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) nonmutant 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 nonmutant 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 area under the plasma concentration–time curve (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 AUC and is negated at high doses of the N292G mutant

Pharmacokinetics and Brain Uptake of an IgG-TNF Decoy Receptor Fusion Protein Following Intravenous, Intraperitoneal, and Subcutaneous Administration in Mice

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine active in the brain. Etanercept, the TNF decoy receptor (TNFR), does not cross the blood-brain barrier (BBB). The TNFR was re-engineered for BBB penetration as a fusion protein with a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse and mediates transport via the endogenous BBB TfR. To support future chronic treatment of mouse models of neural disease with daily administration of the cTfRMAb-TNFR fusion protein, a series of pharmacokinetics and brain uptake studies in the mouse was performed. The cTfRMAb-TNFR fusion protein was radiolabeled and injected into mice via the intravenous, intraperitoneal (IP), or subcutaneous (SQ) routes of administration at doses ranging from 0.35 to 10 mg/kg. The distribution of the fusion protein into plasma following the IP or SQ routes was enhanced by increasing the injection dose from to 3–10 mg/kg. The fusion protein demonstrated long circulation times with high metabolic stability following the IP or SQ routes of injection. The IP or SQ routes produced concentrations of the cTfRMAb-TNFR fusion protein in brain that exceed by 20- to 50-fold the concentration of TNFα in pathologic conditions of the brain. The SQ injection is the preferred route of administration, as the level of cTfRMAb fusion protein produced in brain is comparable to that generated with intravenous injection, and at a much lower plasma area under the concentration curve of the fusion protein as compared to IP administration