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WO1996024065A1 - Procedes de detection de procathepsines et leurs emplois - Google Patents

Procedes de detection de procathepsines et leurs emplois Download PDF

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Publication number
WO1996024065A1
WO1996024065A1 PCT/US1996/001279 US9601279W WO9624065A1 WO 1996024065 A1 WO1996024065 A1 WO 1996024065A1 US 9601279 W US9601279 W US 9601279W WO 9624065 A1 WO9624065 A1 WO 9624065A1
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Prior art keywords
pro
cathepsin
antibody
breast cancer
sample
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PCT/US1996/001279
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English (en)
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WO1996024065A9 (fr
Inventor
James R. Zabrecky
David E. Jarosz
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Oncogene Science, Inc.
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Publication of WO1996024065A1 publication Critical patent/WO1996024065A1/fr
Publication of WO1996024065A9 publication Critical patent/WO1996024065A9/fr

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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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/57415—Specifically defined cancers of breast

Definitions

  • prognostic information Besides tumor size and node status, numerous parameters are currently being used in an attempt to gain prognostic information. These include estrogen and progesterone receptor status, histopathological classification, histologic and nuclear grade, and DNA-ploidy and S-phase fraction.
  • epidermal growth factor receptor oncogenes such as eriB-2/neu and myc
  • tumor suppressor genes like p53 and NM23
  • angiogenic factors like topoisomerase 2, bFGF
  • proteinases such as cathepsin D and urokinase-type plasminogen activator (uPA) .
  • uPA urokinase-type plasminogen activator
  • Proteinases are attractive as prognostic and diagnostic tools since it is well established that proteolytic activity is necessary for the process of invasion and metastasis.
  • Three critical steps in the transition from an in si tu carcinoma to one that is metastatic include the attachment of tumor cells to the basement membrane, localized proteolysis and the eventual migration of the tumor cells through the digested stroma where they gain access to the vascular system (Liotta, 1990) . Circulating tumor cells are then capable of attaching to the endothelial surface and initiating metastatic colonies.
  • An imbalance in localized proteolytic activity may involve increases in the proteolytic enzymes, reduced levels of their endogenous inhibitors or both.
  • Classes of proteinases that have been implicated in the metastatic process include matrix etalloproteinases, cathepsins B and L (cysteine proteinases) , cathepsin D (aspartic proteinase) and plasminogen activator (serine proteinase) .
  • matrix etalloproteinases cathepsins B and L (cysteine proteinases)
  • cathepsin D aspartic proteinase
  • plasminogen activator serine proteinase
  • PSA prostate specific antigen
  • CD cathepsin D
  • Rochefort, 1990 Rochefort, 1992
  • CD is a normal lysosomal proteinase which is synthesized as 52 kDa pro-enzyme and processed to the active, two chain form of 34 and 14 kDa subunits (Rochefort, 1990) .
  • a relationship between CD and cancer was first proposed when it was discovered that the 52 kDa proenzyme was secreted by the hormone-dependent human breast cancer cells, MCF-7 (Westley, 1980) .
  • CB cathepsin B
  • CL cathepsin L
  • pro-CD pro-CD was measured by immunoradioassay in breast cancer cytosols (Brouillet, 1993) . Although pro-CD correlated with total CD, it showed no prognostic value for survival. The authors point out, however, that the proportion of pro-CD recovered in vivo was only 1 to 6% of that produced in cell lines.
  • a key to developing good prognostic markers is to understand the biological changes that occur when a normal cell progresses to a tumor cell and then becomes invasive. Changes in the expression, processing and activity of proteolytic enzymes have been demonstrated for several human cancers where they are thought to play a critical role in the degradation of extracellular matrix proteins.
  • PSA which is a proteinase
  • PSA has proven to be a very useful prognostic marker for prostate cancer (Lilja, 1992) .
  • the fact that transformed cells display changes in the trafficking of cathepsins leading to the overexpression and secretion of the precursor forms represents a significant alteration in cellular processing and one that should be measurable. Reagents with the proper specificity for the pro-forms should be capable of measuring these changes. Since pro-cathepsin D is abnormally secreted by tumor cells this allows for its participation in the degradation of the extracellular matrix and the possibility for its detection in biological fluid such as blood and urine.
  • This invention is directed to a method for making a prognosis for a breast cancer patient involving quantitatively detecting an amount of pro-cathepsin in a sample of a bodily fluid.
  • the method comprises the following steps (a) reacting the sample of the bodily fluid from the patient with an immobilized anti- cathepsin antibody or a suitable cathepsin binding agent as a capture reagent so as to form complexes with any cathepsin present in the sample; (b) reacting the sample of step (a) with an unlabeled antibody capable of specifically binding to an epitope on a pro-cathepsin; (c) reacting the sample of step (b) with a detectable antibody specific for the unlabeled antibody so as to thereby determine the amount of pro-cathepsin in the bodily fluid; and (d) comparing the amount of pro-cathepsin in the bodily fluid with that of a normal subject so as to thereby make a prognosis for the breast cancer patient
  • the pro-cathepsin may be a pro-cathepsin B, D or L.
  • the bodily fluid may be plasma, serum, urine, saliva, lymphatic fluids, whole blood, nipple aspirates or other bodily fluids.
  • the immobilized anti-cathepsin antibody may be a monoclonal or a polyclonal antibody.
  • a suitable cathepsin binding agent may be used such as an enzyme inhibitor e.g. pepstatin.
  • the unlabeled antibody may be a polyclonal antibody.
  • the detectable antibody specific for the unlabeled antibody may be a horseradish peroxidase conjugate.
  • the prognosis may be a prognosis as to the likelihood of recurrence of cancer such as breast cancer.
  • the prognosis may be the prognosis of relapse or of survival.
  • Fig. 1 Immunoblot analysis of cathepsin D antibodies.
  • SK- HEP-1 cell conditioned media was subjected to SDS-PAGE then the separated proteins transferred to nitrocellulose and blotted with the anti-pro-CD polyclonal or the anti-mature- CD monoclonal antibody.
  • the latter antibody detects both pro (52 kDa) and the heavy chain of mature CD (34 kDa) while the pro-specific antibody only detects the pro-form.
  • Fig. 3 Immunoblot analysis of pro-CD in the plasma of breast cancer patients. 0.25 ⁇ l of each plasma sample was subjected to SDS-PAGE then the separated proteins transferred to nitrocellulose and blotted with an anti-CD monoclonal antibody. Each sample is listed by sample number and whether it measured high or low in the pro-CD ELISA. The standard was SK-HEP-1 cell conditioned media. Each blot contains a lane in which a plasma sample that was low in pro-CD was spiked with an amount of SK-HEP-1 standard to result in a total pro-CD level approximately equivalent to the activity measured in a high plasma sample. A) Identical blots developed with an anti-CD or an irrelevant (UPC 10) monoclonal antibody. B) Blot developed with the anti-CD monoclonal antibody.
  • UPC 10 irrelevant
  • Adsorbed proteins were stripped from the beads with pH 9 buffer and SDS sample buffer, separated by SDS-PAGE then blotted with an anti-CD monoclonal antibody.
  • Lane 1 SK-HEP-1 cell conditioned media; lane 2, conditioned media added to a sample of normal plasma; lanes 3-5, plasma samples from breast cancer patients which gave a high value in the pro-CD ELISA, lanes 6-8, plasma samples from normal control individuals.
  • the standard is conditioned media added directly to the gel without prior adsorption to pepstatin agarose.
  • the arrows indicate pro-CD and the heavy chain of the mature form of CD.
  • This invention is directed to a method for making a prognosis for a breast cancer patient involving quantitatively detecting an amount of pro-cathepsin in a sample of a bodily fluid.
  • the method comprises the following steps (a) reacting the sample of the bodily fluid from the patient with an immobilized anti- cathepsin antibody or a suitable cathepsin binding agent as a capture reagent so as to form complexes with any cathepsin present in the sample; (b) reacting the sample of step (a) with an unlabeled antibody capable of specifically binding to an epitope on a pro-cathepsin; (c) reacting the sample of step (b) with a detectable antibody specific for the unlabeled antibody so as to thereby determine the amount of pro-cathepsin in the bodily fluid; and (d) comparing the amount of pro-cathepsin in the bodily fluid with that of a normal subject so as to thereby make a prognosis for the breast cancer patient
  • the pro-cathepsin may be a pro-cathepsin B, D or L.
  • the bodily fluid may be plasma, serum, urine, saliva, lymphatic fluids, whole blood, nipple aspirates or other bodily fluids.
  • the immobilized anti-cathepsin antibody may be a monoclonal or a polyclonal antibody.
  • a suitable cathepsin binding agent may be used such as an enzyme inhibitor e.g.
  • pepstatin calpain inhibitor I or II, acetyl pepstatin, amastatin, bestatin, antipain, deacylleupeptin, chymostatin, conduritol B epoxide, eglin c fragment, elastatin, epiamastatin, epibestin, foroxymithine, leupeptin, peuumamide, pepsinostreptin, pepstatin A or phosphoramidon or suitable functionalized derivative thereof (see Sigma Chemical Company Catalog.)
  • the unlabeled antibody may be a polyclonal antibody.
  • the detectable antibody specific for the unlabeled antibody may be a horseradish peroxidase conjugate.
  • the prognosis may be a prognosis as to the likelihood of recurrence of cancer such as breast cancer. Alternatively, the prognosis may be the prognosis of relapse or of survival.
  • CD cathepsin D
  • the assay is specific for pro-CD and capable of quantitating this antigen in biological samples.
  • Pro-CD levels were measured in plasma samples from 76 breast cancer patients and compared with 36 plasmas from normal control individuals.
  • the breast cancer plasmas showed elevated levels of pro-CD; 12% were more than two standard deviations above the mean for the normal samples.
  • Immunoblots of the plasma samples using a CD monoclonal antibody revealed a band at the appropriate size for pro-CD that corresponded in intensity with the ELISA results.
  • Affinity adsorption of breast cancer plasmas with pepstatin agarose followed by immunoblot analysis revealed a single protein band that corresponded with pro-CD. Only trace amounts were detected in the normal control plasmas.
  • CD cathepsin D
  • CD is a useful prognostic marker for breast cancer (Rochefort, 1992) .
  • Studies using immunoassays showed that CD was a powerful prognostic factor for predicting recurrence and survival and was independent of other traditional prognostic parameters (Spyratos, 1989; Kute, 1992; Seshadri, 1994) .
  • a 5-year prospective study showed that CD has prognostic value in breast cancer (Pujol,
  • ELISA enzyme-linked immunosorbent assay
  • Plasma samples from patients with Stage I-IV breast cancer were obtained from Dianon Systems (Stratford, CT) and those from normal individuals were from the American Red Cross. Samples were stored at -70°C and thawed prior to use.
  • the monoclonal antibody (clone # 6410) made against mature cathepsin D purified from human liver and purified mature cathepsin D standard were obtained from Scripps Laboratories.
  • a second anti-mature CD monoclonal antibody (#IM03) and the Total Cathepsin D ELISA were from Oncogene Science.
  • the UPC 10 monoclonal antibody was obtained from Sigma (#M 9144) .
  • the proteinase inhibitors (Sigma) were made as concentrated stock solutions and used at the following final concentrations: 0.2 mM PMSF, 0.5 ⁇ g/ml Leupeptin, 1.0 mM EDTA and 1.0 ⁇ g/ml Pepstatin. Pepstatin agarose was also obtained from Sigma.
  • Cell Culture Serum-free conditioned media from SK-HEP-1 human liver tumor cells was prepared as follows. Roller bottle cultures were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum(FCS) and nonessential amino acids.
  • DMEM Dulbecco's modified Eagle's medium
  • MAP multiple antigenic peptide
  • New Zealand white rabbits were immunized intra-nodally by injection into the popliteal lymph node (Dresser, 1986) . Animals were primed with 500 ⁇ g of MAP peptide in complete Freund's adjuvant and boosted 3 times with 250 ⁇ g in incomplete Freund's adjuvant.
  • Test bleed titers were monitored by ELISA using microtiter plates coated with MAP peptide (5 ⁇ g/ml) and detection with goat anti-rabbit HRP conjugate (125 ng/ml) .
  • the antisera was purified using Protein A chromatography (Pierce) and antibody concentration determined by absorbance at 280nm. This antibody is commercially available (OSI, #IM04) .
  • Pro-cathepsin D ELISA 96 well microtiter plates were coated by applying lOO ⁇ l/well of anti-cathepsin D monoclonal antibody (clone #6410) at 5 ⁇ g/ml in lOOmM sodium carbonate, pH 9.6; and incubated overnight at room temperature (RT) . Plates were washed with PBS and blocked with 2% BSA, 10% lactose in PBS prior to use. 100 ⁇ l of standard or sample diluted in sample diluent (PBS, 4% BSA and 0.7% NP40) was added to each well and incubated overnight at RT.
  • PBS sample diluent
  • Plates were washed 6 times with wash buffer (10 mM phosphate, pH 7.5, 150 mM NaCl, 0.05% Tween-20) then 100 ⁇ l of the anti- pro-CD rabbit polyclonal detector antibody(4 ⁇ g/ml) was added and incubated for 1 hr at RT. Plates were washed 6 times as before followed by the addition of 100 ⁇ l of goat anti-rabbit horseradish peroxidase conjugate (Kirkegaard and Perry) at 0.25 ⁇ g/ml. After 30 min.
  • Blots were washed 3 times followed by incubation for 1 hr with goat anti-mouse IgG or goat anti- rabbit IgG horse radish peroxidase conjugate (Kirkegaard and Perry) at 30 ng/ml in blocking solution. After 4 washes with TBS-T, the blots were developed using chemiluminescent detection (ECL, Amersham) .
  • Pepstatin affinity adsorption Plasma samples were clarified by brief centrifugation at 15,000xg in a microcentrifuge. 50 ⁇ l of plasma or SK-HEP-1 conditioned media were diluted to 100 ⁇ l with dH 2 0 and added to 200 ⁇ l of binding buffer (100 mM sodium citrate, pH 3.4, 400 mM NaCl) . Samples were added to microcentrifuge tubes containing 50 ⁇ l of packed pepstatin agarose beads previously equilibrated with binding buffer. The samples were allowed to incubate with gentle rocking for 3 hr at 4°C.
  • the beads were pelleted by centrifugation, washed twice in binding buffer containing 6M urea then twice in 100 mM sodium citrate. Proteins were eluted from the beads by the addition of 500 mM Tris, pH 9.0 and reducing SDS-PAGE sample buffer follow by incubation at 100°C. The beads were pelleted by centrifugation and the supernatants subjected to electrophoresis and immunoblotted as described.
  • Pro-CD Specific ELISA Polyclonal antisera was generated by immunizing rabbits with a synthetic peptide from the pro- sequence of pro-CD (Faust, 1985) . The conditioned media from SK-HEP-1 cells was used to confirm that the antisera recognized the pro-form of CD. Immunoblot analysis indicated that SK-HEP-1 cells secrete cathepsin D predominantly in the precursor form. This is shown in Fig. 1 in which a sample of conditioned media is probed with a monoclonal antibody directed against an epitope on mature CD. The antibody recognized a major protein band at 52 kDa and a minor band at about 34 kDa which correspond to pro-CD and the heavy chain of mature CD respectively. An immunoblot of conditioned media probed with the anti-pro- peptide polyclonal antibody showed that only the 52 kDa pro- CD was recognized (Fig. 1) .
  • a pro-CD specific sandwich ELISA was developed in which a monoclonal antibody to mature CD was used as the capture reagent and the anti-pro-CD peptide polyclonal as the detector. The presence of bound detector antibody was determined using a goat anti-rabbit antibody conjugated with HRP. SK-HEP-1 conditioned media, containing pro-CD (see Fig. 1) , was used to prepare standards which were calibrated using the Total Cathepsin D ELISA. The assay produces a linear standard curve in the range of 100 - 1000 femtomoles/ml . A mature cathepsin D standard does not yield a signal in this assay.
  • the pro-CD ELISA is capable of measuring antigen in lysates and conditioned media from human cell lines.
  • the assay must be validated for the measurement of pro-CD in human plasma.
  • about 300 fmol of a pro-CD standard was added to normal plasma samples then measured in the ELISA to determine percent recovery.
  • the results are summarized in Table 2. Plasma caused severe inhibition of the assay signal and, even after a 1:5 dilution in sample diluent, only 18% of the added activity was recovered. Plasma samples were further diluted with sample diluent while maintaining a constant level of pro-CD standard.
  • the ELISA results indicate that a dilution factor of 1:20 to 1:40 is required in order to achieve greater than 90% recovery of added activity.
  • the pro-CD ELISA was used to evaluate 76 plasma samples from breast cancer patients and 36 normal controls. All samples were diluted starting at 1:20. Samples were measured at multiple dilutions and the data displayed good parallelism to the standard curve. The results, expressed in terms of femtomoles/ml of plasma, are presented in Figure 2.
  • SK-HEP-1 cell conditioned media was included as the standard and the band we believe to represent pro-CD is indicated by the arrow.
  • Panel A shows a blot of a high, low and a low sample spiked with an amount of pro-CD standard to result in a total pro-CD level approximately equivalent to the ELISA activity in the high sample.
  • the identical blot developed with the UPC 10 antibody reveals bands that are thought to represent non-specific binding.
  • the band just above the pro-CD marker could be human IgG heavy chain for which the goat anti-mouse detector antibody has a slight affinity.
  • the band at about the molecular weight of the pro-CD standard that appears to vary in intensity consistent with the ELISA results.
  • Panel B shows a blot of more plasma samples developed with the anti- CD monoclonal and again the band at about 52 kDa seems to have an intensity that is consistent with the ELISA analysis.
  • This band clearly migrates slightly above the standard from the SK-HEP-1 cell conditioned media. This may represent the reported observation that pro-CD from cell culture medium undergoes a 1-2 kDa reduction in size (see Discussion) . It is important to point out that in the standard lane of Panel B and in the spiked samples (#33 and #75) , the amount of pro-CD ELISA activity was approximately equal to that contained in the high samples. The resultant band has an intensity comparable to that which we have identified as pro-CD in the plasma samples.
  • the blots in Fig. 3 also reveal a strong band at about 150 kDa that does not appear in the UPC 10 control blot. It appears to be a plasma protein that is recognized by the anti-CD monoclonal but it is much more intense than expected and does not vary in intensity with the ELISA quantitation. This is puzzling since we are unaware of any reports of a form of cathepsin D that migrates in this region on SDS- PAGE. It is possible that this species is responsible for some of interference problems we have experienced with the measurement of plasma samples.
  • Pepstatin affinity adsorption Because of the difficulties associated with direct immunoblot analysis of plasma, we also attempted to enrich for pro-CD by affinity adsorption to pepstatin agarose.
  • Pepstatin is a specific inhibitor of aspartic proteinases and has been used to affinity purify mature and pro-CD (Conner, 1989; Larsen, 1993) . Both forms bind strongly to pepstatin agarose at pH 3.5 and elute at pH 8.3. 50 ⁇ l of pepstatin agarose was incubated with plasma samples that had been adjusted to acidic pH. The beads were washed then the proteins eluted by the addition of pH 9 buffer and SDS-PAGE sample buffer. The entire sample was subjected to SDS-PAGE and immunoblotted using the mature CD monoclonal antibody. Whereas the previous immunoblots were limited to 0.25 ⁇ l of plasma, this procedure allowed for the analysis of 50 ⁇ l of each sample. This greatly improved the likelihood of detecting pro-CD while reducing nonspecific background bands.
  • the pro-CD ELISA was validated for the measurement of pro-CD in blood plasma. Plasma caused an interference problem in the assay but this could be minimized with appropriate dilutions. Spike and recovery tests showed that >90% of added pro-CD antigen could be detected by the ELISA when the plasma was diluted by a factor of 20 or more.
  • the assay is sufficiently sensitive to detect pro-CD in human plasma samples and specificity was confirmed by the fact that the signal was inhibited by excess capture antibody.
  • Pro-CD levels were determined in a series of plasma samples from breast cancer patients and compared to samples from non-cancer controls. The majority of the values were within a narrow range but a portion of the breast cancer plasmas exhibited elevated levels (12% were greater than two standard deviations above the mean of the normal group) . Unfortunately, there is no follow-up information available for these patients so it is not possible to draw any correlations regarding prognosis, although pro-CD levels appeared to be independent of stage.
  • the value of a prognostic marker is in the ability to distinguish a subset of individuals from the total group of cancer patients that differ by a clinically relevant parameter such as probability of relapse, overall survival or response to therapy. Our data shows that a small group of patients have higher then normal levels of pro-CD in their blood.
  • pro-CD The presence of pro-CD in plasma was further verified by taking advantage of its affinity for the aspartic proteinase inhibitor, pepstatin. Affinity adsorption of the breast cancer plasmas with pepstatin agarose followed by immunoblot analysis revealed one band at the correct molecular weight for pro-CD. This experiment confirms that the protein band represents pro-CD based on two criteria, affinity for pepstatin then recognition by a monoclonal antibody. Pro-CD was shown to be prominent in the plasma from breast cancer patients but very low in the normal controls. In fact, the difference in the level of pro-CD between the breast cancer plasmas and normal controls appears to be more dramatic by this method than is indicated by the ELISA results. The ELISA may be underestimating the difference due to the interference problems discussed earlier. It is also important to note that the pepstatin adsorption experiment did not detect any mature CD, indicating that circulating cathepsin D is predominantly in the precursor form.
  • a key to developing good prognostic markers is to understand the biological changes that occur when a normal cell progresses to a tumor cell and then becomes invasive.
  • the fact that tumor cells display changes in the trafficking of cathepsins leading to the overexpression and secretion of the precursor forms represents a significant alteration in cellular processing and one that should be measurable.
  • Secreted pro-CD once activated, could participate in the degradation of the extracellular matrix. Secretion may also allow for its detection in bodily fluids such as blood and urine. Reagents and assays with the proper specificity for pro-CD could provide valuable information regarding tumor status and patient prognosis.
  • CD cathepsin D
  • DMEM Dulbecco's modified Eagle's medium
  • ELISA enzyme-linked immunosorbent assay
  • FCS fetal calf serum
  • HRP horseradish peroxidase
  • MAP multiple antigenic peptide
  • PBS phosphate-buffered saline
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • TBS-T tris-buffered saline-tween.
  • Conner GE The Role of the Cathepsin D Propeptide in Sorting to the Lysosome. JBC, 267 : 21738-21745, 1992.
  • Conner GE Isolation of procathepsin D from mature cathepsin D by Pepstatin affinity chromatography. Biochem. J., 263 : 601-604, 1989.
  • Faust PL, Kornfeld S and Chirgwin JM Cloning and sequence analysis of cDNA for human cathepsin D. PNAS, 82 : 4910- 4914, 1985.
  • Liotta LA and Stetler-Stevenson WG In : Seminars in Cancer Biology. Gottesman, MM. (eds.) Vol. 1(2) : 99-106, 1990.
  • Neville AM Diagnostic Oncology, 1 : 53-63, 1991.
  • Timmons TM and Dunbar BS Protein blotting and immunodetection. In : Methods in Enzymology, Guide to Protein Purification. Deutscher MP. (ed) , San Diego, Academic Press, 1990.
  • the ELISA was performed as described with the usual antibodies and a pro-CD standard as antigen (+) or with irrelevant antibodies or antigens as indicated. Antigen samples were diluted in sample diluent. FCS and BSA were diluted in DMEM. Media represents DMEM alone. The purified mature cathepsin D (matCD) standard was at 4200 fmol/ml. The c-erbB-2 antibody used to replace the capture was a monoclonal and that used to replace the detector was a rabbit polyclonal. na indicates no addition. Table II. Effect of Plasma on the Pro-CD ELISA
  • a fixed amount of a pro-CD standard was added to a series of serially diluted plasma samples which were measured in the ELISA. Percent recoveries are based on the value for the standard alone in the absence plasma. Plasma alone gave negligible activity.
  • the proteinase inhibitor cocktail(PI) as described in Materials was added to a plasma sample containing a standard amount of pro-CD. The percent recovery was determined by ELISA and compared to the same preparation without plasma.

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Abstract

La présente invention concerne un procédé de pronostic concernant une patiente atteinte d'un cancer du sein. Ce procédé consiste en une détection quantitative de la procathepsine B, D ou L dans un échantillon de fluide biologique tel que le plasma, le sérum, l'urine, la salive, la lymphe, le sang total ou le fluide d'aspiration du mamelon. La détection de la procathepsine peut se faire en utilisant comme agent fixateur un agent liant la cathepsine. L'agent liant peut être des anticorps monoclonaux ou polyclonaux spécifiques de la procathepsine ou un inhibiteur d'enzyme tel que la pepstatine. Le pronostic peut porter sur la probabilité de reprise du cancer, de récidive ou de survie.
PCT/US1996/001279 1995-02-03 1996-02-02 Procedes de detection de procathepsines et leurs emplois WO1996024065A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030907A1 (fr) * 1997-01-06 1998-07-16 Wisconsin Alumni Research Foundation Dosage de detection immunohistochimique pour la determination de l'etat de proliferation d'un cancer
WO2001069260A3 (fr) * 2000-03-13 2002-05-10 Res Found Mental Hygiene Proteinase lysosomiale insensible a la pepstatine comme nouveau biomarqueur pour detecter et diagnostiquer le cancer du sein

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY JOURNAL, Volume 263, issued 1989, CONNER, "Isolation of Procathepsin D from Mature Cathepsin D by Pepstatin Affinity Chromatography", pages 601-604. *
BREAST DISEASE, Volume 6, issued 1993, GASPARINI et al., "Immunocytochemical Determination of Pro-Cathepsin D: A Significant Independent Prognostic Indicator in Early-Stage Breast Cancer", pages 85-97. *
CLINICAL CHEMISTRY, Volume 35, No. 2, issued 1969, FREISS et al., "A Two-Site Immunoenzymometric Assay of 52-kDa Pro-Cathepsin D and Its Use in Human Breast Disease", pages 234-237. *
DISSERTATION ABSTRACTS, Volume 52, No. 12, issued June 1992, ACHKAR, "Lysosomal Enzyme Secretion in Tumor Cells and the Purification and Partial Characterization of a 67 kDa Cathepsin B-Like Enzyme Secreted by B16 Melanoma Cells", page 6181-B. *
MEDICAL SCIENCE RESEARCH, Volume 22, No. 1, issued January 1994, HIRANO et al., "Serum Cathepsin B Levels, Urinary Excretion of Cathepsin B and Cancer Tissue Content of Cathepsin B in Patients With Breast Cancer", pages 31-32. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030907A1 (fr) * 1997-01-06 1998-07-16 Wisconsin Alumni Research Foundation Dosage de detection immunohistochimique pour la determination de l'etat de proliferation d'un cancer
JP3336016B2 (ja) 1997-01-06 2002-10-21 ウイスコンシン アラムナイ リサーチ フオンデーシヨン 癌増殖状態の免疫組織化学的検出分析試験
WO2001069260A3 (fr) * 2000-03-13 2002-05-10 Res Found Mental Hygiene Proteinase lysosomiale insensible a la pepstatine comme nouveau biomarqueur pour detecter et diagnostiquer le cancer du sein

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