Interest in combining radiotherapy and immune checkpoint therapy is growing rapidly. In this study, we explored a novel combination of this type to augment anti-tumor immune responses in preclinical murine models of melanoma, neuroblastoma, and head and neck squamous cell carcinoma. Cooperative effects were observed with local radiotherapy and intratumoral injection of tumor-specific antibodies, arising in part from enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We could improve this response by combining radiation with intratumoral injection of an IL-2-linked tumor-specific antibody (termed here an immunocytokine), resulting in complete regression of established tumors in most animals associated with a tumor-specific memory T cell response. Given the T cell response elicited by combined local radiation and intratumoral immunocytokine, we tested the potential benefit of adding this treatment to immune checkpoint blockade. In mice bearing large primary tumors or disseminated metastases, the triple-combination of intratumoral immunocytokine, radiation, and systemic anti-CTLA-4 improved primary tumor response and animal survival compared to combinations of any two of these three interventions. Taken together, our results show how combining radiation and intratumoral immunocytokine in murine tumor models can eradicate large tumors and metastases, eliciting an in situ vaccination effect that can be leveraged further by T cell checkpoint blockade, with immediate implications for clinical evaluation.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 ± 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional (5, 6). However, systemic administration of rIL-12 caused dose-dependent toxicity in mice (7) and in human trials (8). Thus, a delivery mechanism that can provide relatively low levels of IL-12 at the target tissue might be advantageous in that it could generate an antitumor effect without causing systemic toxicity. Indeed, as cancer gene therapy has evolved, recent studies have produced encouraging results, showing that murine fibroblasts (9) or tumor cells (10) transduced in vitro with the IL-12 gene using a retroviral vector were able to induce antitumor immune responses. These data suggest that peritumoral IL-12 delivery may be as efficacious as systemic administration and avoid many undesirable side effects.The particle-mediated method for gene delivery by gene gun utilizes a shock wave to accelerate DNA-coated gold particles into target cells or tissues. At submicrogram quantities of DNA per dose for in vitro or in vivo gene transfer, the gene gun can deliver thousands of DNA copies intracellularly into test tissues, resulting in high level transgene expression (11). As this method is cell surface receptor-independent, it can successfully deliver genes into a wide spectrum of mammalian cell types (12, 13). We have recently demonstrated that a particlemediated, in vivo cytokine gene therapy reduces tumor growth in mice (14). Treatments with interferon-y and tumor necrosis factor a shortly after the implantation of tumor cells inhibited tumor growth and prolonged the survival of tumor-bearing mice. To more closely approximate clinical situations, and to take advantage of the findings that IL-12 more effectively stimulates activated than naive T cells (15, 16), we evaluated the effect of IL-12 gene therapy on the growth of established tumors. In this study, we utilized the gene gun technology for in vivo IL-12 cDNA delivery into the skin overlying the implanted, established tumor tissues. MATERIALS AND METHODSMice. BALB/c, C57BL/6, DBA/2, and A/J female mice between 8 and 12 weeks of age were obtained from HarlanSparagu...

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-γ because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-γ mAb, as well as in IFN-γ-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-γ for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-γ. We confirmed this hypothesis by detecting intracytoplasmic IFN-γ in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-γ production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-γ and mediate tumor cell destruction.