A Genetically Encoded Multifunctional TRAIL Trimer Facilitates Cell-Specific Targeting and Tumor Cell Killing

Department of Surgery, Washington University School of Medicine, and Siteman Cancer Center, St. Louis, Missouri 63110, USA.
Molecular Cancer Therapeutics (Impact Factor: 5.68). 07/2010; 9(7):2142-51. DOI: 10.1158/1535-7163.MCT-10-0225
Source: PubMed


Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L) has been shown to exhibit potent and specific apoptotic activity against tumor cells. Several TRAIL constructs have been tried in patients, and the molecule remains under active clinical investigation. Native and recombinant TRAIL must form a homotrimer to become biologically active. However, noncovalently associated TRAIL displays a high degree of sensitivity to degradation, which limits its therapeutic potential. To enforce trimerization of the recombinant protein, we developed a covalently linked TRAIL trimer (TR3) by genetic fusion. This molecular drug design conferred improved stability without altering the native killing ability of TRAIL. Target specificity was shown by blocking TR3 activity with soluble death receptor 5 (DR5-Fc). In addition, we have shown that TR3 is amenable to further, genetic modifications. The incorporation of additional functional domains to TR3, such as antibody fragments (scFvs) that allow for a more cell-specific delivery of the agent, is stoichiometrically controlled and inconsequential with regard to the bioactivity of TRAIL. As proof of this concept, TR3 activity was targeted to the mouse RBC membrane. TR3-decorated RBCs were effectively capable of target cell killing in a model of pancreatic cancer. TR3 represents a generally applicable platform tool to study basic mechanisms along the death receptor pathway. More importantly, the ability to target TR3 to a cell surface presents the opportunity to create a cancer-selective drug with fewer off-target toxicities and enhanced killing capacities.

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Available from: Peter Goedegebuure, Apr 28, 2014
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    • "The FLAG tag was inserted to distinguish genetically engineered mesothelin from the endogenously expressed mesothelin on ovarian cancer cells. The basic TR3 expression plasmid was described previously [12], modified to include an internal 6 × His tag. Meso-TR3 was generated by N-terminal insertion of soluble mesothelin into the TR3 drug platform. "
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    ABSTRACT: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies.
    Full-text · Article · Jan 2014 · BMC Cancer
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    ABSTRACT: Recently, Spitzer and colleagues (1) reported on a novel recombinant TRAIL variant in which trimerisation of soluble TRAIL, known to be essential for apoptotic activity, is enforced by genetic fusion of three TRAIL monomers. This covalent single-chained TRAIL trimer, designated TR3, was also genetically fused to an erythrocyte-specific scFv antibody fragment, to produce fusion protein scFv-TR3. Erythrocytes decorated with scFv-TR3 seemed to acquire proapoptotic bystander-killing capacity toward Jurkat cancer cells. Previously, we extensively reported on a series of similar scFv-TRAIL fusion proteins with direct tumor targeting and killing capacity, in which we exploit the strong intrinsic capacity of TRAIL to self-associate into stable noncovalent trimers. Spitzer and colleagues referred to part of this body of work (2-4), but wrongfully claim that our homotrimeric scFv-TRAIL fusion proteins are predominantly present as biologically inactive monomers and dimers (1). Contrary to this claim, our size exclusion chromatography data actually showed that scFv-TRAIL fusion proteins are present as active homotrimers, with no detectable inactive monomers or dimers present (2). In addition, Spitzer and colleagues overlooked the fact that our scFv-TRAIL format is capable of direct trivalent target-cell binding by virtue of three scFv-targeting domains per stable trimer. Therefore, target cell-specific binding capacity by our trivalent scFv-TRAIL fusion protein format strongly benefits from the well-established avidity effect. In contrast, the scFv-TR3 format only has monovalent binding capacity, usually too weak for efficient tumor-cell targeting. Finally, Spitzer and colleagues claim an enhanced stability of their eukaryotically produced nontargeted TR3 variant, by comparing it to a prokaryotically produced noncovalent TRAIL preparation of a remarkable low stability (>95% loss of activity at 37°C within 30 minutes). To substantiate this claim, we argue that a eukaryotically produced noncovalent TRAIL trimer should have been used. In fact, using the intrinsic capacity of TRAIL to self-associate into noncovalent trimers, we produced thermo-stable scFv-TRAIL fusion proteins, with an activity half-life of ∼3 days at 37°C (5). Of note, Spitzer and colleagues evaluated thermo-stability of TR3 for a limited time-span of only 6 hours. Therefore, we argue that the data provided by Spitzer and colleagues are insufficient to warrant the claim of superior stability of TR3. Taken together, the conclusions by Spitzer and colleagues that TR3 is more stable and a better platform for scFv-based targeting of TRAIL are, in our opinion, insufficiently substantiated by their data and warrant additional detailed side-by-side comparisons with appropriate control reagents. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
    Full-text · Article · Oct 2010 · Molecular Cancer Therapeutics
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    ABSTRACT: With great interest, we have read the letter by Bremer and Helfrich about our recent report describing the generation and initial characterization of a genetically encoded TRAIL fusion protein (TR3) as a novel platform for cancer therapy (1). We very much appreciate the feedback and acknowledge the expertise of this group in advancing the field of targeted TRAIL therapy. Here, we address the complex nature of TRAIL biosynthesis and the problems that had to be resolved in order to produce bioactive, recombinant TRAIL forms for basic research and biomedical applications. In 2000, Bodmer and colleagues systematically investigated the critical nature of TRAIL's unique cysteine at position 230 (2). It turned out that bioactive material (sTRAIL, amino acids 95-281) could not be produced from mammalian cells because of the formation of intermolecular disulfide bridges identified by Western blotting as covalently linked dimers and trimers. This limitation is the reason we have not been able to do comparative functional studies between mammalian-produced sTRAIL and TR3. It is, however, possible to generate bioactive TRAIL by N-terminal addition of a trimerization domain such as an isoleucine zipper (ILZ; ref. 3). We have produced both TRAIL variants in HEK293T cells and found that sTRAIL was, indeed, nearly completely inactive, whereas ILZ-TRAIL (and TR3) was a potent inducer of cell death (1). With respect to a comparison of scFvC54:sTRAIL (4) and scFv-TR3 (1), we would like to point out that fundamental differences exist between the two concepts. This can be readily deduced from the stoichiometry of the targeting (scFv) and effector domains (TRAIL) employed [1:1 in the former (polyvalent target antigen binding via scFv) and 1:3 in the latter (monovalent)]. Together, the data presented by the authors [Western blotting (predominantly monomers and dimers and some trimers) and size exclusion chromatography (exclusively containing trimers without providing immunostaining data)], Bodmer's studies on Cys(230), and our own experimental results suggest that scFvC54:sTRAIL more closely resembles disulfide-linked multimers that are completely inactive in the absence of ectopically expressed EpCAM on Jurkat targets (4), whereas monomeric scFv-TR3 (as well as all the derivatives described in our study such as TR3 and the spacer-containing scFv-S-TR3) is more closely related to a noncovalently associated ILZ-TRAIL trimer, because these reagents were all potent inducers of apoptosis. It is worth mentioning that studies on the differential activation of death receptors 4 and 5 (DR4/5) suggest that on Jurkat cells (exclusively expressing DR5), only aggregated soluble TRAIL trimers seem to be capable of inducing apoptosis (5). However, this would imply that commercially available, prokaryotically produced TRAIL used in our study [amino acids 114-281, (1)], LZ-TRAIL, ILZ-TRAIL, and all of our TR3 preparations would exist as aggregates, which we think is unlikely, because, for example, TR3 did not differ in its electrophoretic mobility on Western blotting under reducing and nonreducing conditions and did not reveal evidence of higher mole cular weight aggregates on the same blots.(1) Because of the importance of these issues to the entire TRAIL field, we are currently investigating the exact nature of our TR3 variants and look forward to making new and exciting discoveries that will help better understand the complex biology of human TRAIL. With respect to the binding affinities of single-chain antibody fragments, we and many others have shown that monovalent scFvs can successfully deliver a variety of effector proteins to an increasing number of target antigens and exert their functions in vitro and in vivo, without the requirement for higher valencies (1, 6). Thus, future studies that assess the efficacy and mechanistic aspects of the TRAIL-death receptor pathways of both targeting concepts are eagerly awaited. We are optimistic that these results will help us to optimize either targeting strategy and move forward the best concept for the sake of the patients who suffer from cancer. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
    Preview · Article · Oct 2010 · Molecular Cancer Therapeutics
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