The Strong <i>In Vivo</i> Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

<div><p>P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells <i>in vitro</i> almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors <i>in vivo</i> by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our <i>in vitro</i> cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of <i>in vivo</i> UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered <i>in vitro</i> cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive <i>in vivo</i> anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).</p></div

Antibody-dependent cell-mediated cytotoxicity (ADCC, <i>panels A</i> and <i>B</i>) and complement mediated lysis (CDC, <i>panels C</i> and <i>D</i>) induced by UIC2 mAb treatment <i>in vitro</i>.

<p>In the ADCC assay KB-V1 (<i>A</i>) and KB-3-1 (<i>B</i>) tumor cells were labeled with CFDA-SE then mixed with PBMCs freshly isolated from peripheral blood at different target to effector cell ratios. Samples were treated with 10 µM CsA (○), 20 µg/ml UIC2 mAb (▪), 10 µM CsA and 20 µg/ml UIC2 mAb (□) or buffer (•). After 8 h incubation at 37°C, samples were stained with PI and analyzed by flow cytometry. In the CDC assay, cells were incubated with human serum at different dilutions for 4 h. <i>Inset of panel C</i>: Hemolytic effect of the serum (▴) and of heat inactivated serum (▵) on sensitized sheep red blood cells served as positive and negative control, respectively. The percentages of killed cells were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107875#s2" target="_blank">Materials and Methods</a>. Values are means (± SD) of four independent experiments, ***P<0,001.</p

Effect of treatment with DOX combined with UIC2 and/or a low dose of CsA on the weights of the grafted tumors (<i>A</i>) and on the percentage of the detectable tumors in the different treatment groups (<i>B</i>).

<p>Tumor weights were expressed as a percentage of the average weight of the tumors of untreated animals measured at the time of the termination of the experiment (mean values ± SEM, n = 8). Statistically significant differences relative to the untreated control and the DOX-only treated groups are marked with *: P<0.05; **: P<0.01; ***: P<0.001). Grey bars: KB-3-1 (Pgp^-) and empty bars: KB-V1 (Pgp⁺) tumors.</p

Cytotoxic effect of DOX in KB-3-1 (Pgp^-; grey bars) and KB-V1 (Pgp⁺; empty bars) cells and its potentiation by treatments with CsA (used at the indicated concentrations) and/or UIC2 mAb (20 µg/ml).

<p>EC₅₀ values were calculated by fitting the dose-response curves. Values are means (± SD) of 3 independent experiments. Statistically significant differences are shown by * (P<0.05).</p