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WO1993008473A1 - Methode d'analyse d'epanchements malins - Google Patents

Methode d'analyse d'epanchements malins Download PDF

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Publication number
WO1993008473A1
WO1993008473A1 PCT/US1992/009068 US9209068W WO9308473A1 WO 1993008473 A1 WO1993008473 A1 WO 1993008473A1 US 9209068 W US9209068 W US 9209068W WO 9308473 A1 WO9308473 A1 WO 9308473A1
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WIPO (PCT)
Prior art keywords
vpf
antibody
sample
igg
assay
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Application number
PCT/US1992/009068
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English (en)
Inventor
Kiang-Teck Yeo
Harold F. Dvorak
Tet-Kin Yeo
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Beth Israel Hospital Association
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Publication of WO1993008473A1 publication Critical patent/WO1993008473A1/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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • This invention relates to assay methods for determining whether effusion samples from human patients are malignant.
  • Effusions from human patients can be the result of a variety of diseases including congestive heart failure, cirrhosis of the liver, and pneumonia.
  • effusions may also be the result of a malignancy. Consequently, the need exists for a method to determine whether a given effusion sample may be malignant.
  • Vascular permeability factor is a highly conserved 34-42 kD protein secreted by a variety of tumor cells that has been isolated from serum-free culture medium of carcinoma and sarcoma tumor cells and from tumor ascites fluids. Antibodies directed against VPF have also been produced. Dvorak et al., U.S. Patent No. 4,456,550, which is incorporated herein by reference, describes both the isolation of VPF and the creation of antibodies against VPF. VPF was first measured in animals using the Miles assay, Miles and Miles, J. Phvsiol. (Lond) .
  • DELFIA dissociation enhanced lanthanide fluoroimmunoassay
  • DELFIA is a "sandwich” type assay using a time-resolved fluorometer and a lanthanide chelate as a label.
  • VPF is associated with malignant effusions and have developed sensitive and specific assay methods to precisely measure VPF in effusion samples.
  • the VPF immunofluorometric assay of the invention has a minimal detection limit of 0.35 units (as defined below) and is about thirty times more sensitive than the Miles permeability assay.
  • the immunoassay is more precise and simpler to perform, is readily automatable, and can measure large numbers of specimens rapidly and inexpensively.
  • this assay has a potentially important diagnostic utility as a test for tumor metastases by assaying the effusions in the pleural and peritoneal cavities of human patients.
  • the invention features an assay method for determining whether an effusion sample obtained from a human patient is associated with a malignancy, comprising measuring vascular permeability factor (VPF) in the sample, a VPF level greater than about 30 units (as defined below) indicating a likelihood that the sample is a malignant effusion.
  • VPF vascular permeability factor
  • the invention also features a simple, sensitive, and specific immunofluorometric assay for VPF in a human e fusion sample to determine whether the sample is a malignant effusion.
  • This assay employs the following steps i immobilizing a first antibody against a first portion of VPF on a surface, applying the effusion sample to the immobilized first antibody, incubating the sample for a time and at a temperature sufficient to allow this first antibody to bind to VPF in the sample, washing the surface for a time sufficient to remove unbound VPF, applying a second labeled antibody which is generated against a second portion of VPF to the surface and VPF bound to the immobilized first antibody, incubating the sample for a time and at a temperature sufficient to allow the second antibody to bind to VPF bound to the first antibody, washing the surface for a time sufficient to remove any unbound second antibody, and measuring the amount of label that is bound to the surface to determine the amount of VPF in the sample, a level greater than about 30 units indicating that the sample is a malignant effusion.
  • surface includes any microtiter plate well, test tube, plate, or other surface to which the first antibodies of the invention can bind.
  • a pleural or peritoneal effusion is the sample; labeling of the second anti-VPF antibody is carried out with a Europium chelate; and the second antibody used in the test is to a 26-amino acid sequence of the N-terminus of human VPF; i.e., APMAEGGGQNHH-EWKFMDVYQRSYC.
  • the first antibody is to a 20-amino acid sequence of the C-terminus of guinea pig VPF, i.e., YKARQLELNERTCRCDKPRR.
  • Fig. 1 is a chromatographic profile of a Europium labeled antibody to the N-terminus of VPF.
  • Figs. 2A and 2B are graphs showing the optimization curve of the titer of labeled antibody to the C-terminus of VPF.
  • Figs. 3A and 3B are graphs showing the optimization curve of the titer of an antibody to the N- terminus of VPF.
  • Figs. 4A and 4B are graphs showing the sensitivity and intra-assay coefficient of variation (CV) of the immunofluorometric VPF assay of the invention.
  • Fig. 5. is a graph showing the specificity of the immunofluorometric VPF assay of the invention.
  • Fig. 6 is a graph showing the correlation of the VPF immunoassay of the invention and the Miles assay.
  • Fig. 7 is a graph showing the kinetics of VPF production in line 1 tumor cells injected in guinea pigs.
  • Fig. 8 is a graph showing the kinetics of VPF production in line 10 tumor cells injected in guinea pigs.
  • Fig. 9 is a graph showing calibration curves for the VPF fluoroimmunoassay for both human and guinea pig assays.
  • Fig. 10 is a chart showing VPF levels in patients with various pathological conditions of fluid accumula ion.
  • DELFIATM Eu 3+ labeling kits are available from Phar acia-LKB Nuclear Inc. (Gaithersburg, MD) . These kits contain 0.2 ig labeling reagent CN ⁇ -Cp-isothiocyanatobenzyll-diethylenetriamine- N 1 , N 2 , N 3 -tetraacetate-Eu 3+ ) , 100 nmol/L Eu 3+ standard.
  • BSA 75 g/L in Tris-HCL, pH 7.8, 0.5 g/L NaN 3 stabilizer
  • enhancement solution 15 ⁇ -mol/L 2- napthoyltrifluoroacetone, 50 mol/L tri-n-octylphosphine oxide, 100 mmol/1 acetic acid, 6.8 mmol/L potassium hydrogen phthalate, 1.0 g/L Triton X-100 detergent
  • assay buffer Tris-HCl, pH 7.8 solution containing BSA, bovine gamma globulin, Tween 40, diethylenetriamine- pentaacetic acid, 0.5 g/L NaN 3
  • wash concentrate solution 25-fold concentration of Tris-HCl/NaCl, pH 7.8, Tween 20
  • Serum-free media (HL-1) is available from Ventrex Laboratories Inc. (Portland, ME) , and hemoglobin crystals are available from Sigma Chemical Co. (St. Louis, MO) .
  • the "GammaGone”. IgG removal device described below is from Genex Corporation (Gaithersburg, MD) .
  • the labeling buffer is 50 mmol/L NaHC0 3 , pH 8.5, containing 9 g/L NaCl.
  • the elution buffer is 50 mmol/L Tris-HCl, pH 7.8, containing 9 g/L NaCl and 0.5 g/L NaN 3 .
  • the coating buffer is phosphate-buffered saline (PBS), pH 7.0, and the blocking reagent is 30 g/L hemoglobin solution.
  • Polyclonal Antibodies Antibodies were raised against two synthetic peptides that correspond to the N- and C-termini of guinea pig VPF (designated N-IgG and C- IgG, respectively) .
  • the 25- amino acid sequence of the guinea pig N-terminus of VPF is APMAEGEQK- PREWKFMDVYKRSYC
  • the 20-amino acid sequence of the C-terminus of guinea pig VPF is (Y)KARQLELNERTCRCDKPRR (the Y amino acid is attached only for coupling purposes).
  • Keck et al.. Science, 246:1309- 12 (1989) Both peptides were synthesized by Multiple Peptide Systems (San Diego, CA) using standard synthesis techniques and were used to generate antibodies in rabbits as described by Senger et al., Can. Res..
  • Eu 3+ -labeling of N-IgG was performed according to the DELFIATM kit protocol with the following modifications. Affinity-purified antisera were pooled and concentrated to about 0.5 g/L using an Amicon macrosolute concentrator. The PD-10 column was pre-equilibrated with 40 ml of labeling buffer, and 2 ml of the antisera (0.5 g/L) was loaded on the column. The column was rinsed with labeling buffer, 1.0 ml fractions were collected, and the absorbance at 280 nm was measured on a Hitachi U- 2000 spectrophotometer (Hitachi Instruments Inc.,
  • Sepharose CL-6B was poured into a 1.5 cm x 30 cm column to a height of 18 cm. Next preswollen Sephadex G-50 was added to a height of 28 cm and the column was equilibrated with 180 ml of elution buffer. The Eu 3+ -IgG reaction mixture was added and fractionated on this column. The column was rinsed with elution buffer and sixty 1 ml fractions were collected and their absorbances measured at 280 nm.
  • the Sepharose 6B/Sephadex G-50 chromatographic profile in Fig. 1 shows two distinct peaks; the first peak (I) corresponded to Eu 3+ -labeled N-IgG, and the second peak (II) represented unreacted Eu 3+ -chelate.
  • Fig. 1 shows absorbance at 280 nm (0) and fluorescence (•) .
  • Typical labeling yield is approximately 10 Eu 3+ per IgG. For this reason, we showed in a separate experiment that >90% of the fluorescence associated with peak I could be removed by an IgG-removing device (Gam agone device) , indicating that peak I was comprised mainly of Eu 3+ - labeled N-IgG.
  • the specific activity of the Eu 3+ -labeled N- IgG was calculated to be approximately 10 Eu 3+ /IgG, using a 1 nmol/L Eu 3+ standard as described in the DELFIA kit protocol.
  • Coating of microtiter strips 50 ⁇ l of a 50-fold dilution of C-IgG (stock concentration of 0.64 g/L in PBS) was added to each well of the microtiter strips, and the plate incubated overnight at 4°C on a shaker. This is the so-called "first" antibody. Thereafter, the wells were washed six times with DELFIATM wash buffer, and blocked by incubation with a 30 g/L hemoglobin solution at 20°C for 2 h with gentle shaking. Plates were washed six times with DELFIATM wash buffer prior to use.
  • Line 10-cell cultures Guinea pig line 10 tumor cells were grown as suspension cultures in serum-free defined medium HL-1 as described previously in Yeo et al., Biochem. Biophys. Res. Comm.. 179:1568-75 (1991).
  • Conditioned line 10 medium which contains large amounts of VPF, was centrifuged and frozen at -70°C to serve as calibrators.
  • one of the "units" defined herein would correspond to a VPF concentration of approximately 20 picograms/ml.
  • a precise relationship between the units defined herein and the VPF concentration can be determined by one of ordinary skill in the art using standard techniques.
  • signal-to-noise ratio is defined as fluorescence 100 units /fluorescence 0 unit -
  • Fig. 2A shows the calibration curves at 50-fold (0) , 30- fold ( ) , 10-fold ( ⁇ ) , and 5-fold (D) dilutions of N-IgG, respectively.
  • Fig. 2B shows the calibration curves at 50-fold (0) , 30- fold ( ) , 10-fold ( ⁇ ) , and 5-fold (D) dilutions of N-IgG, respectively.
  • VPF Immunoassav a Sensitivity and Intra-assay Coefficient of Variation (CV) of the VPF Immunoassav
  • line 10 conditioned medium corresponding to 0.25 units, 0.50 units, and 1.00 unit were prepared by diluting it with HL-1 medium, and assayed ten times.
  • HL- 1 medium devoid of VPF served as the zero standard.
  • the sensitivity, or minimal detectable dose (defined as +2 SD above the zero standard), was about 0.35 units (Fig. 4A) , by extrapolation from the standard curve.
  • the intra- assay CV was less than 20% at 0.50 units ( Figure 4B) .
  • VPF immunoassay Since the format of this assay depends on the C- IgG as the "first” or “capture” antibody, and the Eu 3+ -N- IgG as the "second" or “detector” antibody, we used peptides corresponding to the N- and C-termini of VPF to demonstrate the specificity of the assay. As shown in Fig. 5, inclusion of C-VPF peptide (final concentration of 80 mg/L) , N-VPF peptide (final concentration of 80 mg/L) , or both peptides in the assay inhibited the binding of VPF in line 10 medium by approximately 80%.
  • peritoneal cavity was then opened by a small incision and 20 ml of Hank's balanced salt solution (HBSS) was injected i.p. and the contents of the peritoneal cavity were mixed by kneading. The peritoneal contents were recovered to the fullest extent possible by syringe. The total peritoneal fluid volume was recorded and the tumor cells counted. Whenever possible urine was recovered from the bladder by syringe.
  • HBSS Hank's balanced salt solution
  • Blood, peritoneal fluid, and urine were kept on ice and the following inhibitors were added: iodoacetamide (final concentration of 0.37 mg/ml), N-ethylmaleimide (final concentration of 0.25 mg/ml), PMSF (final concentration of 0.35 mg/ml) and aprotinin (final concentration of 210 KlU/ml) .
  • Blood, peritoneal fluid, and urine were centrifuged at 160 x g for 20 min at 4°C. The volumes of the resultant plasma, cell-free ascites fluid, and urine were recorded and the samples aliquoted and stored at - 80°C until the time of VPF assay.
  • the two-site time-resolved immunofluorometric assay of the invention was used to assay the guinea pig VPF as described above. As shown in Figs. 7 and 8, the results show a parallel increase in fluid volume ( ⁇ ) , tumor cell number (•) , and VPF (I) in the ascites fluids collected from guinea pigs at various times after injection of tumor line l and tumor line 10 cells, respectively, into the animals. The insets shows that very little VPF is detected in the plasma (ppp) and the urine (u) of these same animals.
  • polyclonal antibodies against the N-terminus of human VPF were produced and labeled with the Europium chelate, and used as the second antibody in the VPF assay method described above.
  • the amino-acid sequence for the N- terminus of human VPF is APMAEGGGQNHHEWKFMDVYQRSYC.
  • Europium labeled human or guinea pig N-IgG (h-Eu 3+ -N-IgG v. g-Eu 3+ -N-IgG) were used to detect VPF concentration in both human and guinea pig sources of VPF, as shown in Fig. 9.
  • Panel A shows that using a guinea pig source for VPF (line 10 medium) , only the guinea pig N-IgG Eu3+ ( ⁇ ) binds 5 fold higher than using the human N-IgG Eu3+ (1) .
  • Panel B shows that using a human source for VPF (human MNNG-HOS cell medium) , the human N-IgG Eu3+ (o) binds about 3 fold higher than guinea pig N-IgG Eu3+ ( ) .
  • Effusion samples from patients with pathological conditions of fluid accumulations were prepared as follows. The patient's skin was disinfected and a local anesthetic was injected. The pleural space was entered posteriorly in the mid-clavicular line superior to the fifth or sixth rib with a sterile 22-gauge needle and fluid was aspirated into a syringe. Similar aseptic techniques were used to remove peritoneal fluids via a puncture in the right lower quadrant of the abdomen. The fluids were then heparinized, and 1.0 ml aliquots were obtained and centrifuged at 15,000 x G for 1.0 min. in an Eppendorf microcentrifuge. The clear supernatant solutions were immediately frozen and stored at -70°C prior to the VPF assay. Analysis of VPF Assay Results
  • VPF levels in the effusion samples were analyzed using a two-sample robust analysis as described in Hoaglin et al., Understanding Robust and Exploratory Data Analysis (John Wiley & Sons, New York, N.Y., 1983). As shown in Fig. 10, VPF levels in patients with malignant cells in effusion fluids are significantly higher than patients without cancer. Overall, these preliminary results suggest that VPF levels in fluids have an important potential diagnostic use to detect cancer. These studies show a strong correlation of high VPF levels with malignant cells in effusion samples, but not necessarily with clinical suspicions of cancer, which may not be definite. Further clinical studies are currently underway to specifically address the use of VPF measurements of effusion samples to diagnose cancer.

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Abstract

Une méthode d'analyse permet de déterminer si un échantillon d'épanchement prélevé sur un homme est associé à une malignité. Ladite méthode consiste à mesurer le facteur de perméabilité vasculaire (VPF) dans l'échantillon, un VPF supérieur à environ 30 unités (selon la définition donnée dans la description) indiquant que l'on est vraisemblablement en présence d'un échantillon d'épanchement malin.
PCT/US1992/009068 1991-10-24 1992-10-21 Methode d'analyse d'epanchements malins WO1993008473A1 (fr)

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US78235091A 1991-10-24 1991-10-24

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5660827A (en) * 1992-03-05 1997-08-26 Board Of Regents, The University Of Texas System Antibodies that bind to endoglin
US5855866A (en) * 1992-03-05 1999-01-05 Board Of Regenis, The University Of Texas System Methods for treating the vasculature of solid tumors
US5863538A (en) * 1992-03-05 1999-01-26 Board Of Regents, The University Of Texas System Compositions for targeting the vasculature of solid tumors
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US6004555A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for the specific coagulation of vasculature
US6036955A (en) * 1992-03-05 2000-03-14 The Scripps Research Institute Kits and methods for the specific coagulation of vasculature
US6093399A (en) * 1992-03-05 2000-07-25 Board Of Regents, The University Of Texas System Methods and compositions for the specific coagulation of vasculature
WO2003082918A1 (fr) * 2002-04-02 2003-10-09 Ark Therapeutics Ltd. Peptides vegf et leur utilisation
US6749853B1 (en) 1992-03-05 2004-06-15 Board Of Regents, The University Of Texas System Combined methods and compositions for coagulation and tumor treatment

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WO1995006131A1 (fr) * 1993-08-23 1995-03-02 Monash University Methode de depistage, de prophylaxie et/ou de traitement de maladies affectant l'homme

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US4376110A (en) * 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies

Non-Patent Citations (6)

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Title
ACTA CYTOLOGICA, Volume 32, issued September-October 1988, W.W. JOHNSTON, "Fine Needle Aspiration Biopsy Versus Sputum and Bronchial Material in the Diagnosis of Lung Cancer. A Comparative Study of 168 Patients", pages 641-646. *
CANCER RESEARCH, Volume 46, issued November 1986, (USA), D.R. SENGER et al., "A Highly Conserved Vascular Permeability Factor Secreted by a Variety of Human and Rodent Tumor Cell Lines", pages 5629-5632. *
SCANDANAVIAN JOURNAL OF CLINICAL LABORATORY INVESTIGATION, Volume 48, issued 1988, I. HEMMILA, "Lanthanides as Probes for Time-Resolved Fluoremetric Immunoassays", pages 389-399. *
SCIENCE, Volume 219, issued 25 February 1983, (Washington, DC, USA), D.R. SENGER et al., "Tumor Cells Secrete a Vascular Permeability Factor that Promotes Accumulation of Ascites Fluid", pages 983-985. *
SCIENCE, Volume 246, issued 08 December 1989, (Washington, DC, USA), P.J. KECK et al., "Vascular Permeability Factor, an Endothelial Cell Mitogen Related to PDGF", pages 1309-1312. *
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6036955A (en) * 1992-03-05 2000-03-14 The Scripps Research Institute Kits and methods for the specific coagulation of vasculature
US6051230A (en) * 1992-03-05 2000-04-18 Board Of Regents, The University Of Texas System Compositions for targeting the vasculature of solid tumors
US5855866A (en) * 1992-03-05 1999-01-05 Board Of Regenis, The University Of Texas System Methods for treating the vasculature of solid tumors
US5863538A (en) * 1992-03-05 1999-01-26 Board Of Regents, The University Of Texas System Compositions for targeting the vasculature of solid tumors
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US5965132A (en) * 1992-03-05 1999-10-12 Board Of Regents, The University Of Texas System Methods and compositions for targeting the vasculature of solid tumors
US6004555A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for the specific coagulation of vasculature
US6004554A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for targeting the vasculature of solid tumors
US5776427A (en) * 1992-03-05 1998-07-07 Board Of Regents, The University Of Texas System Methods for targeting the vasculature of solid tumors
US6093399A (en) * 1992-03-05 2000-07-25 Board Of Regents, The University Of Texas System Methods and compositions for the specific coagulation of vasculature
US5660827A (en) * 1992-03-05 1997-08-26 Board Of Regents, The University Of Texas System Antibodies that bind to endoglin
US6451312B1 (en) 1992-03-05 2002-09-17 Board Of Regents, The University Of Texas System VEGF-gelonin for targeting the vasculature of solid tumors
US7125541B2 (en) 1992-03-05 2006-10-24 The University Of Texas System Board Of Regents Combined methods for tumor vasculature targeting and tumor treatment with radiotherapy
US6749853B1 (en) 1992-03-05 2004-06-15 Board Of Regents, The University Of Texas System Combined methods and compositions for coagulation and tumor treatment
US7112317B2 (en) 1992-03-05 2006-09-26 Board Of Regents, The University Of Texas System Combined methods and compositions for tumor vasculature targeting and tumor treatment
AU2003226515B2 (en) * 2002-04-02 2006-06-08 Ark Therapeutics Ltd. VEGF peptides and their use
WO2003082918A1 (fr) * 2002-04-02 2003-10-09 Ark Therapeutics Ltd. Peptides vegf et leur utilisation

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AU2874292A (en) 1993-05-21
PT101008A (pt) 1994-02-28

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