CpG nucl eotides indicate the position of the 22 CpGs in the CpG island.
  • Figure 5 shows effect of CS-FBS on MAGE-A gene expression by Q-RT-PCR, where only MAGEA2 robustly responds to CS-FBS treatment.
  • Figure 6 shows Q-PCR validation of CT-X antigen expression in PCa specimens.
  • a subset of samples from Figure 2 is used to determine expression by Q-PCR.
  • Relative expression normalized to TATA-binding protein, TBP
  • MAGEA12, CSAG2, MAGEA2, MAGEA6, and PAGE4 are shown for MAGEA12, CSAG2, MAGEA2, MAGEA6, and PAGE4 ( Figure 6A-6E), respectively.
  • Figure 7 shows epigenetic regulation of MAGE A2 expression in PCa cells.
  • Figure 7 A shows extent of methylation of cytosine residues in the androgen-responsive and androgen-independent PCa cell lines.
  • Figure 7B shows relative expression of MAGEA2 normalized to TBP before and after 5AZA treatment.
  • Figure 7C shows fold-change of MAGEA2 expression before and after 5 AZA treatment.
  • Figure 8 shows that MAGEA2 upregulation is associated with chemoresistance in PCa cells.
  • Figure 8A shows effect of docetaxel treatment for 3 days on androgen-responsive LNCaP cells.
  • Figure 813 shows extent of apoptosis as detected by DNA fragmentation determined by flow cytometry.
  • Figure 10 shows both relative AGEA2 expression normalized by TBP and methylation status of the MAGEA2 CpG island (%) for PCal-PCa7, LuCAP23, LuCAP69, and LuCAP96A.
  • Figure 1 1 shows expression of cancer/testis antigens (CTAs) in normal prostate tissue ( P) i n 20 ).
  • CTAs cancer/testis antigens
  • L-PCa clinically localized PCa
  • M-PCa metastatic PCa
  • Figure 1 1A clinically localized PCa
  • NUF2 Figure 1 IB
  • PBK Figure 1 1C
  • TTK Figure 1 ID
  • PAGE4 Figure H E
  • SSX2/3 Figure 1 I F
  • CSAG2 Figure 1 1G
  • MAGEA2 Figure UK
  • MAGEA12 Figure HI.
  • * p ⁇ Q.Q5, **p ⁇ QM, NS not significant.
  • Figure 12 shows expression of cancer/testis antigens (CT As) in clinically localized PCa with recurrence (Rec (+)) (n :::: 43) and without recurrence (Rec (-)) (n :::: 29) for CEP55 ( Figure 12A); NUF2 ( Figure 12B); PAGE4 (Figure 12C); PBK ( Figure 12D); and TTK ( Figure 12E).
  • CT As cancer/testis antigens
  • Figure 13 shows Kaplan-Meier curves illustrating biochemical recurrence-free survival against time after radical prostatectomy stratified by the mRNA expression of CEP55 (Figure 13 A): NUF2 ( Figure 13B); PAGE4 (Figure 13C); PBK ( Figure 13D); and TTK ( Figure 13E) (high versus low groups dichotomized by median value).
  • Figure 14 shows various GSTPl expression data.
  • Rec (+) recurrence
  • Rec (-) recurrence
  • Figure 14C shows Kaplan-Meier curves illustrating biochemical recurrence-free survival against time after radical prostatectomy stratified by the mRNA expression of GSTPl (high versus low groups dichotomized by median value).
  • the disease classification of prostate cancer is useful for determining a therapeutic regimen or treatments suitable for a particular PCa patient.
  • the disease classification includes, but not limited to, primary tumor, hormone-refractory/castrate-resistant PCa, metastasis and/ or various stages of PCa .
  • Potential therapeutic regimes/treatments include, but not limited to, surgery, chemotherapy, radiation, and/or other treatments as described further below.
  • the seminal discovery of this invention allows for a more personalized approach to treating a patient based on the CTA "signature" of that patient.
  • the present invention provides that detection of cancer/testis antigen (CTA) expression can be performed in tissue or biological samples other than directly from the prostate or PCa cells. If increased or decreased expression of selected CTA is observed in, for example, a surgical sample or blood sample not from prostate, the present invention provides that the patient is likely at risk for PCa or PCa recurrence.
  • CTA cancer/testis antigen
  • CTAs Cancer/Testis Antigens
  • the present invention provides that several members of the MAGE-A subfamily are selectively "reactivated” in hormone-refractory but not organ-confined PCa.
  • expression of MAGEA2 but not other MAGE-A genes is dramatically stimulated by a serum constituent that demethylates DNA, The reactivated M AGE-A genes may represent promising biomarkers/therapeutic targets for PCa including hormone-refractory PCa.
  • CTAs are significant predictors of PCa recurrence, for example, after radical prostatectomy (RP).
  • RP radical prostatectomy
  • CTA expression is significantly correlated with the prostatectomy Gieason score indicating that, in addition to the currently utilized clinical information including Gieason score, these CTAs may provide a quantitative readout to predict disease recurrence following RP
  • the present invention provides application of formal in- fixed paraffin-embedded (FFPE) samples for detecting translational relevance to PCa prognosis and treatment.
  • FFPE formal in- fixed paraffin-embedded
  • the CTAs can be broadly divided into two groups based on their chromosomal locations: the CT-X antigens located on the X chromosome and non-X CT antigens located on various autosomes.
  • the CT-X genes are generally expressed in the spermatogonia which are proliferating germ cells.
  • expression of non-X CT genes appears more dominant in the later stages of germ-cell differentiation, such as in spermatocytes.
  • CTAs are known to be associated with advanced disease with poorer outcomes in several types of cancers.
  • the present invention provides that there is an unexpected stage-specific expression of the CTAs in PCa.
  • the CTAs can serve as unique biomarkers that could potentially be used to distinguish men with aggressive disease who need treatment from men with indolent disease not requiring immediate intervention in PCa.
  • Coordinated expression pa tterns of the CT-X antigens appear to be modulated by promoter hypomethylation.
  • the coordinated expression of non-X CT antigens in PCa has not been demonstrated to date.
  • CT-X antigens represent more than half of all CTAs and often constitute multigene families organized in well-defined clusters along the X chromosome.
  • the CT-X antigens are frequently expressed in many types of cancers and several studies have shown that their expression patterns are frequently associated with higher grade lesions and advanced disease with poorer outcome.
  • Numerous studies have also revealed a multitude of CTA functions especially, in tumorigenesis.
  • MAGEA1 and MAGEA4 M-phase phosphoprotein 1 (MPHQSPHl), CAGE, PIWIL2, and PDZ-binding kinase/T-LAK cell -originated protein kinase (PBK/T-LA ) appear to function as putative oncogenes and mediators of proliferative signals, others such as the SYT/SSX oncogene, BRDT, BORIS, and the hi stone H3 4 demethylase JARID1B, function as chromatin modifiers and epigenetic mediators.
  • CT-X antigens MAGEA1 1
  • MAGEA1 CT-X antigens
  • CTA expression profiling samples and cell lines can be performed utilizing a custom microarray containing probes for about two-thirds of all CTAs.
  • the microarray data can also be validated by quantitative PCR (Q-PCR).
  • Functional studies can be carried out by silencing gene expression, for example, with siRNA.
  • DNA methylation can be determined by rnethyiation-specii ic PCR or other techniques well-known in the art.
  • CT-X antigens Several CT-X antigens from the present invention are located on the X chromosome (CT-X antigens).
  • MAGEA/CSAG subfamilies are coordinately upregulated in castrate-resistant PCa (CRPC) but not in primary PCa.
  • CRPC castrate-resistant PCa
  • PAGE4 is highly upregulated in primary PCa but is virtually silent in CRPC.
  • silencing the expression of MAGEA2 the most highly upregulated member significantly impairs proliferation of PCa cells while increasing their chemo-sensitivity.
  • CT-X antigens may serve as unique biomarkers that could potentially be used to distinguish men with aggressive disease who need treatment from men with indolent disease not requiring immediate intervention.
  • CT- X antigens may be novel therapeutic targets for CRPC for which there are currently no effective therapeutics.
  • polynucleotide or “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides and/or deoxyribonucleotides.
  • the backbone of the polynucleotide can include sugars and phosphate groups fas may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • the backbone of the polynucleotide can include a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P- ⁇ ?) or a mixed phosphoramidate- phosphodiester oligomer.
  • a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DN A polymerase with an appropriate primer.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tR A, rRNA, ribozymes, cDNA, recombinant
  • polynucleotides branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fiuororihose and thioate, and nucleotide branches, The sequence of nucleotides may be interaipted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • caps substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
  • promoter refers to the DNA region, usually upstream to the coding sequence of a gene or operon, which binds RN A polymerase and directs the enzyme to the correct transcriptional start site.
  • ti ssue specifi city is particularly relevant in the trea tment of the abnormal counterpart of a normal tissue.
  • Such counterparts include, but are not limited to, cancerous prostate tissue and normal prostate tissue.
  • Tissue specificity also includes the presence of an abnormal tissue type interspersed with normal tissue of a different tissue type, as for example in the case of metastases of PCa, and the like, into tissue such as liver. In this case, the target tissue is the abnormal tissue.
  • tissue specificity in the context of treatment, is particularly relevant in vivo. However, as described herein, ex vivo treatment and tissue replacement also falls within the concept of tissue specificity according to the present invention.
  • AR refers to a protein whose function is to specifically bind to an drogen and, as a consequence of the specific binding, recognize and bind to an androgen response element (ARE), following which the AR is capable of regulating transcriptional activity.
  • the AR is a nuclear receptor that, when activated, binds to cellular androgen-responsive element(s). in normal cells the AR is activated by androgen, but in non- normal cells (including malignant ceils) the AR may be activated by non-androgenic agents, including hormones other than androgens.
  • Mutants include androgen receptors with amino acid additions, insertions, truncations and deletions, as long as the function is sufficiently preserved.
  • a functional androgen receptor is one that binds both androgen and, upon androgen binding, an ARE.
  • a “therapeutically effective amount” refers to an amount at least partially effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result to thereby influence the therapeutic course of a particular disease state.
  • a therapeutically effective amount of an active agent may va ' according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual . Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • terapéuticaally effective amount is an amount sufficient to at least paxtially affect beneficial or desired results, including clinical results.
  • An effective amount can be administered in one or more admmistrations.
  • an effecti ve amoun t of an adenoviral vector is an amount that is sufficient to at least partially palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
  • the active agent according to the methods of the invention is formulated in the composition in a prophylactically effective amount.
  • a prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • treatment includes the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to a ceil or tissue from a subject, who has a diseases or disorder (e.g., cancer), has a symptom of a disease or disorder, or is at risk of (or susceptible to) a disease or disorder, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or disorder, the symptom of the disease or disorder, or the risk of (or susceptibility to) the disease or disorder.
  • a diseases or disorder e.g., cancer
  • suppressing tumor growth refers to at least partially reducing the rate of growth of a tumor, halting tumor growth completely, causing a regression in the size of an existing tumor, eradicating an existing tumor and/or preventing the occurrence of additional tumors upon treatment with the compositions, kits or methods of the present invention.
  • Tumor ceil growth indicates a growth state that is curtailed when compared to growth by ceils treated only with a DNA-damaging agent (e.g., radiation or chemotherapy), without treatment with the siRNA of the invention.
  • Tumor ceil growth can be assessed by any means known in the art, including, but not limited to, directly measuring tumor size, radiographic imaging, utilizing serum biomarkers of disease burden (e.g., serum PS A), determining whether tumor cells are proliferating using a H-thymidine incorporation assay or clonogenic assay, or counting tumor cells.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the type of carrier can be selected based upon the intended route of administration.
  • the carrier is suitable for intravenous, mtrapierioneal, subcutaneous, intramuscular, topical, transdermal or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the acti ve compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • DNA includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, mtemucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5- 10 "C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH. The T ra is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplar ⁇ ' stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1 % SDS, incubating at 42 °C, or, 5x SSC, 1% SDS, incubating at 65 °C, with wash in 0.2x SSC, and 0.1% SDS at 65 °C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical.
  • nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • moderately stringent hybridization conditions include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 U C, and a wash in IX SSC at 45 °C. A positive hybridization is at least twice background.
  • alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.
  • a temperature of about 36 °C is typical for low r stringency amplification, although annealing temperatures may vary betw een about 32 °C and 48 °C depending on primer length.
  • a temperature of about 62 °C is typical, although high stringency annealing temperatures can range from about 50 °C to about 65 °C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90 °C - 95 °C for 30 seconds - 2 minutes., a annealing phase lasting 30 seconds - 2 minutes, and an extension phase of about 72 °C for 1 - 2 minutes. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • the biological sample to be analyzed such as blood or serum
  • the biological sample to be analyzed may be treated, if desired, to extract the nucleic acids contained therein.
  • the resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation.
  • the probes are usually labeled with a detectable label. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes, and chemiluminescent probes.
  • Biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured ceils, e.g., primary' cultures, explants, and transformed cells; stool, urine, ejaculate, or other biological fluids,
  • a biological sample also includes a surgical sample taken from a patient during a surgery, for example, from a tumor or tumor margin.
  • RNA interference RNA interference
  • Caenorhabditis elegaris and plants were first noted in Caenorhabditis elegaris and plants as a novel mechanism of post-transcriptional gene silencing and has since been discovered in many eukaryotes. Rapid progress has been made in the use of RNAi and more specifically siRNAs as a means of attenuating the expression of specific proteins both in vitro and in vivo enabling any protein target, where the cDNA sequence is known, to be inhibited by these sequence- specific, double-stranded RNA molecules.
  • RNA interference (RNAi) is an evoiutionarily conserved sequence-specific post-transcriptional gene silencing mechanism and is thought to function in part as an innate intracellular antiviral immune response.
  • PCa is commonly evaluated according to a scale divided into four lettered stages: A, B, C and D.
  • Tumors in stage A are microscopic; stage Al designates tumors confined to a relatively small area and composed of well-differentiated tissue, while stage A2 tumors are more diffuse and less well differentiated.
  • Stage B tumors are large enough to be felt during a rectal examination, while stage C prostate cancers have spread throughout the gland and typically have pushed past the borders of the prostate into surrounding structures.
  • Stage D tumors have metastasized, e.g., to lymph nodes, bone, or other organs.
  • tumors can be staged by the TNM staging system, in which tumors are ranked on a scale of progressively worsening disease from Tla to T4b (e.g., Tic tumors are non-palpable and non-visible that were detected by elevated blood levels of prostate specific antigen),
  • TNM staging system in which tumors are ranked on a scale of progressively worsening disease from Tla to T4b (e.g., Tic tumors are non-palpable and non-visible that were detected by elevated blood levels of prostate specific antigen).
  • TNM staging system in which tumors are ranked on a scale of progressively worsening disease from Tla to T4b (e.g., Tic tumors are non-palpable and non-visible that were detected by elevated blood levels of prostate specific antigen).
  • Tic tumors are non-palpable and non-visible that were detected by elevated blood levels of prostate specific
  • radical prostatectomy preferably is used with stage A, B and some stage C tumors (i.e., where the tumor growth has not extended considerably beyond the borders of the prostate gland) as well as stage Ti c tumors.
  • Radiation therapy e.g., external or interstitial
  • stage A, B or C tumors as well as Tic tumors.
  • the size of the prostate can be determined by methods known in the art, for example, rectal examination, transrectal ultrasonography or magnetic resonance imaging (MRI).
  • the size or extent of the prostate tumor (and metastatic tumors, if any) can be assessed by known methods including a prostate-specific antigen blood test, bone scanning, X-rays, skeletal survey, intravenous pyelography, CAT-scan, MRI, physical examination, biopsy, and the like.
  • the tumor can also be staged during surgery (e.g., the prostate gland can be examined during surgery and/or a biopsy can be taken and examined).
  • clinical staging and/or surgical staging may be used to evaluate the extent of disease.
  • PSA prostate-specific antigen
  • the PSA blood test is a reasonably specific, sensitive, rapid, and inexpensive tool for screening for PCa.
  • a blood PSA level above 4 ng/ ' ml is considered to be suggestive of the presence of PCa, with levels above 10 ng/ml being particularly indicative of cancer.
  • a pretreatment level of PSA can be established and the efficacy of the treatment assessed by monitoring periodically the PSA level in the subject's blood, wherein decreased PSA levels are used as an indicator of the efficacy of the treatment.
  • the PSA nadir (i.e., the point at which PSA levels do not decrease further even upon further treatment with a peptide compound) can be used as the indicator point for initiation of a second therapy, for example for performance of a procedure that removes or destroys prostatic tumor tissue (including radical prostatectomy, cryosurgery and/or radiation therapy). It is expected that the PSA nadir will be reached sooner using a peptide compound, as compared to treatments which do not include using a peptide compound.
  • sex hormones can be monitored to assess the efficacy of the drug therapy.
  • Concentrations of hormones such as testosterone, dihydrotestosterone, dehydroepiandrosterone (DHEA), DHEA-sulfate, androst-5- ene-3 , 17-diol, and the estrogen 17P-estradiol can all be measured by methods known the skilled artisan.
  • DHEA dehydroepiandrosterone
  • DHEA-sulfate DHEA-sulfate
  • rost-5- ene-3 17-diol
  • 17-diol 17-diol
  • estrogen 17P-estradiol the estrogen 17P-estradiol
  • decreased levels of testosterone and dihydrotestosterone can be used as indicators of treatment efficacy.
  • the methods of the invention can be administered in conjunction with other known treatments for cancer, including, but not limited to, mechanical removal of cancerous cells (e.g., surgical removal of a tumor), and administration of chemotherapeutic agents.
  • chemotherapeutic agents used to treat cancer which act to kill cancer cells and/or slow their growth through other mechanisms.
  • the administrations of such additional treatments and/or agents are intended to be included in the m ethods of the present invention.
  • chemotherapeutic agents include, but are not limited to, antimetabolites such as folate analogs (e.g., methotrexate), purine analogs (e.g., fludarabine, mercaptopurine, and thioguanine (e.g., 6-TG)), adenosine analogs (e.g., cladribine, and pentostatin), pyrimidine analogs (e.g., capecitabine, cytarabine, depocyt, floxuridine, fluorouracil (e.g., 5-FU); and gemcitabine), and substituted ureas (e.g., hydroxyurea); natural products such as antitumor antibiotics (e.g., bleomycin, dactinomycin, actinomycin D, daunorubicin, daunomycin, DaunoXome (liposomal daunorubicin),
  • antimetabolites such as folate analogs (e.g., methotre
  • the antibodies used in the methods of the invention can be prepared via techniques well known to those having ordinary skill in the art (see, e.g., Harlow and Lane (eds.) Antibodies, A Laborator Manual, Cold Spring Harbor Laboratories, 1988).
  • monoclonal antibodies produced against immortal tumor cell lines according to the present invention are useful in the detection and therapy of various cancers, such as PCa.
  • the antibody or antigen binding portion thereof binds to malignant cells.
  • the antibody or antigen binding portion thereof is irnmunoreactive with at least one tumor rejection antigen or with at least one tumor-associated antigen and epitopes thereof.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules or those portions of an immunoglobulin molecule that contain the antigen binding site, including Fab, F(ab) 2 , and F(v).
  • Polyclonal or monoclonal antibodies may be produced by methods known in the art.
  • the antibodies or active fragments thereof may also be produced by genetic engineering including chimeric antibody, single chain antibody.
  • the antibody or an active fragment thereof may be used as an immunotherapeutic.
  • the antibody or an active fragment thereof may be administered alone, or in combination with cbemotherapeutics or immunosuppressive agents as are known in the art.
  • the monoclonal antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and noncompetitive immunoassays in either a direct or indirect format.
  • Examples of such immunoassays are the radioimmunoassay (RJ A) and the sandwich (immunometric) assay.
  • Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immiraohistochemical assays on physiological samples.
  • antibody of the invention can be used to detect CTAs present in electrophoretically dispersed gel protocols such as Western blots and 2-dimensional gels. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • the monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of CTA.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacryiamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation,
  • the incubation medium usually added with the labeled soluble antibody.
  • the "blockers” are added to assure that non-specific proteins, proteases, or anti-heterophilic immunoglobulins to anti-CTA immunoglobulins present in the experimental sample do not cross-ink or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results.
  • the selection of "blockers” therefore may add substantially to the specificity of the assays described in the present invention.
  • nonreievant i.e., nonspecific antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgGl, lgG2a, IgM, etc.) can be used as "blockers".
  • concentration of the "blockers” is important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in the specimen.
  • epitopic determinants includes any determinant capable of specific interaction with the monoclonal antibodies of the invention.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the detectablv labeled monoclonal antibody in using the monoclonal antibodies of the invention for the in vivo, in vitro, or ex vivo detection of antigen, is given in a dose which is diagnostical!y effective.
  • diagnostical effective means that the amount of detectablv labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the CTA antigen for which the monoclonal antibody is specific.
  • the concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having CTA is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of detectably labeled monoclonal antibody for the in vivo, in vitro, or ex vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual.
  • the dosage of monoclonal antibody can vary from about 0.001 mg/m 2 to about 500 mg/m 2 , preferably 0.1 mg/rrf to about 200 mg/rrf , most preferably about 0.1 mg/m 2 to about 10 mg/m z .
  • Such dosages may vary, for example, depending on whether multiple injections are given, tumor burden, and other factors known to those of skill in the art.
  • the type of detection instrument available is a major factor in selecting a given radioisotope.
  • the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
  • Still another important factor in selecting a radioisotope for in vi vo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized.
  • a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140- 250 keV range, which may be readily detected by conventional gamma cameras,
  • radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group.
  • Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to
  • immunoglobulins are the bifunctiona! chelating agents such as diethylenetriaminepentacetic acid (DTP A) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
  • DTP A diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are 1H In, 97 Ru, 67 Ga, 68 Ga, 72 As, 89 Zr, and 20l TI.
  • the monoclonal antibodies of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MR]) or electron spin resonance (ESR).
  • MR magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic imaging can be utilized.
  • gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
  • Elements which are particularly useful in such techniques include 1: “Gd, "Mn, 1 i D , 52 Cr, and "6 Fe.
  • the monoclonal antibodies of the invention can be used to monitor the course of amelioration of CTA associated cell-proliferative disorder.
  • CTA associated cell-proliferative disorder For example, by measuring the increase or decrease in the number of calls expressing a CTA or changes in CTA present in various body fluids, such as ejaculate or urine, it would be possible to determine whether a particular therapeutic regiment aimed at ameliorating the disorder is effective.
  • the monoclonal antibodies of the invention can also be used, alone or in
  • the m onoclonal antibodies of the invention may be unlabeled or labeled with a therapeutic agent. These agents can be coupled either directly or indirectly to the monoclonal antibodies of the invention.
  • One example of indirect coupling is by use of a spacer moiety.
  • spacer moieties can be either insoluble or soluble (Diener, et ah, Science, 231:148, 1986) and can be selected to enable drug release from the monoclonal antibody molecule at the target site.
  • therapeutic agents which can be coupled to the monoclonal antibodies of the invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • non-proteinaceous drugs encompasses compounds which are classically referred to as drugs, for example, mitomycin C, daunorubicin, and vinblastine.
  • the proteinaceous drugs with which the monoclonal antibodies of the invention can be labeled include immunomodulators and other biological response modifiers.
  • biological response modifiers encompasses substances which are involved in modifying the immune response in such manner as to enhance the destruction of a CTA-associated tumor for which the monoclonal antibodies of the invention are specific.
  • immune response modifiers include such compounds as lymphokines. Lymphokines include tumor necrosis factor, the interleukins, lymphoioxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and interferon.
  • Interferons with which the monoclonal antibodies of the invention can be labeled include alpha- interferon, beta- interferon and gamma-interferon and their subtypes.
  • radioisotopically conjugated monoclonal antibodies of the invention for immunotherapy certain isotypes may be more preferable than others depending on such factors as tumor cell distribution as wel l as isotope stability and emission. If desired, the tumor cell distribution can be evaluated by the in vivo diagnostic techniques described above. Depending on the cell proliferative disease some emitters may be preferable to others. In general, alpha and beta particle-emitting radioisotopes are preferred in immunotherapy. [0090] For example, if an animal has solid tumor foci a high energy beta emitter capable of penetrating several millimeters of tissue, such as 90 Y, may be preferable.
  • the cell proliferative disorder consists of simple target ceils, as in the case of leukemia, a short range, high energy alpha emitter, such as 21 "Bi, may be preferable.
  • a short range, high energy alpha emitter such as 21 "Bi
  • radioisotopes which can be bound to the monoclonal antibodies of the invention for therapeutic purposes are l2 % 90 Y, 67 Cu, 2i2 Bi, 2f f At, 2i2 Pb, 47 Sc, f 09 Pd, 65 Zn, and 588 Re.
  • Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. However, ricin is a toxic lectin which has been used imm notherapeutically. This is preferably accomplished by binding the alpha-peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site specific deliver ⁇ ' of the toxic effect.
  • Toxins are poisonous substances produced by plants, animals, or microorganisms, that, in sufficient dose, are often lethal. Diphtheria toxin is a substance produced by
  • Coi nebacterium diphtheria which can be used therapeutically.
  • This toxin consists of an alpha and beta subunit which under proper conditions can be separated.
  • the toxic A component can be bound to an antibody and used for site specific delivery to a CTA expressing cell.
  • Other therapeutic agents which can be coupled to the monoclonal antibodies of the invention are known, or can be easily ascertained, by those of ordinary skill in the art.
  • the labeled or unlabeled monoclonal antibodies of the invention can also be used in combination with therapeutic agents such as those described above. Especially preferred are therapeutic combinations comprising the monoclonal antibody of the invention and
  • the monoclonal antibodies of the invention can be used in combination with alpha-interferon.
  • This treatment modality enhances monoclonal antibody targeting of carcinomas by increasing the expression of monoclonal antibody reactive antigen by the carcinoma cells (Greiner, et ah, Science, 235:895, 1987).
  • the monoclonal antibody of the invention could be used, for example, in combination with gamma-interferon to thereby activate and increase the expression of Fc receptors by effector cells which, in turn, results in an enhanced binding-of the monoclonal antibody to the effector cell and killing of target tumor cells.
  • gamma-interferon to thereby activate and increase the expression of Fc receptors by effector cells which, in turn, results in an enhanced binding-of the monoclonal antibody to the effector cell and killing of target tumor cells.
  • the administration of the monoclonal antibody and the therapeutic agent usually occurs substantially contemporaneously .
  • substantially contemporaneously means that the monoclonal antibody and the therapeutic agent are administered reasonably close together with respect to time.
  • the therapeutic agent can be administered 1 to 6 days before the monoclonal antibody.
  • the administration of the therapeutic agent can be daily, or at any other interval, depending upon such factors, for example, as the nature of the tumor, the condition of the patient and half-l ife of the agent.
  • monoclonal antibodies of the invention it is possible to design therapies combining ail of the characteristics described herein. For example, in a given situation it may be desirable to administer a therapeutic agent, or agents, prior to the administration of the monoclonal antibodies of the invention in combination with effector cells and the same, or different, therapeutic agent or agents. For example, it may be desirable to treat patients with prostate, kidney or bladder carcinoma by first administering ganima-interferon and
  • interleukin-2 daily for 3 to 5 days, and on day 5 administer the monoclonal an tibody of the invention in combination with effector cells as well as ganima-interferon, and interleukin-2.
  • liposomes with the monoclonal antibodies of the invention in their membrane to specifically deliver the liposome to the tumor expressing CTA.
  • These liposomes can be produced such that they contain, in addition to the monoclonal antibody, such immunotherapeutic agents as those described above which would then be released at the tumor site (Wolff, et ai, Biochemical et Biophysical Acta, 802:259, 1984).
  • the dosage ranges for the administration of monoclonal antibodies of the invention are those large enough to produce the desired effect in which the symptoms of the malignant disease are ameliorated.
  • the dosage should not be so large as to cause ad verse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any complication. Dosage ca vary from about 0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one or more dose administrations daily, for one or several days.
  • lower dosages comparable to those used for in vivo diagnostic imaging, can be used.
  • the monoclona l antibodies of the invention can be administered parenterally by injection or by gradual perfusion over time.
  • the monoclonal antibodies of the invention can be administered intravenously, intraperitoneallv, intramuscularly, subcutaneously, intracavity, or transdermal! ' -, alone or in combination with effector cells.
  • the antibody or an active fragment thereof may also be used as an immunotoxin to specifically target and kill malignant primary and metastatic cells.
  • Immunotoxins are characterized by two components and are particularly useful for killing selected cells in vitro or in vivo.
  • One component is a cytotoxic agent which is usually fatal to a cell when attached or absorbed.
  • the second component known as the deliver ⁇ - vehicle, provides a means for delivering the toxic agent to a particular cell type, such a malignant prostate cells.
  • the two components are commonly bonded together by any of a variety of well-known chemical procedures.
  • the linkage to the antibody may be by way of hetero Afunctional crosslinkers, e.g., SPDP, carbodiimide, glutaraldehyde, and the like. Production of various immunotoxins is well-known in the art.
  • Cytotoxic agents include, but are not limited to, radionuclides, such as Iodine 1 ' 1 or other isotopes of iodine. Yttrium 90 , Tlienium 185 , and Bismuth or other alpha emitters; a number of chemotherapeutic drugs, such as vindesine, methotrexate, andriamycin, taxol, and ciplatinum; and cytotoxic proteins such as ribosomal inhibiting proteins like Pseudomonas exotoxin A, riciii, diphtheria toxin, ricin A chain and the like.
  • radionuclides such as Iodine 1 ' 1 or other isotopes of iodine.
  • chemotherapeutic drugs such as vindesine, methotrexate, andriamycin, taxol, and ciplatinum
  • Normal prostrate epithelial (PrEC) cells can be purchased from Lonza Corp. (Waikersvclude, Maryland) and cultured as per the supplier's instructions.
  • the androgen-dependent LNCaP and independent PC3 PCa cell lines are from ATCC and maintained under standard culture conditions. Ceils are treated with 3 ⁇ /L of 5AZA ( Sigma- Aldrich Co., St. Louis, MO) for 72 hours. Fresh medium with drug is changed every 24 hours. R1881 in ethanol is added at a concentration of 10 iiM final directly to CS-FBS- containing medium.
  • RNA extraction and RNA quality assessment Total RNA is isolated with TriZol reagent (Invitrogen, Carlsbad, CA) followed by clean-up using RNeasy mini kit (Qiagen, Valencia, CA). Isolated RNA is quantified with NanoDrop ND-1000 followed by quality assessment with 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) according to manufacturer's protocol.
  • Sample amplification and labeling Sample amplification and labeling procedures are carried out using Low RNA Input Fluorescent Linear Amplification Kit (Agilent
  • RNA spike-in controls are added to RNA samples before amplification and labeling according to manufacturer's protocol.
  • CT Array V2.0 Design & fabrication of CT Array V2.0: The CT Array V2.0 focused microarray contains probes representing 90 CTA. genes [Scanian MJ et ah, (2004)]. Sixty-mer oligonucleotide probes corresponding to each CTA from Agilent Whole Human Genome 44 K expression array and eArray 5.0 probe databases are printed with multiple replicates (20-30), together with standard controls and housekeeping genes used on Agilent catalog expression arrays, [0107] Target fragmentation and microarray hybridization: Fragmentation is carried out by incubating at 60 "C for 30 minutes and stopped by adding equal volume of 2x hybridization buffer (Agilent Technologies).
  • Fragmented targets are added to the CTArray V2.0, assembled into a hybridization chamber (Agilent Technologies) and hybridized at 65 °C for 17 hours in a hybridization oven with rotation. Microarrays are washed and dried according to the Agilent microarray processing protocol.
  • Microarray scanning and data acquisition Microarrays are scanned with Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies for expression microarrays with 100% PMT and 5 mm resolutions. Data are extracted using Feature Extraction Software v8.1 (Agilent Technologies) under default settings for expression microarrays with linear & Lowess normalization. Microarrays with > 5% feature outlier numbers are excluded.
  • Quantitative real-time PGR One microgram RNA is transcribed into first strand cDNA using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's protocol in a total volume of 20 ⁇ .1. Quantitative real-time PGR are carried out with the i-Cycler IQ Real-Time Detection System (Bio-Rad Laboratories, Inc., Hercules, CA). The PGR reaction is performed with 0.2 ⁇ of cDNA template in 25 Lli of reaction mixture containing 12.5 ⁇ of iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) and 0.25 mmoi/L each primer.
  • TATA -binding protein TBP
  • DNA isolation and bisulfite modification Total DNA is extracted from cells using DNeasy Tissue Kit (Qiagen), Sodium bisulfite modification of 1 ,0 or 1.5 g genomic DNA is done using the EZ DNA methylation-Goid Kit (Zynio Research Corp.) or Epitect Bisulfite Kit (Qiagen, Gaithersburg, MD) following the manufacturer's protocol. Bisulfite converted DNA. is eluted with 20 ⁇ L ⁇ of elution buffer. Ten LlL is used as template in PGR reaction using Platinum Taq polymerase and gene-specific primers.
  • PGR conditions are denaturation at 95 °C for 1 minute followed by 35 cycles at 94 °C for 15 seconds, 60 °C for 30 seconds and 72 °C for 45 seconds.
  • PGR products are purified on a 1% agarose gel and cloned into the pCR8 TA vector from Invitrogen (Carlsbad, CA). Plasmid DNA prepared from 10 representative clones is sequenced.
  • MSP Methyl aiion-specifie PGR
  • the normalized index of methylation ( ⁇ ) is defined as the ratio of the amount of methylated templates at the alleles of MAGE A2 to the amount of methylated + unmethylated templates,
  • the NIM serves as an index of the percentage of input copies of DNA that are fully methylated at the primer-binding sites.
  • concentrations are determined by using a protein assay reagent (Pierce, Rockford, IL). Thirty micrograms of protein is loaded and separated by 4-20% SDS-PAGE along with a distinct lane containing 10 ml of Kaleidoscope Prestained Markers (Pierce). The proteins are then transferred to 0,22 mM nitrocellulose membrane (Bio-Rad, Richmond, CA) utilizing a wet transfer apparatus (Bio-Rad). The membrane is incubated at room temperature for 1 hour in 5% non-fat dry milk in phosphate buffered saline (PBS) with 0.1% Tween-20. The membrane is then incuba ted with 1 :500 dilution of primary antibody in PBS with 5% non-fat dry milk and ⁇ ).
  • PBS phosphate buffered saline
  • the present invention provides that CTA expression in prostate cancer (PCa) can be determined using a focused microarray.
  • PCa prostate cancer
  • >20% of the CTAs are up-regulated (>2 to >800 fold) in androgen-responsive LNCaP but not androgen- independent PC-3 cells suggesting the possible in vol vement of androgens in regulating their expression (Table 1).
  • the lack of CTA expression in PC-3 indicates possible repression due to DNA. methylation. Indeed, treating PC-3 cells with the DNA methyltransferase inhibitor 5'- aza-2'-deoxy-azacytidine (5AZA) results in the upregulation of several CTAs.
  • 5AZA DNA methyltransferase inhibitor
  • MAGE-A subfamily consists of 12 members organized into four subclusters (I-IV). While genes from subcluster I remain virtually silent, those from subclusters II-IV are upregulated with MAGEA2 being upregulated (Table 1). Since MAGEA2 is the highest upregulated (800- fold), the expression of MAGEA.2 is also validated by Q- PCR ( Figure 3).
  • Transcription factor binding sites are identified using TRANSFAC 6.0 and stringent parameters allowing 0 mismatch and a score of 100%.
  • SP1 CCCCTCCC SEQ ID NO: 1
  • CCCCACCCCCC (SEQ ID NO: 2) 147 (+) GAGGCGGGCC (SEQ ID NO: 3) 362 (+) GAGGCGGGC (SEQ ID NO: 4)
  • GAMYB CAACGGCC (SEQ ID NO: 7) 287 (+)
  • ADR1 CGGCGGAG (SEQ ID NO: 8) 301 (+)
  • ADR1 GGTCGGGG (SEQ ID NO: 12) 67 (-)
  • promoter analysis is performed using Patch 1.0 (Biobase). Transcription factor binding sites were identified using TRANSFAC 6.0 and stringent parameters allowing 1 mismatch and a score of 90%. Table 4. Summary ' of transcription factor binding sites in the 453 bp region harboring the CpG island in the MAGEA2 promoter.
  • CTGCTTCCTTCC (SEQ ID NO: 29) 305 (-)
  • the present invention provides, as a result of the inadvertent depletion of a constituent from serum, a novel epigenetic phenomenon underlying the reactivation of MAGE A2 in somatic cells.
  • the in vitro experiments described here offer a tractable system to decipher the mechanisms that integrate environmental cues effecting DNA demethylation and gene expression and as such, may serve as a novel paradigm to gain new insight into the reactivation and functions of the CTA genes in cancer.
  • PCa is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths among men. Further, a clinical dilemma in the management of PCa is to distinguish men with aggressive disease who need treatment from men who have indolent disease not requiring immediate intervention.
  • the present invention provides that, in addition to MAGEA2, several members of the MAGE -A subfamily are remarkably specific to HRPC.
  • PAGE4 another member of the CT-X antigens, is highly up-regulated in organ-confined PCa but not HRPC (Table 5).
  • the differential expression of CTAs in PCa can be used as novel biomarkers/therapeutic targets offering new hope for patients with HRPC.
  • Table 5 shows differential expression of CT-X antigens in benign prostate, organ- confined (primary) and hormone-refractory metastatic PCa.
  • Microarray data are obtained from GEO at world wide web ncbi.nlm.nih.gov/geo. Number of samples used in each group is indicated in parenthesis.
  • MAGEA2 expression in PCa is androgeti-independent: Since the data using a focused microarray demonstrate that the CT-X antigens are expressed only in androgen-responsive LNCaP cells, the involvement of androgens in regulating their expression appeared imminent. To determine the potential involvement of androgens, LNCaP cells are cultured in charcoal- stripped serum-containing medium from 4 days and treated them with the synthetic androgen, R 1881. However, no significant stimulation of MAGEA2 expression is observed while that of prostate-specific antigen (PSA), a bona fide androgen-responsive gene, is stimulated -5-fold (Figure 9). There data suggest that androgens may not be involved in MAGEA2 regulation. Table 7. Summary of hormone-refractory PCa samples obtained by rapid autopsy and propagated as xenografts in SOD mice.
  • MAGEA2 expression is determined in additional androgen- responsive and androgen-independent PCa cell lines ( Figure 7). MAGEA2 is upregulated in the androgen-responsive LNCaP and CWR22 cells but not in the androgen-independent PC-3 and DU-145 cells ( Figure 7B). Next the extent of DNA methylation is determined in the MAGEA2 promoter by methylation-specific PCR (MSP) and it is highly methylated (80- 1 00%) in PC-3 and DU-145 ( Figure 7 A).
  • MSP methylation-specific PCR
  • the same region is almost completely (>99%) unmethylated in LNCaP and CWR22 ceils in which MAGEA2 is upregulated.
  • the DN A methyltransferase inhibitor 5 'aza-2'-deoxy-azacytidine (5AZA) is used, Indeed, while the 5AZA treatment results in the upregulation of MAGEA2 expression in PC-3 and DU-145 cells in which the promoter is bypermethylated, it had no significant effect in LNCaP or CWR22 cells in which the promoter is hypomethylated (Figure 7C),
  • the present invention provides that there is a similar correlation in clinical samples.
  • the cytosine residues in the CpG island of the MAGEA2 promoter are highly methyiated(>90%) in primary PCa specimens that do not express the CT-X antigens.
  • CRPC specimens in which the antigens are aberrantly expressed the cytosine residues are severely (>90%) hypomethylated ( Figure 10).
  • MAGEA2 ON-TARGET plus SMART pool siRNA is obtained from Dharmacon.
  • siRNA is transfected with 1 x 10 + LNCaP cells at 100 nM total oligo concentration, using 0.2 ml DharmaFECT-3 transfection reagent in a 96-well plate.
  • Cell proliferation assay Cell proliferation is measured using WST-1 (4-[3 ⁇ 4 ⁇ iodophenyl ⁇ -2- ⁇ 4-nitrophenyl ⁇ -2H-5-tetrazolio]-l ,3-benzene disulfonate) (Roche) following manufacturer's protocol. Two thousand cells per well are seeded in 96-well plates, after 4 hours incubation with WST-1 reagent, WST-1 absorbance at 450 nm is measured.
  • WST-1 4-[3 ⁇ 4 ⁇ iodophenyl ⁇ -2- ⁇ 4-nitrophenyl ⁇ -2H-5-tetrazolio]-l ,3-benzene disulfonate
  • DNA fragmentation is quantified as the percentage of cells with hypodipioid DNA (s ' ub-Gl ). Cells are fixed with 70% ethanol. The nucleic are stained with Guava Cell Cycle Reagent (Guava Technologies, Hayward, CA). DNA content is measured using a Guava system (Guava Technologies). For ever ⁇ ' assay, 5 10 " ' cells are counted.
  • MAGEA2 is silenced in LNCaP cells using siRNA. Knocking down MAGEA2 expression with the specific siRNA results in a 90°/» decrease in the MAGE A2 mRNA when compared to the control siRNA. Furthermore, knocking down MAGEA2 with the specific siRNA significantly impairs cell proliferation compared to the scrambled (control) siRNA, However, given the high degree of sequence similarity among the members of this large subfamily, it is possible that the si RNA. could have "off-target" effects contributing to the observed phenotype. To rule out this possibility, the levels of several members of the MAGEA subfamily, which are also highly upregulated together with MAGEA2, are also determined. The data show that, aside from MAGEA2, the expression of the other MAGEA members are not affected to any significant extent suggesting that the knock down is specific to MAGEA2.
  • MAGEA2 upregulation is associated with chemoresistance in PCa cells: Several studies have demonstrated that aberrant expression of CTAs is associated with
  • MAGEA tumor antigens are shown to confer drug resistance by targeting transactivation of p53 in a variety of cancer cells.
  • the present invention provides that upregulation of MAGEA2 in PCa is also associated with chemoresistance.
  • MAGEA2 expression is knocked down in LNCaP ceils and subsequently treated with docetaxel approved for late-stage PCa.
  • reducing the expression of MAGE A2 with the specific siRNA results in a marked reduction of cell survival by -30% compared to cells treated with the scrambled siRN A especially in the lower concentrations ( ⁇ 1 nM) of the drug used.
  • the unexpected enhancement of MAGEA2 knock down on chemosensitivity is also confirmed by detecting apoptosis by flow cytometry.
  • the present invention provides thai the CT-X antigen identified represent novel biomarkers and therapeutic targets offering new hope for patients with CRPC.
  • Clinical samples and RNA extraction Clinical samples are from 20 normal prostate, 89 clinically localized PCa (29 without recurrence, 43 with recurrence and 17 unknown) and 21 metastatic PCa. All lesions are grossly dissected, rapidly frozen in liquid nitrogen, and stored at -80 °C until RNA extraction. RNA extraction is performed by the Trizol method with DNase treatment.
  • Quantitative singleplex and multiplex real-time PCR First strand cDNA. is made from 0.5 ⁇ RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, inc., Hercules, CA) following the manufacturer's protocol in a total volume of 20 ⁇ , All primers and probes (Tables 8-1 and 8-2) are obtained from Integrated DNA Technologies. Quantitative singleplex and multiplex real-time PCR are carried out with the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.).
  • the singleplex PCR reaction is performed with 0.2 ⁇ of cDNA template in 25 ⁇ of reaction mixture containing 12.5 ⁇ of iQ SYBR Green Supermix (Bio-Rad Laboratories, inc.) and 0.25 ⁇ / ⁇ each primer. PCR reaction is subjected to hot start at 95 °C for 3 minutes followed by 45 cycles of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 1 minute.
  • Quantitative multiplex real-time PCR reaction is carried out with 0.2 ⁇ of cDNA template in 25 ⁇ of reaction mixture containing 12.5 ⁇ of iQ Multiplex Powermix (Bio-Rad Laboratories, Inc.), 0.3 ⁇ /L each primer and 0.2 ⁇ /L probe.
  • the reaction is subjected to hot start at 95 °C for 3 minutes followed by 45 cycles of denaturation at 95 °C for 10 seconds, and annealing and extension at 60 "C for 60 seconds.
  • Amplification of ⁇ -actin is used as a control for RNA integrity for all samples. Almost identical results are obtained when using TATA binding protein as a reference gene instead of ⁇ -actin.
  • a reference RNA from pooled cell line and prostate tissue RNAs are included on each plate to provide standardization across PCR plates. Analysis and fold-change differences are determined using the comparative CT method. Fold change is calculated from the AACT values with the formula 2 ⁇ r ,
  • CTA expression is normalized by dividing each individual value by their respecti ve actin value. Distributions of normalized CTA s are compared among categories of prognostic factors using the Wi!coxon test (for binary factors) or Kruskal-Wallis test (for factors with more than 2 categories). Correlations between the normalized CTAs and other continuous variables are evaluated by linear regression and the Pearson correlation coefficient. Normalized CTAs are dichotomized at the median of the distribution among PCa patients who did not recur. Risk of recurrence associated with CTAs is evaluated in univariate and multi variable proportional hazards models.
  • GSTP1 expression in these samples is also examined, because GSTP1 expression presumably reflects the cancer volume in disease sample.
  • Figure 14 A the highest expression of GSTP1 is observed in normal prostate tissues and lowest in metastatic diseases (normai> clinically localized PCa >metastatic PCa) suggesting that the samples used in the present example are of high quality.
  • CTAs that are associated with disease recurrence after radical prostatectomy (RP)
  • the decision to include CTAs in further analysis is based on the differential expression between normal prostate and clinically localized PCa samples. Based on these criteria, CEP55, NUF2, PBK, TKK and PAGE4 are selected for developing a multiplex PCR assay that can stratify patients with and without PCa recurrence after RP.
  • the CTAs are an important group of proteins thai are typically restricted to the testis in the normal adult but are aberrantly expressed in several types of cancers. Because of their restricted expression pattern, the CTAs can serve as unique biomarkers for cancer diagnosis/prognosis.
  • the aim of this example is to identify promising CTAs that are associated with PCa recurrence following radical prostatectomy (RP).
  • the present invention provides the study of this example to identify a novel, CTA-based, biomarker to differentiate patients with recurrent and non-recurrent disease following RP and underscores its potential impact on PCa prognosis and treatment.
  • CTAs By mining publicly available microarray data in conjunction with data on CTA expression that the inventors obtain using a custom CT microarray, several CTAs are identified to be differentially expressed in normal prostate, primary PCa, and metastatic PCa. The present invention provides that those CTAs are significantly differentially expressed between patients with and without disease recurrence after RP. Thus, the present invention provides a selected set of CTAs representing no vel biomarkers as significant predictors of PCa recurrence.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention est basée sur la découverte déterminante de la corrélation existant entre les antigènes testiculaires/cancéreux (CTA) et la classification des maladies pour le cancer de la prostate (PCa). L'invention fournit présentement des procédés de classification de maladies pour un sujet atteint d'un PCa, basés sur le niveau d'expression du ou des CTA(s) choisi(s). L'invention concerne également des procédés de pronostic de PCa ou de récurrence de PCa chez un sujet sur la base du niveau d'expression du ou des CTA(s) choisi(s).
PCT/US2011/022464 2010-01-26 2011-01-25 Procédés de classification de maladies ou de pronostic du cancer de la prostate basés sur l'expression d'antigènes testiculaires/cancéreux WO2011094233A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US29839010P 2010-01-26 2010-01-26
US61/298,390 2010-01-26

Publications (1)

Publication Number Publication Date
WO2011094233A1 true WO2011094233A1 (fr) 2011-08-04

Family

ID=44319724

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/022464 WO2011094233A1 (fr) 2010-01-26 2011-01-25 Procédés de classification de maladies ou de pronostic du cancer de la prostate basés sur l'expression d'antigènes testiculaires/cancéreux

Country Status (1)

Country Link
WO (1) WO2011094233A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015035146A3 (fr) * 2013-09-05 2015-04-23 Memorial Sloan-Kettering Cancer Center Ddx43 utilisable en tant que biomarqueur de la résistance aux inhibiteurs de mek1/2
WO2015087088A3 (fr) * 2013-12-12 2015-09-17 Almac Diagnostics Limited Classification du cancer de la prostate
WO2017203526A1 (fr) * 2016-05-23 2017-11-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Méthodes de diagnostic du cancer à l'aide d'antigènes testiculaires du cancer
WO2018148180A3 (fr) * 2017-02-07 2018-09-27 Immune Design Corp. Matériaux et méthodes pour l'identification et le traitement du cancer

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050142573A1 (en) * 2002-09-10 2005-06-30 Sidney Kimmel Cancer Center, A California Non- Profit Corporation Gene segregation and biological sample classification methods
US20050255519A1 (en) * 1997-05-02 2005-11-17 Billing-Medel Patricia A Reagents and methods useful for detecting diseases of the prostate
US20090181021A1 (en) * 2006-01-30 2009-07-16 Ludwig Institute For Cancer Research Ltd. Ctsp Cancer-Testis Antigens
US20090215812A1 (en) * 2005-05-13 2009-08-27 Ariad Gene Therapeutics, Inc. Biomarkers for evaluating likelihood of tumor sensitivity to an mtor inhibitor
US20090298082A1 (en) * 2008-05-30 2009-12-03 Klee George G Biomarker panels for predicting prostate cancer outcomes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050255519A1 (en) * 1997-05-02 2005-11-17 Billing-Medel Patricia A Reagents and methods useful for detecting diseases of the prostate
US20050142573A1 (en) * 2002-09-10 2005-06-30 Sidney Kimmel Cancer Center, A California Non- Profit Corporation Gene segregation and biological sample classification methods
US20090215812A1 (en) * 2005-05-13 2009-08-27 Ariad Gene Therapeutics, Inc. Biomarkers for evaluating likelihood of tumor sensitivity to an mtor inhibitor
US20090181021A1 (en) * 2006-01-30 2009-07-16 Ludwig Institute For Cancer Research Ltd. Ctsp Cancer-Testis Antigens
US20090298082A1 (en) * 2008-05-30 2009-12-03 Klee George G Biomarker panels for predicting prostate cancer outcomes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KARPF ET AL.: "Increased Expression of Androgen Receptor Coregulator MAGE-11 in Prostate Cancer by DNA Hypomethylation and Cyclic AMP.", MOL CANCER RES., vol. 7, no. 4, 2009, pages 523 - 35 *
SAMPSON ET AL.: "Inhibition of castration-resistant prostate cancer by the cancer/testis antigen PAGE4, a novel corepressor of the androgen receptor.", FASEB J. MEETING, April 2009 (2009-04-01), Retrieved from the Internet <URL:http://www.fasebj.org/cgi/contenUmeetingabstract/23/1MeetingAbstracts/714.1> [retrieved on 20110307] *
SUYAMA ET AL.: "Expression of cancer/testis antigens in prostate cancer is associated with disease progression.", PROSTATE., vol. 70, no. 16, 1 December 2010 (2010-12-01), pages 1778 - 1787 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015035146A3 (fr) * 2013-09-05 2015-04-23 Memorial Sloan-Kettering Cancer Center Ddx43 utilisable en tant que biomarqueur de la résistance aux inhibiteurs de mek1/2
US10400285B2 (en) 2013-09-05 2019-09-03 Memorial Sloan-Kettering Cancer Center DDX43 as a biomarker of resistance to MEK1/2 inhibitors
WO2015087088A3 (fr) * 2013-12-12 2015-09-17 Almac Diagnostics Limited Classification du cancer de la prostate
US10196697B2 (en) 2013-12-12 2019-02-05 Almac Diagnostics Limited Prostate cancer classification
WO2017203526A1 (fr) * 2016-05-23 2017-11-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Méthodes de diagnostic du cancer à l'aide d'antigènes testiculaires du cancer
WO2018148180A3 (fr) * 2017-02-07 2018-09-27 Immune Design Corp. Matériaux et méthodes pour l'identification et le traitement du cancer

Similar Documents

Publication Publication Date Title
Iwamoto et al. BRCA1 promoter methylation in peripheral blood cells is associated with increased risk of breast cancer with BRCA1 promoter methylation
ES2790823T3 (es) Uso de ARN sin células circulante para el diagnóstico y/o la monitorización de cáncer
Zeng et al. Down-regulation of miRNA-30a in human plasma is a novel marker for breast cancer
Kong et al. Detection of differentially expressed microRNAs in serum of pancreatic ductal adenocarcinoma patients: miR-196a could be a potential marker for poor prognosis
USRE44596E1 (en) Method for the detection of cancer
Kim et al. Molecular changes of epidermal growth factor receptor (EGFR) and KRAS and their impact on the clinical outcomes in surgically resected adenocarcinoma of the lung
Kimura et al. EGFR mutation of tumor and serum in gefitinib-treated patients with chemotherapy-naive non–small cell lung cancer
Liu et al. Diagnostic role of Wnt pathway gene promoter methylation in non small cell lung cancer
Wang et al. Alterations of APC, c-met, and p53 genes in tumor tissue and serum of patients with gastric cancers
Pavlidou et al. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR
Lan et al. MiR-133a is downregulated in non-small cell lung cancer: a study of clinical significance
Molinari et al. Biomarkers and molecular imaging as predictors of response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer
Ines et al. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort
He et al. Extracellular vesicle-associated miRNAs as a biomarker for lung cancer in liquid biopsy
Yang et al. Enhancing detection of circulating tumor cells with activating KRAS oncogene in patients with colorectal cancer by weighted chemiluminescent membrane array method
Qu et al. Clinical value of microRNA-23a upregulation in non-small cell lung cancer
Shaker et al. Impact of micro RNA‐375 and its target gene SMAD‐7 polymorphism on susceptibility of colorectal cancer
Sheu et al. Combined detection of CEA, CK-19 and c-met mRNAs in peripheral blood: a highly sensitive panel for potential molecular diagnosis of non-small cell lung cancer
WO2011094233A1 (fr) Procédés de classification de maladies ou de pronostic du cancer de la prostate basés sur l&#39;expression d&#39;antigènes testiculaires/cancéreux
Ak et al. Microsatellite instability status affects gene expression profiles in early onset colorectal cancer patients
WO2016071477A1 (fr) Procédés pour évaluer la réponse au traitement de patients atteints de cancer et traiter des patients atteints de cancer par analyse de la méthylation de sites cpg
Fouad et al. XRCC1 gene polymorphisms and miR-21 expression in patients with colorectal carcinoma
Okamoto et al. Clinical significance of DNA damage response factors and chromosomal instability in primary lung adenocarcinoma
Moon et al. EGFR, COX2, p-AKT expression and PIK3CA mutation in distal extrahepatic bile duct carcinoma
EP2132330B1 (fr) Procédés et outils pour la détection de la présence de cellules d&#39;adénocarcinome colorectal

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11737529

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11737529

Country of ref document: EP

Kind code of ref document: A1