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. 2011 Jan;39(1):71-6.
doi: 10.1124/dmd.110.036012. Epub 2010 Sep 30.

Brain-penetrating tumor necrosis factor decoy receptor in the mouse

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

Brain-penetrating tumor necrosis factor decoy receptor in the mouse

Qing-Hui Zhou et al. Drug Metab Dispos. 2011 Jan.

Abstract

Biologic tumor necrosis factor inhibitors (TNFIs) include TNF decoy receptors (TNFRs). TNFα plays a pathologic role in both acute and chronic brain disease. However, biologic TNFIs cannot be developed as brain therapeutics because these large molecule drugs do not cross the blood-brain barrier (BBB). To enable penetration of the brain via receptor-mediated transport, the human TNFR type II was re-engineered as an IgG fusion protein, where the IgG part is a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb part of the fusion protein acts as a molecular Trojan horse to ferry the TNFR across the BBB via transport on the endogenous BBB TfR. cTfRMAb-TNFR was expressed by stably transfected Chinese hamster ovary cells and purified by affinity chromatography to homogeneity on electrophoretic gels. The fusion protein reacted with antibodies to both mouse IgG and the human TNFR and bound TNFα with high affinity (K(d) = 96 ± 34 pM). cTfRMAb-TNFR was rapidly transported into mouse brain in vivo after intravenous administration, and the brain uptake of the fusion protein was 2.8 ± 0.5% of injected dose per gram of brain, which is >45-fold higher than the brain uptake of an IgG that does not recognize the mouse TfR. This new IgG-TNFR fusion protein can be tested in mouse models of brain diseases in which TNFα plays a pathologic role.

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Figures

Fig. 1.
Fig. 1.
cTfRMAb-TNFR is comprised of two heavy chains and two light chains. The heavy chain is formed by fusion of the variable region of the heavy chain (VH) of the rat 8D3 MAb against the mouse transferrin receptor (mTfR) to the amino terminus of mouse IgG1 constant (C) region as well as fusion of human TNFR-II ECD to the carboxyl terminus of the heavy chain C region. The light chain is formed by fusion of the variable region of the light chain (VL) of the rat 8D3 MAb to the mouse κ light chain C region (CL). The heavy chain C region is comprised of four domains: CH1, hinge, CH2, and CH3.
Fig. 2.
Fig. 2.
Results of reducing (A) and nonreducing (B) SDS-PAGE of cTfRMAb (lanes 1) and cTfRMAb-TNFR (lanes 2).
Fig. 3.
Fig. 3.
Results of Western blotting with a primary antibody against mouse IgG (A) or human TNFR-II (B). A, the anti-mouse antibody reacts with the HC and LC of both cTfRMAb (lane 2) and cTfRMAb-TNFR (lane 3), but not with the TNFR-II ECD (lane 1). B, the anti-TNFR antibody reacts only with the HC of cTfRMAb-TNFR (lane 3), and the TNFR-II ECD (lane 1), but does not react with cTfRMAb (lane 2).
Fig. 4.
Fig. 4.
A, format of ELISA used to measure binding of cTfRMAb-TNFR to human TNFα. Human TNFα is the capture agent and a conjugate of a goat anti-mouse (GAM) IgG, and alkaline phosphatase (AP) is the detector reagent. B, cTfRMAb-TNFR binds to TNFα, whereas there is no binding to TNFα by mouse IgG1 (mIgG1).
Fig. 5.
Fig. 5.
A, outline of radioreceptor assay binding of TNFα to cTfRMAb-TNFR. A goat anti-mouse (GAM) IgG1 Fc was plated, which bound the Fc region of cTfRMAb-TNFR. The TNFR extracellular domain (ECD) region of the fusion protein then bound the 125I-TNFα, which was displaced by the addition of unlabeled TNFα. B, the saturable binding was analyzed by a nonlinear regression analysis to yield the concentration, Kd, that gave 50% inhibition of TNFα binding to cTfRMAb-TNFR.
Fig. 6.
Fig. 6.
Plasma concentration, expressed as percent ID per milliliter, of the [3H]-cTfRMAb-TNFR after intravenous or intraperitoneal injection in the mouse. Data are means ± S.E. (n = 4 mice/point).
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