The objective of this project is to establish that the naturally occurring asparagine- (Asn)- glycosylation site found in the light chain variable domain (VK) of a monoclonal antibody (MAb), LL2, is a potential site for the universal conjugation of cytotoxic agents (e.g., doxorubicin) for cancer therapy. Phase I is focused on confirming that the carbohydrate moiety found in the LL2 VK domain is a potential conjugation site for indirect dextran mediated drug attachment. LL2 fragment devoid of Fc carbohydrate will be used as the substrate for conjugation. The effect of drug-dextran conjugation to the VK carbohydrate group on the immunoreactivity of the antibody/fragment will also be evaluated. Meanwhile, Asn-glycosylation corresponding to that in murine LL2 will be engineered, by oligonucleotide directed mutagenesis or polymerase chain reaction (PCR), into the VK domains of humanized MN14 and/or LL2 antibody, in the original sequence design of which no Asn-glycosylation site was incorporated. Expression vectors for the humanized MN14 and/or LL2 containing the Asn-glycosylation site will be constructed and expressed in SP2/0 myeloma cells by transfection (electroporation). The efficiency of drug conjugation for the glycosylated humanized antibodies, either in the form of whole IgG or fragments, will be evaluated and compared with that of their non- glycosylated counterparts.
