Mechanism of effective combination radio-immunotherapy against 9464D-GD2, an immunologically cold murine neuroblastoma.

BACKGROUND: Most pediatric cancers are considered immunologically cold with relatively few responding to immune checkpoint inhibition. We recently described an effective combination radio-immunotherapy treatment regimen ( c ombination a daptive- i nnate immunotherapy r egimen (CAIR)) targeting adaptive and innate immunity in 9464D-GD2, an immunologically cold model of neuroblastoma. Here, we characterize the mechanism of CAIR and the role of major histocompatibility complex class I (MHC-I) in the treatment response. METHODS: Mice bearing GD2-expressing 9464D-GD2 tumors were treated with CAIR (external beam radiotherapy, hu14.18-IL2 immunocytokine, CpG, anti-CD40, and anti-CTLA4) and tumor growth and survival were tracked. Depletion of specific immune cell lineages, as well as testing in immunodeficient R2G2 mice, were used to determine the populations necessary for treatment efficacy. Induction of MHC-I expression in 9464D-GD2 cells in response to interferon-γ (IFN-γ) and CAIR was measured in vitro and in vivo, respectively, by flow cytometry and quantitative real-time PCR. A cell line with IFN-γ-inducible MHC-I expression (9464D-GD2-I) was generated by transfecting a subclone of the parental cell line capable of expressing MHC-I with GD2 synthase and was used in vivo to assess the impact of MHC-I expression on responsiveness to CAIR. RESULTS: CAIR cures some mice bearing small (50 mm3) but not larger (100 mm3) 9464D-GD2 tumors and these cured mice develop weak memory responses against tumor rechallenge. Early suppression of 9464D-GD2 tumors by CAIR does not require T or natural killer (NK) cells, but eventual tumor cures are NK cell dependent. Unlike the parental 9464D cell line, 9464D-GD2 cells have uniformly very low MHC-I expression at baseline and fail to upregulate expression in response to IFN-γ. In contrast, 9464D-GD2-I upregulates MHC-I in response to IFN-γ and is less responsive to CAIR. CONCLUSION: Treatment with CAIR cures 9464D-GD2 tumors in a NK cell dependent manner and induction of MHC-I by tumors cells was associated with decreased efficacy. These results demonstrate that the early tumor response to this regimen is T and NK cell independent, but that NK cells have a role in generating lasting cures in the absence of MHC-I expression by tumor cells. Further strategies to better inhibit tumor outgrowth in this setting may require further NK activation or the ability to engage alternative immune effector cells.

Short-course neoadjuvant in situ vaccination for murine melanoma.

BACKGROUND: Surgical resection remains an important component of multimodality treatment for most solid tumors. Neoadjuvant immunotherapy has several potential advantages, including in-situ tumor vaccination and pathologic assessment of response in the surgical specimen. We previously described an in-situ tumor vaccination strategy in melanoma using local radiation (RT) and an intratumoral injection of tumor-specific anti-GD2 immunocytokine (IT-IC). Here we tested whether neoadjuvant in-situ tumor vaccination using anti-GD2 immunocytokine and surgical resection, without RT, could generate immunologic memory capable of preventing recurrence or distant metastasis. METHODS: Mice bearing GD2 expressing B78 melanoma tumors were treated with neoadjuvant radiation, IT-IC, or combined RT + IT-IC. Surgical resection was performed following neoadjuvant immunotherapy. Immune infiltrate was assessed in the resection specimens. Mice were rechallenged with either B78 contralateral flank tumors or pulmonary seeding of non-GD2 expressing B16 melanoma metastasis induced experimentally. Rejection of rechallenge in mice treated with the various treatment regimens was considered evidence of immunologic memory. RESULTS: Neoadjuvant IT-IC and surgical resection resulted in increased CD8 T cell infiltration, a higher CD8:regulatory T cell ratio, and immunologic memory against contralateral flank rechallenge. The timing of resection did not significantly impact the development of memory, which was present as early as the day of surgery. There was less local wound toxicity with neoadjuvant IT-IC compared with neoadjuvant RT +IT IC. Neoadjuvant IT-IC and resection resulted in the rejection of B16 lung metastasis in a CD4 T cell dependent manner. CONCLUSIONS: Neoadjuvant IT-IC generates immunologic memory capable of preventing distant metastasis despite limited efficacy against large primary melanoma tumors. By combining neoadjuvant tumor vaccination and surgery, the toxicity of local RT was avoided. These preclinical data support further investigation regarding the use of neoadjuvant IT-IC in patients with melanoma at high risk for occult distant disease.