title: Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1.
abstract: The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and CH2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.
authors.affiliation: Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
authors.forename: Lai Sum
authors.initials: LS
authors.lastname: Leoh
authors.affiliation: Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
authors.forename: Tracy R
authors.initials: TR
authors.lastname: Daniels-Wells
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Department of Epidemiology, UCLA Fielding School of Public Health, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA; UCLA AIDS Institute, Los Angeles, CA, USA.
authors.forename: Otoniel
authors.initials: O
authors.lastname: Martínez-Maza
authors.affiliation: Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA; UCLA AIDS Institute, Los Angeles, CA, USA; The Molecular Biology Institute, University of California, Los Angeles, CA, USA. Electronic address: penichet@mednet.ucla.edu.
authors.forename: Manuel L
authors.initials: ML
authors.lastname: Penichet
mesh_terms: D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D045744:Cell Line, Tumor; D015922:Complement C1q / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017452:Receptors, IgG / Q000378:metabolism; D011990:Receptors, Transferrin / Q000276:immunology*
chemical_list: D015703:Antigens, CD; C068126:CD71 antigen; C496124:FCGR3A protein, human; D007074:Immunoglobulin G; D050505:Mutant Proteins; D017452:Receptors, IgG; D011990:Receptors, Transferrin; D015922:Complement C1q
keywords: ADCC; Antibodies; CDC; Effector functions; Human IgG3; TfR1