The apparent tumor selective apoptosis-inducing activity of recombinant soluble TNF-related apoptosis-inducing ligand (TRAIL) has aroused much interest for use in clinical application. However, to exploit fully its therapeutic potential, the characteristics of both the TRAIL receptor system and soluble TRAIL (sTRAIL) should be taken into account: first, the widespread expression of the various TRAIL receptors throughout the human body; second, the differential binding affinities and crosslinking requirements of the agonistic receptors TRAIL-R1 and TRAIL-R2; and third, the solution behavior 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 sTRAIL. 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 Key words: single-chain variable fragment; TNF-related apoptosisinducing ligand; targeting; apoptosis; epithelial glycoprotein 2The specific susceptibility of tumor cells to the proapoptotic activity of TNF-related apoptosis-inducing ligand (TRAIL), and the apparent lack of susceptibility of normal cells, makes this molecule a promising anticancer 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 nontagged sTRAIL variants, all of which induce apoptosis in a wide range of human tumor cell lines. 3 Potent antitumor activity of various sTRAIL variants has been demonstrated in several mouse xenograft models of human cancers, including colorectal cancer, 3,4 glioblastoma 4 and breast cancer. 5 Both memTRAIL and sTRAIL can interact with the agonistic TRAIL receptors TRAIL-R1/DR4 and TRAIL-R2/DR5, which initiate apoptosis via their intracellular death domains. 6 -9 TRAIL also binds, albeit with lower affinity, 10 to TRAIL-R3/DcR1and TRAIL-R4/DcR2, 7,11-13 both of which lack a functional death domain. TRAIL-R3 and TRAIL-R4 are considered to act as receptors that potentially modulate TRAIL activity.Expression of the different TRAIL receptors has been demonstrated not only on various tumors, but also on a wide variety of normal human tissues, indicating that apoptosis induction by TRAIL is a delicately regulated mechanism, much of which is still elusive.Clustering of TRAIL-R1 and -R2 by TRAIL leads to formation of the death-inducing signaling complex (DISC) 14 -17 by recruitment of the adapter protein FADD and resultant binding and activation of initiator caspases-8 and -10. 16,18 -21 Activated caspase-8 and -10 subsequently activate downstream effector caspases, including caspase-3, -6 and -7, which cleave cytoskeletal and nuclear proteins e...

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific

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Here, we report on a novel bispecific antibody-derivative, designated RTX-CD47, with unique capacity for CD20-directed inhibition of CD47-SIRPα “don't eat me” signaling. RTX-CD47 comprises a CD20-targeting scFv antibody fragment derived from rituximab fused in tandem to a CD47-blocking scFv. Single agent treatment with RTX-CD47 triggered significant phagocytic removal of CD20pos/CD47pos malignant B-cells, but not of CD20neg/CD47pos cells, and required no pro-phagocytic FcR-mediated signaling. Importantly, treatment with RTX-CD47 synergistically enhanced the phagocytic elimination of primary malignant B cells by autologous phagocytic effector cells as induced by therapeutic anticancer antibodies daratumumab (anti-CD38), alemtuzumab (anti-CD52) and obinutuzumab (anti-CD20). In conclusion, RTX-CD47 blocks CD47 “don't eat me” signaling by cancer cells in a CD20-directed manner with essentially no activity towards CD20neg/CD47pos cells and enhances the activity of therapeutic anticancer antibodies directed to B-cell malignancies.