Ionizable Lipid Nanoparticles for Solid Tumor Chimeric Antigen Receptor Immunotherapy

Chimeric antigen receptor (CAR) monocyte and CAR macrophage therapy have recently emerged as promising new therapies for solid tumor immunotherapies. These therapies utilize viruses to engineer permanent CAR expression in patient-derived macrophages and monocytes that enables these immune cells to recognize and kill cancer cells in an antigen specific manner and simultaneously stimulate a broader anti-tumor immune response. However, virally engineered CARs targeted towards solid tumor antigens have a well-documented clinical history of inducing severe on-target-off-tumor toxicity. Further, due to their immunogenicity, viruses can be challenging to administer directly in vivo to engineer immune cells within the patient themselves. These engineering challenges pose significant barriers both to the successful clinical translation of these exciting therapies and their widespread adoption. Therefore, alternative approaches are needed to engineer these cells for safer tumor-targeted CAR therapies, and to enable their use as “off-the-shelf-therapies” with facile clinical access. In Chapter 2 of this work, we formulated a library of novel ionizable lipids into mRNA lipid nanoparticles (mRNA-LNPs), assess their mRNA delivery to human macrophages and identify a lead candidate LNP whose composition and physicochemical characteristics are optimized using statistical methods and microfluidics. We use the optimized macrophage LNPs to engineer functional patient-derived primary human CAR macrophages in an ex vivo tumor killing assay. In Chapter 3, we synthesized a second-generation structurally analogous ionizable lipid library complimentary to the bioactive lipids identified in Aim 1. We used these libraries to directly study the effect of electronegative spacing groups within the ionizable lipid on LNP physiochemical characteristics, in vitro and ex vivo mRNA delivery to immune cells, and in vivo biodistribution to immune cells. We demonstrate that an LNP with tropism to monocytes can be used to successfully engineer functional CD19-CAR monocytes directly in vivo. Lastly, in Chapter 4 of this work, we combined LNPs and monoclonal antibodies (mAb) to build hierarchical biomaterials with intrinsic immunostimulatory properties. We applied microfluidic droplet emulsions to precisely synthesize mAb-crosslinked LNPs that are capable of agonizing surface receptors of tumor-resident immune cells and can be applied as a novel intratumoral immunotherapy. Taken together, this work highlights materials and approaches that can be applied for robust immune cell engineering with applications to solid tumor immunotherapy and beyond.