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 Fc? 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.
Project description:An interesting format in the development of therapeutic monoclonal antibodies uses the crystallizable fragment of IgG1 as starting scaffold. Engineering of its structural loops allows generation of an antigen binding site. However, this might impair the molecule's conformational stability, which can be overcome by introducing stabilizing point mutations in the CH3 domains. These point mutations often affect the stability and unfolding behavior of both the CH2 and CH3 domains. In order to understand this cross-talk, molecular dynamics simulations of the domains of the Fc fragment of human IgG1 are reported. The structure of human IgG1-Fc obtained from X-ray crystallography is used as a starting point for simulations of the wild-type protein at two different pH values. The stabilizing effect of a single point mutation in the CH3 domain as well as the impact of the hinge region and the glycan tree structure connected to the CH2 domains is investigated. Regions of high local flexibility were identified as potential sites for engineering antigen binding sites. Obtained data are discussed with respect to the available X-ray structure of IgG1-Fc, directed evolution approaches that screen for stability and use of the scaffold IgG1-Fc in the design of antigen binding Fc proteins.
Project description:Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with Fc?RII and Fc?RIII, and complement. These properties render IgG4 relatively "non-inflammatory" and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35 Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the CH3-CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor Fc?RII, Fc?RIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the CH2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc-Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the CH2-CH3 interface and known hydrophobic aggregation motifs. These Fc-Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.
Project description:A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development.
Project description:Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1?<?aglyco-IgG1?<?IgG2?<?IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.
Project description:The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.
Project description:Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis. Antibody aggregation control is also important for therapeutic antibody development. To date, there have been few reports of IgG3 aggregation during protein expression and the low pH conditions needed for purification and virus inactivation. This study explored the potential of IgG3 antibody for therapeutics using anti-CD20 IgG3 as a model to investigate aggregate formation. Initially, anti-CD20 IgG3 antibody showed substantial aggregate formation during expression and low pH treatment. To circumvent this phenomenon, we systematically exchanged IgG3 constant domains with those of IgG1, a stable IgG. IgG3 antibody with the IgG1 CH3 domain exhibited reduced aggregate formation during expression. Differential scanning calorimetric analysis of individual amino acid substitutions revealed that two amino acid mutations in the CH3 domain, N392K and M397V, reduced aggregation and increased CH3 transition temperature. The engineered human IgG3 antibody was further improved by additional mutations of R435H to obtain IgG3KVH to achieve protein A binding and showed similar antigen binding as wild-type IgG3. IgG3KVH also exhibited high binding activity for Fc?RIIIa and C1q. In summary, we have successfully established an engineered human IgG3 antibody with reduced aggregation during bioprocessing, which will contribute to the better design of therapeutic antibodies with high effector function and Fab arm flexibility.
Project description:Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P mutation (in IgG4 core-hinge region) intentionally designed to prevent Fab-arm exchange. A new scaffolding platform for engineering IgG4 antibodies amenable to bioprocessing and bioanalysis is proposed by introducing an "IgG1-like" single-point mutation in the hinge or CH1 region of IgG4S228P. This work offers insight into the design, discovery, and development of innovative therapeutic antibodies that are well suited for robust biomanufacturing and quality control.
Project description:Significant amounts of soluble product aggregates were observed during low-pH viral inactivation (VI) scale-up for an IgG4 monoclonal antibody (mAb IgG4-N1), while small-scale experiments in the same condition showed negligible aggregation. Poor mixing and product exposure to low pH were identified as the root cause. To gain a mechanistic understanding of the problem, protein aggregation properties were studied by varying critical parameters including pH, hold time and protein concentration. Comprehensive biophysical characterization of product monomers and aggregates was performed using fluorescence-size-exclusion chromatography, differential scanning fluorimetry, fluorescence spectroscopy, and dynamic light scattering. Results showed IgG4-N1 partially unfolds at about pH 3.3 where the product molecules still exist largely as monomers owing to strong inter-molecular repulsions and favorable colloidal stability. In the subsequent neutralization step, however, the conformationally changed monomers are prone to aggregation due to weaker inter-molecular repulsions following the pH transition from 3.3 to 5.5. Surface charge calculations using homology modeling suggested that intra-molecular repulsions, especially between CH2 domains, may contribute to the IgG4-N1 unfolding at ? pH 3.3. Computational fluid dynamics (CFD) modeling was employed to simulate the conditions of pH titration to reduce the risk of aggregate formation. The low-pH zones during acid addition were characterized using CFD modeling and correlated to the condition causing severe product aggregation. The CFD tool integrated with the mAb solution properties was used to optimize the VI operating parameters for successful scale-up demonstration. Our research revealed the governing aggregation mechanism for IgG4-N1 under acidic conditions by linking its molecular properties and various process-related parameters to macroscopic aggregation phenomena. This study also provides useful insights into the cause and mitigation of low-pH-induced IgG4 aggregation in downstream VI operation.
Project description:IgG4s are dynamic molecules that undergo a process called Fab-arm exchange. Disulfide bonds between heavy chains are transiently reduced, resulting in half antibodies that reform intact antibodies with other IgG4 half antibodies. In vivo, therapeutic IgG4s can recombine with endogenous IgG4s, resulting in a heterogeneous mixture of bispecific antibodies. A related issue that can occur for any therapeutic protein during manufacturing is interchain disulfide bond reduction. For IgG4s, this primarily results in high levels of half-mAb that persist through purification processes. The S228P mutation has been used to prevent half-mAb formation. However, we demonstrated that IgG4s with the S228P mutation are subject to half-mAb formation and Fab-arm exchange in reducing environments. We identified two novel mutations that stabilize the heavy-heavy chain interaction via incorporation of additional disulfide bonds in the hinge region. Individually, these mutations increase stability toward reduction and lessen Fab-arm exchange. Combination of all three mutations, Y219C, G220C, and S228P, has an additive benefit resulting in an IgG4 with ?7-fold increase in stability toward reduction while preventing Fab-arm exchange. Importantly, the mutations do not affect antigen binding or Fc effector function. These mutations hold great promise for solving mAb reduction during manufacturing and preventing Fab-arm exchange in vivo.
Project description:Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.