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

Abstract

Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumorassociated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N-and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the Nterminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.

Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

“…Compared to Duokines, this results in increased flexibility. Furthermore, conversion into single-chain derivatives can increase the stability of the individual ligands, as shown recently for TRAIL 26 by choosing appropriate linkers and the minimally required THD sequence. Comparing the plasma stability of the different Duokines and scDuokines, we did not observe a drastic improvement of stability for the scDuokines, except for some of the molecules, e.g.…”

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

“…Further linker engineering of scTRAIL led to improved thermal stability and solubility of the Db-scTRAIL molecule. 27 Antibody fusion proteins in the homotrimeric scFv-TNFSF format have also been reported for several members of the mouse TNFSF, including CD40L, FasL, TRAIL, LIGHT, VEGI, lymphotoxin a, lymphotoxin b and lymphotoxin alpha1/beta2, in combination with an ED-A-directed antibody moiety. 34 Biodistribution studies in the F9-tumor mouse model revealed considerable differences in the performance of the scFv-TNFSF fusion proteins.…”

“…20 So far, generation of single-polypeptide chain variants had only been reported for apoptotic members of the conventional family, where improved stability was shown for TNF 25 and TRAIL. 26,27 Here, we report in the antibody-fusion protein context the generation of the single-chain format for TNFSF members of the divergent family, comparing binding and activation potential with corresponding recombinant wild-type proteins. 4-1BBL and OX40L showed comparable receptor binding capacity for both formats in ELISA that was not hampered by the fusion to the scFv EpCAM , indicating adequate linker length and ligand assembly in the single-chain format (Fig.…”

The antibody‐based delivery of interleukin‐12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors

“…Cytokines are a class of immunomodulatory proteins, which have been considered as biopharmaceuticals for cancer therapy . In particular, certain proinflammatory cytokines (such as interleukin [IL]‐2, IL12 and IL15) could potentially be used to boost the activity of T cells and NK cells within the neoplastic mass.…”

“…9 Cytokines are a class of immunomodulatory proteins, which have been considered as biopharmaceuticals for cancer therapy. 10,11 In particular, certain proinflammatory cytokines (such as interleukin [IL]-2, IL12 and IL15) could potentially be used to boost the activity of T cells and NK cells within the neoplastic mass. However, most cytokine products cause side effects at low doses (sometimes even at 1 μg/kg in patients), thus preventing dose escalation to therapeutically active regimens.…”