Eliminating Fc N-linked glycosylation and its impact on dosing consideration for a transferrin receptor antibody-erythropoietin fusion protein in mice.

Erythropoietin (EPO), a hematopoietic growth factor and a promising therapy for Alzheimer's disease has low permeability across the blood-brain barrier. The transferrin receptor antibody fused to EPO (TfRMAb-EPO) is a chimeric monoclonal antibody that ferries EPO into the brain via the transvascular route. However, TfRMAbs have Fc-effector function-related adverse effects including reticulocyte suppression. To overcome this, we recently developed an effectorless TfRMAb-EPO fusion protein, designated TfRMAb-N292G-EPO, by eliminating the Fc N-linked glycosylation site at position 292 of the antibody heavy chain. The mutant fusion protein showed enhanced plasma clearance and dramatically reduced plasma concentrations compared with the wild-type (WT) non-mutant fusion protein. This increased clearance of the aglycosylated TfRMAb is expected to increase the injection dose of the mutant fusion protein. To provide a basis for future therapeutic uses of this IgG-neurotrophin fusion protein, the current study aimed to characterize the pharmacokinetic (PK) profile of this effectorless TfRMAb-N292G-EPO at different doses following different routes of administration in the mouse. Adult C57BL/6J male mice were injected with a single dose (3, 6, 9, or 20 mg/kg; n=3-6 per dose) of TfRMAb-N292G-EPO through either the subcutaneous (SQ) or intraperitoneal (IP) route. TfRMAb-N292G-EPO plasma concentrations were determined using an ELISA. Mice were sacrificed 24 hours after injection, and terminal blood was used for a complete blood count. Brain concentrations in the WT and mutant fusion protein treated mice were compared. We observed stark differences in the plasma PK of TfRMAb-N292G-EPO between the IP and SQ routes of administration. Dose escalation from 3-20 mg/kg increased the plasma Cmax only 3.5-fold for the SQ route, compared with a 35-fold increase for the IP route. The plasma Cmax was 15.0 ± 2.0 ng/mL, 21.3 ± 4.1 ng/mL, 21.3 ± 6.4 ng/mL and 52.8 ± 27.9 ng/mL following SQ injection, and 288 ± 47 ng/mL, 389 ± 154 ng/mL, 633 ± 194 ng/mL and 10,066 ± 7,059 ng/mL following IP injection for 3, 6, 9 and 20 mg/kg dose, respectively. Plasma Cmax following the SQ route was therefore 19- to 190-fold lower compared with the IP route. This finding is consistent with a 31-fold higher apparent clearance following the SQ route compared with the IP route at the highest dose administered. Brain concentrations in the mice treated with a 3 mg/kg dose of the mutant fusion protein were lower than those in the WT treated mice. No reticulocyte suppression was observed at the 3 mg/kg SQ dose of TfRMAb-N292G-EPO. However, reticulocyte suppression increased with an increase in dose and plasma AUC for both the IP and SQ routes. Overall, elimination of Fc N-linked glycosylation, to mitigate TfRMAb effector function side-effects, has a profound effect on the plasma exposure of TfRMAb-N292G-EPO at therapeutic as well as high doses (3-20 mg/kg). This effect is more pronounced following SQ injection. The low plasma concentrations of the mutant fusion protein following a 3 mg/kg dose resulted in negligible brain uptake. The beneficial rescue of reticulocyte reduction by the N292G mutation is a function of plasma AUC and is negated at high doses of the N292G mutant.