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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • 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
  • C12Q2600/112—Disease subtyping, staging or classification

Definitions

• glandular kallikreins are a subgroup of serine proteases which are involved in the post-translational processing of specific polypeptide precursors to their biologically active forms.
• three members of this family have been identified, and some of their properties characterized (Clements, Endoc. Rev.. 10, 343 (1989); Clements, Mol. Cell Endo.. 99, 1 (1994); Jones et al, Acta Endoc. 127. 481 (1992)).
• the hKLKl gene encodes the tissue kallikrein protein, hKl
• the hKLK2 gene encodes the prostate-specific glandular kallikrein protein
• hK2 encodes the prostate-specific antigen protein
• hK3 prostate-specific antigen protein
• Northern blot analysis of mRNA shows that both hK2 and PSA are expressed mainly in the human prostate, while expression of hKl is found in the pancreas, submandibular gland, kidney, and other nonprostate tissues (Chapdelaine et al., FEBS Lett.. 236. 205 (1988); Young et al.. Biochem. 31. 818 (1992)).
• the nucleotide sequence homology between the exons of hKLK2 and hKLK3 is 80%, whereas the nucleotide sequence homology between the exons of hKLK2 and hKLKl is 65%.
• the deduced amino acid sequence homology of hK2 to PSA is 78%, whereas the deduced amino acid sequence homology of hK2 to hKl is 57%.
• the deduced amino acid sequence of hK2 suggests that hK2 may be a trypsin-like protease, whereas PSA is a chymotrypsin-like protease. PSA levels are widely used as a prognostic indicator of prostate carcinoma.
• the invention provides a diagnostic method for detecting hK2 DNA wherein the presence of prostate cancer cells in a physiological sample can be correlated to the detection of hK2 RNA in the sample. Because expression of hK2 is prostate tissue specific, hK2 RNA should theoretically not be detectable in cells present in bodily fluids or non-prostate tissue if there is no locally invasive or metastatic disease, or if all prostate tissue (benign and malignant) has been removed or destroyed.
• the method comprises contacting an amount of DNA obtained by reverse transcription (RT) of RNA from a human physiological sample with a plurality of oligonucleotide primers, preferably at least two oligonucleotide primers, at least one of which an hK2-specific oligonucleotide, in an amplification reaction so as to yield an amount of amplified hK2 DNA.
• amplification reaction is a polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• the presence of amplified hK2 DNA in blood cells, after RT-PCR, is correlated with prostate cancer, i.e., sixty-seven percent (67%) of the prostate cancer patients expressed hK2, ⁇ 7% expressed PSA, and 17% expressed both hK2 and PSA.
• the source of the sample to be tested is human tissue, e.g., prostate, prostate capsule, seminal vesicle, bone marrow or lymph node.
• Another preferred source of the sample to be tested is a human physiological fluid which comprises cells, e.g., blood, serum, or seminal fluid.
• amplified hK2 DNA is defined to mean hK2 DNA in a sample, which was subjected to an amplification reaction, that is present in an amount that is greater than, i.e., 10, preferably 10 4 , and more preferably 10 6 , times greater than, the amount of hK2 DNA which was present in the sample prior to amplification.
• hK2-specific oligonucleotide or "hK2- specific primer” means a DNA sequence that has at least about 80%, more preferably at least about 90%), and more preferably at least about 95%, sequence identity or homology to SEQ ID NO:4 in regions of SEQ ID NO:4 that are divergent from nucleotide sequences which encode hK3 (SEQ ID NO:23).
• An oligonucleotide or primer of the invention has at least about 7-50, preferably at least about 10-40, and more preferably at least about 15-35, nucleotides.
• the oligonucleotide primers of the invention comprise at least 7 nucleotides at the 3' of the oligonucleotide primer which have at least about 80%>, more preferably at least about 85%, and more preferably at least about 90%, identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
• the oligonucleotides of the invention may also include sequences which are unrelated to hK2 nucleic acid sequences, e.g., they may encode restriction endonuclease recognition sequences.
• a preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO: 14.
• Another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO: 17.
• Yet another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:18.
• a preferred diagnostic method of the invention combines RT- PCR detection of hK2 transcripts with RT-PCR detection of transcripts of other gene products associated with prostate cancer. Combined detection of two or more gene products may provide greater diagnostic certainty or yield more informative staging information. Combined detection may also be helpful in differentiating those cells with aggressive growth potential from those that are more indolent.
• RT-PCR detection of hK2 RNA is combined with RT-PCR detection of PSA RNA.
• the invention further provides a diagnostic method for detecting hK2 RNA. The method comprises extracting RNA from a physiological sample obtained from a human. The extracted RNA is reverse transcribed to yield DNA.
• the DNA is contacted with an amount of at least two oligonucleotides effective to amplify the DNA to yield an amount amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide.
• the presence of the amplified hK2 DNA is then detected.
• the presence of the amplified hK2 DNA is indicative of metastatic prostate cancer in the human.
• hK2 RNA or a level of hK2 RNA that rises over time, in bodily fluids or non-prostate tissue may be reasonably expected to indicate the presence of previously undiagnosed metastatic disease.
• Early detection of metastatic disease provides a "lead time" during which alternative therapeutic strategies, including those that may not exist at the time of surgery but are subsequently developed, can be evaluated.
• the present invention provides a method for monitoring the progression of prostate cancer.
• the method comprises contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from a human afflicted with prostate cancer with an amount of at least two oligonucleotides, at least one of which an hK2-specific oligonucleotide, effective to amplify the DNA to yield an amount of amplified hK2 DNA.
• the presence or amount of the amplified hK2 DNA is detected or determined.
• At least one point later in time another sample is taken and the amount of amplified hK2 DNA detected or determined. Then the amounts of amplified hK2 DNA, obtained at least at two different time points, are compared.
• the method comprises contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from the human afflicted with prostate cancer with an amount of at least two oligonucleotides, at least one of which is an hK2-specific oligonucleotide, effective to amplify the DNA to yield an amount of amplified hK2 DNA.
• the presence or amount of the amplified hK2 DNA is then detected or determined.
• the presence or amount of amplified hK2 DNA is indicative of the pathological stage of the prostate cancer.
• hK2 RNA levels in peripheral blood or other bodily tissue or fluid may be used as a marker during intermittent androgen therapy, or during androgen provocative testing, wherein a patient is temporarily placed in a hyperandrogenized state to stimulate a level of hK2 production by any persistent prostate cancer cells sufficient to render them detectable.
• hK2 levels are preferably monitored periodically during the course of hormone therapy. It may be advantageous to also determine hK2 levels before commencement of therapy, and periodically after the conclusion of a therapeutic regimen.
• kits for detecting hK2 RNA in a physiological sample suspected of containing hK2 RNA comprises packaging containing (a) a known amount of a first hK2-specific oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to SEQ ID NO: 4, and (b) a known amount of a second hK2-specific oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to a nucleotide sequence which is complementary to SEQ ID NO:4.
• the invention further provides an isolated, purified peptide comprising SEQ ID NO:22, a biologically active subunit thereof, or a biologically active variant thereof.
• the invention further provides an isolated, purified peptide comprising SEQ ID NO:26, a biologically active subunit thereof, or a biologically active variant thereof.
• an isolated purified antibody or antibody preparation that specifically reacts with a protein or polypeptide which comprises the peptides of the invention described above.
• the term " biologically active subunit" of a peptide of the invention is preferably defined to mean a subunit of a peptide having SEQ ID NO:22, which has at least about 10%>, preferably at least about 50%, and more preferably at least about 90%>, the activity of a peptide having SEQ ID NO:22.
• the activity of a peptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the peptide to elicit a sequence-specific immunologic response when the peptide is administered to an organism, e.g., goat, rabbit, sheep or mice.
• biologically active variant of a peptide of the invention is preferably defined to mean a peptide which has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, identity or homology to SEQ ID NO:22.
• Biologically active variants of the peptides of the invention have at least about 10%, preferably at least about 50%>, and more preferably at least about 90%, the activity of a peptide having SEQ ID NO:22.
• the activity of a variant peptide of the invention can be measured by methods described hereinabove.
• the invention further provides a method for detecting or determining the presence of metastatic prostate cancer in a human non-prostate tissue sample.
• the method comprises mixing an amount of an agent, which binds to an hK2 polypeptide and which does not bind to hK3, with the cells of the mammalian tissue sample so as to form a binary complex comprising the agent and the cells.
• the presence or amount of complex formation in the sample is then detected or determined.
• the presence or amount of the complex provides an indication of the presence of micrometastatic prostate cancer.
• micrometastatic means locally invasive disease, which typically involves penetration of the prostate capsule or seminal vesicle, or occult disease.
• a preferred agent for use in the method is an antibody.
• antibody includes human and animal mAbs, and preparations of polyclonal antibodies, as well as antibody fragments, synthetic antibodies, including recombinant antibodies, chimeric antibodies, including humanized antibodies, anti-idiotopic antibodies and derivatives thereof.
• These antibodies in turn can be used as the basis for direct or competitive assays to detect and quantify hK2 polypeptides (or "protein") in samples derived from tissues such as bone marrow and lymph nodes, and samples of cells such as from physiological fluids which comprise cells.
• Figure 1 depicts the amino acid sequences of wild type mature hK2 (SEQ ID NO:l) and hK3 (SEQ ID NO:7).
• Figure 2 depicts the amino acid sequence, and corresponding nucleic acid sequence, of wild type pphK2 (SEQ ID NO:3 and SEQ ID NO:4, respectively), phK2 (SEQ ID NO:5 and SEQ ID NO:6) and hK2 (SEQ ID NO:l and SEQ ID NO:2). Codon 217 (GCT, Ala) is shown in bold and underlined.
• Figure 3 is a schematic diagram of the pGT expression vectors P GThK2 and pGThK2 v217 .
• Figure 4 depicts chromatographic profiles from the purification of phK2 v217 .
• a sample of the spent medium was applied in bicarbonate buffer, pH 8 and eluted with a salt gradient.
• the A 280 elution profile is represented by a solid line.
• the dotted line represents the results of an ELISA assay of a portion of individual column fractions which was dried onto microtiter plates and developed with a rabbit anti-pphK2 antibody.
• B The hydrophobic interaction profile of pooled DEAE fractions.
• Figure 5 represents an SDS/PAGE analysis of purified hK2 and PSA.
• a 1.5 mg sample of purified phK2 v217 or PSA was boiled in sample buffer with (R) or without (N) 1%> beta-mercaptoethanol. Samples were subjected to SDS/PAGE on a 4-20% gel. The protein bands were visualized by staining the gel with silver.
• Figure 6 depicts Conconavalin A staining of phK2 v217 .
• the predicted position of phK2 is designated by an arrow.
• ZCE an anti-CEA mAb
• BSA was included as examples of glycosylated and non-glycosylated proteins, respectively.
• the presence of a band at the predicted position in the phK2 lane demonstrates that this protein is glycosylated.
• Figure 7 represents the conversion of pro to mature hK2 v2 ' 7 by trypsin cleavage. Trypsin (1% w/w) was incubated with phK2 v217 for 10 minutes at 37°C in 100 mM borate buffer pH 8, and then subjected to HIC-HPLC. The dashed line represents the profile of the phK2 v217 prior to incubation with trypsin.
• FIG. 8 depicts Western blot analysis of seminal fluid using monoclonal antibody (mAb) hKlG 586.1. Processed seminal fluid was diluted
• Protein was transferred to nitrocellulose and incubated with protein-G purified HKIG 586.1 (1 ⁇ g/ml) followed by goat anti-mouse IgG-HRP (1 : 1000). The blot was developed using the ECL detection system (Amersham).
• Figure 9 represents a time course study of hK2 expression in
• AVI 2 cells AV12-hK2 clone #27 was grown to -60-70% confluency, then cells were washed with HBSS and serum free HH4 media was added. Spent medium was withdrawn each day, concentrated and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted and probed with monoclonal antibody
• HK1D 106.4 which detects both phK2 and hK2 (1 :1000) or HKIG 464.3, which detects phK2 (1 : 1000), followed by goat anti-mouse IgG-HRP (1 :500).
• the blot was developed with ECL (Amersham) according to the manufacturer's instructions. Purified phK2 v217 and hK2 v217 were used as controls. The position of hK2 is indicated by the arrow.
• Figure 10 represents a time course study of expression of the variant form of hK2 in transfected AV12 cells.
• AV12- hK2 v217 cells were washed with HBSS and serum free HH4 media was added. Spent media was withdrawn each day, concentrated and subjected to SDS/PAGE on a 12%) gel. Proteins were electroblotted and probed with monoclonal antibodies HK1D 106.4 and HKIG 464.3. Goat anti-mouse IgG-HRP (1 :500) was used as a secondary antibody and the blot was developed with ECL (Amersham) according to the manufacturer's instructions. Purified phK2 v217 and hK2 v217 were used as controls. The position of hK2 is indicated by the arrow. Figure 11 is a plot of hK2 expression and cell viability over time.
• AV12-hK2 clone #27 was grown to 60-70% confluency, washed with HBSS and serum free HH4 media was added. Spent media was withdrawn each day and the hK2 concentration was measured by ELISA using HK1D 106.4 or HKIG 464.3 as a primary antibody, and goat anti-mouse IgG-HRP as a secondary antibody. The reaction was developed with OPD (Sigma, St. Louis, MO). Viable cells were enumerated daily using trypan blue dye exclusion.
• Figure 12 depicts the expression of hK2 in PC3 and DU145 cells.
• PC3 and DU145 cells transfected with pGThK2 were grown to -60-70% confluency, washed and resuspended in serum free HH4 media.
• the spent medium of pGThK2 transfected DU145 cells was collected 3 days after resuspension and the spent medium of pGThK2 transfected PC3 cells was collected 5 days after resuspension.
• Spent media were concentrated and subjected to SDS/PAGE on 12% gels. Proteins were electroblotted and probed with HK1D 106.4 or HKIG 464.3 as described above.
• FIG. 13 depicts the expression of hK2 by selected hK2- containing AVI 2 clones.
• Cells from hK2 containing AVI 2 clone numbers 10, 27, 31 and 32 were grown to -60-70% confluency and washed with HBSS, then serum free HH4 media was added.
• Spent media was withdrawn 7 days after the addition of serum free media, concentrated and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted and probed with HK1D 106.4 or HKIG 464.3.
• Goat anti-mouse IgG-HRP (1 :500) was used as a secondary antibody and the blot was developed with ECL (Amersham) according to manufacturer's instructions.
• Purified phK2 v2I? and hK2 v217 were used as controls. The position of hK2 is indicated by an arrow.
• Figure 14 depicts the expression of phK2 v21? in selected AVI 2- hK2 v217 clones. Cells from AV12 clone numbers 2, 3, 4, 45 and 48 were grown to approximately 60-70% confluency and washed with HBSS, and serum free HH4 media was added.
• Spent media was withdrawn 7 days after the addition of serum free media, concentrated and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted and probed with HK1D 106.4 or HKIG 464.3. Goat anti-mouse IgG-HRP (1 :500) was used as a secondary antibody and the blot was developed with ECL (Amersham) according to manufacturer's instructions. Purified phK2 v217 and hK2 v217 were used as controls. The position of hK2 is indicated by an arrow. Figure 15 depicts the amidolytic specificity of hK2 v217 , hK2, and
• FIG. 16 depicts the specificity of hK2 and PSA for different peptide substrates. Open arrows denote peptide bonds cleaved by PSA; solid arrows denote bonds cleaved by hK2. Peptide #1 represents amino acid residues 210-236 of hK2.
• Peptide #2 represents amino acid residues 1-14 of angiotensinogen, i.e., the renin substrate tetradecapeptide.
• Peptide #3 represents amino acid residues -7 to +7 of phK2.
• Peptide #4 represents amino acid residues 41-56 of hK2.
• Peptide #5 represents the amino acid sequence of the oxidized beta chain of insulin.
• Peptide #6 represents amino acid residues 196-213 of PMSA.
• Figure 17 depicts the activation of phK2 v217 by hK2 but not hK2 v217 .
• phK2 v217 contains the pro leader peptide sequence VPLIQSR, a sequence not present in hK2 v217 .
• Panel A shows phK2 v217 incubated with 1% w/w hK2.
• Panel B is a control with 40% w/w hK2 v217 incubated with phK2 v217 for 6 hours.
• Figure 18 depicts Western blot analysis of hK2 incubated with protease inhibitors. Each sample was separated on a 8-25%> gradient SDS- PAGE, blotted and probed with HKl G586.1. hK2 was incubated for 4 hours at 37°C with the following inhibitors: Lane 1, antichymotrypsin (ACT); lane 2, alpha 2-antiplasmin; lane 3, anti-thrombin III; lane 4, alpha 1 -protease inhibitor (anti-trypsin); lane 5, alpha 2-macroglobulin; lanes 1 and 2 show a covalent complex of the predicted Mr of 90-100 kD.
• ACT antichymotrypsin
• lane 2 alpha 2-antiplasmin
• lane 3, anti-thrombin III anti-thrombin III
• lane 4 alpha 1 -protease inhibitor (anti-trypsin)
• lane 5 alpha 2-macroglobulin
• lanes 1 and 2 show a covalent
• Serpin inhibitors were employed at 20 ⁇ M, macroglobulin at 2.8 ⁇ M, and hK2 at 0.175 uM. Lane 5 shows the higher Mr complexes representing covalent complex formation of hK2 with alpha 2-macroglobulin.
• Figure 19 depicts complex formation of hK2 in human serum.
• Western blots of hK2 and PSA were incubated with human serum.
• hK2 samples were probed with HK1G586.1 and PSA samples with PSM773 anti-PSA mAb
• Lanes 1-6 contain hK2 samples and lanes 7 and 8 are PSA samples.
• Lane 1 represents an hK2 control.
• Lane 2 contains hK2 incubated with ACT for 4 hours.
• Lane 3 represents a serum control with no added protease.
• Lane 4 contains hK2 incubated for 15 minutes with serum.
• Lane 5 contains hK2 incubated with serum for 4 hours.
• Lane 6 contains hK2 incubated with purified alpha-2 macroglobulin for 4 hours.
• Lane 7 contains PSA incubated with serum for 4 hours.
• Lane 8 contains PSA incubated with purified alpha-2 macroglobulin for 4 hours.
• Figure 22 depicts (A) RT-PCR detection of PSA and hK2 mRNAs in RNA extracted from LNCaP cells diluted in whole human blood, and (B) RT-PCR detection of PSA and hK2 mRNAs in RNA extracted from whole human blood of the following patients: patient 17, age 31, male control; patient 21, age 58, clinical stage B; patient 24, age 83, known metastatic disease (D2); patient 26, age 75, pathological stage C (+margins); patient 28, age 57, pathological stage C (+margins); patient 49, age 64, pathological stage C (+seminal vesicles); patient 59, age 31, male control; patient 60, age 73, known metastatic disease.
• D2 known metastatic disease
• hK2 polypeptide includes recombinant pre-pro, pro and mature hK2 polypeptides.
• a mature hK2 polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO:l), as well as "variant" polypeptides which share at least 90% homology with SEQ ID NO: 1 in the regions which are substantially homologous with hK3, i.e., which regions are not identified by bars as shown in Figure 1.
• the variant hK2 polypeptides of the invention have at least one amino acid substitution relative to the corresponding wild type hK2 polypeptide.
• a preferred variant hK2 polypeptide comprises SEQ ID NO:8, i.e., a mature hK2 polypeptide having an alanine to valine substitution at amino acid position 217.
• hK2 polypeptides possess antigenic function in common with the mature hK2 molecule of Figure 1, in that said polypeptides are also definable by antibodies which bind specifically thereto, but which do not cross-react with hK3 (or hKl).
• said antibodies react with antigenic sites or epitopes that are also present on the mature hK2 molecule of Figure 1.
• Antibodies useful to define common antigenic function are described in detail in Serial No. 08/096,946, now U.S. Patent No. 5,516,639, i.e., polyclonal antisera prepared in vivo against hK2 subunit 41-56.
• isolated hK2 nucleic acid is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more, sequential nucleotide bases that are complementary to the non-coding or coding strand of the native hK2 polypeptide RNA or DNA, or hybridizes to said RNA or DNA and remains stably bound under stringent conditions.
• the isolated nucleic acid encodes a biologically active hK2 polypeptide, a variant thereof, or a subunit thereof.
• an hK2 polypeptide can be detected by methods well known to the art including, but not limited to, the ability to react with antibodies specific for hK2 polypeptides, the ability to cleave hK2-specific substrates (see Example 7), or the ability to bind to serum proteins (see Example 9).
• a variant hK2 polypeptide or subunit thereof, or a subunit of an hK2 polypeptide has at least about 10%, preferably at least about 50%, and more preferably at least about 90%, the biological activity of an hK2 polypeptide comprising the amino acid sequence of SEQ ID NO:l.
• the RNA or DNA is isolated in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the nucleic acid and is preferably substantially free of any other mammalian RNA or DNA.
• the phrase "free from at least one contaminating source nucleic acid with which it is normally associated” includes the case where the nucleic acid is reintroduced into the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell.
• isolated hK2 nucleic acid is RNA or DNA that encodes a biologically active hK2 polypeptide sharing at least 90%) sequence identity with the hK3 -homologous regions of the hK2 peptide of Figure 1, as described above.
• isolated, purified as used with respect to an hK2 polypeptide is defined in terms of the methodologies discussed hereinbelow.
• recombinant nucleic acid refers to a nucleic acid, i.e., to DNA that has been derived or isolated from any appropriate tissue source, that may be subsequently chemically altered in vitro, an ⁇ later introduced into target host cells, such as cells derived from animal, plant, insect, yeast, fungal or bacterial sources.
• target host cells such as cells derived from animal, plant, insect, yeast, fungal or bacterial sources.
• An example of recombinant DNA "derived” from a source would be a DNA sequence that is identified as a useful fragment encoding hK2, or a fragment or variant thereof, and which is then chemically synthesized in essentially pure form.
• DNA includes completely synthetic DNA sequence that is excised or removed from said source by chemical means, e.g, by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering. Therefore, "recombinant DNA” includes completely synthetic DNA sequence that is excised or removed from said source by chemical means, e.g, by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering. Therefore, "recombinant DNA” includes completely synthetic DNA sequence that is excised or removed from said source by chemical means, e.g, by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering. Therefore, "recombinant DNA” includes completely synthetic DNA sequence that is excised or removed from said source by chemical means, e.g, by the
• DNA sequences DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from biological sources, and DNA sequences derived from introduced RNA, as well as mixtures thereof.
• the recombinant DNA sequence is not originally resident in the genome of the host target cell which is the recipient of the DNA, or it is resident in the genome but is not expressed, or not highly expressed.
• chimeric means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species.
• Control sequences is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
• the control sequences that are suitable for prokaryotic cells include a promoter, and optionally an operator sequence, and a ribosome binding site.
• Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
• operably linked is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
• Southern analysis or “Southern blotting” is a method by which the presence of DNA sequences in a restriction endonuclease digest of DNA or DNA-containing composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment.
• Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook et al, supra.
• RNA sequences that hybridize to a known probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment.
• the probe is labeled with a radioisotope such as 32 P, by biotinylation or with an enzyme.
• the RNA to be analyzed can be usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.
• “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50°C, or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1 %> bovine serum albumin/0.1 % Ficoll/0.1 %> polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
• SSC sodium lauryl sulfate
• a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1 %> bovine serum albumin/0.1 % Ficoll/0.1 %> polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 m
• Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS. See Sambrook et al., supra, for other examples of stringent conditions.
• An expression cassette or vector comprising a recombinant DNA sequence encoding hK2 which is operably linked to a promoter functional in a host cell may be circular or linear, double-stranded or single-stranded.
• the expression cassette or vector is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the recombinant DNA present in the resultant cell line.
• the expression cassette may itself comprise a promoter that is active in mammalian cells, or may utilize a promoter already present in the genome that is the transformation target.
• promoters include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements).
• a portion of the recombinant DNA may be untranscribed, serving a regulatory or a structural function.
• the expression cassettes or expression vectors to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed.
• the selectable marker may be carried on a separate piece of DNA and used in a co- transformation procedure.
• Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
• Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA and the like. See also Table 1 of Lundquist et al. (U.S. Patent No. 5,554,798).
• Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art.
• a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity.
• Preferred genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E. coli, and the lucif erase gene from firefly Photinus pyralis. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
• elements functional in the host cells such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.
• Transfection can also be accomplished by lipofectin, using commercially available kits, e.g., provided by BRL.
• Suitable host cells for the expression of hK2 polypeptide are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities.
• any higher eukaryotic cell culture can be employed in the practice of the invention, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells.
• a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used, preferably for transfection of Spodoptera frugiperda cells.
• hK2 polypeptide When hK2 polypeptide is expressed in a recombinant cell other than one of human origin, the hK2 polypeptide is completely free of proteins or polypeptides of human origin. However, it is necessary to purify hK2 polypeptide from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to hK2 polypeptide. For example, the culture medium or lysate can be centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The hK2 polypeptide may then be purified from the soluble protein fraction and, if necessary, from the membrane fraction of the culture lysate.
• hK2 polypeptide can then be purified from contaminant soluble proteins and polypeptides by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
• derivatives and variants of the hK2 polypeptide can be readily prepared.
• amides of the hK2 polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide.
• a preferred method for amide formation at the C- terminal carboxyl group is to cleave the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
• Salts of carboxyl groups of the hK2 polypeptide may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
• a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
• a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
• an amine base such as, for example, triethylamine, triethanolamine, and the like.
• N-acyl derivatives of an amino group of the present polypeptides may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptid
• O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.
• the internal hK2 amino acid sequence of Figure 1 can be modified by substituting one or two conservative amino acid substitutions for the positions specified, including substitutions which utilize the D rather than L form.
• Acid addition salts of the polypeptides may be prepared by contacting the polypeptide with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art.
• variant hK2 polypeptides have at least one amino acid substitution relative to SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:5, e.g., SEQ ID NO: 8 has an alanine to valine substitution at position 217 relative to SEQ ID NO: 1.
• amino acids are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of exemplary substitutions. More preferred substitutions are under the heading of preferred substitutions.
• the products are screened for biological activity, e.g., ability to generate hK2-specific antibodies or to specifically react with hK2-specific antibodies, i.e., antibodies that bind to hK2 and not to hK3 (PSA).
• Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• Naturally occurring residues are divided into groups based on common side-chain properties:
• hydrophobic norleucine, met, ala, val, leu, ile
• Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
• Amino acid substitutions are introduced into the DNA molecules of the invention by methods well known to the art.
• hK2 polypeptide and its antigenically active variants, derivatives and fragments thereof can be used in assays for hK2 in samples derived from biological materials suspected of containing hK2 or anti- hK2 antibodies, as disclosed in detail in U.S. Patent No. 5,516,639.
• the hK2 polypeptide can be labeled with a detectable label, such as via one or more radiolabeled peptidyl residues, and can be used to compete with endogenous hK2 for binding to anti-hK2 antibodies, i.e., as a "capture antigen" to bind to anti-hK2 antibodies in a sample of a physiological fluid, via various competitive immunoassay format for hK2 which uses anti-hK2 antibodies which are capable of immobilization is carried out by: (a) providing an amount of anti-hK2 antibodies which are capable of attachment to a solid surface; (b) mixing a physiological sample, which comprises hK2, with a known amount of hK2 polypeptide which comprises a detectable label, to produce a mixed sample; (c) contacting said antibodies with said mixed sample for a sufficient time to allow immunological reactions to occur between said antibodies and said hK2 to form an antibody-hK2 complex, and between said antibodies and said labeled polypeptide to form
• step (f) determining from the result in step (e) the presence or amount of said hK2 in said sample.
• a known amount of anti-hK2 antibody is added to a sample containing an unknown amount of endogenous hK2.
• the known amount is selected to be less than the amount required to complex all of the hK2 suspected to be present, e.g., that would be present in a sample of the same amount of sample material obtained from a patient known to be afflicted with prostate cancer.
• a known amount of the hK2 polypeptide of the invention or a subunit thereof, comprising a detectable label is added.
• endogenous hK2 is present in the sample, fewer antibodies will be available to bind the labeled hK2 polypeptide, and it will remain free in solution. If no endogenous hK2 is present, the added labeled polypeptide will complex with the added anti-hK2 antibodies to form binary complexes. Next, the binary antibody- antigen complexes are precipitated by an anti-mammal IgG antibody (sheep, goat, mouse, etc.). The amount of radioactivity or other label in the precipitate (a ternary complex) is inversely proportional to the amount of endogenous hK2 that is present in the sample, e.g., a pellet containing reduced amounts of radioactivity is indicative of the presence of endogenous hK2.
• monoclonal antibodies against hK2 polypeptide can be prepared using known hybridoma cell culture techniques.
• this method involves prepared an antibody- producing fused cell line, e.g., of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species from which the myeloma cell line used was derived or is compatible.
• Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and sensitive and relatively "pure” immunochemically.
• Immunologically active fragments of the present antibodies are also within the scope of the present invention, e.g., the f(ab) fragment, as are partially humanized monoclonal antibodies.
• Chimeric antibodies comprise the fusion of the variable domains from one immunoglobulin to the constant domains from another immunoglobulin.
• the variable domains are derived from an immunoglobulin gene from a different species, perhaps a human. This technology is well known to the art. See, for example, European Patent
• EP-A-0 125,023 (Cabilly/Genetech) and EP-A-0 120,694 and U.S. Patent No. 4,816,567, which disclose the preparation of variations of immunoglobulin-type molecules using recombinant DNA technology.
• chimeric or modified antibodies Another approach to prepare chimeric or modified antibodies is to attach the variable region of a monoclonal antibody to another non- immunoglobulin molecule, to produce a derivative chimeric molecule (see WO 86/01533, Neuberger and Rabbits/Celltech).
• a further approach is to prepare a chimeric immunoglobulin having different specificities in its different variable regions (see EP 68763 A).
• Yet another approach is to introduce a mutation in the DNA encoding the monoclonal antibody, so as to alter certain of its characteristics without changing its essential specificity. This can be accomplished by site-directed mutagenesis or other techniques known in the art.
• the Winter patent application EP-A-0 239 400 discloses the preparation of an altered, derivative antibody by replacing the complementarity determining regions (CDRs) of a variable region of an immunoglobulin with the CDRs from an immunoglobulin of different specificity, using recombinant DNA techniques ("CDR-grafting").
• CDR-grafting enables "humanization” of antibodies, in combination with the alteration of the framework regions.
• Human antibodies can also be prepared by reconstituting the human immune system in mice lacking their native immune system, then immunizing the mice so as to yield human antibodies which are specific for the immunogen.
• a "humanized” antibody containing the CDRs of a rodent antibody specific for an antigen of interest may be less likely to be recognized as foreign by the immune system of a human. It follows that a "humanized” antibody with the same binding specificity, as e.g., HK1G464, may be of particular use in human therapy and/or diagnostic methods.
• RNA is isolated from a cellular sample suspected of containing hK2 RNA, e.g., total RNA isolated from human prostate tissue.
• RNA can be isolated by methods known to the art, e.g., using TRIZOL TM reagent (GIBCO-BRL/Life Technologies, Gaithersburg, MD).
• Oligo-dT can be employed as a primer in a reverse transcriptase reaction to prepare first-strand cDNAs from the isolated RNA. Resultant first-strand cDNAs are then amplified in PCR reactions.
• Polymerase chain reaction or “PCR” refers to a procedure or technique in which amounts of a preselected fragment of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195.
• sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular
• RNA, bacteriophage or plasmid sequences and the like. See generally Mullis et al., Cold Spring Harbor Svmp. Quant. Biol. 5_1, 263 (1987); Erlich, ed., PCR Technologv. (Stockton Press, NY, 1989).
• amplification of specific nucleic acid sequences by PCR relies upon oligonucleotides or "primers" having conserved nucleotide sequences, wherein the conserved sequences are deduced from alignments of related gene or protein sequences, e.g., a sequence comparison of mammalian hK2 genes.
• one primer is prepared which is predicted to anneal to the antisense strand, and another primer prepared which is predicted to anneal to the sense strand, of a DNA molecule which encodes an hK2 polypeptide.
• the isolated RNA is combined with a primer in a reverse transcriptase (RT) reaction to generate single strand cDNAs.
• RT reverse transcriptase
• Oligo-dT or random sequence oligonucleotides, as well as sequence specific oligonucleotides, can be employed as a primer in the RT reaction. See Sambrook et al., supra.
• the single strand cDNAs are then amplified with sequence specific primers to yield an amplified product.
• the reaction mixture is typically subjected to agarose gel electrophoresis or other convenient separation technique, and the presence or absence of the hK2-specific amplified DNA detected.
• Detection of the amplified hK2 DNA may be accomplished by excising or eluting the fragment from the gel (for example, see Lawn et al., Nucleic Acids Res.. 9, 6103 (1981), and Goeddel et al., Nucleic Acids Res..
• the hK2 amplified DNA may be detected using Southern hybridization with an hK2-specific oligonucleotide probe, or comparing its electrophoretic mobility with DNA standards of known molecular weight.
• a cDNA (approximately 820 bp long) encoding the entire prepro-hK2 (pphK2) (from nucleotide #40 to #858 relative to the start site of the pphK2 transcript), as shown in Figure 2, was synthesized from RNA of human BPH tissue using reverse-transcription polymerase chain reaction (RT-PCR) technology with a pair of hK2 specific oligonucleotide primers (5'ACGCGGATCCAGCATGTGGGACCTGGTTCTCT 3'; SEQ ID NO:9 and 5'ACAGCTGCAGTTTACTAGAGGTAGGGGTGGGAC 3'; SEQ ID NO:10).
• RT-PCR reverse-transcription polymerase chain reaction
• This cDNA was generated such that 5' and 3' ends (with respect to pphK2 sense sequence) were bracketed with BamHl and Pstl sequences, respectively.
• the cDNA was then purified by agarose gel electrophoresis, and digested with BamHl and Pstl restriction enzymes.
• the restricted cDNA was ligated with BamHl-Pstl digested pVL1393 plasmid vector and transformed into the E. coli HB101 strain. E. coli harboring pphK2 cDNA/pVL1393 plasmid vector were selected.
• the pphK2 containing insert was sequenced.
• Plasmid pphK2 cDNA/pVL1393 was mass-produced in E. coli and purified by CsCl gradient ultra-centrifugation. Plasmid pphK2/pVL 1393 in E. coli HB 101 has been deposited in the American Type Culture Collection, Rockville, MD, USA on May 2, 1994 under the provisions of the Budapest Treaty and has been assigned accession number ATCC 69614.
• the mammalian expression vectors, PLNS-hK2 and PLNC-hK2 were obtained by cloning the 0.8 kb wild type hK2 insert from the corresponding TA vector into the plasmids, pLNSX and pLNCX (Miller et al., Biotech.. 9, 980 (1989)), respectively.
• the orientation of the insert in all the mammalian expression vectors was confirmed by DNA sequencing. Generation of recombinant clones
• AVI 2-664 (ATCC CRL-9595), a cell line derived from adeno virus-induced tumors in Syrian hamster, and DU145 cells were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum (D10F). PC3 cells were cultured in Minimal Eagle Medium containing 10%) fetal bovine serum. AVI 2 cells were transfected with the hK2 expression vectors using the calcium phosphate method (Maniatis et al., supra (1989)). Three days after transfection cells were resuspended in D10F + 200 nM methotrexate (MTX).
• MTX methotrexate
• PC3 and DU145 cells were transfected with hK2 mammalian expression vectors using lipofectamine (Gibco-BRL, Gaithersburg, MD) and clones (PC3-hK2 and DU145-hK2) were selected in media containing 400 ⁇ g/ml G418. Purification and sequencing of the protein
• AV12-hK2 clones were grown in D10F + 200 nM MTX. At about 60%) confluency the cells were washed with Hank's balanced salt solution and resuspended in serum-free HH4 medium. The spent medium was collected 7 days after the addition of serum-free spent medium and stored at -20°C. To purify the protein, the serum-free spent medium was concentrated and exchanged into 50 mM sodium bicarbonate pH 8. Samples were filtered with 0.2 ⁇ filters and then pumped directly onto a TSK DEAE-5PW HPLC column, 21 mm X 150 mm, at a flow rate of 5 ml/minute.
• Buffer A contained 50 mM Na bicarbonate pH 7.9 and Buffer B contained 50 mM Na bicarbonate plus 0.5 M NaCl pH 7.6.
• the elution profile was developed with a gradient from 0-50% Buffer B over 35 minutes; 50-100% B from 35-40 minutes and isocratic elution at 100%) B for 5 minutes before re-equilibration in Buffer A.
• the flow rate was 5 mL/minute throughout. In the above procedure, borate buffer could replace bicarbonate buffer with no noticeable difference.
• DEAE fractions were assayed for the presence of hK2 by the dried-down ELISA method (see below) using rabbit anti-pphK2 (Saedi et al., Mol. Cell. Endoc. 109. 237 (1995)).
• Fractions with hK2 activity were pooled and concentrated by ultrafiltration with membranes (10 kD cut off) to approximately 5-8 mL. Solid ammonium sulfate was then added to a final concentration of 1.2 M. This sample was then injected onto a PolyLC, polypropyl aspartamide column, lOOOA pore size, 4.6 mm X 200 mm, to resolve proteins by hydrophobic interaction chromatography (HIC).
• HIC hydrophobic interaction chromatography
• Buffer A was 20 mM Na phosphate, 1.2 M Na sulfate pH 6.3 and Buffer B was 50 mM Na phosphate, 5% 2-propanol, pH 7.4.
• the elution gradient was 0-20% B over 5 minutes; 20-55% B from 5-20 minutes, isocratic at 55% B from 20-23 minutes, 55-100% B from 23-25 minutes; isocratic at 100% B for 2 minutes before re- equilibration Buffer A.
• the flow rate was 1 mL/minute.
• the HIC peak containing hK2 which eluted at about 50% B was exchanged into 50 mM borate buffer pH 8 by repeated concentration with Centricon-10 (Amicon) 10 K MW cutoff ultrafiltration.
• HIC peak containing hK2 was purified further by size exclusion chromatography (SEC) on a 10/30 Pharmacia S12 column. In this case the HIC peak containing hK2 was concentrated by ultrafiltration as above to less than 1 mL and then applied to the size exclusion column equilibrated in 100 mM ammonium acetate pH 7 or sodium borate pH 8. The flow rate was 0.7 mL/minute. The hK2 peak was then concentrated by ultrafiltration.
• HKIG 586.1 (see below) affinity resin was used to purify hK2 by affinity chromatography.
• HKIG 586.1 monoclonal antibody (mAb) was coupled with Pharmacia GammaBind plus Sepharose (cat. no. 17-0886) according to Schneider (J. Biol. Chem.. 257. 10766 (1982)). Briefly, HKIG 586.1 mAb and resin were incubated overnight at 4°C with rotation. Resin was centrifuged (500 X g for 5 minutes at 4°C) and washed twice with 0.2 M triethanolamine, pH 8.2.
• Amine groups were cross-linked in fresh cross linker solution (25 mM dimethyl pimelimidate dihydrochloride in 0.2 M triethanolamine, pH 8.2) for 45 minutes at room temperature (22°C).
• the resin was quenched with 20 mM ethanolamine, pH 8.2, for 5 minutes at room temperature and then washed twice with 1 M NaCl, 0.1 M PO 4 , pH 7.0.
• the resin was washed two more times with PBS and stored at 4°C with 0.05% NaN3 until use.
• Model 477a pulsed liquid phase sequencer was used to sequence the proteins and the peptides.
• the Model 477a employs automated Edman degradation chemistry to sequentially release amino acids from the N-terminus followed by PTH derivatization and chromatography by reversed-phase HPLC.
• the peptide samples were applied to the sequencer on biobrene-treated glass fiber filter supports and whole proteins were applied either to biobrene-treated filters or to pre-activated Porton filters (Beckman, Fullerton, CA).
• mice were injected with 50 ⁇ l of phK2 in complete Freund's adjuvant (CFA) i.p. on day 1, 25 ⁇ g of phK2 in incomplete Freund's adjuvant (IF A) i.p. on day 14 and 25 ⁇ g phK2 in PBS i.p. on day 28.
• CFA complete Freund's adjuvant
• IF A incomplete Freund's adjuvant
• mice Three days prior to fusion, mice were boosted with 10 ⁇ g of phK2 in PBS i.v. Mice were sacrificed and a single cell suspension was prepared from the spleens. Immune B cells were fused with P3.653 myeloma cells.
• Cloned hybridomas were screened by ELISA and selected based on the reactivity of supernatants to hk2 v217 and phK2 v217 and minimal reactivity with PSA.
• Another fusion which employed the same protocol described above except that the immunogen was hK2 v217 , alum was used instead of CFA and IF A, B ALB/c mice were used instead of A/J mice, produced clone HK1H247.
• Hybridoma clones HK1G464.3, HK1G586.1, HK1H247, HK1A523 and HKD106.4 have been deposited with the American Type Culture Collection, in accord with the Budapest Treaty, and granted Accession Nos: HB11983, HB12026, HB12162, HB11876 and HB11937, respectively.
• mice For monoclonal antibodies to peptide immunogens, Balb/c mice were immunized subcutaneously with 100 ⁇ g of KLH-conjugated peptide in complete Freund's adjuvant (CFA) and boosted at three week intervals with 100 ⁇ g of peptide in incomplete Freund's adjuvant (IFA).
• CFA complete Freund's adjuvant
• IFA incomplete Freund's adjuvant
• A/J mice were immunized twice intraperitoneally with 50 ⁇ g of KLH-conjugated peptide and mice with positive titers were boosted intravenously with 25 ⁇ g of conjugate.
• Lymphocytes from the spleens of mice with positive serum titers were fused with myeloma cells to produce hybridoma cells.
• Antibodies produced by clones of these cells were screened as described above. Positive clones were subcloned by limiting dilution and rescreened.
• Monoclonal hybridomas were injected into the peritoneal cavities of pristine primed mice to obtain ascitic fluid.
• the antisera initially were subjected to an IgG separation by precipitation with saturated ammonium sulfate and size chromatography using an ultragel ACA-34 column.
• the polyclonal antisera was further affinity purified using columns produced by cyanogen-bromide coupling of the peptides to Sepharose 4B.
• the purified antibody was eluted from the column with acidic PBS (pH 2.45).
• a dried-down ELISA format was used to measure hK2 in the serum-free spent medium of the clones and in the fractions collected during hK2 purification.
• Microtiter plates (Becton Dickinson Labware, NJ) were coated with 50 ⁇ l of spent media or column fractions overnight at 37°C. The wells were washed with PBS + 0.1% Tween 20 (PBST) and incubated for one hour with 50 ⁇ l of primary antibodies.
• the wells were washed again with PBS+T and incubated for one hour at 37°C with 50 ⁇ l of goat anti mouse-IgG or goat anti rabbit-IgG Fc antibodies coupled with horseradish peroxidase (1 :500, Jackson Immunosearch Laboratories, Inc., West Grove, PA).
• the wells were washed with PBST, incubated with o-phenylenediamine dihydrochloride (OPD, Sigma, MO) for 5 minutes, and the colorimetric reaction was measured at A 490 with an ELISA reader (Biotek Instruments, Inc., model EL310, VT). All samples were assayed in duplicate.
• the serum-free spent medium from AVI 2 cells transfected with vector alone was used as negative control.
• Antibodies were tested in a solution-based ELISA format using biotinylated phK2 v2 ' 7 , hK2 v217 , and PSA.
• PSA was purified by the method of Sensabaugh and Blake (J. Urology. 144. 1523 (1990)).
• biotinylated hK2 v217 or phK2 v217 diluted in 50 ⁇ l Buffer A (8.82 mM citric acid, 82.1 mM sodium phosphate (dibasic), 10% BSA, 0.1 % mannitol, 0.1% Nonidet P-40, pH 7.0) or 0.25 ng biotinylated PSA diluted in 10% horse serum (HS) in PBS was incubated with 50 ⁇ l of hybridoma supematants, negative control supematants (i.e., irrelevant hybridoma supernatant for phK2 v217 and hK2 v2 ' 7 , or 20 ⁇ g/ml irrelevant purified mAb in HS for PSA), or positive control supematants (i.e., 20 ⁇ g/ml purified PSM773 (anti-PSA) mAb in HS for PSA, or HK1D 104 (anti
• HCO514 a mAb against hCG
• ZTG085 a mAb against the tau
• the plate was washed 3 times with 300 ⁇ l of PBS, 0.1% Tween-20 (PBST), and incubated with 100 ⁇ l of gamma-specific goat anti mouse IgG-horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories, Inc., Westgrove, PA), diluted 1 :10,000 in HS, with shaking for 1 hour. After a second PBST washing, color was developed for 30 minutes, with shaking, following the addition of 100 ⁇ l of 1 mg/ml o- phenylenediamine in 50 mM phosphate-citrate buffer, 0.03% sodium perborate, pH 5.0 (Sigma Chemical, St. Louis, MO).
• the reaction was quenched by the addition of 50 ⁇ l 4 N H 2 SO 4 .
• the color intensity was determined by measuring the absorbance at 490 nm and 540 nm using a microtiter plate reader. Absorbances above 2.6 at 490 nm were corrected with 540 nm reading. Sample values are averages ⁇ standard deviation of triplicates. Control values are averages of duplicates.
• hK2 was incubated with 20 ⁇ M of inhibitor at pH 8 in 100 mM borate buffer.
• Inhibitors tested were 1-antichymotrypsin, 1-antitrypsin, 1-antiplasmin, antithrombin and 2-macroglobulin.
• To 5 ⁇ l of hK2 (10 ⁇ g/ml) was added the calculated ⁇ g of inhibitor prepared in 100 mM borate buffer and, if needed, each sample brought up to a total volume of 10 ⁇ l.
• peptides were dissolved in DMSO at 10 mg/ml then diluted 1 :10 into 100 mM borate buffer pH 8 containing PSA, hK2 or trypsin.
• Typical experiments were performed as follows: 1 ⁇ l of peptide was added to 7 ⁇ l of 100 mM borate buffer and then 2 ⁇ l of hK2 (10 ⁇ g/ml), PSA (500 ⁇ g/ml) or trypsin (0.5 ⁇ g/ml) were added. In general, samples were incubated for 16 hours at 37°C.
• Peaks collected off HPLC were concentrated by vacuum centrifugation or lyophilization and then applied to the amino acid sequencer to identify individual fragments. In some cases 10 ⁇ l of the quenched sample mixture was applied directly to the sequencing membrane and, since the sequence was known, the cleavage sites were determined from the distribution of amino acids present in each cycle.
• Protease assays using chromogenic substrates Assays to measure the hydrolysis of para-nitroanilide derivatized substrates were performed using an HP 8452A UV-VIS spectrophotometer equipped with a programmable, thermostated 7-position cell holder. Assays were performed in 100 mM sodium borate pH 8 incubated at 37°C, the absorbance increase monitored at 405 nm.
• Methoxysuccinyl-Arg-Pro-Tyr-para- nitroanilide (MeO-Suc-R-P-Y-pNA) and H-D-pro-phe-arg-para-nitroanilide (P- F-R-pNA) were 1 mM in the assay.
• pphK2 0.8 kb fragment encoding the entire coding sequence of hK2 (pphK2) ( Figure 2) was amplified using PCR, subcloned into the vector PCR II (TA) and several clones were isolated. The nucleotide sequence of the entire pphK2 insert in a few of these clones was determined to detect any mutations that may have been caused by PCR amplification.
• TA-hK2 v217 contains a substitution of T for C at codon 650 of hK2 resulting in a conservative substitution of valine (GTT) for alanine (GCT) at amino acid residue 217 of hK2 ( Figure 2).
• pphK2 inserts of TA-hK2 and TA-hK2 v217 were subcloned into plasmid PGT-d under the control of the GBMT promoter resulting in plasmids pGThK2 and pGThK2 v217 ( Figure 3).
• the GBMT promoter is composed of several regulatory sequences and is activated by the adenovirus El a protein(s) (Berg et al., supra (1992)).
• the plasmid pGThK2 v217 was transfected into AVI 2-664 cells.
• This cell line is derived from a tumor induced in Syrian hamster by adenovirus type 12 and expresses the adenovirus El a protein.
• the Ela protein activates the GBMT promoter which results in the expression of the gene product under the control of this promoter.
• MTX-resistant clonal cells were isolated and their spent medium were analyzed by Western blots. Several clones were identified which secreted into the media a polypeptide immunoreactive to anti-pphK2 antiserum.
• One clone (AV12-pGThK2 v21? #2) was selected for further characterization and protein purification.
• DIG digoxigenin
• phK2 v217 also reacted with RCA (Gal bl-4GlcNAc specificity) and AAA, ( ⁇ (l-6) linked fucose specificity). This pattern of lectin reactivity is consistent with the presence of complex N-linked oligosaccharides.
• the oligosaccharides on phK2 v217 also contain sialic acid since both SNA (sialic acid linked ⁇ (2-6) to galactose) and MAA (sialic acid linked ⁇ (2-6) to galactose were reactive with phK2 v217 .
• the sequence of the pro region of hK2 is VPLIQSR.
• HK1G586.1 was hK2-specific, since it recognized both phK2 v217 and hK2 v217 but not PSA.
• HK1G464 was phK2-specific, since it only recognized phK2 v217 and not hK2 v2,v or PSA.
• the selected hK2 peptides correspond to mature hK2 amino acid residues 8-26 (SEQ ID NO:19), 15-26 (SEQ ID NO:26), 41-56 (SEQ ID NO:20), 43-66 (SEQ ID NO:24), 153-167 (SEQ ID NO:21), 17-71 (SEQ ID NO:22) and 210-235 (SEQ ID NO:25).
• the peptide corresponding to amino acids 17-71 was synthesized in order to increase the likelihood of producing antibodies that recognize the native form of hK2.
• the peptides were synthesized and HPLC purified in the Protein Core Facility at Mayo Clinic/Foundation.
• KLH keyhole limpet hemocyanin
• BSA basic monoclonal antibody
• Sheep, goat and mice were immunized with KLH-hK2 peptides for polyclonal (sheep and goat, SEQ ID Nos:20 and 21, and SEQ ID Nos: 19, 20, 21, 24, 25 and 26, respectively)) and monoclonal antibody (mice; SEQ ID Nos: 19, 20, 21, 24, 25 and 26) production.
• the 17-71 peptide (SEQ ID NO:22) was oxidized to generate an intramolecular disulfide bond between cys 26 and 42 and used to immunize goats and mice for polyclonal and monoclonal antibody production, respectively.
• the hK241-56 Ab from sheep was first purified by hK2 41-56 peptide-affmity column and then used for Western blot analysis.
• the antibody recognized recombinant hK2.
• the detection of hK2 by the hK2 41-56 Ab was abolished by addition of excess hK2 41-56 peptide but not by PSA 41-56 peptide.
• monoclonal anti-hK2 41-56 peptide antibodies were highly specific for hK2 protein in Western analysis.
• Anti-hK2 153-167 peptide antiserum (sheep) recognized recombinant hK2.
• Antisera against hK2 amino acid residues 210-235 showed the highest immunoreactivity.
• Rabbit antiserum to bacterially expressed recombinant hK2 protein which recognizes both PSA and hK2, detected a doublet protein band in concentrated LNCaP cell medium from LNCaP cells which were treated with androgen. In contrast, no immunoreactive protein was detected in LNCaP cell medium from LNCaP cells which were not treated with androgen. Thus, the immunoreactive proteins were induced by androgen.
• the upper band in LNCaP media is a PSA-related protein because a PSA-specific antiserum (rabbit anti-PSA antisera, which was raised against bacterially expressed recombinant PSA) detected mainly the upper band.
• the lower band in LNCaP media is an hK2-related protein because a mouse monoclonal antibody (HKl A523) against hK2 41-56 peptide that has monospecificity for hK2 recognizes the lower protein band.
• Example 5 Expression of hK2 in mammalian cells To express wild type hK2 (hK2) in mammalian cells pGThk2
• Figure 3 was transfected into AVI 2 cells.
• Several clones expressing an hK2 polypeptide were identified by Western analysis using HK1D 106.4 (a hK2- specific mAb raised to a polypeptide corresponding to amino acid residues 17-71 of hK2).
• Clone AV12-hK2#27 was selected for further analysis based on its higher hK2 expression level.
• Cells transfected with vector alone (pGTD) showed no reactivity with HK1D 106.4.
• ELISA using HK1D 106.4 mAb indicated the presence of about 0.5-1 ⁇ g/ml of an hK2 polypeptide in the serum-free spent medium of AV12- hK2 at day 7.
• the same method used in purification of phK2 v217 from AV12- hK2 v217 was used to purify hK2 polypeptides from the day 7 spent medium of AV12-hK2. This resulted in low yields of purified hK2 polypeptides which were unstable to the purification procedures.
• hK2 polypeptides were partially purified using the above method, subjected to SDS/PAGE, electroblotted and subjected to N-terminal amino acid sequencing.
• hK2 polypeptide in the spent medium of AV 12-hK2 at day 7 has the sequence, IVGGWECEK at N-terminus. No competing sequence was evident from the profile of amino acids released sequentially by the Edman degradation procedure. By comparison to PSA, this sequence corresponds to mature hK2 (hK2). Amino acid analysis of this protein was also consistent with that of hK2. This finding demonstrates that phK2 v217 was predominantly present in the serum-free spent medium of AV12-hK2 v217 at day 7, whereas predominantly hK2 was present in the serum-free spent medium of AV12-hK2 at day 7.
• HKID 106.4 recognizes both phK2 and hK2 whereas HKIG 464.3 recognizes only phK2 as its epitope lies in -7 to +7 region of hK2.
• Expression of hK2 polypeptides peaked by day 3 and plateaued thereafter as detected by HKID 106.4 mAbs.
• Two other immunoreactive bands migrating at about 70 kD and about 90 kD were also detected from day 4 onwards.
• hK2 A decrease in hK2 paralleled a decrease in cell viability, suggesting the hK2 is secreted by these cells, as opposed to being released following cell death and lysis. Also, a rise in hK2 corresponded to a drop in phK2, indicating that the pro form of hK2 was automatically converted to the mature form over time.
• hK2 was expressed in DU145 and PC3 cell lines.
• DNA encoding pphK2 was cloned into plasmids pLNCX and pLNSX (Miller and Rosman, BioTechniques. 7, 980 (1989)), under the control of the CMV and SV40 promoters, respectively.
• the resulting plasmids, pLNC-hK2 and pLNS-hK2, respectively were transfected into PC3 and DU145 cells, respectively, and clones were selected in media containing G418. Clones expressing high levels of hK2 were selected (PC3-hK2 and DU145-hK2) by ELISA and Western blots.
• Predominantly phK2 was detected in the spent medium of PC3-hK2 cells even after 7 days, however, predominately hK2 was present in the serum-free medium of DU145-hK2 starting from day 1. This is probably due to abundance of extracellular proteases in DU145 spent medium.
• HKID 106.4 mAb detected not only the major 34 KD band ("hK2") but also the 70 kD and the 90 kD bands that are indicative of the presence of hK2 ( Figure 13).
• HKIG 464.3 detected very low levels of phK2 in all of the AV12-H 2 clones ( Figure 14). This result indicates that predominantly hK2 is present in the spent medium of all the AV12-hK2 clones verifying the biosynthetic mechanism established for AV12-hK2 #27 clone.
• the same analyses were used on AV12-hK2 v217 clones ( Figure 14). Results indicated that only phK2 v217 was present in the spent medium of these clones at day 7 verifying our findings with the AV12-hk2 v217 clone.
• hK2 is expressed as the pro form in mammalian cells and is converted to mature form extracellularly by as yet unknown proteases.
• phK2 may be present in the biological fluids and therefore can be a useful diagnostic marker for pCa and BPH.
• Enzymatic activity and specificity of hK2 and hK2 v2 ⁇ A small amount of hK2 was purified to sufficient purity to determine its enzymatic activity and substrate specificity.
• the general activity of hK2 was measured by determining its amidolytic activity chromogenically on p-nitroanilide derivatives of peptides (Table 3). The p-nitroanilide released by proteolytic digestion of these substrates is measured at absorbance A 405 .
• the substrate methoxysuccinyl-Arg-Pro-Tyr-p ⁇ r ⁇ -nitroanilide is used to measure chymotrypsin-like proteases which cleave at the phenylalanine. This substrate has been used previously to measure the activity of PSA (Christensson et al., Eur. J. Biochem.. 194. 755 (1990)).
• the substrate H-D-Pro-Phe-Arg-p ⁇ r ⁇ -nitroanalide (P-F-R-pNA) is specific for trypsin-like proteases which cleave at arginine (R).
• hK2 was found to have overall activity more than 10 times higher than hK2 v217 on P-F-R-pNA and neither protein showed an ability to hydrolyze MeO-Suc-R-P-Y-pNA, the chymotrypsin substrate.
• Other comparable substrates containing trypsin-like sites for cleavage were also tested and hK2 was found to hydrolyze the substrate P-F-R-pNA with the highest rate.
• CALPEKPAVYTKVVHYRKWIKDTIAAN which has both potential trypsin and chymotrypsin cleavage sites.
• hK2 v217 cleaved at both a trypsin (R-K) and chymotrypsin (Y-R) site with the trypsin-like cleavage at a 2:1 ratio over the chymotrypsin-like cleavage.
• R-K trypsin
• Y-R chymotrypsin
• phK2 v2 ⁇ was also incubated with this peptide and showed no amidolytic activity.
• hK2 showed specificity different than hK2 v2 ⁇ towards this peptide substrate.
• the sequence of peptide #3 in Figure 16 corresponds to amino acid residue -7 to +7 of phK2.
• This region contains the pro peptide, VPLIQSR, which is found as an N-terminal leader peptide in phK2 v217 .
• hK2 was able to cleave this peptide releasing the propeptide region, but hK2 v217 was not.
• To delineate if hK2 can cleave this pro sequence on a native substrate its ability to convert phK2 v217 to hK2 y217 was monitored.
• PSA has been shown to form complexes with ⁇ 2 macroglobulin (MG) and the serine protease inhibitor, antichymotrypsin (ACT).
• MG macroglobulin
• ACT antichymotrypsin
• hK2 was incubated with a series of common proteases present in human plasma (ACT, ⁇ 2-antiplasmin, antithrombin III, and c - antitrypsin (Travis and Salvesen, Ann. Rev. Biochem.. 52, 655 (1983)) and the mixtures were analyzed by Western blot (Figure 18). Any covalent complex of hK2 with these serpins should result in about a 80-100 kD band on SDS/PAGE under reducing conditions.
• ACT and ⁇ 2-antiplasmin formed significant complexes with hK2 ( Figure 18, lane 1 and 2).
• Antithrombin III (lane 3) and ⁇ l-antitrypsin ( ⁇ l protease inhibitor, lane 4) formed no detectable complex with hK2.
• MG a major component of blood plasma, also rapidly complexed with hK2 (lane 5). This complex corresponds to Mr of about 200 kD and 120 kD, which were also formed when PSA was incubated with purified MG ( Figure 18, lane 8, see below).
• hK2 did not form complexes with ⁇ l- antitrypsin, even though this protein inhibits a wide range of trypsin-like proteases (Loebermann et al, J. Mol. Biol. 177. 531 (1984); Carrell and Travis, TIBS, 10, 20 (1985)).
• hK2 formed a complex with ⁇ 2- antiplasmin since this protein has arginine residues in its inhibitor active site (Hunt and Dayhoff, Biochem. Biophv. Res. Comm.. 95, 864 (1980); Chandra et al., Biochemistry. 22, 5055 (1983); Potempa, et al., Science. 241. 699 (1985); Shieh et al, J. Biol. Chem.. 264. 13420 (1989); Mast et al., Biochemistry. 30, 1723 (1991)).
• ACT has a leucine in its inhibitor active site.
• hK2 was very unstable to the purification procedures employed which, as found with other proteases, may be due to its autocatalytic property, and makes it very difficult to purify hK2 or phK2 in quantities sufficient for use as immunogens and calibrators.
• phK2 v217 is highly stable and is converted to hK2 v2 , which was also stable, by trypsin digestion. Purified phK2 v217 and hK2 v217 provided immunogens to generate mAbs specific for hK2 and phK2.
• Example 10 Example 10
• hK2 expression is prostate-specific as other tissues, e.g., kidney and pancreas, showed no staining.
• 264 radical prostatectomy specimens of which 257 were from untreated patients ( Figure 20) and 7 were from androgen deprivation therapy treated patients ( Figure 21), were analyzed in a comparative study. Each specimen was analyzed for the cytoplasmic expression of hK2 in areas with benign epithelium, high grade prostatic intraepithelial neoplasia (PIN) and adenocarcinoma.
• PIN prostatic intraepithelial neoplasia
• Prostate tissue was weighed, measured in three dimensions and inked. The apex and base were amputated at a thickness of 4-5 mm and serially sectioned at 3 mm. The remaining prostate was serially sectioned at 4-5 mm intervals by knife perpendicular to the long axis of the gland from the apex of the prostate to the tip of the seminal vesicles. Transverse sections were prepared and stained with hematoxylin and eosin. A single slice of the radical prostatectomy of each patient which encompassed cancer and benign tissue was fixed in 10% neutral buffered formalin and embedded in paraffin by methods well known to the art.
• Tissue sections on slides were deparaffinized by immersion in xylene and then in 95% ethanol. Endogenous peroxidase activity was blocked by incubating sections for 10 minutes in methanol/H 2 O 2 and then rinsing sections in tap water. Sections were then placed in 10 mM citrate buffer, pH 6.0, and steamed for 30 minutes. Sections were cooled for 5 minutes prior to rinsing in cold running tap water. Nonspecific protein binding was blocked by incubating sections for 10 minutes with 5% goat serum. Slides were then gently drained.
• Specimens with high grade PIN showed intense immunoreactivity throughout the cytoplasm and in the cytoplasmic apical blebs in a majority of cases. There was no apparent difference in the immunoreactivity among the different patterns of PIN except for the cribiform pattern which was usually decreased in intensity centrally when compared to the periphery.
• Carcinoma specimens showed intense cytoplasmic reactivity in virtually all cases.
• Cells with abundant cytoplasmic vacuoles showed less staining, including signet ring cells and areas of mucin; otherwise the cytoplasm was intensely stained.
• the greatest intensity was observed in the highest grade adenocarcinoma (Gleason pattern 4) which showed immunoreactivity in virtually every case.
• Foci with cribiform carcinoma were similar to cribiform PIN in that there was a greater intensity in the periphery than centrally. The peripheral edge and the advancing edge of the carcinomas were always intensely immunoreactive .
• cytoplasmic hK2 expression in prostate tissue is correlated with prostatic neoplasia and prostate cancer.
• the data from prostate obtained from androgen deprivation therapy treated patients is not statistically significant due to the small sample size, there is a decrease in hK2 expression in benign epithelium, high grade PIN and adenocarcinoma in these patients relative to untreated patients.
• an increase in hK2 expression in prostate is a novel marker for high grade PIN and prostate cancer.
• hK2 expressing cells present in tissue biopsies, e.g., prostate capsule, bone marrow or lymph node, or in physiological fluid, e.g., blood, serum or seminal fluid.
• tissue biopsies e.g., prostate capsule, bone marrow or lymph node
• physiological fluid e.g., blood, serum or seminal fluid.
• the method can detect a single hK2 expressing cell in a sample comprising at least about 10 4 , more preferably at least about 10 6 , or even more preferably 10 7 , cells.
• RT-PCR reverse transcriptase-polymerase chain reaction
• cells of the human PSA- and hK2-expressing LNCaP cell line were serially diluted in samples of buffy coat cells.
• the buffy coat cells were isolated from whole blood from normal females and males. Venous blood (5-7 ml) was collected in citrate-dextrose tubes. Samples were centrifuged at 1000 x g for 15 minutes at 4°C. Buffy coat cells were recovered from the top of the cell pellet.
• RNA samples were further extracted with chloroform-butanol (4:1; v/v) to remove residual heme, which can inhibit both reverse transcription and polymerase chain reactions.
• the isolated RNA was then treated with RNase-free DNase. To prepare first strand cDNAs, an aliquot containing 1 ⁇ g of total
• RNA was added to a reverse transcription reaction containing 100 pmol of a PSA-specific oligonucleotide primer (5' TCATCTCTGTATCC 3'; SEQ ID NO: 13) or 100 pmol of an hK2-specific oligonucleotide primer (5' GAGTAAGCTCTA 3'; SEQ ID NO: 14), and Moloney murine leukemia virus reverse transcriptase (GIBCO BRL), and brought to a final volume of 25 ⁇ L (50 mM Tris-HCl, pH 8.3, 75 mM KC1, 3 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM each dNTP, and 800 U Moloney murine leukemia virus reverse tranascriptase).
• a PSA-specific oligonucleotide primer 5' TCATCTCTGTATCC 3'; SEQ ID NO: 13
• an hK2-specific oligonucleotide primer 5
• PSA-specific oligonucleotide primed first strand cDNAs was amplified in a PCR (0.2 mM each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KC1, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 0.1% (w/v) gelatin) with a PSA-specific primer pair.
• PSA PCR employed 50 pmol of PSA-1 (5' GATGACTCCAGCCACGACCT 3'; SEQ ID NO: 15) and 50 pmol of PSA-2 (5' CACAGACACCCCATCCTATC 3'; SEQ ID NO: 16).
• hK2 PSA-specific oligonucleotide primed first strand cDNAs was amplified in a PCR (0.2 mM each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KC1, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 0.1 %> (w/v) gelatin) with an hK2-specific primer pair.
• the hK2 PCR employed 50 pmol of hK2-l (5' GAGGGTTGTGTACAGTCATGGAT 3'; SEQ ID NO: 17) and 50 pmol of hK2-2 (5'
• the cycling parameters employed were: thirty-five to forty cycles of 94°C for 1 minute; 58°C (PSA) or 60°C (hK2) for 90 seconds, and 72°C for 90 seconds. The final cycle was at 72°C for ten minutes. Aliquots of the reaction were electrophoresed on 1.0% agarose gels The gels were stained with ethidium bromide and were viewed under ultraviolet light. Some of the amplified products were excised from the gel and subcloned into a pCRII vector (Invitrogen, San Diego, CA) for sequencing.
• Prostate cancer pathological stages A-C are localized forms of prostate cancer.
• Pathological stage Dl is prostate cancer which has spread to the nodes (nodal metastases).
• Pathological stage D2 is systemic (systemic metastases) prostate cancer.
• hK2 RNA may serve as a useful marker for early detection of micrometastasis of prostate cancer.
• Tyr Arg Lys Trp lie Lys Asp Thr Ile Ala Ala Asn Pro 225 230 235
• CAC AGC TTC CCA CAC CCG CTC TAC AAT ATG AGC CTT CTG AAG CAT CAA 336 His Ser Phe Pro His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gin 95 100 105
• GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC ACC ATC 720 Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile 225 230 235 240
• GGTATCACGT CATGGGGCAG TGAACCATGT GCCCTGCCCG AAAGGCCTTC CCTGTACACC 660

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