BackgroundSurgical 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.MethodsMice 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.ResultsNeoadjuvant 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.ConclusionsNeoadjuvant 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.

BackgroundMost pediatric cancers are considered immunologically cold with relatively few responding to immune checkpoint inhibition. We recently described an effective combination radio-immunotherapy treatment regimen (combination adaptive-innate immunotherapy regimen (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.MethodsMice 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.ResultsCAIR 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.ConclusionTreatment 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.

Introduction: Using an in situ vaccine (ISV) regimen consisting of radiation combined with immunocytokine (tumor-targeting mAb linked to IL2), we can cure mice of well established B78 melanoma tumors (B78s). Mice cured of their B78s with ISV demonstrate long-term immune memory, evidenced by rejection of engraftment of a tumor rechallenge >180 days post initial cure of tumor. Traditionally, immaune memory is thought to be mediated via CD8+ T cells, which require antigen presentation via MHC Class I (MHCI). However, B78s express little to no MHCI but do express MHCII when stimulated with IFNγ. While not commonly expressed on solid tumors, MHCII is expressed on 50-60% of melanomas in humans. Here we explored implications of MHCI and MHCII expression in both the primary and long-term memory anti-tumor responses generated with ISV. Methods: CD4+ or CD8+ T cells were depleted in mice during the primary anti-tumor response (i.e. B78 tumor bearing mice receiving the ISV regimen) or during tumor rechallenge experiments (i.e. B78-cured mice rechallenged with B78s). Tumor growth was monitored. In separate studies, tumors and tumor draining lymphnodes (TDLNs) were harvested during the primary antitumor response and analyzed via flow cytometry to assess T cell activation and immune infiltrate. Finally, TDLNs were harvested from B78-cured mice, 7 days after tumor rechallenge, to define memory T cell subsets. Results: Depletion studies revealed CD4+ T cells are required for both the antitumor response to ISV and the long-term memory response in B78-cured mice, but CD8+ T cells are not required for either of these responses. Increased CD8+ and CD4+ T cell infiltrates are observed in the tumor microenvironment during the primary anti-tumor response. In B78-cured mice, though not required for memory responses, CD8+ central memory T cells are significantly increased in the TDLNs compared to naïve or primary tumor bearing mice. The amount of CD4+ effector memory T cells are significantly increased in the TDLN of B78-cured mice compared to CD8+ effector memory T cells. Conclusion: Often not expressed on solid tumors, MHCII is expressed on some melanoma tumors, and its expression has been correlated with a positive response to immunotherapies. CD4+ cytotoxic T cells can directly engage MHCII on tumors, suggesting this interaction has an important role in the response to immunotherapy for MHCII expressing tumors. Our data suggest that CD4+ T cells drive both the primary anti-tumor and long-term immune memory responses in the B78 model when treating with this effective ISV. We are continuing our efforts to understand the characteristics of the B78 cell line that may be relevant to its response to immunotherapy and its resistance to single agent checkpoint blockade. Understanding the cellular and molecular mechanisms involved in ISV-induced immune recognition and destruction of B78s may guide future improvements of this clinically-relevant immunotherapy regimen. Citation Format: Amy K. Erbe, Arika Feils, Mackenzie Heck, Sabrina VandenHeuvel, Julianna Castillo, Alina Hampton, Lizzie Frankel, Anna Hoefges, Peter Carlson, Alex A. Pieper, Taylor Aiken, Lauren Zebertavage, David Komjathy, Dan Spiegelman, Noah Tsarovsky, Zachary S. Morris, Ravi Patel, Alexander Rakhmilevich, Paul M. Sondel. The influence of MHC class I and II on T cell responses in mouse melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1385.

No abstract

e21561 Background: GD2 is disialoganglioside preferentially expressed in neuroblastoma and melanoma and anti-GD2 directed therapies are used clinically in neuroblastoma, with ongoing clinical trials in melanoma. We are currently developing an in situ vaccination approach using intratumoral (IT) delivery of an immunocytokine (IC) consisting of IL-2 linked to an anti-GD2 monoclonal antibody. While IT-IC monotherapy does not cure mice bearing established B78 melanoma tumors, it is effective when combined with local radiation therapy (RT). Here, we tested whether short course IT-IC monotherapy prior to surgical resection could result in a robust adaptive immune response preventing tumor recurrence following rechallenge after surgery. Methods: Mice bearing 50-100mm3 GD2-expressing melanoma (B78) tumors were treated with a 5-day course of 50μg IT-IC and complete surgical resection was performed 3 days following the final treatment. The immune infiltrate of resected tumors was assessed by flow cytometry. Rechallenge experiments consisted of either 2x106 B78 cells injected into the contralateral flank or 2x105 B16-GD2 cells injected via tail vein for pulmonary metastasis rechallenge. Results: IT-IC treated tumors had fewer viable tumor cells, increased CD8 T-cells, and an improved CD8:Treg ratio. Rejection of B78 contralateral flank rechallenge (implanted 40 days following surgical resection of the primary tumor) was observed in 78% (7/9) of mice treated with IT-IC compared to 50% (5/10) that received surgery alone and 0% (0/5) of naïve mice. Immunologic memory was potent in neoadjuvant-treated mice early after surgery, with all mice (5/5) rejecting contralateral B78 rechallenge that occurred on the day of surgery compared to 0% (0/5) in both surgery-alone and naïve mice. Neoadjuvant IT-IC also prevented the development of B16-GD2 lung metastasis compared to naïve mice or the surgery-alone group (when the IV injected experimental metastases were given 80 days following surgery). Conclusions: While ineffective in curing large B78 melanoma flank tumors as monotherapy, mice receiving neoadjuvant IT-IC developed robust immunologic memory preventing recurrence following surgery. The memory response was present as early as the day of surgery and was sufficient to prevent pulmonary metastasis. IT-IC should be further investigated as a neoadjuvant therapy for preventing recurrence in high-risk settings.