Interleukin-15 after Near-Infrared Photoimmunotherapy (NIR-PIT) Enhances T Cell Response against Syngeneic Mouse Tumors

Abstract

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted NIR-PIT induces rapid necrotic/immunogenic cell death (ICD) that induces antitumor host immunity including re-priming and proliferation of T cells. Interleukin-15 (IL-15) is a cytokine that activates natural killer (NK)-, B- and T-cells while having minimal effect on regulatory T cells (Tregs) that lack the IL-15 receptor. Here, we hypothesized that IL-15 administration with cancer cell-targeted NIR-PIT could further inhibit tumor growth by increasing antitumor host immunity. Three syngeneic mouse tumor models, MC38-luc, LL/2, and MOC1, underwent combined CD44-targeted NIR-PIT and short-term IL-15 administration with appropriate controls. Comparing with the single-agent therapy, the combination therapy of IL-15 after NIR-PIT inhibited tumor growth, prolonged survival, and increased tumor infiltrating CD8+ T cells more efficiently in tumor-bearing mice. IL-15 appears to enhance the therapeutic effect of cancer-targeted NIR-PIT.

Keywords: CD44; cancer; interleukin-15; monoclonal antibodies; near infrared photoimmunotherapy.

Figure 1

In vivo effect of CD44-targeted near infrared photoimmunotherapy (NIR-PIT) and interleukin-15 (IL-15) administration for MC38-luc tumor model. (A) NIR-PIT regimen. Bioluminescence and fluorescence images were obtained at each time point as indicated. i.p., intraperitoneal injection; i.v., intravenous injection. (B) Real-time in vivo IR700 fluorescence imaging of tumor-bearing mice before and approximately 10 min after NIR-PIT. The yellow arrows indicate the tumor locations. (C) In vivo bioluminescence imaging of tumor-bearing mice before and after treatment at the indicated timepoints. (D) Quantitative analysis of luciferase activity before and after treatment in tumor-bearing mice. n = 10/group, mean ± SEM; *, p < 0.05, vs. the other groups; †, p < 0.05, vs. combination group; Tukey–Kramer test. (E) Tumor growth in control and treated groups. n = 10/group, mean ± SEM; *, p < 0.05, vs. the other groups; †, p < 0.05, vs. combination group; Tukey–Kramer test. (F) Survival curves for control and treated groups. n = 10/group; *, p < 0.05; NS, not significant; log-rank test with Bonferroni correction.

Figure 2

Immunohistochemical analysis after CD44-targeted NIR-PIT and/or IL-15 treatment in MC38-luc tumor model. (A) Representative multiplex immunohistochemistry (IHC) images in MC38-luc tumors 4 or 7 days after the treatments indicated. Antibody staining of CD8, CD4, FOXP3, and pan-Cytokeratin (pCK) are shown in magenta, green, yellow, and cyan respectively. Nucleus are stained with DAPI and shown in blue. Tumor areas are indicated in white dotted line. The inset in window d shows examples of CD8 T cell (gray filled arrowhead), CD4 T cell (open arrowhead), and Tregs (white filled arrowhead). Scale bar = 100 µm. (B) Cell density of CD8 T cells, CD4 T cells and Tregs in tumor and stroma calculated from IHC images. No Tx, no treatment; IL-15, IL-15 administration only; 44 PIT, CD44-targeted PIT only; combi, combination therapy of CD44-targeted PIT and IL-15 administration. Bars represent mean, dots represent individual samples, and error bars represent SEM. n = 4/group except n = 2 for CD44 day 4. **, p < 0.05, **, p < 0.01 ***, p < 0.0001; one-way ANOVA (followed by Dunnett’s multiple comparison test, vs. no Tx).

Figure 3

In vivo effect of CD44-targeted NIR-PIT and/or IL-15 administration in LL/2 tumor model. (A) NIR-PIT regimen. IR700 fluorescence images were obtained at each time point as indicated. i.p., intraperitoneal injection; i.v., intravenous injection. (B) Real-time in vivo IR700 fluorescence imaging of tumor-bearing mice before and approximately 10 min after NIR-PIT. The yellow arrows indicate the tumor locations. (C) Tumor growth in control and treated groups. n = 9/group, mean ± SEM; *, p < 0.05, vs. the other groups; †, p < 0.05, vs. combination group; Tukey–Kramer test. (D) Survival curves for control and treated groups. n = 9/group; *, p < 0.05; NS, not significant; log-rank test with Bonferroni correction.

Figure 4

In vivo effect of CD44-targeted NIR-PIT and/or IL-15 administration in the MOC1 tumor model. (A) NIR-PIT regimen. IR700 fluorescence images were obtained at each time point as indicated. i.p., intraperitoneal injection; i.v., intravenous injection. (B) Real-time in vivo IR700 fluorescence imaging of tumor-bearing mice before and approximately 10 min after NIR-PIT. The yellow arrows indicate the tumor locations. (C) Tumor growth in control and treated groups. n = 10/group, mean ± SEM; *, p < 0.05, vs. the other groups; †, p < 0.05, vs. combination group; Tukey–Kramer test. (D) Survival curves for control and treated groups. n = 10/group; *, p < 0.05; NS, not significant; log-rank test with Bonferroni correction.