The novel chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA) promotes stable binding of His-tagged proteins to liposomal membranes: Potent anti-tumor responses induced by simultaneously targeting antigen, cytokine and costimulatory signals to T cells

Recent studies indicate that the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) can be used to engraft T cell costimulatory molecules onto tumor cell membranes, potentially circumventing the need for genetic manipulation of the cells for development of cell- or membrane-based tumor vaccines. Here, we show that a related lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA, which has three NTA moieties in its headgroup instead of one) is several-fold more effective than NTA-DTDA at promoting stable His-tagged protein engraftment. IAsys biosensor studies show that binding of His-tagged B7.1 (B7.1-6H) to NTA₃-DTDA-containing membranes, exhibit a faster on-rate and a slower off-rate, compared to membranes containing NTA-DTDA. Also, NTA₃-DTDA-containing liposomes and plasma membrane vesicles (PMV) engrafted with B7.1-6H and CD40-6H exhibit greater binding to T cells, in vitro and in vivo. Engrafted NTA₃-DTDA- containing PMV encapsulated cytokines such as IL-2, IL-12, GM-CSF and IFN-γ, allowing targeted delivery of both antigen and cytokine to T cells, and stimulation of antigen-specific T cell proliferation and cytotoxicity. Importantly, use of B7.1-CD40-engrafted PMV containing IL-2 and IL-12 as a vaccine in DBA/2J mice induced protection against challenge with syngeneic tumor cells (P815 mammary mastocytoma), and regression of established tumors. The results show that stable protein engraftment onto liposomal membranes using NTA₃-DTDA can be used to simultaneously target associated antigen, costimulatory molecules and cytokines to T cells in vivo, inducing strong anti-tumor responses and immunotherapeutic effect.