Construction and expression of a recombinant antibody-targeted plasminogen activator

Journal Article · Thu Oct 01 04:00:00 EDT 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.19.6904· OSTI ID:6999008

Schnee, J M; Runge, M S; Matsueda, G A; Hudson, N W; Seidman, J G; Haber, E; Quertermous, T

Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin ..beta.. chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the ..gamma..2b constant region and the catalytic ..beta.. chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation.

• Research Organization: Massachusetts General Hospital, Boston (USA)
• OSTI ID: 6999008
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:19; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANTIBODIES
BETA DECAY RADIOISOTOPES
CARDIOVASCULAR DISEASES
CHEMICAL ACTIVATION
CLONING
COAGULANTS
DAYS LIVING RADIOISOTOPES
DISEASES
DNA-CLONING
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
FIBRIN
FIBRINOLYTIC AGENTS
GENES
HEMATOLOGIC AGENTS
HEMOSTATICS
IMMUNOASSAY
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MONOCLONAL ANTIBODIES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLASMINOGEN
PROTEINS
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIOISOTOPES
SCLEROPROTEINS
THROMBOSIS
TRACER TECHNIQUES
VASCULAR DISEASES