Immunotherapeutic synergy between anti-CD137 mAb and intratumoral administration of a cytopathic Semliki Forest virus encoding IL-12

Abstract

Intratumoral injection of Semliki Forest virus encoding interleukin-12 (SFV-IL-12) combines acute expression of IL-12 and stressful apoptosis of infected malignant cells. Agonist antibodies directed to costimulatory receptor CD137 (4-1BB) strongly amplify pre-existing cellular immune responses toward weak tumor antigens. In this study, we provide evidence for powerful synergistic effects of a combined strategy consisting of intratumoral injection of SFV-IL-12 and systemic delivery of agonist anti-CD137 monoclonal antibodies (mAbs), which was substantiated against poorly immunogenic B16 melanomas (B16-OVA and B16.F10) and TC-1 lung carcinomas. Effector CD8(β)(+) T cells were sufficient to mediate complete tumor eradications. Accordingly, there was an intensely synergistic in vivo enhancement of cytotoxic T lymphocytes (CTL)-mediated immunity against the tumor antigens OVA and tyrosine-related protein-2 (TRP-2). This train of phenomena led to long-lasting tumor-specific immunity against rechallenge, attained transient control of the progression of concomitant tumor lesions that were not directly treated with SFV-IL-12 and caused autoimmune vitiligo. Importantly, we found that SFV-IL-12 intratumoral injection induces bright expression of CD137 on most tumor-infiltrating CD8(+) T lymphocytes, thereby providing more abundant targets for the action of the agonist antibody. This efficacious combinatorial immunotherapy strategy offers feasibility for clinical translation since anti-CD137 mAbs are already undergoing clinical trials and development of clinical-grade SFV-IL-12 vectors is in progress.

Figures

Figure 1

Treatment efficacy of SFV-IL-12 + anti-CD137 combination. (a) C57BL/6 female mice were inoculated in the flank with 5 × 105 B16-OVA cells on day 0 and then received an intratumoral injection of saline (top panels), 107 viral particles (vp) (middle panels), or 108 vp of SFV-IL-12 (bottom panels) on day 7. On days 7, 10, and 14 mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) (left panels) or anti-CD137 mAb (right panels). (c) C57BL/6 female mice were inoculated in the flank with 5 × 105 TC-1 cells on day 0 and then received an intratumoral injection of saline (top panels) or 107 vp of SFV-IL-12 (bottom panels) on day 18. On days 18, 22, and 26 mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) (left panels) or anti-CD137 mAb (right panels). (a,c) Each curve represents the evolution of the mean tumor diameter for each individual mouse. The numbers in the right lower corner of each graph indicate the fraction of tumor-free mice on day 60 (for the B16-OVA model) or on day 121 (for the TC-1 model) relative to the total number of animals in each group, and the percentage of complete tumor regressions, respectively. (b,d) Kaplan–Meier plots of mouse survival. The SFV-IL-12 + anti-CD137-treated group (108 vp in b) was compared with the rest of the groups with the log-rank test. n.s., not significant; *P < 0.05; ** P < 0.01;*** P < 0.001. (a,b) The graphs correspond to pooled data of two independent experiments with similar results. α, anti-. SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 2

Treatment efficacy of SFV-IL-12 + anti-CD137 combination on bilateral B16-OVA tumors. (a) C57BL/6 female mice were inoculated in contralateral flanks with 5 × 105 B16-OVA cells on day 0 and then received in the right tumor an injection of saline (top panels), 107 viral particles (vp) (middle panels), or 108 vp of SFV-IL-12 (bottom panels) on day 8. On days 8, 11, and 15 mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) (left panels) or anti-CD137 mAb (right panels). Red curves represent the evolution of SFV-IL-12-treated tumor diameter and green dotted curves represent the evolution of the nontreated tumor diameter for each individual mouse. Red numbers in the right lower corner of each graph indicate the number of completely rejected treated-tumors and green numbers indicate the number of completely rejected contralateral tumors on day 84 relative to the total number of animals in each group, and the percentage of complete tumor regressions, respectively. (b) Kaplan–Meier plot of mouse survival. The SFV-IL-12 (108 vp) + anti-CD137-treated group was compared with the rest of the groups with the log-rank test. n.s., not significant; *P < 0.05; **P < 0.01;***P < 0.001. The graphs correspond to pooled data from two independent experiments with similar results. α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 3

Evaluation of the generation of a protective immune memory response after SFV-IL-12 + anti-CD137 treatment and efficacy in the B16.F10 model. (a) Survivor mice from experiments shown in Figures 1a and 2 were rechallenged in a first step with 5 × 105 B16-OVA cells at day 112 and in a second step with 5 × 105 B16.F10 cells at day 203. When indicated, a group of naive animals were inoculated with the same tumor cells to control each rechallenge experiment. Mice survival was monitored and analyzed by sequential Kaplan–Meier plots. The different groups were compared with the log-rank test. (b) C57BL/6 female mice were inoculated in the flank with 5 × 105 B16.F10 cells on day 0 and then received an intratumoral injection of saline or 107 viral particles (vp) of SFV-IL-12 on day 7. On days 7, 10, and 14 mice received intraperitoneally 100 µg of anti-CD137 mAb as indicated in the figure. (c) Kaplan–Meier plot of mouse survival. The SFV-IL-12 + anti-CD137-treated group was compared with the rest of the groups with the log-rank test. n.s., not significant; *P < 0.05; **P < 0.01. α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 4

CD8+ T cell requirement for SFV-IL-12 + anti-CD137 treatment efficacy. (a) C57BL/6 female mice were inoculated in the flank with 5 × 105 B16-OVA cells on day 0 and then received intratumorally saline (first panel) or 108 viral particles (vp) of SFV-IL-12 (rest of the panels) on day 7. On days 7, 10, and 14 mice treated with saline received intraperitoneally 100 µg of rat immunoglobulin G (IgG) and mice treated with SFV-IL-12 received a course of anti-CD137 mAb as in Figure 1. Depletions of CD4+, CD8β+, and NK1.1+ cells were performed by intraperitoneal injection of 100 µg of specific antibodies administered at days 6, 10, 15, 21, and 28 for αCD4+ and αCD8+ and at days 6, 8, 10, 12, 15, 21, and 28 for αNK1.1+. Each curve represents the evolution of the mean tumor diameter for each individual mouse. The numbers in the right lower corner of each graph indicate the number of tumor-free mice on day 70 relative to the total number of animals in each group, and the percentage of sustained complete tumor regressions, respectively. (b) Kaplan–Meier plot of mouse survival. The different groups were compared with the log-rank test. **P < 0.01;***P < 0.001. The graphs correspond to pooled data from two independent experiments with similar results. α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 5

SFV-IL-12 + anti-CD137 therapy augments the CD8/Treg ratio and the quality of CD8+ T cells. C57BL/6 female mice were flank-inoculated with 5 × 105 B16-OVA cells on day 0 and then received intratumorally saline or 108 viral particles (vp) of SFV-IL-12 on day 7. On days 7 and 10 mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) or anti-CD137 mAb. On day 11 mice were sacrificed and tumors were harvested, processed, and analyzed by flow cytometry. (a) Graphs show the number of CD8+ T cells (left), Tregs (middle), and the ratio between CD8+ T and Treg lymphocytes (right). The results are represented as the mean ± standard error of mean (SEM). (b) Representative dot plots of CD25 and FoxP3 expression on CD4+ T cells. Numbers indicate the percentages of cells in each quadrant analyzed by fluorescence-activated cell sorting (FACS). Tregs are identified in the upper-right quadrants as CD4+CD25+Foxp3+ cells. (c) Plots showing FACS-assessed size and complexity of intratumoral CD8+ T cells. The values on the right upper corner of each panel represent the mean ± SD for FSC or SSC. *P < 0.05; **P < 0.01; α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 6

Combined treatment increases CD8+ T cell and tumor-specific CD8+ T cell numbers. (a–c) C57BL/6 female mice were inoculated in the flank with 5 × 105 B16-OVA cells on day 0 and then received intratumorally saline or 108 viral particles (vp) of SFV-IL-12 on day 7. On days 7, 10, and 14 mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) or anti-CD137 mAb. Mice were bled at days 7, 11, 15, 19, 22, 27, and 35 and percentages of CD8+ T cells (a,b) or the number of CD8+ T cells per 106 peripheral blood mononuclear cells which were OVA- (c, left panel) or tyrosine-related protein-2 (TRP-2)-specific (c, right panel) were determined by flow cytometry. (d) C57BL/6 female mice were inoculated in the flank with 5 × 105 B16-OVA cells on day 0 and then received intratumorally saline or 108 vp of SFV-IL-12 on day 7. On days 7 and 10 mice received intraperitoneally 100 µg of rat IgG or anti-CD137 mAb. On day 11 mice were sacrificed and spleens were harvested, processed, and analysed by flow cytometry. The graph shows the number of OVA-specific CD8+ T cells in the spleens of treated mice. *P < 0.05; **P < 0.01; ***P < 0.001. α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 7

Enhancement of immune responses with SFV-IL-12 + anti-CD137 combined therapy. C57BL/6 female mice were subcutaneously inoculated with 5 × 105 B16-OVA cells on day 0 and then received intratumorally saline or 108 viral particles (vp) of SFV-IL-12 on day 7. On days 7 and 10 mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) or anti-CD137. (a) At day 1 after treatment onset, mice were bled and sera samples were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine interferon γ (IFNγ) concentrations. The results are represented as the mean ± SEM (n = 4 per group). ND, not detected. (b) On day 14, mice were sacrificed and spleens were processed to carry out an IFNγ enzyme-linked immunospot (ELISPOT) assay using OVA or tyrosine-related protein (TRP)-specific peptides for stimulation. The data are represented as number of IFNγ-producing spots per 106 splenocytes. (c) In-vivo killing assay. On day 13, tumor peptide-pulsed splenocytes from naive CD45.1 mice were injected to treated mice and 20 hours later mice were sacrificed and spleens collected. The percentage of specific cell lysis was quantified by flow cytometry. The set histograms in the right show representative cases of this experiment. n.s., not significant; *P < 0.05; **P < 0.01. α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.

Figure 8

SFV-IL-12 + anti-CD137 combined therapy up-regulates CD137 on CD8+ T cells and reduces anti-SFV humoral immune responses. (a–c) B16-OVA tumor-bearing mice were treated intratumorally with saline or 108 viral particles (vp) of SFV-LacZ or SFV-IL-12 on day 7. The same day mice received intraperitoneally 100 µg of rat immunoglobulin G (IgG) or anti-CD137 mAb. Two days later, mice were sacrificed and tumor nodules and draining lymph nodes (DLNs) were processed and analyzed by flow cytometry. (a) Plots showing CD137 expression level on CD8+ T cells from tumor nodules. (b,c) Graphs showing the percentage of CD8+ T cells that upregulate CD137 expression on their surface in tumor nodules or DLNs, respectively. (d) B16-OVA tumor-bearing mice treated with SFV-IL-12 + rat IgG, or anti-CD137 mAb, as described in Figure 1, were bled at day 21 and sera samples were analyzed to determine anti-SFV neutralizing antibody titers. The results are represented as mean ± SEM. *P < 0.05; **P < 0.01. α, anti-; SFV-IL-12, Semliki Forest virus encoding interleukin-12.