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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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In vivo passaging of FusOn-H3-Her2-COL-sFasL results in a virus adapted for enhanced in vitro replication and significantly extends the therapeutic effect of the oncolytic virus in a 4T1 immunocompetent in vivo model(a) Comparative analysis of in vitro replication efficiencies of control and in vivo passaged viruses in 4T1 cells. 4T1 cells were infected in triplicate with designated virus at an MOI of 10. Cells were collected at 24 (black bars) and 48 hours post infection (white bars) and total infectious virus quantified through titration in Vero cells. ns, not significant; *p< 0.01; **p<0.001 as compared to respective 24 or 48 hour FusOn-H3 titer according to student’s T test. (b) Enhanced therapeutic efficacy of FusOn-H3-Her2-COL-sFasL* in 4T1 syngeneic in vivo model. 4T1 subcutaneous tumors were established in right flanks of BALB/c mice by injection of 1×105 cells per mouse. Once tumors reached the average diameter of 4mm, tumors were intratumorally injected with PBS as a control, 1×107 pfu FusOn-H3, or 1×107 pfu FusOn-H3-Her2-COL-sFasL* on day 0 and day 7 (black arrows). Tumors were measured every 3 days with a caliper. Percent change in tumor volume was calculated by dividing the daily tumor volume by the tumor volume measurement at day 0. These measurements were then averaged (n=5 mice per group). No statistical difference between FusOn-H3 and FusOn-H3-Her2-COL-sFasL virotherapy was detected prior to day 20. *p<0.05 on day 20 according to student’s T test. Error bars represent ±SEM.
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Figure 6: In vivo passaging of FusOn-H3-Her2-COL-sFasL results in a virus adapted for enhanced in vitro replication and significantly extends the therapeutic effect of the oncolytic virus in a 4T1 immunocompetent in vivo model(a) Comparative analysis of in vitro replication efficiencies of control and in vivo passaged viruses in 4T1 cells. 4T1 cells were infected in triplicate with designated virus at an MOI of 10. Cells were collected at 24 (black bars) and 48 hours post infection (white bars) and total infectious virus quantified through titration in Vero cells. ns, not significant; *p< 0.01; **p<0.001 as compared to respective 24 or 48 hour FusOn-H3 titer according to student’s T test. (b) Enhanced therapeutic efficacy of FusOn-H3-Her2-COL-sFasL* in 4T1 syngeneic in vivo model. 4T1 subcutaneous tumors were established in right flanks of BALB/c mice by injection of 1×105 cells per mouse. Once tumors reached the average diameter of 4mm, tumors were intratumorally injected with PBS as a control, 1×107 pfu FusOn-H3, or 1×107 pfu FusOn-H3-Her2-COL-sFasL* on day 0 and day 7 (black arrows). Tumors were measured every 3 days with a caliper. Percent change in tumor volume was calculated by dividing the daily tumor volume by the tumor volume measurement at day 0. These measurements were then averaged (n=5 mice per group). No statistical difference between FusOn-H3 and FusOn-H3-Her2-COL-sFasL virotherapy was detected prior to day 20. *p<0.05 on day 20 according to student’s T test. Error bars represent ±SEM.

Mentions: The results in Figure 4b showed that incorporation of Her2-COL-sFasL into the FusOn-H3 backbone affected the virus replication by almost a log. Previous studies by Shah et al have shown that in vivo passage of transgene encoding oncolytic HSVs can improve virus replication.33 We thus passaged FusOn-H3-Her2-COL-sFasL in vivo by injecting the virus into HCT116 tumors and retrieving it 30 days after virus injection. The retrieved virus was then compared with FusOn-H3 and the unpassaged FusOn-H3-Her2-COL-sFasL virus for replication in 4T1 mouse mammary gland tumor cells, which were previously found to be semi-permissive to FusOn-H2 (ref. 34). Figure 6a shows that the passaged virus (herein referred to as FusOn-H3-Her2-COL-sFasL*) replicates closer to the level of FusOn-H3 parental virus in vitro in 4T1 cells, indicating the strategy to improve virus replication through in vivo passage also applies to this sFasL-containing oncolytic HSV.


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

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

In vivo passaging of FusOn-H3-Her2-COL-sFasL results in a virus adapted for enhanced in vitro replication and significantly extends the therapeutic effect of the oncolytic virus in a 4T1 immunocompetent in vivo model(a) Comparative analysis of in vitro replication efficiencies of control and in vivo passaged viruses in 4T1 cells. 4T1 cells were infected in triplicate with designated virus at an MOI of 10. Cells were collected at 24 (black bars) and 48 hours post infection (white bars) and total infectious virus quantified through titration in Vero cells. ns, not significant; *p< 0.01; **p<0.001 as compared to respective 24 or 48 hour FusOn-H3 titer according to student’s T test. (b) Enhanced therapeutic efficacy of FusOn-H3-Her2-COL-sFasL* in 4T1 syngeneic in vivo model. 4T1 subcutaneous tumors were established in right flanks of BALB/c mice by injection of 1×105 cells per mouse. Once tumors reached the average diameter of 4mm, tumors were intratumorally injected with PBS as a control, 1×107 pfu FusOn-H3, or 1×107 pfu FusOn-H3-Her2-COL-sFasL* on day 0 and day 7 (black arrows). Tumors were measured every 3 days with a caliper. Percent change in tumor volume was calculated by dividing the daily tumor volume by the tumor volume measurement at day 0. These measurements were then averaged (n=5 mice per group). No statistical difference between FusOn-H3 and FusOn-H3-Her2-COL-sFasL virotherapy was detected prior to day 20. *p<0.05 on day 20 according to student’s T test. Error bars represent ±SEM.
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Figure 6: In vivo passaging of FusOn-H3-Her2-COL-sFasL results in a virus adapted for enhanced in vitro replication and significantly extends the therapeutic effect of the oncolytic virus in a 4T1 immunocompetent in vivo model(a) Comparative analysis of in vitro replication efficiencies of control and in vivo passaged viruses in 4T1 cells. 4T1 cells were infected in triplicate with designated virus at an MOI of 10. Cells were collected at 24 (black bars) and 48 hours post infection (white bars) and total infectious virus quantified through titration in Vero cells. ns, not significant; *p< 0.01; **p<0.001 as compared to respective 24 or 48 hour FusOn-H3 titer according to student’s T test. (b) Enhanced therapeutic efficacy of FusOn-H3-Her2-COL-sFasL* in 4T1 syngeneic in vivo model. 4T1 subcutaneous tumors were established in right flanks of BALB/c mice by injection of 1×105 cells per mouse. Once tumors reached the average diameter of 4mm, tumors were intratumorally injected with PBS as a control, 1×107 pfu FusOn-H3, or 1×107 pfu FusOn-H3-Her2-COL-sFasL* on day 0 and day 7 (black arrows). Tumors were measured every 3 days with a caliper. Percent change in tumor volume was calculated by dividing the daily tumor volume by the tumor volume measurement at day 0. These measurements were then averaged (n=5 mice per group). No statistical difference between FusOn-H3 and FusOn-H3-Her2-COL-sFasL virotherapy was detected prior to day 20. *p<0.05 on day 20 according to student’s T test. Error bars represent ±SEM.
Mentions: The results in Figure 4b showed that incorporation of Her2-COL-sFasL into the FusOn-H3 backbone affected the virus replication by almost a log. Previous studies by Shah et al have shown that in vivo passage of transgene encoding oncolytic HSVs can improve virus replication.33 We thus passaged FusOn-H3-Her2-COL-sFasL in vivo by injecting the virus into HCT116 tumors and retrieving it 30 days after virus injection. The retrieved virus was then compared with FusOn-H3 and the unpassaged FusOn-H3-Her2-COL-sFasL virus for replication in 4T1 mouse mammary gland tumor cells, which were previously found to be semi-permissive to FusOn-H2 (ref. 34). Figure 6a shows that the passaged virus (herein referred to as FusOn-H3-Her2-COL-sFasL*) replicates closer to the level of FusOn-H3 parental virus in vitro in 4T1 cells, indicating the strategy to improve virus replication through in vivo passage also applies to this sFasL-containing oncolytic HSV.

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