Nonterminal Albumin Fusions for Improved Delivery of Polypeptides

About This Grant

PROJECT SUMMARY/ABSTRACT Polypeptides, due to their high affinity and selectivity for diverse biological targets, hold significant promise as therapeutic agents for treating complex diseases such as cancer, metabolic disorders, and infectious diseases. However, poor pharmacokinetics (PK), including rapid renal clearance and protease degradation, often hinders their clinical applications. This leads to short half-lives and limited bioavailability, necessitating frequent dosing and increasing the burden on the patient and the risk of side effects. Existing strategies to improve PK, such as recombinant fusions to the N- or C-terminus of human serum albumin (HSA) or chemical modifications to constrain conformation like cyclization, frequently compromise the biological activity and manufacturability of these therapeutic agents. This project aims to avoid activity losses due to the steric hindrance from the albumin fusion partner while constraining conformation naturally by developing a novel platform that nonterminally fuses polypeptides to albumin. Nonterminal fusions are relatively unexplored due to the challenges associated with maintaining the native folding and function of the fusion partner. However, recent advancements in structural prediction tools now enable us to accurately predict and mitigate these disruptions. We hypothesize that nonterminally fusing polypeptide cargoes to albumin at externally presented disulfide-constrained loop regions will significantly improve their pharmacokinetic properties without compromising their activity. We will evaluate this strategy for the ability to enhance the stability, half-life, and target localization of therapeutic polypeptides through three specific aims: Aim 1 will establish an expression system and leverage a model peptide to screen potential insertion sites for structural perturbations and availability. Aim 2 will use AlphaFold to develop a computational pipeline to predict structural changes due to nonterminal fusions, establishing rules for generalizing the strategy to any payload. Aim 3 will evaluate the PK properties and therapeutic benefits of nonterminal fusions of Neo-2/15, an engineered IL-2/15 receptor agonist, in cancer models. Successful completion of this project will yield a robust platform for nonterminally fusing polypeptides to albumin, offering a modular and generalizable strategy to improve drug activity, half-life, and target tropism. This approach has the potential to significantly advance the fields of protein engineering and drug delivery. By establishing a precedent and pipeline for nonterminal fusions across any carrier proteins, more effective and durable treatments can be developed in many therapeutic areas and indications, ultimately improving patient outcomes and enhancing public health.