Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an N-glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibited robust tolerance to various buffer conditions and was employed in a column format that can be readily adapted into a typical HDX-MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.

Validation of a de-immunization strategy for monoclonal antibodies using cynomolgus macaque as a surrogate for human.

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human-cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336-672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.

Role of TGF-alpha in the progression of diabetic kidney disease.

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

Generation and activity of a humanized monoclonal antibody that selectively neutralizes the epidermal growth factor receptor ligands transforming growth factor-α and epiregulin.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases.

B cells contribute to multiple aspects of autoimmune disorders, and B cell-targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti-human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell-dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell-targeting therapies in treatment of autoimmune conditions without causing B cell depletion.

Discovery and characterization of a neutralizing pan-ELR+CXC chemokine monoclonal antibody.

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

High-resolution protein structure determination starting with a global fold calculated from exact solutions to the RDC equations.

We present a novel structure determination approach that exploits the global orientational restraints from RDCs to resolve ambiguous NOE assignments. Unlike traditional approaches that bootstrap the initial fold from ambiguous NOE assignments, we start by using RDCs to compute accurate secondary structure element (SSE) backbones at the beginning of structure calculation. Our structure determination package, called RDC-PANDA: (RDC-based SSE PAcking with NOEs for Structure Determination and NOE Assignment), consists of three modules: (1) RDC-EXACT: ; (2) PACKER: ; and (3) HANA: (HAusdorff-based NOE Assignment). RDC-EXACT: computes the global optimal solution of backbone dihedral angles for each secondary structure element by exactly solving a system of quartic RDC equations derived by Wang and Donald (Proceedings of the IEEE computational systems bioinformatics conference (CSB), Stanford, CA, 2004a; J Biomol NMR 29(3):223-242, 2004b), and systematically searching over the roots, each of which is a backbone dihedral varphi- or psi-angle consistent with the RDC data. Using a small number of unambiguous inter-SSE NOEs extracted using only chemical shift information, PACKER: performs a systematic search for the core structure, including all SSE backbone conformations. HANA: uses a Hausdorff-based scoring function to measure the similarity between the experimental spectra and the back-computed NOE pattern for each side-chain from a statistically-diverse rotamer library, and drives the selection of optimal position-specific rotamers for filtering ambiguous NOE assignments. Finally, a local minimization approach is used to compute the loops and refine side-chain conformations by fixing the core structure as a rigid body while allowing movement of loops and side-chains. RDC-PANDA: was applied to NMR data for the FF Domain 2 of human transcription elongation factor CA150 (RNA polymerase II C-terminal domain interacting protein), human ubiquitin, the ubiquitin-binding zinc finger domain of the human Y-family DNA polymerase Eta (pol eta UBZ), and the human Set2-Rpb1 interacting domain (hSRI). These results demonstrated the efficiency and accuracy of our algorithm, and show that RDC-PANDA: can be successfully applied for high-resolution protein structure determination using only a limited set of NMR data by first computing RDC-defined backbones.

Engineered FcRn Binding Fusion Peptides Significantly Enhance the Half-Life of a Fab Domain in Cynomolgus Monkeys.

There is a rapidly growing reinvigoration of the investigation of small proteins, cyclic peptides, and mAb derived domains as biotherapies. The drugability of these structures are challenged by fast peripheral clearance properties that can reduce their potential to be realized as medicines. Engineering strategies have been of limited value because mechanistically the half-life benefit is manifested by increasing the molecular weight and/or the hydrodyanimc radius which slows the molecule's renal elimination, but can result in the inherent loss of activity and target accessibility. The present work evaluated an alternative approach using smaller peptide sequences which bind to the neonatal Fc receptor (FcRn). Results revealed, small linear and cyclic FcRn binding peptides (FcRnBPs) fused to a combination of the N- and C-termini of a Fab can significantly improve the pharmacokinetics of the protein in cynomolgus monkeys relative to the parental Fab. The linear and cyclic conformations, as well as, the number of FcRnBPs fused to the Fab both influence the clearance and the extent of pharmacokinetic benefit. FcRnBP fusion protein kinetics were also affected by a combination of post-translation modifications and non-specific binding properties. The results in this report lay some foundation in fostering the advent of newer technologies toward successfully improving the pharmacokinetics of proteins, peptides, and mAb-derived domains. Additional work in the integration of a variety of factors including the intended site of action, tissue disposition, metabolism, toxicity and pharmacokinetic, and pharmacodynamics relationship of the intended therapeutic modality are key areas for advancement of these approaches.

Polymer-mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery.

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co-purified during Protein A purification, must be removed in a multi-step purification process. Our colleagues have reported the use of a novel polymer-mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel "smart polymer" (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration-dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393-1400, 2017.

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody.

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala41 , Glu44 , and His45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.