Agonistic anti-NKG2D antibody structure reveals unique stoichiometry and epitope compared to natural ligands.

Natural killer (NK) cells are effector cells of the innate immune system that distinguish between healthy and abnormal cells through activating and inhibitory receptor signaling. NKG2D, a homodimeric activating receptor expressed on NK cells, recognizes a diverse class of stress ligands expressed by cells experiencing infection, malignant transformation, chronic inflammation, and other cellular stresses. Despite the variety of NKG2D ligands, they all bind the receptor asymmetrically in a 1:1 ligand to homodimeric NKG2D stoichiometry.

Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.

Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus.

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature.

The emergence of AntibodyPlus: the future trend of antibody-based therapeutics.

To date, close to 100 canonical monoclonal antibody drugs have been approved by the FDA; furthermore, a number of antibody-derived therapeutics in nontraditional formats have reached late development stages and the market, and many more are being evaluated in early-stage development. To better reflect this trend and to set up a framework for forward thinking, we herein introduce the concept of AntibodyPlus, embracing any therapeutics with an antibody component. AntibodyPlus therapeutics contain effector modules, in the form of small molecules, nucleic acids, proteins or even cells, to enhance their therapeutic activities against cancer, virus infection and other diseases.

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping.

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group.

A Mass Spectrometric Characterization of Light-Induced Modifications in Therapeutic Proteins.

During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins.

Light-Controllable Binary Switch Activation of CAR T Cells.

Major challenges to chimeric antigen receptor (CAR) T cell therapies include uncontrolled immune activity, off-tumor toxicities and tumor heterogeneity. To overcome these challenges, we engineered CARs directed against small molecules. By conjugating the same small molecule to distinct tumor-targeting antibodies, we show that small molecule specific-CAR T cells can be redirected to different tumor antigens.

Polyreactivity and polyspecificity in therapeutic antibody development: risk factors for failure in preclinical and clinical development campaigns.

Antibody-based drugs, which now represent the dominant biologic therapeutic modality, are used to modulate disparate signaling pathways across diverse disease indications. One fundamental premise that has driven this therapeutic antibody revolution is the belief that each monoclonal antibody exhibits exquisitely specific binding to a single-drug target. Herein, we review emerging evidence in antibody off-target binding and relate current key findings to the risk of failure in therapeutic development.

Site-specific conjugation of native antibody.

Traditionally, non-specific chemical conjugation, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity determining region (CDR) may reduce binding affinity and specificity.

Site-specific Bioconjugation and Convergent Click Chemistry Enhances Antibody-Chromophore Conjugate Binding Efficiency.

Photosensitizer (PS)-antibody conjugates (photoimmunoconjugates, PICs) enable cancer cell-targeted photodynamic therapy (PDT). Nonspecific chemical bioconjugation is widely used to synthesize PICs but gives rise to several shortcomings. The conjugates are heterogeneous, and the process is not easily reproducible.

Site-Specific Reversible Protein and Peptide Modification: Transglutaminase-Catalyzed Glutamine Conjugation and Bioorthogonal Light-Mediated Removal.

Dynamic photoswitches in proteins that impart spatial and temporal control are important to manipulate and study biotic and abiotic processes. Nonetheless, approaches to install these switches into proteins site-specifically are limited. Herein we describe a novel site-specific method to generate photoremovable protein conjugates.

Detection of Alkynes via Click Chemistry with a Brominated Coumarin Azide by Simultaneous Fluorescence and Isotopic Signatures in Mass Spectrometry.

Alkynes are a key component of click chemistry and used for a wide variety of applications including bioconjugation, selective tagging of protein modifications, and labeling of metabolites and drug targets. However, challenges still exist for detecting alkynes because most 1,2,3-triazole products from alkynes and azides do not possess distinct intrinsic properties that can be used for their facile detection by either fluorescence or mass spectrometry. To address this critical need, a novel brominated coumarin azide was used to tag alkynes and detect alkyne-conjugated biomolecules.

Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.

The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g.

Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method.

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study.

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.

Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification.

When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media.

Synthesis and characterization of -adenosyl-L-selenohomocysteine selenoxide.

Selenium is an essential micronutrient in humans due to the important roles of the selenocysteine-containing selenoproteins. Organoselenium metabolites are generally found to be substrates for the biochemical pathways of their sulfur analogs, and the redox chemistry of selenomethionine and some other metabolites have been previously reported. We now report the first synthesis and characterization of -adenosylselenohomocysteine selenoxide (SeAHO) prepared via hydrogen peroxide oxidation of -adenosylselenohomocysteine (SeAH).

Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody.

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert.

Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization.

The formation of aspartyl succinimide is a common post-translational modification of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry.

Discovery and characterization of a photo-oxidative histidine-histidine cross-link in IgG1 antibody utilizing ¹⁸O-labeling and mass spectrometry.

A novel photo-oxidative cross-linking between two histidines (His-His) has been discovered and characterized in an IgG1 antibody via the workflow of XChem-Finder, (18)O labeling and mass spectrometry (Anal. Chem. 2013, 85, 5900-5908).

Decreased glutathione and elevated hair mercury levels are associated with nutritional deficiency-based autism in Oman.

Genetic, nutrition, and environmental factors have each been implicated as sources of risk for autism. Oxidative stress, including low plasma levels of the antioxidant glutathione, has been reported by numerous autism studies, which can disrupt methylation-dependent epigenetic regulation of gene expression with neurodevelopmental consequences. We investigated the status of redox and methylation metabolites, as well as the level of protein homocysteinylation and hair mercury levels, in autistic and neurotypical control Omani children, who were previously shown to exhibit significant nutritional deficiencies in serum folate and vitamin B₁₂.

Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species.

Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors but still cannot completely account for all heterogeneity.