Development of liposomal Irinotecan (CPT-11) formulation and understanding of its therapeutic potential in combination with immunotherapies in colorectal cancer models

Irinotecan (CPT-11) is a key chemotherapeutic drug for various solid tumors, including colorectal cancer (CRC). However, its clinical use is limited by unfavorable pharmacokinetics, rapid interconversion to inactive form, and dose-limiting gastrointestinal (GI) toxicity. Liposomal drug delivery systems have emerged as effective strategies to improve drug stability, prolong circulation time, and enhance therapeutic effects of encapsulated drugs. Furthermore, growing interest has focused on using liposomal formulations to induce immunogenic cell death (ICD) and modulate the tumor immune microenvironment (TIME), creating opportunities for combination with immunotherapies. In this thesis, a novel liposomal CPT-11 formulation, termed Irinosome High C PEGᵖᵒˢᵗ, was developed and characterized. This formulation was designed to improve drug retention, loading efficiency and pharmacokinetic properties compared with an existing formulation Irinophore C while enhancing therapeutic efficacy and modulating anti-tumor immune responses in murine CRC models. Irinosome High C PEGᵖᵒˢᵗ incorporates and maintains a high internal Cu²⁺ concentration and low internal pH to achieve sustained drug release and a high CPT-11-to-liposomal lipid ratio without the use of ionophores. This new formulation demonstrated superior encapsulation efficiency, improved drug retention in vitro and in vivo, and better storage stability. In immune-competent CT26 CRC model, treatment with Irinosome High C PEGᵖᵒˢᵗ resulted in significant tumor growth inhibition. Mechanistic studies showed Irinosome High C PEGᵖᵒˢᵗ induced ICD in vitro as evidenced by the release of damage associated molecular patterns (DAMPs) markers from Irinosome High C PEGᵖᵒˢᵗ treated cells and increased activation of bone-marrow derived dendritic cells and macrophages following co-culture. Immune profiling of tumors from Irinosome High C PEGᵖᵒˢᵗ treated animals revealed a shift from an immunosuppressive to a more immunostimulatory TIME. Despite these immunomodulatory effects, concurrent treatment with Irinosome High C PEGᵖᵒˢᵗ and anti-PD-1 antibodies did not improve therapeutic outcomes in the syngeneic CT26 model. This may be attributed to transient myelosuppression of Irinosome High C PEGᵖᵒˢᵗ and a reduction of the PD-1/PD-L1 T cell immune checkpoint pathway in treated tumors. These findings highlight the importance of treatment scheduling in chemo-immunotherapy combinations. Overall, this work establishes Irinosome High C PEGᵖᵒˢᵗ as a promising platform with immunomodulatory potential that may support future chemo-immunotherapy strategies.