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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
  • G01N2474/20—Immunohistochemistry assay
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/975—Kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
  • Y10S436/813—Cancer

Definitions

• the invention relates generally to prostate-specific antigen (PSA) and specifically to different forms of PSA and their association with prostate cancer and benign prostate disease
• the measurement of serum PSA is a widely used marker for the early detection of
• BPH benign prostate hyperplasia
• ACT ⁇ jantichymotrypsin
• PSA contains an internal peptide bond cleavage at Lysl45 which renders it inactive (4, 6,
• PSA was isolated from BPH nodules (9), where it was found to contain more enzymatically
• object of the present invention to provide a sensitive method for distinguishing BPH from
• prostate the present invention has examined three different types of prostate tissue 1) non-replasia
• PZ peripheral zone
• PSA free forms of PSA may help distinguish BPH and PCa in patients with prostate disease
• one aspect of the present invention provides a method for determining
• PSA prostate specific antigen
• step (b) determining the amount of BPSA in the sample, and (c) mathematically combining the results of step (a) and step (b)
• Another aspect of the present invention provides a diagnostic method for
• a further aspect of the present invention provides a diagnostic kit for determining
• first and the second agents respectively, comprise a detectable label
• Fig. 1 is a high-performance hydrophobic interaction chromatographic profile of
• transitional zone TZ
• peripheral zone containing 80-
• Fig. 2 is HIC-HPLC profile of PSA purified from TURP prostate tissue
• the BPSA (22%) elutes at 8 min and the other forms of PSA elute at 10 min
• Fig. 3 is HIC-HPLC profile of PSA purified from prostate tissue Fig 3A shows
• Fig. 4 is HIC-HPLC profile of the reaction mixture of PZ-N PSA incubated with excess ACT Peak 1 is inactive ACT which has been cleaved by PSA Peak 2 is the
• Fig. 5 shows dot plots of BPSA and pPSA in 18 matched sets of prostate tissues
• the bottom panel shows the ratio of BPSA/pPSA TZ, transition zone, PZ-N, peripheral
• zone-non cancer PZ-C
• peripheral zone-cancer
• Fig. 6 is the linear sequence of amino acids for PSA The arrows show the sites of
• the present invention is based on the unexpected discovery that different forms of
• PSA are associated with different prostate tissues or different prostate diseases
• the prostate is composed of three zones the central zone, the peripheral zone (PZ)
• the PZ comprises about 70% of the volume of a normal
• the TZ is characterized by small, simple glands embedded in a
• the TZ tissue forms a distinctive boundary with the PZ
• the PZ and TZ are the
• the TZ tissue surrounds the proximal prostate urethra
• the present invention discovers that the ratio of proPSA and BPSA
• one aspect of the present invention provides a method for determining
• PSA prostate specific antigen
• proPSA or "pPSA” as used herein refers to a precursor form of PSA
• a full-length precursor form of PSA includes a propeptide of 7 amino acids, APLILSR,
• proPSA is a proPSA with its terminus starting at -7aa of the
• propeptide It contains the full-length proPSA [-5] proPSA indicates that the terminus of
• the proPSA starts at -5aa of the propeptide, and it contains the last five amino acid sequence of the propeptide sequence of proPSA, etc
• proPSA of the present invention includes both full-length and truncated forms of
• proPSA with its terminus started at any amino acid of the propeptide of the proPSA
• proPSA of the present invention examples include, but are not limited to, [-ljpPSA, [- 2]pPSA, [-4]pPSA, [-5]pPSA and [-7]pPSA
• a proPSA of the present invention particularly [-2]proPSA and [-4]proPSA, exists
• the level of proPSA is elevated if the percentage of the proPSA compared to total PSA is higher than the percentage of the
• proPSA occurring in the transition zone of prostate tissues
• proPSA extracted from prostate tissues contains up
• proPSA of the present invention The proPSA is lower or absent in the
• proPSA may be used as a marker associated with prostate
• the proPSA is inactive, i e , it lacks chymotryp sin-like enzymatic activity and
• a free PSA is a PSA that is not complexed as part of an antichymotrypsin complex
• ProPSA of the present invention may be made by methods commonly known in the
• proPSA of the present invention are discussed in a co-pending U.S patent application entitled “Forms of Prostate Specific Antigen and Methods for Their Detection,” filed
• BPSA BPSA
• PSA has 237 amino acid residues with a molecular mass of 28,400 D (13) and the amino
• a BPSA of the present invention has at least one clip at Lysine
• present invention has the same amino acid sequence of a mature form of PSA, except that
• polypeptide chain of the PSA of the present invention has been hydrolyzed between
• a BPSA of the present invention consists of two clips at Lysl45 and
• a BPSA of the present invention exists at an elevated level in the transition zone of
• the level of BPSA is elevated if the percentage of the
• BPSA compared to total PSA is higher than the percentage of the BPSA occurring in
• peripheral zone cancer and non-cancer prostate tissues are provided.
• PSA extracted from BPH tissues contains from 5 to
• BPSA of the present invention 30%) of BPSA of the present invention
• the BPSA are lower or absent in peripheral zone
• BPSA of the present invention may be specific for BPH
• the BPSA is inactive, i e , it lacks chymotryp sin-like enzymatic activity and
• a free PSA is a PSA that is not complexed as part of an antichymotrypsin complex
• the BPSA of the present invention may be isolated from tissues or seminal plasma
• BPSA of the present invention may be separated from other forms of PSA by
• HIC-HPLC technique a technique that is well known in the art BPSA of the present
• PSA Benign Prostatic Hyperplasia
• an antibody which consists essentially of pooled monoclonal antibodies with
• Monoclonal antibodies are made from an antigen containing the novel form of
• PSA of the present invention or fragments thereof by methods well known in the art (E Harlow et al , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988)
• this method involves preparing an antibody-producing fused cell line, e g , from
• polyclonal antibodies can be further purified, for example, by binding to
• antibody as used in this invention includes intact molecules as well as
• fragments thereof such as Fab, F(ab') 2 and Fv, which are capable of binding the epitopic
• one aspect of the present invention provides an antibody that is
• the antibodies of the present invention preferentially recognize and bind to proPSA or BPSA than other forms of PSA, such as other clipped or non-clipped mature forms of PSA
• present invention bind to proPSA or BPSA of the present invention to a greater extent than
• antibodies that preferentially bind to proPSA include, but are not limited to, PS1Z134,
• PS1Z120, PS1Z125 and PS1Z80 examples include, but are not limited to, PS2C109, PS2C501, PS2C634, PS2C807 and
• Antibodies of the present invention may be used for detecting and determining the
• the proPSA and BPSA may be detected in patient tissue samples by
• an antibody preferably a monoclonal antibody
• a sample may be contacted with an antibody against proPSA and an antibody against BPSA simultaneously, provided that
• the tissue specimen is a tissue specimen obtained
• the prostate tissue may be a normal prostate tissue, a
• immunoassay procedures are also well known in the art and require no repetition herein
• immunoassay procedures are generally described in Paterson et al , Int. J. Can. 37 659 (1986) and Burchell et al , Int. J. Can. 34 763 (1984)
• proPSA and BPSA in a biological sample comprises the steps of (a) contacting an amount
• a sample may be contacted by the first and the second agents
• the agents may be labeled differently or are capable of
• the biological sample can be any human
• physiological fluid sample that contains either proPSA or BPSA of the present invention
• human physiological fluid sample examples include, but are not limited to, serum,
• antibodies may be used as long as such antibodies possess the requisite specificity for the
• antigen provided by the present invention Preferably, monoclonal antibodies are used.
• Monoclonal antibodies can be utilized in liquid phase or bound to a solid phase
• carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,
• the carrier can be either soluble or insoluble for purposes of the invention.
• insoluble carriers include, but are not limited to, a bead and a microtiter plate Those
• the monoclonal antibodies in these immunoassays can be detectably
• monoclonal antibodies of the present invention can be coupled to low molecular weight haptens These haptens can then be specifically
• biotin which reacts with avidin, or dinitrophenyl, pyridoxal and fluorescein, which can
• inventions can also be coupled with a detectable label such as an enzyme, radioactive
• enzymes that can be used as detectable labels are horseradish peroxidase, malate
• dehydrogenase staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,
• alkaline phosphatase alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
• radioactive isotope The presence of the radioactive isotope would then be determined by
• fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
• Fluorescence-emitting metal atoms such as Eu (europium), and other lanthanides
• chelating groups such as DTPA or EDTA
• immunoglobulin is then determined by detecting the presence of luminescence that arises
• labeling compounds are luminol, isoluminol, aromatic acridinium ester, imidazole,
• bioluminescent compound may also be used as a label
• Bioluminescence is a special type of chemiluminescence which is found in biological
• luciferin luciferase
• aequorin luciferase
• invention in a sample may be accomplished by competitive or non-competitive
• immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay
• immunometric assay or "sandwich immunoassay” includes a
• proPSA and BPSA may be used as a serum marker or as an immunohistological
• proPSA and BPSA alone may also be used as a serum marker or as an immunohistological marker for distinguishing BPH from prostate cancer
• BPSA immunohistological marker
• the mathematical combination is a ratio
• ratio of proPSA and BPSA in a sample may be determined by comparing the amount of
• Another aspect of the present invention provides a diagnostic method
• the proPSA and a second binary complex comprising the second agent and the BPSA
• the agents comprise
• antibodies particularly monoclonal antibodies of the present invention
• a monoclonal antibodies of the present invention Preferably, when a monoclonal antibodies of the present invention
• sample is contacted with the first and the second agents, the first and the second agents
• the sample may be contacted
• sample may be contacted with another agent in order to detect another form of PSA
• the sample may be a sample of human
• the sample may be tissue specimen from the sample.
• the agent may be an
• antibody particularly a monoclonal antibody of the present invention.
• the mathematical combination is a ratio
• Another aspect of the present invention also provides a diagnostic kit for
• the kit includes
• first and the second agents respectively, comprise a detectable label or bind to a detectable label
• the sample may be a sample of human
• physiological fluid such as, but not limited to, serum, seminal plasma, urine or plasma.
• sample may also be a tissue specimen coming from the prostate of a patient
• agent a tissue specimen coming from the prostate of a patient
• an antibody particularly a monoclonal antibody of the present invention
• the first and the second agents respectively, comprise a different detectable label or bind to a different detectable label
• proPSA or BPSA alone may be used as a serum marker or as an
• invention also provides a diagnostic method for distinguishing BPH from prostate cancer by
• proPSA or BPSA may be determined in patient tissue samples by
• Prostate tissue was frozen in liquid nitrogen and pulverized to a fine powder in a
• PSA was purified from the filtered supernatant solution by passage over an immunoaffinity column containing bound anti-PSA mAb, PSM773, at 5 mg per ml of resin
• ProPSA was purified from the medium of AN 12 by the use of immunoaffinity
• proPSA were generated as described in the pending U S patent application serial number
• mice were immunized once with 50 ug of blocked immunogen in CFA
• mice were immunized once with 50 ug of mutant proPSA 217 ser-gly immunogen in CFA and twice 25 ug of same immunogen in IFA
• PSA was immunoaffinity purified from tissue extracts using the anti-PSA
• FIG. 1 shows the comparative HIC-HPLC profile of the PSA purified from
• the PSA peak eluting at 8 min contains 28% of the total PSA in the TZ extract, while it is present at only 3% and 8% in the PZ-C and
• the PSA eluting at 8 min has been
• BPSA Since the absolute value of BPSA was seen to vary between 5 and
• FIG. 2 shows the HIC-HPLC profile of the PSA purified from TURP tissue BPSA represented 22% of
• tissues contained high levels of BPSA, with an average of 20% ⁇ 3% of the total PSA
• FIG. 3B shows that the HIC-HPLC purified BPSA and PSA were
• Table 1 shows the percentage of internal clips present in each form of PSA Five
• sequence comparison of these two peaks is distinctive in two ways 1) the 8 min peak contains a high percentage of internal clips including a distinctive clip at Lysl82, and 2) the
• the clip at Lysl82 is the most distinctive feature of the 8 min BPSA peak and
• the clip at Lysl45 is the primary internal cleavage site in PSA purified from seminal
• the PSA from 18 sets of matched prostate tissues was examined 10 from large volume prostates (>50g) and 8 small volume prostates ( ⁇ 20g) The percentage of the total
• PSA present as BPSA is shown in Table 2 In the majority of the samples, the TZ contains
• the inactive forms of PSA in tissue extracts include [-
• Peak 1 in Fig. 4 is the inactive clipped ACT formed after incubation with PSA (7). Peak 2 is the residual excess active ACT remaining after
• Peak 3 is the PSA-ACT complex
• peak 4 is the inactive PSA that did not
• the [-2]pPSA is a truncated form of pPSA which
• the [-2]pPSA contains the serine-arginine dipeptide on the N-terminal isoleucine of mature
• FIG. 5 shows a
• BPSA BPSA has a
• PSA may be a general marker for PSA derived from the TZ
• the forms of PSA in tissues differ from
• PSA derived from pooled seminal plasma contained low levels of BPSA, ranging from 5-10%), while the major fraction of PSA eluting at 10 min by HIC-HPLC was about
• Lys 148 should be Lys 145
• no pPSA in the inactive PSA may be detected from pooled
• the present invention also provides monoclonal antibodies specific for the BPSA
• serum free PSA levels reflect the population of PSA present in prostate tissue disease states, then an immunoassay which measures BPSA and pPSA may improve
• PSA precursor form of PSA

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• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
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• Peptides Or Proteins (AREA)
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• Investigating Or Analysing Biological Materials (AREA)
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• 1999
  • 1999-04-30 US US09/303,339 patent/US6423503B1/en not_active Expired - Lifetime
• 2000
  • 2000-04-12 AT AT00922118T patent/ATE308756T1/de not_active IP Right Cessation
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