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.

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.

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.

Biased Signaling and Allosteric Modulation at the FSHR.

Knowledge on G protein-coupled receptor (GPCRs) structure and mechanism of activation has profoundly evolved over the past years. The way drugs targeting this family of receptors are discovered and used has also changed. Ligands appear to bind a growing number of GPCRs in a competitive or allosteric manner to elicit balanced signaling or biased signaling (i.e., differential efficacy in activating or inhibiting selective signaling pathway(s) compared to the reference ligand). These novel concepts and developments transform our understanding of the follicle-stimulating hormone (FSH) receptor (FSHR) biology and the way it could be pharmacologically modulated in the future. The FSHR is expressed in somatic cells of the gonads and plays a major role in reproduction. When compared to classical GPCRs, the FSHR exhibits intrinsic peculiarities, such as a very large NH2-terminal extracellular domain that binds a naturally heterogeneous, large heterodimeric glycoprotein, namely FSH. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα subunits. G protein-coupled receptor kinases and ß-arrestins are also recruited to this receptor and account for its desensitization, trafficking, and intracellular signaling. Different classes of pharmacological tools capable of biasing FSHR signaling have been reported and open promising prospects both in basic research and for therapeutic applications. Here we provide an updated review of the most salient peculiarities of FSHR signaling and its selective modulation.

MAbTope: A Method for Improved Epitope Mapping.

Abs are very efficient drugs, ∼70 of them are already approved for medical use, over 500 are in clinical development, and many more are in preclinical development. One important step in the characterization and protection of a therapeutic Ab is the determination of its cognate epitope. The gold standard is the three-dimensional structure of the Ab/Ag complex by crystallography or nuclear magnetic resonance spectroscopy. However, it remains a tedious task, and its outcome is uncertain. We have developed MAbTope, a docking-based prediction method of the epitope associated with straightforward experimental validation procedures. We show that MAbTope predicts the correct epitope for each of 129 tested examples of Ab/Ag complexes of known structure. We further validated this method through the successful determination, and experimental validation (using human embryonic kidney cells 293), of the epitopes recognized by two therapeutic Abs targeting TNF-α: certolizumab and golimumab.

Follicle-Stimulating Hormone Receptor: Advances and Remaining Challenges.

Follicle-stimulating hormone (FSH) is produced in the pituitary and is essential for reproduction. It specifically binds to a membrane receptor (FSHR) expressed in somatic cells of the gonads. The FSH/FSHR system presents many peculiarities compared to classical G protein-coupled receptors (GPCRs). FSH is a large naturally heterogeneous heterodimeric glycoprotein. The FSHR is characterized by a very large NH2-terminal extracellular domain, which binds FSH and participates to the activation/inactivation switch of the receptor. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα-subunits. GPCR kinases and ß-arrestins are also recruited to the FSHR and account for its desensitization, the control of its trafficking and its intracellular signaling. Of note, the FSHR internalization and recycling are very fast and involve very early endosomes (EE) instead of EE. All the transduction mechanisms triggered upon FSH stimulation lead to the activation of a complex signaling network that controls gene expression by acting at multiple levels. The integration of these mechanisms not only leads to context-adapted responses from the target gonadal cells but also indirectly affects the fate of germ cells. Depending on the physiological/developmental stage, FSH elicits proliferation, differentiation, or apoptosis in order to maintain the homeostasis of the reproductive system. Pharmacological tools targeting FSHR recently came to the fore and open promising prospects both for basic research and therapeutic applications. This chapter provides an updated review of the most salient aspects and peculiarities of FSHR biology and pharmacology.

G protein-dependent signaling triggers a ß-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

Many interaction partners of ß-arrestins intervene in the control of mRNA translation. However, how ß-arrestins regulate this cellular process has been poorly explored. In this study, we show that ß-arrestins constitutively assemble a p70S6K/ribosomal protein S6 (rpS6) complex in HEK293 cells and in primary Sertoli cells of the testis. We demonstrate that this interaction is direct, and experimentally validate the interaction interface between ß-arrestin 1 and p70S6K predicted by our docking algorithm. Like most GPCRs, the biological function of follicle-stimulating hormone receptor (FSHR) is transduced by G proteins and ß-arrestins. Upon follicle-stimulating hormone (FSH) stimulation, activation of G protein-dependent signaling enhances p70S6K activity within the ß-arrestin/p70S6K/rpS6 preassembled complex, which is not recruited to the FSHR. In agreement, FSH-induced rpS6 phosphorylation within the ß-arrestin scaffold was decreased in cells depleted of Gαs. Integration of the cooperative action of ß-arrestin and G proteins led to the translation of 5' oligopyrimidine track mRNA with high efficacy within minutes of FSH input. Hence, this work highlights new relationships between G proteins and ß-arrestins when acting cooperatively on a common signaling pathway, contrasting with their previously shown parallel action on the ERK MAP kinase pathway. In addition, this study provides insights into how GPCR can exert trophic effects in the cell.-Tréfier, A., Musnier, A., Landomiel, F., Bourquard, T., Boulo, T., Ayoub, M. A., León, K., Bruneau, G., Chevalier, M., Durand, G., Blache, M.-C., Inoue, A., Fontaine, J., Gauthier, C., Tesseraud, S., Reiter, E., Poupon, A., Crépieux, P. G protein-dependent signaling triggers a ß-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

ß-arrestin signalling and bias in hormone-responsive GPCRs.

G protein-coupled receptors (GPCRs) play crucial roles in the ability of target organs to respond to hormonal cues. GPCRs' activation mechanisms have long been considered as a two-state process connecting the agonist-bound receptor to heterotrimeric G proteins. This view is now challenged as mounting evidence point to GPCRs being connected to large arrays of transduction mechanisms involving heterotrimeric G proteins as well as other players. Amongst the G protein-independent transduction mechanisms, those elicited by ß-arrestins upon their recruitment to the active receptors are by far the best characterized and apply to most GPCRs. These concepts, in conjunction with remarkable advances made in the field of GPCR structural biology and biophysics, have supported the notion of ligand-selective signalling also known as pharmacological bias. Interestingly, recent reports have opened intriguing prospects to the way ß-arrestins control GPCR-mediated signalling in space and time within the cells. In the present paper, we review the existing evidence linking endocrine-related GPCRs to ß-arrestin recruitement, signalling, pathophysiological implications and selective activation by biased ligands and/or receptor modifications. Emerging concepts surrounding ß-arrestin-mediated transduction are discussed in the light of the peculiarities of endocrine systems.

Selection and characterization of recombinant dromedary antiovalbumin antibody fragments that do not cross-react with ovalbumin-related protein X: use for immunoaffinity.

Ovalbumin-related protein X (OVAX) and ovalbumin are two very close ovalbumin-related serpins. As primary data on OVAX remain recent, information about possible cross-reaction of available antiovalbumin antibodies with OVAX is still missing. Using labeled purified OVAX and dot ligand blotting, we identified 49 recombinant dromedary antiovalbumin single domain antibody (sdAb) fragments that were unable to bind OVAX. Discrimination between OVAX and ovalbumin was confirmed for two of the corresponding sdAb fragments by surface plasmon resonance and Western ligand blotting (WLB) characterizations. Furthermore, they were covalently linked to Sepharose and used as an affinity matrix for ovalbumin depletion. At least 90% of the original ovalbumin was eliminated from the allantoic fluid of 14 day old chicken embryo in one step. These sdAb fragments, which bind ovalbumin with nanomolar affinity, should also contribute to a better characterization of ovalbumin preparations.

Hypothalamic arcuate neuropeptide Y-neurons decrease periventricular somatostatin-neuronal activity before puberty in the female lamb: morphological arguments.

It is assumed that hypothalamic somatostatin plays a dominant role in the regulation of growth of developing lambs. On the other side, neuropeptide Y (NPY) neurons of the arcuate (ARC) nucleus are potentially involved in the control of gonadotrophins in prepubertal lambs and also of growth hormone (GH) secretion in adults. This study therefore investigated whether the transition from the prepubertal to the peripubertal period is accompanied by changes in NPY-ir and NPY mRNA content in neurons of the ARC nucleus and their putative projections to somatostatin neurons in both the ARC and periventricular (PEV) nuclei. The hypothalami of prepubertal (17-week-old) and peripubertal (32-week-old) female lambs were compared using single and double-labelling immunohistochemistry, and hybridisation in situ for NPY. Single-labelling for NPY mRNA and NPY-ir was quantified by image analysis using a light microscope and expressed as the percent area stained and/or the integral density of the reaction. Double-labelling for NPY-somatostatin relationships was analysed by confocal microscopy. Our data suggest that there are no detectable changes in NPY-ir in the PEV nucleus in the period leading up to puberty, whereas both the distributional area and intensity of NPY-labelling in the ARC are significantly higher in peripubertal compared to prepubertal sheep. In contrast, NPY mRNA levels are higher in prepubertal than in peripubertal ewes in the ARC nucleus. Confocal microscopy suggests the existence of NPY-somatostatin axo-somatic contacts in both PEV and ARC nuclei. In the PEV nucleus, the number of close appositions between NPY-ir fibres and somatostatin-ir perikarya is higher in prepubertal than in peripubertal ewes, but in the ARC no such difference was observed. In conclusion, our observations suggest that there is decreased activity of the NPY neurons of the ARC nucleus closely related to somatostatin neurons in the PEV nucleus at the onset of puberty. The withdrawal of this NPY effect may allow a higher release of somatostatin, which consequently inhibits GH secretion and stops growth. Both peptides are involved in the transmission of signals leading to stop growth at puberty.

A one-step exclusion-binding procedure for the purification of functional heavy-chain and mammalian-type gamma-globulins from camelid sera.

A new approach has recently been proposed for the purification of 'mammalian-type' IgG, consisting of exclusion binding. The technique uses a gel ('Melon gel'; Pierce) that binds to all plasma proteins, but not to IgGs, thus allowing IgGs to be recovered in the FT (flow-through) fraction. Here, the technique was applied to camelid IgGs, which are known to be composed of not only classic mammalian-type IgGs (IgG1) but also HC-IgGs (heavy chain IgGs). Both mammalian type and HC-IgGs can be purified in the FT fraction of dromedary (Camelus dromedarius) plasma samples with less than 8.5% contamination, by making minor improvements to the conditions recommended by the manufacturer. The contaminant proteins, as determined by LC-MS/MS (liquid chromatography-tandem MS), are mainly transferrin and albumin. The recovery rate is elevated for both types of IgGs (95+/-14% and 88+/-25% for IgG1 and HC-IgGs respectively). IgGs thus purified maintain their ability to bind to their antigen, as measured by surface plasmon resonance and Western ligand blotting. Furthermore, IgGs can be purified from plasma samples of all camelid species in a similar manner, although the ratio of HC-IgGs to total IgGs was lower for Lama (llama) and Vicugna (vicuña) than for Camelus species. The 'Melon gel' technique can thus be used to satisfactorily purify IgG1 and HC-IgGs from all camelid species.

A new method to discriminate immunogen-specific heavy-chain homodimer from heterotetramer immunoglobulin G directly in immunized dromedary whole plasma proteins: Western ligand blotting.

The variable domain of heavy-chain camelid antibodies (VHH), exclusively present in the homodimer IgGs (HC IgG), provides valuable ligands for diagnosis, imaging and therapy. These VHHs are efficiently produced from lymphocytes of immunized animals through phage display and recombination biotechnology. For VHH development it is desirable to identify animals with high affinity HC IgG response by monitoring antigen-binding in the course of immunization. The aim of this study was to propose a direct approach on whole plasma samples to distinguish between homodimer IgG and heterotetramer (IgG(1)) responses, and quantify them, using western ligand blotting (WLB). WLB consists here in electrophoretic separation of the target IgG subclasses on the basis of molecular size and binding of (125)I labeled antigen. First, we established the WLB parameters for titration of antigen-binding homodimers in relation to antigen-binding total IgGs in ovalbumin-immunized dromedary plasma samples and demonstrated that the WLB is an alternative to ELISA for IgG subclass titration. As purification of IgG subclasses or availability of IgG subclass-specific antibodies is not necessary, WLB is more direct and practical for screening a large number of samples. Second, WLB was applied to study the pattern of homodimer and heterotetramer responses during the time-course of immunizations against three different types of immunogens. As these patterns can differ between animals and immunogens, the method may be useful for identifying animals displaying the desired antigen-specific homodimer IgG response. Lastly, WLB was also described in its variant form of dot ligand blotting for identifying antigen-binding phages at the final step of a phage display experiment.

Prepubertal changes in the synthesis, storage and release of the somatostatin in the hypothalamus of female lambs: a morphofunctional study.

It is assumed that hypothalamic somatostatin plays a role in the preovulatory phase of the oestrous cycle in sheep. The aim of the study was to investigate the processes of synthesis, storage and release of somatostatin in hypothalamic neurons, in immature female lambs, in the period approaching to puberty. Experiments were carried out on 10 prepubertal (17 weeks old) and 10 peripubertal (32 weeks old) ovary-intact lambs. Morphofunctional changes in the somatostatin neurons were assayed with immunohistochemistry and hybridisation in situ. Computer image analysis was used to determine the density of both reactions and the percentage of the area exhibiting immunohistochemical staining. These parameters express the content of immunoreactive (ir) somatostatin and expression of mRNA for pre-pro-somatostatin (PPS). Two populations of ir somatostatin perikarya were localized in the hypothalamus: a very large number of perikarya in the periventricular (PEV) nucleus, and single cell bodies in the arcuate (ARC) nucleus. Only ir somatostatin fibres, but no perikarya were seen in the ventromedial (VM) nucleus and preoptic area. The analysis of mRNA PPS showed perikarya filled with silver grains localized in the PEV, ARC and VM. There were differences in the content of ir somatostatin and the intensity of the PPS mRNA signal between the two periods investigated. In the median eminence, the content of ir somatostatin in the terminals decreased in the peripubertal compared to the prepubertal group (P<0.001). In the PEV, the content of ir somatostatin in the perikarya and the expression of PPS mRNA decreased in the peripubertal compared to the prepubertal group (P<0.001). In the ARC, the content of ir somatostatin in the perikarya increased (P<0.001), but expression of PPS mRNA decreased (P<0.001) in the peripubertal compared to the prepubertal group. There were no differences in the expression of PPS mRNA in the VM. We concluded, that the different secretory activity of the two hypothalamic populations of somatostatin neurons can be related to their different physiological functions in the prepubertal period of female lambs.

Regulation by estradiol of hypothalamic somatostatin gene expression: possible involvement of somatostatin in the control of luteinizing hormone secretion in the ewe.

In the ewe, the mediobasal hypothalamus (MBH) is the primary central site for estradiol to generate the preovulatory GnRH/LH surges and sexual behavior. This area contains numerous neurons expressing the estradiol receptor alpha, distributed in the ventromedial nucleus (VMN) and the infundibular nucleus (IN). A large proportion of these neurons express somatostatin, making this neuropeptide a potential candidate for transmission of the estradiol signal to the GnRH neurons located in the preoptic area. We tested this hypothesis using ovariectomized ewes that had been subjected to an artificial estrous cycle. In the first experiment, 22 h after progesterone removal, ewes received estradiol (treated ewes) or empty implants (control ewes) for 4 h and then were killed. Using in situ hybridization, we showed that this short estradiol treatment increased the somatostatin mRNA amount by about 50% in the VMN and 42% in the IN. In the second experiment, preovulatory estradiol signal was replaced by somatostatin intracerebroventricular (ICV) administration. This treatment abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion while it did not affect the mean plasma GH concentration. We demonstrated that an increase in somatostatin mRNA occurs at the time of the negative feedback effect of estradiol on LH secretion during the early stage of the GnRH surge induction. As ICV somatostatin administration inhibits the pulsatile LH secretion by acting on the central nervous system, we suggest that somatostatin synthesized in the MBH could be involved in the estradiol negative feedback before the onset of the preovulatory surge.

Early decrease of proopiomelanocortin but not neuropeptide Y mRNA expression in the mediobasal hypothalamus of the ewe, during the estradiol-induced preovulatory LH surge.

In sheep, the mediobasal hypothalamus (MBH) has been shown to be the primary central site of estradiol (E2) action that induces both the preovulatory surge and sexual behaviour. However, the nature of the neurotransmitters or neuromodulators synthesized in the MBH during E2 stimulation remains to be clearly defined. After the cloning of the ovine cDNA sequences and using in situ hybridization, hypothalamic proopiomelanocortin (POMC), and preproneuropeptide Y (preproNPY) mRNA expression was studied in ovariectomized ewes that received a sequential treatment of progesterone and E2. As we showed that an exposition to E2 only for 4h well in advance on the LH surge onset is sufficient to induce the preovulatory surge and estrous behaviour, mRNA expression was evaluated in ewes treated with 6x30-mm E2 implants (experimental group) or with empty implants (control group) and slaughtered 4h after the start of the E2 treatment. Our results demonstrate that this short E2 treatment significantly decreased both the mean number of silver grains per POMC-containing cell (35%) and the mean number of POMC-cells (38%) in the ovine infundibular nucleus, whereas the treatment had no effect on preproNPY mRNA expression. These observations suggest that a reduction of POMC gene transcription could participate to the early neural mechanism of E2 feedback.