Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions

Abstract

Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules.

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

“…To identify effector silencing substitutions that could both improve the production of murine IgG2a-bispecific antibodies and effectively reduce effector function, we screened several antibody variants, including the recently identified combination of L234A, L235A, and P329A (LALA-PG) substitutions that have been shown to effectively silence the effector function of human IgG1 antibodies (32). In contrast to the allosteric effects of aglycosylation, the LALA-PG substitutions directly block the interaction of the Fc with the Fc-␥ receptors and C1q.…”

Novel Drug and Soluble Target Tolerant Antidrug Antibody Assay for Therapeutic Antibodies Bearing The P329G Mutation

“…The high-sequence homology of the Fc region between human and cynomolgus monkey IgG [28] allows the usage of the same assay for the analysis of preclinical and clinical samples [12]. In addition to the PG, several other Fc modifications affect the affinity of mAbs to both the Fc receptors and complement [16,[29][30][31]. We believe that the assay principle can be transferred to other Fc mutations that allow the generation of specific Abs to be used similarly to the anti-PG mAb.…”

Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function

“…Variants with WT binding at pH 6.5 and no detectable binding at pH 7.4 are denoted as 'Yes'. 7 The Tm was measured by deconvolution using a non-2 state model of the first transition in the thermograms shown in Figure 2. 8 Homodimeric trastuzumab with no mutations was used as WT.…”

“…Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity [4][5][6][7]. These strategies include reduction of effector function through modification of glycosylation [8][9][10], use of IgG2/IgG4 scaffolds [11][12][13][14], or the introduction of engineered mutations in the hinge or CH2 domain of the Fc region of the antibody [7,13,[15][16][17].…”

“…These strategies include reduction of effector function through modification of glycosylation [8][9][10], use of IgG2/IgG4 scaffolds [11][12][13][14], or the introduction of engineered mutations in the hinge or CH2 domain of the Fc region of the antibody [7,13,[15][16][17]. Since natural antibodies are homodimeric in nature, so far only symmetric mutations that reduce effector activity have been reported.…”