Daniel R. Higazi

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

Inducing protein aggregation by extensional flow

Significance Proteins are inherently sensitive to environmental factors that include hydrodynamic flow. Flow-induced protein remodeling is used in vivo and can also trigger the aggregation of therapeutic proteins during manufacture. Currently, the relative importance of shear and extensional hydrodynamic flow fields to aggregation remains unclear. Here we develop a flow device that subjects proteins to a defined and quantified flow field that is dominated by extensional flow. We show that extensional flow is crucial to induce the aggregation of globular proteins and that flow-induced aggregation is dependent on both protein structure and sequence. These observations rationalize the diverse effects of hydrodynamic flow on protein structure and aggregation propensity seen in both Nature and in protein manufacture. Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein’s structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β2-microglobulin (β2m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36–1.8 ms, at concentrations as low as 0.5 mg mL−1. In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.

MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential

ABSTRACT Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.

Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan

ABSTRACT Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibodys ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.