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WO2004099780A1 - Dosages immunologiques pour la detection de formes de recepteur d'urokinase - Google Patents

Dosages immunologiques pour la detection de formes de recepteur d'urokinase Download PDF

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Publication number
WO2004099780A1
WO2004099780A1 PCT/DK2004/000324 DK2004000324W WO2004099780A1 WO 2004099780 A1 WO2004099780 A1 WO 2004099780A1 DK 2004000324 W DK2004000324 W DK 2004000324W WO 2004099780 A1 WO2004099780 A1 WO 2004099780A1
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Prior art keywords
upar
cancer
psa
domain
antibody
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Inventor
Timo Piironen
Gunilla HØYER-HANSEN
Nils BRÜNNER
Michael Ploug
Karin List
Birgitte Laursen
Keld DANØ
Hans Liljas
Alexander Haese
Huland Hartwig
Magnus TØRNBLOM
Ove Gustafsson
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Rigshospitalet
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Rigshospitalet
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    • 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
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Definitions

  • This invention relates to the design of novel immunoassays specific for the measurement of various urokinase plasminogen activator receptor forms and their use for the differential prognosis of prostate cancer from healthy control subjects or from subjects suffering from benign conditions in the prostate.
  • Suitable biological specimens for the immunoassay determinations are biological fluids; e.g. serum, plasma, whole blood, urine or cell culture medium samples.
  • the invention reveals diagnostic improvements in that higher clinical sensitivities and/or specificities can be obtained both in relation to the conventionally used determination of total prostate serum antigen, as well as to the more recently introduced method of determining the proportion of free prostate serum antigen (PSA-F) to total prostate serum antigen (PSA-T).
  • proteolytic enzymes plays an essential role in matrix degradation during the process of tissue remodelling associated with cancer invasion and metastasis.
  • the main cause of cancer patient morbidity and mortality is metastatic disease.
  • the proteases and their regulators involved in tumour invasion and metastasis comprise a group of molecules, which can be candidate prognostic or diagnostic tumour markers.
  • uPA urokinase plasminogen activator
  • Plasmin and uPA can cleave uPAR between domains 1 and 2, liberating domain 1, while uPAR domain 2+3 stays on the cell surface [H ⁇ yer-Hansen et al., 1992].
  • uPAR can be released from cells by cleavage at the lipid anchor by phospholipases or proteases, liberating full-length intact uPAR or domain 2+3.
  • Cell-bound, intact and cleaved uPAR are present in several neoplastic cell lines, in transplanted Lewis lung carcinoma in mice, in invasive human mammary carcinoma xenografted into nude mice, and in human ductal breast carcinoma.
  • a soluble circulating form of uPAR has been identified in plasma from healthy individuals and it has been shown that suPAR is elevated in patients with non-small cell lung cancer [Pappot et al., 1997] and colon cancer [Stephens et al., 1997]. Furthermore, a cleaved soluble form, suPAR domain 2+3 has been identified in cystic fluid from ovarian cancer patients [Wahlberg et al., 1998]. In addition, 5 some urine samples have been shown to contain uPAR fragments domain 1 and domain 2+3 by immunoprecipitation and Western blotting methods [Mustjoki et al., 2000].
  • uPAR fragments i.e. domain 1 and domain 2+3
  • the amounts of the cleavage products of uPAR and of suPAR in the circulation may be directly related to the level of uPA catalysed plasminogen activation and thus prove to be a stronger prognostic parameter than the measurement of bulk uPAR with presently available polyclonal immunoassays.
  • PSA prostate specific antigen
  • PSA has been shown to possess chymotrypsin-like protease activity.
  • the active single-chain form of PSA forms stable covalent complexes with several extracellular protease inhibitors, such as alpha-1-antichymotrypsin (ACT), alpha-2-macroglobulin (A G), pregnancy-zone protein
  • PSA-F protein C inhibitor
  • PCI protein C inhibitor
  • alpha-1-antitrypsin alpha-1-antitrypsin
  • the present inventors have designed and validated three novel monoclonal uPAR sandwich assays for the specific detection of 1) full-length intact uPAR, 2) full-length intact uPAR and domain 2+3 fragment, and 3) domain 1 fragment of uPAR.
  • the assays have been applied to sera from carefully characterised prostate cancer patients and control groups in order to study its clinical utility in urological practice. Relationship of these measured uPAR forms to conventional prostate cancer markers, PSA-T and PSA-F, is reported.
  • the present invention thus provides a method for diagnosis and prognosis of cancer such as prostate cancer. It further provides an immunoassay for specific detection of various forms of the urokinase plasminogen activator receptor (uPAR) using several novel monoclonal antibodies specifically binding to the different uPAR forms.
  • uPAR urokinase plasminogen activator receptor
  • a diagnostic or prognostic method, combining the results of the immunoassay and conventional methods for detecting prostate serum antigen (PSA) in a sample is presented.
  • the invention provides a kit for detection of the several different forms of uPAR.
  • This invention relates to the design of immunoassays for specific measurement of soluble and non-soluble urokinase plasminogen activator receptor (uPAR) forms and their use for the differential diagnosis of cancer.
  • uPAR urokinase plasminogen activator receptor
  • the present invention relates to a method of monitoring the presence or stage of a cancer disease in a mammal by specifically detecting, in a sample, the amount of at least one form of uPAR present therein and/or combining the detected amount(s) of the at least one uPAR form with data indicative of the presence of the cancer disease.
  • the combination results in a discriminating value, which indicates whether the patient has said cancer or will develop said cancer.
  • the discriminating value is determined by utilizing the combination on both a healthy control population and a population at risk of having or having said cancer, thereby obtaining the discriminating value.
  • the discriminating value identifies the risk population or cancer population with a predetermined specificity and/or a predetermined sensitivity.
  • the determination of the discriminating value requires the performer to determine the level of specificity and thereby obtaining a certain sensitivity.
  • a certain amount of error can always be expected due to biological diversity and therefore a 100% sensitivity can never be obtained in a prognostic or a diagnostic assay, which at the same time is 100% specific as well.
  • the selected specificity determines the acceptable percentage of false positive calls made by the assay. Normally in assays of this kind, sensitivity is scarified for specificity, so by decreasing the specificity the sensitivity can be increased. The number of patients and healthy individuals tested to obtain the discriminating value also affects the reference value.
  • the method is performed by using an immunoassay, being specific for the measurement of soluble and non-soluble uPAR forms.
  • the immunoassay is designed to detect all complexed and non-complexed forms of uPAR in a biological sample.
  • a composite is formed involving the use of a first antibody, comprising a portion bound to a solid phase and a region being able to bind to a uPAR (catching antibody), the uPAR of interest, a second antibody including a region being able to bind to the uPAR of interest and a portion which is labeled and measuring a fluorescence signal detecting antibody.
  • At least one of the catching and the detecting antibodies are monoclonal antibodies binding specifically to certain parts of the domains of uPAR's.
  • the monoclonal antibodies can also have the property of blocking other uPAR specific monoclonal antibodies to bind certain areas or domains of the uPAR and thereby preventing a detection antibody to bind to certain uPAR domains.
  • the immunoassay can be a time-resolved fluroimmunoassay (TR- FIA) or an enzyme linked immunosorbent assay (ELISA) where the label is a fluorescent compound such as, but not limited to, FITC, phycoerythrin(PE), Texas red, a Cy3 or a Cy5 conjugate, green fluorescence protein (GFP) or modifications thereof.
  • the label can also be a metal substance such as an Eu-chelate, a radioactive substance such as I 125 , S 35 , P 32 etc, or an enzyme such as horseradish peroxidase (HRP) and alkaline phosphatase (AP).
  • the method relates to detecting the presence or stage of a cancer disease such as colon cancer, rectal cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer and gastric cancer. Furthermore, the determination can be performed at several time points at intervals as part of a monitoring of a cancer patient after the treatment for primary cancer.
  • the method can also be used for detecting early stage cancer such as colon cancer Dukes' stage A, colon cancer Dukes' stage B, colon cancer Dukes' stage C, rectal cancer Dukes' stage A, rectal cancer Dukes' stage B and rectal cancer Dukes' stage C.
  • mammal relates to any of the higher vertebrate animals comprising the class mammalia such as but not limited to cats, dogs, pigs, sheep, cattle, horses and primates, wherein the primate is preferably a human.
  • the present invention relates to detecting at least two forms of uPAR selected from the group consisting of intact uPAR (uPAR-123), uPAR domain 2 + 3 fragment
  • uPAR-23 and uPAR domain 1 fragment (uPAR-1), or any combination thereof wherein all three uPAR forms are detected.
  • the uPAR may be soluble (suPAR), or bound to a membrane fragment as well as a complex of uPAR and other proteins.
  • the detected amount of the at least one uPAR form is indicative of the stage of the cancer, indicative for diagnosis of the cancer, or indicative for prognosis of the cancer.
  • the sample is a biological sample selected from the group consisting of blood, serum, plasma, faeces, saliva, urine, a cell lysate, a tissue sample, a biopsy, a tissue lysate, a cell culture and cerebrospinal fluid.
  • the assay can be performed on a sample in solution or in situ on a tissue sample or a biopsy.
  • the detected amount of at least two uPAR forms is combined with data indicative of the presence or stage of a cancer disease selected from the group consisting of colon cancer, rectal cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer and gastric cancer.
  • a cancer disease selected from the group consisting of colon cancer, rectal cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer and gastric cancer.
  • the combination between the detected amount(s) of uPAR form(s) and the data indicative of the presence or stage of the cancer disease is carried out by a method such as logistic regression analysis, artificial neural network analysis, neuro fuzzy network analysis, multilayer perceptron analysis, or learning vector quantization analysis.
  • the detected amount of the at least one uPAR form is combined with data indicative of the presence or stage of prostate cancer, wherein the data being indicative of the presence or stage of prostate cancer are data for total PSA (PSA-T), free PSA (PSA-F) or a value calculated on the basis of PSA data.
  • PSA-T total PSA
  • PSA-F free PSA
  • the specificity of the method of the invention is increased over currently known diagnostic methods for prostate cancer.
  • the diagnostic specificity for prostate cancer relative to diagnostic specificity of detecting PSA-T only, is improved by at least 10%.
  • Comparison of the performance of each analyzed parameter with that of PSA-T and PSA-T + PSA-F at selected sensitivity and specificity levels show that at 80 percent sensitivity level, all logistic regression analysis combinations significantly improved the specificity levels over that obtained by PSA-T alone.
  • the amount of at least one form of uPAR present in the sample is detected specifically using an immunoassay.
  • the immunoassay of the invention is characterized in that a first monoclonal antibody (Mab) is used for specifically binding the at least one form of uPAR to be detected.
  • the first antibody functions as a catcher molecule and it can either be bound to a surface, or it can be in a soluble form.
  • the first antibody is the monoclonal antibody R2, which recognizes domain 3.
  • the second antibody may be selected from the group consisting of R3 and Kl (see material and methods, p8. first paragraph).
  • the second antibody can be selected from the group consisting of R3 and Kl and the uPAR is selected from the group consisting of uPAR-123 and uPAR-23.
  • the second antibody can be labeled with a reporter molecule, which can be a lanthanide such as Eu.
  • the R3 antibody recognizes domain 1, whereas the Kl antibody recognizes domain 2+3. Therefore, in this context, the R3 antibody will only detect uPAR- 123, whereas the Kl antibody will detect both uPAR-123 as well as uPAR-23.
  • the signal from an assay using R2 and R3 or derived fragments thereof is subtracted from the signal from an assay using R2 and Kl.
  • the term antibody refers to whole, intact antibodies as well as derivatives and fragments thereof.
  • the amount of at least one form of uPAR present in the sample is detected specifically using an immunoassay.
  • an immunoassay is characterized in that the first antibody is the monoclonal antibody R5.
  • the second antibody may be R3, wherein the uPAR form detected is domain 1.
  • the second antibody can be labeled with a reporter molecule, including a lanthanide such as Eu.
  • a blocking agent is included in the assay.
  • a suitable blocking agent is a synthetic peptide (AE120), binding to intact uPAR.
  • the invention further comprises two new monoclonal antibodies.
  • the first monoclonal antibody, R5 is capable of binding to uPAR domain 1, and is produced by the hybridoma cell line deposited under the deposition number 03031101 in the European Collection of Animal Cell Cultures under the terms and conditions of the Budapest Treaty.
  • the antibody having immunologically equivalent binding specificity to uPAR domain 1, the R5 antibody, produced by the hybridoma cell line has been deposited under the deposition number 03031101 at the European Collection of Animal Cell Cultures under the terms and conditions of the Budapest Treaty.
  • the second monoclonal antibody, Kl is capable of binding to u-PAR domain 2+3 and is produced by the hybridoma cell line deposited under the deposition number 03031102 in the European Collection of Animal Cell Cultures under the terms and conditions of the Budapest Treaty. Furthermore, the antibody having immunologically equivalent binding specificity to domain 2 + 3 of uPAR, the Kl antibody, produced by the hybridoma cell line has been deposited under the deposition number 03031102 at the European Collection of Animal Cell Cultures under the terms and conditions of the Budapest Treaty.
  • Another embodiment of the present invention relates to detecting the amount of at least one form of uPAR present in the sample detected specifically using a protein microarray assay.
  • a protein microarray assay e.g., a protein microarray assay.
  • the capturing of the at least one form of uPAR is performed using monoclonal antibodies or fragments thereof and wherein the detection is performed using a mass spectrometer, such as a MALDI-TOF spectrometer.
  • a mass spectrometer such as a MALDI-TOF spectrometer.
  • the binding of at least one form of uPAR onto a surface can be utilized as catching mechanism and the detection can be made using a mass spectrometer, such as a SELDI-TOF spectrometer.
  • Another embodiment of the present invention is an immunoassay comprising a first antibody including a portion bound to a solid phase and a region being able to bind to a uPAR form and a second antibody including a region being able to bind to a uPAR and a portion to which has a label.
  • the uPAR form is selected from the group consisting of intact uPAR, uPAR domain 2 + 3 fragment and uPAR domain 1 fragment and any combination thereof.
  • a further embodiment of the present invention is a kit comprising the following components in any combination: A first antibody including a portion bound to a solid phase and a region being able to bind to a uPAR form, including an antibody selected from the group consisting of anti-uPAR Mab R2 or anti-uPAR Mab R5, a second antibody including a region being able to bind to a uPAR and a portion to which has a label, including an antibody selected from the group consisting of anti-uPAR Mab R3 and anti-uPAR Mab Kl and optionally a reference protein.
  • the reference protein can be any uPAR form from a sample or purified as a standard in an immunoassay
  • the uPAR form being selected from the group consisting of intact uPAR, uPAR domain 2 + 3 fragment and uPAR domain 1 fragment and any combination thereof.
  • Fig.l Relative epitope location for the antibodies used in the assays.
  • the arrows indicate the cleavage sites of different proteases.
  • TR- FIAs novel time-resoived fluoroimmunoassays
  • (C.) Domain 1 where domain 1 and intact uPAR are bound to the microtitre well by the domain 1 specific capture Mab R5, followed by the detection of bound domain 1 by the specific Eu-labeled Mab R3, which is not capable of binding to domain 1 within intact uPAR due to the binding of peptide AE120 specifically blocking the binding of detection Mab R3.
  • the curved line (right axis) illustrates the individual CV's for the measured standards (displayed by the straight line, left axis).
  • Fig. 4 Chymotrypsin cleavage of uPAR.
  • Intact recombinant suPAR (aa 1-277), 25 ⁇ g, was incubated with 2.5 ng of chymotrypsin for three days. Samples were taken at different time points (15 min to 3 days) and measured in the TR-FIAs (A.) and analysed by Western blotting (B. and C).
  • the primary antibodies used in the Western blots were R2 for detection of intact suPAR and suPAR-23 (B.), and R3 and R9 for detection of domain 1 and intact suPAR (C).
  • Fig. 5 Fractionated in vitro chymotrypsin cleaved uPAR.
  • suPAR (aa 1-277) was incubated with chymotrypsin for 3 days and the cleavage products were subsequently separated by FPLC size-exclusion column chromatography. The fractions were measured employing the following assays: the intact uPAR assay (black), uPAR-123+23 assay (green), domain 1 assay with (dark-blue) and without (light-blue) the antagonist peptide AE120 and polyclonal ELISA (purple). The amount of uPAR-23 was calculated (red).
  • Fig.6 Fractionated patient serum sample. Serum from a prostate cancer patient (100 ⁇ l) was separated by FPLC size-exclusion column chromatography. The fractions were measured employing the following assays: the intact uPAR assay (black), uPAR-123+23 assay (green), domain 1 assay (dark-blue) and polyclonal ELISA (purple). The amount of uPAR-23 was calculated (red).
  • Fig. 7 Fractionated non-spiked/spiked donor EDTA plasma pool. The plasma was spiked prior to the size-separation by FPLC size-exclusion column chromatography with (B.) 10 ng/mL suPAR-23 (aa 88-277), (C.) 10 ng/mL suPAR (aa 1-277), and (D.) 10 ng/mL uPAR-1 (aa 1-92). Non-spiked plasma is shown in figure (A.).
  • Fig. 8 Soluble uPAR forms in serum. Prostate cancer vs. non-cancer. Box plot showing l:st, 2:nd (median), and 3:rd quartile, as well as the 10:th and 90:th percentile. The serum samples were measured by the uPAR-1 assay, the uPAR-123 assay and the uPAR-123+23 assay. The amount of suPAR-23 was calculated. P-values were calculated using the Mann-Whitney U-test.
  • Fig. 9 Soluble uPAR forms in serum.
  • Prostate cancer vs. non-cancer - PSA-T grey zone 2-10 ng/mL. Box plot showing l:st, 2:nd (median), and 3:rd quartile, as well as the 10:th and 90:th percentile.
  • the serum samples were measured by the uPAR-1 assay, the uPAR-123 assay and the uPAR-123+23 assay.
  • the amount of suPAR-23 was calculated. P-values were calculated using the Mann-Whitney U-test.
  • Fig. 10 Receiver operating characteristics (ROC) curves. Prostate cancer vs. non- cancer - PSA-T grey zone 2-10 ng/mL. Areas under the curves are expressed for PSA- T assay, PSA-T+PSA-F assays, PSA-T+PSA-F+uPAR-123 +UPAR-123+23 assays, and PSA- T+PSA-F+uPAR-123 +uPAR-123+23+uPAR-l assays.
  • Fig. 11 Survival of prostate cancer patients. Box plot showing l :st, 2:nd (median), and 3:rd quartile, as well as the 10:th and 90:th percentile. Patients are divided into three groups (alive, death by prostate cancer, death by other causes) showing concentrations of (A.) suPAR-123, (B.) suPAR-123+23, and (C.) calculated suPAR-23.
  • ROC Receiver operating characteristics
  • R2, R3, and R5 have been raised against human uPAR purified from PMA-stimulated U-937 cells [R ⁇ nne et al., 1991], whereas Mab Kl have been raised against a human suPAR (amino acids 1-277), expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography as described previously [R ⁇ nne et al., 1994].
  • the polyclonal rabbit anti-uPAR used in the polyclonal uPAR ELISA was also raised against recombinant suPAR (amino acids 1-277) [R ⁇ nne et al.,1994].
  • R2 binds an epitope in domain 3 of uPAR with high affinity.
  • R3 and R5 are directed against different non-overlapping epitopes in domain 1 of uPAR, while Kl is directed against domain 2+3 of uPAR. Kl recognizes both intact and domain 2+3 but fails to bind domain 2 or 3 alone.
  • Intact suPAR was produced and purified as described above and domain 2+3 was generated by chymotrypsin cleavage of intact suPAR [Behrendt et al., 1991] and purified by size exclusion chromatography using a Sephadex HR 75 column [Ploug et al., 1993].
  • Recombinant uPAR domain 1 encompassing amino acids 1-92 of the mature protein [Roldan et al., 1990] was produced by use of a Drosophila Expression System (DES) (Invitrogen, Groningen, the Netherlands).
  • DES Drosophila Expression System
  • pMTC-uPAR-DI An expression vector denoted pMTC-uPAR-DI was constructed by generating a domain 1 fragment by standard PCR techniques using Pfu DNA polymerase followed by cloning into the plasmid pMT/V5-His.
  • the uPAR domain 1 was purified from the conditioned medium of S2 cells by immunoaffinity chromatography using the anti-uPAR monoclonal antibody R3 followed by reversed phase HPLC as described previously [Gardsvoll et al., 1999].
  • AE120 is a synthetic peptide antagonist of the uPA-uPAR interaction.
  • the amino acid sequence of AE120 is [D-Cha-F-S-R-Y-L-W-S] 2 - ⁇ A-K. Cha is ⁇ -cyclohexyl-L-alanine.
  • the K d value of AE120 has been assessed in real-time biomolecular interaction analysis (BIA) to 0.57 x 10 "9 M [Ploug et al., 2001].
  • BIOA biomolecular interaction analysis
  • Superdex 75 and 200 HR 10/30 FPLC-column, NAP-5 and NAP-10 gel filtration columns were from Amersham Pharmacia Biotech (Sweden).
  • the FluoStar Galaxy fluorometer was from BMG LabTechnologies (Germany).
  • the DELFIA ® Eu-labeling kit, assay buffer, wash solution, enhancement solution and ProstatusTM assay for PSA-T and PSA-F were from Perkin-Elmer Life Sciences (Finland).
  • White Maxisorp microtitration fluorostrips were obtained from NUNC (Roskilde, Denmark).
  • Antibodies underwent acid treatment before the coating, where one volume of antibody solution and six volumes of 0.01 M HCI solution (pH ⁇ 1.8) were incubated for 5 minutes after which they are transferred into 0.1 M NaH 2 P0 solution (pH ⁇ 4.2). This final solution was added to the plates (200 ⁇ L/well, 5 ⁇ g antibody/mL). The plates were then incubated over night at room temperature in a moisturized container. Following the incubation the plates were washed twice with Delfia Wash Solution added with 0.05% Tween 20.
  • Remaining plastic surface was blocked with a blocking solution containing 50 mM NaH 2 P0 4 , 0.1 % Diazolidinyl urea, 6% Sorbitol, 0.1%DTPA-purified BSA (300 ⁇ L/well). The next day the plates were aspirated and dried for 3 h under fume hood. Sealing tape was added and the plates were stored at 4 °C.
  • Mabs were labelled with Eu-chelates to the extent of 1-5 Eu/IgG and used for specificity, epitope mapping and affinity constant determinations with various uPAR forms by using two-site sandwich (Mab/Mab) time-resolved immunofluorometric assay design. Briefly, in order to remove the TRIS buffer containing amino groups capable of reacting with the later added Eu-chelate, antibody solution was added to the Pharmacia NAP-5 or NAP-10 columns and eluted with 0.05 M carbonate buffer, pH 9.8.
  • the antibody solution was added to 25-200 fold molar excess of Nl-Eu +3 chelate (N 1 -(p-isothiocyanatobenzyl)- diethylenetriamine-N 1 ,N 2 ,N 3 ,N 3 -Tetraacetate-Eu) and incubated over night at 4 °C.
  • the Eu- labeled antibody was separated from the free Eu-chelate by size exclusion chromatography using the Superdex 200 HR 10/30 column according to the instructions in Delfia Eu- labeling kit using TSA-buffer (pH 7.8) for elution.
  • Recombinant intact suPAR (1-277) cleaved for three days with chymotrypsin and subsequently diluted in Assay Buffer, and samples of different biological matrixes e.g. blood plasma, serum, and urine were run through a size separation column (Superdex 75 HR 10/30). TSA-buffer (pH 7.8) containing 1 ⁇ M EDTA was used for elution. The biological matrixes were diluted 1:2 in elution buffer before loading to the column. All samples were filtrated before use through a 0.22 ⁇ m filter. Collected fractions were stored at -80 °C until analysis.
  • biological matrixes e.g. blood plasma, serum, and urine were run through a size separation column (Superdex 75 HR 10/30). TSA-buffer (pH 7.8) containing 1 ⁇ M EDTA was used for elution. The biological matrixes were diluted 1:2 in elution buffer before loading to the column. All samples were filtrated before use through a 0.22
  • Affinity constant determinations of the Eu-labeled Mabs were carried out as described previously [Pettersson et al., 1995J. Briefly, one nanogram per well of purified uPAR-123, uPAR-23, uPAR-1 or standard diluent was incubated to equilibrium in strips coated with various anti-uPAR Mabs. Six concentrations of the labeled antibodies (6.25 to 200 ng/well) were then incubated for 3 hours with immobilized uPAR forms bound to the capture antibody. The signals obtained after subtracting the nonspecific binding from the total binding were used to calculate the affinities of the labeled antibodies according to the method of Scatchard [Scatchard et al., 1949].
  • Novel immunoassays According to the invention, three novel monoclonal uPAR sandwich assays were designed and validated for the measurement of 1) full-length intact uPAR, (uPAR-123), 2) full-length intact uPAR and domain 2+3 fragment, (uPAR-123 + 23) and 3) domain 1 fragment of UPAR (uPAR-1).
  • the results from immunoassays specific for various uPAR forms applied to biological samples from patients to be screened or tested for the presence of malignant or benign prostatic disorders are combined with those obtained from assays of the total and free fraction of PSA, in order to identify a decision point that provides the best or otherwise desired separation of individuals likely to have prostate cancer, benign prostatic hyperplasia, or are likely to present without prostatic lesions.
  • the cut-off concentrations vary depending on whether a high sensitivity or a high specificity is preferred in detecting prostate cancer.
  • the combination of uPAR forms with PSA forms can be accomplished through combination by logistic regression analysis.
  • Combinations by logistic regression analysis such as [PSA-T + PSA-F + uPAR-123 + (uPAR-123+23)] or [PSA-T + PSA-F + uPAR-123 + uPAR-123+23 + uPAR-1] frequently provide even better discrimination than simple algorithms, e.g. ratios calculated from the individual measurements from each patient.
  • Logistic regression analysis is instrumental in providing the basis for various "risk analysis systems that can provide medical decision support".
  • ANN artificial neural networks
  • NFN neuro fuzzy networks
  • MLP multilayer perceptron
  • LVQ learning vector quantization
  • Chymotrypsin cleavage study suPAR (1-277) was cleaved with chymotrypsin in vitro.
  • Reaction mixture (1 mL) contained 25 ⁇ g of suPAR and 2.5 ng (1/10000 w/w) of chymotrypsin ( ⁇ -chymotrypsin, EC 3.4.21.1, Worthington, Freehold, USA).
  • Two samples of 20 ⁇ L were taken from the reaction mixture after incubation for 15 min, 30 min, 1 h, 2 h, 4 h and 3 days.
  • Proteins were separated by SDS-PAGE using a 12% Acrylamide/B ⁇ s-Acrylamide NOVEX pre-cast gel, and then blotted (using semi-dry blotting system) onto a PVDF (Polyvinylidene difluoride) membrane (Immobilon-P Transfer Membranes, pore size 0.45 ⁇ m, Millipore).
  • the primary antibodies used were R2 (5 ⁇ g/mL) for the detection of uPAR- 23 and uPAR-123, and a mixture of 2.5 ⁇ g/mL of R3 and R9 (5 ⁇ g/mL in total) for the detection of uPAR-1 and uPAR-123.
  • Membranes were developed according to the WestemBreeze Chemiluminescent Immunodetection System for detection of mouse primary antibodies (Invitrogen).
  • Analytical detection limit was calculated at 3.3 times the standard deviation (SD) in counts per second (cps) in 20 replicates of Delfia Assay Buffer. The 3.3xSD was converted into concentration units with the corresponding assay standard curve.
  • the intra-assay precision of the immunoassays was determined by measuring 12 replicates of donor EDTA plasma pool. The inter-assay precision was determined by measuring the variation of duplicate measurements on 12 different assay runs. Study populations and specimen handling
  • SP1 consisted of 390 consecutive serum samples from patients referred to the Department of Urology at the University Clinic Hamburg (Germany). In 367 cases, patients were evaluated for the presence of PCa, due to elevated PSA-T concentrations, suspicious digital rectal examination (DRE) or both, to undergo systematic sextant biopsy (6Bx) of the prostate, which was performed using transrectal ultrasound (TRUS) guidance using the Bruel & Kjaer 3535 ultrasound scanner and 7.5 MHz transducer, model 8538. Biopsies were taken from the apex, mid region and base of the left and right peripheral zone. An additional 23 serum samples from patients undergoing transurethral or open surgery for symptomatic benign prostatic hyperplasia in whom pathological workup of the specimen revealed no evidence of cancer were added to the set of patients.
  • TRUS transrectal ultrasound
  • SP2 consisted of 87 serum samples from prostate cancer patients aged 55 to 70 years originally chosen for prostate cancer screening study in Sweden (Sweden) during 1988- 89. All patients were biopsied (triple or quadrant TRUS-guided biopsy) with abnormal findings on either DRE or TRUS or PSA-T value greater than 10 ng/mL. The screening was a nonrecurrent intervention and the survival of prostate cancer patients was followed during a 10-year follow-up period through the Swedish Cancer Registry. The assay for the detection of uPAR domain 1 was not included for the analysis of SP2 due to limited sample volume.
  • Mabs R3 and R5 were bound to domain 1 with affinity constants ranging from 1E8 to
  • the amount of the uPAR-23 is possible to calculate from the results of assays A and B by subtracting B - A.
  • uPAR domain 1 assay we used a recently developed high affinity peptide antagonist reacting with the intact receptor but not with the released domain 1. This peptide at 1 ⁇ M concentration almost totally blocked the binding of Mab R3 to domain 1 contained within the intact uPAR, thus released domain 1 could be measured even in the presence of intact receptor (Figure 2c). Standard curves of the three novel immunoassays are presented in Figure 3a-c.
  • Intact uPAR assay (D123): Monoclonal R2 was selected as coating antibody. Recombinant intact uPAR (1-277) standards from 0.014 to 10 ng/mL, zero standard, controls and samples (in general 1/2 - 1/10 dilutions) were diluted in Delfia Assay Buffer and added (100 ⁇ L/well) in duplicates to the plates followed by incubation with shaking for 1 h at room temperature. The plates were washed six times in Delfia Wash Buffer and Eu-labeled monoclonal R3 (R3-Eu) was added (100 ng/100 ⁇ L/well) succeeded by 2 h incubation at room temperature with shaking. The plates were then washed six times with Delfia Wash Buffer, followed by the addition of Delfia Enhancement Solution (100 ⁇ L/well). Fluorescence was measured after 5 min incubation at room temperature with shaking.
  • Intact uPAR + domain 2+3 assay (D123+23): The protocol is the same as above except that the Eu-labeled monoclonal Kl (Kl-Eu) was used as detecting antibody instead of R3-Eu. Recombinant uPAR-123 was used as standard material since the assay detects uPAR-23 and uPAR-123 with similar affinity constants (Table 1).
  • uPAR domain 1 assay (Dl): Mab R5 was selected as coating antibody. The protocol is the same as above with following exceptions: Recombinant uPAR-1 (1-92) was used as standard material, the sample incubation time is 2 hours, and 1 ⁇ M of the peptide AE120 is added together with R3-Eu to prevent signal from bound uPAR-123. After thorough characterization of in-house Mabs in terms of their domain specificity on the uPAR molecule, it was concluded that none of the antibodies fully discriminated between uPAR-123 and domain 1. Therefore, by using this recently developed high affinity peptide antagonist reacting only with intact receptor and not with the free domain 1 it was possible to block selectively domain 1 epitopes of Mab R3 contained within the intact receptor.
  • Intact suPAR as well as domain 1 and domain 2+3 fragments were identified by the novel immunoassays in the chymotrypsin cleavage study, where recombinant suPAR-123 (1- 277) was cleaved with chymotrypsin under mild conditions. The only observed cleavage after the chymotrypsin treatment was a cleavage at the potential chymotrypsin cleavage site between amino acids 87 and 88 thus liberating domain 1 ( Figure 4).
  • the cleavage study demonstrated the capability of the three novel immunoassays to detect the three different uPAR forms (uPAR-123 , uPAR-23, and uPAR-1), by displaying the progressive cleavage of uPAR-123 over time (Figure 4A.).
  • the increase in the uPAR-1 level was nearly identical to the decrease in the uPAR-123 level.
  • the uPAR-123+23 assay was showing a stable amount of intact uPAR and uPAR-23.
  • the cleavage patterns shown with immunoassays was verified by Western blotting ( Figure 4B and 4C).
  • Analytical detection limits of the D123, D123+23, and Dl assays were 0.01 ng/L, 0.01 ng/L and 0.02 ng/L, respectively.
  • Assay (intra-assay) co-efficient of variation (CV) for the D123, D123+23, and Dl assays were 5.6%, 3.9% and 7.8%, respectively.
  • Corresponding between assay (inter-assay) precision figures were 14.3%, 14,2% and 30.9%, respectively.
  • mean PSA-T was 8.7 ng/mL (range 0.27-87.1 ng/mL).
  • Prostatic biopsy revealed prostate cancer in 225 cases whereas no evidence of malignancy according to sextant biopsy was noted in 165 patients.
  • Mean/median PSA-T concentrations in patients with malignant biopsy result were 10.2/7.8 ng/mL (range 0.27- 87.1 ng/mL) and 6.8/5.5 ng/mL (range 0.65-39.8 ng/mL) in patients with benign biopsy result (Table 2A).
  • Mean/median age of patients in whom prostate biopsy was positive for cancer was 62/63 years (range 43-76 years), and 65/64 years (range 47-85 years) in patients with nega tive biopsy result.
  • SP1 (Hamburg) : Prostate cancer vs. non- cancer; descriptive statistics - no restrictions
  • Table 2B ( HamDL, rg): Prostate cancer vs. non-cancer; descriptiv statistics - PSA-T "grey zone" 2-10 ng/mL
  • the tested parameters resulting in significant sensitivity improvements are those containing uPAR-1 assay, e.g. showing a significant improvement of 19-24% in sensitivity at 80% specificity level.
  • SP1 (Hamburg): Prostate cancer vs. non-cancer; multivariate analysis
  • R2 90101008 at The European Collection of Cell Cultures (ECACC)
  • R3 90101009 at The European Collection of Cell Cultures (ECACC)
  • Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J. Biol. Chem. 1992, 267: 18224-18229.

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Abstract

La présente invention a trait à la conception de trois nouveaux dosages de type sandwich du récepteur de l'activateur du plasminogène de type urokinase (uPAR) monoclonal pour la détection spécifique de 1) de l'uPAR intact pleine longueur, 2) de l'uPAR intact pleine longueur et de fragment 2+3 de domaine de liaison, et 3) de fragment 1 de domaine de liaison à l'uPAR. L'invention a également trait à un procédé combinant les quantités détectées de formes de l'uPAR dans un échantillon avec des données indiquant la présence de maladie cancéreuse. Le procédé peut être utilisé pour la stadification, le pronostic ou diagnostic de cancer de la prostate. L'invention a trait en outre à deux nouveaux anticorps monoclonaux et une trousse et des dosages immunologiques pour la détection d'au moins une forme de l'uPAR.
PCT/DK2004/000324 2003-05-06 2004-05-06 Dosages immunologiques pour la detection de formes de recepteur d'urokinase Ceased WO2004099780A1 (fr)

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US6159330A (en) * 1998-09-18 2000-12-12 Moore U.S.A. Inc. Pressure sealer serrated sealing roll
WO2011047688A1 (fr) * 2009-10-22 2011-04-28 Rigshospitalet Valeur pronostique et prédictive des formes intactes et clivées du récepteur de l'activateur de la plasminogène urokinase dans le cancer de la prostate
US8105602B2 (en) 2006-12-08 2012-01-31 Tactic Pharma, Llc Urokinase-type plasminogen activator receptor epitope, monoclonal antibodies derived therefrom and methods of use thereof
WO2012076812A1 (fr) * 2010-12-08 2012-06-14 bioMérieux Procede et coffret pour le diagnostic in vitro d'un cancer de la prostate
CN112630442A (zh) * 2020-11-03 2021-04-09 浙江大学 一种血浆可溶性尿激酶型纤溶酶原激活物受体及其应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6159330A (en) * 1998-09-18 2000-12-12 Moore U.S.A. Inc. Pressure sealer serrated sealing roll
US8105602B2 (en) 2006-12-08 2012-01-31 Tactic Pharma, Llc Urokinase-type plasminogen activator receptor epitope, monoclonal antibodies derived therefrom and methods of use thereof
WO2011047688A1 (fr) * 2009-10-22 2011-04-28 Rigshospitalet Valeur pronostique et prédictive des formes intactes et clivées du récepteur de l'activateur de la plasminogène urokinase dans le cancer de la prostate
WO2012076812A1 (fr) * 2010-12-08 2012-06-14 bioMérieux Procede et coffret pour le diagnostic in vitro d'un cancer de la prostate
FR2968767A1 (fr) * 2010-12-08 2012-06-15 Biomerieux Sa Procede et coffret pour le diagnostic in vitro du cancer de la prostate
CN112630442A (zh) * 2020-11-03 2021-04-09 浙江大学 一种血浆可溶性尿激酶型纤溶酶原激活物受体及其应用
WO2022095227A1 (fr) * 2020-11-03 2022-05-12 浙江大学 Récepteur de l'activateur du plasminogène de type urokinase soluble dans le plasma et son application

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