Cyclophosphamide augments the efficacy of in situ vaccination in a mouse melanoma model.

Introduction: We have previously shown that an intratumoral (IT) injection of the hu14.18-IL2 immunocytokine (IC), an anti-GD2 antibody linked to interleukin 2, can serve as an in situ vaccine and synergize with local radiotherapy (RT) to induce T cell-mediated antitumor effects. We hypothesized that cyclophosphamide (CY), a chemotherapeutic agent capable of depleting T regulatory cells (Tregs), would augment in situ vaccination. GD2+ B78 mouse melanoma cells were injected intradermally in syngeneic C57BL/6 mice. Methods: Treatments with RT (12Gy) and/or CY (100 mg/kg i.p.) started when tumors reached 100-300 mm3 (day 0 of treatment), followed by five daily injections of IT-IC (25 mcg) on days 5-9. Tumor growth and survival were followed. In addition, tumors were analyzed by flow cytometry. Results: Similar to RT, CY enhanced the antitumor effect of IC. The strongest antitumor effect was achieved when CY, RT and IC were combined, as compared to combinations of IC+RT or IC+CY. Flow cytometric analyses showed that the combined treatment with CY, RT and IC decreased Tregs and increased the ratio of CD8+ cells/Tregs within the tumors. Moreover, in mice bearing two separate tumors, the combination of RT and IT-IC delivered to one tumor, together with systemic CY, led to a systemic antitumor effect detected as shrinkage of the tumor not treated directly with RT and IT-IC. Cured mice developed immunological memory as they were able to reject B78 tumor rechallenge. Conclusion: Taken together, these preclinical results show that CY can augment the antitumor efficacy of IT- IC, given alone or in combination with local RT, suggesting potential benefit in clinical testing of these combinations.

Combined innate and adaptive immunotherapy overcomes resistance of immunologically cold syngeneic murine neuroblastoma to checkpoint inhibition.

BACKGROUND: Unlike some adult cancers, most pediatric cancers are considered immunologically cold and generally less responsive to immunotherapy. While immunotherapy has already been incorporated into standard of care treatment for pediatric patients with high-risk neuroblastoma, overall survival remains poor. In a mouse melanoma model, we found that radiation and tumor-specific immunocytokine generate an in situ vaccination response in syngeneic mice bearing large tumors. Here, we tested whether a novel immunotherapeutic approach utilizing radiation and immunocytokine together with innate immune stimulation could generate a potent antitumor response with immunologic memory against syngeneic murine neuroblastoma. METHODS: Mice bearing disialoganglioside (GD2)-expressing neuroblastoma tumors (either NXS2 or 9464D-GD2) were treated with radiation and immunotherapy (including anti-GD2 immunocytokine with or without anti-CTLA-4, CpG and anti-CD40 monoclonal antibody). Tumor growth, animal survival and immune cell infiltrate were analyzed in the tumor microenvironment in response to various treatment regimens. RESULTS: NXS2 had a moderate tumor mutation burden (TMB) while N-MYC driven 9464D-GD2 had a low TMB, therefore the latter served as a better model for high-risk neuroblastoma (an immunologically cold tumor). Radiation and immunocytokine induced a potent in situ vaccination response against NXS2 tumors, but not in the 9464D-GD2 tumor model. Addition of checkpoint blockade with anti-CTLA-4 was not effective alone against 9464D-GD2 tumors; inclusion of CpG and anti-CD40 achieved a potent antitumor response with decreased T regulatory cells within the tumors and induction of immunologic memory. CONCLUSIONS: These data suggest that a combined innate and adaptive immunotherapeutic approach can be effective against immunologically cold syngeneic murine neuroblastoma. Further testing is needed to determine how these concepts might translate into development of more effective immunotherapeutic approaches for the treatment of clinically high-risk neuroblastoma.

Effective Combination of Innate and Adaptive Immunotherapeutic Approaches in a Mouse Melanoma Model.

Most cancer immunotherapies include activation of either innate or adaptive immune responses. We hypothesized that the combined activation of both innate and adaptive immunity will result in better antitumor efficacy. We have previously shown the synergy of an agonistic anti-CD40 mAb (anti-CD40) and CpG-oligodeoxynucleotides in activating macrophages to induce tumor cell killing in mice. Separately, we have shown that a direct intratumoral injection of immunocytokine (IC), an anti-GD2 Ab linked to IL-2, can activate T and NK cells resulting in antitumor effects. We hypothesized that activation of macrophages with anti-CD40/CpG, and NK cells with IC, would cause innate tumor destruction, leading to increased presentation of tumor Ags and adaptive T cell activation; the latter could be further augmented by anti-CTLA-4 Ab to achieve tumor eradication and immunological memory. Using the mouse GD2+ B78 melanoma model, we show that anti-CD40/CpG treatment led to upregulation of T cell activation markers in draining lymph nodes. Anti-CD40/CpG + IC/anti-CTLA-4 synergistically induced regression of advanced s.c. tumors, resulting in cure of some mice and development of immunological memory against B78 and wild type B16 tumors. Although the antitumor effect of anti-CD40/CpG did not require T cells, the antitumor effect of IC/anti-CTLA-4 was dependent on T cells. The combined treatment with anti-CD40/CpG + IC/anti-CTLA-4 reduced T regulatory cells in the tumors and was effective against distant solid tumors and lung metastases. We suggest that a combination of anti-CD40/CpG and IC/anti-CTLA-4 should be developed for clinical testing as a potentially effective novel immunotherapy strategy.