Accepted Manuscript Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors Ronan Crepin, Gianluca Veggiani, Selma Djender, Anne Beugnet, François Planeix, Christophe Pichon, Sandrine Moutel, Sebastian Amigorena, Franck Perez, Nicolae Ghinea, Ario de Marco PII:
S0006-291X(17)32006-5
DOI:
10.1016/j.bbrc.2017.10.036
Reference:
YBBRC 38653
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 2 October 2017 Accepted Date: 6 October 2017
Please cite this article as: R. Crepin, G. Veggiani, S. Djender, A. Beugnet, Franç. Planeix, C. Pichon, S. Moutel, S. Amigorena, F. Perez, N. Ghinea, A. de Marco, Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors
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Ronan Crepin1,6, Gianluca Veggiani1, Selma Djender1,6, Anne Beugnet1,6, François Planeix2, Christophe Pichon2, Sandrine Moutel1,2, Sebastian Amigorena3,5,6, Franck
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Perez4, Nicolae Ghinea2, Ario de Marco1,5,6,7
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Tumor target and therapeutic antibody - Identification Platform (TAb-IP), PSL Research University, Institut Curie, 26, rue d'Ulm, Paris, France 2
Translational Research Department, PSL Research University, Institut Curie, 26 rue d'Ulm, F75248 Paris Cedex 05, France 3
INSERM U932, PSL Research University, Institut Curie, 26 rue d'Ulm, F75248 Paris
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UMR144, PSL Research University, Institut Curie, 12 Lhomond, 75005, Paris, France SIRIC INCa-DGOS-4654
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CIC IGR Curie 1428
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Dept. of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9 - SI-5261, Vipava, Slovenia
Corresponding author: Ario de Marco, Dept. of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9 - SI-5261, Vipava, Slovenia. [email protected] – Phone: +386 (05) 9099700, Fax: +386 (05) 90 99 722
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ACCEPTED MANUSCRIPT Abstract Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical
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characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so
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far. In this paper we describe the first successful attempt to recover recombinant
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antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the
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proposed biopanning strategy will be profitable to identify useful functional antibodies
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for further members of the GPCR class.
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Keywords: allosteric inhibitor, recombinant antibody production, cAMP-dependent signaling, GPCR, phage display
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ACCEPTED MANUSCRIPT Introduction Follicle-stimulating hormone receptor (FSHR) is a GPCR membrane protein that plays a pivotal role in ovarian follicular maturation in females and in spermatogenesis
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in males. The structure of its ectodomain in complex with its specific ligand FSH was recently described and provided the mechanistic explanation of the two-step interaction between the hormone and its receptor [1]. According to this model, the ligand is first recruited by the high-affinity FSHR subdomain and this binding induces
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a FSH conformational rearrangement that forms the pocket necessary to interact with
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the activating sulfotyrosine at the position 335. Several groups proposed antibodybased therapies aimed at inhibiting the FSHR-dependent signal cascade as a strategy for preventing fertility status [2-4]. The interest for antibodies is justified by their elevated antigen specificity, a condition necessary to prevent unwanted side effects. For instance, whereas FSH, luteinizing and thyrotropin hormones partially
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cross-react with the related receptors, in at least one case some agonist autoantibodies showed absolute receptor specificity [5]. The applicative potential of
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anti-FSHR antibodies was demonstrated in different models. The cAMP production in cell lines overexpressing the FSH receptor was successfully antagonized by
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antibodies raised against its N-term [2] and its activity in vivo was neutralized by antibodies directed against the thumb region close to the active sulfotyrosine [4]. Beside its physiological relevance, FSHR activity has been deeply investigated for its role in osteoporosis and during the development of different tumor pathologies [6-9]. Recently, blocking polyclonal antibodies specific for the FSH β-subunit have been proposed as a remedy against bone resorption due to their success in animal models [10,11]. FSHR antagonist antibodies have been also suggested for inhibiting the development of ovarian cancer [12], whereas neutral anti-FSHR antibodies have 3
ACCEPTED MANUSCRIPT been mainly exploited for the diagnostics of prostate and ovarian tumors [7,13]. Systematic immunohistochemistry analyses allowed for the identification of the receptor overexpression in the peripheral vessel of most of the human tumors and suggest
its
role
in
angiogenesis
[9,14-17].
Nevertheless,
in
pancreatic
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neuroendocrine tumors FSHR seems to localize exclusively inside neoplastic cells, with no expression in tumor blood vessels [18]. Clearly, some contradictory results can rely on the reagent quality and their reliability in the different applications, as it is
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in the case of the antibody used in [18] and that is recommended by the suppliers
suitable for different techniques.
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only for IP and WB. This observation underlines the need to generate new antibodies
Antibodies have been conventionally recovered after animal immunization with the target antigen. This approach exploits the in vivo somatic maturation process and is strongly driven by one or few prevalent immunogenic sequences. This condition
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might prevent the isolation of antibodies directed against functionally relevant but non-immunogenic epitopes. Furthermore, the recombinant protein used for immunization can be itself partially misfolded or aggregated and influence the
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immunogenic response. In contrast, in vitro selection of recombinant antibodies by biopanning against receptors expressed on the surface of eukaryotic cells enables
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the isolation of antibodies that recognize their antigens in their native conformation inside the natural membrane environment and after having undergone the physiological
post-translational
modifications.
Consequently,
the
antibodies
recovered by such approach have increased chances to be functional when used in vivo, as we have demonstrated in more systems [19-21] and exploiting different preimmune nanobody libraries [22,23].
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ACCEPTED MANUSCRIPT We adopted the whole cell-panning strategy and used a synthetic library [23] to isolate several single-domain antibodies (known as VHHs or nanobodies) against FSHR. Their capacity to inhibit the receptor activity and the FSH-dependent signal cascade was evaluated and compared with the activity of the monoclonal antibody
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323 (produced in our laboratory as previously described [24]) that was successfully used in the past to identify FSHR in paraffin tissue samples [9]. This is the first report illustrating the possibility to screen antibody pre-immune libraries for the direct
Cell Culture
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Materials and Methods
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isolation of inhibitory binders of GPCR molecules.
Mammalian adherent cells (L, L-FSHR, HEK293) were grown in DMEM-Glutamax medium (Gibco) containing 10% FBS complemented with penicillin/streptomycin
CO2.
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(Invitrogen) and were cultured at 37°C in a humidified atmosphere enriched in 5%
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Selection of FSHR-specific VHHs by on cell panning
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Humanized nanobodies from the synthetic phage library NaLi-H1 [23] were first preadsorbed on wild type control murine L cells and the unbound fraction was successively incubated with L cells permanently expressing the human FSHR receptor. Two panning protocols were compared. In the first case, 3x1012 phages were saturated in 1 ml of PBS, 2% BSA before incubation in the presence of 5x106 control cells resuspended with accutase (Life Technologies) for 1 hour at 4°C under gentle agitation. The unbound fraction was used for challenging 5x106 L-FSHR cells during one hour at 4°C under gentle agitation in a total volume of 1ml of PBS,
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ACCEPTED MANUSCRIPT 2%BSA. Cells were finally washed 10 times in 10 ml of PBS and recovered each time by centrifugation at 400xg for 5 min. Phages were eluted with 100 mM triethylamine and neutralized with a solution of 1M Tris-HCl, pH7.4. Bound phages were amplified for another round of selection by infection of Escherichia coli TG1 strain. Three
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rounds of selection were performed before clone analysis. In the second case, some modifications were introduced since only 3x1011 phages were used, two depletion cycles on control cells were performed, unbound phages were finally centrifuged
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once more at 1000xg for 10 min to spin down aggregates, and the enrichment step was performed incubating the supernatant in the presence of only 5x104 L-FSHR
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cells. Cells were transferred into a Multiscreen® Durapore 0,65 µm hydrophilic low protein binding plate (Millipore), washed 10 times with 10 ml of PBS and phages were eluted as above. Three rounds of selection were performed also in this second case.
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Positive binders screening
Soluble VHHs were expressed from bacteria single colonies grown in 96-well plates
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and induced with 1 mM IPTG over night as previously described [22]. Two hundred thousands of either control L or L-FSHR cells were detached with Stempro Accutase
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(Invitrogen) and resuspended in 100 µl of PBS, 1%FBS at 4°C (FACS buffer). One hundred µl of crude bacterial culture supernatant were added to the cells and incubated 2 hours at 4°C. Cells were washed with FACS buffer and VHHs bound to cells were detected with an anti-c-myc antibody (9E10, Sigma) followed by goat antimouse immunoglobulins conjugated to phycoerythrin (PE, BD). Cells were analyzed with FACSCalibur Flow Cytometer (BD) using CellQuest Pro Software for mean fluorescence intensity (MFI) calculation. The diversity of selected clones was
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ACCEPTED MANUSCRIPT determined by sequencing with the M13 rev primer: 5’- CAG GAA ACA GCT ATG ACC ATG -3’. Antibody subcloning and production
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The VHH cDNA was NcoI/NotI digested from pHEN2 and subcloned into the pET14brFc/hFc vectors to express reconstituted IgG-like constructs with the Fc fragment of rabbit or human immunoglobulin, respectively [20]. Recombinant antibodies were
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produced in E. coli BL21 (DE3) expressing sulfhydryl oxidase and DsbC and then purified as described in [22]. Antibody concentration was evaluated by UV
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absorbance at 280 nm. SDS-PAGE gels (10%) were Coomassie stained and recorded using a Gel Doc™ EZ System (Bio Rad). Antibody specificity characterization
Antigen-antibody specific recognition was tested by flow cytometry using 2x105
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control and L-FSHR cells detached with Stempro Accutase and resuspended in 100 µl of FACS buffer. Incubation steps were performed at 4°C. Antibody specific binding
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was assessed in 96 well plates using either 0.1 µg/ml of VHH-rFc and Alexa Fluor 647-conjugated goat anti-rabbit conjugated secondary antibody (Invitrogen), or
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antibody-displaying phages saturated in PBS, 10% milk in combination with anti-M13 monoclonal antibody and anti-mouse PE-conjugated antibodies (BD). For competition experiments, cells were pre-incubated with increasing concentrations of either 323 or FSH (3H Biomedical) before the antibody addition. GraphPad software was used for the fitting of the binding data and for calculating the apparent affinity of the titration experiments. Mouse monoclonal antibody 323 [24] was used as the positive control for FSHR in combination with anti-mouse immunoglobulins conjugated to PE. FSHR accumulation at the cell surface was obtained by culturing two days HEK293 cells 7
ACCEPTED MANUSCRIPT transfected with the plasmid pSG5-FSHR [24]. Transfection efficiency was monitored by flow cytometry (FACSCalibur, BD). Soluble FSHR ectodomain expression
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The cDNA of the FSHR ectodomain (amino acids 18-361) was synthesized by Proteogenix and inserted NcoI/NotI into the pFuse-rFc vector [25]. FSHR was collected from the culture medium of HEK293 cell transfected with jetPEI (Polyplus)
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and grown 4 days in serum-free medium. FSHR-Fc was purified from the medium on HiTrap MabSelect SuRe (1 ml) column and its concentration was evaluated by
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reading the UV absorbance at 280nm. SDS-PAGE gels (10%) were Coomassie stained and recorded using a Gel Doc™ EZ System (Bio Rad). Evaluation of cyclic AMP response after stimulation with FSH The activation of the FSHR signaling pathway in HEK293 cells was monitored by
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quantifying cAMP accumulation with the cAMP pGloSensor™-22F kit (Promega) according to manufacturer’s recommendations. Briefly HEK293 cells were co-
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transfected with plasmids pSG5-FSHR and pGloSensor™-22F. After 2 days, cells were resuspended with accutase and seeded in a white 96 well plate at 5x105 cells
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per well. After 4 hours incubation at 37°C, cells were equilibrated with GloSensor™ reagent (Promega) for 1 hour at RT before antibody addition. Cells were incubated 30 min before stimulating the receptor activity by adding 10 nM FSH (3H Biomedical) and luminescence was measured after 60 or 90 min. ELISA test, Western blot and Surface Plasmon Resonance (SPR) analyses
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ACCEPTED MANUSCRIPT ELISA, Western Blot and SPR analyses are described in details in the Supplementary Materials. Results
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The antibodies specific for L cell epitopes were depleted from the synthetic NaLi-H1 phage display library by incubation in the presence of 5 million of wild type cells. The unbound fraction was challenged with the same amount of L cells overexpressing FSHR to select anti-FSHR specific antibodies. Three biopanning cycles were
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performed and two different specific antibodies were isolated by screening 96
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colonies. The result was encouraging but inferior to the average outputs obtained in our lab using other cell systems. We reasoned that the low expression level of FSHR at the surface of L cells in comparison to other major cell surface proteins (data not shown) decreased the chance to bind specific antibodies. Consequently, their dilution among the antibodies binding to irrelevant antigens would have made difficult their
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recovery. To overcome this drawback, we introduced some modifications in the panning protocol intended to reduce the proportion of non-specific antibodies present
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in the final elution fraction: the initial number of phages was decreased to 3x1011, the depletion steps were doubled, and the number of cells used for enrichment was
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limited to 50 000. Three further clones were recovered by applying this strategy. The target specificity of the five selected VHHs was confirmed by FACS using wt and FSHR-overexpressing L cells (Fig. 1). The monoclonal antibody 323 and HGX44, a VHH antibody directed against the GFP protein, were used as a positive and negative control, respectively. Recombinant VHHs can be easily engineered into reagents suitable for specific applications. The five validated antibodies were sub-cloned into a vector that enables the expression of VHH-rFc constructs which dimerize into IgG-like antibodies when 9
ACCEPTED MANUSCRIPT produced in the bacterial cytoplasm in the presence of sulfhydryl oxidase and DsbC (Fig. 2A). Such reconstituted dimeric antibodies are detectable using conventional anti-rabbit secondary antibodies, bind to Protein A, and have higher avidity than the monovalent VHHs from which they are generated [25]. The yield of such antibodies
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was in the range of 5-15 mg/l culture and their specificity was validated by ELISA using the purified ectodomain of FSHR (Figs. 2B and 2C) and by FACS using cells overexpressing FSHR (Suppl. Figs 1).
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The apparent affinity of the IgG-like antibodies was calculated by FACS and found to be in the middle-higher nanomolar range (Fig. 2D). We considered this approach
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more indicative of an in vivo condition than using methods based on ELISA in which the antigen structure may be altered by the interaction with the plastic surface or on SPR performed using the recombinant ectodomain of FSHR-Fc which is prone to lose its native conformation during the purification procedure. This is because VHHs
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–mostly when selected under native conditions, as in this case- preferentially recognize conformational epitopes that can be compromised by coating recombinant antigens on surfaces. As a comparison, the apparent affinity of the monoclonal
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antibody 323 measured by FACS was in the low nanomolar range (Fig. 2D), whereas its affinity estimated using a 2:1 model applied to the SPR results and that take into
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account the IgG bivalency was 4x10-8 (Fig. 2E). These values are in agreement with previous measurements that showed a 10 fold avidity-dependent increase of the apparent binding capacity for bivalent antibodies [20]. This characterization step allowed confirming the binding specificity of the selected antibodies and their effectivity after reconstitution into IgG-like format. However, since their apparent affinity for FSHR seemed significantly lower than that of the monoclonal 323, the practical utility of such antibodies could be questioned. To
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ACCEPTED MANUSCRIPT address this issue, we measured the effects of the recombinant antibodies on the FSH-dependent cAMP accumulation. A significant reduction was observed for three of the tested clones (Fig. 3A). The recombinant antibody with the highest inhibitory effect (B1) was used to establish a concentration-dependent inhibition curve of cAMP
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accumulation. The incubation time was reduced to enable lower initial cAMP concentrations and to evidence minor amount deviations. Surprisingly, the monovalent VHH performed even better than the IgG-like antibody (Figs. 3B and 3C).
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This result suggested that nanobodies have a different inhibition mechanism.
Previous results reported that 323 competed with the natural ligand FSH [20] for the
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same binding site and this would explain its inhibitory antagonist activity. In contrast, our experimental data (Fig. 4) clearly showed that none of the recombinant antibodies recognized an epitope overlapping with that of 323. Cells were preincubated with 323 at 50 µg/ml (~0.33 µM) at 4°C before the addition of the VHH-Fc
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at 0.5 µg/mL (~6.25 nM). Considering the apparent KD of 323 (Fig. 2D), at this concentration the cell receptors should be totally saturated by the monoclonal antibody and consequently the contemporary detection of both 323 and VHH-Fc
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without MFI decrease indicates the absence of competition. Similarly, no clear competition was observed on L-FSHR cells between the recombinant antibodies and
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FSH. Cells were pre-incubated at increasing FSH concentrations (0-1.32 µM) and the phage-displayed antibodies (monovalent) were added successively (Suppl. Fig. 2). Despite FSH affinity for FSHR is in the low nanomolar range [26] and therefore probably almost three logs higher than that of the VHHs, nanobodies were not significantly displaced by FSH. Altogether, these results suggest that the inhibitory mechanism of the recombinant antibodies might be allosteric rather than antagonistic or inverse agonistic.
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ACCEPTED MANUSCRIPT Discussion Mouse immunization with FSHR ectodomain resulted in the production of sets of monoclonal antibodies all directed against an immunodominant region initially identified in the amino acid sequence 172-358 [24]. Since these antibodies have
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usually antagonist behavior, it is probable that their actual epitope overlaps the sequence 285-309 that corresponds to the region bound by the hormone FSH [4]. Only the immunization with decapeptides corresponding to the N-terminal FSHR
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ectodomain allowed for the production of agonist antibodies with clear stimulatory effect [2]. Otherwise, despite the increasing interest for FSHR in oncological
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diagnostic for both immunohistochemistry and imaging applications [9,14-17,27], no other antibody has been characterized for its binding features and has been proposed for in vivo treatment or to deliver either drugs or imaging probes. The main limit of treatments performed using inverse agonist or antagonist binders is
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that administrations must be repeated frequently to maintain a sufficient concentration to compete with natural binders. For this reason, allosteric molecules
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are preferred. In the case of FSHR, small molecules and peptide that act as positive allosteric modulators [28-30] as well as a biased negative allosteric antagonist [31]
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have recently been described. Since for allosteric molecules the specificity is more critical than affinity, the antibodies described in this work would appear good candidates for more rigorous functional tests also considering that the possibility to produce them either as mono- or bivalent molecules could be useful to assess the role of glycoprotein hormone receptor dimerization [32]. Furthermore, producing functional IgG-like antibodies in bacteria [20] enables to cut significantly the costs for the pre-clinical characterization of lead binders. The present yields obtained using simple flasks and not-optimized medium could be largely increased under rigorous 12
ACCEPTED MANUSCRIPT fermentation conditions. Our data represent also the first report in which antibodies able to modulate the activity of a GPCR protein have been isolated in vitro from a synthetic library and it will be worthy to demonstrate that this strategy is effective also
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for GPCRs with smaller ectodomains. Acknowledgments
The authors acknowledge COPIO/ITMO-TS/Aviesan, Région île de France, CEST-
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95020 (Institut Curie), CIC IGR-Curie 1428, and Labex DCBIOL (ANR-10-IDEX0001-02 PSL* - ANR-11-LABEX-0043) for their financial support and Nathalie
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Amzallag for her management. The authors would like to thank Jean-Christophe Rain (Hybrigenics Service, Paris, France) for the kind gift of HGX44. This work has received support under the program «Investissements d’Avenir » launched by the French Government and implemented by ANR with the references ANR-11-LABEX-
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0043 and ANR-10-IDEX-0001-02 PSL. Literature
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[2] L. Abdennebi, L. Couture, D. Grebert, et al., Generating FSH antagonists and agonists through immunization against FSH receptor N-terminal decapeptides. J. Mol. Endocrinol. 22 (1999) 151-159. [3] A.J. Rao, S.G. Ramachandra, V. Ramesh, et al., Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor. Reprod. Biomed. Online 8 (2004) 385-391.
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ACCEPTED MANUSCRIPT [4] J.D. Ghosalkar, S.J. Dharma, T.D. Nandedkar, S.D. Mahale, Identification of the region 285-309 of follicle stimulating hormone receptor as a bioneutralizing epitope. J. Reprod. Immunol. 74 (2007) 24-33. [5] M. Tonacchera, E. Ferrarini, A. Dimida, et al., Effects of a thyroid-stimulating
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ACCEPTED MANUSCRIPT [13] S. Heublein, T. Vrekoussis, D. Mayr, et al., Her-2/neu expression is a negative prognosticator in ovarian cancer cases that do not express the follicle stimulating hormone receptor (FSHR). J. Ovarian Res. 6 (2013) 6. [14] M.A Siraj, C. Pichon, A. Radu, N. Ghinea, Endothelial follicle stimulating
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ACCEPTED MANUSCRIPT [21] C. Ronan, D. Gentien, A. Duché, et al., Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma Patient Derived Xenografts. Pigment Cell Melanoma Res. 30 (2017) 317-327. [22] A. Monegal, D. Ami, C. Martinelli, et al. Immunological applications of single
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[24] B. Vannier, H. Loosfelt, G. Meduri, C. Pichon, E. Milgrom, Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor. Biochemistry 35 (1996) 1358-1366. [25] S. Moutel, A. El Marjou, O. Vielemeyer, et al., A multi-Fc species system for
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recombinant antibody production. BMC Biotechnol. 9 (2009) 14. [24] J.L Tilly, T. Aihara, K. Nishimori, et al., Expression of recombinant human folliclestimulating hormone receptor: species-specific ligand binding, signal transduction,
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ACCEPTED MANUSCRIPT [27] B.J. Arey, S.D. Yanofsky, M.C. Pérez, et al., Differing pharmacological activities of thiazolidinone analogs at the FSH receptor. Biochem. Biophys. Res. Commun. 368 (2008) 723-728. [28] C.J. van Koppen, P.M. Verbost, R. van de Lagemaat, et al., Signaling of an
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(2005) 1954-1964.
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Recombinant VHH antibodies bind to the FSH receptor expressed on L cells Five antibodies recovered by biopanning (A8, B1, C1, E6, E8) were screened by
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FACS for their specific recognition of the FSH receptor by assessing their binding to control cells (L) and cells overexpressing the receptor (L-FSHR). HGX44 is an antiGFP VHH (negative control) and 323 is an anti-FSHR monoclonal antibody (positive
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control)
Figure 2. Characterization of the reconstituted IgG-like recombinant anti-FSHR
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antibodies
A) The engineered VHH-Fc fusion was purified and analyzed by PAGE in both reducing and oxidizing conditions to show the molecule dimerization. B) Recombinant FSHR produced as a soluble ectodomain fused to an Fc domain and as full-length
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protein. The apparent mass of the full-length FSHR (>200 kDa) indicates probable glycosylation and stable dimerization. C) Antigen specificity of five anti-FSHR antibodies and one anti-GFP control antibody measured by ELISA using purified
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recombinant FSHR-Fc ectodomain and control proteins (HER2-Fc and BSA); bars represent standard deviations. D) FACS-assessed apparent affinity of anti-FSHR
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antibodies compared with negative (HGX44, anti-GFP) and positive (323, monoclonal anti-FSHR) controls. E) SPR-measured affinity of the monoclonal 323 anti-FSHR antibody
Figure 3. Antibody-induced inhibition of FSH-dependent cAMP accumulation in transfected HEK293 cells A) The FSH-dependent cAMP accumulation was measured in the presence of five anti-FSHR antibodies. The cAMP concentration measured in the absence of antibody
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ACCEPTED MANUSCRIPT addition (only DMEM medium) represented the absolute reference. The anti-GFP HGX44 was the negative control and the monoclonal anti-FSHR 323 the positive control. B) Concentration-dependent inhibition of the cAMP accumulation in the presence of the monovalent VHH B1 antibody. C) Inhibition of FSH-dependent
(B1-Fc) antibody. Bars represent standard deviations
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signaling assessed by cAMP accumulation in the presence of the bivalent IgG-like B1
Figure 4. Recombinant antibodies and monoclonal 323 recognize independent
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epitopes on the FSHR surface
The bidimensional FACS analysis indicates that none of the five recombinant
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antibodies competes with the monoclonal antibody 323 for the same epitope on the FSHR surface. A) L-FSHR cells incubated with 323 at 50 µg/ml. B) L-FSHR cells incubated with Fc-VHH at 0.50 µg/ml. C) L-FSHR cells pre-incubated with 323 for
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Highlights
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First successful panning against a GPCR using whole cells and a synthetic library Isolation of nanobodies which inhibit FSH-dependent cAMP accumulation Identification of nanobodies which are not competitors of FSH Immunoreagents for studying FSHR-induced angiogenesis in cancer
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• • • •
S0006-291X(17)32006-5
DOI:
10.1016/j.bbrc.2017.10.036
Reference:
YBBRC 38653
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 2 October 2017 Accepted Date: 6 October 2017
Please cite this article as: R. Crepin, G. Veggiani, S. Djender, A. Beugnet, Franç. Planeix, C. Pichon, S. Moutel, S. Amigorena, F. Perez, N. Ghinea, A. de Marco, Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors
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Ronan Crepin1,6, Gianluca Veggiani1, Selma Djender1,6, Anne Beugnet1,6, François Planeix2, Christophe Pichon2, Sandrine Moutel1,2, Sebastian Amigorena3,5,6, Franck
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Perez4, Nicolae Ghinea2, Ario de Marco1,5,6,7
1
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Tumor target and therapeutic antibody - Identification Platform (TAb-IP), PSL Research University, Institut Curie, 26, rue d'Ulm, Paris, France 2
Translational Research Department, PSL Research University, Institut Curie, 26 rue d'Ulm, F75248 Paris Cedex 05, France 3
INSERM U932, PSL Research University, Institut Curie, 26 rue d'Ulm, F75248 Paris
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5
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UMR144, PSL Research University, Institut Curie, 12 Lhomond, 75005, Paris, France SIRIC INCa-DGOS-4654
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7
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CIC IGR Curie 1428
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Dept. of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9 - SI-5261, Vipava, Slovenia
Corresponding author: Ario de Marco, Dept. of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9 - SI-5261, Vipava, Slovenia. [email protected] – Phone: +386 (05) 9099700, Fax: +386 (05) 90 99 722
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ACCEPTED MANUSCRIPT Abstract Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical
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characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so
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far. In this paper we describe the first successful attempt to recover recombinant
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antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the
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proposed biopanning strategy will be profitable to identify useful functional antibodies
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for further members of the GPCR class.
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Keywords: allosteric inhibitor, recombinant antibody production, cAMP-dependent signaling, GPCR, phage display
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ACCEPTED MANUSCRIPT Introduction Follicle-stimulating hormone receptor (FSHR) is a GPCR membrane protein that plays a pivotal role in ovarian follicular maturation in females and in spermatogenesis
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in males. The structure of its ectodomain in complex with its specific ligand FSH was recently described and provided the mechanistic explanation of the two-step interaction between the hormone and its receptor [1]. According to this model, the ligand is first recruited by the high-affinity FSHR subdomain and this binding induces
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a FSH conformational rearrangement that forms the pocket necessary to interact with
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the activating sulfotyrosine at the position 335. Several groups proposed antibodybased therapies aimed at inhibiting the FSHR-dependent signal cascade as a strategy for preventing fertility status [2-4]. The interest for antibodies is justified by their elevated antigen specificity, a condition necessary to prevent unwanted side effects. For instance, whereas FSH, luteinizing and thyrotropin hormones partially
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cross-react with the related receptors, in at least one case some agonist autoantibodies showed absolute receptor specificity [5]. The applicative potential of
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anti-FSHR antibodies was demonstrated in different models. The cAMP production in cell lines overexpressing the FSH receptor was successfully antagonized by
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antibodies raised against its N-term [2] and its activity in vivo was neutralized by antibodies directed against the thumb region close to the active sulfotyrosine [4]. Beside its physiological relevance, FSHR activity has been deeply investigated for its role in osteoporosis and during the development of different tumor pathologies [6-9]. Recently, blocking polyclonal antibodies specific for the FSH β-subunit have been proposed as a remedy against bone resorption due to their success in animal models [10,11]. FSHR antagonist antibodies have been also suggested for inhibiting the development of ovarian cancer [12], whereas neutral anti-FSHR antibodies have 3
ACCEPTED MANUSCRIPT been mainly exploited for the diagnostics of prostate and ovarian tumors [7,13]. Systematic immunohistochemistry analyses allowed for the identification of the receptor overexpression in the peripheral vessel of most of the human tumors and suggest
its
role
in
angiogenesis
[9,14-17].
Nevertheless,
in
pancreatic
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neuroendocrine tumors FSHR seems to localize exclusively inside neoplastic cells, with no expression in tumor blood vessels [18]. Clearly, some contradictory results can rely on the reagent quality and their reliability in the different applications, as it is
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in the case of the antibody used in [18] and that is recommended by the suppliers
suitable for different techniques.
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only for IP and WB. This observation underlines the need to generate new antibodies
Antibodies have been conventionally recovered after animal immunization with the target antigen. This approach exploits the in vivo somatic maturation process and is strongly driven by one or few prevalent immunogenic sequences. This condition
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might prevent the isolation of antibodies directed against functionally relevant but non-immunogenic epitopes. Furthermore, the recombinant protein used for immunization can be itself partially misfolded or aggregated and influence the
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immunogenic response. In contrast, in vitro selection of recombinant antibodies by biopanning against receptors expressed on the surface of eukaryotic cells enables
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the isolation of antibodies that recognize their antigens in their native conformation inside the natural membrane environment and after having undergone the physiological
post-translational
modifications.
Consequently,
the
antibodies
recovered by such approach have increased chances to be functional when used in vivo, as we have demonstrated in more systems [19-21] and exploiting different preimmune nanobody libraries [22,23].
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ACCEPTED MANUSCRIPT We adopted the whole cell-panning strategy and used a synthetic library [23] to isolate several single-domain antibodies (known as VHHs or nanobodies) against FSHR. Their capacity to inhibit the receptor activity and the FSH-dependent signal cascade was evaluated and compared with the activity of the monoclonal antibody
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323 (produced in our laboratory as previously described [24]) that was successfully used in the past to identify FSHR in paraffin tissue samples [9]. This is the first report illustrating the possibility to screen antibody pre-immune libraries for the direct
Cell Culture
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Materials and Methods
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isolation of inhibitory binders of GPCR molecules.
Mammalian adherent cells (L, L-FSHR, HEK293) were grown in DMEM-Glutamax medium (Gibco) containing 10% FBS complemented with penicillin/streptomycin
CO2.
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(Invitrogen) and were cultured at 37°C in a humidified atmosphere enriched in 5%
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Selection of FSHR-specific VHHs by on cell panning
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Humanized nanobodies from the synthetic phage library NaLi-H1 [23] were first preadsorbed on wild type control murine L cells and the unbound fraction was successively incubated with L cells permanently expressing the human FSHR receptor. Two panning protocols were compared. In the first case, 3x1012 phages were saturated in 1 ml of PBS, 2% BSA before incubation in the presence of 5x106 control cells resuspended with accutase (Life Technologies) for 1 hour at 4°C under gentle agitation. The unbound fraction was used for challenging 5x106 L-FSHR cells during one hour at 4°C under gentle agitation in a total volume of 1ml of PBS,
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ACCEPTED MANUSCRIPT 2%BSA. Cells were finally washed 10 times in 10 ml of PBS and recovered each time by centrifugation at 400xg for 5 min. Phages were eluted with 100 mM triethylamine and neutralized with a solution of 1M Tris-HCl, pH7.4. Bound phages were amplified for another round of selection by infection of Escherichia coli TG1 strain. Three
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rounds of selection were performed before clone analysis. In the second case, some modifications were introduced since only 3x1011 phages were used, two depletion cycles on control cells were performed, unbound phages were finally centrifuged
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once more at 1000xg for 10 min to spin down aggregates, and the enrichment step was performed incubating the supernatant in the presence of only 5x104 L-FSHR
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cells. Cells were transferred into a Multiscreen® Durapore 0,65 µm hydrophilic low protein binding plate (Millipore), washed 10 times with 10 ml of PBS and phages were eluted as above. Three rounds of selection were performed also in this second case.
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Positive binders screening
Soluble VHHs were expressed from bacteria single colonies grown in 96-well plates
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and induced with 1 mM IPTG over night as previously described [22]. Two hundred thousands of either control L or L-FSHR cells were detached with Stempro Accutase
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(Invitrogen) and resuspended in 100 µl of PBS, 1%FBS at 4°C (FACS buffer). One hundred µl of crude bacterial culture supernatant were added to the cells and incubated 2 hours at 4°C. Cells were washed with FACS buffer and VHHs bound to cells were detected with an anti-c-myc antibody (9E10, Sigma) followed by goat antimouse immunoglobulins conjugated to phycoerythrin (PE, BD). Cells were analyzed with FACSCalibur Flow Cytometer (BD) using CellQuest Pro Software for mean fluorescence intensity (MFI) calculation. The diversity of selected clones was
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ACCEPTED MANUSCRIPT determined by sequencing with the M13 rev primer: 5’- CAG GAA ACA GCT ATG ACC ATG -3’. Antibody subcloning and production
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The VHH cDNA was NcoI/NotI digested from pHEN2 and subcloned into the pET14brFc/hFc vectors to express reconstituted IgG-like constructs with the Fc fragment of rabbit or human immunoglobulin, respectively [20]. Recombinant antibodies were
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produced in E. coli BL21 (DE3) expressing sulfhydryl oxidase and DsbC and then purified as described in [22]. Antibody concentration was evaluated by UV
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absorbance at 280 nm. SDS-PAGE gels (10%) were Coomassie stained and recorded using a Gel Doc™ EZ System (Bio Rad). Antibody specificity characterization
Antigen-antibody specific recognition was tested by flow cytometry using 2x105
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control and L-FSHR cells detached with Stempro Accutase and resuspended in 100 µl of FACS buffer. Incubation steps were performed at 4°C. Antibody specific binding
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was assessed in 96 well plates using either 0.1 µg/ml of VHH-rFc and Alexa Fluor 647-conjugated goat anti-rabbit conjugated secondary antibody (Invitrogen), or
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antibody-displaying phages saturated in PBS, 10% milk in combination with anti-M13 monoclonal antibody and anti-mouse PE-conjugated antibodies (BD). For competition experiments, cells were pre-incubated with increasing concentrations of either 323 or FSH (3H Biomedical) before the antibody addition. GraphPad software was used for the fitting of the binding data and for calculating the apparent affinity of the titration experiments. Mouse monoclonal antibody 323 [24] was used as the positive control for FSHR in combination with anti-mouse immunoglobulins conjugated to PE. FSHR accumulation at the cell surface was obtained by culturing two days HEK293 cells 7
ACCEPTED MANUSCRIPT transfected with the plasmid pSG5-FSHR [24]. Transfection efficiency was monitored by flow cytometry (FACSCalibur, BD). Soluble FSHR ectodomain expression
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The cDNA of the FSHR ectodomain (amino acids 18-361) was synthesized by Proteogenix and inserted NcoI/NotI into the pFuse-rFc vector [25]. FSHR was collected from the culture medium of HEK293 cell transfected with jetPEI (Polyplus)
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and grown 4 days in serum-free medium. FSHR-Fc was purified from the medium on HiTrap MabSelect SuRe (1 ml) column and its concentration was evaluated by
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reading the UV absorbance at 280nm. SDS-PAGE gels (10%) were Coomassie stained and recorded using a Gel Doc™ EZ System (Bio Rad). Evaluation of cyclic AMP response after stimulation with FSH The activation of the FSHR signaling pathway in HEK293 cells was monitored by
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quantifying cAMP accumulation with the cAMP pGloSensor™-22F kit (Promega) according to manufacturer’s recommendations. Briefly HEK293 cells were co-
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transfected with plasmids pSG5-FSHR and pGloSensor™-22F. After 2 days, cells were resuspended with accutase and seeded in a white 96 well plate at 5x105 cells
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per well. After 4 hours incubation at 37°C, cells were equilibrated with GloSensor™ reagent (Promega) for 1 hour at RT before antibody addition. Cells were incubated 30 min before stimulating the receptor activity by adding 10 nM FSH (3H Biomedical) and luminescence was measured after 60 or 90 min. ELISA test, Western blot and Surface Plasmon Resonance (SPR) analyses
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ACCEPTED MANUSCRIPT ELISA, Western Blot and SPR analyses are described in details in the Supplementary Materials. Results
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The antibodies specific for L cell epitopes were depleted from the synthetic NaLi-H1 phage display library by incubation in the presence of 5 million of wild type cells. The unbound fraction was challenged with the same amount of L cells overexpressing FSHR to select anti-FSHR specific antibodies. Three biopanning cycles were
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performed and two different specific antibodies were isolated by screening 96
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colonies. The result was encouraging but inferior to the average outputs obtained in our lab using other cell systems. We reasoned that the low expression level of FSHR at the surface of L cells in comparison to other major cell surface proteins (data not shown) decreased the chance to bind specific antibodies. Consequently, their dilution among the antibodies binding to irrelevant antigens would have made difficult their
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recovery. To overcome this drawback, we introduced some modifications in the panning protocol intended to reduce the proportion of non-specific antibodies present
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in the final elution fraction: the initial number of phages was decreased to 3x1011, the depletion steps were doubled, and the number of cells used for enrichment was
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limited to 50 000. Three further clones were recovered by applying this strategy. The target specificity of the five selected VHHs was confirmed by FACS using wt and FSHR-overexpressing L cells (Fig. 1). The monoclonal antibody 323 and HGX44, a VHH antibody directed against the GFP protein, were used as a positive and negative control, respectively. Recombinant VHHs can be easily engineered into reagents suitable for specific applications. The five validated antibodies were sub-cloned into a vector that enables the expression of VHH-rFc constructs which dimerize into IgG-like antibodies when 9
ACCEPTED MANUSCRIPT produced in the bacterial cytoplasm in the presence of sulfhydryl oxidase and DsbC (Fig. 2A). Such reconstituted dimeric antibodies are detectable using conventional anti-rabbit secondary antibodies, bind to Protein A, and have higher avidity than the monovalent VHHs from which they are generated [25]. The yield of such antibodies
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was in the range of 5-15 mg/l culture and their specificity was validated by ELISA using the purified ectodomain of FSHR (Figs. 2B and 2C) and by FACS using cells overexpressing FSHR (Suppl. Figs 1).
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The apparent affinity of the IgG-like antibodies was calculated by FACS and found to be in the middle-higher nanomolar range (Fig. 2D). We considered this approach
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more indicative of an in vivo condition than using methods based on ELISA in which the antigen structure may be altered by the interaction with the plastic surface or on SPR performed using the recombinant ectodomain of FSHR-Fc which is prone to lose its native conformation during the purification procedure. This is because VHHs
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–mostly when selected under native conditions, as in this case- preferentially recognize conformational epitopes that can be compromised by coating recombinant antigens on surfaces. As a comparison, the apparent affinity of the monoclonal
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antibody 323 measured by FACS was in the low nanomolar range (Fig. 2D), whereas its affinity estimated using a 2:1 model applied to the SPR results and that take into
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account the IgG bivalency was 4x10-8 (Fig. 2E). These values are in agreement with previous measurements that showed a 10 fold avidity-dependent increase of the apparent binding capacity for bivalent antibodies [20]. This characterization step allowed confirming the binding specificity of the selected antibodies and their effectivity after reconstitution into IgG-like format. However, since their apparent affinity for FSHR seemed significantly lower than that of the monoclonal 323, the practical utility of such antibodies could be questioned. To
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ACCEPTED MANUSCRIPT address this issue, we measured the effects of the recombinant antibodies on the FSH-dependent cAMP accumulation. A significant reduction was observed for three of the tested clones (Fig. 3A). The recombinant antibody with the highest inhibitory effect (B1) was used to establish a concentration-dependent inhibition curve of cAMP
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accumulation. The incubation time was reduced to enable lower initial cAMP concentrations and to evidence minor amount deviations. Surprisingly, the monovalent VHH performed even better than the IgG-like antibody (Figs. 3B and 3C).
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This result suggested that nanobodies have a different inhibition mechanism.
Previous results reported that 323 competed with the natural ligand FSH [20] for the
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same binding site and this would explain its inhibitory antagonist activity. In contrast, our experimental data (Fig. 4) clearly showed that none of the recombinant antibodies recognized an epitope overlapping with that of 323. Cells were preincubated with 323 at 50 µg/ml (~0.33 µM) at 4°C before the addition of the VHH-Fc
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at 0.5 µg/mL (~6.25 nM). Considering the apparent KD of 323 (Fig. 2D), at this concentration the cell receptors should be totally saturated by the monoclonal antibody and consequently the contemporary detection of both 323 and VHH-Fc
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without MFI decrease indicates the absence of competition. Similarly, no clear competition was observed on L-FSHR cells between the recombinant antibodies and
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FSH. Cells were pre-incubated at increasing FSH concentrations (0-1.32 µM) and the phage-displayed antibodies (monovalent) were added successively (Suppl. Fig. 2). Despite FSH affinity for FSHR is in the low nanomolar range [26] and therefore probably almost three logs higher than that of the VHHs, nanobodies were not significantly displaced by FSH. Altogether, these results suggest that the inhibitory mechanism of the recombinant antibodies might be allosteric rather than antagonistic or inverse agonistic.
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ACCEPTED MANUSCRIPT Discussion Mouse immunization with FSHR ectodomain resulted in the production of sets of monoclonal antibodies all directed against an immunodominant region initially identified in the amino acid sequence 172-358 [24]. Since these antibodies have
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usually antagonist behavior, it is probable that their actual epitope overlaps the sequence 285-309 that corresponds to the region bound by the hormone FSH [4]. Only the immunization with decapeptides corresponding to the N-terminal FSHR
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ectodomain allowed for the production of agonist antibodies with clear stimulatory effect [2]. Otherwise, despite the increasing interest for FSHR in oncological
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diagnostic for both immunohistochemistry and imaging applications [9,14-17,27], no other antibody has been characterized for its binding features and has been proposed for in vivo treatment or to deliver either drugs or imaging probes. The main limit of treatments performed using inverse agonist or antagonist binders is
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that administrations must be repeated frequently to maintain a sufficient concentration to compete with natural binders. For this reason, allosteric molecules
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are preferred. In the case of FSHR, small molecules and peptide that act as positive allosteric modulators [28-30] as well as a biased negative allosteric antagonist [31]
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have recently been described. Since for allosteric molecules the specificity is more critical than affinity, the antibodies described in this work would appear good candidates for more rigorous functional tests also considering that the possibility to produce them either as mono- or bivalent molecules could be useful to assess the role of glycoprotein hormone receptor dimerization [32]. Furthermore, producing functional IgG-like antibodies in bacteria [20] enables to cut significantly the costs for the pre-clinical characterization of lead binders. The present yields obtained using simple flasks and not-optimized medium could be largely increased under rigorous 12
ACCEPTED MANUSCRIPT fermentation conditions. Our data represent also the first report in which antibodies able to modulate the activity of a GPCR protein have been isolated in vitro from a synthetic library and it will be worthy to demonstrate that this strategy is effective also
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for GPCRs with smaller ectodomains. Acknowledgments
The authors acknowledge COPIO/ITMO-TS/Aviesan, Région île de France, CEST-
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95020 (Institut Curie), CIC IGR-Curie 1428, and Labex DCBIOL (ANR-10-IDEX0001-02 PSL* - ANR-11-LABEX-0043) for their financial support and Nathalie
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Amzallag for her management. The authors would like to thank Jean-Christophe Rain (Hybrigenics Service, Paris, France) for the kind gift of HGX44. This work has received support under the program «Investissements d’Avenir » launched by the French Government and implemented by ANR with the references ANR-11-LABEX-
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0043 and ANR-10-IDEX-0001-02 PSL. Literature
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[2] L. Abdennebi, L. Couture, D. Grebert, et al., Generating FSH antagonists and agonists through immunization against FSH receptor N-terminal decapeptides. J. Mol. Endocrinol. 22 (1999) 151-159. [3] A.J. Rao, S.G. Ramachandra, V. Ramesh, et al., Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor. Reprod. Biomed. Online 8 (2004) 385-391.
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ACCEPTED MANUSCRIPT [4] J.D. Ghosalkar, S.J. Dharma, T.D. Nandedkar, S.D. Mahale, Identification of the region 285-309 of follicle stimulating hormone receptor as a bioneutralizing epitope. J. Reprod. Immunol. 74 (2007) 24-33. [5] M. Tonacchera, E. Ferrarini, A. Dimida, et al., Effects of a thyroid-stimulating
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express functional follicle-stimulating hormone receptor (FSHR). J. Urol. 161 (1999) 970-976.
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ACCEPTED MANUSCRIPT [13] S. Heublein, T. Vrekoussis, D. Mayr, et al., Her-2/neu expression is a negative prognosticator in ovarian cancer cases that do not express the follicle stimulating hormone receptor (FSHR). J. Ovarian Res. 6 (2013) 6. [14] M.A Siraj, C. Pichon, A. Radu, N. Ghinea, Endothelial follicle stimulating
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hormone receptor in primary kidney cancer correlates with subsequent response
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Immunohistochemical detection of FSH receptors in pituitary adenomas and adrenal tumors. Folia Histochem. Cytobiol. 50 (2012) 325-330.
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[16] M. Renner, B. Goeppert, M.A Siraj, et al., Follicle-stimulating hormone receptor expression in soft tissue sarcomas. Histopathology 63 (2013) 29-35. [17] A. Siraj, V. Desestret, M. Antoine, et al., Expression of follicle-stimulating hormone receptor by the vascular endothelium in tumor metastases. BMC Cancer
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[18] C. Sardella, D Russo, F. Raggi, et al., Ectopic expression of FSH receptor isoforms in neoplastic but not in endothelial cells from pancreatic neuroendocrine
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tumors. J. Endocrinol. Invest. 36 (2013) 174-179. [19] P.E. Massa, A. Paniccia, A. Monegal, A. de Marco, M. Rescigno, Salmonella
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ACCEPTED MANUSCRIPT [21] C. Ronan, D. Gentien, A. Duché, et al., Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma Patient Derived Xenografts. Pigment Cell Melanoma Res. 30 (2017) 317-327. [22] A. Monegal, D. Ami, C. Martinelli, et al. Immunological applications of single
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domain llama recombinant antibodies isolated from a naïve library. Prot. Engineer. Des. Sel. 22 (2009) 273-280.
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humanized nanobodies providing highly functional antibodies and intrabodies. eLife (2016) 10.7554/eLife.16228
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recombinant antibody production. BMC Biotechnol. 9 (2009) 14. [24] J.L Tilly, T. Aihara, K. Nishimori, et al., Expression of recombinant human folliclestimulating hormone receptor: species-specific ligand binding, signal transduction,
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[25] Y. Xu, D. Pan, C. Zhu, et al., Pilot study of a novel 18F-labeled FSHR probe for tumor imaging. Mol. Imaging Biol. 16 (2014) 16:578-585. [26] S.D. Yanofsky, E.S. Shen, F. Holden, et al. Allosteric activation of the folliclestimulating hormone (FSH) receptor by selective, nonpeptide agonists. J. Biol. Chem. 281 (2006) 13226-13233.
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ACCEPTED MANUSCRIPT [27] B.J. Arey, S.D. Yanofsky, M.C. Pérez, et al., Differing pharmacological activities of thiazolidinone analogs at the FSH receptor. Biochem. Biophys. Res. Commun. 368 (2008) 723-728. [28] C.J. van Koppen, P.M. Verbost, R. van de Lagemaat, et al., Signaling of an
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[30] E. Urizar, L. Montanelli, T. Loy, et al., Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity, EMBO J. 24
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(2005) 1954-1964.
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Recombinant VHH antibodies bind to the FSH receptor expressed on L cells Five antibodies recovered by biopanning (A8, B1, C1, E6, E8) were screened by
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FACS for their specific recognition of the FSH receptor by assessing their binding to control cells (L) and cells overexpressing the receptor (L-FSHR). HGX44 is an antiGFP VHH (negative control) and 323 is an anti-FSHR monoclonal antibody (positive
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control)
Figure 2. Characterization of the reconstituted IgG-like recombinant anti-FSHR
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antibodies
A) The engineered VHH-Fc fusion was purified and analyzed by PAGE in both reducing and oxidizing conditions to show the molecule dimerization. B) Recombinant FSHR produced as a soluble ectodomain fused to an Fc domain and as full-length
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protein. The apparent mass of the full-length FSHR (>200 kDa) indicates probable glycosylation and stable dimerization. C) Antigen specificity of five anti-FSHR antibodies and one anti-GFP control antibody measured by ELISA using purified
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recombinant FSHR-Fc ectodomain and control proteins (HER2-Fc and BSA); bars represent standard deviations. D) FACS-assessed apparent affinity of anti-FSHR
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antibodies compared with negative (HGX44, anti-GFP) and positive (323, monoclonal anti-FSHR) controls. E) SPR-measured affinity of the monoclonal 323 anti-FSHR antibody
Figure 3. Antibody-induced inhibition of FSH-dependent cAMP accumulation in transfected HEK293 cells A) The FSH-dependent cAMP accumulation was measured in the presence of five anti-FSHR antibodies. The cAMP concentration measured in the absence of antibody
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ACCEPTED MANUSCRIPT addition (only DMEM medium) represented the absolute reference. The anti-GFP HGX44 was the negative control and the monoclonal anti-FSHR 323 the positive control. B) Concentration-dependent inhibition of the cAMP accumulation in the presence of the monovalent VHH B1 antibody. C) Inhibition of FSH-dependent
(B1-Fc) antibody. Bars represent standard deviations
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signaling assessed by cAMP accumulation in the presence of the bivalent IgG-like B1
Figure 4. Recombinant antibodies and monoclonal 323 recognize independent
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epitopes on the FSHR surface
The bidimensional FACS analysis indicates that none of the five recombinant
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antibodies competes with the monoclonal antibody 323 for the same epitope on the FSHR surface. A) L-FSHR cells incubated with 323 at 50 µg/ml. B) L-FSHR cells incubated with Fc-VHH at 0.50 µg/ml. C) L-FSHR cells pre-incubated with 323 for
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one hour at 4°C before the addition of VHH-Fc at 0.5 µg/ml
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350
L-FSHR
300
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Figure 1
L-FSHR L
L
200 150
50 0 C1
E6
EP
B1
VHH
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A8
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100
E8
800 600 MFI
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250 MFI
1000
400 200 0
HGX44
323
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Figure 2
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170 130
200
100
130 100
70
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70 55
55
35
25
25
15
15
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
FSHR-Fc
A8
B1
C1
E6
Her2-Fc
E8
HGX44
33,3 nM 11,1 nM 3,7 nM
323
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B1-Fc
Apparent KD (M) C1-Fc E6-Fc
E8-Fc
BSA
E
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D
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40 35
OD 450 nm
+
L
β-Mercaptoethanol
C
L-FSHR
B
FSHR-Fc
A
323
1,55E-07 1,48E-07 2,02E-07 9,65E-08 6,10E-08 1,50E-09
ka
kd
6,60E+05
2,76E-02
KD (M)
Chi2
4,18E-08
8,01
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A
600
- FSH
+ FSH
400
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cAMP (RLU)
500
300 200
0
160 140 120 100 80 60 40 20 0
B1
E6
E8
HGX44
11.1
3.70
1.23
0.41
0.14
concentration (µg/ml)
B1-Fc
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160 140 120 100 80 60 40 20 0
33.3
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100
C
C1
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cAMP (RLU)
B
B1
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A8
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100
cAMP (RLU)
Figure 3
100
33.3
11.1
3.70
1.23
concentration (µg/ml)
0.41
0.14
323
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Figure 4 A
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VHHrFc (FL-4)
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323
B1-Fc
A8-Fc
B1-Fc
VHHrFc (FL-4)
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C
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FSHR323 (FL-2)
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A8-Fc
VHHrFc (FL-4)
B
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FSHR323 (FL-2)
FSHR323 (FL-2)
C1-Fc
E6-Fc
E8-Fc
C1-Fc
E6-Fc
E8-Fc
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Highlights
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First successful panning against a GPCR using whole cells and a synthetic library Isolation of nanobodies which inhibit FSH-dependent cAMP accumulation Identification of nanobodies which are not competitors of FSH Immunoreagents for studying FSHR-induced angiogenesis in cancer
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