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. 2010 Apr;38(4):566-72.
doi: 10.1124/dmd.109.031534. Epub 2010 Jan 14.

Monoclonal antibody-glial-derived neurotrophic factor fusion protein penetrates the blood-brain barrier in the mouse

Qing-Hui Zhou  1 , Ruben J Boado, Jeff Zhiqiang Lu, Eric Ka-Wai Hui, William M Pardridge
Affiliations

Monoclonal antibody-glial-derived neurotrophic factor fusion protein penetrates the blood-brain barrier in the mouse

Qing-Hui Zhou et al. Drug Metab Dispos. 2010 Apr.

Abstract

Glial-derived neurotrophic factor (GDNF) is a potent neuroprotective agent for multiple brain disorders, including Parkinson's disease. However, GDNF drug development is difficult because GDNF does not cross the blood-brain barrier (BBB). To enable future drug development of GDNF in mouse models, the neurotrophin was re-engineered as an IgG fusion protein to enable penetration through the BBB after intravenous administration. The 134-amino acid GDNF was fused to the heavy chain of a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR) designated the cTfRMAb. This antibody undergoes receptor-mediated transport across the BBB and acts as a molecular Trojan horse to ferry the GDNF into mouse brain. The cTfRMAb-GDNF fusion protein was expressed by stably transfected Chinese hamster ovary cells, affinity-purified, and the biochemical identity was confirmed by mouse IgG and GDNF Western blotting. The cTfRMAb-GDNF fusion protein was bifunctional and bound with high affinity to both the GDNF receptor alpha1, ED(50) = 1.7 +/- 0.2 nM, and the mouse TfR, ED(50) = 3.2 +/- 0.3 nM. The cTfRMAb-GDNF fusion protein was rapidly taken up by brain, and the brain uptake was 3.1 +/- 0.2% injected dose/g brain at 60 min after intravenous injection of a 1-mg/kg dose of the fusion protein. Brain capillary depletion analysis showed the majority of the fusion protein was transcytosed across the BBB with penetration into brain parenchyma. The brain uptake results indicate it is possible to achieve therapeutic elevations of GDNF in mouse brain with intravenous administration of the cTfRMAb-GDNF fusion protein.

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Figures

Fig. 1.
Fig. 1.
The cTfRMAb-GDNF fusion protein is composed of two HCs and two LCs. The HC is formed by fusion of the VH of the rat 8D3 MAb against the mouse TfR (mTfR) to the amino terminus of mouse IgG1 constant (C) region and fusion of human GDNF to the carboxyl terminus of the HC C-region. The LC is formed by fusion of the VL of the rat 8D3 MAb to the mouse κ LC C-region (CL). The HC C-region is composed of four domains: CH1, hinge, CH2, and CH3.
Fig. 2.
Fig. 2.
The TV expressing the cTfRMAb-GDNF fusion protein is composed of a single strand of DNA, which includes separate expression cassettes for the LC of the cTfRMAb, the HC gene formed by fusion of the cDNA encoding the GDNF to the 3′-end of the cDNA encoding the HC of the cTfRMAb, and murine DHFR, to allow for amplification of cell lines with methotrexate treatment. The HC and LC genes are 5′-flanked by the cytomegalovirus (CMV) promoter and 3′-flanked by the bovine growth hormone (BGH) polyA sequence; the DHFR gene is 5′-flanked by the SV40 promoter and 3′-flanked by the hepatitis B virus (HBV) polyA sequence. The plasmid also contains genes for neomycin (neo) resistance and ampicillin resistance (ampR).
Fig. 3.
Fig. 3.
Reducing (A) and nonreducing (B) SDS-PAGE of molecular mass standards (lane 1) and the cTfRMAb-GDNF fusion protein (lane 2). The HC doublet reflects differential glycosylation, which is also seen on Western blotting (Fig. 4).
Fig. 4.
Fig. 4.
Western blotting with a primary antibody against mouse IgG (A) or against GDNF (B). The anti-mouse antibody reacts with the HC and LC of both the cTfRMAb and the cTfRMAb-GDNF fusion protein but does not react with GDNF. The anti-GDNF antibody reacts only with the HC of the cTfRMAb-GDNF fusion protein and with GDNF but does not react with the HC or LC of the cTfRMAb.
Fig. 5.
Fig. 5.
Radio-receptor assay of the mouse TfR uses mouse fibroblasts as the source of the mouse TfR and 125I-8D3 as the binding ligand. Binding is displaced by unlabeled 8D3 MAb (A) or the cTfRMAb-GDNF fusion protein (B). The KD of 8D3 self-inhibition and the KI of cTfRMAb-GDNF cross-inhibition were computed by nonlinear regression analysis.
Fig. 6.
Fig. 6.
Binding of the cTfRMAb-GDNF fusion protein to the GFRα1 extracellular domain is saturable. The ED50 of cTfRMAb-GDNF fusion protein binding was determined by nonlinear regression analysis. There is no binding to the GFRα1 by mouse IgG1 (mIgG1).
Fig. 7.
Fig. 7.
The cTfRMAb-GDNF fusion protein activates luciferase gene expression in cultured SK-N-MC neural cells, which are cotransfected with the c-ret kinase and the luciferase gene under the influence of the TH promoter. Data are mean ± S.E. (n = 4 wells/point). The Amax and ED50 were determined by nonlinear regression analysis.
Fig. 8.
Fig. 8.
Plasma concentration, expressed as percentage of ID/ml, of the [3H]cTfRMAb-GDNF fusion protein after intravenous or intraperitoneal injection in the mouse. Data are mean ± S.E. (n = 3 mice/point).
Fig. 9.
Fig. 9.
A, brain uptake, expressed as percentage of ID/g brain, for the cTfRMAb-GDNF fusion protein, compared with the mouse brain uptake for the OX26 MAb against the mouse TfR, which is a brain blood volume marker in the mouse (Lee et al., 2000). B, VD of the cTfRMAb-GDNF fusion protein in the brain homogenate (H), the postvascular supernatant (S), and the vascular pellet (P). Data are mean ± S.E. (n = 3 mice).
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