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Development of a Humanized Disulfide-stabilized Anti-p185^HER2 Fv-ß-Lactamase Fusion Protein for Activation of a Cephalosporin Doxorubicin Prodrug

Department of Cell Genetics [M. L. R., P. C.], Protein Engineering [L. P.], Bio-Analytical Technology [C. E. K., C. W., W. L. T. W., A. N.], Experimental Therapeutics [J. M., G. O.], and Bioorganic Chemistry [B. B.], Genentech Inc, South San Francisco, CA 94080-4990

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) ß-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 ß-lactamase, RTEM-1, is joined to the carboxy terminus of V_H. The dsFv3-ß-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, k_cal/k_m = 1.5 x 10⁵ 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-ß-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-ß-lactamase fusion protein is an important step toward targeted prodrug therapy of p185^HER2-overexpressing tumors.

¹ To whom requests for reprints should be addressed, at Genentech Inc, Department of Cell Genetics, 460 Point San Bruno Boulevard, South San Francisco, CA 94080-4990.

Received 7/ 5/94. Accepted 10/25/94.

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