Humanization of anti‐human insulin receptor antibody for drug targeting across the human blood–brain barrier

Abstract

A murine monoclonal antibody (MAb) to the human insulin receptor (HIR) has been engineered for use as a brain drug delivery system for transport across the human blood-brain barrier (BBB). The HIRMAb was humanized by complementarity determining region (CDR) grafting on the framework regions (FR) of the human B43 IgG heavy chain and the human REI kappa light chain. A problem encountered in the humanization process was the poor secretion of the CDR-grafted HIRMAb by myeloma cells. This problem was solved by the production of human/mouse hybrids of the engineered heavy chain variable region (VH), which led to the replacement of five amino acids in the FR3 of the VH with original murine amino acids. No replacement of FR amino acids in the light chain variable region (VL) was required. The affinity of the humanized HIRMAb for the HIR was decreased 27% relative to the murine HIRMAb. The humanized HIRMAb avidly bound to the HIR of isolated human brain capillaries, which are used as an in vitro model system of the human BBB. The HIRMAb cross reacts with the HIR of Old World primates such as the Rhesus monkey. The humanized HIRMAb was radiolabeled with 125-iodine, and injected intravenously into an adult, anesthetized Rhesus monkey. Brain scanning showed the humanized HIRMAb was rapidly transported into all parts of the primate brain after intravenous administration. The humanized HIRMAb may be used as a brain drug and gene delivery system for the targeting of large molecule therapeutics across the BBB in humans.

Developing Therapeutic Antibodies for Neurodegenerative Disease

“…Like TfR, antibodies to InsR have also been generated for large molecule delivery across the BBB. Among the most studied is the mouse monoclonal 83-14 antibody against the human InsR, where trace doses of radiolabeled antibody showed an increase in brain uptake, but also a fast clearance, suggesting that this antibody has high affinity for InsR [125,126]. Nevertheless, 83-14 has been characterized in vivo using a number of fusion proteins, including Aβ 40 for amyloid imaging [127], single-chain anti-Aβ [128,129], tissue-specific gene targeting, and glial cell-derived neurotrophic factor [130].…”

Biologic TNFα-inhibitors that cross the human blood-brain barrier

“…The most potent Trojan horse for the human brain is a genetically engineered MAb against the human insulin receptor (HIR). 11 The engineering of the chimeric or humanized HIRMAb enables the subsequent genetic engineering of IgG fusion proteins, wherein the biologic drug, which is normally not transported across the BBB, is fused to the HIRMAb. Figure 1 illustrates the structure of two classes of HIRMAb fusion proteins that have been re-engineered to cross the human BBB.…”

Designing the future of nanomedicine: current barriers to targeted brain therapeutics

“…Essential to this is the species-specificity of currently studied antibodies: a rat mAb, OX26, to the mouse TfR is not functional as a Trojan horse for therapeutic delivery in a mouse model, while two engineered rat monoclonal antibodies, 8D3 and RI7-217, to the mouse TfR succeeded in transcytosing the BBB (76). A murine mAb to the human insulin receptor (HIR) can be tested in rats, but not in humans due to immunological response to a mouse antibody (77). For these approaches to be applicable in human studies, chimeric and humanized antibodies should be engineered and evaluated.…”