Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor.
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.
Boado
Ruben J
RJ
ArmaGen Technologies, Inc., Santa Monica, California 90401, USA.
Zhou
Qing-Hui
QH
Lu
Jeff Zhiqiang
JZ
Hui
Eric Ka-Wai
EK
Pardridge
William M
WM
D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000493:pharmacokinetics*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000493:pharmacokinetics*; D001812:Blood-Brain Barrier; D001921:Brain / Q000276:immunology* / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering; D011869:Radioligand Assay; D051381:Rats; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics; D014162:Transfection
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv