Enhancement of electroporation facilitated immunogene therapy via T-reg depletion

Abstract

Regulatory T cells (T-regs) can negatively impact tumor antigen-specific immune responses after infiltration into tumor tissue. However, depletion of T-regs can facilitate enhanced anti-tumor responses, thus augmenting the potential for immunotherapies. Here we focus on treating a highly aggressive form of cancer using a murine melanoma model with a poor prognosis. We utilize a combination of T-reg depletion and immunotherapy plasmid DNA delivered into the B16F10 melanoma tumor model via electroporation. Plasmids encoding murine granulocyte macrophage colony-stimulating factor and human B71 were transfected with electroporation into the tumor and transient elimination of T-regs was achieved with CD25-depleting antibodies (PC61). The combinational treatment effectively depleted T-regs compared to the untreated tumor and significantly reduced lung metastases. The combination treatment was not effective in increasing the survival, but only effective in suppression of metastases. These results indicate the potential for combining T-reg depletion with immunotherapy-based gene electrotransfer to decrease systemic metastasis and potentially enhance survival.

Figure 1

Tumor growth for anti-CD25 monoclonal antibody (mAb) treatments. (a) Experimental protocol. C57/BJ6 mice (n=6) were treated, that is, with anti-CD25 mAb, irrelevant IgG mAb or untreated 1 day prior to tumor inoculation and 4 days post tumor inoculation, 5 × 10⁵ B16F10 cells were inoculated subcutaneously to the mice and tumour volume was monitored. (b) Tumor growth curves. Tumor growth from anti-CD25 mAb, irrelevant IgG mAb and untreated is presented as tumor volume measurements from day 0 to day 12. Tumor volume was calculated using the formula V=ab²/6. Data are presented as the means±standard error of the mean. *At days 6, 8, 10 and 12, compared anti-CD25 mAb with untreated and IgG mAb, P<0.05. (c) Survival of tumor-bearing mice. Survival is presented using Kaplan–Meier survival curves. *Compared anti-CD25 mAb with untreated B16F10 and IgG mAb, P<0.02.

Figure 2

Treatment of preestablished B16F10 using immunogene therapy. (a) Tumor growth of pGT141 GM-CSF-b7.1, pMG and untreated of preestablished B16F10 tumor is presented from day 0 to day 12. Data are presented as the means±standard error of the mean. *At days 4, 6, 8, 10 and 12, compared pGmCSF-b7.1 with untreated B16F10 tumor, P<0.05. (b) Survival of immuogene-treated tumor-bearing mice. Survival is presented using Kaplan–Meier survival curves. *Compared pGmCSF-b7.1 with untreated B16F10 tumor, P<0.02.

Figure 3

Combination treatments with anti-CD25 and immunogene therapy. (a) Protocol of antibody administration, immunogene therapy and tumor inoculation. C57/BJ6 mice (n=6) were treated using the antibody regime previously described and combined with immunogene when tumor reached approximately 100 mm³ in volume, 5 × 10⁵ B16F10 cells were inoculated subcutaneously to the mice and tumor was monitored. (b) Tumor volume was measured and presented as means±standard error of the mean. *Combinational treatment (pGmCSF-b7.1 and anti-CD25 mAb) compared with untreated B16F10, P<0.005. (c) Survival is presented using Kaplan–Meier survival curves. *Compared pGmCSF-b7.1/anti-CD25 mAb with untreated B16F10 tumor, P<0.02. mAb, monoclonal antibody.

Figure 4

Suppression of pulmonary metastasis from B16F10 tumor. (a) Schematic representation of treatment schedule: C57/BJ6 mice (n=6) were treated, that is, with anti-CD25 mAb, irrelevant IgG mAb or untreated 1 day prior to tumor inoculation and 4 days post tumor inoculation, 5 × 10⁵ B16F10 cells were inoculated subcutaneously to the mice and tumor volume was monitored. Tumors were treated with±pGmCSF-b7.1 or pMG when the tumors reached approximately 100 mm³ in volume. (b) Macroscopic image of lungs. (c) The number of surface metastases counted. The results represent those from four animals per group. (d) Lung weight measurements are presented as means±standard error of the mean. *pGmCSF-b7.1 and anti-CD25 mAb combinational treatment compared with pMG and anti-CD25, P<0.05.

Figure 5

T-regs inactivation. Anti-CD25 mAb intraperitoneal administration inactivates T-regs locally and systemically at the tumor site and spleen. (a) The CD4⁺CD25⁺FoxP3⁺ cell populations were significantly reduced. Compared anti-CD25 mAb with untreated B16F10 and IgG mAb in tumor, P<0.004. (b) The CD4⁺CD25⁺FoxP3⁺ cell populations were significantly reduced. Compared anti-CD25 mAb with untreated B16F10 and IgG mAb in the spleen, **P<0.001. (c) The immunotherapy with pGmCSF-b7.1 alone had no effect on the CD4⁺CD25⁺FoxP3 tumor cell populations. (d) Combinational treatment reduced the CD4⁺CD25⁺FoxP3⁺ cell population. (e) Combinational treatment also reduced the CD4⁺CD25⁺FoxP3⁻ cell population compared with the untreated tumor, *P<0.05. Data are presented as % respective cell population in the respective tissue±standard error of the mean.