Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 Å resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR

Phase I study of 131I-chimeric(ch) TNT-1/B monoclonal antibody for the treatment of advanced colon cancer

Purpose: The primary aim of this study was to evaluate the biodistribution and toxicity of 131I-chimeric(ch) TNT-1/B monoclonal antibody (MAB), which binds to intracellular antigens of necrotic regions within tumors, in patients with advanced colon or colorectal cancer. The rationale for targeting areas of tumor necrosis is the observation that necrotic lesions are more abundant in cancer lesions than in surrounding tissues. Patients and Methods: Cohorts of patients with advanced colon or colorectal cancer were administered a one-time 30-60-minute intravenous (i.v.) infusion of 131I-ChTNT-1/B at doses ranging from 12.95 to 66.23 MBq/kg (0.35-1.79 mCi/kg). Results: The dose-limiting toxicity, experienced at 66.23 MBq/kg (1.79 mCi/kg) 131I-ChTNT-1/B MAB, was myelosuppression. Two (2) patients at the 66.23-MBq/kg (1.79 mCi/kg) dose level had both grade 3 thrombocytopenia and grade 3 neutropenia that persisted for at least 2 weeks but were reversible. The maximum tolerated dose was 58.09 MBq/kg (1.57 mCi/kg) 131I-chTNT-1/B MAB. Of the 21 patients, one developed a moderate human antichimeric antibody (HACA) response and 6 developed low HACA responses. Conclusions: The infusion of 131I-chTNT-1/B MAB was well tolerated, without significant nonhematological toxicity. No patient obtained a complete or partial response, based on tumor cross-product response criteria. Tumor localization was seen in patients with dose levels at, and exceeding, 50.23 MBq/kg (1.36 mCi/kg) 131I-chTNT-1/B MAB. © Mary Ann Liebert, Inc.link_to_subscribed_fulltex