IL-12 in preclinical tumor models

The tumor-protective role of IL-12 has been established for both the cytokine and the receptor. Mice lacking the p35 subunit display increased numbers of chemically induced papillomas and increased incidence of N-methyl-N-nitrosourea-induce T cell lymphomas [166, 181, 182], while the lack of the p40 subunit led to accelerated growth of MCA-induced sarcomas [183]. The absence of IL12Rβ2 subunit expression has been shown to predispose to malignancy increasing the incidence of spontaneous tumors and accelerating the growth of transplantable tumors [184].

Preclinical models investigating the anti-tumor immune response induced by IL-12 include the B16 melanoma model, the CT26 colon carcinoma model, the TSA mammary carcinoma model and the 4T1 breast cancer model amongst others (IL-12 in preclinical glioma models will be described in section 5.4.2.1). However, its mechanism of action has been shown to differ among tumor models. Factors influencing tumor-rejection are dose, timing, and location of IL-12 release/injection. In the B16 melanoma model, overexpression of IL-12 in tumor cells led to tumor suppression mediated by a subset of

Rorγt-dependent innate lymphoid cells [185, 186]. While other studies using established s.c. B16 tumors and i.p. injection of IL-12 led to a CD8⁺ T cell-dependent tumor rejection [187]. In contrast, increasing the dose of IL-12 for the treatment of established B16 tumors required NK and NKT cell-dependent mechanisms of tumor suppression [183, 188].

The transplantable BALB/c colon carcinoma CT26 model has also been extensively used within IL-12 treatment approaches. However, IL-12 given systemically did not have any effect on the s.c. injected primary tumor, most likely due to the absence of responding cell types [189]. In contrast, it has been shown that IL-12 significantly reduces liver metastasis, a mechanism most likely involving liver-resident NKT cells [190]. When CT26 tumor cells were modified to overexpress IL-12, inhibition of tumor take and tumor rejection were dependent on the level of IL-12 expression. Amounts of IL-12 in the pg range led to delayed tumor onset and reduced number of lung metastasis upon s.c. and i.v. injection [189]. However, tumors were only rejected when simultaneous depletion of CD4⁺ T cells was performed, presumably due to IL-12 receptor expression on regulatory T cells [191]. In contrast, amounts of IL-12 released by CT26 in the ng range did not form tumors, unless injected cell numbers were increased to 10-50 fold of the minimal lethal dose of the parental CT26 cell line [189]. Interestingly, this mechanism was found to be IFN-γ independent but required GM-CSF production by CD4⁺ T cells for tumor rejection [192].

The TSA cell line, initially derived from a spontaneous mouse mammary carcinoma, has been modified to overexpress distinct cytokines, amongst which IL-12 was found to be the most potent in inducing tumor protection [193, 194]. This mechanism was dependent on cytotoxic CD8⁺ T cells secreting IFN-γ [195]. In the 4T1 tumor model, considered to be a less immunogenic model for breast cancer, IL-12 did not affect the primary tumor but induced a significant reduction of lung metastasis. This mechanism was found to be partially dependent on IFN-γ-producing NK cells [166, 196-198].

5.4.2.1. IL-12 in preclinical glioma models

Regarding preclinical models for glioma, IL-12 mediated glioma rejection has been claimed to be T cell and NK cell-dependent [120, 199-205]. Strategies in delivering IL-12 include systemic administration, intratumoral delivery, viral transfer systems and cells overexpressing IL-12.

Early studies with a GL-26 glioma model and intratumoral administration using adenoviral delivery of IL-12 at day four post tumor cell injection led to the survival of 50% of mice accompanied by the infiltration of CD8⁺ and CD4⁺ T cells [199]. However, these studies lacked functional experiments showing the necessity of T cells for the anti-tumor immune response [199]. Injection of neural stem cells expressing IL-12 on day two post tumor cell injection only led to tumor rejection in 20% of mice. Here, IL-12 led to an influx of T cells. However, this study also lacked functional data to undermine the requirement of T cells for tumor rejection [200]. In another C57BL/6 model, mice were injected i.c. with 203 glioma cells. In this study, treatment consisted of systemic injection of IL-12 and IL-18, together with vaccination of dendritic cells pulsed with Semliki Forest virus (SFV) and 203 glioma cDNA [201]. Here, tumor rejection was induced in a T and NK cell-dependent fashion and required IFN-γ [201].

In the GL-261 tumor model, an approach of using concentrated DNA/PPC (polyethylenimine covalently modified with methoxypolyethyleneglycol and cholesterol) complexes delivering a murine plasmid encoding IL-12 (pmIL-12) in combination with biodegradable carmustine (BCNU) chemotherapy led to survival in 40% of mice [202].

Moreover, Vetter et al. used the GL261 glioma cell model and injected cells into the cerebellum of transgenic mice that constitutively expressed IL-12 under the control of the GFAP promoter in astrocytes, leading to mainly CD8⁺ T cell-dependent glioma rejection [203]. Our lab has previously shown that local IL-12 delivery combined with systemic blockade of the co-inhibitory receptor CTLA-4 in the GL-261 tumor model leads to tumor rejection in 80% of mice when initiating treatment at day 21 post tumor cell injection.

Moreover, tumor rejection was T cell- and perforin-dependent and elicited immunological memory [120].

In studies using a recombinant adeno-associated virus (rAAV) as a vehicle for local delivery of IL-12 in athymic mice bearing DBTRG gliomas, tumor rejection was found to be NK cell-dependent [204]. In a follow-up study, the mechanism was further complemented in that tumor rejection required the activation of microglia expressing TRAIL [205]. Another recent approach used oncolytic herpes simplex virus (HSV) delivering angiostatin (G47Δ-mAngio) and IL-12 (G47Δ-mIL12). Angiostatin, a potent inducer of tumor vasculature regression co-delivered with IL-12, significantly prolonged survival of mice bearing U87 gliomas [206]. While a recent study implicated the importance of macrophages in a triple treatment setup of anti-CTLA-4, anti-PD-1 and

G47Δ-mIL12 in a glioblastoma stem-like cell model (GSC005), leading to tumor rejection in 50% of mice [207].

In summary, the anti-tumorigenic effects of IL-12 are not only tissue-specific but are also time- and dose-dependent, leading to the involvement of distinct responsive effector cell subsets.