Shirley Jane Peters

A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells but reported yields from Chinese hamster ovary (CHO) cells are lower at ∼300 mg/L. We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X‐box binding protein (XBP‐1S) and endoplasmic reticulum oxidoreductase (ERO1‐Lα) and has been named CHOS‐XE. CHOS‐XE cells produced increased antibody (MAb) yields (5.3– 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L.

Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability

Background: IgG1 and IgG4 have different inter-light chain-heavy chain disulfide bond (DSB) arrangements. Results: IgG4 mutants with an IgG1-like DSB and a S241P hinge mutation showed increased Fab thermal stability and reduced DSB heterogeneity compared with IgG4 WT. Conclusion: Fab domain thermal stability and DSB heterogeneity of IgG4 can be improved. Significance: Such engineered IgG4 molecules offer potential advantages during therapeutic antibody production. The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (CH1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227–230) at the C terminus of CH1. An inter-LC-CH1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-CH1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227–230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.

Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

The aggregation of protein‐based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation‐prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc‐hinge fragments by using variable‐temperature ion‐mobility mass spectrometry (VT‐IM‐MS). We observed changes in the conformations of isolated proteins as a function of temperature (300–550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher‐order structural organization mitigated by the hinge region. VT‐IM‐MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal‐stability studies for proteins of therapeutic interest.

The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation

Background: IgG4 antibodies are able to undergo a process termed Fab arm exchange (FAE). Results: A novel method for quantifying FAE in physiologically relevant matrices was developed. Conclusion: A hinge mutation in IgG4 antibodies inhibits FAE to undetectable levels in the immunoassays described herein. Significance: These methods are valuable for investigating and assessing the factors affecting and controlling IgG4 FAE. Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices.

Engineered hexavalent Fc proteins with enhanced Fc-gamma receptor avidity provide insights into immune-complex interactions

Autoantibody-mediated diseases are currently treated with intravenous immunoglobulin, which is thought to act in part via blockade of Fc gamma receptors, thereby inhibiting autoantibody effector functions and subsequent pathology. We aimed to develop recombinant molecules with enhanced Fc receptor avidity and thus increased potency over intravenous immunoglobulin. Here we describe the molecular engineering of human Fc hexamers and explore their therapeutic and safety profiles. We show Fc hexamers were more potent than IVIG in phagocytosis blockade and disease models. However, in human whole-blood safety assays incubation with IgG1 isotype Fc hexamers resulted in cytokine release, platelet and complement activation, whereas the IgG4 version did not. We used a statistically designed mutagenesis approach to identify the key Fc residues involved in these processes. Cytokine release was found to be dependent on neutrophil FcγRIIIb interactions with L234 and A327 in the Fc. Therefore, Fc hexamers provide unique insights into Fc receptor biology.Tania Rowley et al. present multivalent Fc molecules with enhanced avidity for Fc gamma receptors in order to improve the treatment of autoantibody-mediated human diseases. They found several key amino acids involved in Fc receptor binding interactions.