Engineering red blood cells (RBCs) to carry biotherapeutics can overcome the challenges of poor pharmacokinetics, undesirable biodistribution, and immunogenicity common to a disappointingly large number of drugs, particularly biologic agents[1-4]. Surface loading of RBCs via affinity ligands coupled to therapeutic cargoes is a particularly attractive strategy due to its simplicity of manufacture, biocompatibility, and versatility[5, 6]. These properties greatly enhance its potential for translation to clinical use. A major barrier to the use of surface-coupled RBC therapeutics is the limited knowledge of how human RBC ligands alter RBC physiology, such as membrane deformability, and how these changes interrelate with efficacy and pharmacodynamics. We recently described how fusion proteins targeted to different human RBC epitopes differed significantly with respect to their membrane effects, specific activity, and effects on susceptibility to osmotic and mechanical stress (Villa et al. 2017, submitted). Furthermore, an antibody previously used for coupling of therapeutics to mouse RBCs (Ter119), while efficacious[7, 8], also demonstrated rigidifying effects. These observations raise several important questions regarding how exogenous ligands may be used to tune RBC membrane properties towards the appended therapeutic cargo, not only to avoid undesirable effects and enhance safety, but also to change pharmacodynamics and enhance efficacy. Given the importance of several of these antigens as targets for autoantibodies or alloantibodies, their ligand-dependent effects on RBC biology are of additional clinical significance. RBC membrane rigidity is especially responsive to exogenous ligands[9-11] and has significant effects on circulation and blood flow[12-15], interaction with cellular components of the blood, interface with the immune system, and margination within the vascular lumen. These behaviors are likely to be especially impactful for RBC-delivered thromboprophylactic and hemostatic agents. Therefore, engineering deformability via appended ligands is a particularly attractive approach to optimize the efficacy of these therapies. The goal of this proposal is to develop and characterize a library of human and mouse RBC ligands with varying membrane modulatory effects, show how they alter flow dynamics, and determine their efficacy in vitro and in vivo when fused to anti-thrombotic (thrombomodulin) and hemostatic (factor VIII) agents. These studies will define how RBC rigidity interrelates with the efficacy of surface-coupled RBC drug carriers.
Our goal is to characterize the ways in which proteins bound to red blood cells (RBCs) change their membrane properties and show how these changes impact the effectiveness of RBC-bound therapeutic proteins. These findings are especially relevant to diseases of blood clotting or bleeding, because RBCs, which are naturally within blood vessels, are particularly effective carriers of drugs targeting these conditions.