WO2015189162A1

WO2015189162A1 - Single-domain antibodies directed against a cys-loop receptor and methods for obtaining them

Info

Publication number: WO2015189162A1
Application number: PCT/EP2015/062755
Authority: WO
Inventors: Ghérici HASSAINE; Lamia MEBARKI; Hugues NURY
Original assignee: Theranyx
Priority date: 2014-06-09
Filing date: 2015-06-09
Publication date: 2015-12-17

Abstract

The instant invention relates to single domain antibodies directed against a Cys-loop receptor, preferably 5-HT3 receptor. Said single -domain antibodies have a dissociation constant Kd for the Cys-loop receptor of at least 10^-6 M and can modulate its activity. The invention also relates to a method for obtaining said single-domain antibodies as well as the uses of said single -domain antibodies in therapy or in the biochemical field.

Description

SINGLE-DOMAIN ANTIBODIES DIRECTED AGAINST A CYS-LOOP RECEPTOR AND

METHODS FOR OBTAINING THEM

FIELD OF THE INVENTION

The present invention relates to single-domain antibodies (sdAbs) directed against Cys-loop receptors, in particular functional sdAbs, pharmaceutical compositions comprising thereof and their uses in biological research and in therapy. A method for obtaining said single -domain antibodies is also provided.

TECHNOLOGICAL BACKGROUND

Ion channels are pore -forming membrane proteins which control and gate the flow of ions across cell membrane. Ion channels are involved in various cellular functions. They allow calcium influx, modulate membrane potential and affect intracellular calcium concentration. They are also involved in regulation of cellular functions such as gene transcription, muscle contraction and hormone secretion. Up to now, at least 400 human ion channel genes have been identified. Ion channels may be classified depending on the nature of the gating. One can distinguish ligand-gated ion channels, and in particular Cys-loop ligand- gated ion channels (also called hereafter Cys-loop receptors).

The Cys-loop receptors are named after a characteristic loop formed by a disulfide bond between two cysteine residues in the N terminal extracellular domain. They are subdivided with respect to their ion selectivity (anion or cation) and further into families defined by their endogenous ligand. These receptors share a common structure of five subunits which are pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Each receptor has an extracellular domain that contains the ligand- binding sites, a transmembrane domain that allows ions to pass across the membrane, and an intracellular domain that plays a role in channel conductance and receptor modulation.

Cys-loop receptors encompass cation selective receptors such as serotonin 5-HT3 receptor and acetylcholine (nACh) receptor as well as anion selective receptors such as GABA_A receptor and Gly (glycine) receptor.

Cys-loop receptors constitute potential therapeutic targets for the treatment of various disorders such as emesis, epilepsy, itching, pain, depression, analgesia, sleep disorders, Parkinson's disease, Alzheimer's disease and irritable bowel syndrome, as well as for the management of substance abuse and addiction such as smoking addiction.

Extensive investigations have thus been conducted in order to identify therapeutic drugs targeting Cys- loop receptors. Despite these efforts, only few Cys-loop receptor modulators are currently available. These drugs encompass, for instance, 5-HT3R antagonists dedicated for the treatment of chemotherapy or radiation-induced nausea and vomiting (e.g. ondansetron, tropisetron, granisetron) or for the treatment of irritable bowel syndrome (alosetron and cilansetron). Partial agonists of nicotinic receptor, such as varenicline, are also available for smoking cessation therapy. Some other drugs have been hampered by shortcomings such as poor selectivity and dose-limiting side effects.

The limited number of available drugs can be explained by the structural complexity of Cys-loop receptors which are composed of five subunits. Cys-loop receptors have generally more than one type of subunits and may display several heteromeric stoechiometries with distinct physiological and pharmacological properties. Their activity is under the control of allosteric modulation and their ligand- binding sites are at the interface with adjacent subunits. Structural data for Cys-loop receptors remain extremely limited. High-resolution structure of vertebrate Cys-loop receptors are still missing so that the current structural models rely on high resolution structures of non-vertebrate homologs only. How endogenous ligand binds the extracellular domain and gates the intramembranous channel remains an open question (Thompson et al., Quaterly Reviews of Biophysics, 2010,43(4),449-499).

Even for the 5-HT3 receptors for which several antagonists are known, the identification of new ligands by in silico docking remains problematic because of the wide range of possible ligand orientations.

Noteworthy, available drugs directed against 5-HT3 receptors are chemical compounds. No functional antibody directed to a Cys loop receptor seems to have been described up to now. For instance, the sole antibodies described for 5HT3-receptors are polyclonal antibodies dedicated to cell immuno-staining. Indeed, the identification of immoglobulins directed to a membrane receptor and which can further modulate its activity remains a real challenge. To that respect, patent application US 2010/0173799 relates to a method for generating immunoglobulin sequences directed to a transmembrane anchored antigen, such as P2X7 or CXCR7, by DNA vaccination. Conrath et al. (Protein Science, 2009, 18:619- 628) describes the generation of nanobodies directed against integral membrane nitric oxide reductase (NOR) by dromedary immunization with purified NOR following by phage display performed on VHH library.

However, functional antibodies, including single-domain antibodies and derivatives thereof, directed to a Cys-loop receptor have never been described so far.

There is thus a need for novel antibodies, in particular single-domain antibodies such as VHH, directed against Cys-loop receptor.

SUMMARY OF THE INVENTION

The present invention relates to a single domain antibody directed against a Cys-loop receptor. Preferably, said single-domain antibody has a dissociation constant (Kd) for said Cys-loop receptor of at most 10^"6 M, said Kd being preferably determined by SPR as shown in the below Example 1. In some embodiments, the single-domain antibody is a modulator of said Cys-loop receptor, e.g. an agonist, a potentiator or an antagonist of said Cys-loop receptor. The Cys-loop receptor may be selected from the group consisting of a serotonin (5-HT3) receptor, an acetylcholine (nicotinic ACh or nACh) receptor, a glycine (Gly) receptor, a γ-aminobutyric acid (GABA_A,GABA_C) receptor and a zinc-activated (ZAC) receptor. A preferred Cys-loop receptor is 5-HT3 receptor subtype such as 5HT3A and 5-HT3AB subtypes. The Cys-loop receptor is preferably human. The single-domain antibody may be selected from the group consisting of isolated VHHs, preferably from Camelidae, humanized VHHs and fragments thereof. In some embodiments, the single domain antibody comprises an amino acid sequence having a sequence identity of at least 60%, preferably at least 70% with an amino acid sequence selected from the group consisting of SEQ ID N°l, 2, 3, 4, 5, 6, 42, 43, 44, 45, 46, and 47.

In some further embodiments, the single domain antibody is directed against a 5-HT3 receptor and has 3 complementarity determining regions, CDR1 to CDR3, wherein:

- CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:7, 10, 13, 16

,19, 22, 48, 51, 54, 57, 60 and 63 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO:7, 10, 13, 16 ,19, 22, 48, 51, 54, 57, 60 and 63 in virtue of one, two, or three amino acid modifications;

CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:8, 11, 14, 17, 20, 23, 49, 52, 55, 58, 61 and 64 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO:8, 11, 14, 17, 20, 23, 49, 52, 55, 58, 61 and 64in virtue of one, two, or three amino acid modifications; and,

CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 12, 15, 18, 21, 24, 50, 53, 56, 59, 62, and 65 ; or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 9, 12, 15, 18, 21, 24, 50, 53, 56, 59, 62, and 65, in virtue of one, two, or three acid modifications.

In particular, said single domain antibody may comprise the sequence

FR1 -CDR1 -FR2-CDR2- FR3-CDR3-FR4 wherein:

- FR1, FR2, FR3 and FR4 are Framework regions, preferably selected from framework regions as depicted in Figure 1A, Figure 8 A and variants thereof, humanized framework regions of a VHH from Camelidae and camelized framework regions of a human VH, and

said single domain antibody comprises one of the following combinations of CDRs:

CDR1 is, or consists essentially of, SEQ ID NO:7, CDR2 is, or consists essentially of, SEQ ID NO:8 and CDR3 is, or consists essentially of, SEQ ID NO:9;

CDR1 is, or consists essentially of, SEQ ID NO: 10, CDR2 is, or consists essentially of, SEQ ID NO: 11 and CDR3 is, or consists essentially of, SEQ ID NO: 12;

CDR1 is, or consists essentially of, SEQ ID NO: 13, CDR2 is, or consists essentially of, SEQ ID NO: 14 and CDR3 is, or consists essentially of, SEQ ID NO: 15;

- CDR1 is, or consists essentially of, SEQ ID NO: 16, CDR2 is, or consists essentially of,

SEQ ID NO: 17 and CDR3 is, or consists essentially of, SEQ ID NO: 18; CDR1 is, or consists essentially of, SEQ ID NO: 19, CDR2 is, or consists essentially of, SEQ ID NO:20 and CDR3 is, or consists essentially of, SEQ ID NO:21 ;

CDR1 is, or consists essentially of, SEQ ID NO:22, CDR2 is, or consists essentially of, SEQ ID NO:23 and CDR3 is, or consists essentially of, SEQ ID NO:24, CDR1 is, or consists essentially of, SEQ ID NO:48, CDR2 is, or consists essentially of, SEQ ID NO:49 and CDR3 is, or consists essentially of, SEQ ID NO:50;

CDR1 is, or consists essentially of, SEQ ID NO:51, CDR2 is, or consists essentially of, SEQ ID NO:52 and CDR3 is, or consists essentially of, SEQ ID NO:53;

CDR1 is, or consists essentially of, SEQ ID NO:54, CDR2 is, or consists essentially of, SEQ ID NO:55 and CDR3 is, or consists essentially of, SEQ ID NO:56;

CDR1 is, or consists essentially of, SEQ ID NO:57, CDR2 is, or consists essentially of, SEQ ID NO:58 and CDR3 is, or consists essentially of, SEQ ID NO:59;

CDR1 is, or consists essentially of, SEQ ID NO:60, CDR2 is, or consists essentially of, SEQ ID NO:61 and CDR3 is, or consists essentially of, SEQ ID NO:62; and CDR1 is, or consists essentially of, SEQ ID NO:63, CDR2 is, or consists essentially of, SEQ ID NO:64 and CDR3 is, or consists essentially of, SEQ ID NO:65.

In some embodiments, the single domain antibody comprises at least one of the following features:

the single -domain antibody is a potentiator of 5-HT3A receptor subtype,

the single -domain antibody is a potentiator of 5-HT3AB receptor subtype,

the single -domain antibody is an antagonist of 5-HT3A receptor subtype,

the single -domain antibody is an antagonist of 5-HT3AB receptor subtype,

the single -domain antibody is an agonist of 5-HT3A receptor subtype,

the single -domain antibody is an agonist of 5-HT3AB receptor subtype,

the single -domain antibody binds both 5-HT3AB receptor subtype and 5-HT3A receptor subtype, the single -domain antibody specifically binds 5-HT3AB receptor subtype as compared to 5- HT3A receptor subtype,

the single -domain antibody specifically binds 5-HT3A receptor subtype as compared to 5- HT3AB receptor subtype,

the single -domain antibody is able to bind a human 5-HT3 receptor, such as human 5-HT3AB receptor subtype and human 5-HT3A receptor subtype.

A further object of the invention is an anti-Cys loop receptor polypeptide comprising at least one single- domain antibody as defined above. In some embodiments, the anti-Cys loop receptor polypeptide further comprises an additional entity selected from the group consisting of PEG moieties, N-linked or O-linked glycosylation moieties, Fc domain from human immunoglobulins, variants and fragments thereof, a single-domain antibody directed against a human serum protein, a labeling mean, an affinity tag, a drug, a toxin and combinations thereof. Another object of the invention is an isolated nucleic acid comprising a sequence encoding a single- domain antibody or an anti-Cys loop receptor polypeptide of the invention as well as vector containing it. The invention also relates to a host cell comprising said nucleic acid and to a method for producing a single-domain antibody or an anti-Cys loop receptor polypeptide of the invention comprising culturing said host cell and recovering said single domain antibody or said polypeptide from the cell culture.

A further object of the invention is a pharmaceutical composition comprising a polypeptide or a single- domain antibody (sdAb) of the invention with a pharmaceutically acceptable excipient. The polypeptide and the sdAb of the invention may be used as a drug. They may also be used in the crystallization of a Cys-loop receptor, in the purification of a Cys-loop receptor, in cell immuno-staining, in a process for screening a candidate ligand to a Cys-loop receptor, in in vivo imaging, in electron microscopy imaging or as biological reagents in an immunoassay.

Another object of the invention is a method for obtaining a single-domain antibody directed against a Cys-loop receptor, and optionally able to modulate said Cys-loop receptor, wherein said method comprises the steps of:

a) preparing a library of biological recipients selected from cells, viruses, ribosomes, mRNA and

DNAs, each biological recipient displaying thereon a single -domain antibody, from an antibody- expressing tissue or cells isolated from a non-human animal, preferably belonging to a Camelidae species, which has been immunized with an immunogenic composition comprising a purified Cys-loop receptor,

b) screening the library of step a) for biological recipients displaying a single-domain antibody able to bind the said Cys-loop receptor, and

c) selecting a single-domain antibody directed against the Cys-loop receptor, preferably displaying a Kd for said Cys-loop receptor of at most 10^~6 M, from the biological recipients identified in step b), and

wherein step b) optionally comprises contacting the biological recipients with the Cys-loop receptor in the presence of a modulator of said Cys-loop receptor.

In a preferred embodiment, the library of step a) is a library of bacteriophages displaying thereon VHHs and step b) is performed by phage display.

In some other embodiments, the method of the invention comprises one or several (1, 2 ,3, 5, 6 or 7) additional steps selected from the group of:

a step of assessing or screening the capability of the single -domain antibodies for modulating the activity of said Cys-loop receptor,

a step of assessing the affinity of the single-domain antibodies towards the Cys-loop receptor, a step of isolating and/or sequencing the nucleic acid sequence encoding for the selected single- domain antibody

a step of expressing a selected single-domain antibody in a suitable host cell, a step of removing single-domain antibodies which cross-react or cross-binds with another Cys- loop receptor,

a step of selecting single-domain antibodies which bind to the corresponding human Cys-loop receptor, if a non-human Cys-loop receptor has been used for immunization.

- a step of selecting a single-domain antibody which modulates the activity of the said Cys-loop receptor and

a step of introducing one or more amino acid modifications into the sequence of a selected single- domain antibody so as to optimize it, e.g. so as to obtain a humanized single-domain antibody. At last, the invention also relates to the use of a single-domain antibody obtained by the hereabove method or the nucleic acid encoding it, for the preparation of a polypeptide according to the invention.

FIGURES

Figure 1A shows the amino acid sequences (SEQ ID NO: 1-6) of 6 functional VHHs directed against 5- HT3A receptor. CDRs and FRs regions are indicated for each VHH.

Figure IB shows the sequence alignment of SEQ ID NO: 1-6.

Figure 2A shows in vivo surface labeling using a dye (Tri.NTA-Atto) for T-Rex CHO cell line expressing 5HT3 receptor (5HT3R+) incubating with VHH7 (SEQ ID NO:3). Control: T-Rex CHO cell line which do not express 5-HT3R (5-HT3R -).

Figure 2B shows the profile of elution by size -exclusion chromatography for 5-HT3A receptor alone (dashed curve), VHH7 (SEQ ID NO:3) alone and 5-HT3A receptor + VHH7. Figure 2 also provides the SDS electrophoresis gel for 5-HT3 receptor + VHH7 complex.

Figure 3 shows the response obtained by SPR for the binding of 5-HT3R at different concentrations (0.05, 0.5, 2, 5, 20, 50, 100 and 200 nM) for immobilized VHH7 and the resulting dose-response curve. Figure 4 shows the response obtained by SPR for the binding of 5-HT3R at different concentrations (0.1, 0.5, 1, 2.5, 5, 10, 20.5, 41, 82, 164, 328, 492 nM) for immobilized VHH 15 (SEQ ID NO:4) and the resulting dose-response curve.

Figure 5A and Figure 5B show the whole -cell electrophysiology recordings obtained for T-Rex Hek293 cells expressing 5-HT3R pre -incubated with VHH7 (9 nM and 90 nM) after induction with serotonin (5- HT). The pre -incubation with VHH7 enables to inhibit the serotonin-elicited currents in a dose-dependent manner.

Figure 6A and Figure 6B show the FlexStation responses of CHO cells stably transfected with 5-HT3A receptors. Increase in fluorescence in cells loaded with membrane potential dye is observed when said cells are contacted with 1 μΜ of serotonin (5-HT). The incubation of CHO cells with 40 μΜ of VHH15 or VHH16 inhibits the increase of fluorescence induced by 5-HT (1 μΜ).

Figure 7A illustrates the patch clamp protocol for the assessment of the ability of each VHH to modulate the activity of 5-HT3A receptor expressed on Xenopus oocytes. The modulation ability of each VHH was determined as the ratio of the maximum of current measured in the presence of 1 μΜ of VHH (test pulse) to the maximum of current measured for the first control pulse. The second control pulse was performed in order to determine whether the VHHs had a long-lasting effect on 5-HT3 receptor.

Figure 7B shows the percentage of modulation exerted by BSA or 1 μΜ of each VHH in the patch-clamp assay.

Figure 7C shows the response curve obtained for 0.8 μΜ of VHH7. Figure 7D shows the response curve obtained for 1 μΜ of VHH5. Figures 7E and 7F show the response curves obtained for 1.5 μΜ and 15 nM of VHH15 respectively. For figures 7C-7F : X-axis: Current (μΑ), Y-axis : time (s).

Figure 8A shows the amino acid sequences (SEQ ID NO:42-47) of 6 VHHs directed against 5-HT3AB receptor. CDRs and FRs regions are indicated for each VHH.

Figure 8B shows the sequence alignment of SEQ ID NO:42-47.

Figures 9A-9F show the results of the FACS analysis performed for VHHAB-5, VHHAB-13, VHHAB- 9, VHHAB-10, VHHAB-18 and VHHAB-21. In each Figure, Curve 1 corresponds to a negative control using CHO T-Rex cells which do not express 5-HT3ABR or 5-HT3AR. Curve 2 refers to a negative control in which the supernatant containing the VHH was replaced by a TGI periplasmic extract free of VHH. Curve 3 refers to the response obtained from CHO T-rex cells expressing m5-HT3AR and finally Curve 4 refers to the response obtained from CHO T-rex cells expressing m5-HT3ABR. The protocol of the FACS analysis is described in Example 2 (see section 2).

Figure 10A and Figure 10B show the FlexStation responses of CHO cells transfected with 5-HT3AB receptor. Increase in fluorescence in cells loaded with membrane potential dye is observed when said cells are contacted with 4 μΜ of serotonin (5-HT - curve 1). The incubation of CHO cells with 35 μΜ of VHHAB-5 or VHHAB-13, in the absence of serotonin, also induces an increase of the fluorescence (curve 2). By contrast, the incubation of the cells with the buffer (negative control - curve 3) does not induce any change in fluorescence. DETAILED DESCRIPTION OF THE INVENTION

■ Definitions

As used herein, the verb "to comprise" is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Cys-loop receptors: Cys-loop receptor (also called Cys-loop ligand-gated ion channel) family comprises membrane-spanning-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. Cys-loop receptors are divided with respect to the type of ion that they conduct (anion or cation) and further into families defined by the endogenous ligand. Cys- loop receptors share a common structure consisting of five pseudo-symmetrically arranged subunits surrounding a central ion-conducting pore. Each subunit comprises a large extracellular domain, which corresponds to the binding site of the endogenous ligand, four transmembrane regions M1-M4 and an intracellular loop, in particular between M3 and M4. Their name derives from a 13-amino acid loop within the extracellular domain which is enclosed by a pair of disulphide-bonded Cys residues. The Cys- loop receptors are described in Thompson et al. Quaterly Reviews of Biophysics, 2010, 43(4), 449-499, the disclosure of which being incorporated by reference.

As used herein, Cys-loop receptors may derive from vertebrates or invertebrates. Invertebrate Cys-loop receptors encompass EXP-1, MOD-1, pHCl, HisCl, RDL, GluCl and SsCl. Vertebrate members of this family include, without being limited to, serotonin (5-HT3) receptors, acetylcholine (nicotinic ACh or nACh) receptor, glycine (Gly) receptor, γ-aminobutyric acid (GABA_A,GABA_C) receptor and zinc- activated (ZAC) receptor.

Cys-loop receptors generally display several subtypes.

For instance, subtypes of 5-HT3 receptor encompass 5-HT3A, 5-HT3AB, 5-HT3AC, 5-HT3AD and 5- HT3AE receptors. Subtypes of Gly receptor encompass Glycine al, Glycine a2, Glycine a3, Glycine a4, Glycine αΐβ, Glycine α2β, Glycine α3β and Glycine α4β receptors. Subtypes of γ-aminobutyric acid receptors include Gaba al, Gaba β3, Gaba pl,Gaba α1β3, Gaba βΐ, Gaba p2, Gaba p3, Gaba plp2, Gaba plp3, and Gaba p2p3 receptors.

An example of ZAC receptor is ZacN receptor. In the context of the present invention, the Cys-loop receptors are preferably from mammals, in particular from human or murine origin.

A preferred Cys-loop receptor is a 5-HT3 receptor, in particular a human or murine 5-HT3 receptor. Up to know, five subunits for 5-HT3 receptors have been identified, namely subunits 5-HT3 A, B, C, D and E.

Noteworthy, 5-HT3-A subunits can form a functional homomeric receptor. The other functional 5-HT3 receptors are hetero-pentamers which comprise one or several 5-HT3A subunits. 5-HT3 receptors differ from other serotonin receptors in that its action is not mediated via G proteins. For review about 5-HT3 receptors, see Thompson and Lummis, Curr Pharm Des, 2006; 12(28): 3615-3630, the disclosure of which being incorporated by reference.

Heavy-chain antibodies (HCAb): HCAbs refer to immunoglobulins which are devoid of light chains and consist in two heavy chains. Each heavy chain comprises a constant region (CH) and a variable domain which enables the binding to a specific antigen, epitope or ligand. As used herein, HCAbs encompass heavy chain antibodies of the camelid-type in which each heavy chain comprises a variable domain called VHH and two constant domains (CH2 and CH3). Noteworthy, camelid HCAbs lack the first constant domain (CHI). Such heavy-chain antibodies directed against a specific antigen can be obtained from immunized camelids. Camelids encompass dromedary, camel lama and alpaca. Camelid HCAbs have been described by Hamers-Casterman et al., Nature, 1993, 363:446. Other examples of HCAb are immunoglobulin-like structures (Ig-NAR) from cartilaginous fishes. Single-domain antibody: As used herein, a single-domain antibody (also herein called single domain antibody, sdAb or nanobody) refers to a single-variable domain, derived from a heavy-chain antibody, which is able to bind an antigen, an epitope or a ligand alone, that is to say, without the requirement of another binding domain. A single domain antibody may derive from VHH and V-NAR. V-NAR refers to the variable domain found in immunoglobulin-like structures (Ig-NAR) discovered in cartilaginous fishes such as sharks. As an alternative, single-domain antibody may be obtained from human VH by camelization. For review about single -domain antibodies, one may refer to Saerens et al, Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference. The single -domain antibody of the invention comprises at least one complementarity determining region (CDR) which determines its binding specificity. Preferably, the single -domain antibody comprises several CDRs which are distributed between framework regions (FRs). CDRs and FRs are preferably fragments or derivatives from a naturally-occurring antibody variable domain.

In the context of the present invention, the single domain antibody preferably derives from the variable domain (VHH) of camelid heavy-chain antibodies. Thus, single-domain antibodies of the invention encompass isolated naturally-occurring VHH, VHH variants such as humanized VHH, fragments thereof or polypeptide constructs comprising them.

In the context of the invention, the CDRs of a single domain-antibody directed to a 5-HT3 receptor can be determined by computer modeling and docking based on the X-ray structure of the 5-HT3A receptor with VHH 15 (SEQ ID N°4) described in Hassaine et al., Nature, 2014, 512, 276-281.

Alternatively, the CDRs may be determined by sequence alignments based on standard algorithms.

Single-domain antibody directed to a Cys-loop receptor. As used herein, a single domain antibody directed to a Cys-loop receptor refers to a single domain antibody able to bind a Cys-loop receptor.

As used herein, a sdAb is able to bind a Cys-loop receptor, if the sdAb is able to bind at least one subtype of the Cys-loop receptor (if any), for instances 1, 2, 3, 4, 5 or 6 subtypes of the Cys-loop receptor.

The affinity of the sdAb for the Cys-loop receptor may be represented by its dissociation constant (Kd) for said receptor.

Typically, single domain antibodies of the present invention (or polypeptides comprising them) have a dissociation constant Kd for their Cys-loop receptor of at most 10^~6 M and more preferably of at most 10^" ⁷, 10^"8, or 10^"9 M. The Kd of the sdAbs of the invention for their receptor is preferably from 1.10^"12 M to 1.10^"6 M

The affinity of a single domain antibody for a given Cys-loop receptor may be determined by the well- known methods described in the prior art. Preferably, in the context of the invention, the Kd of a sdAb refers to its binding strength to a given purified Cys-loop receptor. Such a Kd may be determined by surface plasmon resonance assay in which the sdAb is immobilized on the biosensor chip and the Cys- loop receptor is passed over the immobilized sdAb under flow conditions leading to the measurements of kon and k_off and thus Kd. For illustration, one can refer to the Examples of the instant application (see Example 1 section 3). Functional single domain antibody: As used herein, a functional single -domain antibody refers to a sdAb able to modulate the activity of the Cys-loop receptor. Modulators of a Cys-loop receptor encompass antagonists and agonists. In other words, a functional sdAb may act as an antagonist, an agonist or a potentiator on the Cys-loop receptor. Typically, the ability of a single domain antibody to modulate the activity of a Cys-loop receptor may be determined by standard patch clamp study performed on whole cells expressing functional Cys-loop receptors or on reconstituted vesicles.

Another alternative is the FlexStation analysis which is based on the use of fluorescent voltage-sensitive dyes to detect changes in the membrane potential as described in Price and Lummis, 2005, J Neurosci Methods, 149:172-177.

In order to characterize the functionality of the single -domain antibodies of the invention, the skilled artisan may adapt the patch clamp and the FlexStation Analysis assays described in Lummis et al, 2011, the Journal of Pharmacology and Experimental Therapeutics, 339, 125-131, see in particular, Material and methods section - Electrophysiology and FleXstation. The skilled artisan may further refer to the Examples of the instant application - Example 1 section 4.

Sequence identity: The "percentage identity" between two amino acid sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle" for pairwise sequence alignment of proteins providing by EMBL-EBI and available on

http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=emboss needle&context=protein, using default settings : (I) Matrix : BLOSUM62, (ii) Gap open : 10, (iii) gap extend : 0.5, (iv) output format : pair, (v) end gap penalty : false, (vi) end gap open : 10, (vii) end gap extend : 0.5.

"Consists essentially of: in the context of amino acid sequences, the expression "consists essentially of refers to an amino acid sequence which differs from another amino acid sequence in virtue of one, two or three amino acid modifications, preferably in virtue of one or two amino acid modifications..

Amino acid modification: as used herein, by "amino acid modification" is meant a change in the amino acid sequence of a polypeptide. "Amino acid modifications" which may be also termed "amino acid changes", herein include amino acid mutations such as substitution, insertion, and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.

The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and/or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:

Table 1— Amino Acid Residue

Table 2 - Alternative Conservative Amino Acid Residue Substitution Groups

Table 3 - Further Alternative Physical and Functional Classifications of Amino Acid Residues

Additional groups for conservative substitutions are: valine-leucine -isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine -glutamine.

Parent polypeptide or polypeptide parent: as used herein, it is meant an unmodified polypeptide that is subsequently modified to generate a variant. In the context of the invention, the parent polypeptide may be a VHH from a naturally-occurring HCAb.

Variant polypeptide. Polypeptide variant or Variant: as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification. In the context of the invention, a variant is a variant of a VHH from a naturally-occurring HCAb. Typically, a variant comprises from 1 to 50 amino acid modifications, preferably from 1 to 40 amino acid modifications. In particular, the variant may have from 1 to 30 amino acid changes, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid changes as compared to its parent. The variants may comprise one or several amino acid substitutions, and/or, one or several amino acid insertions, and/or one or several amino acid deletions. In some embodiments, the variant may comprise one or several conservative substitutions, e.g. as shown hereabove. In some other embodiments, the variant comprises one or several amino acid modifications in the framework domains.

^■ Method for obtaining single domain antibodies directed against Cys-loop receptors

Surprisingly, the Applicant showed that single-domain antibodies directed to 5-HT3 receptors may be obtained by immunizing camels with purified 5-HT3A receptors following by the preparation of VHH library and then the selection and/or identification of specific VHHs by phage display. The resulting VHHs may be functional and act as antagonists, agonists, allosteric modulators, e.g. as potentiators of 5- HT3 receptors, in particular 5-HT3A receptors. The Applicant also obtained VHHs from the immunization of llamas with purified full 5-HT3AB receptor. The resulting VHHs were able to modulate the activity of 5-HT3AB. Moreover, some of them showed specificity to 5-HT3ABR subtype as compared to 5-HT3AR subtype.

It was the first time that functional antibodies, in particular single-domain antibodies, against a Cys-loop receptor were obtained. Thus, the Applicant provides therein, for the first time, a method for preparing functional single-domain antibodies directed to a Cys-loop receptor and the characterization of such antibodies.

Noteworthy, the immunization with purified 5-HT3R was sufficient and effective to induce functional antibodies. Such a result was quite surprising since it was thought that the generation of functional antibodies against a cell membrane protein required the immunization with whole cells expressing the said protein or with a nucleic acid construct encoding for said protein, so as to expose the immune system of the host animal to the cell membrane-anchored proteins in their original conformation and environment (for instance see patent application US 2010/0173799).

The present invention relates to a method for obtaining and/or selecting a single-domain antibody (sdAb), preferably a VHH, directed against a Cys-loop receptor.

The method comprises the steps of:

a) preparing a library of biological recipients, each biological recipient displaying thereon a single- domain antibody wherein the library is prepared from an antibody-expressing tissue or cells isolated from a non-human animal which has been immunized with an immunogenic composition comprising a purified Cys-loop receptor,

b) screening the library of step a) for biological recipients displaying a sdAb able to bind said Cys-loop receptor, and c) obtaining and/or selecting a sdAb directed against said Cys-loop receptor from the biological recipients identified in step b).

The method according to the invention may be performed for obtaining sdAbs directed against any kind of Cys-loop receptors, preferably from mammals such as human or murine Cys-loop receptors. In some embodiments, the Cys-loop receptor is selected from the group consisting of a serotonin (5-HT3) receptor, an acetylcholine (nicotinic ACh or nACh) receptor, a glycine (Gly) receptor, a γ-aminobutyric acid (GABA_A,GABA_C) receptor and a zinc-activated (ZAC) receptor. Gly receptors encompass Glycine al, Glycine a2, Glycine a3, Glycine a4, Glycine αΐβ, Glycine α2β, Glycine a3fia and Glycine α4β receptors, γ-aminobutyric acid receptors include Gaba al, Gaba β3, Gaba pl,Gaba α1β3, Gaba βΐ, Gaba p2, Gaba p3, Gaba plp2, Gaba plp3, and Gaba p2p3 receptors. An example of ZAC receptor is ZacN receptor. In some further embodiments, the Cys-loop receptor is selected from the group consisting of 5- HT3A receptor, 5-HT3AB receptor, Glycine al receptor, and Glycine β3 receptor.

Step c) is preferably performed so as to select a sdAb having a Kd for the Cys-loop receptor of at most 10^" ⁶ M, e.g. by performing SPR or ELISA assay.

The non-human animal has been immunized with an immunogenic composition comprising the Cys-loop receptor in a purified form. Cys-loop receptor is thus not essentially present as a trans-membrane protein embedded in the membrane of whole cells. Noteworthy, the method of the invention does not require the immunization of the non-human animal with whole cells expressing said Cys-loop receptor. Moreover, the method of the invention may preclude the use of genetic immunization i.e. the administration to the non-human animal with a nucleic acid able to induce the expression of said Cys-loop receptor by the cells of said non-human animal.

Accordingly, in some embodiments, the immunogenic composition is devoid of Cys-loop receptor in a form embedded in the membrane of whole cells.

The purified Cys-loop receptor is preferably homopentameric or heteropentameric. The purified Cys-loop receptor is thus composed of five subunits. For instance, 5-HT3A receptor comprises five 5-HT3A protein subunits.

The purified Cys-loop receptor may be present in the immunogenic composition as monomers or as agglomerates. Preferably, the Cys-loop receptor is mostly present as monomers, which means that at least 80% of Cys-loop receptors present in the immunogenic composition is present as monomers.

At least 80% of Cys-loop receptor encompasses at least 85%, at least 90%, at least, 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

The purified Cys-loop receptor may comprise one or several affinity tags useful for purification or immobilization purposes. Preferably, the affinity tag(s) is present at the N-terminal or C-terminal extremity of a Cys-loop receptor subunit. Appropriate affinity tags encompass, without being limited to, FLAG-Tag, His-tag, Strep-tag, Avi-tag, HA-tag, S-tag, and the like. Preferably, the affinity is selected from His-tag, FLAG-tag and Strep-tag. The purified Cys-loop receptor may be obtained by conventional methods described in the prior art. Typically, the Cys-loop receptor may be obtained by culturing cells expressing the Cys-loop receptor, preparing cell membrane extract, solubilizing Cys-loop receptor from cell membrane extract, preferably with a detergent solution, and then purifying the resulting Cys-loop receptor. The cells expressing the Cys-loop receptor may be a cell naturally expressing the Cys-loop receptor, a host cell system transiently expressing the Cys-loop receptor or a recombinant stable cell line expressing the Cys-loop receptor. For instance, Cys-loop receptors may be expressed using the Semliki Forest virus system in mammalian cells as described in Blazey et al, Cytotechnology, 2000, 32, 199-208 or in a tetracycline -inducible stable mammalian cell line as described in Dostalova et al, Protein Science, 2010 19:1728-1738 or in Tol et al, Journal of Biological Chemistry, 2013, 288, 5756-5769. The solubilization of Cys-loop receptor from cell membranes may be performed by incubating cell membranes in a detergent solution, preferably comprising nonaethyleneglycol monodecyl ether (C12E9) following by centrifugation. The Cys-loop receptor may be then purified from the supernatant by affinity chromatography, preferably by tag-affinity chromatography. For illustration, see the below Example - section 1 which describes the production and the purification of 5-HT3A receptor.

This method can be adapted for the preparation of any other Cys-loop receptor.

The immunogenic composition comprises the purified Cys-loop receptor as described above and a pharmaceutically acceptable vehicle or carrier such as a buffer. The immunogenic composition may further contain an immunoadjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant. The amount of immunogen (namely the Cys-loop receptor), the buffer and the amount of immunoadjuvant may be determined by the skilled artisan by routine experiments.

The immunization of the non-human animal, preferably from Camelidae, may comprise repeated administrations, e.g. 2, 3, 4, 5 or 6 administrations of an immunogenic composition, in suitable time intervals. Such intervals are preferably days to weeks, e.g. 3 days to 4 weeks, more preferably 5 days to three weeks. As illustrated in the below example, suitable intervals may comprise administrations on 0, 21, 42, 52 and 63 days. The one skilled in the art is able to determine, upon routine experiments, the number of administrations and the suitable intervals so as to induce an appropriate immune response. Typically, the immunization sequence is repeated until an adequate antibody response is elicited in the non-human animal. The suitable dosage of purified Cys-loop receptor to administer depends on the non- human animal to immunize as well as the scheduled immunization sequences. Typically, an amount of Cys-loop receptor ranging from 0.1-10 mg, preferably from 0.2-5mg, can be used for each administration. The immunogenic composition can be administered by any appropriate route, e.g., by intradermal, subcutaneous, intraperitoneal, intranasal, or intramuscular route.

In a preferred embodiment, the non-human animal is selected from animal species expressing Heavy- chain antibodies (HCAb). Preferably, the non-human animal belongs to Camelidae. The Camelidae family encompasses camel, dromedary, llama, vicuna, alpaca and guanaco. In other embodiments, the non-human animal is a cartilaginous fish such as nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates). The skilled artisan may refer to Dooley et al. Mol Immunol, 2003, 40:25-30 which describes the selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

Once a suitable antibody response has been elicited in the animal, a library of single -domain antibodies such as VHHs library can be prepared. The skilled person is well acquainted with techniques for establishing suitable libraries of immunoglobulin sequences, including V-NAR and VHH sequences. The preparation of the library firstly comprises the isolation of antibody-expressing tissue or cells from the non-human animal. Said tissues or cells encompass peripheral blood monocytes (PBMCs), peripheral blood lymphocytes (PBLs), peripheral lymph nodes, in particular lymph nodes draining the site of immunization, the spleen, bone marrow, or other immunologically relevant materials. Said tissue or cells may be collected and isolated by standard methods. Once said biological material is isolated from the immunized non-human animal, the nucleic acid sequences encoding for single-domain antibodies are extracted, isolated and transferred into appropriate biological recipients able to express thereon the desired single-domain antibodies, whereby the desired library of single -domain antibodies is obtained.

As used herein, a library of single-domain antibodies refers to a set or a collection of a plurality of single- domain antibodies, each single -domain antibody being displayed on an appropriate biological recipient. As used herein, each biological recipient is a display system which couples a given protein (herein a single-domain antibody) with its encoding nucleic acid, e.g its cognate mRNA, cDNA or gene. In other words, each biological recipient comprises a nucleic acid encoding the single -domain antibody which is displayed thereon. Biological recipients encompass, without being limited to, cells, virus such as phages, ribosomes, DNAs including plasmids, and mRNAs.

As used herein, cells encompass prokaryotic cells and eukaryotic cells. Thus, the biological excipient may be selected from mammalian cells, insect cells, yeasts such as S.cerevisiae and bacteria such as E. coli.

In some other embodiments, the biological recipient may be a virus, for instance a bacteriophage.

In some further embodiments, the biological recipient may be a nucleic acid, such as DNAs, including plasmids, and mRNAs. In other embodiments, the biological recipient is a ribosome. As non-limiting examples of biological recipients, one may cite the following display systems: a mRNA display system wherein the single -domain antibody is covalently linked to its cognate mRNA, for instance by puromycin ; a plasmid display system wherein the single -domain antibody is displayed on its encoding plasmid through, for instance, NF-kB homodimer-DNA template complex ; and a ribosome display system wherein the stalled ribosome mediates the non-covalent coupling of the single-domain antibody with its cognate mRNA,

In other words, the method for obtaining and/or selecting a sdAb according to the invention may be based on a protein selection technology such as, but without being limited to, cell display, phage display, ribosome display, ni NA display, DNA display or plasmid display. These techniques are well-described in the state in the art.

In a more general aspect, the skilled person is indeed familiar with suitable techniques for extraction of immunoglobulin sequences and manipulating these sequences for creating a library of cells, phages, DNAs or ribosomes displaying said immunoglobulin sequences or a fragments thereof.

In the context of the invention, the library is preferably a library of VHHs.

For instance, in order to generate a library of VHHs displayed on ribosomes, the skilled artisan may refer to Perruchini et al., Acta Neuropathol, 2009,118:865-695, the disclosure of which being incorporated herein by reference. For illustration about DNA display, one can refer to Yonezawa et al. Nucleic Acids Research, 2003, Vol.31, 19, el 18.

In some embodiments, the biological recipients are bacteriophages.

For instance, in order to generate a library of VHHs displayed on bacteriophages, the skilled artisan can refer to Muydermans et. al, Molecular Biotechnology, 2001, 74, 277-302, in particular to the section entitled Recombinant VHH, the disclosure of which being incorporated therein by reference. In order to generate a library of V-NARs displayed on bacteriophages, the skilled artisan may refer to Dooley et al. Mol Immunol, 2003, 40:25-30.

A non-limiting example for preparing a bacteriophage library of VHHs comprises the extraction of total RNA from isolated peripheral blood lymphocytes. The extracted RNAs serve as template for the synthesis of cDNA, e.g. by RT-PCR. The nucleic acid sequences encoding for VHHs may be then selectively amplified from the total cDNAs by using PCR primers that anneal specifically on the sequence encoding the hinge of the HCAbs as described in van der Linden et al., 2000, J. Immunol. Methods, 240,185-195. An alternative method for obtaining nucleic acid sequences encoding for VHHs is based on an amplification step from cDNA using a pan-annealing primer that binds to a conserved region of all constant γ genes in combination with a primer that anneals at the leader signal sequence of VH and VHH, so that both nucleic acid fragments encoding CH2-VHH and CH2-CH1-VH are amplified. These two type of fragments are separated, e.g. on agarose gel, and CH2-VHH encoding sequences are recovered for a further amplification, e.g. by nested PCR so as to specifically amplify nucleic acid fragments encoding VHHs (for more details, see e.g. Saerens et al, 2004, J Biol Chem, 50, 51965-51972).

The nucleic acid sequences encoding VHHs can be then cloned into phagemid vectors, e.g. into pHEN. The library of VHHs can be subsequently obtained by transformation of a suitable host such as E.coli with the phagemid vectors followed by infection with phage helpers.

The library obtained in step a) preferably displays a high diversity of independent clones. Accordingly, the library may contain at least 1.10⁵, more preferably at least 1.10⁶ distinct clones. A diversity of at least 10⁶ encompass a diversity of at least 2.10⁶, of at least 4.10⁶, of at least 6.10⁶, of at least 8. 10⁶, of at least 1. 10⁷, of at least 5.10⁷,of at least 1.10^s and at least 5.10⁸. Once the library of sdAbs is obtained, the step b) of screening the library for sdAbs directed to the Cys- loop receptor can be performed. The screening step may comprise one or several rounds of selection (also called rounds of panning).

One or several rounds of selection encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 rounds of selection. Typically step b) may comprise from 2 to 6 rounds of selections, e.g 3 rounds of selection.

Step b) of screening may be performed by any method suitable for identifying single-domain antibodies able to bind the Cys-loop receptor. In particular, the rounds of selection may comprise incubating the library in the presence of said Cys-loop receptor in its pentameric native form, a subunit or a fragment thereof, so as to select the single-domain antibodies which bind said Cys-loop receptor, a subunit or a fragment thereof. In some embodiments, a human Cys-loop receptor, a subunit or a fragment thereof is used in step b).

A fragment of Cys-loop receptor may be selected from the N-terminal extremity of the cys-loop receptor, an extracellular loop of the Cys-loop receptor, in particular M2-M3 loop, or an intracellular loop of the Cys-loop receptor. In preferred embodiments, at least one round of selection, more preferably all the rounds of selection, are performed with Cys-loop receptor, in particular with full-length Cys-loop receptor. The Cys-loop receptor may be present as an isolated form or may be incorporated in the membrane of a vesicle (e.g. liposomes) or present as a membrane receptor on appropriate cells. The Cys- loop receptor or said vesicle, cell, fragments of subunit thereof, may be immobilized on a suitable support such as plate or magnetic beads. For this aim, the Cys-loop receptor may comprise at least one affinity tag. Preferably, the rounds of selection are performed with purified Cys-loop receptor, which may be obtained as described hereabove.

In certain embodiments, in order to select a functional single-domain antibody, i.e. a sdAb able to modulate the Cys-loop receptor, the single-domain antibodies of the library may be contacted with the Cys-loop receptor in the presence of a known modulator of said Cys-loop receptor, in at least one round of selection.

In other words, in at least one round of selection of step b), one screens for biological recipients displaying a sdAb able to bind said Cys loop-receptor in the presence of a modulator. Several rounds of selection may be performed in the presence of at least one modulator. The modulators used in the rounds of selection may be the same or may be distinct.

Said modulator of the Cys-loop receptor may be selected from the group consisting of an antagonist, an agonist, an allosteric modulator or an endogenous ligand of said Cys-loop receptor.

For instance, in order to select single -domain antibodies which inhibit the activity of the Cys-loop receptor, the Cys-loop receptor modulator may be an antagonist. Alternatively, in order to identify single- domain antibodies acting as allosteric modulators, the selection may be performed in the presence of an agonist of the Cys-loop receptor.

Several modulators of Cys-loop receptors are described in the prior art. For instance, agonists of 5-HT3 receptor encompass, without being limited to, 2-methyl-5-HT, ethanol, alpha-methyltryptamine, varenicline, m-Chlorophenylbiguanide, quipazine, N-Methylquipazine dimaleate, 1-Phenylbiguanide hydrochloride, Quipazine dimaleate, RS-56812, SR-57227, YM-31636 and salts thereof.

Antagonists of 5-HT3 receptor encompass, without being limited to, AS-8112, granisetron, ondansetron, tropisetron, dolasetron, palonosetron, ramosetron, tropisetron alosetron, batanopride, 3-AQC, B-HT 920, MDL 72222, mirtazapine, mosapride citrate, Tropanyl-3,5-dimethylbenzoate, VUF 10166 and Y-25130 and salts thereof. In some embodiments, step b) comprises several rounds of selections and at least one round of selection is performed in the presence of the Cys-loop receptor modulator or combination of modulators. In other embodiments, step b) comprises several rounds of selection and all the rounds of selection are performed in the presence of said Cys-loop receptor modulator.

For instance, step b) may comprise 3 rounds of selection performed in the presence of a Cys-loop receptor modulator. In a particular embodiment, the library of single-domain antibodies obtained in step a) is a virus library, for instance a bacteriophage library. The step b) of screening may be thus performed by virus display, such as phage display. Phage display is a standard laboratory technique which has been extensively used, in particular in the field of VHH screening. For review about this matter, see for instance Muyldermans, Reviews in Molecular Biotechnology, 2001, 74, 277-302.

For instance, in this embodiment, step b) may comprise the following sub-steps of:

bl) contacting the library of bacteriophages obtained in step a) with the Cys-loop receptor or said fragment or subunit thereof,

b2) recovering the bacteriophages displaying single -domain antibodies which have bound said immobilized Cys-loop receptor, or said fragment or subunit thereof,

- b3) optionally, cloning the bacteriophages recovered in step b2) so as to obtain a library enriched in bacteriophages displaying single -domain antibodies which bind the Cys-loop receptor, and b4) optionally, repeating steps bl) to b3) until an appropriate enrichment of the library is obtained. The single-domain antibodies are preferably VHHs from camelids, and thus the immunized non-human animal belongs to Camelidae family. In step bl) the bacteriophages of the library are preferably contacted with the Cys-loop receptor or fragment or subunit thereof in the presence of a Cys-loop receptor modulator or a combination of modulators. The incubation in the presence of the Cys-loop receptor modulator may enable the selection of functional single-domain antibodies. The Cys-loop receptor, or the fragment or the subunit thereof, is preferably immobilized on an appropriate surface. In step b4), the enrichment of the library may be determined by bacteria colonies number. Step b) may further comprise a step b5) of screening for VHHs binding the Cys-loop receptor by ELISA assay. Said ELISA assay may be performed on periplasmic extracts recovered from induced bacteria colonies corresponding to the selected bacteriophages. For illustration, see the below Examples. In additional embodiments, in step c), the nucleic acid encoding for the selected sdAbs can be determined by sequencing. The corresponding single-domain antibodies can be then recombinantly produced in a suitable host cell. Accordingly, the step c) of the method of the invention may thus comprise recovering the biological recipients selected in step b), isolating the nucleic acid sequences encoding for the corresponding single -domain antibodies and expressing them in a suitable host cell.

The method may further comprise a step of assessing the binding affinity of the sdAb to the Cys-loop receptor. The skilled artisan may assess such affinity by ELISA assay, by SPR analysis or by competitive binding against the endogenous ligand or a modulator of the Cys-loop receptor. The method may also contain a step of selecting sdAbs able to modulate the Cys-loop receptor. This step may be performed e.g. by patch clamp or by FlexStation analysis and may be carried out typically after step b). Criteria to determine whether a sdAb is able to modulate a Cys-loop receptor are described here- below in the section entitled "Single domain antibodies according to the invention". Typically, a sdAb is a modulator of a Cys-loop receptor if said sdAb is able to modulate the response induced by the endogenous ligand by at least 10%, preferably by at least 20% or if said sdAb is able to induce a response in the absence of the endogenous ligand which is equal to at least 10%, preferably to at least 20% of the response induced by the endogenous ligand alone, in similar conditions.

At least 20% encompasses at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%.

The method may also contain a step of assessing the binding specificity of the single -domain antibodies or a step of selecting sdAbs which specifically bind said Cys-loop receptor as compared to another Cys- loop receptor. This latter step may typically comprise the removal of single-domain antibodies which cross-react or cross-bind with another Cys-loop receptor.

The method may further contain a step of selecting single-domain antibodies which bind to the corresponding human Cys-loop receptor, in particular if a non-human Cys-loop receptor has been used in step a) or in step b).

The method may optionally further comprise one or more other suitable steps, such as, for example and without limitation, a step of affinity maturation, a step of screening for binding and/or for activity against the Cys-loop receptor, a step of introducing one or more amino acid modifications into the sequence of the sdAb so as to optimize it, e.g. so as to obtain a humanized sdAb, a step of humanizing the selected single-domain antibody, a step of introducing the CDRs from a selected single-domain antibody into a humanized scaffold, a step of preparing an anti-Cys loop receptor polypeptide as described herein, and/or any suitable combination of one or more of such steps, in any suitable order.

In a further aspect the invention relates to a method for producing a single-domain antibody, preferably a VHH, comprising the step of:

a) preparing a library of biological recipients, preferably cells, viruses, DNAs, RNAs or ribosomes, each biological recipient displaying thereon a single -domain antibody, preferably a VHH, wherein the library is prepared from an antibody-expressing tissue or cells isolated from a non- human animal which has been immunized with an immunogenic composition comprising a purified Cys-loop receptor,

b) screening the library of step a) for biological recipients displaying a sdAb able to bind said Cys- loop receptor, preferably in the presence of a modulator of said Cys-loop receptor,

c) selecting a sdAb directed to said Cys-loop receptor from the biological recipients identified in step b), said sdAb preferably displaying a Kd of at most 10^"6 M,

d) identifying the nucleic acid sequence encoding from a sdAb selected in step c),

e) optionally, modifying the nucleic acid sequence obtained in step d) so as to obtain a modified nucleic acid encoding for a variant of a sdAb selected in step c),

f) transfecting a host cell with an expression vector comprising the modified nucleic acid of step e) or the nucleic acid of step d)

g) culturing the transfected host cell under conditions which cause the expression of the sdAb or the variant thereof; and

h) isolating the sdAb or the variant thereof produced by the transfected host cell. ^■ Specific embodiments of the process according to the invention

In some embodiments, the method for selecting and/or obtaining a single -domain antibody and preferably a VHH, directed against a Cys-loop receptor comprises the step of:

immunizing a non-human animal, preferably belonging to a Camelidae species, with an immunogenic composition comprising a purified Cys-loop receptor, and optionally an immunoadjuvant, isolating from said non-human animal an antibody-expressing tissue or cells such as peripheral blood monocytes (PBMCs) peripheral blood lymphocytes (PBLs), and peripheral lymph nodes

preparing a library of biological recipients, preferably a library of cells,viruses, DNAs, RNAs, or ribosomes displaying thereon a single-domain antibody, from the isolated antibody-expressing tissue or cells,

screening said library for biological recipients displaying a single-domain antibody able to bind the said Cys-loop receptor, said step optionally comprises contacting the biological recipients with the Cys-loop receptor in the presence of a modulator of said Cys-loop receptor so as to screen biological recipients displaying a sdAb able to bind the Cys-loop receptor in the presence of said modulator, and selecting a single -domain antibody which binds the Cys-loop receptor preferably with a Kd of at most 10^~6 M from the identified biological recipients identified by the previous step of screening.

In some further embodiments, the invention relates to a method for obtaining and/or selecting a sdAb, preferably a VHH, directed against a Cys-loop receptor, preferably a 5-HT3 receptor, which comprises the steps of:

a) preparing a library of bacteriophages, each bacteriophage displaying thereon a sdAb, from an antibody-expressing tissue or cells isolated from a non-human animal, preferably a Camelidae, which has been immunized with an immunogenic composition comprising a purified Cys-loop receptor, b) screening the library of step b) for bacteriophages displaying sdAb able to bind said Cys-loop receptor, said step b) is preferably performed by phage display and optionally comprises contacting the bacteriophages with the Cys-loop receptor in the presence of a modulator of said Cys-loop receptor so as to screen bacteriophages displaying thereon a sdAb able to bind the Cys-loop receptor in the presence of said modulator and

c) selecting a sdAb which binds the Cys-loop receptor preferably with a Kd of at most 10^~6 M, from the bacteriophages identified in step b).

In some specific embodiments, said methods comprising a step of assessing the ability of the sdAbs to modulate the activity of the Cys-loop receptor. Such assessment may be performed by electrophysiology assays and may be carried out, for instance, after step c).

In some alternate or additional embodiments, step b) of screening comprises several rounds of selection and at least one round of selection is performed in the presence of a modulator of the Cys-loop receptor such as an antagonist.

In some other preferred embodiments, step b) may comprise the following sub-steps of:

bl) contacting the Cys-loop receptor with bacteriophages of the library obtained in step a), preferably in the presence of a modulator of said Cys-loop receptor,

b2) recovering the bacteriophages displaying VHHs which have bound said receptor in step bl), b3) optionally, cloning the bacteriophages recovered in step b2) so as to obtain a library enriched in bacteriophages displaying VHHs which bind said Cys-loop receptor, and

b4) optionally, repeating steps bl) to b3) until an appropriate enrichment of the library is obtained. In step bl) the Cys-loop receptor is preferably immobilized on a surface.

Step bl) is preferably performed in the presence of an antagonist of said Cys-loop receptor. For instance, antagonists of 5-HT3 receptor encompass AS-8112, granisetron, ondansetron, tropisetron, alosetron, and batanopride. In some alternate or additional embodiments, the method may further comprise a step of removing VHHs which bind a distinct Cys loop receptor. For instance, if the method is for obtaining VHHs directed against 5-HT3 receptor, VHHs which bind nAch receptor are removed. This step may be performed before, within or after step b).

In some more specific embodiments, the invention relates to a method for obtaining and/or selecting a sdAb, preferably a VHH, directed against a Cys-loop receptor, preferably a 5-HT3 receptor such as 5- HT3A or 5-HT3AB receptor subtypes, and able to modulate the activity of said Cys-loop receptor, comprising the steps of:

a) preparing a library of bacteriophages, each bacteriophage displaying thereon a sdAb, from an antibody-expressing tissue or cells isolated from a non-human animal, preferably from Camelidae, which has been immunized with an immunogenic composition comprising a purified Cys-loop receptor,

b) screening the library of step b) for bacteriophages displaying sdAb able to bind said Cys-loop receptor, said step b) comprises contacting the bacteriophages with the Cys-loop receptor in the presence of a modulator of said Cys-loop receptor,

c) selecting sdAbs which bind the Cys-loop receptor preferably with a Kd of at most 10^~6 M, from the bacteriophages identified in step b).,

d) selecting sdAbs obtained in step c) able to modulate the activity of said Cys-loop receptor and e) recovering the sdAbs selected in step d).

In preferred embodiments, step b) is performed by phage display and/or comprises the sub-steps bl)-b4) as described hereabove.

Each sdAb from bacteriophages selected in step c) is preferably identified, produced by recombinant means or by chemical synthesis and then isolated, before carrying out step d). Criteria to determine whether a sdAb is able to modulate a Cys-loop receptor are described here-below in the section entitled "Single domain antibodies according to the invention".

Typically, a sdAb is a modulator of a Cys-loop receptor if said sdAb is able to modulate the response induced by the endogenous ligand by at least 10%, preferably by at least 20% or if said sdAb is able to induce a response in the absence of the endogenous ligand which is equal to at least 10%, preferably to at least 20% of the response induced by the endogenous ligand alone, in the similar conditions.

It goes without saying that these specific embodiments of the methods of the invention may further comprise any additional steps as described in the above section entitled "Method for obtaining single domain antibodies directed against Cys-loop receptors".

^■ Single domain antibodies according to the invention

The instant invention also relates to a single -domain antibody directed to a Cys-loop receptor. In a preferred embodiment, the single -domain antibody is functional, that means that the single -domain antibody is able to modulate the activity of the Cys-loop receptor, upon certain conditions. For instance, the single -domain antibody can modulate the opening and/or closing of said Cys-loop receptor, and thus modulate the flow of ions through said Cys-loop receptor (i.e. to increase or to decrease such flow, or to partially or fully block such flow).

Accordingly, as fully-detailed hereunder, the single -domain antibodies according to the invention and derivatives thereof can be used to modulate the biological functions, pathways, responses, effects, mechanisms and actions in which the activation of a Cys-loop receptor is involved.

In some embodiments, the single-domain antibody according to the invention may act as an agonist of a Cys-loop receptor. In other embodiments, the single-domain antibody is an antagonist (also called blocker) of the Cys-loop receptor.

In further embodiments, the single -domain antibody may be an allosteric modulator. In other words, the single-domain antibody may amplify or attenuate the effect of the endogenous ligand on the Cys-loop receptor.

The ability of the single-domain antibody according to the invention to modulate the activity of the Cys- loop receptor may be determined by well-known methods according to the prior art. Appropriate methods encompass patch clamp electrophysiology and FlexStation analysis.

The functional activity of the single-domain antibody is preferably determined by patch clamp assay which enables to compare inhibition or increase of ion flux penetrating the cell under activation of the targeted receptor.

According to this assay, the single -domain antibody is considered as an antagonist of the Cys-loop receptor if the response elicited by the endogenous ligand of the Cys-loop receptor in the presence of the single-domain antibody is no more than 90% of the response elicited by the endogenous ligand in the absence of said single-domain antibody. No more than 90% encompasses no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 30%, no more than 10%.

The single-domain antibody is considered as an agonist if said single-domain antibody is able to elicit a response in the absence of the endogenous ligand of the Cys-loop receptor, said response being at least 10% of the response elicited by the endogenous ligand. At least 10%, encompasses at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 90%, at least 100%, at least 150%, at least 200%. Noteworthy, the single-domain antibody is a full-agonist when the elicited response is at least 100% of the response elicited by the endogenous ligand and a partial agonist when the elicited response is lower than the response induced by the endogenous ligand.

The single -domain antibody may be an allosteric modulator if said single -domain antibody is able to modulate the response induced by the endogenous ligand by at least 10%, preferably by at least 20%, such as by at least 30%, 40% or 50%.

The single -domain antibody may be a negative allosteric modulator when the response is attenuated. The single domain-antibody may be a positive allosteric modulator when the response is enhanced. The percentage of modulation exerted by the single-domain antibody on the activity of the Cys-loop receptor may be assessed by patch clamp electrophysiology assay on Xenopus oocytes expressing a recombinant Cys-loop receptor.

In such an assay, the modulation percentage of a single-domain antibody acting as an agonist may be determined by comparing:

(1) the maximum of the current elicited by the endogenous ligand of the Cys-loop receptor, said endogenous ligand being present at a concentration ranging from 0.1 *EC₅₀ to 10.EC₅₀, typically at a concentration of around EC₅₀ in the absence of the single -domain antibody with

(2) the maximum of the current elicited by the single-domain antibody being present at a concentration of at most 100 μΜ, preferably at most 10 μΜ, in the absence of the endogenous ligand.

On the other hand, the modulation percentage of a single -domain antibody acting as an inhibitor (or antagonist) or as a potentiator (or positive allosteric modulator) of the Cys-loop receptor may be determined by comparing:

(1) the maximum of the current elicited by the endogenous ligand of the cys-loop receptor, said endogenous ligand being present at a concentration ranging from 0.1 *EC₅₀ to 10.EC₅₀, typically at a concentration of around EC_50, in the absence of the single -domain antibody with

(2) the maximum of the current elicited by the endogenous ligand (at the same concentration as (1)) in the presence of the single -domain antibody, the single -domain antibody being present at a concentration of at most 100 μΜ, preferably at most 10 μΜ.

As used herein, the EC₅₀ of the endogenous ligand refers to the concentration of the endogenous ligand which elicits a response halfway between the baseline and maximum response, in said patch clamp electrophysiology assay.

In a preferred embodiment, the single domain antibody is an antagonist of a Cys-loop receptor. For instance, the single -domain antibody is an antagonist of a 5-HT3 receptor, i.e. an antagonist of at least one 5-HT3 receptor subtype.

In another embodiment, the single domain antibody is an agonist of a Cys-loop receptor. For instance, the single-domain antibody is an agonist of a 5-HT3 receptor i.e. an agonist of at least one 5-HT3 receptor subtype.

In some other embodiments, the single-domain antibody is a potentiator of a Cys-loop receptor. For instance said single-domain antibody is a potentiator of a 5-HT3 receptor, i.e. a potentiator of at least one 5-HT3 receptor subtype.

Preferably, the at least one 5-HT3 receptor subtype encompasses 5-HT3AR and/or 5-HT3ABR subtypes. In other embodiments, the single -domain antibody is a specific modulator of a member of Cys-loop receptor family. For instance, the single domain-antibody may modulate 5-HT3 receptors without significantly modulating other Cys-loop receptors such as nACh receptors. Moreover, the single-domain antibody may display distinct activities towards distinct subtypes of a Cys- loop receptor member. For instance, the single -domain antibody may be a full antagonist of 5-HT3A receptor while being a partial antagonist of 5-HT3AB receptor

The single-domain antibody may display a constant dissociation (Kd) for the Cys-loop receptor of at most 10^~6 M, preferably of at most 10^~7 M and even at most 10^~8 M. Typically, the Kd of the sdAbs of the invention for their receptor may be from 1.10 ¹² M to 1.10 ⁶ M. Kd is preferably determined by surface plasmon resonance assay. For instance, one may refer to the SPR assay described in the below examples for assessing the binding affinity of the single domain antibody to 5-HT3 receptor.

The single-domain antibody may bind any epitope, including linear epitopes and conformational epitopes of a Cys-loop receptor. Among others, the single-domain antibody may bind an epitope present or formed by the extracellular domain of the Cys-loop receptor.

Noteworthy, the single-domain antibody of the invention may bind the same site as the endogenous ligand of the Cys-loop receptor. In some other embodiments, the sdAb may bind at least partially the binding site of the endogenous ligand. In such cases, the single-domain antibody can block and/or compete with the binding of the endogenous ligand of said Cys-loop receptor. In other embodiments, the sdAb of the invention may bind a site other than the binding site of the endogenous ligand. For instance, the endogenous ligand of 5-HT3 receptors is serotonin (5-HT). Alternatively, the single -domain antibody may interact and/or bind a site present within the channel of the Cys-loop receptor or on the opening of said channel.

As mentioned above, the single-domain antibodies may be directed against any Cys-loop receptor, preferably from a vertebrate, more preferably from a mammal and still more preferably a human Cys-loop receptor.

Cys-loop receptors encompass serotonin (5-HT3) receptors, acetylcholine (nicotinic ACh or nACh) receptors, glycine (Gly) receptors, γ-aminobutyric acid (GABA_A,GABA_C) receptors and zinc-activated (ZAC) receptors. In some embodiments, the single-domain antibody according to the invention is directed against 5-HT3 receptors, in particular against 5-HT3A receptor and/or 5-HT3AB receptor. In some more preferred embodiments, the single-domain antibody according to the invention is directed against a human Cys-loop receptor, in particular a human 5-HT3 receptor such as human 5-HT3A receptor and/or human 5-HT3AB receptor.

In some embodiments, the single domain antibody directed against a Cys-loop receptor from one mammal species may cross-react with a homologous receptor from another species. For instance, the single domain antibody directed to a murine Cys-loop receptor (e.g. mouse 5-HT3A receptor or mouse 5-HT3AB receptor) may cross-react with the corresponding human Cys-loop receptor (human 5-HT3A receptor or human 5-H3AB receptor).

As used herein, a cross-reacting single -domain antibody refers to a single domain antibody able to bind two Cys-loop receptors, preferably with a Kd of at most 10^~6 M for said two Cys-loop receptors. In some other embodiments, the single-domain antibody against a Cys-loop receptor does not cross-react with a homologous receptor from another species. In such a case, the Kd of the single domain antibody for said homologous Cys-loop receptor is undetectable or of at least 10^~6 M, preferably of at least 10^~5 M. In some further embodiments, a single -domain antibody may specifically binds, or is specific to, a first Cys-loop receptor, as compared to a second Cys-loop receptor.

As used herein, "specific binding" refers to the ability of the single -domain antibody to detectably bind an epitope present on a given Cys-loop receptor while having little detectable reactivity with another Cys- loop receptor. Typically, a single -domain antibody specifically binds a first Cys-loop receptor as compared to a second Cys-loop receptor when the ratio of its Kd for the second Cys-loop receptor to the Kd for the first Cys-loop receptor is at least 10², more preferably at least 10³. It goes without saying that the Kd of the sdAb for the first Cys-loop receptor is preferably at most 10^~6M.

In some other additional embodiments, the single-domain antibody is able to specifically bind a Cys-loop receptor but not another Cys-loop receptor. For instance, the single domain antibody can bind 5-HT3 receptors, in particular human 5-HT3 receptors, without binding any other Cys-loop receptors, in particular other human Cys-loop receptors, such as nACh receptors, Gly receptors or GABA receptors.

The ability of a sdAb of the invention to bind a Cys-loop receptor and the corresponding Kd are preferably assessed by SPR as shown in the Examples.

Alternatively, the binding specificity may be evidenced by FACS analysis as shown in Example 2.

In some other embodiments, the single -domain antibody may be specific to a Cys-loop receptor subtype. For instance, the single-domain antibody may specifically bind 5-HT3A receptor as compared to 5- HT3AB receptor. In another non-limiting example, the single -domain antibody may be specific to 5- HT3AB receptor as compared to 5-HT3A receptor. Typically, a single -domain antibody specifically binds a first Cys-loop receptor subtype as compared to a second Cys-loop receptor subtype when the ratio of its Kd for the second Cys-loop receptor subtype to the Kd for the first Cys-loop receptor subtype is at least 10², more preferably at least 10³. It goes without saying that the Kd of the sdAb for the first Cys- loop receptor subtype is preferably at most 10^~6M.

In some other embodiments, the single -domain antibody may be able to bind several subtypes of a given Cys-loop receptor. In other words, the single-domain antibody can cross-react with several subtypes of a given Cys-loop receptor. For instance, the single -domain antibody may bind 5-HT3A and 5-HT3AB subtypes. It goes without saying that the Kd of the sdAb for the both subtypes are preferably at most 10^" ⁶M.

As mentioned above, a single-domain antibody according to the invention comprises a single variable domain derived from an antibody able to bind an antigen or an epitope alone, that is to say, without the requirement of another binding domain. In particular, the single -domain antibody according to the invention is devoid of light chain or fragment thereof. The single-domain antibody according to the invention may derive from the single variable domain of a heavy-chain antibody (HCAb). Heavy-chain antibodies - which comprise two heavy chains and are naturally devoid of light chains - may be obtained by immunization of camelids or sharks. Alternatively, the single -domain antibody according to the invention may be an engineered form of a heavy variable domain of an antibody.

In some embodiments, the single -domain antibody may comprise an amino acid sequences selected from a V-NAR from Ig-NAR, engineered V-NAR and fragments thereof.

In some preferred embodiments, the single-domain antibody is selected from the group consisting of VHHs, namely the variable domains of heavy-chain antibodies from Camelidae species, VHH variants, in particular humanized VHHs, and fragments thereof.

As used herein, a fragment refers to a portion of a VHH able to bind said Cys-loop receptor with a Kd of at most 10^"6 M.

A humanized VHH refers to a VHH variant which comprises one or several amino acid modifications (hereunder "humanizing amino acid modifications") as compared to a naturally-occurring VHH, said modifications enabling to decrease its immunogenicity with respect to a human subject without significantly decreasing the affinity for the Cys-loop receptor.

A humanized VHH according to the present invention may be obtained by replacing one or more of the amino acids in the Camelidae VHH sequence by their human counterpart as found in the human consensus sequence, with proviso that said amino acid modifications do not significantly affect the antigen binding capacity of the resulting VHH.

Such a method is well-known by the skilled artisan. The state in the art provides several examples of humanized scaffold for VHHs which can be used in the context of the invention. Humanized VHHs encompass partially humanized VHHs and fully-humanized VHHs.

Potentially useful humanizing amino acid modifications, in particular substitutions, can be determined by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled artisan. As an alternative, the one skilled in the art may graft the CDRs of a VHH within a humanized scaffold of VHH described in the state in the art, so as to obtain the desired humanized VHH directed against the Cys-loop receptor.

Method for humanizing VHH as well as humanized VHH scaffolds are provided, for instance, in patent application US 2010/0215664, WO2011/117423, in Conrath et al, Journal of Molecular Biology, 2005, 350:112-125 and in Vincke, Journal of Biological Chemistry, 2009, 284, 3273-3284.

As an alternative, the single-domain antibody of the invention may be a camelized human VH, namely a variant of human VH comprises amino acid modifications, in particular amino acid substitutions in the VH/VL interface so as to have a structure and biochemical properties close to that of naturally-occurring VHHs. To that respect, the skilled artisan may refer to Riechmann and Muyldermans, Journal of Immunological Methods, 1999, 231, 25-38.

The single -domain antibody of the invention comprises at least one, preferably three, complementarity determining regions (CDR) which determine its binding specificity. Preferably, the single -domain antibody comprises several, preferably 3, CDRs which are distributed between framework regions (FRs), the CDRs determining its binding specificity.

In some embodiments, the single domain antibody comprises four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1 " or "FR1 "; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively. These framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region 1 " or "CDR1 "; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. These framework regions and complementary determining regions are preferably operably linked in the following order:

FR 1 -CDR 1 -FR2-CDR2-FR3 -CDR3 -FR4 (from amino terminus to carboxy terminus).

In preferred embodiments, CDR1, CDR2 and CDR3 derives from a naturally-occurring VHH directed against a Cys-loop receptor and FR1, FR2, FR3 and FR4 are selected from naturally-occurring framework domains from camelids, humanized framework domains from camelids and camelized framework domains of a human VH.

In some further embodiment, the single-domain antibody comprises a VHH or a humanized version of a VHH, said VHH being identified or obtained by a method according to the invention.

^■ Single domain antibodies directed against a 5-HT3 receptor

A further object of the instant invention is a single-domain antibody directed against a 5-HT3 receptor, preferably a human 5-HT3 receptor. In other words, the single -domain antibody of the invention preferably binds at least one subtype of 5-HT3 receptor. Preferably the single-domain antibody of the invention binds at least one subtype of human 5-HT3 receptor.

In some embodiments, the single -domain antibody binds 5-HT3A receptor subtype without binding 5- HT3AB receptor subtype, or vice versa. In some other embodiments, the single-domain antibody binds both 5-HT3A and 5-HT3AB receptor subtypes.

The single -domain antibody preferably displays a Kd of at most 10^~6 M for a 5-HT3 receptor in particular a human 5-HT3 receptor, preferably for a 5-HT3A receptor and/or for a 5-HT3AB receptor. Kd may be determined by SPR assay as described in the below examples.

In some embodiments, the binding site of the single domain antibody overlaps or is comprised in the binding site of serotonin. The binding site of serotonin for 5-HT3 receptor has been predicted by in silico docking and is described, for instance in Thompson et al., Quaterly Reviews of Biophysics, 2010, page 1- 51. In some further embodiments, the single -domain antibody is a modulator of a 5-HT3 receptor. In other words, the single -domain antibody of the invention modulates the activity of at least one subtype of 5- HT3-receptor. For instance, said single -domain antibody can modulate 5-HT3A subtype or/and 5-HT3AB subtype.

The ability of the VHH to modulate the activity of 5-HT3 receptor may be determined by a patch clamp assay or by FlexStation analysis.

For instance, the patch clamp assay may be performed on Xenopus oocytes expressing a recombinant 5- HT3, as described in Example 4. The ability of the single-domain antibody to modulate 5-HT3 receptor may be determined by comparing the maximum of the current elicited by serotonin (e.g. at a concentration around its EC₅₀, e.g. 2.5μΜ) in the absence of the single-domain antibody with the maximum of the current elicited by serotonin in the presence of the single -domain antibody (preferably at a concentration of at most 10 μΜ, e.g. at a concentration of 1 μΜ).

In some embodiments, the single -domain antibody is an antagonist of a 5-HT3 receptor, for instance an antagonist of the 5-HT3ABR or/or 5-HT3AR.

Preferably, the maximum of current elicited by serotonin in the presence of the single-domain antibody is no more than 60%, preferably no more than 50% of the maximum of the current elicited by the endogenous ligand in the absence of said single -domain antibody. No more than 50% encompasses no more than 80%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%.

In some other embodiments, the single-domain antibody is a potentiator of a 5-HT3 receptor, for instance a potentiator of the 5-HT3ABR and/or 5-HT3AR subtype. Preferably, the maximum of current elicited by serotonin in the presence of the single -domain antibody is more than 110%, preferably more than 120% of the maximum of the current elicited by the endogenous ligand in the absence of said single-domain antibody. More than 120% encompasses more than 130%, more than 140%, more than 150%.

In some other embodiments, the single-domain antibody is an agonist of a 5-HT3 receptor, for instance an agonist of the 5-HT3ABR and/or 5-HT3AR subtype. Preferably said single -domain antibody is able to elicit a response in the absence of serotonin which is at least 50%, preferably at least 70%, more preferably at least_80% of the response elicited by serotonin alone.

In some specific embodiments, the single -domain antibody comprises an amino acid sequence having at least 60%, preferably at least 70%, more preferably at least 80% of identity with an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID N°42, SEQ ID N°43, SEQ ID N°44, SEQ ID N°45, SEQ ID N°46, and SEQ ID N°47.

In some other embodiments, the single -domain antibody comprises an amino acid sequence having at least 60%, preferably at least 70%, more preferably at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the single-domain antibody is a potentiator of a 5-HT3 receptor and comprises an amino acid sequence having at least 60% of sequence identity with SEQ ID NO: l. Such a single-domain antibody is preferably a potentiator of 5-HT3A receptor.

In some other embodiments, the single-domain antibody is an agonist of a 5-HT3 receptor and comprises an amino acid sequence having at least 60% of identity with SEQ ID N°42 or SEQ ID N°43. Such a single-domain antibody is preferably an agonist of 5-HT3AB receptor subtype and may also bind, and even modulate, 5-HT3A receptor subtype.

In some further embodiments, the single-domain antibody is comprises an amino acid sequence having at least 60% of identity with a sequence selected from SEQ ID N°44-47 and specifically binds 5-HT3AB receptor subtype as compared to 5-HT3A receptor subtype.

In some other embodiments, the single-domain antibody is an antagonist of a 5-HT3 receptor and comprises an amino acid sequence having at least 60% of sequence identity with a sequence selected from SEQ ID NO:2-6. Such a single-domain antibody is preferably an antagonist of 5-HT3A receptor. As used herein, at least 60% amino acid sequence identity encompasses at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity.

In some embodiments, the single -domain antibody is a variant of a VHH selected from VHHs of SEQ ID NO: 1-6 or SEQ ID N°42-47. The sequence of the single-domain antibody may differ from the sequence of its parent VHH in virtue of at least one amino acid modification, preferably in virtue of 1 to 50 amino acid modifications. In some embodiments, the single-domain antibody does not contain more than 30, preferably no more than 20 amino acid modifications as compared to its parent VHH. Preferably, the single-domain antibody is a variant of a VHH of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID N°42, SEQ ID N°43, SEQ ID N°44, SEQ ID N°45, SEQ ID N°46, and SEQ ID N°47, preferably a variant of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, which differs from its parent VHH in virtue of 1 to 20 amino acid modifications, preferably in virtue of 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

In some embodiments, the variant has a profile of activity (e.g. agonist, antagonist or potentiator) for the 5-HT3 receptor similar to that of its parent.

Preferably, the amino acid modifications are present within the framework regions, which means that the CDRs of the single -domain antibody are identical (i.e. has 100% amino acid sequence identity) to the CDRs of its VHH parent

Said amino acid modifications can be conservative amino acid modifications.

When present in Framework regions, the amino acid modifications may correlate with humanizing amino acid modifications.

In some specific embodiment, the single domain-antibody has at least 60%, preferably at least 70%, more preferably at least 80% of amino acid sequence identity with at least one VHH of SEQ ID NO: 1-6 or SEQ ID N°42-47. In some other embodiments, the single-domain antibody is selected from the group of VHHs of SEQ ID NO:l, 2, 3, 4, 5, 6, 42, 43, 44, 45, 46 and 47.

In some further embodiments, the single-domain antibody comprises at least 1, preferably 2 or 3, complementarity determining regions (CDR) selected from the group of CDRl, CDR2 and CDR3 of VHHs of SEQ ID NO: l-6, as shown in Figure 1A and that of CDRl, CDR2 and CDR3 of VHHs of SEQ ID NO:42-47 as shown in Figure 8A. More precisely, the single -domain antibody may comprise at least one CDR selected in the group consisting of SEQ ID:7-24 and SEQ ID NO:48-65.

In a more specific aspect, the single -domain antibody of the invention comprises 3 complementarity determining regions, CDRl to CDR3, wherein:

CDRl has an amino acid sequence selected from the group consisting of SEQ ID NO:7, 10, 13, 16, 19, and 22 or an amino acid sequence that differs from an amino acid sequence selected SEQ ID NO:7, 10, 13, 16, 19 and 22 in virtue of one, two, or three amino acid modifications;

CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:8, 11, 14, 17, 20 and 23 or an amino acid sequence that differs from an amino acid sequence selected NO:8,

11, 14, 17 , 20 and 23 in virtue of one, two, or three amino acid modifications; and,

CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 12, 15, 18, 21 and 24 ; or an amino acid sequence that differs from an amino acid sequence selected SEQ ID NO: 9, 12, 15, 18, 21 and 24, in virtue of one, two, or three acid modifications.

In some other embodiments, the single -domain antibody of the invention comprises three complementary determining regions CDRl, CDR2 and CDR3, wherein:

- CDRl is selected from the group consisting of CDRls of SEQ ID NO:7, 10, 13, 16, 19, and 22,

- CDR2 is selected from the group consisting of CDR2s of SEQ ID NO:8, 11, 14, 17, 20, and 23; and

- CDR3 is selected from the group consisting of CDR3s of SEQ ID NO: 9, 12, 15, 18, 21 and 24.

Said single -domain antibody may further have at least 60%, preferably at least 70%, more preferably at least 80%, e.g. at least 90%, of amino acid sequence identity with at least one amino acid sequence among SEQ ID NO: 1-6. Preferably, said single -domain antibody is able to bind and to modulate 5-HT3A receptor subtype.

In another embodiment, the single-domain antibody of the invention comprises 3 complementarity determining regions, CDRl to CDR3, wherein:

CDRl has an amino acid sequence selected from the group consisting of SEQ ID NO:48, 51, 54, 57, 60, and 63 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 48, 51, 54, 57, 60, and 63 in virtue of one, two, or three amino acid modifications; CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:49, 52, 55, 58, 61 and 64 or an amino acid sequence that differs from an amino acid sequence selected from NO:49, 52, 55, 58, 61 and 64 in virtue of one, two, or three amino acid modifications; and, CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 53, 56, 59, 62 and 65 ; or an amino acid sequence that differs from an amino acid sequence selected from

SEQ ID NO: 50, 53, 56, 59, 62 and 65, in virtue of one, two, or three acid modifications.

In some other embodiments, the single -domain antibody of the invention comprises three complementary determining regions CDR1, CDR2 and CDR3, wherein:

CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:48, 51, 54, 57, 60, and 63;

CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:49, 52, 55,

58, 61 and 64; and,

CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 53, 56,

59, 62 and 65.

Said single -domain antibody may further have at least 60%, preferably at least 70%, more preferably at least 80%, e.g. at least 90%, of amino acid sequence identity with at least one amino acid sequence among SEQ ID NO:42-47. Preferably, said single -domain antibody is able to bind and to modulate 5- HT3AB receptor subtype. In some other aspects, the single-domain antibody of the invention comprises one of the following combinations of CDRs:

CDR1 is, or consists essentially of, SEQ ID NO: 16, CDR2 is, or consists essentially of, SEQ ID NO: 17 and CDR3 is, or consists essentially of, SEQ ID NO: 18;

- CDR1 is, or consists essentially of, SEQ ID NO: 19, CDR2 is, or consists essentially of, SEQ ID

NO:20 and CDR3 is, or consists essentially of, SEQ ID NO:21 ;

CDR1 is, or consists essentially of, SEQ ID NO:22, CDR2 is, or consists essentially of, SEQ ID NO:23 and CDR3 is, or consists essentially of, SEQ ID NO:24,

CDR1 is, or consists essentially of, SEQ ID NO:48, CDR2 is, or consists essentially of, SEQ ID NO:49 and CDR3 is, or consists essentially of, SEQ ID NO:50;

CDR1 is, or consists essentially of, SEQ ID NO:51, CDR2 is, or consists essentially of, SEQ ID NO:52 and CDR3 is, or consists essentially of, SEQ ID NO:53; CDR1 is, or consists essentially of, SEQ ID NO:54, CDR2 is, or consists essentially of, SEQ ID NO:55 and CDR3 is, or consists essentially of, SEQ ID NO:56;

CDR1 is, or consists essentially of, SEQ ID NO:60, CDR2 is, or consists essentially of, SEQ ID NO:61 and CDR3 is, or consists essentially of, SEQ ID NO:62;

CDR1 is, or consists essentially of, SEQ ID NO:63, CDR2 is, or consists essentially of, SEQ ID

NO:64 and CDR3 is, or consists essentially of, SEQ ID NO:65,

Said single -domain antibody may further have at least 60%, preferably at least 70%, more preferably at least 80%, e.g. at least 90% of amino acid sequence identity with at least one amino acid sequence among SEQ ID NO: 1-6 and SEQ NO:42-47. Alternatively or in addition, said single domain antibody is able to bind and/or to modulate 5-HT3AB receptor subtype and/or 5-HT3A receptor subtype.

In a more specific aspect, the single -domain antibody of the invention comprises one of the following combinations of features:

i. The single domain antibody is a potentiator of 5-HT3A receptor subtype and comprises a

combination of CDRs wherein : CDR1 is, or consists essentially of, SEQ ID NO:7, CDR2 is, or consists essentially of, SEQ ID NO:8 and CDR3 is, or consists essentially of, SEQ ID NO:9; ii. The single domain antibody is an antagonist of 5-HT3A receptor subtype and comprises one of the following combinations of CDRs :

SEQ ID NO: 17 and CDR3 is, or consists essentially of, SEQ ID NO: 18;

CDR1 is, or consists essentially of, SEQ ID NO: 19, CDR2 is, or consists essentially of, SEQ ID NO:20 and CDR3 is, or consists essentially of, SEQ ID NO:21 ;

CDR1 is, or consists essentially of, SEQ ID NO:22, CDR2 is, or consists essentially of, SEQ ID NO:23 and CDR3 is, or consists essentially of, SEQ ID NO:24, iii. The single domain antibody is an antagonist of 5-HT3AB receptor subtype and comprises one of the following combinations of CDRs :

- CDR1 is, or consists essentially of, SEQ ID NO:51, CDR2 is, or consists essentially of,

SEQ ID NO:52 and CDR3 is, or consists essentially of, SEQ ID NO:53; iv. The single domain antibody specifically binds 5-HT3AB receptor subtype as compared to 5- HT3A receptor subtype and comprises one of the following combinations of CDRs

CDRl is, or consists essentially of, SEQ ID NO:54, CDR2 is, or consists essentially of, SEQ ID NO:55 and CDR3 is, or consists essentially of, SEQ ID NO:56;

- CDRl is, or consists essentially of, SEQ ID NO:57, CDR2 is, or consists essentially of,

SEQ ID NO:58 and CDR3 is, or consists essentially of, SEQ ID NO:59;

CDRl is, or consists essentially of, SEQ ID NO:60, CDR2 is, or consists essentially of, SEQ ID NO:61 and CDR3 is, or consists essentially of, SEQ ID NO:62;

CDRl is, or consists essentially of, SEQ ID NO:63, CDR2 is, or consists essentially of, SEQ ID NO:64 and CDR3 is, or consists essentially of, SEQ ID NO:65,

Preferably, the single -domain antibody of the invention binds and modulates a human 5-HT3 receptor.

In some alternate embodiments or in combination with any previous embodiments, the single -domain antibody of the invention comprises 4 framework regions, FR1 to FR4, and 3 complementarity determining regions, CDRl to CDR3, that are operably linked in the order FR 1 -CDR1-FR2-CDR2-FR3 - CDR3-FR4 wherein CDRl, CDR2 and CDR3 are complementary regions as previously described and FR1, FR2, FR3 and FR4 are framework regions 1 to 4.

For instance, said FR1, FR2, FR3 and FR4 may be selected from FR1, FR2, FR3 and FR4 of SEQ ID NO: l-6, as depicted in Figure 1A, FR1, FR2, FR3 and FR4 of SEQ ID N°42-47 as depicted in Figure 8A, humanized framework regions of a VHH from Camelidae and camelized framework regions of a human VH.

In some alternate embodiments or in combination with a previous embodiment, the single -domain antibody of the invention comprises 4 framework regions, FR1 to FR4, and 3 complementarity determining regions, CDRl to CDR3, that are operably linked in the order FR1 -CDRl -FR2-CDR2- FR3- CDR3-FR4, (from amino terminus to carboxy terminus) wherein:

CDRl has an amino acid sequence selected from the group consisting of SEQ ID NO:7, 10, 13,

16, 19, and 22 or an amino acid sequence that differs from an amino acid sequence selected SEQ ID NO:7, 10, 13, 16, 19 and 22 in virtue of one, two, or three amino acid modifications;

- CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:8, 11, 14,

17, 20 and 23 or an amino acid sequence that differs from an amino acid sequence selected NO:8, 11, 14, 17, 20 and 23 in virtue of one, two, or three amino acid modifications; and, CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 12, 15,

18, 21 and 24 ; or an amino acid sequence that differs from an amino acid sequence selected SEQ ID NO: 9, 12, 15, 18, 21 and 24, in virtue of one, two, or three acid modifications.

In said embodiment, the framework regions FR1 to FR4 may be selected from the group consisting of: Framework regions having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% amino acid sequence identity with the framework amino acid sequence of any one of SEQ ID NO: 1-6 as shown in Figure 1 (e.g. SEQ ID 25-41), ;

Framework regions from a camelized human VH andFramework regions from a humanized VHH, including partially or fully-humanized VHH, such as humanized versions of the framework regions of

SEQ ID NO: 1-6 as shown in Figure 1, in particular humanized versions of the framework regions of SEQ

ID N° 19, 20 and 21.

In some other embodiments, the single-domain antibody of the invention comprises 4 framework regions, FRl to FR4, and 3 complementarity determining regions, CDRl to CDR3, that are operably linked in the order FRl -CDRl -FR2-CDR2- FR3-CDR3-FR4, wherein:

CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:8, 11, 14, 17, 20 and 23 or an amino acid sequence that differs from an amino acid sequence selected NO: 8,

11, 14, 17, 20 and 23 in virtue of one, two, or three amino acid modifications; and,

CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 12, 15, 18, 21 and 24 ; or an amino acid sequence that differs from SEQ ID NO: 9, 12, 15, 18, 21 and 24, in virtue of one, two, or three acid modifications;

- FRl has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence from SEQ ID NO:25, 26 or 27, ;

FR2 has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:28-33 ;

FR3 has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:34-38 , and

FR4 has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:39-41.

FRl may differ from SEQ ID NO:25, 26 or 27 in virtue of one, two, three, four, five, six, seven, eight or nine amino acid modifications.

FR2 may differ from SEQ ID NO:28-33 in virtue of one, two or three amino acid modifications.

FR3 may differ from SEQ ID NO: 34-38 in virtue of one, two, three, four, five, six, seven, or eight amino acid modifications.

FR4 may differ from SEQ ID NO:39-41 in virtue of one, two or three amino acid modifications. In some further embodiments, the single domain antibody comprises the sequence

FRl -CDRl -FR2-CDR2- FR3-CDR3-FR4 wherein:

- the single-domain antibody comprises one of the following combinations of CDRs: - CDRl is SEQ ID NO:7, CDR2 is SEQ ID NO:8 and CDR3 is SEQ ID NO:9;

- CDRl is SEQ ID NO: 10, CDR2 is SEQ ID NO: 11 and CDR3 is SEQ ID NO: 12;

- CDRl is SEQ ID NO: 13, CDR2 is SEQ ID NO: 14 and CDR3 is SEQ ID NO: 15;

- CDRl is SEQ ID NO: 16, CDR2 is SEQ ID NO: 17 and CDR3 is SEQ ID NO: 18;

- CDRl is SEQ ID NO: 19, CDR2 is SEQ ID NO:20 and CDR3 is SEQ ID NO:21 ; and

- CDRl is SEQ ID NO:22, CDR2 is SEQ ID NO:23 and CDR3 is SEQ ID NO:24.; and

- the framework regions FRl, FR2 and FR3 are as defined in any one preceding embodiments. In particular:

FRl may be an amino acid sequence selected from the group consisting of SEQ ID NO:25-27 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 25-

27 in virtue of one, two, three, four, five, six, seven, eight or nine amino acid modifications;

FR2 may be an amino acid sequence selected from the group consisting of SEQ ID NO:28-33 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO:28-

33 in virtue of one, two or three amino acid modifications;

- FR3 may be an amino acid sequence selected from the group consisting of SEQ ID NO:34-38 and an amino acid sequence that differs from an amino acid sequence selected from SEQ ID

NO:34-38 in virtue of one, two, three, four, five, six, seven, or eight amino acid modifications; and

FR4 may be an amino acid sequence selected from the group consisting of SEQ ID NO:39-41, ID or may differ from an amino acid sequence selected from SEQ ID NO:39-41 in virtue of one, two or three amino acid modifications.

In some other embodiments or in combination with a previous embodiment, the single -domain antibody of the invention comprises 4 framework regions, FRl to FR4, and 3 complementarity determining regions, CDRl to CDR3, that are operably linked in the order FRl -CDRl -FR2-CDR2- FR3-CDR3-FR4, (from amino terminus to carboxy terminus) wherein:

CDRl has an amino acid sequence selected from the group consisting of SEQ ID NO:48, 51, 54,

57, 60, and 63 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 48, 51, 54, 57, 60, and 63 in virtue of one, two, or three amino acid modifications; - CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:49, 52, 55,

58, 61 and 64 or an amino acid sequence that differs from an amino acid sequence selected from NO:49, 52, 55, 58, 61 and 64 in virtue of one, two, or three amino acid modifications; and, CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 53, 56,

59, 62 and 65 ; or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 50, 53, 56, 59, 62 and 65, in virtue of one, two, or three acid modifications.

In said embodiment, the framework regions FRl to FR4 may be selected from the group consisting of: Framework regions having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% amino acid sequence identity with the framework amino acid sequence of any one of SEQ ID NO:42-47 as shown in Figure 8A

Framework regions from a camelized human VH and

- Framework regions from a humanized VHH, including partially or fully-humanized VHH, such as humanized versions of the framework regions of SEQ ID NO:42-47 as shown in Figure 8A. In some other embodiments, the single-domain antibody of the invention comprises 4 framework regions, FRl to FR4, and 3 complementarity determining regions, CDRl to CDR3, that are operably linked in the order FRl -CDRl -FR2-CDR2- FR3-CDR3-FR4, wherein:

- CDRl has an amino acid sequence selected from the group consisting of SEQ ID NO:48, 51, 54,

57, 60, and 63 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 48, 51, 54, 57, 60, and 63 in virtue of one, two, or three amino acid modifications; CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:49, 52, 55,

58, 61 and 64 or an amino acid sequence that differs from an amino acid sequence selected from NO:49, 52, 55, 58, 61 and 64 in virtue of one, two, or three amino acid modifications; and,

59, 62 and 65 ; or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 50, 53, 56, 59, 62 and 65, in virtue of one, two, or three acid modifications. FRl has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence from SEQ ID NO:66 or 67;

FR2 has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:68-73 ;

FR3 has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:74-79 , and

- FR4 has at least 80%, preferably at least 90% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO: 80-82.

FRl may differ from SEQ ID NO:66 or 67 in virtue of one, two, three, four, five, or six amino acid modifications.

FR2 may differ from an amino acid sequence selected from SEQ ID NO:68-73 in virtue of one, two, three amino or four amino acid modifications.

FR3 may differ from an amino acid sequence selected from SEQ ID NO: 74-79 in virtue of one, two, three, four, five, six, seven, or eight amino acid modifications.

FR4 may differ from an amino acid sequence selected from SEQ ID NO: 80-82 in virtue of one, two or three amino acid modifications.

In some further embodiments, the single domain antibody comprises the sequence FR1 -CDR1 -FR2-CDR2- FR3-CDR3-FR4 wherein:

- the single-domain antibody comprises one of the following combinations of CDRs:

- CDR1 is, or consists essentially of, SEQ ID NO:51, CDR2 is, or consists essentially of, SEQ ID

NO:52 and CDR3 is, or consists essentially of, SEQ ID NO:53;

CDR1 is, or consists essentially of, SEQ ID NO:63, CDR2 is, or consists essentially of, SEQ ID NO:64 and CDR3 is, or consists essentially of, SEQ ID NO:65, and

- the framework regions FR1, FR2 and FR3 are as defined in any one preceding embodiments. In particular:

FR1 may be an amino acid sequence selected from the group consisting of SEQ ID NO:66-67 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO:66- 67in virtue of one, two, three, four, five, or six amino acid modifications;

- FR2 may be an amino acid sequence selected from the group consisting of SEQ ID NO:68-73 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 68- 73 in virtue of one, two or three amino acid modifications;

FR3 may be an amino acid sequence selected from the group consisting of SEQ ID NO: 74-79 and an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 74- 79 in virtue of one, two, three, four, five, six, seven, or eight amino acid modifications; and

FR4 may be an amino acid sequence selected from the group consisting of SEQ ID NO: 80-82 or may differ from an amino acid sequence selected from SEQ ID NO: 80-82 in virtue of one, two or three amino acid modifications. ^■ Anti-Cys loop receptor polypeptides according to the invention

In a further aspect, the present invention relates to a polypeptide construct, also called therein an anti-Cys loop receptor polypeptide, comprising at least one sdAb of the invention or a fragment thereof. As used herein, a fragment refers to a portion of a sdAb able to bind said Cys-loop receptor with a Kd of at most 10^"6 M. Said anti-Cys loop receptor polypeptide may further comprise an additional entity which is fused or conjugated to the at least one sdAb. It goes without saying that the anti-Cys-loop receptor polypeptide is able to bind said Cys-loop receptor and may modulate its activity. The additional entity conjugated or fused to the sdAb may be of any type. The additional entity and the sdAb may be linked to each other directly or via a spacer. The spacer is can be any standard linker commonly used for the preparation of polypeptide constructs. In some embodiments, the linker is a polypeptides comprising from 1 to 50 amino acid residues. Some preferred examples are Gly-Ser linkers such as tetraglycyl-seryl-triglycyl-serine peptide or polyalanine linkers. The additional moiety may be a chemical moiety enabling the immobilization of the sdAb on a surface. The additional entity may be selected from streptavidin, biotin, avidine, affinity tags such as His-tag, aviTag, calmodulin-tag, HA-tag, Myc -tag, strep-tag, thioredoxin tag and the like. In other embodiments, the additional entity may be a chemical or a biochemical entity useful for detection, in other words, a labeling mean. Said labeling mean may vary based on the detection method and the intended use of the polypeptide. As explained below, the polypeptides of the invention may be used, among others, for cell immunostaining, in vitro assay such as ELISA, in vivo imaging and the like. Accordingly, the polypeptide of the invention may comprise a sdAb of the invention fused or conjugated to a labeling mean, e.g. a molecule or a protein selected from an enzyme such as horseradish peroxidase or alkaline phosphatase, a fluorescent protein such as GFP, a fluorescent label such as fluorescein rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine, a phosphorescent label, a chemiluminescent label or bioluminescent label such as luminal or isoluminol, a chromophore, a radio-isotope e.g. suitable for in vivo imaging or a heavy-metal ion. When the anti-Cys loop receptor polypeptide is used as a drug, the additional entity may be selected among entities which may enhance, promote or extend the biological activities of sdAb in vivo. In some embodiments, the anti-Cys loop receptor polypeptide may comprise one or several moieties enable to increase the serum half -life of the sdAb in a human patient. This moiety may be selected from the group consisting of poly(ethyleneglycol) (PEG) molecules, N-linked or O-linked glycosylation moieties, Fc domain from human immunoglobulins, in particular the Fc domain from human IgG, and variants thereof which may display enhanced binding to FcRn receptor, Fragments from Fc domain of human Ig, in particular CH3 and/or CH2 domains, a single-domain antibody directed against a human serum protein such as human serum albumin, thyroxine -binding protein, transferrin, or fibrinogen. Preferably, the at least one additional entity is a sdAb directed against human serum protein. Appropriate sdAbs directed against human serum albumin are described, for instance, in Patent applicationWO04/062551, the disclosure of which being incorporated therein by reference.

In some further embodiments, the anti-Cys-loop receptor polypeptide of the invention is multivalent. In such a case, the polypeptide of the invention comprises at least two sdAbs of the invention directed against said Cys-loop receptor. Accordingly, the polypeptide of the invention may be divalent, trivalent, tetravalent, pentavalent or hexavalent. The polypeptide may comprise identical sdAbs or distinct sdAbs. Multivalent polypeptides may display improved functionality as compared to monovalent polypeptides, due to avidity.

In some additional embodiments, the anti-Cys-loop receptor polypeptide may be multispecific (i.e. bispecific) and multivalent. For instance, the anti-Cys loop receptor polypeptide may comprise at least two sdAbs directed against said Cys-loop receptor and at least one sdAb directed against a human serum protein such as the human serum albumin.

In some additional or alternative embodiments, the additional entity may be a drug or a toxin, for instance, a drug or a toxin acting on the nervous system. The toxin or the drug may have the same biological activity or the same therapeutic target as the sdAb. In other embodiments, the toxin or the drug may have a distinct therapeutic target. Examples of drugs encompass, without being limited to, anxiolytics, antidepressants, anti-inflammatories, analgesics, anti-emetics, drugs for the treatment of Parkinson's disease or Alzheimer's disease and the like.

Another object of the invention is an anti-5HT3 receptor polypeptide comprising a sdAb as described within the present section. Said polypeptide may comprise one or several additional entities as described in the hereabove section untitled "Anti-Cys loop receptor polypeptides according to the invention".

In a preferred embodiment, the polypeptide of the invention comprises at least one sdAb directed against 5-HT3 receptor as described in the above section Single domain antibodies directed against a 5-HT3 receptor such as VHHs having at least 60%, preferably at least 80% e.g. at least 90% of amino acid sequence identity with at least one sequence among SEQ ID NO: l, 2, 3, 4, 5,6, 42, 43, 44, 45, 46 and 47. In some further embodiments, the polypeptide of the invention is a heavy-chain antibody.

^■ Uses according to the invention

The single-domain antibodies and the polypeptides according to the invention may be used in various fields, including biological research, biochemical industry or medicine.

For instance, the single-domain antibodies may be used as ligands for the purification of Cys-loop receptors. They can also be used as crystallization chaperone so as to promote the crystallization of a Cys- loop receptor. Single domain-antibodies for the crystallization of a 5-HT3 receptor may be selected from single-domain antibodies having at least 60%, preferably at least 70%, more preferably at least 80%, e.g. at least 90% sequence identity with at least one sequence among SEQ ID NO: l, 2, 3, 4, 5, 6, 42, 43, 44, 45, 46 and 47. A preferred single -domain antibody for the crystallization of a 5-HT3 receptor, in particular 5-HT3A receptor, is of SEQ ID NO:5.

The sdAbs and the polypeptides of the invention may also be used in cell immuno-staining, in in vivo imaging and for diagnosis purposes.

They may also be used as biological reagents in in vitro assays, e.g. as test compounds or competitive binders for the identification, the screening or the characterization of potential drugs targeting a Cys-loop receptor.

The invention also relates to a kit for screening drug candidates for a disorder involving a Cys-loop receptor comprising a sdAb or a polypeptide according to the invention. The polypeptides and the sdAbs of the invention may be used in various immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays". Since Cys-loop receptors are relevant targets for the treatment of various diseases and disorders, including addictions, the sdAbs and related polypeptides, in particular the functional ones, may be used as a drug. In particular, the sdAb or the polypeptide of the invention may be used in the treatment or the prevention of a disorder involving a Cys-loop receptor. The invention also relates to the use of said sdAb or said polypeptide for the manufacture of a medicament for the treatment of a disorder involving a Cys-loop receptor.

For instance, the sdAbs against a 5-HT3 receptor and polypeptides comprising them can be used in the treatment, the management and/or the prevention of chemotherapy-induced, radiotherapy-induced and/or postoperative nausea and vomiting, irritable bowel syndrome, addiction, pruritis, emesis, fibromyalgia, migraine, rheumatic diseases, neurological disorders such as anxiety, psychosis, nociception and cognitive dysfunction.

sdAbs and polypeptides of the invention acting as an antagonist of 5-HT3 receptor, in particular as an antagonist of 5-HT3A receptor, may be used for the treatment or the prevention of a disorder selected from emesis, chemotherapy-induced, radiotherapy-induced and/or postoperative nausea and vomiting, and irritable bowel disorder syndrome.

sdAbs and polypeptides of the invention directed to a nACh receptor may be used in the treatment, the management and/or the prevention of schizophrenia, Alzheimer's disease, pain, Parkinson's disease, attention deficit-hyperactivity disorder, Crohn's disease, myasthenia gravis, nicotine dependence, inflammation, sporadic amyotrophic lateral sclerosis, Escobar syndrome, glaucoma, rheumatoid arthritis, inflammation and analgesia. These compounds may be also used in smoking cessation therapy.

sdAbs and polypeptides directed to a GABA_A receptor may have anxiolytic, anticonvulsant, amnesic, sedative, hypnotic, euphoriant, antinociceptive and/or muscle relaxant properties. sdAbs and polypeptides directed to a GABA_A receptor may be used in the treatment, the management and/or the prevention of psychomotor disorders, nervous disorders, and many others nervous systems pathology such as depression, bipolar syndrome, epilepsy, dyskinesia, anxiety, schizophrenia, anxiety, cognitive disorders alcoholism, ataxia, dependence on psychoactive substances, obesity and insomnia. They can be used as anaesthetic compounds. They may also be used in the treatment or the prevention of cancers associated with an overexpression of GABRA3 gene, such as certain lung cancer, endometrial cancer and glioma. sdAbs and polypeptides directed to Glycine receptor may be used in the treatment, the management and/or the prevention of neurologic disorders such as hyperekplexia for inflammatory pain, immunomodulation, spasticity, epilepsy and hyperalgesia.

sdAbs and polypeptides directed to a Zinc-activated receptor may be used in the treatment or the prevention of pathologies such as ischemia, epilepsy and traumatic brain injury.

The invention further relates to a pharmaceutical composition comprising a sdAb or a polypeptide of the invention together with a pharmaceutically acceptable excipient. The pharmaceutical composition of the invention may be formulated according to standard methods such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; Twenty first Edition, 2005).

Pharmaceutically acceptable excipients that may be used are, in particular, described in the Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; 6th revised edition, 2009). The pharmaceutical composition of the invention may be obtained by admixing a sdAb or a polypeptide of the invention with an appropriate degree of purity with at least one customary excipient (or carrier) such as (a) fillers or diluents such as for example, starch, lactose, sucrose, glucose, mannitol, microcrystalline cellulose and silicic acid; (b) binders, such as, carboxymethylcellulose, gelatin, polyvinylpyrrolidone, sucrose; (c) humectants, as for example, glycerol; (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, sodium croscarmellose and sodium carbonate; (e) solution retarders, as for example paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as glycerol monostearate; (h) adsorbents such as kaolin and bentonite; (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, (j) antioxidant agents, (k) buffering agents such as sodium citrate or sodium phosphate, (1) preservatives, (m) flavours and perfumes, etc. It goes without saying that (i) the excipient(s) to be combined with (ii) the active ingredient may vary upon (i) the physico-chemical properties including the stability of the said active ingredient, (ii) the pharmacokinetic profile desired for said active ingredient, (iii) the galenic form and (iv) the route of administration.

The pharmaceutical composition may comprise:

from 0,01% to 90% by weight of a compound of the invention, and

from 10% to 99,99% by weight of excipients,

the percentage being expressed as compared to the total weight of the composition.

Preferably, the pharmaceutical composition may comprise:

from 0, 1 % to 50% by weight of a compound of the invention, and

from 50% to 99,9% by weight of excipients.

The pharmaceutical compositions of the invention may be administered by any conventional route, including by enteral route (i.e. oral) e.g. in the form of tablets, capsules, by parenteral, intramuscular, transdermal, intravenous route e.g. in the form of injectable solutions or suspensions and by topical route e.g. in the form of gels, ointments, gels, lotions, patches, suppositories and the like.

In some particular embodiments, the pharmaceutical composition may be a lyophilisate or a freeze-dried powder which may be dissolved in an appropriate vehicle just before being administered to the patient. A further object of the invention is a method for treating a patient suffering from a disorder involving a Cys-loop receptor, wherein said method comprises administering to said patient a therapeutically effective amount of a polypeptide or a sdAb according to the invention. By "therapeutically effective amount" herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by the skilled artisan using known techniques. Dosages may range from 0.001 to 100 mg/kg of body weight or greater, for example 0.1, 1.0, 10, or 50 mg/kg of body weight, with 1 to lOmg/kg being preferred. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by the skilled artisan. Administration of the pharmaceutical composition of the invention may be done by various routes, including, but not limited to, oral, subcutaneous, intravenous, parenteral, intranasal, intraortical, intraocular, rectal, vaginal, transdermal, topical (e.g., gels), intraperitoneal, intramuscular, or intrapulmonary route.

The polypeptides and sdAbs described herein may be administered with other therapeutics concomitantly, i.e., the therapeutics described herein may be co-administered with other therapies or therapeutics, including for example, small molecules, radiation therapy, surgery, etc.

The invention also relates to the therapeutic use of a nucleic acid encoding a sdAb according to the invention, e.g. for treating or preventing a disorder as described above. Said nucleic acid may be administered to the patient in a form suitable to promote the delivery of the sdAb in a cell of interest in the patient, whereby the cell is able to express and release the sdAb. For instance, the nucleic acid encoding the sdAb may be present within a viral vector or a plasmid. Alternatively, the nucleic acid may be formulated with a synthetic vector for gene transfection such as polycationic polymers or liposome- forming phospholipids e.g. DOPC.

^■ Further objects according to the invention

In a further aspect, the invention relates to an isolated nucleic acid comprising a sequence encoding a single-domain antibody or a polypeptide according to the instant invention.

The invention also relates to a vector comprising a nucleic acid which comprises a sequence encoding a single-domain antibody or a polypeptide according to the invention. Preferably, the vector is such that a nucleic acid sequence encoding said single -domain antibody or polypeptide is operably linked to a promoter and optionally to other regulatory elements such as e.g. terminators, enhancers, polyadenylation signals, signal sequences for secretion, and the like. Such vectors are particularly useful for the recombinant production of the single-domain antibody or polypeptide according to the invention. For instance, the vector is transfected in an appropriate host cell and the host cell is then cultured in conditions allowing the production of the single-domain antibody or the polypeptide according to the invention. For review about the recombinant expression of a given protein, one may refer for instance to Ausubel et al, "Current Protocols in Molecular Biology ", Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3(rd) edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York). As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

The invention also pertains to a host cell comprising a nucleic acid or a vector as defined above. The host cell may be used for the production of a single-domain antibody or a polypeptide of the invention. The host cell may be any host cell capable of producing an sdAb or a polypeptide of the invention, including e.g. a prokaryotic host cell, such as e.g., E. coli, or a (cultured) mammalian, plant, insect, fungal or yeast host cell, including e.g. CHO-cells, BHK-cells, human cell lines (including HeLa, COS and PER C6), Sf9 cells and Sf+ cells. An appropriate host cell encompasses a cell of an eukaryotic microorganism such as yeasts and filamentous fungi. Preferred yeast host cell include Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and Kluyveromyces lactis. For illustration of appropriate strains, constructs and fermentation conditions for production of the sdAb or the polypeptide of the invention, see, for instance, van de Laar et al., {Biotechnology and Bioengineering, 2007, 96, 3:483-494).

A further object of the invention is a method for producing a sdAb or a polypeptide according to the invention, wherein the method comprises the steps of:

a) culturing a host cell as previously-defined and

b) recovering the said single domain antibody or the said polypeptide from the cell culture.

It goes without saying that step a) is performed under conditions allowing the expression of the desired sdAb or polypeptide by the host cell. Suitable expression conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled artisan.

Under such conditions, the sdAb or the polypeptide of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced. The sdAb and the polypeptide may be produced, for instance, as inclusion bodies or secreted in the culture medium.

The sdAb or the polypeptide of the invention may then be isolated from the host cell and/or from the culture medium in which said host cell was cultivated, using protein isolation and/or purification techniques known per se, such as chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques and the like.

As an alternative, the sdAb or the polypeptide of the invention may be produced by a transgenic mammal such as of transgenic rabbits, goats, or sheeps. For instance, they may be recovered from the milk of said transgenic animal. Another option is the production of the sdAb and the polypeptide of the invention in a transgenic plant, such as a transgenic tabacco. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. EXAMPLES

EXAMPLE 1: VHHs directed to 5-HT3A receptor

1. Preparation of purified 5-HT3 receptors

5-HT3A Mouse 5-HT3A receptor cDNA was cloned into the pcDNA5 TO vector and use to generate stable recombinant T-Rex CHO cell line as described in Tol et al, Journal of Biological Chemistry, 2013, 288, 5756-5769. A Strep-tag was engineered to the N-terminus of the 5-HT3A receptor. The T-Rex CHO cell lines were obtained by transfection of the corresponding plasmid DNA for 5-HT3A with Lipofectamine (Invitrogen). After 1-2 days cells were trypsinized and diluted 1 :20 and 1 : 100 in petri dishes containing 15 ml of DMEM-F12, 10% FBS, 200 μg/ml hygromycin and 10 μg/ml blasticidin. Colonies containing approximately 100 cells were detached after 10-14 days from the surface with trypsin-EDTA applying glass cloning cylinders and single colonies were harvested and replated on 12- and 24-well plates two weeks later. Recombinant 5-HT3 expression was obtained by induction with 2 μg/ml tetracycline and 4 mM sodium butyrate, respectively, for 36-48 h. The expression of 5-HT3 was confirmed by radioligand binding assays as previously described in Hovius et al. (J. Neurochem., 1998, 70, 824-834) except that membranes were replaced by 104 intact cells.

Large-scale production of 5-HT3 was achieved by culturing the stable CHO cell line in roller flasks. Induced CHO cells were harvested after 40 h and cells removed, pelleted and stored at -80°C. Generally yields in the range of 30 g of cell pellet containing 50 mg of active receptor. Cell pellets were subjected to membrane preparation by resuspension in 10 mM HEPES pH 7.4, 1 mM EDTA and a protease inhibitor cocktail (Roche) followed by homogenization with an Ultra-Turrax T25 homogenizer. Homogenates were centrifuged for 1 h at 110,000 x g, resuspended in 50 mM Tris/HCl, 500 mM NaCl pH7.4 and frozen in liquid nitrogen or subjected immediately to solubilization. The C12E9 detergent was added to a final concentration of 1 % and membranes were solubilized by gentle agitation for at least 1 h. Large particles were then removed by low speed (3200 x g) centrifugation for 10 min followed by high speed (110,000 x g) centrifugation for 1 h to clear insoluble material. The solution was passed through a 0.22 μιη filter and subjected to affinity purification. Solubilized 5-HT3 receptor was subjected to Streptacin Superflow High Capacity Column (IBA, Germany) purification overnight and equilibrated with 50 mM Tris/HCl, 500 mM NaCl, 0.005% C12E9, pH 7.4 using a continuous flow of 1 ml/min. The columns were washed with 2 column volumes of 50 mM Tris/HCl, 125 mM NaCl, 0.005% C12E9, pH 7.4 and eluted with 3 column volumes of the same buffer including 5 mM d-desthiobiotin at a flow rate of approximately 3 column volumes per hour. Purified 5-HT3 receptor was concentrated using 100,000 MWCO Concentrators (Millipore) to 1-5 mg/ml, depending on further applications. If higher purity was required, 5-HT3 receptor was loaded onto a Superdex 200 10/300 GL gel filtration column (GE Healthcare) in 50 niM Tris/HCl, 125 niM NaCl, 0.005% C12E9, pH 7.4. The concentration of 5-HT3 receptor during the purification steps was determined by one or more of the following methods: radioligand binding, by a BCA assay (Sigma Aldrich) (in the presence of 0.1% Triton X-100), or by measuring A280 absorbance with a Nanodrop 2000 (Thermo Scientific) assuming 1 Abs = 1 mg/ml. Another appropriate protocol for preparing 5-HT3 receptor is described in Hassaine et al., Biochim Biophys Acta. 2013, 1828, 2544-2552.

2. Identification of VHHs directed against m5-HT3A receptor

Llamas (n = 4) were subjected to immuninuzation with purified 5-HT3 receptor (1 mg receptor per immunization + immunoadjuvant) according to the following schedule:

Day 0: Immunization (Complete Freund's adjuvant - CFA), Pre -bleed

Day 21 : Immunization (Incomplete Freund's adjuvant - IF A)

Day 42: Immunization (IF A)

Day 52: Test Bleed

Day 63: Immunization (IF A)

Day 73: Production Bleed

Lymphocytes from immunized llama blood were purified on Leucosep (Greiner Bio One). Total RNA was extracted from peripheral blood lymphocytes and used as a template for first strand cDNA synthesis with oligo(dT) primers. VHH encoding sequences were amplified by PCR as previously described by Saerens, et al. (2004), J. Biol. Chem., 2004 50, 51965-51972, digested with PstI and NotI, and cloned into the PstI and NotI sites of the phage-display phagemid vector pHEN4. A Vi l l i library of approximately 10⁷ independent transformants was obtained. Approximately 82% of the transformants harbored inserts corresponding to the expected size of the V i l l i gene. Three consecutive rounds of phage display and panning (10¹¹ phages per well of microliter plate) were performed on streptactin plates (IBA and Nunc) coated with 100 μΐ (100 ii / ml) purified 5-HT3A receptor in 8 wells. Three rounds of selection were conducted in the presence of a 5HT3A receptor antagonist (Granisetron) at a concentration of 10 μΜ. The enrichment of antigen-specific phages within the pools at each round of panning was assessed by polyclonal phage LI ISA. A clear enrichment was obtained after the second and third rounds of panning. A total of 192 individual colonies (96 from the second round and 96 from third round) were randomly selected and analyzed by ELISA for the presence of VHH specific to antigens in periplasmic extracts. The VHH sequences from the LI .ISA-positive colonies were subjected to sequence determination and allowed the identification of several different VI I I Is (see Figure 1).

3. Characterization of selected VHHs

■ Preparation o f VHHs

VHH were sub-cloned into a standard pLIC vector construct with a His tag at de C-terminal and transformed into BL21 E. coli strain. Scale-up was conducted in shaker flasks at 24° in auto inducing ZYP medium overnight. Cell pellets were sonicated in lysate buffer (PBS, 1% Triton X-100, 5 mM Imidizole pH 8.0, 0.1% PMSF). Purification was conducted on a Ni-NTA resin followed by gel filtration and analyzed by SDS-page gel. ■ Binding of VHHs to 5-HT3a receptor

The abilities of the selected VHHs to bind 5-HT3a receptor were checked by immuno-staining and size- exclusion chromatography.

Each VHH was incubated with recombinant T-Rex CHO cell line expressing 5HT3-receptor (5HT3-R+) and with T-Rex CHO cell line which does not express 5-HT3 receptor (5-HT3R-). The binding of VHH to 5HT3-recepror was revealed with a dye (Tri.NTA-Atto), which specifically targets the poly-histidine tag of the VHH (Tris-NTA-Atto647).

All the identified VHHs were verified by in vivo surface labeling using a dye (Tri.NTA-Atto), which specifically targets the poly-histidine tag fused to the VHH (Tris-NTA-Atto647). Incubation of 500 nM of VHH with the T-Rex CHO cell line expressing high levels of the 5HT3 receptor showed clear labeling on the surface (Fig. 2) contrary to CHO cell which did not express 5-HT3. For illustration, Figure 2A shows the in vivo surface labeling of CHO cells obtained with VHH7. Similar labeling was obtained with the other VHHs of the invention. Furthermore, size-exclusion chromatography was applied on 100 μg purified 5-HT3 receptor incubated with VHH7 (SEQ ID NO:3). After a few minutes incubation the mixture was loaded on a Superose 6 10/300 column. The receptor peak was imperceptibly shifted toward a higher apparent molecular mass, which was confirmed by SDS gel electrophoresis where the corresponding fractions confirmed the presence of the VHH7 in this peak (Figure 2A).

^■ Binding affinity of VHHs for 5-HT3A receptor

Method:

Immobilization and binding experiments were performed in TNC buffer (50mM Tris-Hcl, pH 7.4, 125 mM NaCl, 2 mM C₁₂E₉) at a flow rate of 10 μΐ/min. The VHH was coupled to the surface of a sensor chip NTA (GE Healthcare) by injecting the reagents in the following order: 20 μΐ NiCl₂ 0.5 mM in HNC buffer and 50 μΐ VHH 10μΜ in TNC buffer. The amount of immobilized VHH was approximately 100 RU. Different concentrations of purified 5-HT₃ receptors ranging from 0 to 200 nM were applied in a random order to the surface for 10 minutes. Surface regeneration was achieved with sequential injection of 100 μΐ EDTA 0.1M in TNC buffer and 100 μΐ SDS 0.5% at 50 μΐ/min to dissociate bound proteins. One channel without VHH probes was used as reference in all experiments to correct the binding response for bulk refractive index changes and unspecific binding. Apparent affinity constants were obtained by plotting the responses at equilibrium versus 5-HT₃ receptors concentration. BiaEvaluation Software Version 4.1 (Biacore) and IGOR Pro Version 6.22A (Wavemetrics) were used for data processing.

Results: The results obtained for VHH7 and VHH15 are shown in Figures 3 and 4 respectively.

The Kd of VHH7 for 5-HT3AR was in the nanomolar range and was compatible with the binding of more than one VHH7 per 5-HT3 receptor.

The Kd of VHH7 is about 7.8 nM. The Kd of VHH15 (SEQ ID NO:4) is about 142 nM.

4. Assessment of the ability of VHHs to modulate the activity of 5-HT3 receptor

^■ Whole-cell electrophysiology

Method:

Whole -cell electrophysiology recordings were conducted to demonstrate the inhibition of serotonin- elicited currents after pre -incubation with VHH7. The T-Rex Hek293 stable cell line expressing the 5- HT3A receptor incubated with two concentrations (9 and 90 nM) of VHH7

Measurements were performed on an inverted fluorescent microscope (AxiovertlOO, Zeiss, Jena, Germany) at room temperature on cells at least 24h after induction. The bath solution at pH 7.4 contained 147 niM NaCl, 12 niM glucose, 10 niM HEPES, 2 niM KC1, 2 niM CaCl₂ and 1 niM MgCl₂. Borosilicate patch pipettes (GB 150TF-8P, Science Products, Basel, Switzerland) were pulled with a P-87 niicropipette puller (Sutter Instruments, Novato, CA) and had a resistance of 3-15 ΜΩ when filled with a pipette solution at pH 7.4 containing 140 niM NaCl, 10 niM EGTA/EDTA and 10 niM HEPES. Electrical currents were recorded with an EPC9 patch-clamp amplifier (HEKA, Germany) and the holding potential used was -60 mV with the ground electrode connected to the bath solution. Data were sampled at 400 Hz and filtered with a Bessel filter at 2.9 kHz using the A/D converter of the EPC-9 and the Pulse software (HEKA, Germany). Ligand and VHH solutions were applied with a solution changer (RSC200, Bio- Logic, Claix, France) at an approximate flow rate of 250 μΐ/minutes. A typical pulse to test the pharmacology of the VHH consisted of 30 s of serotonin ligand, followed by an incubation of 3-5 minutes of VHH terminated by 30 s of a mix of serotonin and VHH. To end the pulse, the cells were washed for 3-5 minutes with bath solution. This pulse was repeated in varying the concentration of the VHH. The IC₅₀ may be determined by baseline correction, normalization and fitting of the peak currents using IGOR Pro (WaveMetrics, USA) with a Hill equation.

Results:

Figure 5 shows that VHH7 exhibits an inhibitory effect on 5-HT3A receptor in a dose -response experiment. VHH7 does act as a blocker of 5-HT3A receptor.

^■ FlexStation analysis

Method: Fluorescence-based membrane potential assays

Membrane potential assays were done with the red dye of the FLIPR membrane potential assay kit (Molecular Devices) on a FlexStation fluorescence plate reader (Molecular Devices). Adherent cells (CHO) expressing the 5-HT3R were seeded in black clear-bottomed 96-wells. When cell density reached 80% confluence the cell medium was replaced with 100 wl red dye solution (prepared by dissolving FLIPR red dye in 10 ml DPBS+, followed by a three-fold dilution into 150mMTris/HCl, pH 7.4 to give a final concentration of lOOmM Tris/HCl and 50 mM salts from DPBS+. Cells in red dye solution were incubated for 45 minutes at 37°C then the VHH were added and incubated for 20 minutes at 37°C, before measurements were started. Using the FlexStation, the time course of fluorescence intensity was measured for 3 minutes, with excitation at 535 nm, emission at 565 nm, and a bandpass of 550 nm. After 20 seconds, 5-HT3R expressing cells were activated by the addition 1 wM serotonin (final concentration). Data was analyzed with Softmax Pro (Molecular Devices) and Igor Pro, by calculating the Ymax from the linear part of the initial slope (first 20 seconds after serotonin addition) and fitting the percentage of inhibition with a 4-parameter or Hill curve.

Results:

Figure 6A and Figure 6B show the FlexStation responses of CHO cells transfected with 5-HT3A receptors. Increase in fluorescence in cells loaded with membrane potential dye is observed when said cells are contacted with 1 μΜ of serotonin (5-HT). The incubation of CHO cells with 40 μΜ of VHH15 or VHH16 inhibits the increase of fluorescence induced by 5-HT (1 μΜ). Such results show that VHH15 and VHH16 act as antagonist of 5-HT3a receptor.

^■ Patch clamp on Xenopus oocyte

Method

- Preparation of oocytes transiently expressing 5-HT3 receptor. :

Recordings were obtained with the Hi-Clamp automate (Multi-Channel Systems) using Xenopus oocytes microinjected with 2 ng of phenol/chloroform purified mRNA obtained by in vitro transcription. Oocytes were surgically removed from anesthetized Xenopus laevis females using procedures that conformed to European regulations for animal handling and experiments, and were approved by governmental services and the Institutional Ethical Committee

- Patch clamp assay

The buffer used is modified from the standard ND96 buffer: 91 mM Na+, 99 mM C1-, 2 mM K+, 1.8 mM Ca2+, 1 mM Mg2+, 5 mM HEPES, pH 7.4. The two borosilicate pipettes were filled with 3 M KC1. Oocytes selected on their response to 2.5 μΜ serotonin were subjected to serial incubations with increasing concentrations of VHH. Solution changes are performed by moving the impaled oocytes in continuously-stirred 200-μ1 reservoirs of a 96-well plate. Recordings are performed at -50 mV and data are analyzed using the Hi-Clamp software DataMining. Currents generated by 5 μΜ of 5-HT after VHH incubation, are normalized, averaged between several oocytes and fitted using a Hill equation.

A three -pulse protocol was chosen in order to determine the action exerted by each VHH on 5-HT3 receptor. The oocyte was tested for response in the presence of 2.5 μΜ 5-HT a first time (first control pulse). Then, the oocyte was incubated in the presence of the VHH (1 μΜ) for 5 minutes and tested a second time in the presence of 2,5 μΜ of 5-HT. The oocyte was washed for 5 minutes and tested a third time. Note that before the beginning of this sequence, the robot applied a short 5-HT pulse (not depicted) that allowed sorting of the oocytes with correct expression.

The modulation activity of the VHH refers to the percentage of remaining current after 5 minutes of incubation with VHH and was determined as the ratio of the maximum of current measured in the presence of the VHH, at the end of the incubation (test pulse) to the maximum of current measured for the first control pulse. The second control pulse was performed in order to determine whether the VHHs had a long-lasting effect on 5-HT3 receptor. The general protocol is depicted in Figure 7A. Other concentrations were tested for VHH7 (0.7 μΜ) and VHH 15 (1.5 μΜ and 15 nM).

Bovine serum albumin (BSA) at mass concentration corresponding to 1 μΜ of VHH was used as negative control.

Results

The results are depicted in Figure 7B which shows the percentage of modulation induced by each VHH (1 μΜ) and by BSA.

BSA induced a non-specific modulation of the activity of 5-HT3 receptor. The modulation measured for all the tested VHHs was significantly higher than that obtained with BSA, which demonstrated that the VHHs exerted a specific action on 5-HT3 receptor.

Noteworthy, VHH4 acted as a potentiator, as it amplified the current induced by the endogenous ligand, i.e. serotonin. By contrast, VHH5, VHH7, VHH15, and VHH 16 acted as antagonists of 5-HT3 modulators. VHH5 and VHH 16 attenuated the response elicited by 5-HT and thus acted as partial antagonists (or negative modulators) of 5-HT3 receptor.

As shown in Figures 7C, 7D and 7E, VHH7 and VHH15 exerted a strong and long-lasting inhibition of 5- HT3 receptor. Noteworthy, serotonin was not able to activate 5-HT3 receptor, even after washing the oocyte during 5 minutes in a flow of buffer.

EXAMPLE 2: VHHs directed to 5-HT3AB receptor

1. Identification of VHHs directed against m5-HT3AB receptor

The protocol used for the identification of VHHs directed to, and able to modulate, m5-HT3AB receptor subtype was similar to that used for the identification of VHHs directed to m5-HT3A receptor except that the llamas were immunized with purified 5-1 1 1 ΛΒ receptor. The V i l l i sequences from the Fl . ISA- positive colonies were subjected to sequence determination and allowed the identification of several different VHHs. Some of them are depicted in Figures 8A and 8B : VHHAB-5 (SEQ ID N°42), VI I I IAB- 13 (SEQ ID N°43), VHHAB-9 (SEQ ID N°44), VHH-AB-18 (SEQ ID N°46), and VHHAB-21 (SEQ ID N°47).

The ability of the selected VHHs to bind m5-HT3AB was confirmed by standard methods (see above).

2. Characterization of the selected VHHs " Binding specificity : FACS (Fluorescence-activated cell sorting)

A standard FACS analysis was conducted so as to determine whether the identified VHHs are specific to the 5-HT3AB receptor subtype or cross-react with 5-HT3A receptor subtype. Protocol:

For the aim of the study, recombinant ( I K ) T Rex cells expressing either ni5-I i Γ3ΛΚ or m5-HT3ABR were used.

Each V i l l i of interest (namely V I I I I AB-5 (SEQ I I ) N°42), VHHAB-13 (SEQ I I ) N°43), VHHAB-9 (SEQ I I ) N°44), VHH-AB-18 (SEQ I I ) N°46), and VHHAB-21 (SEQ I I ) N°47)) was sub-cloned into a standard pi I EN I vector construct with a Myc tag at the ( ^'-termi nal extremity and transformed into TGI E. coli strain. The cells were cultured i n standard conduction (cell culture (4 ml) prepared from a pre -culture (1/100) volume: 4 ml . incubation: 3h at 37°C followed by induction with I iiiM of I P I G after and then incubation). After incubation, the ceil culture was subjected to the following treatment:

centrifugation : 15 mi n at 3200 g,

- freezing : 30 min / thawing 15 min

addition of 400 u l of PBS, incubation during 45 min at room temperature under stirring centrifugation during 1 5 min at 3200 g

recovery of the supernatant containing the V i l l i

FACS Protocol:

- FACS buffer : PBS IX, BSA 0.5%, EDTA 5 niM

- PFA 4% : 10 ml of PFA 16% + 30 niL PBS IX

The following protocol was followed:

For a plate of 24 wells containing CHO T Rex cell culture

1. After the removal of the supernatant by flicking, 1 mi of FACS Buffer was added. The plate was then incubating during 5 min.

2. The ceils were transferred into a 96 well round bottom plate (150 μ Ι per well which corresponds to about 100 000 cells per well).

3. A centrifugation (400G, 1 min, 21 °C) was carried out. The supernatant was eliminated by flicking.

4. ΙΟΟμΙ of the supernatant containing the V i l l i of interest was added i n each well. (For the control experience, a periplasmic extract of TGI culture was added instead)

5. The plate was incubated during 45 min at 4°C

6. 150 μ Ι of FACS Buffer was added in each well. The plate was centrifuged (400G, 1 min, 21 °C) and then, the supernatant was removed.

7. 100 μ Ι of a mouse anti-cMyc antibody (1/100) was added to each well.

8. The plate was incubated during 15 min.

9. 10 μΐ of FACS Buffer was added in each wel l. The plate was centrifuged (400G, 1 min, 21 °C) and then, the supernatant was removed. 10. 100 μ Ι of an anti-mouse antibody coupled to Phycoerythrin (PE) (1/100) was added to each well.

I 1. 150 μ Ι of I ACS Buffer was added in each well. The plate was centrifuged (400G, 1 min, 21°C) and then, the supernatant was removed.

12. 100 μΐ of PFA 4% was added to each well to fix the cells.

13. The plate was incubating during 10 min at room temperature away from light.

14. ΙΟΟμΙ of FACS buffer was added. The plate was centrifuged (400G, I min, 21°C) and then, the supernatant was removed.

1 . Step 14 was repeated with 150 μ Ι of FACS buffer.

16. 150μ1 of FACS buffer was then added in each well and the FACS analysis was performed.

Results

The results of FACS analysis are shown in Figure 9A-9F. In each Figure, Curve 1 corresponds to a negative control using CHO T-Rex cells which do not express 5-HT3ABR or 5-HT3AR. Curve 2 refers to a negative control in which the supernatant containing the VHH was replaced by a TGI periplasmic extract free of VHH, Curve 3 refers to the response obtained from CHO T-rex cells expressing m5-HT3- AR and finally Curve 4 refers to the response obtained from CHO T-rex cells expressing m5-HT3-ABR As depicted in Figures 9 A and 9B, VHHAB-5 and VHHAB-13 are able to bind the both subtypes of receptors.

By contrast, the four other VHHs, namely VHHAB-9, VHHAB-10, VHHAB-18 and VHHAB-21 showed very good specificity for the 5-HT3ABR subtype receptor as compared to 5-HT3AR subtype.

3. Assessment of the ability of VHHs to modulate the activity of 5-HT3AB receptor

The ability of VHHAB-5 and VHHAB-13 to modulate the activity of 5-HT3AB receptor was determined by FlexStation analysis as described above.

Results:

Figure 10A and Figure 10B show the FlexStation responses of CHO cells transfected with 5-HT3AB receptor. Increase in fluorescence in cells loaded with membrane potential dye is observed when said cells are contacted with 4 μΜ of serotonin (5-HT - curve 1). The incubation of CHO cells with 35 μΜ of VHHAB-5 or VHHAB-13, in the absence of serotonin, also induces an increase of the fluorescence (curve 2). By contrast, the incubation of the cells with the buffer (negative control - curve 3) does not induce any change in fluorescence.

Such results show that VHHAB-5 (SEQ ID N°42) and VHHAB-13 (SEQ ID N°43) act as agonists of 5- HT3AB receptor. Table 4: list of amino acid sequences

SEQ ID N° Description

1 VHH4

2 VHH5

3 VHH7

4 VHH15

5 VHH15s

6 VHH16

7 CDR1 of VHH4

8 CDR2 of VHH4

9 CDR3 of VHH4

10 CDR1 of VHH5

11 CDR2 of VHH5

12 CDR3 of VHH5

13 CDR1 of VHH7

14 CDR2 of VHH7

15 CDR3 of VHH7

16 CDR1 of VHH15

17 CDR2 of VHH15

18 CDR3 of VHH15

19 CDR1 of VHH15s

20 CDR2 of VHH15s

21 CDR3 of VHH15s

22 CDR1 of VHH16

23 CDR2 of VHH16

24 CDR3 of VHH16

25 FRl of VHH4, VHH15 and VHH15s

26 FRl of VHH5

27 FRl of VHH7 and VHH16

28 FR2 of VHH4

29 FR2 of VHH5

30 FR2 of VHH7

31 FR2 of VHH15

32 FR2 of VHH15s

33 FR2 of VHH16

34 FR3 of VHH4

35 FR3 of VHH5

36 FR3 of VHH7

37 FR3 of VHH15 and VHH15s

38 FR3 of VHH16

39 FR4 of VHH4, VHH15, VHH15s and VHH16

40 FR4 of VHH5

41 FR4 of VHH7 SEQ ID N° Description

42 VHH-m5HT3 AB -5 (also called herein VHHAB-5)

43 VHH-m5HT3AB-13 (also called herein VHHAB-13)

44 VHH-m5HT3 AB -9 (also called herein VHHAB -9)

45 VHH-m5HT3AB-10 (also called herein VHHAB-10) _ς

46 VHH-m5HT3AB-18 (also called herein VHHAB-18)

47 VHH-m5HT3AB-21 (also called herein VHHAB-21)

48 CDR1 of VHHAB-5

49 CDR2 of VHHAB-5

50 CDR3 of VHHAB-5

51 CDR1 of VHHAB-13

52 CDR2 of VHHAB-13

53 CDR3 of VHHAB-13

54 CDR1 of VHHAB -9

55 CDR2 of VHHAB -9

56 CDR3 of VHHAB -9

57 CDR1 of VHHAB-10

58 CDR2 of VHHAB-10

59 CDR3 of VHHAB-10

60 CDR1 of VHHAB-18

61 CDR2 of VHHAB-18

62 CDR3 of VHHAB-18

63 CDR1 of VHHAB-21

64 CDR2 of VHHAB-21

65 CDR3 of VHHAB-21

66 FRl of VHHAB-5, VHHAB-13, VHHAB-10, VHHAB-18, VHHAB-21

67 FRl of VHHAB -9

68 FR2 of VHHAB-5

69 FR2 of VHHAB-13

70 FR2 of VHHAB -9

71 FR2 of VHHAB-10

72 FR2 of VHHAB-18

73 FR2 of VHHAB -21

74 FR3 of VHHAB-5

75 FR3 of VHHAB-13

76 FR3 of VHHAB -9

77 FR3 of VHHAB-10

78 FR3 of VHHAB-18

79 FR3 of VHHAB -21

80 FR4 of VHHAB-5

81 FR4 of VHHAB-13, VHHAB-18 and VHHAB-10

82 FR4 of VHHAB -9 and VHHAB-21

Claims

1. A single domain antibody directed against a Cys-loop receptor which has a dissociation constant Kd for said Cys-loop receptor of at most 10^~6 M.

2. The single domain antibody of claim 1 , which is a modulator of said Cys-loop receptor.

3. The single domain antibody of claim 1 or 2, wherein the said Cys-loop receptor is selected from the group of a serotonin (5-HT3) receptor, an acetylcholine (nicotinic ACh or nACh) receptor, a glycine (Gly) receptor, a γ-aminobutyric acid (GABA_A,GABA_C) receptor and a zinc-activated (ZAC) receptor, preferably a serotonin (5-HT3) receptor .

4. The single domain antibody of any one of claims 1-3 which is selected among an agonist, an antagonist or a potentiator of a 5-HT3 receptor.

5. The single -domain antibody according to any one of claims 1-4 being selected from the group consisting of isolated VHHs, preferably from Camelidae, humanized VHHs and fragments thereof.

6. The single domain antibody of any one of claims 1-5, said single domain antibody being directed against a serotonin (5-HT3) receptor and comprises an amino acid sequence having a sequence identity of at least 60%, preferably at least 70% with an amino acid sequence selected from the group consisting of SEQ ID N°l, 2, 3, 4, 5, 6, 42, 43, 44, 45, 46, and 47..

7. The single domain antibody of any one of claims 1-6, said single domain antibody being directed against a serotonin (5-HT3) receptor and comprising 3 complementarity determining regions, CDR1 to

CDR3, wherein:

CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:7, 10, 13, 16 ,19, 22, 48, 51, 54, 57, 60 and 63 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO:7, 10, 13, 16 ,19, 22, 48, 51, 54, 57, 60 and 63 in virtue of one, two, or three amino acid modifications;

CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:8, 11, 14,

17, 20, 23, 49, 52, 55, 58, 61 and 64 or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO:8, 11, 14, 17, 20, 23, 49, 52, 55, 58, 61 and 64in virtue of one, two, or three amino acid modifications; and,

- CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 12, 15,

18, 21, 24, 50, 43, 56, 59, 62, and 65 ; or an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 9, 12, 15, 18, 21, 24, 50, 43, 56, 59, 62, and 65, in virtue of one, two, or three acid modifications.

8. The single domain antibody of claim 7 comprises the sequence

FRl -CDR1 -FR2-CDR2- FR3-CDR3-FR4 wherein:

FRl, FR2, FR3 and FR4 are framework regions preferably selected from framework regions as depicted in Figure 1A, in Figure 8 A, and variants thereof, humanized framework regions of a VHH from Camelidae and camelized framework regions of a human VH ; and

- CDR1 is, or consists essentially of, SEQ ID NO:7, CDR2 is, or consists essentially of, SEQ ID

NO:8 and CDR3 is, or consists essentially of, SEQ ID NO:9;

- CDR1 is, or consists essentially of, SEQ ID NO:22, CDR2 is, or consists essentially of, SEQ ID

NO:23 and CDR3 is, or consists essentially of, SEQ ID NO:24,

- CDR1 is, or consists essentially of, SEQ ID NO:60, CDR2 is, or consists essentially of, SEQ ID

NO:61 and CDR3 is, or consists essentially of, SEQ ID NO:62;

CDR1 is, or consists essentially of, SEQ ID NO:63, CDR2 is, or consists essentially of, SEQ ID NO:64 and CDR3 is, or consists essentially of, SEQ ID NO:65,

9. The single domain antibody of claim 6 which comprises at least one of the following features:

the single -domain antibody is a potentiator of 5-HT3A receptor subtype,

the single -domain antibody is a potentiator of 5-HT3AB receptor subtype, the single -domain antibody is an antagonist of 5-HT3A receptor subtype,

the single -domain antibody is an antagonist of 5-HT3AB receptor subtype,

the single -domain antibody is an agonist of 5-HT3A receptor subtype,

the single -domain antibody is an agonist of 5-HT3AB receptor subtype,

- the single -domain antibody binds both 5-HT3AB receptor subtype and 5-HT3A receptor subtype, the single-domain antibody specifically binds 5-HT3AB receptor subtype as compared to5-HT3A receptor subtype,

10. An anti-Cys loop receptor polypeptide comprising at least one single -domain antibody as defined in any one of claims 1 to 9 and optionally an additional entity selected from the group consisting of PEG, relinked or O-linked glycosylation moieties, Fc domain from human immunoglobulins, and variants thereof, fragments from Fc domain of human Ig, a single-domain antibody directed against a human serum protein, a labeling mean, an affinity tag, a drug, a toxin and combinations thereof

11. An isolated nucleic acid comprising a sequence encoding a single-domain antibody as defined in any one of claims 1 to 9 or an anti-Cys loop receptor polypeptide as defined in claim 10.

12. A host cell containing the nucleic acid of claim 11.

13. A method for producing the single-domain antibody as defined in any one of claims 1-9 or an anti- Cys loop receptor polypeptide as defined in claim 10 comprising the steps of:

- culturing a host cell as defined in claim 12 and

- recovering said single domain antibody or said polypeptide from the cell culture.

14. A pharmaceutical composition comprising a polypeptide as defined in claim 10 or a single -domain antibody as defined in any one of claims 1-9 together with a pharmaceutically acceptable excipient.

15. The polypeptide as defined in claim 10 or the single-domain antibody as defined in any one of claims 1-9 for use as a drug.

16. Use of a single-domain antibody as defined in claims 1-9 or a polypeptide as defined in claim 10 in the crystallization of a Cys-loop receptor, in the purification of a Cys-loop receptor, in cell immuno- staining, in in vivo imaging, in a process for screening a candidate ligand to a Cys-loop receptor, or as a biological reagent in an immunoassay.

17. A method for obtaining a single -domain antibody directed against a Cys-loop receptor, wherein said method comprises the steps of:

a) preparing a library of biological recipients selected from cells, viruses, DNAs, RNAs and ribosomes, each biological recipient displaying thereon a single -domain antibody, from an antibody-expressing tissue or cells isolated from a non-human animal, preferably belonging to a Camelidae species, which have been immunized with an immunogenic composition comprising a purified Cys-loop receptor,

b) screening the library of step a) for biological recipients displaying a single-domain antibody able to bind said Cys-loop receptor, and

c) selecting a single-domain antibody directed against the Cys-loop receptor from the biological recipients identified in step b), and

wherein :

the Cys loop receptor is preferably a 5-HT3 receptor such as 5-HT3A receptor or 5-HT3AB receptor subtype,

the single -domain antibodies are preferably VHHs, and

step b) preferably comprises contacting the biological recipients with the Cys-loop receptor in the presence of a modulator of said Cys-loop receptor.