• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • SC 560 br The cells were incubated during the


    The SC 560 were incubated during the experiment with escalating doses of ADS, TubA-PEG20kDa-SpG, or Trz alone. The HER2-specific ADS (TubA-PEG20kDa-SpG/Trz) exhibited high cytotoxic potency in the HER2-expressing SK-BR-3 cancer cells, with IC50 values of 0.006 μg/ ml ± 0.001 (drug equiv.) (Fig. 7). The same ADS was more than 16-fold less potent in the HER2/neu negative MDA-MB-231 human breast cancer cells with IC50 values of 0.101 μg/ml ± 0.015, thus confirming that Trz in the ADS plays an active role in binding HER2/neu receptor 
    and yields an improved cytotoxic effect. TubA, instead, exhibited a si-milar cytotoxic efficacy in both cell lines (SK-BR-3, IC50 = 0.045 μg/ ml ± 0.001; MDA-MB-231, IC50 = 0.085 μg/ml ± 0.012), while Trz had no cytotoxic effect in any culture. In addition, the IC50 values of the non-targeted counterpart (TubA-PEG20kDa-SpG) and the non-HER2-specific ADS (TubA-PEG20kDa-SpG/Rtx) in the SK-BR-3 and MDA-MB-231 cancer cells were very similar, which again supports the relevance of Trz for selective cytotoxicity of its ADS construct in cancer cells HER/ neu+. The reduced cytotoxic sensitivity of MDA-MB-231 to the ADS is due to the lack of HER2/neu expression in this cell line on the one hand and on the other, it further supports the HER2/neu mediated binding and internalization of the ADS in the SK-BR-3 cell line (HER2/neu+).
    This in vitro cytotoxicity investigation clearly showed that the non-covalently bound Trz in the ADS constructs preserved its ability to re-cognize its target, that the ADS was internalized by cells expressing the target, and that after cell internalization the drug was released and carried out its cytotoxic activity.
    4. Conclusions
    This study focused on designing and developing a more homo-geneous ADC product in the effort to achieve a versatile and adaptable drug delivery system. A new drug delivery platform based on a non-covalent interaction between mAbs and an Fc-binding molecule (FcBM), which also acts as drug-carrier, was investigated here. As it is well known that SpA and SpG are able to bind the Fc region of many immunoglobulins with high affinity, they were chosen as the FcBM models. The proteins were successively PEGylated at the N-terminal
    amino acid with a heterobifunctional PEG that was in turns conjugated to a drug or a dye. ITC and DLS analyses showed that the PEGylated FcBMs non-covalently bound mAbs with high affinity by simply mixing, thus creating the PEG-FcBM/mAbs complex. After the complexation with a model mAb (Trz or Rtx), Cy5 labeled ADSs were tested in vitro via cytometry analysis that showed high selectivity for the targeted antigen expressing cells. The preformed ADS proved to be stable even after incubation with a competitive antibody, maintaining the ability to recognize the target cells. From the study, SpG emerged as promising Fc-binding molecule for such application thanks to the feature of its PEGylated form to bind the target mAb with a binding ratio of 1:1, allowing a reduction of the heterogeneity of the system. Consequently, the toxin TubA was conjugated to PEG-SpG to get the TubA-PEG20kDa-SpG/Trz ADS. As expected this ADS showed a preferential cytotoxic activity against the HER2/neu+ cell line (SK-BR-3) with respect to the HER2/neu− cell line (MDA-MB-231), thus supporting its application for both therapeutic purposes.
    To conclude, an ADS approach was used to create a non-covalent drug delivery system utilizing an antibody as the targeting moiety that preserved an in vitro affinity for antigen presenting cells. Using this approach means that the number of drug molecules delivered by a single antibody can be strictly controlled, and as only the Fc region is involved in FcBM binding, this ensures that the Fab regions remain free to interact with the target antigen. As different antibodies can be used with the same FcBM construct, it is possible to select the most oppor-tune mAb depending on cancer that is being treated.