Targeting of multidrug-resistant human ovarian carcinoma cells with anti-P-glycoprotein antibody conjugates

Abstract

A monoclonal antibody (mAb) to P-glycoprotein (Pgp), UIC2, is used as a targeting moiety for N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer/drug [(meso chlorin e(6) mono(N-2-aminoethylamide) (Mce(6)) or doxorubicin (DOX)] conjugates to investigate their cytotoxicity towards the Pgp-expressing human ovarian carcinoma cell line A2780/AD. The binding, internalization, and subcellular trafficking of a fluorescein labeled UIC2 targeted HPMA copolymer are studied and show localization to the plasma membrane with limited internalization. The specificity of the UIC2-targeted HPMA copolymer/drug conjugates are confirmed using the sensitive cell line A2780 that does not express Pgp.

Figure 1

Chemical structure of HPMA copolymer/drug conjugates.

Figure 2

Flow cytometry analysis of P-glycoprotein expression on the human ovarian cancer cell line A2780/AD: (—) A2780 control cells; (···) A2780 control cells exposed to fluorescein labeled anti-mouse IgG; (---) A2780/AD cells exposed to P-(AP-FITC)-mAb; and () A2780/AD cells exposed to UIC2 mAb followed by fluorescein labeled anti-mouse IgG. FL1-Height is the relative intensity of fluorescein.

Figure 3

Fluorescent microscopy images of A2780/AD cells incubated with UIC2 antibodies followed by fluorescein labeled anti-mouse IgG for 16 h at 4 (A) or 37°C (B), with conjugate 11 [P-(AP-FITC)-mAb] for 16h at 4 (C) or 37°C (D), and with conjugate 11 [P-(AP-FITC)-mAb] for 48h at 37°C (E) and conjugate 10 [P-(AP-FITC)] for 16 h at 37°C (F).

Figure 4

Viability of A2780/AD cells as a function of Mce₆ concentration. The cytotoxicity of targeted and non-targeted HPMA copolymer/drug conjugates was determined using a modified MTT assay. Typical dose-response curves are shown for free Mce₆ (-♦-); P-(G-Mce₆) (-▲-); P-(GG-Mce₆) (-□-); P-(GFLG-Mce₆) (-○-); P-(GG-Mce₆)-mAb (-■-); and P-(GFLG-Mce₆)-mAb (-●-).