Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug.
The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli. One variant, dsFv3 (VL L46C VH D101C0, has similar affinity for antigen (Kd = 0.7 nM) as the wild-type Fv and was used to construct a fusion protein in which beta-lactamase, RTEM-1, is joined to the carboxy terminus of VH. The dsFv3-beta-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, kcat/km = 1.5 x 10(5) s-1 M-1). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cell lines SK-BR-3 and MCF7, which express p185HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-beta-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-beta-lactamase fusion protein is an important step toward targeted prodrug therapy of p185HER2-overexpressing tumors.
Rodrigues
M L
ML
Department of Cell Genetics, Genetech Inc, South San Francisco, CA 94080-4990.
Presta
L G
LG
Kotts
C E
CE
Wirth
C
C
Mordenti
J
J
Osaka
G
G
Wong
W L
WL
Nuijens
A
A
Blackburn
B
B
Carter
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001483:Base Sequence; D001943:Breast Neoplasms; D004220:Disulfides; D004317:Doxorubicin / Q000378:metabolism*; D005260:Female; D018796:Immunoconjugates / Q000494:pharmacology*; D007140:Immunoglobulin Fab Fragments; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011355:Prodrugs / Q000378:metabolism*; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D014407:Tumor Cells, Cultured; D001618:beta-Lactamases
D000911:Antibodies, Monoclonal; D004220:Disulfides; D004317:Doxorubicin; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D011355:Prodrugs; D018719:Erb-b2 Receptor Tyrosine Kinases; D001618:beta-Lactamases