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Enhancing the bystander killing effect of an oncolytic HSV by arming it with a secretable apoptosis activator.

Loya SM, Zhang X - Gene Ther. (2015)

Bottom Line: The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles.When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability.Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA [2] Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
Although oncolytic viruses have shown great promise as cancer therapeutics, results from a recent phase III clinical trial indicate that their potency may need further improvement for a clear clinical benefit. Here, we report a novel strategy to increase the bystander effect of virotherapy by arming an oncolytic virus with a secreted form of a Her2 single chain antibody linked to a self-multimerizing Fas ligand extracellular domain (Her2-COL-sFasL). The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles. When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability. Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model. Our data suggest that arming an oncolytic virus with a secretable and self-multimerizing apoptosis inducer is a feasible strategy to improve the potency of virotherapy.

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Design and Western blot analysis of secreted multimerized sFasL chimeric molecules(a) Schematic representation of the sFasL chimeric molecules used in this study. A portion of the FasL extracellular domain (amino acids 139–281) was used to make constructs. Secr, optimal secretion signal; HA, hemagglutinin tag; COL, collagen-like trimerization domain; Her2 scFv, FRP5 Her2 single chain variable fragment; TM, transmembrane domain; AA, amino acids. (b and c) Western blot detection of chimeric sFasL molecules and control constructs under reducing and non-reducing conditions. HCT116 cells were transfected with mammalian expression constructs encoding the respective chimeric molecules. After 48 hours cell debris was removed from media and equal amounts of media subjected to Western blot analysis (b) with 25mM DTT and (c) without DTT. HA tag antibody was used for detection and data shown at two different exposure times for clarity. Western blot results are representative of three independent experiments.
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Figure 1: Design and Western blot analysis of secreted multimerized sFasL chimeric molecules(a) Schematic representation of the sFasL chimeric molecules used in this study. A portion of the FasL extracellular domain (amino acids 139–281) was used to make constructs. Secr, optimal secretion signal; HA, hemagglutinin tag; COL, collagen-like trimerization domain; Her2 scFv, FRP5 Her2 single chain variable fragment; TM, transmembrane domain; AA, amino acids. (b and c) Western blot detection of chimeric sFasL molecules and control constructs under reducing and non-reducing conditions. HCT116 cells were transfected with mammalian expression constructs encoding the respective chimeric molecules. After 48 hours cell debris was removed from media and equal amounts of media subjected to Western blot analysis (b) with 25mM DTT and (c) without DTT. HA tag antibody was used for detection and data shown at two different exposure times for clarity. Western blot results are representative of three independent experiments.

Mentions: We first designed and constructed a panel of chimeric molecules with differing combinations of functional domains. A schematic of the constructs is shown in Figure 1a. First, an optimal secretion signal and hemagglutinin tag (HA) was fused to the amino-terminus of sFasL (amino acids 139–281 of the extracellular domain) forming Secr-sFasL. Second, for the purpose of multimerization, a previously optimized collagen-based multimerization domain (COL) was inserted between the HA tag and sFasL domain, forming COL-sFasL. The COL domain consists of the hinge region of human IgG at the N terminus, a collagen-like scaffold (glycine-proline-proline)10 repeat flanked at the C terminus by the NC1 domain of type XXI collagen (see Supplementary Figure S1 for sequence details).28 Furthermore, Her2-COL-sFasL was constructed by inserting the Her2 scFv between the secretion signal and HA tag of COL-sFasL. For comparison, Her2-sFasL was constructed by inserting the Her2 scFv between the secretion signal and HA tag of Secr-sFasL. In addition two control constructs, HA-COL and Her2-COL, were made that contain differing functional domains without sFasL.


Enhancing the bystander killing effect of an oncolytic HSV by arming it with a secretable apoptosis activator.

Loya SM, Zhang X - Gene Ther. (2015)

Design and Western blot analysis of secreted multimerized sFasL chimeric molecules(a) Schematic representation of the sFasL chimeric molecules used in this study. A portion of the FasL extracellular domain (amino acids 139–281) was used to make constructs. Secr, optimal secretion signal; HA, hemagglutinin tag; COL, collagen-like trimerization domain; Her2 scFv, FRP5 Her2 single chain variable fragment; TM, transmembrane domain; AA, amino acids. (b and c) Western blot detection of chimeric sFasL molecules and control constructs under reducing and non-reducing conditions. HCT116 cells were transfected with mammalian expression constructs encoding the respective chimeric molecules. After 48 hours cell debris was removed from media and equal amounts of media subjected to Western blot analysis (b) with 25mM DTT and (c) without DTT. HA tag antibody was used for detection and data shown at two different exposure times for clarity. Western blot results are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352111&req=5

Figure 1: Design and Western blot analysis of secreted multimerized sFasL chimeric molecules(a) Schematic representation of the sFasL chimeric molecules used in this study. A portion of the FasL extracellular domain (amino acids 139–281) was used to make constructs. Secr, optimal secretion signal; HA, hemagglutinin tag; COL, collagen-like trimerization domain; Her2 scFv, FRP5 Her2 single chain variable fragment; TM, transmembrane domain; AA, amino acids. (b and c) Western blot detection of chimeric sFasL molecules and control constructs under reducing and non-reducing conditions. HCT116 cells were transfected with mammalian expression constructs encoding the respective chimeric molecules. After 48 hours cell debris was removed from media and equal amounts of media subjected to Western blot analysis (b) with 25mM DTT and (c) without DTT. HA tag antibody was used for detection and data shown at two different exposure times for clarity. Western blot results are representative of three independent experiments.
Mentions: We first designed and constructed a panel of chimeric molecules with differing combinations of functional domains. A schematic of the constructs is shown in Figure 1a. First, an optimal secretion signal and hemagglutinin tag (HA) was fused to the amino-terminus of sFasL (amino acids 139–281 of the extracellular domain) forming Secr-sFasL. Second, for the purpose of multimerization, a previously optimized collagen-based multimerization domain (COL) was inserted between the HA tag and sFasL domain, forming COL-sFasL. The COL domain consists of the hinge region of human IgG at the N terminus, a collagen-like scaffold (glycine-proline-proline)10 repeat flanked at the C terminus by the NC1 domain of type XXI collagen (see Supplementary Figure S1 for sequence details).28 Furthermore, Her2-COL-sFasL was constructed by inserting the Her2 scFv between the secretion signal and HA tag of COL-sFasL. For comparison, Her2-sFasL was constructed by inserting the Her2 scFv between the secretion signal and HA tag of Secr-sFasL. In addition two control constructs, HA-COL and Her2-COL, were made that contain differing functional domains without sFasL.

Bottom Line: The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles.When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability.Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA [2] Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
Although oncolytic viruses have shown great promise as cancer therapeutics, results from a recent phase III clinical trial indicate that their potency may need further improvement for a clear clinical benefit. Here, we report a novel strategy to increase the bystander effect of virotherapy by arming an oncolytic virus with a secreted form of a Her2 single chain antibody linked to a self-multimerizing Fas ligand extracellular domain (Her2-COL-sFasL). The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles. When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability. Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model. Our data suggest that arming an oncolytic virus with a secretable and self-multimerizing apoptosis inducer is a feasible strategy to improve the potency of virotherapy.

Show MeSH
Related in: MedlinePlus