Human IgG isotype‐specific amino acid residues affecting complement‐mediated cell lysis and phagocytosis

Abstract

In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) mouse/human immunoglobulin (Ig) G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described. Gln-268 is substituted by His in gamma 4 Q268H, Ser-331 is substituted by Pro in gamma 4 S331P, and in gamma 4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by Fc gamma RII- and Fc gamma RIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, gamma 4 Q268H was inactive, but both gamma 4 S331P and gamma 4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants gamma 4 S331P and gamma 4 Q268H/S331P were both active only when complement was introduced. gamma 4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by Fc gamma RII and/or Fc gamma RIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect Fc gamma RII/Fc gamma RIII-mediated phagocytosis. In this manner the mutants gamma 4 S331P and gamma 4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.

Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc

“…Because most of these sites are conserved in human IgG isotypes that are deficient in C1q binding and complement activation (5), it is evident that there are other factors that influence complement activation. Also, other amino acid residues in human IgG1 have been shown to be important for C1q binding and complement activation (9,10,15). Perhaps, the differences in function between these IgG isotypes is due to the conformation of the Ab modulated by a few residues, the hinge (26), and/or carbohydrate (27).…”

“…Because these three residues, E318, K320, and K322, are conserved in human IgG and IgGs of several other species, they have been designated as the C1q binding motif (7). However, several studies implicate that the contact sites for C1q in murine IgG2b may be different from that of human IgG1 (9,10,13,15,16). In one of these studies, a K320A mutation in a human IgG1 Ab was shown to have little or no effect on C1q binding or CDC activity (10).…”

“…In one of these studies, a K320A mutation in a human IgG1 Ab was shown to have little or no effect on C1q binding or CDC activity (10). Also, a few residues, L235, D265 and P331, which are important for C1q binding and CDC activity in a human IgG background (9,10,13,15,16), are of minimal importance for complement activation in a murine IgG2b background (7). Substitution of P331 with serine in human IgG1 resulted in a 60% decrease in binding to C1q and complete loss of complement activity (15).…”

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

“…2 Thus, studies that attempt to explain this disparity have focused on the hinge region, where the sequence variation is highest. 2,[4][5][6][7][8][9][10][11][12][13][14] The unique lower hinge residues from an IgG2 have been placed into both an IgG1 and IgG3 antibody background 4,11,12,15 and assayed for binding to Fc receptors. The IgG1 mutant showed reduced binding to all tested Fc gamma receptors, however, the reduction observed was well below that of the wild-type IgG2/Fc receptors affinities.…”

IgG2m4, an engineered antibody isotype with reduced Fc function

“…Further studies have identified individual amino acid residues that interact with Fcγ receptors and complement C1q protein. For example, residues H268, 13,14 A330 and P331 were shown to be critical for Fcγ receptors and C1q bindings. [15][16][17][18][19][20] IgG1, IgG2 and IgG4 isotypes are the choices for therapeutic antibodies currently in clinical use.…”