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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 represents an effective treatment strategy for low immunogenic and radioresistant LLC tumor. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with EGFR mRNA vaccine (32 μg) or with buffer. 7 days after completed vaccination splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to EGFR- or Control-peptide library using an ELISpot assay. ** - p = 0.0036 (B) C57BL/6 mice (n = 10 per group) were challenged subcutaneously on the right limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated either with local radiation (36 Gy total, divided into 3 equal fractions on 3 consecutive days) or with radioimmunotherapy as indicated (with first vaccination given 6 h before second radiation). Vaccination therapy without radiation started on day 14 post tumor challenge. Untreated mice served as a control. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. (C) Median survival time of mice analyzed in Figure 3B.
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Fig3: Combination of RNA immunotherapy with radiation represents an effective treatment strategy for low immunogenic and radioresistant LLC tumor. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with EGFR mRNA vaccine (32 μg) or with buffer. 7 days after completed vaccination splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to EGFR- or Control-peptide library using an ELISpot assay. ** - p = 0.0036 (B) C57BL/6 mice (n = 10 per group) were challenged subcutaneously on the right limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated either with local radiation (36 Gy total, divided into 3 equal fractions on 3 consecutive days) or with radioimmunotherapy as indicated (with first vaccination given 6 h before second radiation). Vaccination therapy without radiation started on day 14 post tumor challenge. Untreated mice served as a control. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. (C) Median survival time of mice analyzed in Figure 3B.

Mentions: Having demonstrated the strong synergistic effect of radiotherapy and mRNA immunotherapy in the treatment of highly immunogenic E.G7-OVA tumors, we sought to determine whether this therapeutic strategy is also effective against one of the most challenging tumor models: LLC. This tumor model is resistant to different kinds of therapeutic regimens (Avastin, radiation, adoptive T cell transfer) [6–8]. It is characterized by nearly complete MHC class I down regulation, a lack of known tumor rejection antigens, very aggressive growth kinetics, frequent ulceration in small tumors and rapid formation of spontaneous lung metastases from the primary solid tumor. We chose human EGFR and Connexin as target antigens because of the evidence for their immunogenicity [9]. As shown in Figure 3A, application of the EGFR mRNA vaccine (5 vaccinations in total, twice a week, 32 μg per vaccination) resulted in a strong induction of EGFR-specific T cell responses compared to control animals (p = 0.0036). In corroboration with the literature data [10] dose finding studies revealed that LLC is strongly resistant to radiation (Additional file 1: Figure S1). As shown in Figure 3B, to achieve a weak and transient inhibitory effect on tumors with a volume of just 50 mm3, a total dose of 36 Gy was required. This dose was split into three fractions, given on consecutive days, starting on day 18 after tumor challenge. As shown in Figure 3B, immunotherapy alone was not able to mediate any tumor growth inhibition and radiation therapy alone resulted in only transient inhibition. In sharp contrast, mRNA vaccines combined with a high dose of radiotherapy resulted in a strong synergistic anti-tumor effect. In conclusion, our data show the efficacy of radioimmunotherapy in controlling tumor growth of a low immunogenic carcinoma.Figure 3


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 represents an effective treatment strategy for low immunogenic and radioresistant LLC tumor. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with EGFR mRNA vaccine (32 μg) or with buffer. 7 days after completed vaccination splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to EGFR- or Control-peptide library using an ELISpot assay. ** - p = 0.0036 (B) C57BL/6 mice (n = 10 per group) were challenged subcutaneously on the right limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated either with local radiation (36 Gy total, divided into 3 equal fractions on 3 consecutive days) or with radioimmunotherapy as indicated (with first vaccination given 6 h before second radiation). Vaccination therapy without radiation started on day 14 post tumor challenge. Untreated mice served as a control. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. (C) Median survival time of mice analyzed in Figure 3B.
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Fig3: Combination of RNA immunotherapy with radiation represents an effective treatment strategy for low immunogenic and radioresistant LLC tumor. (A) C57BL/6 mice (n = 5 per group) were vaccinated 2 times (1 vaccination/week) either with EGFR mRNA vaccine (32 μg) or with buffer. 7 days after completed vaccination splenocytes from vaccinated mice were analyzed for IFN-γ secretion in response to EGFR- or Control-peptide library using an ELISpot assay. ** - p = 0.0036 (B) C57BL/6 mice (n = 10 per group) were challenged subcutaneously on the right limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated either with local radiation (36 Gy total, divided into 3 equal fractions on 3 consecutive days) or with radioimmunotherapy as indicated (with first vaccination given 6 h before second radiation). Vaccination therapy without radiation started on day 14 post tumor challenge. Untreated mice served as a control. Tumor growth was monitored by measuring the tumor size in 3 dimensions using calipers. (C) Median survival time of mice analyzed in Figure 3B.
Mentions: Having demonstrated the strong synergistic effect of radiotherapy and mRNA immunotherapy in the treatment of highly immunogenic E.G7-OVA tumors, we sought to determine whether this therapeutic strategy is also effective against one of the most challenging tumor models: LLC. This tumor model is resistant to different kinds of therapeutic regimens (Avastin, radiation, adoptive T cell transfer) [6–8]. It is characterized by nearly complete MHC class I down regulation, a lack of known tumor rejection antigens, very aggressive growth kinetics, frequent ulceration in small tumors and rapid formation of spontaneous lung metastases from the primary solid tumor. We chose human EGFR and Connexin as target antigens because of the evidence for their immunogenicity [9]. As shown in Figure 3A, application of the EGFR mRNA vaccine (5 vaccinations in total, twice a week, 32 μg per vaccination) resulted in a strong induction of EGFR-specific T cell responses compared to control animals (p = 0.0036). In corroboration with the literature data [10] dose finding studies revealed that LLC is strongly resistant to radiation (Additional file 1: Figure S1). As shown in Figure 3B, to achieve a weak and transient inhibitory effect on tumors with a volume of just 50 mm3, a total dose of 36 Gy was required. This dose was split into three fractions, given on consecutive days, starting on day 18 after tumor challenge. As shown in Figure 3B, immunotherapy alone was not able to mediate any tumor growth inhibition and radiation therapy alone resulted in only transient inhibition. In sharp contrast, mRNA vaccines combined with a high dose of radiotherapy resulted in a strong synergistic anti-tumor effect. In conclusion, our data show the efficacy of radioimmunotherapy in controlling tumor growth of a low immunogenic carcinoma.Figure 3

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