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1.
Bioconjug Chem ; 32(8): 1834-1844, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34369158

RESUMO

Antibody-drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody-drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.


Assuntos
Especificidade de Anticorpos , Antineoplásicos/química , Imunoconjugados , Compostos de Sulfidrila/química , Anticorpos Monoclonais , Sítios de Ligação , Desenho de Fármacos , Modelos Moleculares , Conformação Proteica
2.
ACS Cent Sci ; 10(8): 1548-1561, 2024 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-39220705

RESUMO

Great progress has been made in the detection of large biomolecular analytes by native mass spectrometry; however, characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can separate overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on a Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications, the UHMR instrument can detect charge-reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

3.
J Am Soc Mass Spectrom ; 34(7): 1330-1341, 2023 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-37265400

RESUMO

Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography-mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.


Assuntos
Anticorpos Monoclonais , Imunoconjugados , Humanos , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Imunoconjugados/química , Cisteína/química , Compostos de Sulfidrila , Dissulfetos/química
4.
Chem Sci ; 14(35): 9316-9327, 2023 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-37712025

RESUMO

Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution. Here, we demonstrate that this paradigm breaks down for several types of macromolecular protein complexes due to the intrinsic heterogeneity induced by the stochastic nature of their assembly. Utilizing several protein assemblies of adeno-associated virus capsids and ferritin, we demonstrate that these particles can produce a variety of unexpected spectral appearances, some of which appear superficially similar to a resolved charge state distribution. When interpreted using conventional charge inference strategies, these distorted spectra can lead to substantial errors in the calculated mass (up to ∼5%). We provide a novel analytical framework to interpret and extract mass information from these spectra by combining high-resolution native mass spectrometry, single particle Orbitrap-based charge detection mass spectrometry, and sophisticated spectral simulations based on a stochastic assembly model. We uncover that these mass spectra are extremely sensitive to not only mass heterogeneity within the subunits, but also to the magnitude and width of their charge state distributions. As we postulate that many protein complexes assemble stochastically, this framework provides a generalizable solution, further extending the usability of native mass spectrometry in the characterization of biomacromolecular assemblies.

5.
MAbs ; 14(1): 2095701, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35799328

RESUMO

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.


Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Sítios de Ligação
6.
Commun Biol ; 4(1): 1241, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725463

RESUMO

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.


Assuntos
Antineoplásicos/farmacologia , Vesículas Extracelulares/fisiologia , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Linhagem Celular Tumoral , Humanos
7.
Nat Commun ; 11(1): 1816, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286330

RESUMO

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.


Assuntos
Agregados Proteicos , Sequência de Aminoácidos , Substituição de Aminoácidos , Regiões Determinantes de Complementaridade/química , Escherichia coli/metabolismo , Humanos , Imunoglobulina G/química , Viabilidade Microbiana , Mutação/genética , Anticorpos de Cadeia Única/química , beta-Lactamases/química
8.
J Am Soc Mass Spectrom ; 28(9): 1855-1862, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28484973

RESUMO

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein's size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstract ᅟ.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Proteínas/química , RNA/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Gases/química
9.
Nat Struct Mol Biol ; 23(9): 786-793, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27455461

RESUMO

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Cinética , Chaperonas Moleculares/fisiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína
10.
Sci Rep ; 6: 38644, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27995962

RESUMO

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais/farmacocinética , Fenômenos Biofísicos , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Hidrogênio , Camundongos , Mutação/genética , Especificidade de Órgãos , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Ratos , Espectrometria de Massas por Ionização por Electrospray , Propriedades de Superfície , Viscosidade
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