A bifunctional murine::human chimeric antibody with one antigen-binding arm replaced by bacterial beta-lactamase.
We here report the genetic engineering of a murine::human chimeric antibody--directed against the tumor marker human placental alkaline phosphatase--in which one antigen-binding arm (Fab) has been replaced by Escherichia coli beta-lactamase (Bla). A mutated Bla gene in which the termination codon had been replaced by GAG, was fused in-phase to the cDNA sequence encoding the hinge region, CH2 and CH3 of the human IgG3 heavy chain. The resulting BlaHG3f fusion gene was placed under control of the Simian Virus 40 late promoter, and transiently expressed in COS-1 cells together with the genes encoding the murine light and murine::human chimeric heavy chains. Approximately 200 ng/ml of correctly assembled bifunctional antibody-Bla immunoconjugates were detected in the culture supernatant. This observation indicates that Bla (with its own leader peptide) can efficiently direct secretion into the culture medium of adventitious sequences fused at its C-terminus. Furthermore, the assembly in the Fc region was not affected by steric hindrance due to a Bla moiety and an Fab arm in close proximity. The antibody-Bla immunoconjugate could be of therapeutic value for the activation of cephalosporin-based anti-cancer prodrugs at the tumor site. Moreover, the expression strategy adopted here is particularly suitable for a quick and convenient analysis of newly designed gene products in which the Bla moiety has been replaced by other enzymes or by antigen-binding fragments in order to engineer bispecific antibodies.
De Sutter
K
K
Laboratory of Molecular Biology, Gent University, Belgium.
Fiers
W
W
D000469:Alkaline Phosphatase / Q000276:immunology*; D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D014408:Biomarkers, Tumor / Q000276:immunology*; D005260:Female; D005818:Genetic Engineering; D006801:Humans; D000922:Immunotoxins / Q000235:genetics*; D051379:Mice; D010920:Placenta / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
D000912:Antibodies, Neoplasm; D001426:Bacterial Proteins; D014408:Biomarkers, Tumor; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D001618:beta-Lactamases