The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic ...collaborations. The path from the initial applications probing biochemical interaction in vitro to “state-of-the-art” cellular assays toward clinical applications using homogeneous time-resolved fluorescence technology is described. An overview of the luminescent lanthanide macrocyclic compounds developed at Cisbio in the recent past is given with an emphasis on specific constraints required by specific applications. Recent assays for drug-discovery and diagnostic purposes using both antibody-based and suicide-enzyme-based technology are illustrated. New perspectives in the field of molecular medicine and time-resolved microscopy are discussed.
Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. ...Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.
Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated ...the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence.
We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival.
The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003).
Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.
The epidermal growth factor receptor (EGFR) is a cell-surface receptor with a single transmembrane domain and tyrosine kinase activity carried by the intracellular domain. This receptor is one of the ...four members of the ErbB family including ErbB2, ErbB3, and ErbB4. Ligand binding, like EGF binding, induces a conformational rearrangement of the receptor and induces a homo/hetero dimerization essentially with ErbB family receptors that leads to the phosphorylation of the kinase domain, triggering a signaling cascade. EGFR can also form inactive dimers in a ligand-independent way through interactions between cytoplasmic domains. To date, the conformation of EGFR extracellular domain engaged in these inactive dimers remains unclear. In this study, we describe the successful selection and characterization of llama anti-EGFR nanobodies and their use as innovative conformational sensors. We isolated three different specific anti-EGFR clones binding to three distinct epitopes. Interestingly, the binding of all three nanobodies was found highly sensitive to ligand stimulation. Two nanobodies, D10 and E10, can only bind the ligand-free EGFR conformation characterized by an intramolecular tether between domains II and IV, whereas nanobody G10 binds both ligand-free and ligand activated EGFR, with an 8-fold higher affinity for the extended conformation in the presence of ligand. Here we took advantage of these conformational probes to reveal the existence of tethered EGFR in EGFR/ErbB2 predimers. These biosensors represent important tools allowing the determination of EGFR conformations and should help the design of relevant inhibitors.
In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not ...fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.
Phospholipase C β (PLC-β)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca
2+ triggered by
d-
myo-inositol 1,4,5-trisphosphate (IP
3), a PLC-β ...hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP
3 production was also established using IP
3 binding proteins. The short lifetime of IP
3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP
3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to
d-
myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP
3 metabolic cascade. We show here that IP
1 can be used as a surrogate of IP
3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP–One assay was validated on various GPCR models. It has the advantage over the traditional Ca
2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-β activity in any nontransfected primary cultures. Finally, the high assay specificity for
d-
myo-inositol 1 monophosphate (IP
1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.
Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their ...expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients' treatment in oncology.
Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water‐soluble, and ...negatively charged sulfonic‐ or carboxylic acid derivatives of para‐substituted aryl–alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell‐surface proteins with time‐resolved Förster resonance energy transfer (TR‐FRET) assays in plate‐based format and using TR‐FRET microscopy.
Bright and selective: Hydrophilic EuIII complexes have been synthesized that suppress nonspecific binding and permit their use in bioconjugates using time‐resolved assays, as exemplified for G protein‐coupled receptor antagonists.
Synthesis, absorption spectra and luminescebce properties of a series of lanthanide trisbipyridine cryptates Ln within R-Bpy x R-Bpy x R-Bpy, where Ln = Eu, Gd and R = H, COOH, COOCH3, CONH(CH2)2NH2 ...are described. Comparison of the unsubstituted parent compound with the substituted compounds shows that bipyridine substitution doesn't alter significantly the photophysical properties of the lanthanide cryptate. The absorption maximum is slightly red-shifted when three bipyridines are substituted, whereas substituting one bipyridines has a negligible effect on the absorption spectra. The experimental triplet state energy is between 21600 and 22 100 cm(-1) for the series of compounds and the luminescence lifetimes at 77 K are between 0.5 and 0.8 ms in HO2 and equal to 1.7 ms in D2O. The experimental characterizations are completed by DFT and TD-DFT calculations to assess the ability of these approaches to predict absorption maxima, triplet state energies and structural parameters of lanthanide cryptates and to characterize the electronic structure of the excited states. The calculations on the unsubstituted parent and substituted compounds show that absorption maxima and lowest 3pipi* triplet state energies can be accurately determined from density functional theory (DFT) and time-dependent (TD) DFT calculations.
We previously developed a technique known as homogeneous time-resolved fluorescence (HTRF) which has become a reference method in the design of bioassays. The technique makes use of the fluorescence ...resonance energy transfer (FRET) depending of the close proximity between europium cryptate, as a long-lived luminescent donor, and a matching fluorescent acceptor. The extension of this technique to
in vivo cellular imaging is possible by the labeling of specific molecules within a living cell, each labeled either with the donor or acceptor and taking images of fluorescence resonance energy transfer (FRET). The time-resolved mode allows to discriminate the signal arising from molecules in close proximity from the background fluorescence of excess amounts of unbound fluorescent marker and from cell autofluorescence. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay upon direct excitation. The decay is also much slower than cell autofluorescence, and interference of cell autofluorescence is reduced under a time-resolved fluorescence microscope with a time-gated function equipped camera. The technique was evaluated both under a time-resolved fluorescence microscope and a microplate TRF reader.
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