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.

An Antibody-Drug Conjugate That Selectively Targets Human Monocyte Progenitors for Anti-Cancer Therapy

“…We first evaluated the cytotoxic activity of H22-dPBD against THP-1 cells, a monocytic leukemia cell line expressing CD64 ( Figure 2B ). In addition to H22-dPBD humanized with IgG1 (H22(IgG1)-dPBD) and the isotype matched control ADC [DNP(IgG1)-dPBD], we also tested the nullbody H22-dPBD [H22(Null)-dPBD], in which H22 is humanized with an IgG4 analogue having three mutations that stabilize the Ab and minimize non-specific binding to Fcγ receptors ( 39 ). Compared with DNP(IgG1)-dPBD, H22(IgG1)-dPBD had a more effective level of CD64-specific cytotoxicity.…”

Profiling the Biophysical Developability Properties of Common IgG1 Fc Effector Silencing Variants

“…The alternative use of detergent is less straightforward and may affect yield to a greater extent than low pH [ 61 ]. While stabilizing mutations for IgG4 are known [ 62 ], IgG1 has significantly higher representation in marketed therapeutic antibodies; approximately 59% are IgG1 whereas IgG2 and IgG4 comprise 7% and 21%, respectively [ 63 ]. This may reflect a more straightforward manufacturing process and the favorable developability and functional profile of IgG1.…”

Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

“…The scFv regions of KT112 (phage clone) were cloned in‐frame into an expression vector with a signal peptide and the human IgG4 Fc region (S228P/S235E/R409K) 17 . Immunoglobulin G4 (S228P/S235E/R409K) is a low‐effector‐activity Ab format that shows increased stability.…”

“…The scFv regions of KT112 (phage clone) were cloned in-frame into an expression vector with a signal peptide and the human IgG4 Fc region (S228P/S235E/R409K). 17 Immunoglobulin G4 (S228P/S235E/ R409K) is a low-effector-activity Ab format that shows increased stability. The V H and V L regions of KT112, AM1 (AbbVie), and DNP (negative control Ab) were each cloned in-frame into an expression vector with a signal peptide and human IgG4 constant region (S228P/S235E/ R409K).…”