Blood-Brain Barrier Transport, Plasma Pharmacokinetics, and Neuropathology Following Chronic Treatment of the Rhesus Monkey with a Brain Penetrating Humanized Monoclonal Antibody Against the Human Transferrin Receptor

Abstract

A monoclonal antibody (mAb) against the blood-brain barrier (BBB) transferrin receptor (TfR) is a potential agent for delivery of biologic drugs to the brain across the BBB. However, to date, no TfRMAb has been tested with chronic dosing in a primate model. A humanized TfRMAb against the human (h) TfR1, which cross reacts with the primate TfR, was genetically engineered with high affinity (ED50 = 0.18 ± 0.04 nM) for the human TfR type 1 (TfR1). For acute dosing, the hTfRMAb was tritiated and injected intravenously (IV) in the Rhesus monkey, which confirmed rapid delivery of the humanized hTfRMAb into both brain parenchyma, via transport across the BBB, and into cerebrospinal fluid (CSF), via transport across the choroid plexus. For chronic dosing, a total of 8 adult Rhesus monkeys (4 males, 4 females) were treated twice weekly for 4 weeks with 0, 3, 10, or 30 mg/kg of the humanized hTfRMAb via a 60 min IV infusion for a total of 8 doses prior to euthanasia and microscopic examination of brain and peripheral organs. A pharmacokinetics analysis showed the plasma clearance of the hTfRMAb in the primate was nonlinear, and plasma clearance was increased over 20-fold with chronic treatment of the low dose, 3 mg/kg, of the antibody. Chronic treatment of the primates with the 30 mg/kg dose caused anemia associated with suppressed blood reticulocytes. Immunohistochemistry of terminal brain tissue showed microglia activation, based on enhanced IBA1 immuno-staining, in conjunction with astrogliosis, based on increased GFAP immuno-staining. Moderate axonal/myelin degeneration was observed in the sciatic nerve. Further studies need to be conducted to determine if this neuropathology is induced by the antibody effector function, or is an intrinsic property of targeting the TfR in brain. The results indicate that chronic treatment of Rhesus monkeys with a humanized hTfRMAb may have a narrow therapeutic index, with associated toxicity related to microglial activation and astrogliosis of the brain.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human

“…TfR antibodies with various characteristics have been developed for delivery of biotherapeutic cargos across the BBB, notably TfR/BACE1 bispeci c antibody [7] and an anti-Aβ antibody fused to a single TfR-binding Fab fragment [37]. TfR is highly expressed in reticulocytes causing on-target toxicity of effector-function competent antibodies [38]; high-a nity TfR antibodies also exhibit poor pharmacokinetics due to peripheral target-mediated clearance [42,53]. Due to lack of species cross-reactivity of TfR antibodies, the development of transgenic mouse expressing human TfR extracellular domain [7,12,13,37], use of surrogate antibody or of transgenic mouse expressing human TfR [14,15,37] were necessary in pre-clinical studies.…”

Blood-brain barrier transport using a high-affinity, brain-selective VNAR (Variable Domain of New Antigen Receptor) antibody targeting transferrin receptor 1

“…Reducing receptor binding affinity or monovalent formatting of these antibodies restored intracellular trafficking and maintained TfR1 levels in the CNS. Furthermore, exposure to TfR1 antibodies with full effector function caused severe anemia in mice due to the hemolysis [4] and immune-mediated CNS toxicity in addition to anemia in primates [5]. However, it was previously shown that direct antibodymedicated cytotoxicity was related to effector function, which can be attenuated by engineering the antibody Fc region [4,18,41].…”

“…Despite clear advances, several features of monoclonal anti-TfR1 antibodies as BBB carriers have hampered their clinical development. Anti-TfR1 antibodies can cause anemia by target-mediated lysis of TfR1-rich reticulocytes [4,5]. Off-target binding to TfR1 expressed in other peripheral tissues such as liver and kidney, likely accounts for the relatively short plasma half-life of some antibodies targeting TfR1 [4,6,7].…”

A differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human

“…The protein expression of RMT receptors IGF1R, LRP1 and TfR, was analyzed by Western blot and To facilitate translational studies of BBB RMT carriers (shuttles) developed against various RMT receptors described in the literature (Table 1) high-affinity TfR antibodies also exhibit poor pharmacokinetics due to peripheral target-mediated clearance [52]. Due to lack of species cross-reactivity of TfR antibodies the development of transgenic mouse expressing human TfR extracellular domain [7,36], or use of surrogate antibody were necessary in pre-clinical studies.…”