Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-13
2001-04-03
Smith, Lynette R. F. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S242000, C530S388220
Reexamination Certificate
active
06210904
ABSTRACT:
BACKGROUND OF THE INVENTION
Drug-induced thrombocytopenia contributes to morbidity and, occasionally, mortality in patients treated with a wide range of medications (Karpatkin, (1971)
Am. J. Med. Sci
. 262, p. 68). More than 100 different medications have been implicated in drug-induced thrombocytopenia, including heparin, quinine, quinidine and sulfonamide antibiotics (Shulman et al. “Hemostasis and Thrombosis” (ed 2) Philadelphia, Pa., Lippincott (1987) p.452, and Kracke et al. (1943)
JAMA
122, p. 168.
Drug-dependent antibodies reactive with platelets have been identified in only a few instances. Curtis et al., (1984)
Blood
v. 84, n.1, 176-183, applied flow cytometry to the detection of such antibodies induced by sulfonamide antibiotics. Visentin et al. (1990)
Transfusion
Oct. v. 30 n. 8, 694-700 describes detection of drug-dependent, platelet-reactive antibodies by antigen-capture ELISA and flow cytometry.
The occurrence of fibrinogen receptor antagonist induced-thrombocytopenia has prompted a search for monoclonal antibodies (mAbs) specific for GP IIb/IIIa receptor:receptor antagonist complexes. Monoclonal antibodies capable of detecting alterations in glycoprotein IIb/IIIa upon binding of fibrinogen receptor antagonists to glycoprotein IIb/IIIa are useful in diagnosing patients at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia. The monoclonals can be tagged with fluorescent or radioactive tracers for ease in diagnosis.
The present invention includes monoclonal antibodies which recognize induced binding sites formed on GPIIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GPIIb/IIIa receptor, and a means for identifying, in a patient, the presence of one or more antibodies in GP IIb/IIIa receptor (fibrinogen receptor) antagonist-induced thrombocytopenia which recognize GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complexes formed with purified platelets or purified GPIIb/IIIa receptor and a selected GPIIb/IIIa receptor antagonist. Identification of such antibodies identifies the patient as being at risk to development of thrombocytopenia resulting from administration to the patient of the selected GP IIb/IIIa receptor antagonist.
Diagnosis of patients at risk to developing fibrinogen receptor antagonist induced-thrombocytopenia can be implemented using monoclonal antibodies, having fluorescent, nuclear magnetic or radioactive tags, which are specific for GP IIb/IIIa receptor:receptor antagonist complexes. These monoclonal antibodies will associate with the complex unless a patient's plasma sample, when mixed with the complex and the monoclonal antibody, prevents association of the complex with the monoclonal antibody.
SUMMARY OF THE INVENTION
The invention includes isolated monoclonal antibodies or detectable monoclonal antibodies which recognize induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor. Exemplary monoclonal antibodies of the invention include mAb10-758 and mAb15-758. The invention also includes humanized forms of the monoclonal antibodies.
The invention is also a hybridoma cell line selected from the group consisting of DD6 and FB3, which cell lines produce monoclonal antibodies of the invention, e.g. mAb10-758, and mAb15-758.
The invention is also a method for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, which comprises combining
i) a plasma sample of the patient;
ii) detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor; and
iii) GP IIb/IIIa receptor:GP IIb/III receptor antagonist complex,
and determining association of the detectable monoclonal antibody with the complex in the presence of the plasma. The monoclonal antibodies, e.g. detectable mAb10-758 and detectable mAb15-758, are made detectable by labeling, e.g., with a fluorescence label.
In one class of the method for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, the fibrinogen receptor antagonist is selected from the group consisting of
Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH
2
(SEQ. ID NO.
1),
Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH
2
(SEQ. ID NO.
2),
Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH
2
(SEQ. ID NO.
3),
Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH
2
(SEQ. ID NO.
4),
N-Methyl-D-phenylalanyl-N-[(1S)-1-formyl-4-guanidinobutyl]-L-prolinamide,
((1-(2-((4-(aminoiminomethyl)benzoyl)amino)-3-(4-hydroxyphenyl)-1-oxopropyl)-4-piperidinyl)oxy)-(S)-acetic acid,
Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,
N-(2-(2-(((3-((aminoiminomethyl)amino)propyl)amino)carbonyl)-1-piperidnyl)-1-(cyclohexylmethyl)-2-oxoethyl)-(R,S)-glycine,
(2-S-(n-Butylsulfonylamino)-3[4-(piperidin-4-yl)butyloxyphenyl]propionic acid, hydrochloride, Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,
2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4-yl)ethyl]-4H-pyrazolo-[1,5-a][1,4]diazepin-2-yl]carbonyl]-amino]propionic acid,
(R)-methyl-3-[[[3-[4-(aminoiminomethyl)phenyl]-4,5-dihydro-5-isoxazolyl]acetyl]amino]-N-(butoxycarbonyl)-L-alanine monoacetate, and
[3(R)-[2-Piperidin-4-yl)ethyl]-2-piperidone-1]acetyl-3(R)-methyl-&bgr;-alanine,
and pharmaceutically acceptable salts thereof.
The invention is also a test kit for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, which comprises a GP IIb/IIIa receptor:GP IIb/III receptor antagonist complex, and a detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor.
The invention is also a method for identifying a patient not at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which comprises combining
i) patient plasma;
ii) a detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor; and
iii) a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex,
to form a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:detectable monoclonal antibody complex, and detecting the presence of the detectable monoclonal antibody in the GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:detectable monoclonal antibody complex.
The invention is also a method for identifying a patient at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which comprises combining
i) patient plasma;
ii) a detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor; and
iii) a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex,
to form a reaction product, and detecting the absence of the detectable monoclonal antibody in the reaction product.
DETAILED DESCRIPTION OF THE INVENTION
The anti-GPIIb/IIIa antibodies against purified GPIIb/IIIa associated with a potent fibrinogen receptor antagonist are specific to the &bgr;
3
subunit and are able to recognize this subunit on endogenous &agr;
II
&bgr;
3
and &agr;
v
&bgr;
3
integrins as shown by immunoprecipitation.
These antibodies recognize ligand-induced binding sites formed on the receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor. Specific examples of such monoclonal antibodies include mAb10-758 and mAb15-758.
mAb10-758 binding affini
Bednar Bohumil
Bollag Daniel M.
Gould Robert J.
Merck & Co. , Inc.
Parr Richard S.
Portner Ginny Allen
Smith Lynette R. F.
Winokur Melvin
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