Development of a Humanized Disulfide-stabilized Anti-p185HER2 Fv-β-Lactamase Fusion Protein for Activation of a Cephalosporin Doxorubicin Prodrug
1995
Abstract The humanized anti-p185 HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185 HER2 -overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) β-lactamase fusion protein for use in antibody-dependent enzymemediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the V L /V H interface and secreted from Escherichia coli . One variant, dsFv3 (V L L46C V H D101C), has similar affinity for antigen ( K d = 0.7 nm) as the wild-type Fv and was used to construct a fusion protein in which β-lactamase, RTEM-1, is joined to the carboxy terminus of V H . The dsFv3-β-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, k cal / k m = 1.5 × 10 5 s -1 M -1 ). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cells lines SK-BR-3 and MCF7, which express p185 HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-β-lactamase fusion protein specifically enhances the toxicity level of PRODOX to that of doxorubicin against SK-BR-3 but not MCF7 cells. The fusion protein retains both antigen-binding plus kinetic activity in murine serum and is cleared rapidly as judged by pharmacokinetic analysis in nude mice (initial and terminal half-lives of 0.23 and 1.27 h, respectively). Development and characterization of the dsFv3-β-lactamase fusion protein is an important step toward targeted prodrug therapy of p185 HER2 -overexpressing tumors.
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