Targeting Doxorubicin to Epidermal Growth Factor Receptors by Site-specific Conjugation of C225 to Poly(L-glutamic Acid) Through a Polyethylene Glycol Spacer

Purpose: Targeted delivery of chemotherapeutic agents through antibody-polymer conjugates has met with limited success. One of the limiting factors is the loss of antibody's binding affinity upon conjugation with polymeric carriers because of lack of control over the number and site of attachment. This study aims to synthesize monovalent polymeric immunoconjugates through site-specific conjugation and to evaluate the in vitro binding activities of the resulting construct.

Methods: Antibody C225 against epidermal growth factor receptors was coupled to the terminus of a doxorubicin-bound block copolymer, poly(L-glutamic acid)-co-polyethylene glycol (PG-PEG). Western blot analysis, confocal fluorescent microscopy, and cytotoxicity assay were performed to confirm the specific binding of C225-PEG-PG-Dox to EGFR.

Results: C225 was conjugated to PEG-PG-doxorubicin conjugates by reacting sulfhydryl group introduced to C225 with vinylsulfone group introduced at the terminus of PEG-PG block copolymer. Polymeric immunoconjugate C225-PEG-PG-Dox, but not control (i.e., conjugate without antibody), selectively bound to human vulvar squamous carcinoma A431 cells that overexpress epidermal growth factor receptors. Receptor-mediated uptake of C225-PEG-PG-Dox occurred rapidly (within 5 min), whereas nonspecific uptake of PEG-PG-Dox required an extended period of time (24 h) to internalize. Binding of C225-PEG-PG-Dox to A431 cells could be blocked by pretreatment with C225 antibody. C225-PEG-PG-Dox was more potent than free doxrubicin in inhibiting the growth of A431 cells after a 6-h exposure period.

Conclusion: Site-specific conjugation of a monoclonal antibody to the terminus of a polymeric carrier enhances receptor-mediated delivery of anticancer agents.