TRAIL is a member of the TNF superfamily and is well known for its ability to cause cancer-selective apoptosis. A number of different approaches have been utilized in the past to produce biologically active TRAIL trimers, and are all based on the expression of monomeric cDNAs. In this study, recombinant human TRAIL trimers (TR3-family) were generated based on a single polypeptide format. We demonstrate potent apoptosis-inducing activity of TR3, similar to rTRAIL but with an enhanced stability profile compared to the latter. We further believe that the genetic approach to trimerization will extend to other TNF family members. In fact, we found that a similar concept has been reported for TNF, and similar to TR3, recombinant TNF trimers also demonstrated increased stability compared to its non-covalently associated form (19
). But the most important finding of our current work is the fact that TR3 can be further genetically modified while TRAIL activity remains fully preserved.
In this report, we demonstrate the feasibility of such modifications by incorporating cell-targeting epitopes to the parental TR3 molecule. As an example, we have demonstrated that an antibody fragment (scFv) with specificity for mouse RBCs to the N-terminus of TR3 allowed us to deliver bioactive TRAIL to a native cell membrane in a stoichiometrically-controlled fashion. This latter point represents another key feature of the TR3 platform technology and means, that one targeting molecule (scFv) could deliver a prearranged, bioactive TRAIL trimer (TR3) to a defined target site. Therefore, changing the scFv fragment to one which specifically targets a cancer cell may reduce some of the off-target toxicity associated with other rTRAIL formulations (9
). A similar strategy has been recently applied using a monomeric TRAIL-encoding scFv fusion constructs (20
), which generated predominantly monomeric as well as dimeric and only a minor fraction of biologically active trimeric complexes (22
). Therefore, we believe that the steric control provided by our assembly strategy (scFv:TR3 = 1:3) would significantly expand on the size and nature of attachments to TR3 than we would envision to encounter by combining three TRAIL molecules each carrying a cell targeting antibody fragment.
Another important modification we explored is the elongated spacer domain, inserted between the targeting scFv and TR3. This configuration was expected (1) to better enable binding to the RBC via scFv and (2) to elevate the TR3 domain farther away from the RBC surface thereby better allowing an interaction with target cell-expressed death receptors. While we do not present evidence for the spacer being advantageous for optimal target cell binding, we found that, when RBC-bound, this spacer substantially enhanced the activity of the drug compared to its spacer-deficient analog. The fact that such a spacer-containing TR3 molecule was capable of bridging two unrelated cell types (TR3-decorated RBCs and Jurkat/BxPC3 cells) and could induce apoptosis via cell-cell contact, suggests that a future anti-cancer therapeutic based on this design should be as capable of bridging tumor-specific antigens and DR4/5 located on the same cell (cis-effect) as well as adjacent cells (trans-effect) causing apoptosis of both tumor cells.
And finally, the nearly complete absence of artificial linker sequences of TR3 and the incorporation of spacer sequences with human origin, human DAF and CR1 domains (scFv-S-TR3), has the theoretical advantage of reducing potential immunogenicity of a novel therapeutic based on this technology. However, this aspect and immunogenicity due to generation of neoepitopes need to be evaluated in a small animal model, i.e. using mouse TRAIL for the construction of murine TR3 and the respective murine-derived spacer sequences.
Secreted TRAIL, expressed from a monomeric cDNA in mammalian cells is mostly inactive (23
). This has been attributed mainly to the formation of interchain disulfide bridges, which in turn causes this mixture of TRAIL monomers, dimers and trimers to have reduced affinity for its cell surface receptors. We have been able to confirm these results and only the enforced trimer formation via an isoleucine-zipper (ILZ)-domain could restore TRAIL activity (data not shown). Therefore, trimer enforcement via generation of TR3 resembles more closely an ILZ-TRAIL configuration in that formation of these disulfide bridges is likely being disabled since TR3 is biologically highly active when produced from mammalian cells. These findings have important consequences if biochemical post-production manipulations are not an option. One example represents gene therapy in which the patient's own (mammalian) cells are the source of the therapeutic protein. Even though such a concept has been investigated (24
), it seems likely that TR3 could represent a more powerful alternative over TRAIL produced from a monomer-encoding cDNA.
A potential activity-enhancing modification relates to the location where the additional fusion partners of TR3 were attached. We initiated our studies by fusing these domains to the N-terminus of TR3. However, we have recently shown that switching the scFv from the N- to the C-terminus of an RBC-targeted mouse complement regulator resulted in a substantial improvement of its activity (25
). Theoretically, those types of variations could also improve the properties of tumor-targeted TR3 variants, considering that both termini of the TR3 fusion protein should be accessible in light of several reported crystallographic studies on non-covalently associated TRAIL trimers (26
). Furthermore, TRAIL binding specificity can be tailored by genetic engineering (by introducing several amino acid substitutions) toward either of the two death-inducing receptors DR4 or DR5 (30
), depending on which death receptor would represent the more promising therapeutic target. These types of alterations could be easily applied to TR3.
One unexpected observation requires further exploration. Addition of non-targeted TRAIL forms (TR3 and rTRAIL) to the Jurkat target cells consistently resulted in a plateau in killing at ~40-60%. This plateau effect has been considered a limitation of TRAIL-based monotherapies (31
). With TRAIL immobilized on a solid matrix (the RBC surface), this plateau was consistently overcome. Our initial observations indicate that there may be cell signaling advantages to a surface-based TR3 delivery platform which warrant further investigation.
In summary, we present a new method to generate recombinant human TRAIL (TR3-family) based on a single polypeptide format featuring potent apoptosis-inducing activity and enhanced stability. Importantly, TR3 can be further modified in a stoichiometrically-controlled fashion without interfering with TRAIL function. In order to achieve higher target cell specificity and/or killing activity, we are currently adopting this platform to a variety of different tumor-associated Ags. This approach will take advantage of two ligand-receptor interactions facilitated by a single, multifunctional drug - tumor Ag:scFv and TR3:death receptor. As a consequence, we predict that the resulting multi-domain therapeutic would have greater affinity for the tumor and a reduced affinity toward normal host cells. The predicted advantages of such a concept would be a stronger and more sustained induction of the death receptor pathway in the targeted tumor cells. As TRAIL has been shown to augment the effects of standard therapies, a tumor-targeted TR3 might further enhance the effectiveness of other chemotherapies while limiting off-target toxicities to the patients.