br An analysis of the interaction between the
3.4. An analysis of the interaction between the ADS and cancer cell lines
The polymer moiety was labeled with the Cy5 fluorophore in order to assess the capacity of a PEG-protein/mAb ADS to bind the target antigen expressed on the cell surface. Cy5-PEG5kDa-SpA/Rtx was tested on Jurkat CD20 negative cell line and BL-41, LCL and Raji CD20 po-sitive cells, while Cy5-PEG20kDa-SpG/Trz on HER2/neu− (IGROV-1 and MDA-MB-231) and HER2/neu+ (SKOV-3 and SK-BR-3) cells. The re-sults showed that the Cy5-PEG5kDa-SpA/Rtx ADS was bound to the CD20+ targeted cancer Ac-DEVD-CHO as well as the free Rtx. Indeed, the geo-metric mean of the positive cells was significantly higher with respect to that of the ADS prepared with the non-specific mAb (Bevacizumab) (Fig. 5, A). The Cy5-PEG20kDa-SpG/Trz was also bound to the HER2/ neu+ cancer cell line, and the geometric mean was similar to that of the respective free Trz (Fig. 5, B). Neither of the three negative cell lines interacts with the mAbs and the ADSs.
Competition experiments were also performed to assess the stability of a preformed Cy5-PEG-protein/mAb ADS complex after incubation with a competitive mAb that did not recognize the tumor cell line; this was done to verify if the mAb in the starting ADS could be displaced by another one. The incubation used a non-specific mAb, Bevacizumab,
Fig. 5. ADS binding evaluation in diﬀerent tumor cell lines. Interaction experiments in (A) B-cells and (B) ovarian adenocarcinoma and breast cancer cell lines. The grey shaded areas depict autofluorescence control. The values in the upper-right corner of each panel represent the geometric mean. Data are representative of three independent experiments.
Fig. 6. ADS competition experiments in target tumor cell lines. The stability of ADS complex was evaluated in B-cells and ovarian adenocarcinoma cell line, (A) and (B), respectively. The grey shaded areas depict autofluorescence control. The respective geometric mean values are indicated at the upper-right corner of each panel. Data are representative of three independent experiments.
which is unable to recognize the cancer cell lines here tested. Cytofluorimetric analysis disclosed that the non-specific mAb did not perturb the strong, specific binding of the preformed ADSs of Rtx or Trz (Fig. 6, A and B). Indeed, the geometric mean values were comparable when B-cells were incubated with Cy5-PEG5kDa-SpA/Rtx or with Cy5-PEG5kDa-SpA/Rtx mixed with Bevacizumab (Raji 62.7 and 74.8; LCL
96.9 and 92.2, respectively); similar results were obtained for the cells incubated with Cy5-PEG20kDa-SpG/Trz or with the Cy5-PEG20kDa-SpG/ Trz and Bevacizumab mixture (524 and 475, respectively). Although these experiments cannot replicate the real in vivo context, where the host antibodies can displace the selected mAb in an ADS construct, the results here presented are encouraging because they did not show any
Fig. 7. Survival curves of human breast cancer cell lines in response to ADSs, TubA-PEG20kDa-SpG/Trz and TubA-PEG20kDa-SpG/Rtx and to non-targeted TubA-PEG20kDa-SpG. The growth inhibition eﬀect was evaluated by ATPlite assay. The table shows the mean ± SD of two independent experiments. For each experiment, the IC50 was calculated from each single semi-logarithmic dose-response curve by linear interpolation, and the values that were obtained, reported in μg/ml in free drug equivalents, were averaged.
perturbation of the ability of the specific prepared ADS to recognize the target cells after challenging with an unspecific antibody.
3.5. In vitro inhibition of tumor growth by ADS
For further developments we selected SpG as Fc-binding molecule because its PEGylated form showed a 1:1 binding ratio with mAbs, thus allowing a reduction of heterogeneity and better reproducibility. The in vitro cytotoxicity of ADS was investigated using TubA-PEG20kDa-SpG/ Trz against the targeted cancer line. TubA was chosen as the active cargo because of its potent cytotoxicity. Antimicrotubular agents, in fact, which block cell growth by inhibiting mitosis, are considered some of the most appropriate payloads for ADC development given their high activity levels (within the sub-nanomolar range). TubA was tethered by means of a disulfide bond to one PEG end. Disulfide bridges have, in fact, been largely exploited in drug delivery systems to promote the drug’s release after cellular internalization . Drug release is trig-gered by the reducing environment of the cytoplasm; the concentra-tions of reduced glutathione are 100–1000 times higher than those in the plasma .