FcγRIIIa, which is predominantly expressed on the surface of natural killer cells, plays a key role in antibody-dependent cell-mediated cytotoxicity (ADCC), a major effector function of therapeutic IgG antibodies that results in the death of aberrant cells. Despite the potential uses of aglycosylated IgG antibodies, which can be easily produced in bacteria and do not have complicated glycan heterogeneity issues, they show negligible binding to FcγRIIIa and abolish the activation of immune leukocytes for tumor cell clearance, in sharp contrast to most glycosylated IgG antibodies used in the clinical setting. For directed evolution of aglycosylated Fc variants that bind to FcγRIIIa and, in turn, exert potent ADCC effector function, we randomized the aglycosylated Fc region of full-length IgG expressed on the inner membrane of Escherichia coli. Multiple rounds of high-throughput screening using flow cytometry facilitated the isolation of aglycosylated IgG Fc variants that exhibited higher binding affinity to FcγRIIIa-158V and FcγRIIIa-158F compared with clinical-grade trastuzumab (Herceptin®). The resulting aglycosylated trastuzumab IgG antibody Fc variants could elicit strong peripheral blood mononuclear cell-mediated ADCC without glycosylation in the Fc region.

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Complement dependent cytotoxicity (CDC), which removes aberrant target cells through the assembly and complex formation of serum complement molecules, is the main effector function of anticancer therapeutic antibodies. In this study, we found that the CDC activity of anti-CD20 antibody increased remarkably when the symmetry of natural Immunoglobulin G (IgG) antibodies was broken. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in an in-house constructed rituximab-resistant cell line, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance. Based on these findings, we developed an asymmetric bispecific antibody (SBU-CD55×CD20) that targets both CD55 and CD20 simultaneously to effectively remove rituximab-resistant cancer cells. In various cancer cell lines, including rituximab-resistant lymphoma cells, the SBU-CD55×CD20 antibody exhibited significantly higher CDC activity than either anti-CD20 IgG antibody alone or a combination of anti-CD20 IgG antibody and anti-CD55 IgG antibody. Furthermore, compared with other bispecific antibodies with symmetric features, the asymmetric bispecific antibody (SBU-CD55×CD20) exhibited significantly higher CDC activity against rituximab-resistant cancer cells. These results demonstrate that improving CDC using an asymmetric CD55-binding bispecific antibody could be a new strategy for designing therapeutics to treat patients with relapsed or refractory cancers.

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