3E). liver following unmodified- or CBD- CPI treatment were analyzed. fig. S8 Conjugation of CBD to CPI is usually indispensable for B16F10 tumor growth suppression. fig. S9 EMT6 immune-excluded tumor is not very responsive to CBD-CPI and CBD-IL-2. fig. S10 CBD-CPI treatment decreases immune suppressive MDSCs within B16F10 tumor. fig. S11 Immune cells within B16F10 tumor and spleen were analyzed after CBD-IL-2 treatment. fig. S12 CBD-IL-2 treatment increases the number of CD8+ T cells and NK cells within MMTV-PyMT tumor but not EMT6 tumor. table S1 Protein sequences. NIHMS1028087-supplement-Figures.pdf (908K) GUID:?8FB9266D-0DD5-4502-BBDA-6072638989BD Abstract Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T-lymphocyte antigen 4 antibody (CTLA4) + anti-programmed death-ligand 1 antibody (PD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain name (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain name, harnessing 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. CBD conjugation or fusion decreases the systemic toxicity of both CTLA4+PD-L1 combination therapy and IL-2, Decloxizine for example eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells. In an orthotopic breast tumor model, combination treatment with CPI Decloxizine and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain name of VWF can be used to improve safety and efficacy of systemically-administered tumor drugs with high translational promise. One Sentence Summary: An engineered cancer immunotherapy using a collagen-binding domain name enhances efficacy and reduces adverse events. INTRODUCTION Immune checkpoint inhibitors (CPI) have demonstrated clinical efficacy in cancer immunotherapy (1, 2). Immune Decloxizine checkpoints are inhibitory pathways used by the immune system to protect cells from excessive immune responses (3). Cytotoxic T-lymphocyte antigen 4 (CTLA4, CD152) is expressed on regulatory T cells (Tregs) and activated T cells (4, 5). In the clinic, anti-CTLA4 antibody (CTLA4) treatment prolonged survival of melanoma patients (5). Some tumor cells express programmed death-ligand 1 (PD-L1, CD274). Association of PD-L1 with its ligand programmed death 1 (PD-1, CD279) results in inactivation of T cells. Anti-PD-L1 (PD-L1) blocking antibodies have shown efficacy against several types of cancer (6, 7). Moreover, combination therapy using aPD-1 (nivolumab) and CTLA4 (ipilimumab) shows prolongation of survival (8) and has been approved by the US Food and Drug Administration (FDA) for treatment of advanced GluN1 melanoma and renal cell carcinoma. However, CPI treatment also shows severe side effects, including immune-related adverse events (8C10). In combination therapy, 96% of patients experienced adverse events, and 36% of patients discontinued therapy due to adverse events (8). Interleukin-2 (IL-2: aldesleukin) is usually a cytokine that induces proliferation and activation of T cells and natural killer (NK) cells (11). Administration of IL-2 has exhibited antitumor effects in the clinic (12), and aldesleukin has been approved by the US FDA for treatment of metastatic melanoma and renal cell carcinoma. In clinical studies, 19% of patients responded to aldesleukin with prolonged survival, but almost all patients experienced treatment-related adverse events, including 1.1% of treatment-related death (13). Aldesleukin has a narrow therapeutic window due to induction of severe adverse events such as pulmonary edema (14). Because such immunotherapeutics serve to activate immune responses, their side effects are caused by immune.