Binding specificity and internalization properties of an antibody-avidin fusion protein targeting the human transferrin receptor.
The human transferrin receptor (hTfR1) is a membrane-bound protein involved in transferrin (Tf)-mediated iron uptake and is highly expressed on malignant cells. A second version of the receptor (hTfR2) also mediates Tf-dependent iron import. We previously developed a protein composed of avidin fused to a mouse/human chimeric IgG3 specific for hTfR (anti-hTfR IgG3-Av) that was originally designed to deliver biotinylated drugs into cancer cells. We have now found that anti-hTfR IgG3-Av does not cross-react with hTfR2 and binds hTfR1 expressed on the surface of cells, attached to a solid surface, and in solution. We also found that the hemochromatosis protein (HFE), another ligand of the TfR, does not inhibit the binding of anti-hTfR IgG3-Av to the receptor. In addition, using live cell laser scanning confocal microscopy (LCLSCM) we demonstrated that anti-hTfR IgG3-Av and anti-hTfR IgG3 are internalized into cells expressing hTfR1 at a similar rate. Furthermore, our proliferation and morphological studies demonstrated the effective cytotoxicity of a biotinylated toxin delivered by anti-hTfR IgG3-Av only into cells expressing hTfR1. Our results better define the properties of anti-hTfR IgG3-Av and pave the way for the rational design of future in vitro and in vivo studies for the treatment of human malignancies.
Rodríguez
José A
JA
Division of Surgical Oncology, Department of Surgery, University of California, Los Angeles, CA 90095-1782, USA.
Helguera
Gustavo
G
Daniels
Tracy R
TR
Neacato
Isabel I
II
López-Valdés
Héctor E
HE
Charles
Andrew C
AC
Penichet
Manuel L
ML
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D015703:Antigens, CD / Q000096:biosynthesis / Q000276:immunology; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D001360:Avidin / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D019921:Biotinylation; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D008024:Ligands; D051379:Mice; D018613:Microscopy, Confocal; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000970:Antineoplastic Agents; C068126:CD71 antigen; D018796:Immunoconjugates; D007074:Immunoglobulin G; D008024:Ligands; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D001360:Avidin; D007501:Iron