Engineering Erythrocyte Affinity for Improved Pharmacokinetics and Immune Tolerogenesis

The clinical success of recombinant protein therapeutics in the treatment of disease is a hallmark of modern medical research, yet there remain numerous unaddressed issues regarding their delivery and immunogenicity. Regardless of in vivo efficacy, the biological fate of administered protein drugs often dictates their therapeutic potential. Secondary side effects induced due to high-dose requirements for of short-lived drugs frequently halt further development of therapeutics. As such, engineering proteins to display improved pharmacokinetic behavior in vivo has become an increasingly prolific theme in pharmaceutical research. A vast majority of protein therapeutics additionally induce a detectable undesired immune response, which may lead to secondary symptoms during treatment and hypersensitivity reactions upon re-exposure. In this work, we describe the development of a novel platform technology that improves the pharmacokinetic behavior of proteins by exploiting natural circulation and immune function of the circulating erythrocyte. Present at ∼5×106 / µL and circulating for 120 days in humans, erythrocytes offer opportune drug carrier characteristics due to their high blood concentration, long circulation time, and relatively large 5 µm diameter. Using phage display, we discovered a unique 12-residue peptide (ERY1) that binds mouse erythrocytes with high specificity in vivo. We demonstrate that recombinant fusion of ERY1 to the terminus of a model protein increases the protein‚s circulation half-life 3.2- to 6.3-fold, decreases clearance 2.15-fold, and improves bioavailability 1.67-fold in mice. The ERY1-fused protein exhibited enhanced pharmacokinetics due to its capacity to non-covalently bind circulating erythrocytes in the blood, thereby delaying glomerular filtration in the kidneys. Furthermore, we demonstrate that high-affinity erythrocyte binding by a protein induces immune tolerance towards the antigen, by hijacking natural tolerogenesis mechanisms driven by apoptotic cells. Using ovalbumin (OVA) as a model protein antigen, we created a variant containing several grafted ERY1 peptides (ERY1-OVA) that binds to circulating healthy and eryptotic erythrocytes with high affinity and specificity. In an adoptive transfer mouse model using major histocompatibility complex I (MHC-I) transgenic CD8+T cells specific for OVA, we show that ERY1-OVA induces enhanced antigen-specific T-cell proliferation to a tolerogenic phenotype. Moreover, mice administered ERY1-OVA failed to induce an inflammatory immune response following antigen-specific challenge with a strong adjuvant, whereas mice administered OVA exhibited robust responses. As ERY1-OVA binds healthy and eryptotic erythrocytes, our results align with current hypotheses in the field that emphasize the importance of apoptotic cell-derived antigen in tolerogenesis. Next-generation therapeutics capable of effective and enhanced bioavailability while maintaining immunological stealth will profoundly impact the quality of therapy, and potentially broaden the eligible patient base for treatment. We conclude engineering proteins to bind erythrocytes in vivo improves their pharmacokinetic behavior and induces immune tolerance, and may serve as a modular enabling technology to bring experimental molecules to clinical consideration, and to further improve current therapeutics.