The representative sensorgrams are depicted inFigure S1. == Table1.Affinity for FcRIIIaF158and TMof antibody variants. and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use Narirutin of substitutions that reduce the TMof the CH2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens. Keywords:A/I ratio, ADCC, Fc engineering, FcR, antibody engineering == Introduction == Monoclonal antibodies (mAbs) have enormous potential as anticancer therapeutics. MAbs promote elimination of tumor cells by Fab-dependent and Fc-dependent mechanisms, such as interference with signaling pathways, apoptosis induction, complement-dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is induced when effector cells are recruited by the Fc domain engaging with a member of the Fc receptor family, which is comprised in humans of FcRI, FcRIIa, FcRIIb, FcRIIc, FcRIIIa and FcRIIIb isoforms. FcRI, FcRIIa, FcRIIc and FcRIIIa are activating receptors characterized by the immunoreceptor tyrosine-based activation motif (ITAM), and FcRIIb is the only inhibitory receptor characterized by ITIM. The receptors are expressed on a variety of immune cells, such as NK cells, monocytes, macrophages and dendritic cells.1 Increasing affinity for FcR enhances ADCC, so Fc engineering is considered to be a promising means of increasing the antitumor potency of mAbs.2Previous reports described that follicular lymphoma patients treated with rituximab had, on average, significantly prolonged progression-free survival if they possessed two copies of the high-affinity FcRIIIa allele, FcRIIIaV158.3,4This result suggests that the efficacy of rituximab is mediated by FcRIIIa-expressing cells, such as NK cells, and that higher affinity to FcRIIIa improves the efficacy. Several strategies have been employed to enhance the FcR binding of mAbs. The first strategy was engineering the glycan moiety attached to Asn297 residue in the Fc domain. Afucosylated IgG1 antibody, which is a mAb without fucose in the N-linked glycan at Asn297, binds to FcRIIIa with higher affinity and mediates superior ADCC compared with wild-type fucosylated IgG1 antibody.5,6The second strategy was introducing amino acid substitutions into the Fc domain. mAbs with triple substitutions, S239D/A330L/I332E, bind to FcRIIIa with higher affinity and have shown superior ADCC activity than wild-type IgG.7In addition to enhancing the binding to activating FcRs, minimizing the interaction with inhibitory FcR, namely FcRIIb, is another strategy to enhance the potential of the antibody.8Enhanced cancer elimination was observed in FcRIIb knockout mice compared with that observed in mice expressing FcRIIb when they were treated with anti-Her2/neu mAb or anti-E-cadherin mAb,9,10demonstrating that the ratio of activating FcR binding to inhibitory FcR binding (A/I ratio) is an important factor determining the therapeutic efficacy of antitumor antibody.11mAb with five substitutions, L235V/F243L/R292P/Y300L/P396L, showed enhanced binding to FcRIIIa, but not to FcRIIb, which improved the A/I ratio.8In terms of improving the selectivity for a specific FcR, antibody variants with selectively enhanced binding for FcRI were reported.12 Although these glyco- and protein-engineering approaches have successfully enhanced the effector function of mAbs, each technology has issues to overcome. First, afucosylated antibodies bind with lower affinity to FcRIIIaF158than to FcRIIIaV158. As a consequence, the resulting ADCC mediated by NK cells bearing the lower-affinity FcRIIIa allotype is lower than that mediated Hbg1 by NK cells bearing the higher-affinity allotype, suggesting that afucosylated Fc may not achieve maximum ADCC activity for patients having the lower-affinity allotype.13Second, because activating and inhibitory FcRs have high homology, the Narirutin S239D/A330L/I332E variant also increased binding affinity against inhibitory FcRIIb, which Narirutin would be undesirable for achieving maximum antitumor efficacy considering the A/I ratio. Moreover, the TMin the CH2 domain of the S239D/A330L/I332E variant was significantly reduced (by more than 20C), which could be an issue when the variant is developed as a pharmaceutical product.14Third, although the L235V/F243L/R292P/Y300L/P396L variant did not increase the binding affinity against inhibitory FcRIIb, it had only 10-fold increased binding affinity to FcRIIIa, which is substantially less than the S239D/A330L/I332E variant, thereby achieving only a moderate A/I ratio. To date, neither glyco- nor protein-engineering has been able to overcome all these issues. Ideal therapeutic use requires an antibody Fc variant that has higher binding affinity to both FcRIIIaF158and FcRIIIaV158and better stability of the CH2 domain, but that does not increase binding affinity to inhibitory FcRIIb to maintain a higher.