Pharmacokinetics and safety in rhesus monkeys of a monoclonal antibody-GDNF fusion protein for targeted blood-brain barrier delivery

Abstract

Purpose: Glial-derived neurotrophic factor (GDNF) is a potential therapy for stroke, Parkinson's disease, or drug addiction. However, GDNF does not cross the blood-brain barrier (BBB). GDNF is re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR), which acts as a molecular Trojan horse to deliver the GDNF across the BBB. The pharmacokinetics (PK), toxicology, and safety pharmacology of the HIRMAb-GDNF fusion protein were investigated in Rhesus monkeys.

Methods: The fusion protein was administered as an intravenous injection at doses up to 50 mg/kg over a 60 h period to 56 Rhesus monkeys. The plasma concentration of the HIRMAb-GDNF fusion protein was measured with a 2-site sandwich ELISA.

Results: No adverse events were observed in a 2-week terminal toxicology study, and no neuropathologic changes were observed. The PK analysis showed a linear relationship between plasma AUC and dose, a large systemic volume of distribution, as well as high clearance rates of 8-10 mL/kg/min.

Conclusions: A no-observable-adverse-effect level is established in the Rhesus monkey for the acute administration of the HIRMAb-GDNF fusion protein. The fusion protein targeting the insulin receptor has a PK profile similar to a classical small molecule.

Fig. 1

Plasma glucose in 4 treatment groups of Rhesus monkeys at the pre-test, and various times after the bolus IV injection of 0–10 mg/kg of the HIRMAb-GDNF fusion protein. Data are mean ± S.D. (n = 6–10 animals per group). There are no significant changes in plasma glucose among the treatment groups.

Fig. 2

(A) Structure of the 2-site ELISA used to measure the concentration of the HIRMAb-GDNF fusion protein. A mouse monoclonal antibody (mMAb) against the HIR is plated, which captures the HIR ECD derived from CHO cells. The HIRMAb part of the fusion protein binds the HIR ECD, and the GDNF part of the fusion protein is bound by a goat anti-GDNF antibody, which is bound by a conjugate of a rabbit anti-goat (RAG) antibody and alkaline phosphatase (AP). (B) Control Rhesus monkey plasma was spiked with 5 different doses of the HIRMAb-GDNF reference standard. There is a linear correlation between the observed concentration and the predicted concentration of fusion protein.

Fig. 3

Linear relationship between the plasma AUC and the dose of HIRMAb-GDNF fusion protein. Data are mean ± S.D. (n = 6–10 animals/group).

Fig. 4

(A) Structure of the 2-site ELISA for detection of antibodies against the HIRMAb-GDNF fusion protein. The HIRMAb or the HIRMAb-GDNF fusion protein is used as the capture reagent, and the biotinylated HIRMAb-GDNF fusion protein is used as the detector reagent, along with a complex of streptavidin (SA) and horseradish peroxidase (HRP); the biotin moiety is designated, ‘B’. (B) Absorbance at various dilutions of a rabbit antiserum prepared against the HIRMAb-GDNF fusion protein, and the respective pre-immune serum (triangles). Either the HIRMAb (squares) or the HIRMAb-GDNF fusion protein (diamonds) was used as the capture reagent. Data are means of duplicates that varied  < 10%. (C) Absorbance at 1:50 dilutions of Rhesus monkey plasma taken at day 13, following the administration of 0, 0.4, 2, or 10 mg/kg of the HIRMAb-GDNF fusion protein every 12 h for 5 consecutive doses over days 1–3 of the study. The total dose of fusion protein given over the 60-hr period was 0, 2, 10, and 50 mg/kg (Methods). A significant immune reaction is registered with an (A490–A655) signal > 0.3.