(567f) Spatiotemporal Delivery and Activation of Nanobody-Sting Agonist Conjugate Cancer Immunotherapies

This talk will address the current limitations in cancer therapeutic delivery of immunotherapies and offer a strategy to overcome these challenges through spatiotemporally controlled delivery and activation of immune modulating drug payloads. Here, we focus on the development of a drug delivery platform that enables an elongated pharmacokinetic profile – with selective biodistribution to tumor tissue – for controlled activation of the STING (stimulator of interferon genes) signaling pathway in the tumor microenvironment (TME), leading to an upregulated inflammatory response (interferon production). This strategy relies on two elements: (1) spatial control of delivery of the STING agonist through selective binding of immune checkpoint inhibitor receptors (CD274, PD-L1) expressed on the surface of tumor cells, as well as (2) temporal control through systemic delivery by albumin hitchhiking. We generated a recombinant fusion protein consisting of two antibody fragments that bind to both PD-L1 and serum albumin and performed site-specific payload conjugation to place a single agonist on each fusion protein to yield a homogeneous, atomically precise 1:1 protein-agonist conjugate. In vitro evaluation of the protein-agonist conjugate showed nanomolar activity to stimulate the STING pathway, without sustaining cellular cytotoxicity. In vivo, we found that albumin hitchhiking enabled a half-life extension of the small molecule payload from 90 minutes to 55 hours (36x improvement), and preferential accumulation of the protein-agonist conjugate within the tumor microenvironment, making albumin hitchhiking a powerful approach to selective spatial targeting of tumor cells. Importantly, in both EMT6 and B16.F10 cancer models, we found that our conjugate platform offered a lower administration dose at 2.5 µg – compared to 30 µg of free drug used in literature – and an enhanced therapeutic response when compared to standard immune checkpoint blockade (ICB). Using this approach, we demonstrate the synthesis, in vitro, and in vivo evaluation of an immunostimulatory drug delivery system with controlled tumor-selective STING agonist delivery and activation in the TME.