Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor

Abstract

Biologic tumor necrosis factor (TNF)-α inhibitors do not cross the blood-brain barrier (BBB). A BBB-penetrating TNF-α inhibitor was engineered by fusion of the extracellular domain of the type II human TNF receptor (TNFR) to the carboxyl terminus of the heavy chain of a mouse/rat chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb-TNFR fusion protein and etanercept bound human TNF-α with high affinity and K(D) values of 374 ± 77 and 280 ± 80 pM, respectively. Neuroprotection in brain in vivo after intravenous administration of the fusion protein was examined in a mouse model of Parkinson's disease. Mice were also treated with saline or a non-BBB-penetrating TNF decoy receptor, etanercept. After intracerebral injection of the nigral-striatal toxin, 6-hydroxydopamine, mice were treated every other day for 3 weeks. Treatment with the cTfRMAb-TNFR fusion protein caused an 83% decrease in apomorphine-induced rotation, a 67% decrease in amphetamine-induced rotation, a 82% increase in vibrissae-elicited forelimb placing, and a 130% increase in striatal tyrosine hydroxylase (TH) enzyme activity. In contrast, chronic treatment with etanercept, which does not cross the BBB, had no effect on neurobehavior or striatal TH enzyme activity. A bridging enzyme-linked immunosorbent assay specific for the cTfRMAb-TNFR fusion protein showed that the immune response generated in the mice was low titer. In conclusion, a biologic TNF inhibitor is neuroprotective after intravenous administration in a mouse model of neurodegeneration, providing that the TNF decoy receptor is reengineered to cross the BBB.

Figures

Fig. 1.

A, the cTfRMAb-TNFR fusion protein is formed by fusion of the ECD of the type II human TNFR to the carboxyl terminus of each heavy chain of the cTfRMAb. B, the etanercept fusion protein is formed by fusion of the same ECD of the type II human TNFR to the amino terminus of the Fc fragment of human IgG1 heavy chain.

Fig. 2.

Radioreceptor assay shows saturation of binding of human TNF-α to either the cTfRMAb-TNFR fusion protein or to etanercept. The binding dissociation constant, KD, was determined by nonlinear regression analysis. The horizontal line at 1.5% binding represents the nonspecific binding observed when either human IgG1 or mouse IgG1 was plated in lieu of the fusion protein.

Fig. 3.

Contralateral rotation (over 20 min) after the administration of apomorphine to PD mice treated with saline, etanercept, or the cTfRMAb-TNFR fusion protein at 1, 2, and 3 weeks after toxin injection. Data are means ± S.E. (n = 10 mice/group). Statistical differences from the saline-treated animals: *, p < 0.01 at weeks 1, 2, and 3, as determined by ANOVA with Bonferroni correction.

Fig. 4.

Ipsilateral rotation (over 20 min) after the administration of amphetamine to PD mice treated with saline, etanercept, or the cTfRMAb-TNFR fusion protein at 1, 2, and 3 weeks after toxin injection. Data are means ± S.E. (n = 10 mice/group). Statistical differences from the saline-treated animals: *, p < 0.01; ‡, p < 0.05, at weeks 1, 2, and 3, as determined by ANOVA with Bonferroni correction.

Fig. 5.

Vibrissae-elicited forelimb placing test scores for the right side, which is ipsilateral to the toxin lesion, and for the left side, which is contralateral to the toxin lesion, for the saline-, etanercept-, and the cTfRMAb-TNFR fusion protein-treated mice. All scores were measured at 3 weeks after toxin injection. Data are means ± S.E. (n = 10 mice/group). Statistical differences from the saline-treated animals: *, p < 0.01, as determined by ANOVA with Bonferroni correction.

Fig. 6.

TH enzyme activity on the lesioned side (right) and the nonlesioned side (left) in the striatum and in the frontal cortex of mice treated with saline, etanercept, or the cTfRMAb-TNFR fusion protein. Brain TH activity was measured at 3 weeks after toxin administration. Data are means ± S.E. (n = 10 mice/group). Statistical differences from the saline-treated animals in the right striatum: *, p < 0.01, as determined by ANOVA with Bonferroni correction.

Fig. 7.

TH immunocytochemistry for three mice treated with the cTfRMAb-TNFR fusion protein (A–C) and three mice treated with saline (D–F). The lesioned side of the brain corresponds to the right side of the figure.

Fig. 8.

A, structure of the two-site ELISA for detection of antibodies against the cTfRMAb-TNFR fusion protein. The cTfRMAb-TNFR fusion protein is used as the capture reagent, and the biotinylated cTfRMAb-TNFR 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, Western blot of nonbiotinylated cTfRMAb-TNFR fusion protein (lane 1) and biotinylated cTfRMAb-TNFR fusion protein (lane 2) with a conjugate of avidin and biotinylated peroxidase. C, absorbance at 1:50 dilutions of mouse plasma taken preinjection or postinjection after 3 weeks of either intravenous injections with either etanercept or the cTfRMAb-TNFR fusion protein. Data are shown for all 10 mice in each group.