Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases. Summary Brain delivery, biodistribution and pharmacodynamics of a lysosomal enzyme fused to a moderate-affinity transferrin receptor-directed blood-brain barrier enzyme transport vehicle are superior to a traditional high-affinity anti-TfR monoclonal antibody fusion. ### Competing Interest Statement Funding: This study was funded by Denali Therapeutics Inc. Conflicts of interest: A.A., C.S.M., M.E.K.C, D.C., J.C.D., M.E.P., E.R.T., R.C., L.A.D., J.D., T.E., M.F., T.G., D.J.K., N.L., I.A.L., H.N.N., H.S., B.T., J.W., M.S.D., D.D., K.G., K.R.H., J.W.L., P.E.S., M.D.T., J.M.H., K.S., L.S., R.J.W., R.G.T., A.G.H., and M.S.K are paid employees of Denali Therapeutics Inc. Denali Therapeutics Inc. has filed patent applications related to the subject matter of this paper.