Intratumoral administration of mRNA encoding a fusokine consisting of IFN-β and the ectodomain of the TGF-β receptor II potentiates antitumor immunity

Abstract

It is generally accepted that the success of immunotherapy depends on the presence of tumor-specific CD8⁺ cytotoxic T cells and the modulation of the tumor environment. In this study, we validated mRNA encoding soluble factors as a tool to modulate the tumor microenvironment to potentiate infiltration of tumor-specific T cells. Intratumoral delivery of mRNA encoding a fusion protein consisting of interferon-β and the ectodomain of the transforming growth factor-β receptor II, referred to as Fβ², showed therapeutic potential. The treatment efficacy was dependent on CD8⁺ T cells and could be improved through blockade of PD-1/PD-L1 interactions. In vitro studies revealed that administration of Fβ² to tumor cells resulted in a reduced proliferation and increased expression of MHC I but also PD-L1. Importantly, Fβ² enhanced the antigen presenting capacity of dendritic cells, whilst reducing the suppressive activity of myeloid-derived suppressor cells. In conclusion, these data suggest that intratumoral delivery of mRNA encoding soluble proteins, such as Fβ², can modulate the tumor microenvironment, leading to effective antitumor T cell responses, which can be further potentiated through combination therapy.

Figure 1. Fβ² mRNA is translated to a functional protein

(A) Schematic representation of the Fβ² mRNA construct. (B) Splenocytes were cultured for 5 hours in Fβ², control (Ctrl) supernatants or exposed to increasing amounts of recombinant IFN-β. qPCR was performed to determine Mx1 expression. The graph depicts the amount of fusokine (pg/ml). The quantity was determined based on the expression of Mx1 upon treatment with recombinant IFN-β and normalized to 2^−Δct for Ppia (n=3). (C) The TGF-β reporter HEK293T cell line was cultured for 24 hours in 0 to 400 pg/ml of recombinant TGF-β (rec. TGF-β). The histogram overlay depicts the eGFP expression. Representative plots are shown (n=4) (D) The TGF-β reporter HEK293T cell line was cultured for 24 hours in Fβ², Ctrl supernatants or exposed to 20 ng of a commercial available anti-TGF-β antibody, and supplemented with 200 pg of recombinant TGF-β. The histogram overlay depicts the eGFP expression. Representative plots are shown (n=3).

Figure 2. The Fβ² fusokine modulates myeloid cells to improve CD8⁺ T cell responses

(A-E) DCs were cultured for 48 hours in Fβ² or Control (Ctrl) supernatants. (A) Expression of MHC II, CD80, CD86 and MHC I was evaluated by flow cytometry. The column graph depicts the median fluorescence intensity (MFI) (n=6). (B) Supernatants were analyzed for the presence of IL-6 and TNF-α (n=3-12). (C-E) DCs were pulsed with the peptide SIINFEKL and co-cultured for 6 days with CD8⁺ OT-I cells at a 1:10 ratio. (C) The production of IFN-γ by OT-I cells was determined by ELISA (n=9). (D) The production of IFN-γ by OT-I cells was confirmed via flow cytometry. Representative plots are show. (E) The column graph depicts the results of the experiments (n=6). (F) The TGF-β reporter HEK293T cell line was cultured for 24 hours in the conditioned medium used to generate MDSCs or in supernatants of in vitro generated day 5 MDSCs (n=3). (G-I) MDSCs were cultured for 3 days in Fβ² or Ctrl supernatants. (G) These MDSCs were co-cultured for 3 days with CD8⁺ splenocytes that were activated with anti-CD3/anti-CD28 beads. Activated CD8⁺ splenocytes cultured in the absence of MDSCs served as a positive control (Pos). Supernatants were screened for the presence of IFN-γ (n=4). (H) MDSC viability was assessed using a colorimetric assay. The colorimetric assay reflects the amount of viable cells in the plate, and is measured as absorbance. The graph depicts the absorbance (n=3). (I) Flow cytometry was performed to assess the percentage of CD11b and sca-1⁺ cells (n=3).

Figure 3. Tumors treated with Fβ² show lower proliferative rates and express increased levels of MHC I and PD-L1

(A) 4T1, E.G7-OVA and TC-1 tumor cells were cultured for 4 days in Fβ² or Ctrl supernatants. Cell proliferation was assessed using a colorimetric assay (n=3). (B-C) E.G7-OVA, MO4 and TC-1 cells were cultured for 24 hours in Fβ² or Ctrl supernatants, after which expression of (B) MHC I and (C) PD-L1 was evaluated by flow cytometry. The column graphs depict the MFI (n=3). (D-F) E.G7-OVA tumor cells were cultured for 3 days in Fβ² or Ctrl supernatants, after which they were co-cultured with CD8⁺ OT-I spleen cells in the presence of anti-CD107 antibodies for 4 hours at a tumor:T cell ratio of 1:10 (E) and 1:1 (F). Four hours later cells were additionally stained for CD3 and CD8. The graphs depict the amount of CD107a and CD8⁺ T cells (n=3).

Figure 4. Simultaneous exposure of tumor cells and CD8 T cells to Fβ² significantly enhances the killing capacities of antigen-specific T cells

(A) CD8⁺ OT-I spleen cells were cultured for 3 days in Fβ² or Ctrl supernatants. Simultaneously, E.G7-OVA tumor cells were cultured for 3 days in these supernatants. Subsequently, both cell populations were washed and mixed together as depicted. These were co-cultured in the presence of anti-CD107 antibodies for 4 hours at a tumor:T cell ratio of (C) 10:1 or (D) 1:1, after which the T cells were labeled with anti-CD3 and anti-CD8 antibodies. (B) The flow cytometry graphs are representative for the experiments. (C-D) The graphs depict the percentage of CD107a within CD8⁺ T cells in the co-cultures (n=3 and n=4, respectively).

Figure 5. Intratumoral delivery of Fβ² mRNA to E.G7-OVA bearing mice delays tumor growth

(A-C) E.G7-OVA cells (5 × 10⁶) were transplanted subcutaneously in C57BL/6 mice. (A) Mice were treated with a single intratumoral injection of 10 μg mRNA encoding tNGFR (circle) or Fβ² (triangle). The graph shows the average tumor size at the day of treatment and 3 days later for a total of 11 mice and summarizes the results of 4 independent experiments. (B) Mice (10 per group) were treated with 3 intratumoral injections of 10 μg of tNGFR (circle) or Fβ² (triangle) mRNA at three-day intervals. The graph depicts the size of the tumor of individual mice. (C) Mice (8 per group) were treated with consecutive intratumoral injections of 0.8 RL (square), 10 or 50 μg of tNGFR (circle) or Fβ² (triangle) mRNA at three-day interval. The graph depicts the size of the tumor of individual mice.

Figure 6. Intratumoral delivery of Fβ² mRNA to TC-1 bearing mice delays tumor growth

(A-G) TC-1 cells were transplanted subcutaneously in C57BL/6 mice. Once the tumors reached an average size of 100 mm³ the mice were treated according to the scheme shown in A. Tumor growth was monitored. (B) The graph shows the average tumor size at the day of treatment and 3 days later (n=11). (C-D) Mice (5-6 per group) were treated with 3 intratumoral injections of 0.8 RL (square), 50 μg of tNGFR (circle) or Fβ² (triangle) mRNA at three-day intervals. (C) The graph depicts the median survival. (D) The graph depicts the growth of tumors in individual mice. (E) Treatment regimen with CD8⁺ T cell depletion. (F) The graph depicts the survival of the mice treated as in E. (G) The graph depicts the mean of the tumor sizes of the respective groups (5 mice per group).

Figure 7. Blockade of PD-1/PD-L1 interactions combined with Fβ² mRNA treatment improves therapeutic responses

TC-1 cells were transplanted subcutaneously in C57BL/6 mice (5 per group). (A-C) Once the tumors reached a size of 100-500 mm³ mice were treated according to the scheme shown in A. Subsequently, tumor growth was monitored. (B) The graph depicts the mean of the tumor sizes of the respective groups when applying a less aggressive model using 2 × 10⁴ TC-1 cells. (C) The graph depicts the mean of the tumor sizes of the respective groups when applying a more aggressive model using 2 × 10⁵ TC-1 cells.