An intracellular VHH targeting the Luteinizing Hormone receptor modulates G protein-dependent signaling and steroidogenesis.

Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor ß-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.

A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling.

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR.

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

Highly-Sensitive In Vitro Bioassays for FSH, TSH, PTH, Kp, and OT in Addition to LH in Mouse Leydig Tumor Cell.

We demonstrate here that highly sensitive in vitro bioassays for FSH, TSH, and PTH can be set up in mouse Leydig Tumor Cells (mLTC), in addition to the normal LH/CG bioassay, after they were transfected with expression vectors encoding the corresponding Gs Protein-Coupled Receptors (GsPCR), such as FSHR, TSHR, or PTHR. Although the ß2 adrenergic receptor is also a GsPCR, its expression in mLTC led to a significant but very low cAMP response compared to those observed with FSH, TSH, or PTH. Similarly, after transfection of the GiPCR MT1 melatonin receptor, we did not observe any inhibitory effect by melatonin of the LH or hCG stimulation. Interestingly, after transfection of mLTC with the human kisspeptin receptor (hKpR), which is a GqPCR, we observed a dose-dependent synergy of 10-12-10-7 M kisspeptin variants with a fixed concentration of 0.3 nM LH or hCG. Without any exogenous receptor transfection, a 2 h preincubation with OT or AVP led to a dose-dependent cAMP response to a fixed dose of LH or hCG. Therefore, highly sensitive in vitro bioassays for various hormones and other GPCR ligands can be set up in mLTC to measure circulating concentrations in only 3-10 µL of blood or other body fluids. Nevertheless, the development of an LHRKO mLTC cell line will be mandatory to obtain strict specificity for these bioassays to eliminate potential cross-reaction with LH or CG.

Intracellular VHHs to monitor and modulate GPCR signaling.

Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or ß-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery.

A New in Silico Antibody Similarity Measure Both Identifies Large Sets of Epitope Binders with Distinct CDRs and Accurately Predicts Off-Target Reactivity.

Developing a therapeutic antibody is a long, tedious, and expensive process. Many obstacles need to be overcome, such as biophysical properties (issues of solubility, stability, weak production yields, etc.), as well as cross-reactivity and subsequent toxicity, which are major issues. No in silico method exists today to solve such issues. We hypothesized that if we were able to properly measure the similarity between the CDRs of antibodies (Ab) by considering not only their evolutionary proximity (sequence identity) but also their structural features, we would be able to identify families of Ab recognizing similar epitopes. As a consequence, Ab within the family would share the property to recognize their targets, which would allow (i) to identify off-targets and forecast the cross-reactions, and (ii) to identify new Ab specific for a given target. Testing our method on 238D2, an antagonistic anti-CXCR4 nanobody, we were able to find new nanobodies against CXCR4 and to identify influenza hemagglutinin as an off-target of 238D2.

Serodolin, a ß-arrestin-biased ligand of 5-HT7 receptor, attenuates pain-related behaviors.

G protein­coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT7 receptor (5-HT7R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a ß-arrestin­dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT7R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT7R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT7R stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. In this study, using a ß-arrestin­biased agonist, we provided insight into the molecular mechanism triggered by 5-HT7R and revealed its therapeutic potential in the modulation of pain response.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

Accurate determination of epitope for antibodies with unknown 3D structures.

MAbTope is a docking-based method for the determination of epitopes. It has been used to successfully determine the epitopes of antibodies with known 3D structures. However, during the antibody discovery process, this structural information is rarely available. Although we already have evidence that homology models of antibodies could be used instead of their 3D structure, the choice of the template, the methodology for homology modeling and the resulting performance still have to be clarified. Here, we show that MAbTope has the same performance when working with homology models of the antibodies as compared to crystallographic structures. Moreover, we show that even low-quality models can be used. We applied MAbTope to determine the epitope of dupilumab, an anti- interleukin 4 receptor alpha subunit therapeutic antibody of unknown 3D structure, that we validated experimentally. Finally, we show how the MAbTope-determined epitopes for a series of antibodies targeting the same protein can be used to predict competitions, and demonstrate the accuracy with an experimentally validated example.3D: three-dimensionalRMSD: root mean square deviationCDR: complementary-determining regionCPU: central processing unitsVH: heavy chain variable regionVL: light chain variable regionscFv: single-chain variable fragmentsVHH: single-chain antibody variable regionIL4Rα: Interleukin 4 receptor alpha chainSPR: surface plasmon resonancePDB: protein data bankHEK293: Human embryonic kidney 293 cellsEDTA: Ethylenediaminetetraacetic acidFBS: Fetal bovine serumANOVA: Analysis of varianceEGFR: Epidermal growth factor receptorPE: PhycoerythrinAPC: AllophycocyaninFSC: forward scatterSSC: side scatterWT: wild typeKeywords: MAbTope, Epitope Mapping, Molecular docking, Antibody modeling, Antibody-antigen docking.