IL-12 and IL-27 sequential gene therapy via intramuscular electroporation delivery for eliminating distal aggressive tumors

Abstract

Eradication of residual malignancies and metastatic tumors via a systemic approach is the key for successfully treating cancer and increasing cancer patient survival. Systemic administration of IL-12 protein in an acute large dose is effective but toxic. Systemic administration of IL-12 gene by persistently expressing a low level of IL-12 protein may reduce the systemic toxicity but only eradicates IL-12-sensitive tumors. In this study, we discovered that sequential administration of IL-12- and IL-27-encoding DNA, referred to as sequential IL-12-->IL-27 (IL-12 administration followed by IL-27 administration 10 d after) gene therapy, not only eradicated IL-12-sensitive CT26 tumors from 100% of mice but also eradicated the highly malignant 4T1 tumors from 33% of treated mice in multiple independent experiments. This IL-12-->IL-27 sequential gene therapy is not only superior to IL-12-encoding plasmid DNA given a total of two times at a 10-d interval sequential gene therapy for eliminating tumors but also for inducing CTL activity, increasing T cell infiltration into tumors, and yielding a large number of tumor-specific IFN-gamma-positive CD8 T cells. Notably, depletion of either T or NK cells during the IL-27 treatment phase reverses tumor eradication, suggesting an NK cell requirement for this sequential gene therapy-mediated tumor eradication. Both reversal of the administration sequence and coadministration of IL-12 and IL-27 impaired tumor eradication in 4T1 tumor-bearing mice. This IL-12-->IL-27 sequential gene therapy, via sequential administration of IL-12- and IL-27-encoding plasmid DNA into tumor-bearing mice through i.m. electroporation, provides a simple but effective approach for eliminating inaccessible residual tumors.

FIGURE 1. Images of tumor eradication and strong inhibition of tumor growth by IL12-IL27 sequential gene therapy

pCtr, pIL12, and pIL27 represent control, IL12, and IL27 encoding plasmid DNA, respectively. pCtr-pCtr indicates that both the first and the second administration were control plasmid DNA. The same definition applied to pIL12-pIL12, pIL27-pIL27, pIL27-pIL12, and pIL12+pIL27-pIL12+pIL27. A total of 10 μg plasmid DNA was used for each administration with a 10 day interval between two administrations. The first administration was performed on day 4 after inoculation of tumor cells. The picture was taken 18 days after the final (second) intramuscular injection.

FIGURE 2. 4T1 Tumor growth inhibition and mouse survival by IL12-IL27 sequential gene therapy

The same dose, number of administrations, and treatment combinations described in Fig. 1 legend applied to this Figure. A. Inhibition of tumor growth by different IL12 and IL27 sequential gene therapy combinations (n=5). Arrows labeled 1^st and 2^nd represent the first and the second treatment times, with the first administration performed on day 4 after inoculation of tumor cells. The p- value indicates the significant difference between IL12-IL27 sequential gene therapy and the other treatment groups. B. Kaplan-Meier survival curves for IL12-IL12 and IL12-IL27 sequential gene therapy (n=9). p<0.05 for IL12-IL12 vs. IL12-IL27 treatments.

FIGURE 3. Comparison of CT26 tumor regression by IL12-IL12 and IL12-IL27 sequential gene therapy

The same dose, administration number and Schedule for IL12-IL12 and IL12-IL27 treatment were used as described in Fig. 1 legend applied to this Figure. The first treatment for the early treatment model was initiated on day 4 (3A, 3C), whereas the first treatment for the late-treatment model was initiated on day 11 (3B, 3D). A, C. Eradication of tumors by IL12-IL12 sequential gene therapy. B, D. Eradication of tumors by IL12-IL27 sequential gene therapy. E. Aggressive tumor growth after administering control plasmid DNA. F. Impairing IL12-IL27 sequential gene therapy-mediated tumor eradication by administering anti-wsx1 antibody. p<0.05 for IL12-IL27 vs (IL12-IL27)+anti-wsx1 antibody.

FIGURE 4

Kinetics of cytokine expression during the IL12-IL12 and IL12-IL27 sequential gene therapy. The same treatment dose and administration number described in Fig. 1 were used in this Figure. Blood was collected on days 1, 4, and 8 after the first and the second administration. D1, D4, and D8 were the days after the first and the second administration. 1^st and 2^nd indicates the first and the second administrations at an interval 10 days between two treatments. Ctr-Ctr, administration of control plasmid DNA for both administrations. A, B, and C. Expression kinetics of IL12, IFNγ and IL27. P>0.05 for the level of IL12 and IFNγ between IL12-IL12 and IL12-IL27 treatments on the same blood collection dates; p<0.05 for the level of IL27 expression after the second treatment between the same two treatment groups. P<0.001 for the cytokines between control groups and the treatment groups.

FIGURE 5

Comparison of the anti-tumor immune responses between IL12-IL12 and IL12-IL27 gene therapy. The same dose, treatment schedule and sequence described in Figure 1 were used in this experiment. Ten days after the final (the second) administration, anti-tumor immune responses between these two treatments were analyzed. The first administration was performed on day 11 after inoculation of tumor cells. A. NK cell cytolytic activity using homologous tumor cells and NK cell depleted spleen cells. B. NK cell cytolytic activity using Yac1 cells. C. Difference in T cell cytolytic activity (CTL) between IL12-IL12- and IL12-IL27-treated mice. CT26 tumor cells were used as target cells. *, represent significant difference at p<0.01. D. Difference in infiltration of T cells in CT26 tumors between IL12-IL12 and IL12-IL27 sequential gene therapy (p=0.036). E. Effect of depletion of CD8 T and NK cells on IL12-IL27 sequential gene therapy mediated anti-tumor efficacy. P<0.001 between injection of depletion antibodies against CD8 T cells or NK cells and injection of murine IgG on the indicated days. Antibodies were administered twice a week at a dose of 50 μg per mouse for each administration. Arrows indicates the plasmid DNA administrgation time. F. Kinetics of challenged tumor growth between the IL12-IL12 and IL12-IL27-cured mice.

FIGURE 6

Induction of anti-tumor immune memory by IL12-IL27 sequential gene therapy. CT26 tumor model was used for this study in vivo and the spleen cells collected from the treated mice were used for ELISPOT assay, detecting the difference in induction of acute tumor-specific IFNγ positive CD8 cells between IL12-IL12 and IL12-IL27 sequential gene therapy. To ensure the effector cells were primarily CD8 T cells, both NK and CD4 T cells were depleted using neutralization antibody one day prior to euthanizing mice. E and T represent effector and target cells. Two treatments were performed (see detail in Fig. 1 legend with the first treatment on day 11 after inoculation of tumor cells). A. IFNγ positive spots from different incubations. B. Spot number as counted under dissecting microscope. ***, represents significant difference at a p<0.001.