The IgG Fc Contains Distinct Fc Receptor (FcR) Binding Sites: The Leukocyte Receptors FcγRI and FcγRIIa Bind to a Region in the Fc Distinct from That Recognized by Neonatal FcR and Protein A

Abstract

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (FcγRIIb). The FcγRI and FcγRII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with FcγRI and FcγRIIa. We demonstrate that recombinant soluble murine FcγRI and human FcγRIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in FcγRI and FcγRIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble FcγRIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between FcγRIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.

Boosting antibody developability through rational sequence optimization

“…1): a G1m17 allotype EEM version (named mAb1_WT) and a hinge LL->AA version (named mAb1*_WT). 29 Expression levels of both Fc versions in a stably transfected recombinant Chinese hamster ovary (CHO) cell line was extremely low, with a titer below 1 mg/l. Hence, the poor expression was independent of the constant region.…”

“…The wild-type protein was constructed bearing 2 different Fc parts; heavy chain Fc of G1m17 allotype EEM (white boxes) or a heavy chain hinge LL->AA version (striped boxes). 29 The variants with engineered variable regions were constructed as G1m17 allotype EEM Fc version. Engineered positions in the variable regions are highlighted by arrows.…”

IgG2m4, an engineered antibody isotype with reduced Fc function

“…It is interesting to point out that the introduction of L234F/L235E/P331S triple mutation into the Fc of human IgG1 resulted in an almost complete loss of binding to CD32A (FcγRIIa), 28 consistent with earlier work that showed that L234/L235 play an important role in IgG1 binding to FcγRIIa. 36 It is interesting to see if the triple mutation (L234F/L235E/P331S) can bind to FcγRIIb/c. 28 The marketed IgG2 and IgG4 hybrid (IgG2/4, 26 ) eculizumab, which closely resembles the IgG2m4 described in this work, has been successfully developed.…”

Cleavage of IgGs by proteases associated with invasive diseases

“…[6][7][8] Of particular interest is that proteolytic sensitivity has been mapped primarily to short stretches of amino acids within either the upper, or perhaps more importantly, within the lower hinge/CH2 region, a highly conserved stretch of amino acids critical for binding to the Fc family of receptors. [9][10][11][12][13][14][15][16][17][18][19][20][21] Several of the proteases that are capable of cleaving IgGs within the lower hinge/CH2 are also associated with either pathogenic bacteria, e.g., glutamyl endopeptidase V8 (GluV8) of Staphylococcus aureus or immunoglobulin-degrading…”

“…31,32 All of the Fcγ family of receptors can interact with human IgG1, and these interactions perhaps function to further stabilize the lower hinge region allowing an assessment of amino acid contact points between FcRs and the Fc. 33 Multiple points of interaction within the CH2 domain and FcγRs have been documented, but there is also a critical stretch in the lower hinge/CH2 region required for FcγR-binding ranging from E233-L234-L235-G236-G237-P238, [9][10][11][12][13][14][15][16][17][18][19][20][21] (EU numbering). 13 Although contact between each of these amino acids and FcRs have not been visualized directly by crystallography, mutational studies implicate a requirement for each member of this sequence.…”