Abstract
Fusion proteins combining hexavalent TRAIL with antibody fragments allow for a targeted delivery and efficient apoptosis induction in tumor cells. Here, we analyzed scFv-Fc-scTRAIL molecules directed against EGFR, HER2, HER3, and EpCAM as well as an untargeted Fc-scTRAIL fusion protein for their potentials to induce cell death both in vitro and in a xenograft tumor model in vivo. The scFv-Fc-scTRAIL fusion protein directed against EGFR as well as the fusion protein directed against EpCAM showed targeting effects on the two tested colorectal carcinoma cell lines Colo205 and HCT116, while a fusion protein targeting HER3 was more effective than untargeted Fc-scTRAIL only on Colo205 cells. Interestingly, another anti-HER3 scFv-Fc-scTRAIL fusion protein exhibiting approximately 10-fold weaker antigen binding as well as the HER2-directed molecule were unable to increase cytotoxicity compared to Fc-scTRAIL. A comparison of EC50 values of cell death induction and antigen binding supports the assumption that high affinity antigen binding is one of the requirements for in vitro targeting effects. Furthermore, a minimal number of expressed target antigens might be required for increased cytotoxicity of targeted compared to non-targeted molecules. In a Colo205 s.c. xenograft tumor model, strongest antitumor activity was observed for the anti-HER3 scFv-Fc-scTRAIL fusion protein based on antibody 3-43, with complete tumor remissions after six twice-weekly injections. Surprisingly, a similar in vivo activity was also observed for untargeted Fc-scTRAIL in this tumor model, indicating that additional factors contribute to the potent efficacy of targeted as well as untargeted hexavalent Fc-scTRAIL fusion proteins in vivo.
Highlights
Pro-apoptotic receptor agonists (PARAs) are powerful molecules to kill cancer cells independently of p53 and the intrinsic apoptosis pathway
Five different antibody moieties directed against four distinct tumorassociated antigens were employed, including the EGFRtargeting antibody hu225 derived from antibody C225 used in cetuximab [29] and humanized by CDR grafting [30], the trastuzumab-derived 4D5 directed against human epidermal growth factor receptor 2 (HER2) [31], the HER3-targeting antibodies 3M6 and 3-43 [33], as well as the humanized version 323/A3hu3 [34] of the anti-epithelial cell adhesion molecule (EpCAM) antibody 323/A3 [35, 36]
High molecular weight species were found for scFv4D5-fragment crystallizable (Fc)-single-chain variants of TNF-related apoptosis-inducing ligand (TRAIL) (scTRAIL), whereas fractions of smaller size were detected for scFv3M6-FcscTRAIL and scFv323/A3hu3-Fc-scTRAIL (Figure 2C)
Summary
Pro-apoptotic receptor agonists (PARAs) are powerful molecules to kill cancer cells independently of p53 and the intrinsic apoptosis pathway. Despite promising anti-tumor effects in preclinical models [1, 2], soluble TRAIL (sTRAIL) as well as agonistic antibodies against the two death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) did not show therapeutic activity in clinical trials [3] This deficiency has been attributed to intrinsic or acquired resistance of many human tumors towards TRAILinduced apoptosis [4,5,6] as well as to a limited efficacy of the applied therapeutics. This insufficient activity has been linked to the lacking ability of sTRAIL and agonistic antibodies to induce higher order clustering of TRAIL receptors [7,8,9]. Based on the required clustering of death receptors for efficient apoptosis induction, several www.impactjournals.com/oncotarget strategies have been developed to enhance the activity of TRAIL-based therapeutics [8]
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