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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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Efficacy of RNA immunotherapy is strongly dependent on the tumor burden and time available for the induction of immune response. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (32 μg) or buffer. After 7 days splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to Ovalbumin- or Connexin-derived epitope using an ELISpot assay. * - p = 0.0154 (B) C57BL/6 mice (n = 8 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (64 μg), control vaccine (64 μg) or buffer. 6 days after the second vaccination, mice were challenged subcutaneously with 1 × 106 syngenic E.G7-OVA tumor cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. *** - p < 0.0001 (C) Expression of Ovalbumin in tumors escaping the control of the immune system, following prophylactic vaccination, was analyzed. Total RNA was isolated and OVA expression was quantified via qRT-PCR in relation to mGAPDH. ** - p = 0.0034 (D) C57BL/6 mice (n = 8 per group) were challenged s.c. with 0.3 × 106 syngenic E.G7-OVA tumor cells on day 0. On day 3 mice were treated either with OVA vaccine (32 μg), control vaccine (32 μg) or buffer. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. ** - p = 0.0091, ***- p < 0.001, ****- p < 0.0001. All presented data show representative results of at least two independent experiments.
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Fig1: Efficacy of RNA immunotherapy is strongly dependent on the tumor burden and time available for the induction of immune response. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (32 μg) or buffer. After 7 days splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to Ovalbumin- or Connexin-derived epitope using an ELISpot assay. * - p = 0.0154 (B) C57BL/6 mice (n = 8 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (64 μg), control vaccine (64 μg) or buffer. 6 days after the second vaccination, mice were challenged subcutaneously with 1 × 106 syngenic E.G7-OVA tumor cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. *** - p < 0.0001 (C) Expression of Ovalbumin in tumors escaping the control of the immune system, following prophylactic vaccination, was analyzed. Total RNA was isolated and OVA expression was quantified via qRT-PCR in relation to mGAPDH. ** - p = 0.0034 (D) C57BL/6 mice (n = 8 per group) were challenged s.c. with 0.3 × 106 syngenic E.G7-OVA tumor cells on day 0. On day 3 mice were treated either with OVA vaccine (32 μg), control vaccine (32 μg) or buffer. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. ** - p = 0.0091, ***- p < 0.001, ****- p < 0.0001. All presented data show representative results of at least two independent experiments.

Mentions: To demonstrate the potential of two-component mRNA-based cancer vaccines to mount strong immune responses, we vaccinated mice twice with a vaccine encoding the model antigen ovalbumin (OVA). Six days after the boost vaccination, high frequencies of OVA-specific T cells were detected in the splenocytes of vaccinated mice (Figure 1A). Moreover, prophylactic immunization with the OVA-encoding vaccine significantly delayed the growth of E.G7-OVA tumors (Figure 1B). Interestingly, qRT-PCR analysis of escaping tumors revealed a complete loss of OVA expression in the recurring tumors. These data suggest that the vaccination enhanced tumor surveillance resulting in the tumor outgrowth due to either antigen loss or the selection of OVA-negative cells (Figure 1C). Next, we tested the efficacy of the mRNA-based OVA vaccine in treating small, established tumors. We showed that despite the very limited time window for the induction of the immune response, frequent vaccination with two injections per week was able to induce a statistically significant delay in tumor growth compared to control animals (p = 0.0091) (Figure 1D). However, the effect was less pronounced compared to the prophylactic treatment. In summary, our results show that our two-component mRNA vaccine induces strong protective immunity and delays tumor growth in therapeutic settings.Figure 1


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)

Efficacy of RNA immunotherapy is strongly dependent on the tumor burden and time available for the induction of immune response. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (32 μg) or buffer. After 7 days splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to Ovalbumin- or Connexin-derived epitope using an ELISpot assay. * - p = 0.0154 (B) C57BL/6 mice (n = 8 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (64 μg), control vaccine (64 μg) or buffer. 6 days after the second vaccination, mice were challenged subcutaneously with 1 × 106 syngenic E.G7-OVA tumor cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. *** - p < 0.0001 (C) Expression of Ovalbumin in tumors escaping the control of the immune system, following prophylactic vaccination, was analyzed. Total RNA was isolated and OVA expression was quantified via qRT-PCR in relation to mGAPDH. ** - p = 0.0034 (D) C57BL/6 mice (n = 8 per group) were challenged s.c. with 0.3 × 106 syngenic E.G7-OVA tumor cells on day 0. On day 3 mice were treated either with OVA vaccine (32 μg), control vaccine (32 μg) or buffer. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. ** - p = 0.0091, ***- p < 0.001, ****- p < 0.0001. All presented data show representative results of at least two independent experiments.
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Fig1: Efficacy of RNA immunotherapy is strongly dependent on the tumor burden and time available for the induction of immune response. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (32 μg) or buffer. After 7 days splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to Ovalbumin- or Connexin-derived epitope using an ELISpot assay. * - p = 0.0154 (B) C57BL/6 mice (n = 8 per group) were vaccinated 2 times (1 vaccination/week) either with OVA mRNA vaccine (64 μg), control vaccine (64 μg) or buffer. 6 days after the second vaccination, mice were challenged subcutaneously with 1 × 106 syngenic E.G7-OVA tumor cells. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. *** - p < 0.0001 (C) Expression of Ovalbumin in tumors escaping the control of the immune system, following prophylactic vaccination, was analyzed. Total RNA was isolated and OVA expression was quantified via qRT-PCR in relation to mGAPDH. ** - p = 0.0034 (D) C57BL/6 mice (n = 8 per group) were challenged s.c. with 0.3 × 106 syngenic E.G7-OVA tumor cells on day 0. On day 3 mice were treated either with OVA vaccine (32 μg), control vaccine (32 μg) or buffer. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. ** - p = 0.0091, ***- p < 0.001, ****- p < 0.0001. All presented data show representative results of at least two independent experiments.
Mentions: To demonstrate the potential of two-component mRNA-based cancer vaccines to mount strong immune responses, we vaccinated mice twice with a vaccine encoding the model antigen ovalbumin (OVA). Six days after the boost vaccination, high frequencies of OVA-specific T cells were detected in the splenocytes of vaccinated mice (Figure 1A). Moreover, prophylactic immunization with the OVA-encoding vaccine significantly delayed the growth of E.G7-OVA tumors (Figure 1B). Interestingly, qRT-PCR analysis of escaping tumors revealed a complete loss of OVA expression in the recurring tumors. These data suggest that the vaccination enhanced tumor surveillance resulting in the tumor outgrowth due to either antigen loss or the selection of OVA-negative cells (Figure 1C). Next, we tested the efficacy of the mRNA-based OVA vaccine in treating small, established tumors. We showed that despite the very limited time window for the induction of the immune response, frequent vaccination with two injections per week was able to induce a statistically significant delay in tumor growth compared to control animals (p = 0.0091) (Figure 1D). However, the effect was less pronounced compared to the prophylactic treatment. In summary, our results show that our two-component mRNA vaccine induces strong protective immunity and delays tumor growth in therapeutic settings.Figure 1

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