Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.     

Site‐Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne–Azide Cycloaddition Reactions

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine‐reactive probes that target surface‐exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)‐binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site‐specifically labeled product. Z₃₂BPA was synthesized by solid‐phase peptide synthesis and further functionalized to give alkyne‐Z₃₂BPA and azide‐Z₃₂BPA for Cu^I‐catalyzed cycloaddition, as well as DBCO‐Z₃₂BPA for Cu‐free strain‐promoted cycloaddition. The Z₃₂BPA variants were conjugated to the human IgG1 antibody trastuzumab and site‐specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2‐expressing cells in immunofluorescence microscopy.     

Tuning a Protein‐Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site‐selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light‐chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50–70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro‐substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site‐selective lysine labeling of antibodies and other immunoglobulin‐type proteins.     

Fluorescent Visualization of Src by Using Dasatinib‐BODIPY

Many biological experiments are not compatible with the use of immunofluorescence, genetically encoded fluorescent tags, or FRET‐based reporters. Conjugation of existing kinase inhibitors to cell‐permeable fluorophores can provide a generalized approach to develop fluorescent probes of intracellular kinases. Here, we report the development of a small molecule probe of Src through conjugation of BODIPY to two well‐established dual Src‐Abl kinase inhibitors, dasatinib and saracatinib. We show that this approach is not successful for saracatinib but that dasatinib‐BODIPY largely retains the biological activity of its parent compound and can be used to monitor the presence of Src kinase in individual cells by flow cytometry. It can also be used to track the localization of Src by fixed and live‐cell fluorescence microscopy. This strategy could enable generation of additional kinase‐specific probes useful in systems not amenable to genetic manipulation or could be used together with fluorescent proteins to enable a multiplexed assay readout.     

DNA Primer Extension with Cyclopropenylated 7‐Deaza‐2′‐deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1‐methylcyclopropene (1MCP) group at the 7‐position of 7‐deaza‐2′‐deoxyadenosine and applied for primer extension experiments. The real‐time kinetic data reveals that this 1MCP‐modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP‐modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site‐specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.     

Tagging Glycoproteins with Fluorescently Labeled GDP‐Fucoses by Using α1,3‐Fucosyltransferases

Fucose‐containing glycans mediate a variety of biological processes, but there is little information on reaction processes and mechanisms mediated by fucosyltransferases. We recently reported on fluorescently labeled GDP‐β‐L ‐fucose‐ATTO 550, which enabled monitoring of α1,3‐fucosyltransferase activity. Here we present an extension to the previously described results, based on the synthesis of a fluorescein‐isothiocyanate (FITC)‐labeled and two carboxyfluorescein‐labeled (FAM‐labeled) NDP‐β‐L ‐fucose derivatives, and applied all four compounds in labeling of different glycoproteins with the aid of four different fucosyltransferases. The labeling processes were analyzed by in‐gel fluorescence and fluorescence polarization measurements. Comparison with the ATTO‐labeled sugar revealed that the FITC‐labeled fucose was the best of these substrates, and that the bacterial enzyme HP‐FucT tolerated the fluorescent substrates better than human fucosyltransferases.     

Efficient Acid‐Catalyzed 18F/19F Fluoride Exchange of BODIPY Dyes

Fluorine‐containing fluorochromes are important validation agents for positron emission tomography imaging compounds, as they can be readily validated in cells by fluorescence imaging. In particular, the ¹⁸F‐labeled BODIPY‐FL fluorophore has emerged as an important platform, but little is known about alternative ¹⁸F‐labeling strategies or labeling on red‐shifted fluorophores. In this study we explore acid‐catalyzed ¹⁸F/¹⁹F exchange on a range of commercially available N‐hydroxysuccinimidyl ester and maleimide BODIPY fluorophores. We show this method to be a simple and efficient ¹⁸F‐labeling strategy for a diverse span of fluorescent compounds, including a BODIPY‐modified PARP‐1 inhibitor, and amine‐ and thiol‐reactive BODIPY fluorophores.     

Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group

Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology.     

SNAP‐Tag‐Based Subcellular Protein Labeling and Fluorescent Imaging with Naphthalimides

Genetically encoded technologies provide methods for the specific labeling and imaging of proteins, which is essential to understand the subcellular localization of these proteins and their function. Herein, we employed naphthalimide, an efficient two‐photon fluorophore, to develop O⁶‐benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP‐tag. Three naphthalimide–BG derivatives, TNI‐BG, QNI‐BG, and ONI‐BG, were conveniently synthesized through modular “click chemistry” in high yields. All of them showed high labeling efficiency with SNAP‐tag in solution (≈1–2×10³ s^?1 m ^?1) and in bacteria. Among them, ONI‐BG showed high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP‐tag in mammalian cells. The protein‐labeled naphthalimides exhibited high two‐photon absorption cross‐sections, which indicated their potential application in protein‐specific two‐photon fluorescent imaging, such as two‐photon fluorescent lifetime imaging and two‐photon multicolor imaging. Therefore, ONI‐BG is a versatile tool that can be used to track subcellular proteins through multiple fluorescent techniques.     

Biosynthetic Labeling and Two‐Color Imaging of Phospholipids in Cells

Phospholipids with a choline head group are abundant components of all biological membranes, performing critical functions in cellular structure, metabolism, and signaling. In spite of their importance, our ability to visualize choline phospholipids in vivo remains very limited. We present a simple and robust chemical strategy to image choline phospholipids, based on the metabolic incorporation of azidocholine analogues, that accurately reflects the normal biosynthetic incorporation of choline into cellular phospholipids. Azidocholine‐labeled phospholipids can be imaged in cells with high sensitivity and resolution, following derivatization with fluorophores, by bio‐orthogonal chemical reactions compatible with live‐cell imaging. We used this method to visualize the subcellular localization of choline phospholipids. We also demonstrate that double metabolic labeling with azidocholine and propargylcholine allows sensitive two‐color imaging of choline phospholipids. Our method represents a powerful approach to directly image phospholipids, and to study their dynamics in cells and tissues.     

Antibody affinity maturation using bacterial surface display

A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A heavy-chain mutant having wild-type affinity (KD = 0.91+/-0.22 nM) and an expected representation frequency of less than 1 x 10(6), was selected to homogeneity after three rounds utilizing increasingly stringent selection conditions. The isolated clone possessed two distinct point mutations relative to the wild-type DNA sequence, yet still coded for the wild-type amino acid sequence, suggesting that the wild-type residues may be optimal at the randomized positions. An affinity improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant approximately threefold lower than the wild-type antibody, was isolated from a smaller light-chain library in a single sorting step. Flow cytometry was shown to be a simple and rapid method for the determination of the relative hapten dissociation rate constants of selected clones without requiring subcloning. The relative rate constants estimated by FACS were confirmed by producing the scFv antibodies in soluble form and measuring hapten binding kinetics by surface plasmon resonance (SPR). These results demonstrate that E.coli surface display, coupled with quantitative selection and analysis using FACS, has the potential to become a powerful tool for rapid isolation and characterization of desirable mutants from large polypeptide libraries.      

Biocompatible Azide–Alkyne “Click” Reactions for Surface Decoration of Glyco‐Engineered Cells

Bio‐orthogonal copper (I)‐catalyzed azide–alkyne cycloaddition (CuAAC) has been widely used to modify azide‐ or alkyne‐bearing monosaccharides on metabolic glyco‐engineered mammalian cells. Here, we present a systematic study to elucidate the design space for the cytotoxic effects of the copper catalyst on NIH 3T3 fibroblasts and on HEK 293‐F cells. Monitoring membrane integrity by flow cytometry and RT‐PCR analysis with apoptotic and anti‐apoptotic markers elucidated the general feasibility of CuAAC, with exposure time of the CuAAC reaction mixture having the major influence on biocompatibility. A high labeling efficiency of HEK 293‐F cells with a fluorescent alkyne dye was rapidly achieved by CuAAC in comparison to copper free strain‐promoted azide–alkyne cycloaddition (SPAAC). The study details effective and biocompatible conditions for CuAAC‐based modification of glyco‐engineered cells in comparison to its copper free alternative.     

Synthesis of pH‐ and solvent‐responsive smart core crosslinked star polymer by atom transfer radical polymerization

A highly fluorescent fluorescein dye labeled star shaped random copolymer with precise energy distribution was synthesized using atom transfer radical polymerization. The arm‐first strategy was utilized to achieve star architecture of the polymer. Dye labeling was carried out by azide‐alkyne cycloaddition. Acrylic acid and fluorescein dye both contributed to achieve a pH response. The synthesized polymer showed an attractive pH‐ and solvent‐sensitive fluorescence owing to efficient energy transfer from one fluorescent center to another. © 2014 Society of Chemical Industry     

DNA‐Templated Introduction of an Aldehyde Handle in Proteins

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site‐specific conjugates. This can rarely be achieved by simple residue‐specific random labeling, but generally requires genetic engineering. Using site‐selective DNA‐templated reductive amination, we created DNA–protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal‐binding functionality facilitates site‐selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal‐binding site. We demonstrate DNA‐templated reductive amination for His₆‐tagged proteins and metal‐binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps.     

A Practical NMR‐Based High‐Throughput Assay for Screening Enantioselective Catalysts and Biocatalysts

Two NMR‐based approaches for high‐throughput screening of enantioselective catalysts and biocatalysts are described. One version makes use of pseudo‐enantiomers or pseudo‐meso‐compounds based on ¹³C‐labeling. A throughput of at least 1400 ee determinations per day is possible by using an appropriate flow‐through cell and an autosampler. The other approach is based on traditional diastereomer formation using a chiral reagent or complexing agent. The ee values are accurate to within ±2% and ±5% of the true values.     

Covalent Protein Labeling by SpyTag–SpyCatcher in Fixed Cells for Super‐Resolution Microscopy

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag–SpyCatcher system as an epitope‐like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super‐resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform‐specific labeling.     

A Nanobody Activation Immunotherapeutic that Selectively Destroys HER2‐Positive Breast Cancer Cells

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti‐dinitrophenyl (anti‐DNP) antibodies to the surface of HER2‐positive breast cancer cells, resulting in their targeted destruction by antibody‐dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease‐relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.     

HER2‐ and EGFR‐Specific Affiprobes: Novel Recombinant Optical Probes for Cell Imaging

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell‐surface receptors/tyrosine kinases. EGFR‐ and HER2‐positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR‐ or HER2‐negative cancers. The details of HER2 proto‐oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009 , 315, 683–696). We have created photostable and relatively simple‐to‐produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2‐ or EGFR‐specific Affibody® molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell‐surface antigens/ receptors.     

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.     

Evaluation of UDP‐GlcN Derivatives for Selective Labeling of 5‐(Hydroxymethyl)cytosine

5‐(hydroxymethyl)cytosine (5‐hmC) is a newly identified oxidative product of 5‐methylcytosine (5‐mC) in the mammalian genome, and is believed to be an important epigenetic marker influencing a variety of biological processes. In addition to its relatively low abundance, the fluctuation of 5‐hmC levels over time during cell development poses a formidable challenge for its accurate mapping and quantification. Here we describe a specific chemoenzymatic approach to 5‐hmC detection in DNA samples by using new uridine 5′‐diphosphoglucosamine (UDP‐GlcN) probes. Our approach requires modification of the glucose moiety of UDP‐Glc with small amino groups and transfer of these glucose derivatives to the hydroxy moiety of 5‐hmC by using T4 phage glucosyltransferases. We evaluated the transfer efficiencies of three glucosyltransferases (wild‐type α‐ and β‐GTs and a Y261L mutant β‐GT) with five different UDP‐Glc derivatives containing functionalized groups for subsequent bioconjugation and detection. Our results indicate that UDP‐6‐N₃‐Glc, UDP‐6‐GlcN, and UDP‐2‐GlcN can be transferred by β‐GT with efficiencies similar to that seen with the native UDP‐Glc cofactor. 6‐N₃‐Glc‐ and 6‐GlcN‐containing oligonucleotides were selectively labeled with reactive fluorescent probes. In addition, a 2 kb DNA fragment modified with 2‐GlcN groups was specifically detected by use of a commercially available antiglucosamine antibody. Alternative substrates for β‐GT and correlated glycosyltransferases might prove useful for the study of the function and dynamics of 5‐hmC and other modified nucleotides, as well as for multiplex analysis.