Immunogenic Polymer Lipid Nanodiscs

With the major progress made in cancer immunology in the last 50 years, research has shed light on the biological mechanisms of cancer immune escape, identifying the molecular pathways which are responsible for immune dysfunction and disease progression. This has opened up the opportunity to develop new classes of drugs to target these dysfunctional processes and restore physiological immune control. Among these, drug activators of Toll-Like Receptors 7/8 have shown to be potent immune stimulators which could trigger inflammation in tumors to ultimately raise an adaptive tumor-killing immune response. However, their clinical translation was considerably limited by severe toxicity induced by the uncontrolled activation of the TLR7/8 pathway when the small-molecule drugs could spread freely in systemic circulation. This has drawn considerable efforts in developing drug formulation solutions to control the pharmacokinetics of TLR7/8 agonists. This thesis describes the exploration of the nanodisc-forming polymer poly(Acrylic Acid-co-Styrene) (AASTY) as a drug delivery platform for TLR7/8 agonists and in vivo imaging modalities. This amphiphilic polymer can interact with lipid bilayers and can isolate membrane-bound proteins into discoidal nanoparticles named polymerlipid nanodiscs. While this technology was initially developed for structural characterization of membrane proteins, this thesis reports the first in vivo translation of AASTY. The polymer was synthetized through Radical Addition-Fragmentation Chain Transfer and covalently linked with a TLR7/8 agonist and other functionalities using common conjugation chemistry techniques. The biodistribution and pharmacokinetic profile of the various conjugates was explored on mice and then tested in murine cancer models as an immunotherapy platform. Thanks to its unique interaction with lipids, AASTY could locally bind to biological tissues for durations ranging from weeks to months, and slowly got cleared through the liver, making it an ideal platform for loco-regional drug delivery. At higher concentrations, it could even diffuse through tissues and exhibit permeation enhancement properties. When attached with a TLR7/8 agonist and injected intratumorally in a CT26 murine colon adenocarcinoma model, the conjugate could then successfully trigger a strong innate inflammation locally with limited systemic side effects, which overall inhibited tumor growth and increased survival. Through tumor rechallenge and bilateral cancer model experiments, the therapy appeared to involve an adaptive immune response which showed abscopal therapeutic effects and induction of cancer-specific immune memory. By exploring various parameters of the treatment’s dose regimen, therapeutic efficacy was demonstrated for a single highly concentrated injection of the compound. The therapy was then extended to other syngenic murine cancer models as a monotherapy or in combination with Immune Checkpoint Inhibitors and also showed antitumor effects over a large spectrum of cancer immunogenicity, ranging from mildly immunologically hot cancer models to extensively cold ones. Following the profiling of serum cytokine levels, immune cell transporters of the polymer and of the antigens captured in nanodiscs, the polymer conjugate is understood to have a multimodal mode of action following a concentration gradient. From a high concentration at the injection center to lower concentration at distal diffusion points, the polymer is believed to induce cell death, nanodisc solubilization of all tumor antigens and depot in cell membranes, while, at all concentration levels, activating or repolarizing immune cells through TLR7/8a stimulation. This leads to a complete in situ immune activation and antigen presentation driven by immunogenic polymerlipid nanodiscs, ultimately raising personalized antitumor responses. The polymer platform is a new avenue for personalized cancer immunotherapy and more broadly for attaining prolonged tissue retention of immune modulators and other covalently attached drugs.