Advances in mass spectrometry-based epitope mapping of protein therapeutics.

Monoclonal antibody (mAb) has grown to be the major asset in protein therapeutics market since its initial introduction in 1980s. To identify a suitable mAb as a drug candidate for development requires deep understanding of disease biology and attribute sciences, including epitope-paratope binding recognition. Mass spectrometry (MS) has become a critical technology platform in epitope mapping. MS-based approaches utilize chemical labeling to assess the changes of solvent accessibilities during binding interactions, and the labeling can be either reversible or irreversible. Reversible labeling is represented by hydrogen/deuterium exchange (HDX), which probes the changes via exchange between backbone amide hydrogen and deuterium in the solvent. Irreversible labeling targets amino acid residue side chains and involves chemical based labeling such as glycine ethyl ester labeling, radical based labeling such as fast photochemical oxidation of proteins (FPOP), and chemical cross-linking. All these methods have been developed extensively for characterization of binding interface within an immunocomplex. This review covers the fundamentals of these different MS-based methods and highlights recent case studies to illustrate unique capabilities of MS-based approaches in epitope mapping of protein therapeutics.

Enzymatic removal of sialic acid enables iCIEF stability monitoring of charge variants of a highly sialylated bispecific antibody.

Antibody-based therapeutic proteins have highly complex molecular structures. The final therapeutic protein product may contain a wide range of charge variants. Accurate analysis of this charge variant composition is critical to determine manufacturing process consistency and protein stability and ultimately helps to ensure that patients receive a safe and efficacious product. Here, a highly sialylated bispecific antibody (bsAb-1) challenged the ability to monitor stability by imaged capillary isoelectric focusing (iCIEF). This challenge was overcome by optimization of the iCIEF master mix buffer (adjustment of urea concentration, addition of l-arginine) and enzymatic removal of sialic acid. The method was qualified by assessing linearity, precision, LOD, LOQ, accuracy, and robustness in accordance with ICH guidance. Main species loss detectability increased up to approximately fivefold compared to the iCIEF method without desialylation when monitoring changes in stressed samples. Importantly, the results of the iCIEF method with desialylation correlated with results obtained through LC-MS tryptic peptide mapping and enabled analysis of formulation development stability samples. Finally, this analytical method shows the potential to assess low-concentration formulation development samples down to a sample concentration of 0.1 mg/ml.

Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in a fully immunocompetent in vivo model.

T-cell engagers (TCEs) are a growing class of biotherapeutics being investigated in the clinic for treatment of a variety of hematological and solid tumor indications. However, preclinical evaluation of TCEs in vivo has been mostly limited to xenograft tumor models in human T-cell reconstituted immunodeficient mice, which have a number of limitations. To explore the efficacy of human TCEs in fully immunocompetent hosts, we developed a knock-in mouse model (hCD3E-epi) in which a 5-residue N-terminal fragment of murine CD3-epsilon was replaced with an 11-residue stretch from the human sequence that encodes for a common epitope recognized by anti-human CD3E antibodies in the clinic. T cells from hCD3E-epi mice underwent normal thymic development and could be efficiently activated upon crosslinking of the T-cell receptor with anti-human CD3E antibodies in vitro. Furthermore, a TCE targeting human CD3E and murine CD20 induced robust T-cell redirected killing of murine CD20-positive B cells in ex vivo hCD3E-epi splenocyte cultures, and also depleted nearly 100% of peripheral B cells for up to 7 days following in vivo administration. These results highlight the utility of this novel mouse model for exploring the efficacy of human TCEs in vivo, and suggest a useful tool for evaluating TCEs in combination with immuno-oncology/non-immuno-oncology agents against heme and solid tumor targets in hosts with a fully intact immune system.

Structure of the human marker of self 5-transmembrane receptor CD47.

CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47's ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.

Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation.

ABSTACT: The growing prevalence of synthetically modified proteins in pharmaceuticals and materials has exposed the need for efficient strategies to enable chemical modifications with high site-selectivity. While genetic engineering can incorporate non-natural amino acids into recombinant proteins, regioselective chemical modification of wild-type proteins remains a challenge. Herein, we use photoredox catalysis to develop a site-selective tyrosine bioconjugation pathway that incorporates bioorthogonal formyl groups, which subsequently allows for the synthesis of structurally defined fluorescent conjugates from native proteins. A water-soluble photocatalyst, lumiflavin, has been shown to induce oxidative coupling between a previously unreported phenoxazine dialdehyde tag and a single tyrosine site, even in the presence of multiple tyrosyl side chains, through the formation of a covalent C-N bond. A variety of native proteins, including those with multiple tyrosines, can successfully undergo both tyrosine-specific and single-site-selective labelling. This technology directly introduces aldehyde moieties onto native proteins, enabling rapid product diversification using an array of well-established bioorthogonal functionalization protocols including the alkyne-azide click reaction.

Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.

Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy.

Higher-Order Structure Characterization of NKG2A/CD94 Protein Complex and Anti-NKG2A Antibody Binding Epitopes by Mass Spectrometry-Based Protein Footprinting Strategies.

NK group 2 member A (NKG2A), an immune checkpoint inhibitor, is an emerging therapeutic target in immuno-oncology. NKG2A forms a heterodimer with CD94 on the cell surface of NK and a subset of T cells and recognizes the nonclassical human leukocyte antigen (HLA-E) in humans. Therapeutic blocking antibodies that block the ligation between HLA-E and NKG2A/CD94 have been shown to enhance antitumor immunity in mice and humans. In this study, we illustrate the practical utilities of mass spectrometry (MS)-based protein footprinting in areas from reagent characterization to antibody epitope mapping. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the higher-order structure characterization of NKG2A in complex with CD94 provides novel insights into the conformational dynamics of NKG2A/CD94 heterodimer. To fully understand antibody/target interactions, we employed complementary protein footprinting methods, including HDX-MS and fast photochemical oxidation of proteins (FPOP)-MS, to determine the binding epitopes of therapeutic monoclonal antibodies targeting NKG2A. Such a combination approach provides molecular insights into the binding mechanisms of antibodies to NKG2A with high specificity, demonstrating the blockade of NKG2A/HLA-E interaction.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

Site-Selective Functionalization of Methionine Residues via Photoredox Catalysis.

Bioconjugation technologies have revolutionized the practice of biology and medicine by allowing access to novel biomolecular scaffolds. New methods for residue-selective bioconjugation are highly sought to expand the toolbox for a variety of bioconjugation applications. Herein we report a site-selective methionine bioconjugation protocol that uses photoexcited lumiflavin to generate open-shell intermediates. This reduction-potential-gated strategy enables access to residues unavailable with traditional nucleophilicity-based conjugation methods. To demonstrate the versatility and robustness of this new protocol, we have modified various proteins and further utilized this functional handle to append diverse biological payloads.

Chemoselective Methionine Bioconjugation: Site-Selective Fluorine-18 Labeling of Proteins and Peptides.

Chemoselective methionine bioconjugation with alkyne-bearing oxaziridine and alkyne-bearing iodonium salts was investigated as a new platform for site-selective radiolabeling of proteins and peptides with fluorine-18. Alkyne-bearing sulfimide conjugates, resulting from oxaziridine modification, underwent copper-assisted alkyne-azide cycloaddition (CuAAC) with an 18F-labeled PEGylated azide to afford 18F-labeled triazoles in excellent radiochemical yields. Diazoester sulfonium salt bioconjugates, formed from alkyne-bearing 2-diazoiodonium salts, gave low yields of 18F-labeled triazoles and were shown to be unstable to CuAAC conditions. Photolytic removal of the diazo group, however, afforded the trialkylsulfonium salt which smoothly underwent CuAAC with the 18F-labeled PEGylated azide to afford high radiochemical yields of the desired 18F-labeled click product. Overall, the results establish the viability of chemoselective methionine bioconjugation as a method for preparing site-selective 18F-labeled PET radioligands.

Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting.

Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs' epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA). MS-based protein footprinting strategies, including hydrogen/deuterium exchange MS, fast photochemical oxidation of proteins, and carboxyl group footprinting with glycine ethyl ester, were further applied to determine BsAb's binding epitopes. This combination approach provides molecular details on the binding mechanisms of BsAb to the two distinct antigens with rapid output and high resolution.

Epitope and Paratope Mapping of PD-1/Nivolumab by Mass Spectrometry-Based Hydrogen-Deuterium Exchange, Cross-linking, and Molecular Docking.

Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen-deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking. HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the C'D-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.

Mapping binding epitopes of monoclonal antibodies targeting major histocompatibility complex class I chain-related A (MICA) with hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry.

Major histocompatibility complex class I chain-related A and B (MICA/B) are cell-surface proteins that act as ligands to natural killer cell receptors, NKG2D, expressed on immune cells. Prevention of proteolytic shedding of MICA/B to retain their integrity on the cell surface has become a therapeutic strategy in immuno-oncology. Given the unique mechanism of MICA/B shedding, structural characterization of MICA/B and therapeutic agent interaction is important in the drug discovery process. In this study, we describe the practical utility of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in epitope mapping studies of a cohort of four monoclonal antibodies targeting MICA in a rapid manner. HDX-MS followed by electron-transfer dissociation allows high-resolution refinement of binding epitopes. This integrated strategy offers, for the first time, molecular-level understanding of MICA's conformational dynamics in solution as well as the unique mechanism of actions of these antibodies in targeting MICA. Graphical abstract.

Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain with a Small Molecule Inhibitor.

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

An Integrated Approach for Determining a Protein-Protein Binding Interface in Solution and an Evaluation of Hydrogen-Deuterium Exchange Kinetics for Adjudicating Candidate Docking Models.

We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7Rα but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.

VISTA is an acidic pH-selective ligand for PSGL-1.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Conformational Assessment of Adnectin and Adnectin-Drug Conjugate by Hydrogen/Deuterium Exchange Mass Spectrometry.

Higher-order structure (HOS) characterization of therapeutic protein-drug conjugates for comprehensive assessment of conjugation-induced protein conformational changes is an important consideration in the biopharmaceutical industry to ensure proper behavior of protein therapeutics. In this study, conformational dynamics of a small therapeutic protein, adnectin 1, together with its drug conjugate were characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) with different spatial resolutions. Top-down HDX allows detailed assessment of the residue-level deuterium content in the payload conjugation region. HDX-MS dataset revealed the ability of peptide-based payload/linker to retain deuterium in HDX experiments. Combined results from intact, top-down, and bottom-up HDX indicated no significant conformational changes of adnectin 1 upon payload conjugation. Graphical Abstract ᅟ.

Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples.

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.