Abstract
Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically “silent”.
Highlights
The biological functionality of therapeutic antibodies depends on the interactions of two regions of the protein with components of its external environment: the antigen binding region (Fab) interacting with an antigen, and the fragment crystallizable (Fc) region interacting with components of the immune system
We describe here the functional and structural characteristics of three novel silent Fc designs: huIgG1 sigma (IgG1σ), which is a variant of huIgG1, and huIgG4 sigma1 (IgG4σ1) and huIgG4 sigma2 (IgG4σ2), which are variants of huIgG4
Fc engagement with FcγR can activate myeloid cell and NK cell activity as well as the generation of reactive species that induce apoptosis and release of inflammatory cytokines which are important for eliminating unwanted target cells [52,53,54,55]
Summary
The biological functionality of therapeutic antibodies depends on the interactions of two regions of the protein with components of its external environment: the antigen binding region (Fab) interacting with an antigen, and the fragment crystallizable (Fc) region interacting with components of the immune system. Fc-mediated effector functions are best avoided for some applications such as systemic neutralization of cytokines, targeting cell surface antigens on immune cells, or when engineering bispecific molecules to bring target diseased cells within proximity of effector immune cells to provide a more specific immune receptor engagement [3,8,9]. In each of these cases, it is best not to stimulate unwanted cell and tissue damage or risk undesired effector cell activation, immune cell depletion, or FcγR cross-linking that might induce cytokine release through engagement of Fc-mediated effector functions [3]. Development of completely silent Fc domains can be critical for biologics that do not require FcγR or C1q mediated effector functions [11]
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