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mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.

Fotin-Mleczek M, Zanzinger K, Heidenreich R, Lorenz C, Kowalczyk A, Kallen KJ, Huber SM - Radiat Oncol (2014)

Bottom Line: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect.Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated.Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: CureVac GmbH, CureVac GmbH, Paul-Ehrlich-Str, 15, Tübingen 72076, Germany. mf@curevac.com.

ABSTRACT

Background: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics.

Methods: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells.

Results: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors.

Conclusion: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

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Combination of RNA immunotherapy with radiation allows for the eradication of large established tumors and induction of epitope spreading. (A) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.3 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 150 mm3) mice were treated either with OVA mRNA vaccine (32 μg), radiation (6 Gy total, divided into 3 equal fractions on 3 consecutive days) or radioimmunotherapy as indicated (with first vaccination given 6h before first radiation). Untreated mice served as a control. *** - p < 0.001 (B) Median survival time of mice analysed in Figure 2A. (C) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.5 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 300 mm3) mice were treated either with local radiation (8 Gy total, divided into 4 equal fractions on 4 consecutive days) or radioimmunotherapy as indicated (64 μg/vaccination, with first vaccination given 6 h before first radiation). Untreated mice served as a control. ** - p < 0.01 (D) All complete responders, which were tumor free after combination therapy (day 106), were re-challenge subcutaneously with 1 × 105 parenteral OVA-negative EL-4 cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. All presented data show representative results of at least two independent experiments.
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Fig2: Combination of RNA immunotherapy with radiation allows for the eradication of large established tumors and induction of epitope spreading. (A) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.3 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 150 mm3) mice were treated either with OVA mRNA vaccine (32 μg), radiation (6 Gy total, divided into 3 equal fractions on 3 consecutive days) or radioimmunotherapy as indicated (with first vaccination given 6h before first radiation). Untreated mice served as a control. *** - p < 0.001 (B) Median survival time of mice analysed in Figure 2A. (C) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.5 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 300 mm3) mice were treated either with local radiation (8 Gy total, divided into 4 equal fractions on 4 consecutive days) or radioimmunotherapy as indicated (64 μg/vaccination, with first vaccination given 6 h before first radiation). Untreated mice served as a control. ** - p < 0.01 (D) All complete responders, which were tumor free after combination therapy (day 106), were re-challenge subcutaneously with 1 × 105 parenteral OVA-negative EL-4 cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. All presented data show representative results of at least two independent experiments.

Mentions: Therapeutic vaccines alone are not effective in curing bulky, large tumors due to the time required to induce a potent immune response as well as tumor-mediated inhibitory mechanisms. We, therefore, asked whether the combination of RNA immunotherapy with local irradiation could result in the successful treatment of the established tumors. E.G7-OVA tumor cells were transplanted subcutaneously into the right limb and mice were left untreated until the tumor reached a volume of about 150 mm3. Since immunotherapy and radiation therapy can successfully treat high percentage of small tumors, the large tumor size as well as the delayed start of the therapy was chosen to enable demonstration of the synergistic effect of radioimmunotherapy in the settings when neither of the monotherapies was sufficient. Tumors of mice in the radiation and the combined radioimmunotherapy groups were irradiated with a total dose of 6 Gy, administered in 3 equal fractions for three consecutive days. Mice in the immunotherapy and the radioimmunotherapy groups received two vaccinations per week starting at the first day of radiation. Immunotherapy alone showed only a marginal effect on tumor growth (Figure 2A), which was expected due to the limited time during which an immune response can develop while tumors are in the exponential growth phase. Similarly, radiation alone induced only a transient inhibition of tumor growth for about 7 days. In contrast, combined radioimmunotherapy significantly improved the anti-tumor effect (Figure 2A). All mice treated with this regimen showed pronounced tumor regression, resulting in complete and sustained eradication of the tumor in 3 out of 7 mice. Median survival in the combination group increased significantly to 45 days after the start of treatment, compared to 9 days for untreated mice (p = 0.0002), 11 days for mice receiving immunotherapy (p = 0.0176) and 17.5 days for mice receiving radiation therapy (p = 0.045) (Figure 2B).Figure 2


mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.

Fotin-Mleczek M, Zanzinger K, Heidenreich R, Lorenz C, Kowalczyk A, Kallen KJ, Huber SM - Radiat Oncol (2014)

Combination of RNA immunotherapy with radiation allows for the eradication of large established tumors and induction of epitope spreading. (A) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.3 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 150 mm3) mice were treated either with OVA mRNA vaccine (32 μg), radiation (6 Gy total, divided into 3 equal fractions on 3 consecutive days) or radioimmunotherapy as indicated (with first vaccination given 6h before first radiation). Untreated mice served as a control. *** - p < 0.001 (B) Median survival time of mice analysed in Figure 2A. (C) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.5 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 300 mm3) mice were treated either with local radiation (8 Gy total, divided into 4 equal fractions on 4 consecutive days) or radioimmunotherapy as indicated (64 μg/vaccination, with first vaccination given 6 h before first radiation). Untreated mice served as a control. ** - p < 0.01 (D) All complete responders, which were tumor free after combination therapy (day 106), were re-challenge subcutaneously with 1 × 105 parenteral OVA-negative EL-4 cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. All presented data show representative results of at least two independent experiments.
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Related In: Results  -  Collection

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Fig2: Combination of RNA immunotherapy with radiation allows for the eradication of large established tumors and induction of epitope spreading. (A) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.3 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 150 mm3) mice were treated either with OVA mRNA vaccine (32 μg), radiation (6 Gy total, divided into 3 equal fractions on 3 consecutive days) or radioimmunotherapy as indicated (with first vaccination given 6h before first radiation). Untreated mice served as a control. *** - p < 0.001 (B) Median survival time of mice analysed in Figure 2A. (C) C57BL/6 mice (n = 8 per group) were challenged subcutaneously on the right limb with 0.5 × 106 syngenic E.G7-OVA tumor cells. 13 days after tumor challenge (at a median tumor volume of 300 mm3) mice were treated either with local radiation (8 Gy total, divided into 4 equal fractions on 4 consecutive days) or radioimmunotherapy as indicated (64 μg/vaccination, with first vaccination given 6 h before first radiation). Untreated mice served as a control. ** - p < 0.01 (D) All complete responders, which were tumor free after combination therapy (day 106), were re-challenge subcutaneously with 1 × 105 parenteral OVA-negative EL-4 cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. All presented data show representative results of at least two independent experiments.
Mentions: Therapeutic vaccines alone are not effective in curing bulky, large tumors due to the time required to induce a potent immune response as well as tumor-mediated inhibitory mechanisms. We, therefore, asked whether the combination of RNA immunotherapy with local irradiation could result in the successful treatment of the established tumors. E.G7-OVA tumor cells were transplanted subcutaneously into the right limb and mice were left untreated until the tumor reached a volume of about 150 mm3. Since immunotherapy and radiation therapy can successfully treat high percentage of small tumors, the large tumor size as well as the delayed start of the therapy was chosen to enable demonstration of the synergistic effect of radioimmunotherapy in the settings when neither of the monotherapies was sufficient. Tumors of mice in the radiation and the combined radioimmunotherapy groups were irradiated with a total dose of 6 Gy, administered in 3 equal fractions for three consecutive days. Mice in the immunotherapy and the radioimmunotherapy groups received two vaccinations per week starting at the first day of radiation. Immunotherapy alone showed only a marginal effect on tumor growth (Figure 2A), which was expected due to the limited time during which an immune response can develop while tumors are in the exponential growth phase. Similarly, radiation alone induced only a transient inhibition of tumor growth for about 7 days. In contrast, combined radioimmunotherapy significantly improved the anti-tumor effect (Figure 2A). All mice treated with this regimen showed pronounced tumor regression, resulting in complete and sustained eradication of the tumor in 3 out of 7 mice. Median survival in the combination group increased significantly to 45 days after the start of treatment, compared to 9 days for untreated mice (p = 0.0002), 11 days for mice receiving immunotherapy (p = 0.0176) and 17.5 days for mice receiving radiation therapy (p = 0.045) (Figure 2B).Figure 2

Bottom Line: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect.Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated.Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: CureVac GmbH, CureVac GmbH, Paul-Ehrlich-Str, 15, Tübingen 72076, Germany. mf@curevac.com.

ABSTRACT

Background: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics.

Methods: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells.

Results: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors.

Conclusion: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

Show MeSH
Related in: MedlinePlus