Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity

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.

An optimized antibody-single-chain TRAIL fusion protein for cancer therapy

“…3A, B). 20 For both variants, an increased portion of the dimeric, full-length form was obtained, compared with Db-scTRAIL-95L8 used in previous studies ( Fig. 3D, E; 72% for Db-Glyco-scTRAIL-FAVSGAA and 82% for Db-GlycoscTRAIL-FAVSGIA vs. 54% for the reference Db-scTRAIL-95L8).…”

“…For comparison, homotrimeric soluble TRAIL (sTRAIL 115-281 ) exhibited a melting point of 46 C. The apoptosis-inducing activity of the new scTRAIL variants was tested in a cytotoxicity assay using Colo205 colon carcinoma cells sensitized with 250 ng/ml bortezomib. 20 All scTRAIL variants were bioactive with EC 50 values in the range of 77 to 199 pM (Table 1, Fig. S2).…”

“…18 Thus, unlike the naturally processed soluble TRAIL and recombinant therapeutics derived thereof, 19 a scTRAIL lacks inactivating dissociation at dilute concentrations, i.e., is stable as a trimer at physiologic temperatures. 18,20 ScTRAIL molecules have been successfully used to generate antibody TRAIL fusion proteins to achieve specific targeting and to improve pharmacokinetic properties with the aim to enrich the therapeutic protein at the tumor site. 18,20,21 Moreover, depending on the antibody format used as fusion partner for scTRAIL, we recently showed that a controlled oligomerization of the scTRAIL module can be achieved, for example, through fusion of a diabody or a dimerization module such as the heavy chain domain 2 of IgE, resulting in substantially increased biological activity compared to sTRAIL, scTRAIL, and targeted scTRAIL in a monomeric scFv fusion protein.…”

“…18,20 ScTRAIL molecules have been successfully used to generate antibody TRAIL fusion proteins to achieve specific targeting and to improve pharmacokinetic properties with the aim to enrich the therapeutic protein at the tumor site. 18,20,21 Moreover, depending on the antibody format used as fusion partner for scTRAIL, we recently showed that a controlled oligomerization of the scTRAIL module can be achieved, for example, through fusion of a diabody or a dimerization module such as the heavy chain domain 2 of IgE, resulting in substantially increased biological activity compared to sTRAIL, scTRAIL, and targeted scTRAIL in a monomeric scFv fusion protein. 20,21 Because we noted a rather low thermal stability of scTRAIL with a melting temperature of approximately 46 C, as determined by dynamic light scattering, we set out to develop derivatives of scTRAIL with increased biophysical and biochemical properties as a prerequisite for clinical development of tumor-targeted TRAIL-based therapeutics.…”

Increased Apoptosis Induction in Hepatocellular Carcinoma by a Novel Tumor-Targeted TRAIL Fusion Protein Combined With Bortezomib

“…This TRAIL version can be further endowed with target-dependent activity by fusion to an Ab derivate recognizing tumor-associated antigens. 28,29 In the present study, we investigated the proapoptotic effects of a novel scTRAIL fusion protein targeting EGFR, in combination with BZB, in primary hepatocytes and organ-like tissues from patients with HCC, nonalcoholic fatty liver disease (NAFLD), and healthy control individuals. We demonstrate that EGFR-targeted TRAIL in combination with BZB selectively induces apoptosis in HCC, but not in healthy or nontumor liver cells and is superior to nontargeted TRAIL.…”