Abstract
TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. We have recently developed the genetically stabilized TRAIL platform TR3 in efforts to improve the limitations associated with currently available drug variants. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. However, cell death proceeded exclusively via a bystander mechanism and protected the mesothelin-positive targets from apoptosis rather than leading to their elimination. Incorporation of a spacer-into the mesothelin surface antigen or the cancer drug itself-converted SS-TR3 into a cis-acting phenotype. Further experiments with membrane-anchored TR3 variants and the native cytokine confirmed our hypothesis that membrane-proximal TRAIL species lack the capacity to physically engage their cognate receptors coexpressed on the same cell membrane. Our findings not only provide an explanation for the “peaceful” coexistence of ligand and receptor of a representative member of the TNF superfamily but give us vital clues for the design of activity-enhanced TR3-based cancer therapeutics.
Highlights
tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells
We have previously reported on TR3 fusion proteins tethered to mouse red blood cells (RBC) using the single chain antibody fragment Ter[119], essentially converting soluble biomolecules into membrane-bound counterparts [Fig. S1a and refs 8,24]
The molecular spacer used to separate the scFv targeting domain from the TR3 effector domain is comprised of four short consensus repeats (SCRs), ~60 amino acid globular and flexible domains conserved among complement regulatory proteins (CRPs)
Summary
TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. Apoptosis is an evolutionarily well-conserved process for the coordinated removal of undesired cells from a multicellular organism As such, it serves important functions ranging from early embryologic development to the eradication of senescent and potentially cancerous cells throughout our lives[1,2]. Soluble TRAIL from monomeric cDNAs in mammalian cells have failed due to intermolecular disulfide bridge formation[6] This limitation prompted us to combine the three TRAIL protomers www.nature.com/scientificreports/. Membrane tethering of MUC16-targeted Meso-TR3 to ovarian cancer cells was capable of overriding the therapeutic plateau of non-targeted TR3 (ref. 7) caused by an overexpression of the prosuvival factor cFLIP14
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