Abstract
Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC50 ∼2 × 10−12 M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance.
Highlights
Resistance to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be caused by the expression levels and composition of proapoptotic and decoy TRAIL receptors as well as by intracellular mechanisms
For generation of functionally improved scFvscTRAIL fusion proteins, single-chain TRAIL (scTRAIL) was first genetically optimized for mammalian expression
A humanized single-chain Fv fragment ‘huC225’ (RE Kontermann, unpublished data) derived from cetuximab was fused to the N-terminus of scTRAIL, yielding scFvaEGFR-scTRAIL (Figure 1a)
Summary
Resistance to TRAIL-induced apoptosis can be caused by the expression levels and composition of proapoptotic and decoy TRAIL receptors as well as by intracellular mechanisms. TRAIL mutants, acting predominantly on either DR4 or DR5, facilitate cell death induction, for example on DR5-positive tumor cells, without the need for membrane immobilization or crosslinking of soluble TRAIL.[12,13] Alternatively, TRAIL can be fused to antibodies directed against tumor markers It has been shown for CD33-directed scFv-TRAIL that such fusion proteins are superior over antibody-targeted chemotherapeutics because of stronger cytotoxic activity and the minimization of side effects on normal nontransformed cells.[14] Correct trimerization and zinc coordination of recombinant TRAIL therapeutics seem to be crucial for biological activity and for nonreactivity toward nontransformed cells, in particular hepatocytes.[15] Our previous results showed that three extracellular domains of human TRAIL can be fused by peptide linkers to yield highly active single-chain TRAIL (scTRAIL) that can be further endowed with target-dependent activity by fusion to an antibody derivative.[16] Such antigen-binding scFv-scTRAIL molecules functionally mimic the natural membraneexpressed TRAIL and are more effective therapeutics than scTRAIL alone.[16] We here sought to develop a concept for controlled oligomerization of scFv-scTRAIL fusion proteins leading to improved expression and stability as well as increased functional activity under retention of tumor selectivity
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