A novel combination immunotherapy for cancer by IL-13Rα2-targeted DNA vaccine and immunotoxin in murine tumor models

Abstract

Optimum efficacy of therapeutic cancer vaccines may require combinations that generate effective antitumor immune responses, as well as overcome immune evasion and tolerance mechanisms mediated by progressing tumor. Previous studies showed that IL-13Rα2, a unique tumor-associated Ag, is a promising target for cancer immunotherapy. A targeted cytotoxin composed of IL-13 and mutated Pseudomonas exotoxin induced specific killing of IL-13Rα2(+) tumor cells. When combined with IL-13Rα2 DNA cancer vaccine, surprisingly, it mediated synergistic antitumor effects on tumor growth and metastasis in established murine breast carcinoma and sarcoma tumor models. The mechanism of synergistic activity involved direct killing of tumor cells and cell-mediated immune responses, as well as elimination of myeloid-derived suppressor cells and, consequently, regulatory T cells. These novel results provide a strong rationale for combining immunotoxins with cancer vaccines for the treatment of patients with advanced cancer.

Figure 1

Combination therapy with IL13-PE and IL-13Rα2 DNA vaccine inhibited tumor growth and prolonged survival in 4T1 breast carcinoma and MCA304 sarcoma models. A, Tumor cells (1 × 10⁴/well) were cultured in leucine-free medium containing various concentrations of IL13-PE38, with or without 1 µg/ml of IL-13, for 20–22 h. Cells were then pulsed with 1 µCi of [³H]leucine for 4 h. The amount of radioactivity incorporated into cells was measured. B, Treatment schedule in tumor-bearing mice. 4T1 (C) or MCA304 (E) tumors implanted were intratumorally treated with IL13-PE (on days 7, 9, and 11) and vaccinated with four subsequent DNA vaccines (on days 13, 18, 23, and 28). Kaplan–Meier survival curves of 4T1 (D) and MCA304 (F) tumor models were plotted. Each group contained six mice. Tumor volumes were measured by Vernier caliper, and OST was calculated based on the sacrifice of mice when tumors reached >2 cm. Experiments were repeated twice. Bars represent SD.

Figure 2

Inhibition of lung and lymph node metastasis in orthotopic 4T1 breast carcinoma by combination therapy with IL13-PE and IL-13Rα2 DNA. Mice were sacrificed on day 33 posttumor implantation. Lungs and lymph nodes were harvested. Each group had five or six mice. A, Lungs were inoculated with india ink, as described in Materials and Methods. A representative lung from each group is shown. Original magnification ×100. B, Representative H&E-stained sections of lung from each group. Total surface pulmonary lung nodules (C) and lymph nodes >5 mm in size (D) were counted. Statistical analysis was performed using one-way ANOVA.

FIGURE 3

Measurement of CTL activity and IFN-γ release in mice treated with IL13-PE and IL-13Rα2 DNA. Splenocytes restimulated with mitomycin C-treated 4T1 (A) or MCA304 (B) tumor cells for 1 wk in culture medium containing IL-2 (20 IU/ml) were used as effector cells. 4T1 or MCA304 target cells were labeled with [⁵¹Cr] for 2 h, washed thrice, and then plated into 96-well plates with effector cells. Specific lysis was calculated, as described in Materials and Methods, after 4 h of culture. Culture supernatants of splenocytes restimulated with mitomycin C-treated 4T1 (C) or MCA304 (D) tumor cells for 48 h were assessed by ELISA for murine IFN-γ production. D5 melanoma and CT-26 were used as negative control. Spleens from five mice were harvested. Data presented are representative of two independent experiments with similar results. Bars, SD. Statistical analysis was performed using oneway ANOVA. The difference between DNA vaccine and combination therapy group in IFN-γ ELISA assay in the 4T1 tumor model was statistically significant at p < 0.001 (C). Similarly, the difference between either DNA vaccine alone or IL13-PE alone and combination therapy was statistically significant at p < 0.001 in the MCA304 tumor model (D).

FIGURE 4

Detection of CD4⁺ and CD8⁺ T cells and chemokines in regressing tumors of treated mice. The 4T1 tumor samples in mice receiving PBS, IL13-PE alone, IL-13Rα2 DNA alone, and the combination therapy of IL13-PE and IL-13Rα2 DNA were collected on day 33 from the experiment shown in Fig. 1B. A, The immunofluorescence microscopic analyses of tumors from three mice were done using Abs specific for CD4, CD8, CXCL9, CXCL10 (IP-10), and CXCR3. The representative images of a tumor from each group are shown. Scale bars, 50 µm. Original magnification ×400. B, The number of CD4⁺ (upper panel) or CD8⁺ (lower panel) cells (per ×400 field of view) was counted in each group. A total of six fields was counted for each area. C–E, Role of host-immune cells in combination therapy-induced 4T1 and MCA304 tumor growth inhibition. CD4⁺ and/or CD8⁺ T cells or NK cells were depleted in the mice receiving combination therapy with IL13-PE and IL-13Rα2 DNA in 4T1 (C) and MCA304 (D) tumor models. For CD4⁺ and CD8⁺ depletion, mice were injected i.p. with 0.25 mg of anti-CD4 Ab (GK1.5) and/or 0.2 mg of anti-CD8 Ab (2.43) on days −2, −1, (Figure legend continues) 5, 11, and 17 relative to the tumor implantation. NK cells were depleted with rabbit anti–asialo-GM1 Ab (50 µg/injection) on the same days as CD4⁺, CD8⁺ T cell depletion (E). RAG-2 knockout or wild-type C57BL/6 mice bearing MCA304 tumor received combination therapy. Each group contained five or six mice.

FIGURE 5

Measurement of serum autoantibodies generated by combination therapy with IL13-PE and IL-13Rα2 DNA. Blood serum samples were collected on day 33 from the experiment shown in Fig. 2B or 2D. A and C, Autoantibody against IL-13Rα2 was quantified by ELISA. To assess whether Ab against IL-13Rα2 generated in treated mice inhibited tumor growth, the sera from the mice were incubated with 4T1 or MCA304 tumor cells for 48 h, and cell proliferation was evaluated. B, At 1:500 dilution, serum taken from 4T1 tumor-bearing mice receiving combination therapy showed a modest, but significant, inhibition of 4T1 tumor cell proliferation. D, Less inhibition was seen for the MCA304 tumor. Each group contained five or six mice. Data are representative of two independent experiments with similar results.

FIGURE 6

Combination therapy reduces the number of Tregs. A, Splenocytes from five mice were harvested on day 30 and stained for CD3, CD4, CD25, and Foxp3. Stained cells were analyzed by flow cytometry for Treg marker expression. One representative image is shown. B, Gating on CD3⁺CD4⁺ cells and analysis for the expression of CD25 and Foxp3. The combination therapy decreased the population of Tregs compared with the other groups. The difference between PBS control and combination therapy group and combination therapy was statistically significant at p < 0.001. C, The number of Tregs infiltrated into tumors by an immunofluorescence microscopic analysis. Tumors of three mice from each group were harvested on day 30 and stained with CD4 and Foxp3. Representative images from each group are shown. Scale bar, 50 mm. Original magnification ×400. D, The ratio of CD4⁺ and Foxp3⁺ cells was calculated. A total of six fields was counted for each sample. The difference between either DNA vaccine alone or IL13-PE alone and combination therapy was statistically significant at p < 0.005.

FIGURE 7

Combination therapy suppresses the MDSCs in spleen and tumors. A and B, Mice splenocytes were harvested 1 d before 4T1 injection (day −1), 1 d after IL13-PE treatment (day 12), and 2 d after IL-13Rα2 DNA vaccination (day 30) and stained for Gr-1 and CD11b. Stained cells were analyzed by flow cytometry for MDSC expression. C, Representative flow profiles on day 30. Combination therapy dramatically decreased the population of MDSCs on day 30. Statistical analysis was performed using one-way ANOVA, and synergism was assessed by the least-squares regression test and REML approach. The difference between DNA vaccine-alone group and combination therapy group was statistically significant at p < 0.001. D, The number of MDSCs infiltrated into tumors by an immunofluorescence microscopic analysis. Tumors from three mice were harvested from each group on day 30 and stained for CD11b, Gr-1, arginase-1, and NO synthase-2. A representative tumor staining is shown. Scale bars, 50 µm. Original magnification ×400. E, The numbers of MDSCs (per ×400 field of view) were counted in each group. A total of six fields was counted for each area. The difference between DNA vaccine-alone group and combination therapy group was statistically significant at p < 0.001.