WO2000061638A2 - Detection de cancer et de grossesse anormale au moyen d'anticorps monoclonaux specifiques pour des isoformes de la hcg - Google Patents

Detection de cancer et de grossesse anormale au moyen d'anticorps monoclonaux specifiques pour des isoformes de la hcg Download PDF

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Publication number
WO2000061638A2
WO2000061638A2 PCT/US2000/009776 US0009776W WO0061638A2 WO 2000061638 A2 WO2000061638 A2 WO 2000061638A2 US 0009776 W US0009776 W US 0009776W WO 0061638 A2 WO0061638 A2 WO 0061638A2
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Prior art keywords
hcg
nicked
hcgn
isoforms
antibodies
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PCT/US2000/009776
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English (en)
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WO2000061638A3 (fr
Inventor
Alexander Krichevsky
Steven Birken
John O'connor
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Trustees Of Columbia University In The City Of New York
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Priority to AU42349/00A priority Critical patent/AU4234900A/en
Priority to CA002369923A priority patent/CA2369923A1/fr
Publication of WO2000061638A2 publication Critical patent/WO2000061638A2/fr
Publication of WO2000061638A3 publication Critical patent/WO2000061638A3/fr

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    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors

Definitions

  • hCG is comprised of and ⁇ subunits, the ⁇ subunit being heavily glycosylated.
  • a variety of isoforms of hCG, including free and ⁇ subunits, heterodimeric (or whole) hCG with peptide bond cleavages in loop 2 of the ⁇ fragment (referred to as nicked hCG), free nicked I CG subunits, and the ⁇ core fragment are known. See S. Birken, et al., 129 Endocrinology 1551-1558 (1991), S. Birken, et al, 131 Endocrinology 1390-1397 (1993), and S. Birken, et al, 125 Mol. Cell Endocrinol. 121-131 (1996).
  • the nicked isoform of heterodimeric hCG has been reported to be present in blood as well as urine and is known to have much lower recognition by antibodies directed to heterodimeric hCG, its subunits, and fragments, as well as greatly reduced biological activity.
  • Hyperglycosylated hCG occurs in multiple isoforms and has been detected in blood and urine, and in cell surface membranes, and its chemical and immunological properties are summarized in M.M.
  • the present invention therefore provides a method for detecting certain hCG isoforms by providing two monoclonal antibodies, B151 and B152, each with unique specificity for hCG isoforms, for use in immunoassays for diagnosis of certain malignancies and characterization of normal and problem pregnancies.
  • Binding curves are shown for various forms of hCG-related hormones as detailed infra. Each panel represents a separate assay in which all ligands were introduced in the same assay. Points were connected by straight lines, although regression analysis (four-parameter logistic) indicated excellent fit to logistic or sigmoidal curve-shaped model. Panels A and B represent two distinct assays with similar results.
  • the present invention is based on the conception, production, identification, and characterization of two monoclonal antibodies with distinct specificity using a nicked, hyperglycosylated form of hCG purified from a single choriocarcinoma patient as an immunogen.
  • the two antibodies designated as B151 and B152, were selected by the use of radiolabeled C5 and CR 127 (see infra).
  • Fig. 1 shows potency comparisons of liquid phase competitive radioimmunoassays of both B151 and B152 antibodies comparing competitors: (1) standard CR 127 pregnancy hCG (which has a 20% content of nicked hCG), (2) C5 chorio CG (100% nicked and hyperglycosylated), (3) nicked CG made from CR 127 by purification, and (4) non-nicked hCG derived from CR 127.
  • the labeled ligand was C5 chorio hCG. It is apparent from Fig.
  • B151 shows a preference for nicked forms of hCG.
  • C5 chorio hCG or CR 127 hCGn bind with similar affinities.
  • the slightly lower potency of CR 127 hCGn may be ascribed to its 20% contamination with non-nicked hCG.
  • B152 only shows a preference to C5, the hyperglycosylated chorio CG.
  • CR 127 hCGn is no more potent a competitor than nick-free CR 127 hCG.
  • B151 binds simultaneously with antibodies directed towards the determinant which exists in heterodimeric hCG as represented by antibody B109 (see A. Krichevsky, et al. (1988), Id.).
  • a general beta antibody which binds to the most common and potent hCG antigenic site such as B 108 or B207 (see A. Krichevsky, et al, 2 Endocrine 511-520 (1994)) binds well to both B151 and B152 antibodies.
  • B152 binds simultaneously to all antibodies tested except those to the ⁇ COOH-terminal region (CTP).
  • CTP ⁇ COOH-terminal region
  • B152 as capture and B207 or B108 as detection antibody produces an assay which measures all normal pregnancy forms of hCG (both intact and nicked and ⁇ subunit) to a similar extent but prefers binding to the form of hCG or ⁇ subunit to its C5 immunogen.
  • This assay does not prefer nicked forms of hCG but hyperglycosylated forms such as C5.
  • B152 and CTP 104, as well as several other monoclonal antibodies to the COOH-terminal region of hCG ⁇ cannot bind simultaneously to C5, implying that this region is part of or very close to the epitope of 1- 152.
  • the isoforms of hCG utilized were (1) C5, the choriocarcinoma hCG which is glycosylated and has hexasaccharide carbohydrate structures in its beta COOH-terminal region, (2) 814, which is non- nicked hCG prepared from the hCG reference preparation CR127 derived from normal, late first trimester hCG, (3) 813, which is nicked hCG (80% nicked) produced from CR127 by hydrophobic chromatography, (4) M4 hCG, which is nicked hCG with regligible hyperglycosylation, and (5) MIA hCG, which is non-nicked and not significantly hyperglycosylated, but which is missing 80% of the hCG ⁇ COOH- terminus.
  • the B152 two-site assay prefers to bind to C5, its immunogen, but shows nearly equal recognition of both 813 and 814, nicked and non-nicked hCG of normal pregnancy, confirming that B152 does not display significant preference for the nicked form of hCG but rather for the form with carbohydrate differences.
  • This lack of preference is also confirmed by the potency of M4, which is also 100% nicked as is C5, but which is not hyperglycosylated and displays a potency similar to CR 127 hCG whether nicked or non-nicked.
  • MIA is the least potent ligand and is the only one r ssing most of its beta COOH-terminal peptide, confirming the role of this region in the B 152 epitope.
  • antibody B151 prefers binding to nicked forms of hCG, regardless of the origin of the hCG molecule (normal pregnancy or choriocarcinoma). This preference is shown by similar binding to both preparations of nicked hCG, C5 and nicked CR127. The latter preparation is not hyperglycosylated as is C5.
  • Nick-free CR127 has the lowest affinity to this antibody as would be expected for a nick-directed antibody. This finding led to the conclusion that B151 was directed towards an epitope dependent upon peptide bond cleavages in hCG beta subunits while binding to B152 was not affected by peptide bond cleavages within this loop.
  • B152 was a carbohydrate-directed antibody.
  • B151 is used as capture antibody and virtually any general beta antibody as detection antibody in a t vo site assay (see Table II), little cross-reaction with hLH is observed.
  • B151 cannot bind simultaneously with antibodies that are directed chiefly to the hCG beta core region (such as B201 and B204), nor can the antibody bind with antibodies directed to heterodimeric hCG such as B109 or A 109, but it can bind at the same time as antibodies to the beta COOH-region. B151 therefore represents a new hCG epitope revealed after nicking of the beta subunit.
  • the second antibody, B152 preferentially binds with hCG with the type of carbohydrate modifications prevalent in choriocarcinoma CG C5 regardless of the state of the nicking of the polypeptide chain.
  • Antibody B152 appears to be specific for the hCG beta COOH-terminal region. This specificity is shown by failure of a monoclonal antibody to bind simultaneously with beta COOH terminal antibodies such as CTP 104. In two site assays with B152, it was shown that hCG isoform MIA which is missing n.ost of its COOH-terminal region, binds poorly to B152.
  • Each antibody has a different application in accordance with its specificity.
  • immunoassays are designed for detection of hCG isoforms for use in the diagnosis of certain malignancies and characterization of normal and abnormal pregnancy.
  • B151 permits formulation of direct assays for nicked hCG which have been applied in studies of early pregnancy. These measurements have diagnostic applications for certain cancers and in the detection of Down's syndrome.
  • the B152 antibody is used in assays which detect differences in the carbohydrate portion of hCG. Major potential applications of this antibody include detection of Down's syndrome and recognition of pregnancies destined for early pregnancy loss.
  • body fluid samples such as blood from the patient are tested for the presence of the nicked hCG isoforms characteristically produced during Down's syndrome pregnancy and certain malignancies with B151 and for the presence of the hCG isoforms characteristically produced during problem pregnancies such as Down's syndrome and preeclampsia.
  • an assay utilizing B152 is used to quantify the carbohydrate variant characteristically produced during healthy pregnancies since pregnancies which are likely to fail early have proportionately little of this hCG isoform.
  • Such immunoassays are known to those skilled in the art and need not be described in detail here.
  • immunoassays assays for the detection of the hCG isoforms characteristically produced by these various conditions and to note that such assays may be conducted by, for instance, i'l mobilizing the hCG and labeling the antibodies for detection of the antibodies or by the immunoassays described herein.
  • mice were immunized intraperitoneally with choriocarcinoma hCG preparation C5 (Kardana, A, et al, 129 Endocrinology 1541-1550 (1991)) diluted in saline (600 ⁇ g ml) and mixed 1:1 with Freund's adjuvant. After three consecutive immunizations
  • Example 2 Liquid Phase Assays Liquid phase assays were performed using a solution of 80 ⁇ l 0.3M PBS with
  • mice whose antisera had the greatest discrimination between binding of radiolabeled C5 and radiolabeled CR 127 hCG were sacrificed and their spleens were used for hybridoma production by the methods described in A. Krichevsky, et al.
  • Immulon 4TM microtiter wells are coated with capture antibody (as described above) at a titer determined to provide the best combination of sensitivity and range.
  • the antibody in coating buffer sodium bicarbonate 0.2M, pH 9.5, 200 ⁇ l/well
  • wash buffer was PBS with 0.05% TWEEN 20TM.
  • the plates were then blocked with 1% bovine serum albumin (BSA) in PBS overnight at 4°C and again aspirated and washed.
  • BSA bovine serum albumin
  • Standards, clinical samples and controls were added to the coated wells (200 ⁇ l/well), incubated 24 hrs at 4°C, after which well contents were aspirated, washed (5X) and labeled antibody (either I labeled antibody (50K-100K CPM well) or peroxidase-labeled) added and then again incubated 24 hrs at 4°C.
  • the wells were aspirated, washed (5X), and counted by ⁇ - counter (Packard Instruments COBRA, Meridien, CT) or by absorption spectrophotometry as appropriate.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La gonadotrophine chorionique humaine (hCG) existe dans le sang et l'urine en tant que variété d'isoformes. L'une d'elles, qui contient des clivages de liaisons peptidiques à l'intérieur de sa sous unité bêta, est désignée hCG coupée (hCGn). Cette isoforme de la hCGn est plus courante dans l'urine des patients présentant certaines malignités et éventuellement dans d'autres maladies de grossesse. L'invention porte sur deux anticorps monoclonaux à une isoforme de la hCGn isolée chez un patient choriocarcinome. Deux analyses immunométriques sur site ont été élaborées à l'aide de ces anticorps, désignés B151 et B152. Le premier présente une bonne spécificité pour hCGn indépendante de la source de la hCGn, forme évacuée par des patients choriocarcinomes, ou la forme de la hCGn provenant de grossesses normales. L'autre anticorps, B152, est exceptionnellement sensible aux fractions glucidiques de hCG choriocarcinome, coupée ou non, étant donné que sa reconnaissance de ligand est fonction des différences glucidiques plutôt que des différences de liaisons peptidiques. Ces deux analyses immunométriques apportent de nouveaux outils de diagnostic fondés sur le mesurage direct de ces isoformes de la hCG.
PCT/US2000/009776 1999-04-12 2000-04-12 Detection de cancer et de grossesse anormale au moyen d'anticorps monoclonaux specifiques pour des isoformes de la hcg WO2000061638A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU42349/00A AU4234900A (en) 1999-04-12 2000-04-12 Detection of cancer and abnormal pregnancy using monoclonal antibodies specific for hcg isoforms
CA002369923A CA2369923A1 (fr) 1999-04-12 2000-04-12 Detection de cancer et de grossesse anormale au moyen d'anticorps monoclonaux specifiques pour des isoformes de la hcg

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US12884599P 1999-04-12 1999-04-12
US60/128,845 1999-04-12

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095458A1 (fr) * 2004-04-02 2005-10-13 The Talwar Research Foundation Anticorps monoclonal chimerique destine a se lier selectivement a hcg avec une haute affinite
US7439026B2 (en) * 2001-07-30 2008-10-21 Quest Diagnostics Investments Incorporated Methods and kits for detecting ITA in a biological sample

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010282A1 (fr) * 1996-09-06 1998-03-12 Yale University Depistage prenatal du syndrome de down a l'aide de gonadotrophine hyperglycosylee
WO1999041584A2 (fr) * 1998-02-03 1999-08-19 The Trustees Of Columbia University In The City Of New York METHODES DE PREVISION DE L'ISSUE D'UNE GROSSESSE CHEZ UN SUJET PAR DOSAGE BIOLOGIQUE DE hCG

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010282A1 (fr) * 1996-09-06 1998-03-12 Yale University Depistage prenatal du syndrome de down a l'aide de gonadotrophine hyperglycosylee
WO1999041584A2 (fr) * 1998-02-03 1999-08-19 The Trustees Of Columbia University In The City Of New York METHODES DE PREVISION DE L'ISSUE D'UNE GROSSESSE CHEZ UN SUJET PAR DOSAGE BIOLOGIQUE DE hCG

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
A. KRICHEVSKY ET AL.: "Development, characterization, and application of monoclonal antibodies to the native and synthetic betaCOOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG." ENDOCRINOLOGY, vol. 134, no. 3, March 1994 (1994-03), pages 1139-1145, XP000939398 Philadelphia, PA, USA cited in the application *
G. KOVALEVSKAYA ET AL.: "Early pregnancy human chorionic gonadotropin (hCG) isoforms measured by an immunometric assay for choriocarcinoma-like hCG." THE JOURNAL OF ENDOCRINOLOGY, vol. 161, no. 1, April 1999 (1999-04), pages 99-106, XP000939395 Bristol, GB *
G. KOVALEVSKAYA ET AL.: "Evaluation of nicked human chorionic gonadotropin content in clinical specimens by a specific immunometric assay." CLINICAL CHEMISTRY, vol. 45, no. 1, January 1999 (1999-01), pages 68-77, XP000946000 Washington, DC, USA *
J. BIDART ET AL.: "Monoclonal antibodies to the free beta-subunit of human chorionic gonadotropin define three distinct antigenic domains and distinguish between intact and nicked molecules." ENDOCRINOLOGY, vol. 131, no. 4, October 1992 (1992-10), pages 1832-1840, XP000939365 Philadelphia, PA, USA *
J. O'CONNOR ET AL.: "Recent advances in the chemistry and immunochemistry of human chorionic gonadotropin: impact on clinical measurement." ENDOCRINE REVIEWS, vol. 15, no. 5, 1994, pages 650-683, XP000939376 Baltimore, MD, USA *
L. COLE ET AL.: "Hyperglycosylated hCG, a potential alternative to hCG in Down syndrome screening." PRENATAL DIAGNOSIS, vol. 18, no. 9, September 1998 (1998-09), pages 926-933, XP000939392 Chichester, GB cited in the application *
P. BERGER ET AL.: "Variants of human chorionic gonadotropin from pregnant women and tumor patients recognized by monoclonal antibodies." THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 77, no. 2, August 1993 (1993-08), pages 347-351, XP000939386 Philadelphia, PA, USA *
S. BIRKEN ET AL.: "Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient." ENDOCRINE, vol. 10, no. 2, April 1999 (1999-04), pages 137-144, XP000939394 Totowa, NJ, USA *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7439026B2 (en) * 2001-07-30 2008-10-21 Quest Diagnostics Investments Incorporated Methods and kits for detecting ITA in a biological sample
US7897362B2 (en) 2001-07-30 2011-03-01 Quest Diagnostics Investments Incorporated Methods and kits for detecting ITA in a biological sample
WO2005095458A1 (fr) * 2004-04-02 2005-10-13 The Talwar Research Foundation Anticorps monoclonal chimerique destine a se lier selectivement a hcg avec une haute affinite

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CA2369923A1 (fr) 2000-10-19
AU4234900A (en) 2000-11-14
WO2000061638A3 (fr) 2001-04-19

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