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

Noah Tsarovsky; Mildred Felder; Mackenzie Heck; Jacob Slowinski; Kayla Rasmussen; Sabrina VandenHeuvel; Jen Zaborek; Zachary S Morris; Amy K Erbe; Paul M Sondel; Alexander L Rakhmilevich

doi:10.3389/fonc.2023.1200436

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

Front Oncol. 2023 Sep 6:13:1200436. doi: 10.3389/fonc.2023.1200436. eCollection 2023.

Authors

Noah Tsarovsky¹, Mildred Felder¹, Mackenzie Heck¹, Jacob Slowinski¹, Kayla Rasmussen¹, Sabrina VandenHeuvel¹, Jen Zaborek², Zachary S Morris^{1

3}, Amy K Erbe^{1

3}, Paul M Sondel^{1

3

4}, Alexander L Rakhmilevich^{1

3}

Affiliations

¹ Department of Human Oncology, Madison, WI, United States.
² Department of Biostatistics and Medical Informatics, Madison, WI, United States.
³ Paul P. Carbone Comprehensive Cancer Center, Madison, WI, United States.
⁴ Department of Pediatrics, University of Wisconsin, Madison, WI, United States.

Abstract

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.

Keywords: cyclophosphamide; immunocytokine; in situ vaccine.; melanoma; radiation.

Grants and funding

This work was supported by Midwest Athletes Against Childhood Cancer; Stand Up 2 Cancer; the St. Baldrick’s Foundation; the Crawdaddy Foundation; Alex’s Lemonade Stand Foundation, The Cancer Research Institute, and the University of Wisconsin Carbone Cancer Center. This research was also supported in part by public health service grants U54-CA232568, R35-CA197078, 1DP5OD024576, U01-CA233102, P50 DE026787, and P01 CA250972 from the National Cancer Institute, the National Institutes of Health and the Department of Health and Human Services. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Schematic diagrams in Figures 1A and 4A were created with Biorender.com.