A Novel Affibody-Auristatin E Conjugate With a Potent and Selective Activity Against HER2+ Cell Lines

Abstract

Targeted therapy is a new type of cancer treatment that most often uses biologically active drugs attached to a monoclonal antibody. This so called antibody-drug conjugate strategy allows the use of highly toxic substances that target tumor cells specifically, leaving healthy tissues largely unaffected. Over the last few years, antibody-drug conjugates have become a powerful tool in cancer treatment. We developed and characterized a novel cytotoxic conjugate against HER2 tumors in which the antibody has been substituted with a much smaller molecule: the affibody. The conjugate is composed of the ZHER2:2891 affibody that recognizes HER2 and a highly potent cytotoxic drug auristatin E. The ZHER2:2891 molecule does not contain cysteine(s) in its amino acid sequence. We generated 3 variants of ZHER2:2891, each containing a single cysteine to allow conjugation through the maleimide group that is present in the cytotoxic component. In 2 variants, we introduced single S46C and D53C substitutions. In the third variant, a short Drug Conjugation Sequence (DCS) containing a single cysteine was introduced at the C-terminus of ZHER2:2891, resulting in ZHER2:2891-DCS. The latter variant exhibited a significantly higher conjugation yield, and therefore its cytotoxicity has been studied more thoroughly. The ZHER2:2891-DCS-MMAE conjugate killed the HER2-overexpressing SK-BR-3 and MDA-MB-453 cells efficiently (IC50 values of 5.2 and 24.8 nM, respectively). The T-47-D and MDA-MB-231 cells that express normal levels of HER2 were significantly less sensitive to the conjugate (IC50 values of 135.6 and 161.5 nM, respectively). Overall, we have demonstrated for the first time that proteins other than antibodies/antibody fragments can be successfully combined with a linker-drug module, resulting in conjugates that eliminate cancer cells selectively.

A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells

“…The fluorescently labeled anti-HER2 diaffibody was used to stain the SK-BR-3 cells, which strongly overexpress HER2, and the control U-87 MG cells, which have physiological levels of HER2. The HER2 status of these cell lines was previously confirmed by SDS-PAGE analysis [41]. A similar experiment was also performed with commercially available anti-HER2 mouse monoclonal antibodies, followed by donkey anti-mouse polyclonal antibodies conjugated with FITC.…”

“…Importantly, we previously reported that all these cell lines are similarly sensitive to free MMAE [41,51]. The cytotoxicity of (Z HER2:4 ) 2 DCS-MMAE was measured by the MTT proliferation assay (Figure 9 and Figure 10).…”

“…To further evaluate the selectivity of (Z HER2:4 ) 2 DCS-MMAE in the colorimetric MTT assay, the following cell lines were used; SK-BR-3 and MDA-MB-453 HER2-positive (HER2+) cell lines; T-47D cells that exhibit a slight increase in HER2 levels (HER2+/−); and three HER2-negative cell lines (HER2−), U-87 MG, SK-MES-1, and MDA-MB-231 [41,42]. The above-mentioned cells were treated with increasing doses of the conjugate, and their viability was monitored after 72, 96, and 120 h. According to the obtained results, 0.8 nM (Z HER2:4 ) 2 DCS-MMAE reduced the SK-BR-3 cells’ viability to 15% within 120 h (Figure 9a).…”

“…The above-mentioned cells were treated with increasing doses of the conjugate, and their viability was monitored after 72, 96, and 120 h. According to the obtained results, 0.8 nM (Z HER2:4 ) 2 DCS-MMAE reduced the SK-BR-3 cells’ viability to 15% within 120 h (Figure 9a). This concentration of the conjugate had a minor effect on the viability of MDA-MB-453 cells, which express lower levels of HER2 than SK-BR-3 cells [41,42], and no effect on the viability of T-47D cells (Figure 9). A 5-fold increase of (Z HER2:4 ) 2 DCS-MMAE concentration caused a decrease of MDA-MB-453 viability to 15% within 120 h (Figure 9b).…”

“…Affibody is an example of a potential non-antibody drug carrier [41,44,45,46]. This small protein has been engineered to bind to a large number of target proteins or peptides with high affinity [29].…”