Tumor necrosis factor receptor-IgG fusion protein for targeted drug delivery across the human blood-brain barrier.
The tumor necrosis factor-alpha receptor (TNFR) extracellular domain (ECD) is a decoy receptor that could be developed as a neurotherapeutic for stroke, brain injury, or chronic neurodegeneration. However, the TNFR ECD is a large molecule therapeutic that does not cross the blood-brain barrier (BBB). Human TNFR ECD was re-engineered by fusion of the receptor protein to the carboxyl terminus of the chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR). The HIRMAb-TNFR fusion protein is bifunctional, and binds both the HIR, to trigger receptor-mediated transport across the BBB, and TNFalpha, to sequester this cytotoxic cytokine. COS cells were dual transfected with the heavy chain (HC) and light chain fusion protein expression plasmids, and the HC of the fusion protein was immunoreactive with antibodies to both human IgG and TNFR. The HIRMAb-TNFR fusion protein bound to the extracellular domain of the HIR with an affinity comparable to the HIRMAb, and bound TNFalpha with a K(D) of 0.34 +/- 0.17 nM. Both the TNFR:Fc fusion protein and the HIRMAb-TNFR fusion protein blocked the cytotoxic actions of TNFalpha on human cells in a bioassay. In conclusion, these studies describe the re-engineering of the TNFR ECD to make this decoy receptor transportable across the human BBB.
Hui
Eric Ka-Wai
EK
ArmaGen Technologies, Inc., Santa Monica, California 90401, USA.
Boado
Ruben J
RJ
Pardridge
William M
WM
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000502:physiology; D001927:Brain Diseases / Q000628:therapy; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D016503:Drug Delivery Systems; D006801:Humans; D007142:Immunoglobulin gamma-Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D066298:In Vitro Techniques; D007700:Kinetics; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017931:DNA Primers; D007142:Immunoglobulin gamma-Chains; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C099374:Ro 45-2081; D011972:Receptor, Insulin