Selective targeting of a TNFR decoy receptor pharmaceutical to the primate brain as a receptor-specific IgG fusion protein

Abstract

Decoy receptors, such as the human tumor necrosis factor receptor (TNFR), are potential new therapies for brain disorders. However, decoy receptors are large molecule drugs that are not transported across the blood-brain barrier (BBB). To enable BBB transport of a TNFR decoy receptor, the human TNFR-II extracellular domain was re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the TNFR therapeutic decoy receptor across the BBB. The HIRMAb-TNFR fusion protein was expressed in stably transfected CHO cells, and was analyzed with electrophoresis, Western blotting, size exclusion chromatography, and binding assays for the HIR and TNFalpha. The HIRMAb-TNFR fusion protein was radio-labeled by trititation, in parallel with the radio-iodination of recombinant TNFR:Fc fusion protein, and the proteins were co-injected in the adult Rhesus monkey. The TNFR:Fc fusion protein did not cross the primate BBB in vivo, but the uptake of the HIRMAb-TNFR fusion protein was high and 3% of the injected dose was taken up by the primate brain. The TNFR was selectively targeted to brain, relative to peripheral organs, following fusion to the HIRMAb. This study demonstrates that decoy receptors may be re-engineered as IgG fusion proteins with a BBB molecular Trojan horse that selectively targets the brain, and enables penetration of the BBB in vivo. IgG-decoy receptor fusion proteins represent a new class of human neurotherapeutics.

Figure 1

(A) SDS-PAGE under reducing conditions for the chimeric HIRMAb and the HIRMAb-TNFR fusion protein. (B) SDS-PAGE under reducing conditions for the TNFR:Fc fusion protein. Molecular weight standards are shown on the left and right sides of the gels.

Figure 2

Western blot with either a primary antibody against human IgG (hIgG) (left panel) or against the human TNFR (right panel). Immunoreactivity is shown for the HIRMAb or the HIRMAb-TNFR fusion protein. Molecular weight standards are shown on the right side.

Figure 3

Binding of either the HIRMAb or the HIRMAb-TNFR fusion protein to the HIR is saturable. The ED50 was determined by non-linear regression analysis. Data are mean ± SE (n=3 replicates per point). The molecular weights of the HIRMAb and the HIRMAb-TNFR fusion protein are 150 kDa and 210 kDa, respectively.

Figure 4

(A) A radio-receptor assay is used to quantitate the saturable binding of TNFα to the HIRMAb-TNFR fusion protein. A mouse anti-human (MAH) IgG1 Fc is plated, which binds the Fc region of the HIRMAb-TNFR fusion protein. The TNFR extracellular domain (ECD) region of the fusion protein binds the [¹²⁵I]-TNFα, which is displaced by unlabeled TNFα. (B) The saturable binding was analyzed by a non-linear regression analysis to yield the concentration, ED50, that yields 50% inhibition of TNFα binding to the HIRMAb-TNFR fusion protein.

Figure 5

TNFα causes cytotoxicity in actinomycin D-treated human WEHI-13VAR cells with an ED50 of about 10 pg/mL. In the presence of either 1 nM TNFR:Fc or 1 nM HIRMAb-TNFR, there is no cytotoxicity caused by the high concentrations of TNFα.

Figure 6

(A) The plasma concentration of [¹²⁵I]-TNFR:Fc fusion protein and [³H]-HIRMAb-TNFR fusion protein is plotted vs the time after a single intravenous injection of the proteins in the adult Rhesus monkey. Data are expressed as % injected dose (I.D.)/mL. (B) The % of plasma radioactivity that is precipitable by 10% trichloroacetic acid (TCA) is plotted vs. the time after injection for both proteins. Data are mean ± SE (n=3 replicates per point).

Figure 7

The plasma area under the concentration curve or AUC (A), the brain uptake or % injected dose (ID) per 100 gram brain (B), and the BBB permeability-surface area (PS) product (C), are plotted for the TNFR:Fc fusion protein, for the HIRMAb-TNFR fusion protein, and a brain plasma volume marker, human IgG1 (hIgG1). The IgG1 data are from Boado and Pardridge (2009a). All measurements were made at 2 hours after intravenous administration of the protein. Data are mean ± SE (n=3 replicates per point).

Figure 8

Ratio of the organ PS product for the HIRMAb-TNFR fusion protein, relative to the organ PS product for the TNFR:Fc fusion protein, is plotted for each organ. Data are mean ± SE (n=3 replicates per point). The ratio for brain is the mean of the values for frontal gray matter, frontal white matter, cerebellar gray matter, and cerebellar white matter, which varied between 22-37.