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Immunological data from cancer patients treated with Ad5/3-E2F-Δ24-GMCSF suggests utility for tumor immunotherapy.

Hemminki O, Parviainen S, Juhila J, Turkki R, Linder N, Lundin J, Kankainen M, Ristimäki A, Koski A, Liikanen I, Oksanen M, Nettelbeck DM, Kairemo K, Partanen K, Joensuu T, Kanerva A, Hemminki A - Oncotarget (2015)

Bottom Line: Accumulating data suggests that virus induced oncolysis can enhance anti-tumor immunity and break immune tolerance.To capitalize on the immunogenic nature of oncolysis, we generated a quadruple modified oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GMCSF).The virus was tested preclinically in vitro and in vivo and then 13 patients with solid tumors refractory to standard therapies were treated.

View Article: PubMed Central - PubMed

Affiliation: Cancer Gene Therapy Group, Transplantation Laboratory & Haartman Institute, University of Helsinki, Helsinki, Finland.

ABSTRACT
Oncolytic viruses that selectively replicate in tumor cells can be used for treatment of cancer. Accumulating data suggests that virus induced oncolysis can enhance anti-tumor immunity and break immune tolerance. To capitalize on the immunogenic nature of oncolysis, we generated a quadruple modified oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GMCSF). Ad5/3-E2F-Δ24-GMCSF (CGTG-602) was engineered to contain a tumor specific E2F1 promoter driving an E1 gene deleted at the retinoblastoma protein binding site ("Δ24"). The fiber features a knob from serotype 3 for enhanced gene delivery to tumor cells. The virus was tested preclinically in vitro and in vivo and then 13 patients with solid tumors refractory to standard therapies were treated. Treatments were well tolerated and frequent tumor- and adenovirus-specific T-cell immune responses were seen. Overall, with regard to tumor marker or radiological responses, signs of antitumor efficacy were seen in 9/12 evaluable patients (75%). The radiological disease control rate with positron emission tomography was 83% while the response rate (including minor responses) was 50%. Tumor biopsies indicated accumulation of immunological cells, especially T-cells, to tumors after treatment. RNA expression analyses of tumors indicated immunological activation and metabolic changes secondary to virus replication.

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Related in: MedlinePlus

(A) A schematic representation of the Ad5/3-E2F-Δ24-GMCSF virusThe E1A promoter is replaced by a human E2F1 promoter, which controls the transcription of the E1A gene. The E1A gene features a 24 base-pair deletion to avoid the self-activation of the promoter by E2F released by E1A-Rb interaction, a critical fault in previous designs with an intact E1A gene. Gp19k and 6.7K in E3 have been replaced with the cDNA of human GM-CSF. The serotype 5 (Ad5) fiber knob has been replaced by the serotype 3 (Ad3) knob. (B) Top panel: Ad5/3-E2F-Δ24-GMCSF -expressed GMCSF is functionally active. TF1 cells, which require human GMCSF for viability, were cultured in the presence of human recombinant GMCSF or filtered supernatant from virus infected cells. As a control, supernatant from cells infected with a virus not expressing GM-CSF was used (Ad5/3-Δ24). The viability of TF1-cells treated with growth medium (GM) only was set as 100% hGMCSF control and filtered supernatant showed significantly (P < 0.001) higher viability over GM, while no difference was seen between other groups. Middle panel: Less infective virus particles were produced by Ad5/3-E2F-Δ24-GMCSF in human primary hepatocytes at 48 and 72 h time points compared to the control viruses, indicating low replication in normal human cells. Bottom panel: to evaluate tumor selectivity of the virus, livers of non-tumor-bearing Syrian hamsters were injected and no viral E4 copy number increase was detected with qPCR. In contrast, E4 copy number increased in HapT-1 tumors. Replacing the native E1A promoter with the E2F-1 promoter does not impair virus replication and cell killing effect in vitro(C) or in vivo(D). (c) Results of a cell viability assay shows the cell killing efficiency of Ad5/3-E2F-Δ24-GMCSF compared to Ad5/3-Δ24-GMCSF - a control virus bearing the genetically intact E1A promoter - a wild type serotype 5 virus and a non-replicating control virus Ad5/3luc1 in A549 lung cancer cells, SKOV3ip.1 ovarian cancer cells and PC3-MM2 prostate cancer cells. (d) Ad5/3-E2F-Δ24-GMCSF significantly (P < 0.01) slowed down tumor progression compared to the mock (growth medium only) animals in Syrian Hamsters bearing pancreatic cancer tumors. 3 × 108 VP of virus was administered intratumorally on days 0, 3 and 6. Concomitant low dose cyclophosphamide did not significantly improve the anti-tumor effect of Ad5/3-E2F-Δ24-GMCSF.
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Figure 1: (A) A schematic representation of the Ad5/3-E2F-Δ24-GMCSF virusThe E1A promoter is replaced by a human E2F1 promoter, which controls the transcription of the E1A gene. The E1A gene features a 24 base-pair deletion to avoid the self-activation of the promoter by E2F released by E1A-Rb interaction, a critical fault in previous designs with an intact E1A gene. Gp19k and 6.7K in E3 have been replaced with the cDNA of human GM-CSF. The serotype 5 (Ad5) fiber knob has been replaced by the serotype 3 (Ad3) knob. (B) Top panel: Ad5/3-E2F-Δ24-GMCSF -expressed GMCSF is functionally active. TF1 cells, which require human GMCSF for viability, were cultured in the presence of human recombinant GMCSF or filtered supernatant from virus infected cells. As a control, supernatant from cells infected with a virus not expressing GM-CSF was used (Ad5/3-Δ24). The viability of TF1-cells treated with growth medium (GM) only was set as 100% hGMCSF control and filtered supernatant showed significantly (P < 0.001) higher viability over GM, while no difference was seen between other groups. Middle panel: Less infective virus particles were produced by Ad5/3-E2F-Δ24-GMCSF in human primary hepatocytes at 48 and 72 h time points compared to the control viruses, indicating low replication in normal human cells. Bottom panel: to evaluate tumor selectivity of the virus, livers of non-tumor-bearing Syrian hamsters were injected and no viral E4 copy number increase was detected with qPCR. In contrast, E4 copy number increased in HapT-1 tumors. Replacing the native E1A promoter with the E2F-1 promoter does not impair virus replication and cell killing effect in vitro(C) or in vivo(D). (c) Results of a cell viability assay shows the cell killing efficiency of Ad5/3-E2F-Δ24-GMCSF compared to Ad5/3-Δ24-GMCSF - a control virus bearing the genetically intact E1A promoter - a wild type serotype 5 virus and a non-replicating control virus Ad5/3luc1 in A549 lung cancer cells, SKOV3ip.1 ovarian cancer cells and PC3-MM2 prostate cancer cells. (d) Ad5/3-E2F-Δ24-GMCSF significantly (P < 0.01) slowed down tumor progression compared to the mock (growth medium only) animals in Syrian Hamsters bearing pancreatic cancer tumors. 3 × 108 VP of virus was administered intratumorally on days 0, 3 and 6. Concomitant low dose cyclophosphamide did not significantly improve the anti-tumor effect of Ad5/3-E2F-Δ24-GMCSF.

Mentions: The E1A region, E3 region and fiber are genetically modified in Ad5/3-E2F-Δ24-GMCSF (Figure 1A). Virally produced GMCSF was tested functional by analyzing the growth of GMCSF dependent TF-1 erythroleukemia cells upon addition of filtered supernatant from Ad5/3-E2F-Δ24-GMCSF infected A549 cells (Figure 1B top).


Immunological data from cancer patients treated with Ad5/3-E2F-Δ24-GMCSF suggests utility for tumor immunotherapy.

Hemminki O, Parviainen S, Juhila J, Turkki R, Linder N, Lundin J, Kankainen M, Ristimäki A, Koski A, Liikanen I, Oksanen M, Nettelbeck DM, Kairemo K, Partanen K, Joensuu T, Kanerva A, Hemminki A - Oncotarget (2015)

(A) A schematic representation of the Ad5/3-E2F-Δ24-GMCSF virusThe E1A promoter is replaced by a human E2F1 promoter, which controls the transcription of the E1A gene. The E1A gene features a 24 base-pair deletion to avoid the self-activation of the promoter by E2F released by E1A-Rb interaction, a critical fault in previous designs with an intact E1A gene. Gp19k and 6.7K in E3 have been replaced with the cDNA of human GM-CSF. The serotype 5 (Ad5) fiber knob has been replaced by the serotype 3 (Ad3) knob. (B) Top panel: Ad5/3-E2F-Δ24-GMCSF -expressed GMCSF is functionally active. TF1 cells, which require human GMCSF for viability, were cultured in the presence of human recombinant GMCSF or filtered supernatant from virus infected cells. As a control, supernatant from cells infected with a virus not expressing GM-CSF was used (Ad5/3-Δ24). The viability of TF1-cells treated with growth medium (GM) only was set as 100% hGMCSF control and filtered supernatant showed significantly (P < 0.001) higher viability over GM, while no difference was seen between other groups. Middle panel: Less infective virus particles were produced by Ad5/3-E2F-Δ24-GMCSF in human primary hepatocytes at 48 and 72 h time points compared to the control viruses, indicating low replication in normal human cells. Bottom panel: to evaluate tumor selectivity of the virus, livers of non-tumor-bearing Syrian hamsters were injected and no viral E4 copy number increase was detected with qPCR. In contrast, E4 copy number increased in HapT-1 tumors. Replacing the native E1A promoter with the E2F-1 promoter does not impair virus replication and cell killing effect in vitro(C) or in vivo(D). (c) Results of a cell viability assay shows the cell killing efficiency of Ad5/3-E2F-Δ24-GMCSF compared to Ad5/3-Δ24-GMCSF - a control virus bearing the genetically intact E1A promoter - a wild type serotype 5 virus and a non-replicating control virus Ad5/3luc1 in A549 lung cancer cells, SKOV3ip.1 ovarian cancer cells and PC3-MM2 prostate cancer cells. (d) Ad5/3-E2F-Δ24-GMCSF significantly (P < 0.01) slowed down tumor progression compared to the mock (growth medium only) animals in Syrian Hamsters bearing pancreatic cancer tumors. 3 × 108 VP of virus was administered intratumorally on days 0, 3 and 6. Concomitant low dose cyclophosphamide did not significantly improve the anti-tumor effect of Ad5/3-E2F-Δ24-GMCSF.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: (A) A schematic representation of the Ad5/3-E2F-Δ24-GMCSF virusThe E1A promoter is replaced by a human E2F1 promoter, which controls the transcription of the E1A gene. The E1A gene features a 24 base-pair deletion to avoid the self-activation of the promoter by E2F released by E1A-Rb interaction, a critical fault in previous designs with an intact E1A gene. Gp19k and 6.7K in E3 have been replaced with the cDNA of human GM-CSF. The serotype 5 (Ad5) fiber knob has been replaced by the serotype 3 (Ad3) knob. (B) Top panel: Ad5/3-E2F-Δ24-GMCSF -expressed GMCSF is functionally active. TF1 cells, which require human GMCSF for viability, were cultured in the presence of human recombinant GMCSF or filtered supernatant from virus infected cells. As a control, supernatant from cells infected with a virus not expressing GM-CSF was used (Ad5/3-Δ24). The viability of TF1-cells treated with growth medium (GM) only was set as 100% hGMCSF control and filtered supernatant showed significantly (P < 0.001) higher viability over GM, while no difference was seen between other groups. Middle panel: Less infective virus particles were produced by Ad5/3-E2F-Δ24-GMCSF in human primary hepatocytes at 48 and 72 h time points compared to the control viruses, indicating low replication in normal human cells. Bottom panel: to evaluate tumor selectivity of the virus, livers of non-tumor-bearing Syrian hamsters were injected and no viral E4 copy number increase was detected with qPCR. In contrast, E4 copy number increased in HapT-1 tumors. Replacing the native E1A promoter with the E2F-1 promoter does not impair virus replication and cell killing effect in vitro(C) or in vivo(D). (c) Results of a cell viability assay shows the cell killing efficiency of Ad5/3-E2F-Δ24-GMCSF compared to Ad5/3-Δ24-GMCSF - a control virus bearing the genetically intact E1A promoter - a wild type serotype 5 virus and a non-replicating control virus Ad5/3luc1 in A549 lung cancer cells, SKOV3ip.1 ovarian cancer cells and PC3-MM2 prostate cancer cells. (d) Ad5/3-E2F-Δ24-GMCSF significantly (P < 0.01) slowed down tumor progression compared to the mock (growth medium only) animals in Syrian Hamsters bearing pancreatic cancer tumors. 3 × 108 VP of virus was administered intratumorally on days 0, 3 and 6. Concomitant low dose cyclophosphamide did not significantly improve the anti-tumor effect of Ad5/3-E2F-Δ24-GMCSF.
Mentions: The E1A region, E3 region and fiber are genetically modified in Ad5/3-E2F-Δ24-GMCSF (Figure 1A). Virally produced GMCSF was tested functional by analyzing the growth of GMCSF dependent TF-1 erythroleukemia cells upon addition of filtered supernatant from Ad5/3-E2F-Δ24-GMCSF infected A549 cells (Figure 1B top).

Bottom Line: Accumulating data suggests that virus induced oncolysis can enhance anti-tumor immunity and break immune tolerance.To capitalize on the immunogenic nature of oncolysis, we generated a quadruple modified oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GMCSF).The virus was tested preclinically in vitro and in vivo and then 13 patients with solid tumors refractory to standard therapies were treated.

View Article: PubMed Central - PubMed

Affiliation: Cancer Gene Therapy Group, Transplantation Laboratory & Haartman Institute, University of Helsinki, Helsinki, Finland.

ABSTRACT
Oncolytic viruses that selectively replicate in tumor cells can be used for treatment of cancer. Accumulating data suggests that virus induced oncolysis can enhance anti-tumor immunity and break immune tolerance. To capitalize on the immunogenic nature of oncolysis, we generated a quadruple modified oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GMCSF). Ad5/3-E2F-Δ24-GMCSF (CGTG-602) was engineered to contain a tumor specific E2F1 promoter driving an E1 gene deleted at the retinoblastoma protein binding site ("Δ24"). The fiber features a knob from serotype 3 for enhanced gene delivery to tumor cells. The virus was tested preclinically in vitro and in vivo and then 13 patients with solid tumors refractory to standard therapies were treated. Treatments were well tolerated and frequent tumor- and adenovirus-specific T-cell immune responses were seen. Overall, with regard to tumor marker or radiological responses, signs of antitumor efficacy were seen in 9/12 evaluable patients (75%). The radiological disease control rate with positron emission tomography was 83% while the response rate (including minor responses) was 50%. Tumor biopsies indicated accumulation of immunological cells, especially T-cells, to tumors after treatment. RNA expression analyses of tumors indicated immunological activation and metabolic changes secondary to virus replication.

Show MeSH
Related in: MedlinePlus