Therapeutic monoclonal antibodies have become molecules of choice to treat autoimmune disorders, inflammatory diseases, and cancer. Moreover, bispecific/multispecific antibodies that target more than one antigen or epitope on a target cell or recruit effector cells (T cell, natural killer cell, or macrophage cell) toward target cells have shown great potential to maximize the benefits of antibody therapy. In the past decade, many novel concepts to generate bispecific and multispecific antibodies have evolved successfully into a range of formats from full bispecific immunoglobulin gammas to antibody fragments. Impressively, antibody fragments such as bispecific T-cell engager, bispecific killer cell engager, trispecific killer cell engager, tandem diabody, and dual-affinity-retargeting are showing exciting results in terms of recruiting and activating self-immune effector cells to target and lyse tumor cells. Promisingly, crystallizable fragment (Fc) antigen-binding fragment and monomeric antibody or half antibody may be particularly advantageous to target solid tumors owing to their small size and thus good tissue penetration potential while, on the other hand, keeping Fc-related effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis, and extended serum half-life via interaction with neonatal Fc receptor. This review, therefore, focuses on the progress of Fc engineering in generating bispecific molecules and on the use of small antibody fragment as scaffolds for therapeutic development.

Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic.

Today, monoclonal immunoglobulin gamma (IgG) antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs) is achieved through both its antigen-binding fragment (Fab) and crystallizable fragment (Fc). Fab can specifically recognize tumor-associated antigen (TAA) and thus modulate TAA-linked downstream signaling pathways that may lead to the inhibition of tumor growth, induction of tumor apoptosis, and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis. Moreover, Fc is the region interacting with the neonatal Fc receptor in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anticancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce the cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering-based mAbs under clinical trials.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from 9 early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 hours, and were no longer detectable at 24 hours. Bortezomib therefore appears promising for treating MG and possibly other antibody-mediated autoimmune or allergic disorders, especially if given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

The role of Th17 cells in the pathogenesis of autoantibody-mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR-specific CD4+ T cells is producing IL-17. ko and WT mice (n = 15/group) as described in the Materials and methods. The cumulative results of (A) EAMG disease score and (B) the percentage of incidence are shown as mean ± SEM and are pooled from two independent experiments. * = p < 0.05, *** = p < 0.001 (two-tailed Mann-Whitney U test).autoimmunity has been clearly demonstrated, their contribution to autoantibody-mediated diseases has only been indirectly suggested [2,3]. Myasthenia gravis (MG) is a prototypic antibodymediated autoimmune disease caused by binding of antibodies to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction, which eventually leads to muscle weakness [4,5] Results Less EAMG symptoms in IL-17 ko miceIn order to assess the role of IL-17 in EAMG disease development, we immunized WT and IL-17 ko mice with tAChR in CFA and evaluated disease symptoms. While some animals already developed mild transient signs of EAMG after the 2nd immunization, more severe muscle weakness did not appear until after the 3rd immunization and then only in WT mice. Altogether, muscle fatigability became evident in the majority, i.e. around 75%, of WT mice (Fig. 1A and B) after the 3rd immunization, while most IL-17 ko mice remained symptom-free and mild muscle weakness was detected in only about 25% of the immunized IL-17 ko mice ( Fig. 1A and B). These findings clearly confirm the contribution of IL-17 to EAMG pathogenesis. T cell differentiation in EAMG does not differ between WT and IL-17 ko miceNext, we investigated whether significant numbers of tAChRspecific Th17 cells can be detected in EAMG. At 2 weeks after each immunization, blood was taken from WT mice and stimulated with a tAChR peptide pool containing the immunodominant tAChR 146-162 peptide [20]. Then the resulting tAChR-specific cytokine production was analyzed. In peripheral blood a significant frequency of CD4 + T cells expressing IL-17 after stimulation with the tAChR peptide pool was already identified at two weeks after the first immunization (Supporting Information Fig. 1). While these cells were still observed at later time points, no further increase was detected after the booster immunizations. Confirming earlier reports [21,22], even after three immunizations we were not able to detect a specific expression of CD40L, IFN-γ, or IL-17 after stimulation with murine AChR peptides (Supporting Information Fig. 2).Since the complete absence of IL-17 might have an impact on T-cell priming in our model, we then analyzed in detail the generation of anti tAChR-specific CD4 + T cells in WT in relation to IL-17 ko mice in EAMG.Splenocytes isolated from WT and IL-17 ko mice that had ...