Tumor-targeted IL-12 combined with local irradiation leads to systemic tumor control via abscopal effects in vivo.

NHS-IL12 is an immunocytokine, a fusion protein of IL12's functional domains and a necrosis-targeting antibody, which has shown significant effects against human rhabdomyosarcoma xenografts in a humanized tumor model, including terminal growth arrest and differentiation of the tumor cells. Here, we locally irradiated the tumors, increasing necrosis and consequently intratumoral immune cytokine availability, and asked whether this effect may surmount efficacy of single treatment modality. Humanized mice bearing bilateral rhabdomyosarcoma xenografts were evaluated for tumor burden and survival after irradiation, systemic NHS-IL12 therapy or a combination of both. Intratumoral immune compartments were characterized by immunohistochemistry and molecular methods. TH1-cytokine dependency of underlying effector mechanisms were investigated in vitro in several human tumor cell lines. NHS-IL12 when combined with irradiation terminally arrested tumor growth and significantly improved survival. Combination treatment induced dense intratumoral T-cell infiltrates, clonal epitope-specific T-cell expansions, expression of cytotoxins, decreased pro-tumorigenic cytokines and induced senescence and differentiation in the cancer cells. Senescence and differentiation were reproduced in vitro and confirmed to be dependent on TH1 cytokines IFNγ and TNF-α. NHS-IL12 and irradiation together induced broad intratumoral TH1 biased NK and T-cell compartments, established antitumoral cytokine profiles and irreversibly growth arrested tumor cells, leading to systemic cancer control and improved survival. For the first time, we describe immune-induced senescence as a novel mechanism resulting from a treatment regimen combining irradiation with immunotherapy.

Enhanced binding of necrosis-targeting immunocytokine NHS-IL12 after local tumour irradiation in murine xenograft models.

PURPOSE: NHS-IL12 is an immunocytokine targeting necrotic tumour areas. IL12 shows anti-tumour activity. As local irradiation might induce additional necrosis in solid tumours, we aimed to evaluate the increase in intratumoural accumulation of NHS-IL12 after irradiation and correlate the findings with diffusion-weighted MRI studies in two xenograft models. METHODS: Human rhabdomyosarcoma (A204) and prostate cancer (PC3) cells were studied in vitro and as subcutaneous xenografts. Radiation sensitivity of the cell lines was assessed in vitro by colony formation assays. In vivo tumour necrosis was assessed based on apparent diffusion coefficients (ADC). Biodistribution of NHS-IL12 was evaluated with and without tumour irradiation using in vivo small-animal PET and ex vivo biodistribution. RESULTS: A204 and PC3 differed in their intrinsic radiation sensitivity. Accordingly, radiation-induced tumour necrosis was found only in A204 xenografts. In comparison with control, ADC was significantly increased after irradiation of A204 tumours with 1 × 8.0 Gy and 5 × 2.0 Gy, whereas no change in ADC was observed in PC3 xenografts in all irradiation regimes. ADC correlated with histology. An enhanced uptake of radiolabelled NHS-IL12 in A204 tumours was detected by PET and ex vivo biodistribution after tumour irradiation. In PC3 tumours, no increase in NHS-IL12 uptake was observed. CONCLUSIONS: In dependence of the tumour model, tumour irradiation enhanced tumour necrosis measured in MRI and histology. In vivo PET and ex vivo biodistribution showed enhanced binding of NHS-IL12 in rhabdomyosarcoma xenografts. Thus, enhanced binding of necrosis-targeting immunocytokines might be a novel mechanism of additive effects in combination with irradiation.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation.

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside.

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.