21 research outputs found
Development and Applications of Well-Defined Antibody and Antibody Fragment Bioconjugates
Antibodies have been widely used in many biological applications, including reseach tools, imaging, and therapeutics, due to their excellent binding specificity and affinity. As therapeutics, the ability to elicit immune effector functions and plasma half-life extension through the function of the constant domain (Fc) render antibodies superior to small molecules in these aspects. This work describes the use of chemical-based bioconjugation techniques to develop novel bioconjugates from antibody fragments and full-sized antibodies for therapeutic and imaging purposes. The complex structures of this class of biomolecules -- with the presence of multiple polypeptide chains, extensive disulfide networks, and critically important glycosylation patterns -- make it difficult to manipulate these biomolecules in a controlled manner. Thus, protein modification techniques play important roles in enabling the development of these constructs. In this work, we discuss the generation of Fc-synthetic molecule hybrids as antibody mimics, full-sized antibody-synthetic molecule conjugates as bispecific antibodies, and MS2-antibody conjugates as targeted delivery vehicles. The ability to conjugate different moieties into one molecule provides the ability to shuffle and combine synergistic advantages of the component molecules
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Development and Applications of Well-Defined Antibody and Antibody Fragment Bioconjugates
Antibodies have been widely used in many biological applications, including reseach tools, imaging, and therapeutics, due to their excellent binding specificity and affinity. As therapeutics, the ability to elicit immune effector functions and plasma half-life extension through the function of the constant domain (Fc) render antibodies superior to small molecules in these aspects. This work describes the use of chemical-based bioconjugation techniques to develop novel bioconjugates from antibody fragments and full-sized antibodies for therapeutic and imaging purposes. The complex structures of this class of biomolecules -- with the presence of multiple polypeptide chains, extensive disulfide networks, and critically important glycosylation patterns -- make it difficult to manipulate these biomolecules in a controlled manner. Thus, protein modification techniques play important roles in enabling the development of these constructs. In this work, we discuss the generation of Fc-synthetic molecule hybrids as antibody mimics, full-sized antibody-synthetic molecule conjugates as bispecific antibodies, and MS2-antibody conjugates as targeted delivery vehicles. The ability to conjugate different moieties into one molecule provides the ability to shuffle and combine synergistic advantages of the component molecules
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Targeted Molecular Imaging of Cancer Cells Using MS2-Based (129)Xe NMR.
We have synthesized targeted, selective, and highly sensitive (129)Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized (129)Xe NMR signal contrast and hyper-CEST (129)Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized (129)Xe NMR nanoscale biosensor for future applications in cancer detection in vivo
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Native and Denaturing MS Protein Deconvolution for Biopharma: Monoclonal Antibodies and Antibody-Drug Conjugates to Polydisperse Membrane Proteins and Beyond.
Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive [M + nH]n+ or negative [M - nH]n- depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of N-acetylglucosamine (GlcNAc; -203.1 Da)
Site-Specific Protein Transamination Using N‑Methylpyridinium-4-carboxaldehyde
The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-Methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild-type antibody or to modify both the heavy and the light chains after N-terminal sequence mutation to add additional glutamate residues
Optimization and Expansion of a Site-Selective <i>N</i>‑Methylpyridinium-4-carboxaldehyde-Mediated Transamination for Bacterially Expressed Proteins
Site-selective
bioconjugation methods are valuable because of their
ability to confer new properties to proteins by the chemical attachment
of specific functional groups. Well-defined bioconjugates obtained
through these methods have found utility for the study of protein
function and the creation of protein-based materials. We have previously
reported a protein modification strategy to modify the N-terminus
of peptides and proteins using <i>N</i>-methylpyridinium-4-carboxaldehyde
benzenesulfonate (Rapoport’s salt, RS) as a transamination
reagent, which oxidizes the N-terminal amino group to provide a uniquely
reactive aldehyde or ketone. This functional handle can subsequently
be modified with an alkoxyamine reagent of choice. Previous work had
found glutamate terminal sequences to be highly reactive toward RS-mediated
transamination. However, proteins of interest are often recombinantly
expressed in <i>E. coli</i>, where the expression of a glutamate-terminal
protein is rendered difficult because the N-terminal methionine derived
from the start codon is not cleaved when Glu is in the second position.
In this work, we describe a way to overcome this difficulty via the
insertion of a Factor Xa proteolytic cleavage site to acquire the
optimal glutamate residue at the N-terminus. Additionally, we present
studies on alternative high-yielding sequences containing N-terminal
residues that can be expressed directly. We have used site-directed
mutagenesis to validate these findings on a model cellulase enzyme,
an endoglucanase from the thermophilic <i>Pyrococcus horikoshii</i>. Activity assays performed with these mutants show that RS transamination
and subsequent modification with alkoxyamines have no negative impact
on cellulolytic ability
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High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond
Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z
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Biodistribution of Antibody-MS2 Viral Capsid Conjugates in Breast Cancer Models.
A variety of nanoscale scaffolds, including virus-like particles (VLPs), are being developed for biomedical applications; however, little information is available about their in vivo behavior. Targeted nanoparticles are particularly valuable as diagnostic and therapeutic carriers because they can increase the signal-to-background ratio of imaging agents, improve the efficacy of drugs, and reduce adverse effects by concentrating the therapeutic molecule in the region of interest. The genome-free capsid of bacteriophage MS2 has several features that make it well-suited for use in delivery applications, such as facile production and modification, the ability to display multiple copies of targeting ligands, and the capacity to deliver large payloads. Anti-EGFR antibodies were conjugated to MS2 capsids to construct nanoparticles targeted toward receptors overexpressed on breast cancer cells. The MS2 agents showed good stability in physiological conditions up to 2 days and specific binding to the targeted receptors in in vitro experiments. Capsids radiolabeled with 64Cu isotopes were injected into mice possessing tumor xenografts, and both positron emission tomography-computed tomography (PET/CT) and scintillation counting of the organs ex vivo were used to determine the localization of the agents. The capsids exhibit surprisingly long circulation times (10-15% ID/g in blood at 24 h) and moderate tumor uptake (2-5% ID/g). However, the targeting antibodies did not lead to increased uptake in vivo despite in vitro enhancements, suggesting that extravasation is a limiting factor for delivery to tumors by these particles
Targeted Molecular Imaging of Cancer Cells Using MS2-Based (129)Xe NMR.
We have synthesized targeted, selective, and highly sensitive (129)Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized (129)Xe NMR signal contrast and hyper-CEST (129)Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized (129)Xe NMR nanoscale biosensor for future applications in cancer detection in vivo
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Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting
The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics