R409K mutation prevents acid-induced aggregation of human IgG4

Abstract

Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability.

Fig 1. Schematic of domain subclass-switched/swapped and/or substituted derivative antibodies constructed in this study.

Shaded and hollow rectangles represent domains derived from human IgG1 and IgG4, respectively, whereas gray bars depict amino acid substitutions. All antibodies shared variable regions of anti-CD20 antibody and light-chain constant region of the kappa isotype.

Fig 2. Identification of an amino acid responsible for low-pH-induced aggregation in human IgG4.

(A) Identification of the domain critical for aggregate formation through domain swapping between IgG1 and IgG4. SEC analysis of the percentage of aggregation following incubation at pH 3.4 for 10 and 60 min at 37°C. (B) Alignment of the CH3 domains of human IgG1 and IgG4. (C) Identification of the amino acid in CH3 domain that is responsible for acid-induced aggregation. (D) Effect of amino acid substitution at position 409 on acid-induced aggregation. (E) Effect of S228P or L235E mutation on acid-induced aggregation. (F) Effect of K370E mutation on acid-induced aggregation. Data are presented as means ± SD of experiments performed in triplicate. Black bars: initial data; gray and white bars: pH 3.4 for 10 and 60 min at 37°C, respectively.

Fig 3. DSC analysis of thermodynamic stability.

(A) DSC charts of anti-CD20 IgG1, IgG4, IgG4PE, and IgG4PE R409K antibodies at pH 7.4 with PBS buffer. (B) Post-deconvoluted DSC charts of IgG1, IgG4, IgG4PE, and IgG4PE R409K antibodies at pH 7.4 with PBS buffer. (C) Post-deconvoluted DSC charts of IgG4PE and IgG4PE R409K antibodies at different pH conditions including 5.0, 6.0, and 7.4.

Fig 4. Effect of R409K mutation on Fab-arm exchange in vitro and in vitro.

(A) Effect of R409K mutation on Fab-arm exchange in vitro. Bispecific anti-CD20 antibodies composed of kappa/lambda light chains were detected using ELISA. Mixtures of anti-CD20 IgG1, IgG4, IgG4PE, IgG4PE_K370E, or IgG4PE_R409K containing kappa light chain and anti-CD20 IgG4 containing lambda light chain were incubated with 1 mM reduced glutathione at 37°C for 12 h. Goat anti-human kappa antibodies were coated on 96-well immunoplates, which were then incubated with diluted mixtures of antibodies; the bispecific antibody was detected using peroxidase-labeled goat F(abʹ)2 anti-human lambda antibodies. Results are shown as means ± SD of experiments performed in triplicate. (B) Effect of R409K mutation on Fab-arm exchange in vivo. Nude mice (n = 5) were injected with a mixture of anti-VLA4 IgG1, IgG2AAAS, IgG4, IgG4PE, or IgG4PE_R409K containing kappa light chain and anti-CD20 IgG4 containing lambda light chain. IgG2AAAS: IgG2 variant harboring V234A/G237A/P331S substitutions. Blood samples were collected at 10 days after antibody administration and were used for the detection of kappa/lambda bispecific antibodies or whole IgG. Data bar and error bar: average and standard error, respectively.

Fig 5. Effect of R409K mutation on antigen (FcγR) binding and CDC activity.

(A) Binding of IgG4 mutants to Raji cells, measured using flow cytometry; cells were stained with IgG4 (open circles), IgG4PE (closed squares), IgG4PE_R409K (open squares), or isotype control IgG (closed circles), and the binding was detected using an Alexa Fluor 488-labeled secondary antibody. (B) ELISA for measuring the binding activity of IgG4 mutants toward human FcRs. Each FcγR was coated on 96-well immunoplates by using anti-tetra-His antibodies, and the plates were incubated with the indicated concentrations of IgG1 (closed circles), IgG4 (open circles), IgG4PE (closed squares), or IgG4PE_R409K (open squares); binding was detected using a peroxidase-labeled secondary antibody. (C) CDC of IgG4 mutants against Raji cells. After 2-h incubation, cytotoxicity was measured using CellTiter-Glo. Means ± SD of triplicates are shown.

Fig 6. Structure of the Fc region of human IgG4.

The model was built using published structural data of human IgG4 (Protein Data Bank accession code: 4C54). Amino acids of human IgG4 described in the Discussion are highlighted in a different color.