Target cell-restricted and -enhanced apoptosis induction by an scFv:sTRAIL fusion protein with specificity for the pancarcinoma- associated antigen EGP2

The apparent tumour selective apoptosis inducing activity of recombinant soluble TRAIL has aroused much interest for use in clinical application. However, to fully exploit its therapeutic potential the characteristics of both the TRAIL receptor system and sTRAIL should be taken into account. Firstly, the wide spread expression of the various TRAIL receptors throughout the human body; secondly, the differential binding affinities and crosslinking requirements of the agonistic receptors TRAIL-R1 and TRAIL-R2; and thirdly, the solution behaviour of particular sTRAIL preparations. Therefore, we constructed a novel TRAIL fusion protein, designated scFvC54:sTRAIL, comprising the human scFv antibody fragment C54 genetically linked to the N-terminus of human soluble TRAIL. The scFvC54:sTRAIL fusion protein was designed to induce apoptosis by crosslinking of agonistic TRAIL receptors only after specific binding of scFvC54:sTRAIL to the abundantly expressed carcinoma-associated cell surface antigen EGP2 (alias EpCAM). Target antigen restricted apoptosis induction was demonstrated for various EGP2-positive tumour cells and could be inhibited by an EGP2 competing antibody. Target antigen binding converted soluble scFvC54:sTRAIL into a membrane bound form of TRAIL that was capable of signalling apoptosis not only through TRAIL-R1, but also through TRAIL-R2. Size-exclusion FPLC indicated that scFvC54:sTRAIL was produced as stable and homogeneous trimers in the absence of detectable TRAIL aggregates. The favourable characteristics of the scFvC54:sTRAIL fusion protein potentially reduce the amount of sTRAIL required for anti-tumour activity and may be of value for the treatment of various human carcinomas. Introduction The specific susceptibility of tumour cells to the pro-apoptotic activity of TRAIL, and the apparent lack of susceptibility of normal cells, makes this molecule a promising anti-cancer therapeutic agent. Native TRAIL is expressed as a homotrimeric type II transmembrane protein (memTRAIL), but can also be proteolytically cleaved to form a soluble trimer (sTRAIL)1,2. To date, various forms of soluble recombinant TRAIL have been generated, including FLAG-, HIS-, and non-tagged sTRAIL variants, all of which induce apoptosis in a wide range of human tumour cell lines3. Potent anti-tumour activity of various sTRAIL variants has been demonstrated in several mouse xenograft models of human cancers, including colorectal cancer3,4, glioblastoma4, and breast cancer5. International Journal of Cancer. 2004 Mar 20; 109(2):281-90