Abstract 1553: Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies
Authors: Ishihara Jun , Ishihara Ako , Mansurov Aslan , Sasaki Koichi , Lee Steve S. , Williford John-Michael , Potin Lambert , Hosseinchi Peyman , Gray Laura T. , Katsumata Kiyomitsu , Kron Stephen J. , Swartz Melody A. , Hubbell Jeffrey A. 
Published: August 2019

Abstract
Cancer immunotherapy with immune checkpoint inhibitors (CPI), interleukin (IL)-2, and IL-12 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-CTLA4 (αCTLA4) + anti-PD-L1 (αPD-L1) and the cytokines IL-2 and IL-12 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokines) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, with 57% of total injected dose depositing in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2/IL-12. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. CBD-fusion to IL-12 significantly decreased hepatotoxicity and systemic IFNg concentration. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. CBD-IL-12 therapy induced remission of B16F10 melanoma and EMT6 breast cancer, along with increased intratumoral IFNg concentration compared to normal IL-12 therapy. Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokines) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. Unlike other active tumor-targeting approaches, CBD-based targeting is novel in a sense that it exploits abnormal tumor vessel structure; this characteristic affords binding to the ubiquitous extracellular matrix protein in the tumor, while limiting exposure to other tissues. CBD-immunotherapeutics may efficiently activate tumor-antigen-specific T cells and increase intratumoral downstream cytokines while maintaining systemic immune homeostasis by avoiding influencing non-tumor antigen specific T cells and systemic cytokine release.
Citation Format: Jun Ishihara, Ako Ishihara, Aslan Mansurov, Koichi Sasaki, Steve S. Lee, John-Michael Williford, Lambert Potin, Peyman Hosseinchi, Laura T. Gray, Kiyomitsu Katsumata, Stephen J. Kron, Melody A. Swartz, Jeffrey A. Hubbell. Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1553.