Avidity as a Tool for Cancer Drug Delivery Systems

Abstract

Despite extensive research conducted over the last century, cancer therapeutics are rarely curative and are associated with debilitating side effects that limit their dosing and efficacy. To reduce off-target toxicities and optimize drug exposure to malignant cells, researchers have implemented drug delivery strategies to encapsulate, increase the circulation half-life, sustain the release and improve the targeting of the drug. Many of these platforms incorporate high-affinity biomolecules, which can function either as targeting domains to improve tumor localization or as a strategy to sustain the release of a therapeutic from a delivery material. The interaction between two proteins can be further modulated by increasing the valency of the binding domain, which alters the avidity of the drug delivery vehicle for its binding partner. The avidity, or the accumulated strength of all the binding interactions, can likewise impact targeting or drug release. Herein, we elucidate precisely how avidity can be optimized in a drug delivery vehicle to improve the biodistribution and pharmacokinetics of cancer therapeutics. In the first part of this dissertation, we systematically dissect the impact of avidity on tumor-targeted drug delivery. Targeting moieties are frequently incorporated onto drug delivery vehicles to promote preferential accumulation of the therapeutic at the tumor while mitigating exposure in healthy tissue. Many strategies attempt to maximize tumor localization by increasing the overall binding strength of the drug delivery vehicle for a receptor on the tumor. However, due to the binding site barrier effect, this strategy may prevent the therapeutic’s access to malignant cells in the tumor core. In this work, we design drug delivery carriers with a different number of binding moieties but the same apparent dissociation constant to precisely evaluate how avidity impacts tumor accumulation and penetration. Once these combinations were selected from a library of fusion proteins, we adapted our carriers for intravenous administration and non-invasively tracked their accumulation at the tumor site. At the point where specific binding was maximal in the tumor, we conducted immunofluorescent analysis of the tumor to assess how deeply the carrier had penetrated. We demonstrated that, independent of valency, KD,app-matched fusion proteins exhibited the same tumor accumulation and penetration. We further evaluated the impact of avidity on tumor targeting by creating fusion proteins with a range of KD,app values, and demonstrated that accumulation and penetration depends on overall binding strength rather than the number of binding moieties. In the second part of this dissertation, we evaluate how avidity can be tuned to modulate the release of antibody therapeutics from a depot with the goal of understanding how sustained release could impact the efficacy of immune checkpoint blockade (ICB) antibodies. ICB antibodies are clinically limited by their poor accumulation at the tumor and lymphoid tissues, as well as their high off-tumor toxicity. To improve their efficacy and reduce side effects, ICB antibodies would benefit from localized, sustained delivery at the tumor or ipsilateral site. Herein, we design a sustained release platform with high avidity with the ability to prolong the release of therapeutic antibodies for over two weeks. We recombinantly fused multimers of the Z domain – a protein with high affinity for the Fc region of immunoglobulins – to a thermally responsive biopolymer with tunable depot forming behavior. Our platform enables sequence-level control over the phase behavior of the biopolymer and the binding behavior of the fusion protein for the Fc. After showing that this platform can entrap antibody therapeutics into a depot at physiological temperature, we evaluate how avidity, thermal behavior, and antibody to protein mixing ratio can be precisely tuned to modulate release of fluorescently labeled antibodies when injected as subcutaneous depot. Using our platform, we achieve antibody release rates ranging from one to eighteen days. We demonstrate that avidity – controlled by the number of Z domains present on the fusion and the mixing ratio – can significantly impact the rate of antibody release from a depot.