Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor

Abstract

Monoclonal antibodies (MAbs) are potential new therapeutics for brain diseases. However, MAbs do not cross the blood-brain barrier (BBB). The present work describes the genetic engineering of a fusion protein composed of a therapeutic single chain Fv (ScFv) antibody and a mouse/rat chimeric MAb against the mouse transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the therapeutic ScFv across the BBB in vivo in the mouse. The ScFv is fused to the carboxyl terminus of the heavy chain of the chimeric TfRMAb, and this fusion protein is designated cTfRMAb-ScFv. Chinese hamster ovary cells were permanently transfected, and a high secreting cell line in serum free medium was cloned. The cTfRMAb-ScFv fusion protein was purified to homogeneity on gels and Western blotting with protein G affinity chromatography. The cTfRMAb-ScFv fusion protein was bifunctional and bound both the target antigen, as determined by ELISA, and the mouse TfR, as determined with a radio-receptor assay. The cTfRMAb-ScFv fusion protein was radio-iodinated with the Bolton-Hunter reagent, and a pharmacokinetics study in mice showed that the fusion protein was rapidly cleared from blood with a median residence time of 175 +/- 32 min. The fusion protein was avidly taken up by brain with a % injected dose (ID)/g of 3.5 +/- 0.7, as compared to an MAb with no receptor specificity, which was 0.06 +/- 0.01% ID/g. These studies demonstrate that therapeutic MAbs may be re-engineered as fusion proteins with BBB molecular Trojan horses for targeted delivery across the BBB in vivo.

Figure 1

The cTfRMAb-ScFv fusion protein is formed by fusion of the variable region of the heavy chain (VH) of the rat 8D3 MAb against the mouse transferrin receptor (mTfR) (yellow) to the amino terminus of mouse IgG1 constant-region (green), and fusion of a single chain Fv (ScFv) antibody against the Aβ amyloid peptide to the carboxyl terminus of the heavy chain C-region. The light chain is comprised of the variable region of the light chain (VL) of the rat 8D3 MAb (light blue) and the mouse kappa light chain C-region (CL) (dark red). The heavy chain constant-region is comprised of 4 domains: CH1, hinge, CH2, and CH3. The CH2–CH3 interface is the binding site for the neonatal Fc receptor (FcRn). The ScFv is comprised of the VH (dark blue) and the VL (light red) derived from the anti-Aβ MAb.

Figure 2

The tandem vector expressing the cTfRMAb-ScFv fusion protein is comprised of separate expression cassettes on a single strand for the LC of the cTfRMAb, the HC gene formed by fusion of the cDNA encoding the ScFv to the 3’ end of the cDNA encoding the HC of the cTfRMAb, and murine dihydrofolate reductase (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, and the DHFR gene is 5’-flanked by the SV40 promoter and the hepatitis B virus (HBV) polyA sequence. The plasmid also contains genes for neomycin (neo) resistance and ampicillin resistance (ampR).

Figure 3

Reducing SDS-PAGE of molecular weight standards (lane 1) and the cTfRMAb-ScFv fusion protein (lane 2). The size of the heavy chain is about 85 kDa and the size of the light chain is about 26 kDa.

Figure 4

Western blotting with a primary antibody against mouse IgG and biotinylated molecular weight standards (lane 1), the cTfRMAb (lane 2), and the cTfRMAb-ScFv fusion protein (lane 3). Both the cTfRMAb and the cTfRMAb-ScFv fusion protein share the same light chain. The size of the heavy chain of the cTfRMAb-ScFv fusion protein is about 28 kDa larger than the size heavy chain of the cTfRMAb owing to fusion of the ScFv.

Figure 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-ScFv fusion protein at concentrations up to 30 nM (B). The KD of 8D3 self-inhibition and the KI of chimeric TfRMAb cross-inhibition were computed by non-linear regression analysis.

Figure 6

(A) The ELISA used to measure cTfRMAb-ScFv binding to the Aβ^1–40 employs a complex of streptavidin (SA) and N-biotinyl Aβ^1–40 as the capture reagent and a goat anti-mouse (GAM) conjugate of alkaline phosphatase (AP) as the detector reagent. (B) Binding of the cTfRMAb-ScFv fusion protein to the Aβ^1–40 is linear, whereas there is no binding to the Aβ^1–40 of mouse (m) IgG1κ.

Figure 7

Plasma concentration, expressed as % of injected dose (I.D.)/mL, of the [^125I]-cTfRMAb-ScFv fusion protein after either intravenous or intra-peritoneal injection in the mouse. Data are mean ± S.E. (n=3 mice/point).

Figure 8

Brain uptake, expressed as % of injected dose (ID)/gram brain, for the cTfRMAb-ScFv fusion protein, in comparison with the brain uptake values for the rat 8D3 MAb against the mouse TfR, the mouse OX26 MAb against the rat TfR, and the cTfRMAb reported previously,. Data are mean ± S.E. (n=3 mice).