Project Portugal 2030

Summary

The clinical potential of therapeutic Abs with intracellular targets is severely compromised by the impaired crossing of the cellular membrane and escape from the endolysosomal compartment, and required spaciotemporal release of functional Ab. So far, the majority of HA-based nanosystems for intracellular delivery of therapeutic proteins are based on nanogels with proteins entrapped within the aqueous matrix or complexed by non-covalent interactions. Although such systems improve the pharmacokinetic profile and stability of therapeutic cargo, they are associated with protein leakage. An alternative based on using protein as a template for self-assembly of synthetic polymers by covalent grafting via stimuli-sensitive linkers and/or the addition of moieties for noncovalent stabilization of the protein demonstrated improved delivery and offered valuable guidelines for future development. However, this proof-of-concept was never applied to Ab delivery nor to biodegradable natural polymers like HA. Apart from antifouling properties, HA provides active targeting of cancer cells overexpressing CD44 receptors. Although previous reports remain unanimous about the efficiency of HA nanosystems for intracellular delivery of proteins, the influence of HA Mw on protein functionality or cell uptake was not considered. Although HA can internalize nanosystems by endocytosis, its tumor accumulation is based on a highly heterogeneous EPR effect. To improve tumor accumulation, HA will be decorated with additional transcytosis ligands. So far, such a combination of complementary ligands has not been described for protein-loaded nanosystems. Therefore, to accost the full potential of HA-based intracellular Ab delivery, we aim to combine several polyfunctional elements for the design of the all-in-one nanosystem for on-site triggerable Ab release. Detailed structure-activity analysis will provide information on the key structural parameters necessary for efficient cytosol delivery of this class of bioactive molecules, by addressing the following: 1. Design and optimization of biocompatible/degradable Ab-HA nanoconjugates based on self-immolative stimuli-sensitive linker. The influence of HA Mw on the overall performance of HA-Ab conjugates will be evaluated. Different Ab feeds will be investigated. 2. Introduction of carbamoylated guanidine moieties for complementary non-covalent interactions to improve Ab stability and endosomal release of Ab-HA conjugates. The influence of guanidine density on Ab stability and endosomal release will be evaluated. 3. Improve TNBC tumor and cell targeting/penetrability by combining CD44 targeting by HA with additional tumor-targeting moiety. The transcytosis mechanism of iRGD tumor penetrating ligand will be exploited to complement the delivery of Ab-HA conjugates. Finally, the development of a new multicellular TNBC spheroid able to recapitulate interactions between key constituents of the tumor microenvironment (cancer cells, tumor-associated macrophages, and fibroblasts) will provide us with a tool for improved pre-clinical screening of new precision therapies. The overall outcomes of this project will provide valuable guidelines for future development and optimization of antibody-polymer conjugates for intracellular epitope targeting in TNBC.