Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Mar 1;102(4):1251-8.
doi: 10.1002/bit.22135.

Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse

Ruben J Boado  1 , Yun Zhang, Yuntao Wang, William M Pardridge
Affiliations

Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse

Ruben J Boado et al. Biotechnol Bioeng. .

Abstract

Protein therapeutics may be delivered across the blood-brain barrier (BBB) by genetic fusion to a BBB molecular Trojan horse. The latter is an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) against a BBB receptor, such as the insulin receptor or the transferrin receptor (TfR). Fusion proteins have been engineered with the MAb against the human insulin receptor (HIR). However, the HIRMAb is not active against the rodent insulin receptor, and cannot be used for drug delivery across the mouse BBB. The rat 8D3 MAb against the mouse TfR is active as a drug delivery system in the mouse, and the present studies describe the cloning and sequencing of the variable region of the heavy chain (VH) and light chain (VL) of the rat 8D3 TfRMAb. The VH and VL were fused to the constant region of mouse IgG1 heavy chain and mouse kappa light chain, respectively, to produce a new chimeric TfRMAb. The chimeric TfRMAb was expressed in COS cells following dual transfection with the heavy and light chain expression plasmids, and was purified by protein G affinity chromatography. The affinity of the chimeric TfRMAb for the murine TfR was equal to the 8D3 MAb using a radio-receptor assay and mouse fibroblasts. The chimeric TfRMAb was radio-labeled and injected into mice for a pharmacokinetics study of the clearance of the chimeric TfRMAb. The chimeric TfRMAb was rapidly taken up by mouse brain in vivo at a level comparable to the rat 8D3 MAb. In summary, these studies describe the genetic engineering, expression, and validation of a chimeric TfRMAb with high activity for the mouse TfR, which can be used in future engineering of therapeutic fusion proteins for BBB drug delivery in the mouse.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Agarose gel electrophoresis and ethidium bromide staining of PCR cloning of 0.4 kb TfRMAb VH (A), 0.4 kb TfRMAb VL (B), 1.4 kb mouse IgG1 C-region (C), and 0.7 kb mouse kappa C-region (D). The PCR generated cDNA is shown in lane 1, and DNA size standards are shown in lanes 2 and 3 for each panel.
Figure 2
Figure 2
Genetic engineering of the eukaryotic heavy chain (HC) expression plasmid, pCD-HC and the light chain (LC) expression plasmid, pCD-LC, is shown in Panels A and B, respectively. The variable region of the HC (VH) of the chimeric TfRMAb is fused to the C-region of mouse IgG1 (mIgG1) in pCD-HC, and the variable region of the LC (VL) of the chimeric TfRMAb is fused to the C-region of mouse kappa (mKappa) in pCD-LC.
Figure 3
Figure 3
Deduced amino acid sequence of the chimeric TfRMAb heavy chain (A) and light chain (B). The individual complementarity determining regions (CDR) and framework regions (FR) of the VH and VL are shown. The HC C-region is comprised of four sub-domains: CH1, hinge, CH2, and CH3. The LC C-region is denoted as CL.
Figure 4
Figure 4
Western blot shows identical reactivity with an anti-mouse antibody of the chimeric TfRMAb (lane 1) and the 8D3 rat hybridoma-generated TfRMAb (lane 2).
Figure 5
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 or the chimeric TfRMAb. The KD of 8D3 self-inhibition and the KI of chimeric TfRMAb cross-inhibition were computed by nonlinear regression analysis (see Materials and Methods Section).
Figure 6
Figure 6
A: Plasma concentration of [125I]-cTfRMAb in the mouse is expressed as % of injected dose (ID)/mL, and is plotted versus time after a single intravenous injection in the anesthetized mouse. B: Plasma radioactivity that is precipitable by trichloroacetic acid (TCA) is plotted versus time after intravenous injection. Data are mean ± SE (n = 3 mice per time point). Some error bars are too small to be visible.
Figure 7
Figure 7
The organ volume of distribution (VD) in the mouse at 60 min after intravenous injection is shown for brain, heart, liver, and kidney for the [125I]-cTfRMAb (open bars), the [125I]-OX26 TfRMAb (solid bars), and the [125I]-8D3 TfRMAb (gray bars). The VD data for the [125I]-OX26 TfRMAb and the [125I]-8D3 TfRMAb are from Lee et al. (2000). Data are mean ± SE (n = 3 mice per time point). Some error bars are too small to be visible.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Boado RJ, Zhang Y, Zhang Y, Pardridge WM. Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier. Biotechnol Bioeng. 2007a;97:1376–1386. - PubMed
    1. Boado RJ, Zhang Y, Zhang Y, Xia CF, Pardridge WM. Fusion antibody for Alzheimer's disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation. Bioconjug Chem. 2007b;18:447–455. - PMC - PubMed
    1. Boado RJ, Zhang Y, Zhang Y, Wang Y, Pardridge WM. GDNF fusion protein for targeted-drug delivery across the human blood-brain barrier. Biotechnol Bioeng. 2008a;100:387–986. - PubMed
    1. Boado RJ, Zhang Y, Zhang Y, Xia CF, Wang Y, Pardridge WM. Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier. Biotechnol Bioeng. 2008b;99:475–484. - PubMed
    1. Boado RJ, Zhang Y, Zhang Y, Xia CF, Wang Y, Pardridge WM. Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans. Bioconjug Chem. 2008c;19:731–739. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources