Chimeric proteins and methods to screen for compounds and ligands binding to gpcr

Abstract

To provide a combination of a GPCR and a VHH against the GPCR, which can be used in assay and screening techniques.SOLUTION: A composition comprising (i) a chimeric GPCR having the structure: [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence], wherein the ECs and TMs form a functional ligand binding site, the ECs and TMs are derived from a first GPCR, and the ICs are derived from a second GPCR different from the first GPCR, and (ii) an immunoglobulin single variable domain that is capable of binding to at least one of the ICs derived from the second GPCR.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to chimeric proteins that can be used to discover and develop compounds and ligands that bind to GPCRs.

[0002] The present invention also relates to methods and arrangements in which said chimeric proteins are used.

[0003] In particular, the present invention relates to chimeric proteins, methods and arrangements that can be used to identify compounds and ligands capable of binding to GPCRs, and to test compounds and ligands capable of binding to GPCRs.

[0004] The screening and assay techniques provided by the present invention can be used, inter alia, to identify, generate, optimize, and / or develop compounds and ligands that can bind to GPCRs and that can be used and / or developed as therapeutic, prophylactic, and diagnostic agents. As further described herein, such compounds or ligands can be any desired and / or suitable compound or ligand, including, but not limited to, small molecules, small peptides, biomolecules, or other chemical entities; examples of such compounds will be apparent to those skilled in the art based on the further disclosure herein.

[0005] For example, small molecules or molecular fragments identified and / or generated using the chimeric proteins, methods, and arrangements of the present invention (i.e., "hits" from such screens) can be used as starting points for further drug discovery and development efforts (e.g., using well-known techniques of so-called "hits-to-leads" chemistry), which may also involve the use of assays in which the chimeric proteins of the present invention are employed (e.g., functional assays, or assays used for quality control purposes). Compounds identified using the methods and techniques of the present invention (i.e., "hits"), and any compounds generated or developed using such hits as a starting point, are collectively referred to herein as "compounds of the present invention" and form a further aspect of the present invention. It will be apparent to those skilled in the art that such compounds may be so-called "hits," "leads," "development candidates," "preclinical compounds," "clinical candidates," or commercial compounds or products, depending, for example, on their stage of development and the specific terminology used by the company or entity developing and / or commercializing them.

[0006] Additionally, as further described herein, the methods and assays of the present invention may allow for the identification and / or characterization of allosteric agonists, antagonists and / or inverse agonists (depending on the particular target and assay used).

[0007] Other features, aspects, embodiments, uses and advantages of the present invention will become apparent from the further description herein. [Background technology]

[0008] Assay and screening techniques for GPCRs are well known in the art. It is estimated that more than half of all modern pharmaceuticals target membrane proteins, and approximately one-third of all modern pharmaceuticals target GPCRs. Please refer to standard handbooks and the further prior art cited herein. [In this regard, it should be noted that, in general, the terms "7TM receptor" and "7TM" are often used interchangeably with "GPCR" in the art. However, according to the IUPHAR database, there are some 7TM receptors that do not signal through G proteins. For the purposes of this specification and claims, the terms "GPCR" and "7TM" are used interchangeably herein and include all transmembrane proteins, particularly transmembrane receptors, that have seven transmembrane domains, regardless of their intracellular signaling cascade or signaling mechanism. However, it should be understood that throughout this specification and claims, 7TM receptors that signal through G proteins are a preferred embodiment of the present invention.

[0009] As is well known, GPCRs are not static entities whose function is determined solely by their primary, secondary, or tertiary structure, but are often flexible structures that can transition between different conformational states (also referred to as "conformational changes") such that the GPCR may exist in equilibrium between these different states. Some of these states may be functional and / or active, while other states may be basal states (which may or may not exhibit a level of constitutive activity), essentially inactive states, and / or less active states relative to relatively more functional or active states. The geometries of different epitopes, binding sites (including ligand-binding sites), and / or catalytic sites that may be present in or on the GPCR may also differ between these different conformations; for example, in some conformational states, the binding site may not be available / accessible for ligand binding and / or the affinity for interaction between the binding site and the associated ligand(s) is reduced compared to the more active conformational states.

[0010] It is also known that binding of a ligand to a GPCR can change its conformation (e.g., from an inactive / less active conformation to an active / more active conformation) and / or shift its equilibrium from an inactive / less active conformation to an active / more active conformation. Binding of a ligand to one binding site of a GPCR can make another binding site on the GPCR more accessible to its associated ligand(s) and / or result in an increase in the affinity of the other binding site for said ligand(s) and / or shift the equilibrium from a conformation in which the other binding site has a lower affinity for said ligand(s) to a conformation in which the other binding site has a higher affinity for said ligand(s). For example, binding of an extracellular ligand to an extracellular binding site on a GPCR can result in a conformational change in the cytoplasmic side that can, for example, increase the affinity of the intracellular binding site for the intracellular ligand (e.g., increase the affinity for the interaction between a G protein and a G protein binding site), or vice versa. This change in binding affinity for the intracellular ligand following binding of the extracellular ligand, and subsequent binding of the intracellular ligand to the intracellular binding site, is part of the mechanism by which GPCRs transduce extracellular signals.

[0011] Generally, as further described herein, with respect to a GPCR, an "agonist" can be said to shift the conformational equilibrium from an inactive state (or one or more less active states) to an active state (or one or more more active states), while an "inverse agonist" of a GPCR can be said to shift the conformational equilibrium in the opposite direction.

[0012] Without being limited to any particular hypothesis or mechanism, it is also postulated that GPCRs can form complexes with an extracellular ligand (which binds to the extracellular binding site of the GPCR) and an intracellular ligand (which binds to the intracellular binding site of the GPCR), and that the interaction between the GPCR and each ligand is stabilized by the binding of the other ligand (in other words, the complex is stabilized by the binding of both ligands). Again, in this case, binding of one or both of the ligands can shift the conformational equilibrium of the GPCR toward (the formation and / or stabilization of) this complex. See, e.g., WO 2012 / 007593, cited below.

[0013] Given that the perceived "global" state of a GPCR is largely governed by the (statistical) distribution of the GPCR across its various possible conformations, and thus by the equilibrium that exists between these conformational states, when, in this specification or claims, a GPCR is said to undergo a conformational change to a particular conformation (i.e., from one or more other conformations), this should be understood to include mechanisms or circumstances by which the conformational equilibrium of the GPCR shifts toward said conformation (i.e., under the particular conditions employed, e.g., those used for screening or related assays). Similarly, when a ligand is said to induce a conformational change to a particular conformation of a GPCR (i.e., from one or more other conformations), this includes mechanisms or circumstances by which binding of the ligand shifts the conformational equilibrium of the protein toward said conformation (i.e., under the particular conditions employed, e.g., those used for screening or related assays).

[0014] However, it should also be noted that while any one of the mechanisms described herein (or any combination thereof) may be involved in the practice of the present invention at any given time (e.g., depending on the particular GPCR and / or ligand(s) to which the present invention is applied), the present invention in its broadest sense is not limited to any particular mechanism, explanation, or hypothesis, so long as application of the present invention to a particular GPCR results in the technical effect(s) outlined herein.

[0015] One challenge in screening compounds directed at GPCRs is that the correct conformation of the GPCR may be lost when the GPCR is expressed or used in isolation from its native environment (even when it is feasible or possible to express the GPCR and ensure proper folding outside of its cellular environment). It may also be difficult to ensure that the GPCR is in its desired conformation (often a functional conformation, such as its active conformation) under the conditions used for screening. There may also be a need or advantage in achieving a shift in the conformational equilibrium of the GPCR to a conformational state that is more suitable for screening or assay purposes (such as an active state, or a state in which the relevant binding site is more accessible and / or has a better geometry for assay or screening purposes). As described further herein, such conformations are also referred to as "druggable" conformations, and in accordance with preferred embodiments of the present invention, measures are applied (as further described herein) to ensure that the intended druggable format of the GPCR is provided.

[0016] For example, WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 175643, WO 2014 / 118297, WO 2014 / 122183, and WO 2014 / 118297 are directed to protein binding domains that can be used to stabilize specific conformational states of GPCRs for the purposes of determining their structure and for drug screening and discovery. In these references, VHH domains are used that can stabilize GPCRs in desired, particularly more druggable, conformations, e.g., functional and / or active states, e.g., conformations that arise when an activating ligand (agonist) binds to the extracellular side of the GPCR so that the GPCR can activate heterotrimeric G proteins. See, for example, Pardon et al., Angew Chem Int Ed Engl. 2018, 57(19):5292-5295; Che et al., Cell. 2018, 172(1-2):55-67; ​​Manglik et al., Annu Rev Pharmacol Toxicol. 2017; 57:19-37; Pardon et al., Nat Protoc. 2014, 674-93; Kruse et al., Nature, 2013, 504(7478); Steyaert and Kobilka, Curr Opin Struct Biol., 2011, 567-72; Eglen and Reisine, Pharmaceuticals 2011, 4, 244-272; and Rasmussen et al., Nature, 2011, 469(7329):175-180, and further references cited therein. VHH domains that can be used to stabilize a desired conformation of membrane proteins such as GPCRs are also referred to herein as Confobodies (Confobody™ is a registered trademark of Confo Therapeutics, Ghent, Belgium).

[0017] Some non-limiting examples of conformobodies that can bind to intracellular epitopes of GPCRs and can be used to stabilize GPCRs in a desired conformation (and can be used in the present invention) include CA2764, CA3431, CA3413, CA2780, CA2765, CA2761, CA3475, CA2770, CA3472, CA3420, CA3433, CA343 VHHs designated CA3484, CA2760, CA2773, CA3477, CA2774, CA2768, CA3424, CA2767, CA2786, CA3422, CA2763, CA2772, CA2771, CA2769, CA2782, CA2783, and CA2784 (see, e.g., WO 2012 / 007593, Tables 1 and 2, and SEQ ID NOs: 1-29); CA5 VHHs designated as Nb9-1, Nb9-8, XA8633, and CA4910 (see, e.g., Tables 1 and 2 and SEQ ID NOs: 15, 16, 17, 19, and 20 of WO 2014 / 118297); Nb9-11, Nb9-7, Nb9-7, Nb9-22, Nb9-17, Nb9-24, Nb9-9, Nb9-14, Nb9-2, Nb9-20, Nb_C3, NbH-4, Nb-E The VHHs designated Nb_1, Nb_A2, Nb_B4, Nb_D3, Nb_D1 and Nb_H1 (see, for example, Tables 1 and 2 and SEQ ID NOs: 1-19 in WO 2014 / 122183), and the VHHs designated XA8639, XA8635, XA8727 and XA9644 (see, for example, WO 2015 / 121092, Tables 2 and 3 and SEQ ID NOs: 2-6 and 74).

[0018] Some non-limiting examples of VHHs capable of binding to G proteins are CA4435, CA4433, CA4436, CA4437, CA4440 and CA4441 (see, for example, Tables 2 and 3 and SEQ ID NOs: 1-6 of WO 2012 / 175643007593). Summary of the Invention

[0019] In general, the methods described in the prior art for raising such VHHs require that the desired GPCR be available and used in an appropriate conformation (i.e., a conformation that will allow the VHH to be produced). This is not only the case for immunization purposes (i.e., to generate an immune library), but also for selection and screening purposes (where it is necessary to appropriately express the GPCR in the desired conformation in the phage, ribosome, or other display system used to screen the immune library), as well as for screening and selection using natural or synthetic libraries. If these limitations lead to a situation where it is not possible to obtain an appropriate VHH for the desired conformation(s) of a GPCR, these prior art methods may be of limited use when they are applied to said GPCR.

[0020] In general, the present invention aims to provide alternative methodologies for providing assay techniques and compound / ligand screening methods that can be used for GPCRs. In particular, the present invention aims to provide such methodologies that avoid the need to generate VHHs specific to a desired conformation of a native GPCR, and therefore avoid any difficulties or limitations that may be associated therewith.

[0021] More particularly, the present invention aims to provide a combination of a GPCR and a VHH against said GPCR that can be used in assays and screening techniques.

[0022] The assay and screening techniques provided by the present invention can be used to discover and develop (e.g., identify, generate, test, and optimize) compounds directed against related GPCRs (i.e., that have specificity for and / or are intended to target one or more GPCRs, e.g., for therapeutic, prophylactic, and / or diagnostic purposes). Preferably, such compounds are specific for (i.e., selective for) one particular GPCR compared to other (closely related) GPCRs.

[0023] Compounds identified and / or developed using the assay and screening techniques provided by the present invention can be used (as defined herein) to modulate the associated GPCR, its signaling, and / or the biological functions, pathways, and / or mechanisms in which said GPCR or its signaling is involved. For example, the present invention can be used to discover and develop compounds that are agonists, antagonists, inverse agonists, inhibitors, or modulators (such as allosteric modulators) of GPCRs and / or signaling, pathways in which GPCRs are involved, and / or physiological and / or biological mechanisms.

[0024] Typically, compounds discovered and / or developed using the present invention will be directed to GPCRs that are expressed by or in cells present in the body of a subject to be treated with the compound or ligand discovered or developed using the methods and techniques of the present invention.

[0025] The present invention can be used to discover and / or develop any type of compound suitable for its intended use, which is often as a therapeutic, diagnostic, or prophylactic agent. Thus, these compounds can be small molecules, peptides, biomolecules, or other chemical entities. Examples of suitable biomolecules include, for example, antibodies and antibody fragments (such as Fab, VH, VL, and VHH domains) and compounds based on antibody fragments (such as ScFvs and diabodies, as well as other compounds or constructs comprising one or more VH, VL, and / or VHH domains), Alphabodies™ and avimer-based scaffolds, compounds based on other protein scaffolds such as PDZ domains, Protein A domains (such as Affibodies™), ankyrin repeats (such as DARPins™), fibronectins (such as Adnectins™), and lipocalins (such as Anticalins™), as well as DNA- or RNA-based binding moieties, including, but not limited to, DNA or RNA aptamers. See further description herein and, for example, Simeon and Chen, Protein Cell 2018, 9(1):3-14; Binz et al., Nat. Biotech 2005, 23:1257; and Ulrich et al., Comb Chem High Throughput Screen 2006, 9(8):619-32.

[0026] The methods and techniques of the invention can be used, for example, to screen libraries of such compounds to identify one or more "hits" that are specific for the relevant GPCR (in particular, that are specific for a desired conformation of the GPCR and / or that are capable of inducing a desired conformation of the GPCR, e.g., a ligand-binding conformation, and in particular an agonist-binding conformation), and / or as part of a strategy to improve the affinity and / or potency of compounds directed against GPCRs, and / or as assays used to improve the pharmacological and / or other properties of such compounds (e.g., in the case of small molecules, as part of a "hit-to-lead" campaign).

[0027] The chimeric proteins, methods, and techniques of the present invention can also be used for the purpose of so-called "fragment-based drug discovery" or "FBDD" (also known as "fragment-based lead discovery" or "FBLD"). See, for example, basic books such as Lamoree and Hubbard, Essays in Biochemistry (2017) 61, 453-464; Jahnke and Erlanson, "Fragment-based approaches in drug discovery", 2006; Zartler and Shapiro, "Fragment-based drug discovery: a practical approach", 2008; and Kuo, "Fragment-based drug design: tools, practical approaches, and examples", 2011. [Brief explanation of the drawings]

[0028] [Figure 1]1 shows a schematic representation of a first arrangement of the present invention, in which a second ligand (shown as (4) in FIG. 1 ) forms part of a second fusion protein (which in the embodiment shown in FIG. 1 is formed by the second ligand (4), a linker (11) and the second member (7) of the binding pair (6 / 7)) and is directly (as defined herein) bound to the layer-spanning protein (2). In the setup shown in FIG. 1 : the boundary layer is shown as (1); the first environment is shown as [A]; the second environment is shown as [B]; the layer-spanning protein (i.e., the chimeric GPCR of the present invention) is shown as (2); the first ligand is shown as (3); and the first binding site of the layer-spanning protein (2), exposed to the first environment [A] and to which the first ligand (3) can bind, is shown as (8). As described herein, the first binding site (8) of the layer-spanning protein is the extracellular binding site (as defined herein) of the chimeric GPCR of the present invention; the second ligand is designated as (4). As described herein, the second ligand is preferably a binding domain or binding unit capable of binding to a binding site of the chimeric GPCR comprising at least one intracellular loop (as described herein), more preferably a conformation-induced binding domain or binding unit (as described herein), and may in particular be an ISVD, more particularly a conformation-induced ISVD; the second binding site of the layer-spanning protein (2), exposed to a second environment [B] and capable of binding the second ligand (4), is designated as (9).As described herein, said second binding site (9) of the layer-spanning protein is an intracellular binding site (as defined herein) of the chimeric GPCR of the present invention and comprises at least one ICL from the second GPCR; the binding pair capable of generating a detectable signal is shown as (6 / 7) and consists of a first binding member (6) linked (directly or via a linker or spacer (10)) to the layer-spanning protein (2) and a second binding member (7) linked (directly or via a linker or spacer (11)) to a second ligand (4); the first fusion protein comprises the layer-spanning protein (2) fused directly or via a linker (10) to the first binding member (6); the second fusion protein comprises the second ligand (4) fused directly or via a linker (11) to the second binding member (7); The first and second fusion proteins are positioned relative to each other and the boundary layer (1) in such a manner that when the second ligand (4) binds to the layer-spanning protein (2) (i.e., directly via the binding site (9)), the first binding member (6) and the second binding member (7) can contact or come into close proximity to each other (or otherwise suitably associate) to generate a detectable signal (shown by the flash symbol in Figure 1). [Figure 2]2 is a schematic representation of a second arrangement of the present invention, in which the second ligand (shown as (4) in FIG. 2 ) is separated from the second fusion protein (formed in the embodiment shown in FIG. 2 by the binding domain (5), the linker (11) and the second member (7) of the binding pair (6 / 7)), and the binding domain (5) present in the second fusion protein binds indirectly (as defined herein, in the case of FIG. 2 , via the second ligand (4)) to the layer-spanning protein (2). In the setup shown in FIG. 2 : the boundary layer is shown as (1); the first environment is shown as [A]; the second environment is shown as [B]; the layer-spanning protein (i.e., the chimeric GPCR of the present invention) is shown as (2); the first ligand is shown as (3); and the first binding site of the layer-spanning protein (2), exposed to the first environment [A] and to which the first ligand (3) can bind, is shown as (8). As described herein, said first binding site (8) of the transmembrane protein is the extracellular binding site (as defined herein) of the chimeric GPCR of the present invention; the second ligand is designated as (4); the second binding site of the transmembrane protein (2), which is exposed to a second environment [B] and to which the second ligand (4) can bind, is designated as (9).As described herein, said second binding site (9) of the layer-spanning protein is an intracellular binding site (as defined herein) of the chimeric GPCR of the present invention and comprises at least one ICL from the second GPCR; a binding domain or binding unit capable of binding to a second ligand (4) is designated as (5); a binding pair capable of generating a detectable signal is designated as (6 / 7) and consists of a first binding member (6) linked (directly or via a linker or spacer (10)) to the layer-spanning protein (2) and a second binding member (7) linked (directly or via a linker or spacer (11)) to the binding domain or binding unit (5); a first fusion protein comprises a layer-spanning protein (2) fused directly or via a linker (10) to the first binding member (6); a second fusion protein comprises a binding domain (5) fused directly or via a linker (11) to the second binding member (7); The first and second fusion proteins are positioned relative to each other and the boundary layer (1) in such a manner that when the binding domain (5) binds to the layer-spanning protein (2) (i.e., indirectly by binding to a second ligand (4) which in turn binds to the layer-spanning protein (2) via the binding site (9)), the first binding member (6) and the second binding member (7) can contact or come into close proximity to each other (or otherwise appropriately associate) to generate a detectable signal (shown by a flash symbol in Figure 2). [Figure 3]Schematically illustrates a third arrangement of the present invention, in which the second ligand (shown as (4) in FIG. 3 ) is separated from the second fusion protein and forms part of a protein complex (12) formed by the second ligand (4) and one or more additional proteins (in the case of FIG. 3 , for illustrative purposes, the complex is illustrated as comprising the second ligand (4) and two additional proteins (4a) and (4b); see also the insert of FIG. 3 ). In the embodiment shown in FIG. 3 , the second ligand (4) is also separated from the second fusion protein (formed in the embodiment shown in FIG. 3 by the binding domain (5), the linker (11), and the second member (7) of the binding pair (6 / 7)), and the binding domain (5) present in the second fusion protein binds indirectly (as defined herein, in the case of FIG. 3 , via the protein complex (12)) to the layer-spanning protein (2). In the setup shown in Figure 3: the boundary layer is shown as (1); the first environment is shown as [A]; the second environment is shown as [B]; the layer-spanning protein (i.e., the chimeric GPCR of the present invention) is shown as (2); the first ligand is shown as (3); and the first binding site of the layer-spanning protein (2), exposed to the first environment [A] and to which the first ligand (3) can bind, is shown as (8). As described herein, said first binding site (8) of the layer-spanning protein is the extracellular binding site (as defined herein) of the chimeric GPCR of the invention; a second ligand is shown as (4) and forms a complex (12) with one or more other proteins (in Figure 3, for illustrative purposes, complex (12) is represented as a complex comprising three proteins / subunits, namely, the second ligand (4) and two further subunits (4a) and (4b); see also the insert of Figure 3); a second binding site of the layer-spanning protein (2), exposed to the second environment [B] and to which complex (12) can bind, is shown as (9).As described herein, said second binding site (9) of the layer-spanning protein is an intracellular binding site (as defined herein) of the chimeric GPCR of the present invention and comprises at least one ICL from the second GPCR; a binding domain or binding unit capable of binding to the complex (12) is designated as (5); a binding pair capable of generating a detectable signal is designated as (6 / 7) and consists of a first binding member (6) linked (directly or via a linker or spacer (10)) to the layer-spanning protein (2) and a second binding member (7) linked (directly or via a linker or spacer (11)) to the binding domain or binding unit (5); a first fusion protein comprises the layer-spanning protein (2) fused directly or via a linker (10) to the first binding member (6); a second fusion protein comprises the binding domain (5) fused directly or via a linker (11) to the second binding member (7); The first and second fusion proteins are positioned relative to each other and to the boundary layer (1) in such a manner that when the binding domain (5) binds to the layer-spanning protein (2) (i.e., indirectly by binding to a complex (12) which in turn binds to the layer-spanning protein (2) via the binding site (9)), the first binding member (6) and the second binding member (7) can contact or be in close proximity to each other (or otherwise suitably associated) to generate a detectable signal (shown by a flash symbol in Figure 3). [Figure 4] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant MC4R screening assay described in Example 2. [Figure 5] 1 is a graph showing assay results obtained using the recombinant MC4R screening assay described in Example 2. [Figure 6] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 3. [Figure 7] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 3. [Figure 8] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 3. [Figure 9] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 3. [Figure 10] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 3. [Figure 11A] 11A-11C are graphs showing assay results obtained using two recombinant APJ receptor screening assays described in Example 4. Figure 11A shows results obtained with a recombinant apelin receptor bearing the ICL of the mu-opioid receptor (MOR). [Figure 11B] Figure 11B shows the assay results obtained using two recombinant APJ receptor screening assays described in Example 4. Figure 11B shows the results obtained with a recombinant apelin receptor bearing an ICL from the beta-2AR receptor. [Figure 12] 1 shows the results of the compound library screening carried out in Example 5. [Figure 13] 1 shows the results of the compound library screening carried out in Example 5. [Figure 14] 1 shows the results of the compound library screening carried out in Example 5. [Figure 15] 1 shows the results of the compound library screening carried out in Example 5. [Figure 16A] Shown are the two MC4R chimeras referred to in the experimental section (SEQ ID NO: 11 and 12) and an alignment of the amino acid sequences of the human MC4R (SEQ ID NO: 13) and beta-2-adrenergic receptor (SEQ ID NO: 14) from which they are derived. [Figure 16B]Shown are the two MC4R chimeras referred to in the experimental section (SEQ ID NO: 11 and 12) and an alignment of the amino acid sequences of the human MC4R (SEQ ID NO: 13) and beta-2-adrenergic receptor (SEQ ID NO: 14) from which they are derived. [Figure 16C] Shown are the two MC4R chimeras referred to in the experimental section (SEQ ID NO: 11 and 12) and an alignment of the amino acid sequences of the human MC4R (SEQ ID NO: 13) and beta-2-adrenergic receptor (SEQ ID NO: 14) from which they are derived. [Figure 17] 1 is a schematic representation of the tertiary structure of the amino acid sequence of MC4R-B2AR chimera 1 (SEQ ID NO: 11), showing the N-terminal and C-terminal sequences, ECL, ICL, and TM. The amino acid residues of the chimeric GPCR derived from B2AR (ICL and some ICL-adjacent residues) are shaded gray. [Figure 18] 1 is a graph showing a schematic diagram of testing a series of compounds (A2-F11, indicated on the x-axis) in a radioligand assay using wild-type MC4R (SEQ ID NO: 13) and MC4R-B2AR chimera 1 (SEQ ID NO: 11). For each compound, displacement was measured relative to the wild-type (values ​​indicated by dots) and relative to the chimera (values ​​indicated by squares) (see Example 6). [Figure 19] Graph showing the results obtained with a series of compounds in a radioligand assay using an MC4R / B2AR chimera and the assay setup shown in FIG. 1 (each data point on the graph represents a different compound). The results of the radioligand assay are presented along the y-axis ("Conforatio @ 10 micromolar"), and the results of the ConfoSensor assay are presented along the x-axis ("Confosensor Ratio"). Compounds selected for further testing in the cAMP cellular assay using wild-type MC4R are designated A-I. [Figure 20] 19 is a graph from two separate experiments showing the readout in a cAMP cellular assay using wild-type MC4R for Alpha-MSH (reference), unstimulated cells ("Unstim"), and compounds A-I selected for testing in the cAMP assay based on the data shown in FIG. [Figure 21] 19 is a graph from two separate experiments showing the readout in a cAMP cellular assay using wild-type MC4R for Alpha-MSH (reference), unstimulated cells ("Unstim"), and compounds A-I selected for testing in the cAMP assay based on the data shown in FIG. [Figure 22] The amino acid sequence of human β2AR is shown (UniProt P07550, see SEQ ID NO: 17 and FIG. 22). [Figure 23] 1 is a plot obtained in Example 9 comparing the results of an OX2 assay of the present invention (using recombinant OX2 fusions) and an OX2 IP-One assay, where the x-axis represents data obtained in the assay of the present invention and the y-axis represents data obtained in the IP-One assay, and each dot represents the result of a single compound. [Figure 24A] 24A shows plots obtained when a large compound library was screened against recombinant OX2 receptors using the assay of the present invention in Example 10. Figure 24A shows the results obtained when compounds were tested at 30 μM, the x-axis represents the ratio of the signal obtained by the tested compound ("sample") to the signal obtained by the carrier solvent ("blank"), and each dot represents the result obtained with a single compound. [Figure 24B] 24B shows the plots obtained when a large compound library was screened against recombinant OX2 receptors using the assay of the present invention in Example 10. Figure 24B shows the results obtained when compounds were tested at 200 μM, with the x-axis representing the ratio of the signal obtained by the tested compound ("sample") to the signal obtained by the carrier solvent ("blank"), and each dot representing the result obtained with a single compound. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is described herein with reference to particular embodiments and with reference to certain non-limiting examples and drawings. Any reference signs in the claims should not be construed as limiting the scope. The drawings described are schematic only and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. When the term "comprising" is used in the specification and claims, it does not exclude other elements or steps. When an indefinite or definite article is used when referring to a singular noun (e.g., "a," "an," or "the"), it also includes the plural of that noun unless something specifically stated otherwise. Furthermore, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements and are not necessarily used to describe an order or chronology. It should be understood that terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may operate in orders other than those described or illustrated herein.

[0030] Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with molecular and cellular biology, structural biology, biophysics, pharmacology, genetics, and protein and nucleic acid chemistry described herein is that which is well known and commonly used in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991), provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements through 2002); up, Biomolecular crystallography: principles, practice and applications to structural biology, 1st ed., Garland Science, Taylor & Francis Group, LLC, an information business, NY (2009); Limbird, Cell Surface Receptors, 3rd ed., Springer (2004).

[0031] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably herein and refer to polymeric forms of amino acids of any length, and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Standard single-letter amino acid abbreviations are used throughout the application. Typically, the term "amino acid" refers to "proteinogenic amino acids," i.e., amino acids that occur naturally in proteins. In particular, amino acids are in the L-isomer form, although D-amino acids are also contemplated.

[0032] As used herein, the terms "nucleic acid molecule," "polynucleotide," "polynucleic acid," and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0033] Any of the peptides, polypeptides, nucleic acids, compounds, etc. disclosed herein may be "isolated" or "purified." "Isolated" is used herein to indicate that the referenced material (i) has been separated from one or more materials with which it naturally occurs (e.g., separated from at least some cellular material, separated from other polypeptides, or separated from its natural sequence context), and / or (ii) has been produced by a process involving the hand of man, such as recombinant DNA technology, protein engineering, chemical synthesis, etc., and / or (iii) has a sequence, structure, or chemical composition not found in nature. "Isolated" is intended to include compounds within a sample that have been substantially enriched in the compound of interest and / or from which the compound of interest has been partially or substantially purified. As used herein, "purified" indicates that the referenced material has been removed from its natural environment and is at least 60% free, at least 75% free, or at least 90% free from other components with which it is naturally associated, also referred to as "substantially pure."

[0034] As used herein, the term "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison.

[0035] Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Li, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Determining the percentage of sequence identity can be performed manually or by using computer programs available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989)), BLAST and BLAST 2.0 (Altschul et al., J. Mol. Biol., 215:403 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0036] "Similarity" refers to the percentage of amino acids that are identical or that constitute conservative substitutions. Similarity can be determined using sequence comparison programs such as GAP (Deveraux et al., 1984). In this method, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, and such gaps are determined, for example, by the comparison algorithm used by GAP. As used herein, a "conservative substitution" refers to the substitution of an amino acid with another amino acid whose side chain has similar biochemical properties (e.g., aliphatic, aromatic, or positively charged), and is well known to those skilled in the art. A non-conservative substitution refers to the substitution of an amino acid with another amino acid whose side chain does not have similar biochemical properties (e.g., substitution of a hydrophobic residue with a polar residue). Typically, a conservative substitution results in a sequence that is no longer identical but still very similar. By conservative substitutions, combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp are intended.

[0037] A "deletion" is defined herein as a change in either the amino acid sequence or the nucleotide sequence in which one or more amino acid residues or nucleotide residues are absent compared to the amino acid sequence or nucleotide sequence of a parent polypeptide or nucleic acid, respectively. In the context of a protein, a deletion can involve the deletion of about 2, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or fragment thereof may contain two or more deletions. In the context of a GPCR, a deletion may be a loop deletion, or an N-terminal and / or C-terminal deletion. As will be apparent to one of skill in the art, an N-terminal and / or C-terminal deletion of a GPCR is also referred to as a truncation of the amino acid sequence of the GPCR or a truncated GPCR.

[0038] An "insertion" or "addition" is a change in an amino acid sequence or nucleotide sequence in which one or more amino acids or nucleotide residues have been added, respectively, compared to the amino acid sequence or nucleotide sequence of a parent protein. An "insertion" generally refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, and an "addition" can refer to the insertion or addition of amino acid residues at the N-terminus, C-terminus, or both termini. In the context of a protein or fragment thereof, an insertion or addition is typically an insertion or addition of about 1, about 3, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

[0039] "Substitution," as used herein, results from the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid sequence or nucleotide sequence of a parent protein or fragment thereof. It is understood that a protein or fragment thereof may have conservative amino acid substitutions that do not substantially affect the activity of the protein. By conservative substitutions, combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp are intended.

[0040] A "mutation" is defined herein as a change in either an amino acid sequence or a nucleotide sequence that is a deletion, insertion, or substitution, as described herein. When an amino acid or nucleotide sequence contains two or more such mutations, each of these mutations may independently be a deletion, insertion, or substitution.

[0041] The term "amino acid difference" refers to the total number of amino acid residues in a sequence that are changed (i.e., by substitution, insertion, and / or deletion) compared to a starting or reference sequence. The number of amino acid differences between a sequence and a reference sequence can usually be determined by aligning and comparing the sequences, for example.

[0042] The term "ortholog," when used in reference to an amino acid or nucleotide / nucleic acid sequence from a given species, refers to an identical amino acid or nucleotide / nucleic acid sequence from a different species. Two sequences should be understood to be orthologs of one another if they are derived from a common ancestral sequence through direct lineage and / or are otherwise closely related in terms of both their sequence and their biological function. Orthologs typically have a high degree of sequence identity, but do not necessarily (and often do not) share 100% sequence identity.

[0043] The term "recombinant" when used with reference to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a nucleic acid or polypeptide that is not found within the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise aberrantly expressed, repressibly expressed, over-expressed, or not expressed at all.

[0044] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. This process includes both transcription and translation.

[0045] As used herein, the term "operably linked" refers to a linkage in which a regulatory sequence is contiguous with a gene of interest to regulate the gene of interest, and a linkage in which the regulatory sequence acts in trans or at a distance to regulate the gene of interest. For example, a DNA sequence is operably linked to a promoter when it is ligated downstream of the promoter with respect to the transcription initiation site of the promoter, allowing transcription elongation to proceed through the DNA sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide when it is expressed as a preprotein involved in the transport of the polypeptide. Linking of a DNA sequence to a regulatory sequence is typically achieved by ligation at appropriate restriction sites using restriction endonucleases known to those skilled in the art, or by inserting adapters or linkers instead.

[0046] As used herein, the term "regulatory sequence," also referred to as "control sequence," refers to a polynucleotide sequence necessary to affect the expression of a coding sequence to which it is operably linked. Regulatory sequences are sequences that control the transcription, post-transcriptional events, and translation of a nucleic acid sequence. Regulatory sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., ribosome binding sites); sequences that improve protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. The term "regulatory sequence" is intended to include at least all components whose presence is essential for expression, and may include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0047] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. A vector can be any suitable type of vector, including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid, or even an artificial chromosome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of a specific gene of interest. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Suitable vectors contain regulatory sequences, such as promoter, enhancer, and terminator sequences, as desired and depending on the particular host organism (e.g., bacterial cells, yeast cells). Typically, a recombinant vector according to the present invention comprises at least one "chimeric gene" or "expression cassette." An expression cassette is generally a DNA construct, preferably comprising (5' to 3' in the direction of transcription) a promoter region, a polynucleotide sequence of the present invention or a homolog, variant, or fragment thereof operably linked to a transcription initiation region, and a termination sequence comprising an RNA polymerase termination signal and a polyadenylation signal. It is understood that all of these regions should be operable in the living cell, such as a prokaryotic or eukaryotic cell, to be transformed. The promoter region, including the transcription initiation region, which preferably comprises an RNA polymerase binding site, and the polyadenylation signal, may be native to the living cell to be transformed, or the regions may be derived from another source that is functional in the living cell.

[0048] As used herein, the term "host cell" is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A host cell may be an isolated cell or a cell line grown in culture, or may be a cell present in a living tissue or organism. In particular, host cells are cells of bacterial or fungal origin, but may also be cells of plant or mammalian origin. The terms "host cell," "recombinant host cell," "expression host cell," "expression host system," and "expression system" are intended to have the same meaning and are used interchangeably herein.

[0049] In the present invention, as is common in the art, amino acid sequences are given using the single-letter amino acid code starting from the N-terminus and ending at the C-terminus. Also, in the present specification and claims, if a position or residue is closer to the N-terminus than a given position or residue, the first-mentioned position or residue is said to be "upstream" of the given position or residue, and if it is closer to the C-terminus than a given position or residue, the first-mentioned position or residue is said to be "downstream" of the given position or residue.

[0050] "G-protein coupled receptors" or "GPCRs" are polypeptides that share a common structural motif: an extracellular amino terminus (N-terminus), an intracellular carboxy terminus (C-terminus), and seven hydrophobic transmembrane regions of 22-24 hydrophobic amino acids each forming seven alpha helices that span the membrane. Each span is identified by a number, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are connected by regions of amino acids between transmembrane-2 and transmembrane-3, between transmembrane-4 and transmembrane-5, and between transmembrane-6 and transmembrane-7, designated "extracellular" regions 1, 2, and 3 (EC1, EC2, and EC3), respectively, on the outer, or "extracellular," side of the cell membrane. The transmembrane helices are also connected by stretches of amino acids between transmembrane-1 and transmembrane-2, between transmembrane-3 and transmembrane-4, and between transmembrane-5 and transmembrane-6, referred to as "intracellular" regions 1, 2, and 3 (IC1, IC2, and IC3), respectively, on the inside, or "intracellular," side of the cell membrane. The "carboxy" ("C") terminus of the receptor is in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor is in the extracellular space outside the cell.The structure and classification of GPCRs are generally well known in the art, and further discussion of GPCRs can be found in Cvicek et al., PLoS Comput Biol. 2016 Mar 30;12(3):e1004805.doi:10.1371 / journal.pcbi.1004805; Ventakakrishnan, Current Opinion in Structural Biology, 2014, 27:129-137; Isberg, Trends Pharmacol. Sci., 2015 Jan 22-13; Probst, DNA Cell Biol. 1992, 11:1-20; Marchese et al., Genomics 23:609-618, 1994; and the following books: Jurgen Wess (ed.) Structure-Function Analysis of G Protein-Coupled Receptors, Wiley Liss (1st ed., October 15, 1999); Kevin R. Lynch (ed.), Identification and Expression of G Protein-Coupled Receptors, published by John Wiley & Sons (March 1998); and Tatsuya Haga (ed.), G Protein-Coupled Receptors, published by CRC Press (September 24, 1999); and Steve Watson (ed.), G-Protein Linked Receptor Factsbook, published by Academic Press (1st ed., 1994). As described in the prior art and other scientific literature, in naturally occurring GPCRs, the N- and C-terminal portions, TM domains, intracellular loops, and extracellular loops are usually arranged (from N- to C-terminus) as follows: [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence].

[0051] The International Union of Basic and Clinical Pharmacology (IUPHAR) maintains a database of receptors (including GPCRs) and their known endogenous ligands and signaling mechanisms (http: / / www.guidetopharmacology.org / targets.jsp). According to this database, as of January 2019, approximately 800 GPCRs have been identified in humans, of which approximately half have sensory functions (e.g., olfaction, taste, light perception, and pheromone signaling), and approximately half mediate signaling associated with ligands ranging in size from small molecules to peptides and large proteins. As of January 2019, the IUPHAR database describes two systems for classifying GPCRs, one of which is based on six classes of GPCRs: class A (rhodopsin-like), class B (secretin receptor family), class C (metabotropic glutamate), class D (fungal mating pheromone receptors, not found in vertebrates), class E (cyclic AMP receptors, also not found in vertebrates), and class F (frizzled / smoothened).The IUPHAR database also refers to an alternative classification scheme known as "GRAFS," which divides vertebrate GPCRs into five classes (overlapping with the AF nomenclature): the glutamate family (overlapping with "Class C" above), which includes, among others, metabotropic glutamate receptors, calcium-sensing receptors, and GABAB receptors; the rhodopsin family (overlapping with "Class A" above), which includes receptors for a wide variety of small molecules, neurotransmitters, peptides, and hormones, as well as olfactory receptors, visual pigments, taste type 2 receptors, and five pheromone receptors (V1 receptors); and the adhesion family GPCRs (Class B receptors). the Frizzled family, which consists of 10 Frizzled proteins (FZD(1-10)) and Smoothened (SMO); and the secretin family, which are receptors for peptide ligands / hormones with 27-141 amino acid residues, including glucagon, glucagon-like peptides (GLP-1, GLP-2), glucose-dependent insulinotropic polypeptide (GIP), secretin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and growth hormone-releasing hormone (GHRH). In this specification and the appended claims, the classification of types A-F will be used unless expressly stated otherwise. See also Cvicek et al., cited herein.

[0052] The term "biologically active" with respect to a GPCR refers to a GPCR that has the biochemical function of a naturally occurring GPCR (e.g., a binding function, a signaling function, or the ability to change conformation as a result of ligand binding).

[0053] Generally, the term "naturally occurring" with respect to a GPCR refers to a GPCR that is naturally produced (e.g., by a wild-type mammal such as a human). Such GPCRs are found in nature. "Non-naturally occurring" with respect to a GPCR refers to a GPCR that is not naturally occurring. Naturally occurring GPCRs that are constitutively activated by mutation and variants of naturally occurring transmembrane receptors, such as epitope-tagged GPCRs and GPCRs lacking their natural N-termini, are examples of non-naturally occurring GPCRs. Non-naturally occurring variants of naturally occurring GPCRs are often activated by the same ligand as the naturally occurring GPCR. Non-limiting examples of naturally occurring or non-naturally occurring GPCRs in the context of the present invention are further provided herein.

[0054] As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least four, five, six, or seven such amino acids, and more usually, at least eight, nine, or ten such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a "conformational epitope" refers to an epitope comprising amino acids in a spatial conformation unique to the folded three-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in a linear sequence and that are clustered in the folded structure of a protein. However, a conformational epitope may also be comprised of a linear sequence of amino acids that adopts a conformation unique to the folded three-dimensional conformation of a polypeptide (and that does not exist in the denatured state).

[0055] The term "conformation" or "conformational state" of a protein (such as a GPCR) generally refers to the spatial arrangement, structure, or range of structures that a protein can adopt at any given time. Those skilled in the art will recognize that determinants of conformation or conformational state include the protein's primary structure, as reflected in the protein's amino acid sequence (including modified amino acids), and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structure (e.g., alpha helix, beta sheet, among others), tertiary structure (e.g., the three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions of the polypeptide chain with other protein subunits). Post-translational and other modifications of the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, among others, can affect the conformation of a protein. Additionally, environmental factors such as the pH, salt concentration, ionic strength, and osmolality of the surrounding solution, as well as interactions with other proteins and cofactors, among others, can affect the conformation of a protein. The conformational state of a protein can be determined by functional assays for activity or binding to another molecule, or by physical methods such as X-ray crystallography, NMR, or spin labeling, among others. For a general discussion of protein conformation and conformational states, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0056] As used herein, "functional conformation" or "functional conformational state" refers to the fact that proteins (such as GPCRs) have various conformational states with a dynamic range of activity, particularly ranging from inactive to maximally active. It will be apparent that "functional conformational state" is intended to include any conformational state of a protein with any activity, including inactive, and is not intended to include a denatured state of a protein. Non-limiting examples of functional conformations include an active conformation, an inactive conformation, or a basal conformation (as further defined herein). As noted above, a particular class of functional conformations is defined as a "druggable conformation," which generally refers to a therapeutically relevant conformational state(s) of a protein. See, e.g., Johnson and Karanicolas, PLoS Comput Biol 9(3):e1002951.doi:10.1371 / journal.pcbi.1002951. Also see, for example, WO 2014 / 122183, which describes that the agonist-binding conformation of the muscarinic acetylcholine receptor M2 corresponds to the druggable conformation of this receptor associated with pain and glioblastoma, and describes VHHs that can stabilize the druggable conformation for assay and screening purposes. Therefore, it will be understood that druggability is limited to certain conformations depending on the therapeutic indication. Further details are provided further herein.

[0057] As used herein with respect to proteins that are receptors (such as GPCRs), the term "active conformation" refers more specifically to a conformation or set of receptor conformations that permits signal transduction to intracellular effector systems, such as G protein-dependent signaling and / or G protein-independent signaling (e.g., β-arrestin signaling). Thus, "active conformation" encompasses a set of ligand-specific conformations, including agonist-specific, partial agonist-specific, or biased agonist-specific active state conformations, that result in the cooperative binding of intracellular effector proteins.

[0058] In addition to the above, the terms "active conformation" and "active form" as used herein with respect to GPCRs refer to a GPCR that is folded in a manner that is (functionally) active. GPCRs can be placed into an active conformation using an activating ligand (agonist) for the receptor; such a conformational change generally allows the receptor to activate a heterotrimeric G protein. For example, a GPCR in its active conformation binds to a heterotrimeric G protein and catalyzes nucleotide exchange in the G protein, activating downstream signaling pathways. Activated GPCRs bind to inactive, GDP-bound heterotrimeric G proteins, causing the G protein to release GDP so that GTP can bind. This process creates a transient "nucleotide-free" state that allows GTP to bind. Upon GTP binding, the receptor and G protein dissociate, allowing the GTP-bound G protein to activate downstream signaling pathways, such as adenylyl cyclase, ion channels, and RAS / MAPK. The terms "inactive conformation" and "inactive form" refer to a GPCR that is folded in such a manner that it is inactive. A GPCR can be placed in an inactive conformation using an inverse agonist of the receptor. For example, a GPCR in its inactive conformation does not bind with high affinity to a heterotrimeric G protein. The terms "active conformation" and "inactive conformation" are further explained herein. As used herein, the term "basal conformation" refers to a GPCR that is folded in such a manner that it exhibits activity toward a specific signaling pathway even in the absence of an agonist (also referred to as basal activity or constitutive activity). An inverse agonist can inhibit this basal activity. Thus, the basal conformation of a GPCR corresponds to a stable conformation or distinct structural species in the absence of a ligand or accessory protein.

[0059] Similarly, the term "inactive conformation," as used herein with respect to proteins that are receptors (such as GPCRs), refers to a range of receptor conformations that do not permit or block signal transmission to intracellular effector systems. Thus, "inactive conformations" encompass a range of ligand-specific conformations, including inverse agonist-specific inactive state conformations, thereby preventing cooperative binding of intracellular effector proteins. It will be understood that the binding site of the ligand is not critical for obtaining an active or inactive conformation. Thus, orthosteric ligands as well as allosteric modulators can similarly stabilize receptors in active or inactive conformations.

[0060] The term "binder," as used herein, refers to all or part of a proteinaceous (protein, protein-like, or protein-containing) molecule capable of binding to a membrane protein (such as a GPCR) using specific intermolecular interactions. In certain embodiments, the term "binder" is not intended to include naturally occurring binding partners of the relevant membrane protein, such as G proteins, arrestins, endogenous ligands, or variants or derivatives (including fragments) thereof. More specifically, the term "binder" refers to a polypeptide, particularly a protein domain. Suitable protein domains are elements of the overall protein structure that are self-stable and fold independently of the rest of the protein chain, and are often referred to as "binding domains." Such binding domains vary in length from about 25 amino acids up to 500 and more amino acids. Many binding domains can be classified into folds, which are recognizable and distinguishable three-dimensional structures. Some folds are common to many different proteins and therefore have been given special names. Non-limiting examples are binding domains selected from three- or four-helix bundles, armadillo repeat domains, leucine-rich repeat domains, PDZ domains, SUMO or SUMO-like domains, cadherin domains, immunoglobulin-like domains, phosphotyrosine-binding domains, pleckstrin homology domains, src homology 2 domains, among others. Thus, binding domains may be derived from naturally occurring molecules, e.g., components of the innate or adaptive immune system, or may be entirely artificially designed.

[0061] In general, binding domains may be based on immunoglobulins or may be based on domains present in proteins such as, but not limited to, microbial proteins, protease inhibitors, toxins, fibronectin, lipocalins, single-stranded antiparallel coiled-coil proteins, or repeat motif proteins. Specific examples of binding domains known in the art include, but are not limited to, antibodies, heavy chain antibodies (hcAbs), single domain antibodies (sdAbs), minibodies, variable domains derived from camelid heavy chain antibodies (VHHs or nanobodies), variable domains of novel antigen receptors derived from shark antibodies (VNAs), alphabodies, protein A, protein G, designed ankyrin repeat domains (DARPins), fibronectin type III repeats, anticalins, knottins, engineered CH2 domains (nanobodies), engineered SH3 domains, affibodies, peptides, and proteins, lipopes, and the like. Examples of suitable binding domains include peptides (e.g., pepducins) (see, e.g., Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., 1997; Binz et al., 2004; Koide et al., 1998; Dimitrov, 2009; Nygren et al., 2008; WO 2010066740). When selection methods are used to generate specific types of binding domains, combinatorial libraries containing consensus or framework sequences with randomized potential interacting residues are often used to screen for binding to a molecule of interest, e.g., a protein.

[0062] According to a preferred embodiment, it is specifically envisaged that the binding agent of the present invention is derived from the innate or adaptive immune system. Preferably, the binding agent is derived from an immunoglobulin. Preferably, the binding agent according to the present invention is derived from an antibody or an antibody fragment. The term "antibody" (Ab) generally refers to a polypeptide encoded by an immunoglobulin gene or a functional fragment thereof that specifically binds to and recognizes an antigen, and is known to those skilled in the art. Antibody is intended to include conventional four-chain immunoglobulins, which comprise a pair of two identical polypeptide chains, each having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50 kDa). Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. The term "antibody" is intended to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments. In some embodiments, the antigen-binding fragment may be, but is not limited to, Fab, Fab', and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), and fragments comprising or consisting of either a VL or VH domain, and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen. The term "antibody" is also intended to include heavy-chain antibodies or fragments thereof comprising an immunoglobulin single variable domain, as further defined herein.

[0063] The term "immunoglobulin single variable domain" or "ISVD" defines a molecule in which the antigen-binding site is present in and formed by a single immunoglobulin domain (this differs from conventional immunoglobulins or fragments thereof in which typically two immunoglobulin variable domains interact to form the antigen-binding site). However, it will be clear that the term "immunoglobulin single variable domain" includes fragments of conventional immunoglobulins in which the antigen-binding site is formed by a single variable domain. Preferably, binding agents within the scope of the present invention are immunoglobulin single variable domains.

[0064] In general, an immunoglobulin single variable domain comprises four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), and preferably comprises an amino acid sequence according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1), or any suitable fragment thereof (usually comprising at least a portion of the amino acid residues forming at least one of the complementarity-determining regions). ISVDs comprising four FRs and three CDRs are known to those skilled in the art, and are described, by way of non-limiting example, in Wesolowski et al., 2009. Typical, but non-limiting, examples of immunoglobulin single variable domains include light chain variable domain sequences (e.g., VL domain sequences) or suitable fragments thereof, or heavy chain variable domain sequences (e.g., VH domain sequences or VHH domain sequences) or suitable fragments thereof, as long as they are capable of forming a single antigen-binding unit. Thus, according to a preferred embodiment, the binding agent is an immunoglobulin single variable domain which is a light chain variable domain sequence (e.g. a VL domain sequence) or a heavy chain variable domain sequence (e.g. a VH domain sequence), more particularly the immunoglobulin single variable domain is a heavy chain variable domain sequence derived from a traditional four chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. The immunoglobulin single variable domain can be a domain antibody, or a single domain antibody, or a "dAB" or dAb, or a nanobody (as defined herein), or another immunoglobulin single variable domain, or any suitable fragment of any one thereof. For a general description of single domain antibodies see the following book: "Single domain antibodies", Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, Vol. 911. An immunoglobulin single variable domain generally comprises a single amino acid chain which can be considered to comprise four "framework sequences" or FRs and three "complementarity determining regions" or CDRs (as defined herein above).It is evident that the framework regions of immunoglobulin single variable domains can also contribute to their antigen binding (Desmyter et al., 2002; Korotkov et al., 2009).

[0065] As further described herein, the total number of amino acid residues in a VHH, Nanobody or Confobody may be in the region of 110-120, preferably 112-115, and most preferably 113. It should be noted, however, that parts, fragments, analogs or derivatives of a VHH or Nanobody (as further described herein) are not particularly limited with respect to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are suitable for the purposes described herein.

[0066] In the present application, the amino acid residues / positions within the immunoglobulin heavy chain variable domain are determined by the V sequences derived from camel in the literature as described by Riechmann and Muyldermans, J. Immunol. Methods, 2000 Jun. 23; 240(1-2):185-195. HH As applied to domains (see, e.g., Figure 2 therein), they are shown using the Kabat numbering system ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). See also, e.g., Figure 1 of International Application WO 2108 / 134235, which provides a table listing some of the amino acid positions in VHHs and their numbering according to several alternative numbering systems (e.g., Aho and IMGT; it should be noted that, unless expressly indicated otherwise, for the purposes of this specification and claims, the Kabat numbering system is definitive for amino acid residues / positions in VHHs, nanobodies or confobodies, and other numbering systems are given for reference only).

[0067] Regarding CDRs, as is well known in the art, there are several rules for defining and describing CDRs of VH or VHH fragments, such as the Kabat definition (based on sequence variability and is the most commonly used) and the Chothia definition (based on the location of structural loop regions). See, for example, the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, although Kabat CDRs may also be referred to, it is most preferred that CDRs be defined based on the Abm definition (based on Oxford Molecular's AbM antibody modeling software), as this Abm definition is considered to be the best compromise between the Kabat and Chothia definitions. Again, see the website http: / / www.bioinf.org.uk / abs / .

[0068] Therefore, in this specification and claims, all CDRs or VHHs, nanobodies or confobodies are defined according to the Abm rules unless expressly stated otherwise herein.

[0069] It should be noted that immunoglobulin single variable domains as binding agents in the broadest sense are not limited to a particular biological source or a particular preparation method. The term "immunoglobulin single variable domain" or "ISVD" encompasses variable domains of various origins, including mouse, rat, rabbit, donkey, human, shark, and camelid variable domains. According to a particular embodiment, the immunoglobulin single variable domain is derived from a shark antibody (so-called immunoglobulin novel antigen receptor or IgNAR), more particularly from a naturally occurring heavy-chain shark antibody without a light chain, known as a VNAR domain sequence. Preferably, the immunoglobulin single variable domain is derived from a camelid antibody. More preferably, the immunoglobulin single variable domain is derived from a naturally occurring heavy-chain camelid antibody without a light chain, known as a VHH domain sequence or nanobody.

[0070] According to a particularly preferred embodiment, the binding agents of the invention are immunoglobulin single variable domains that are nanobodies (as further defined herein and including, but not limited to, VHHs). As used herein, the term "nanobody" (Nb) refers to a single domain antigen-binding fragment. It particularly refers to a single variable domain derived from a naturally occurring heavy chain antibody and is known to those skilled in the art. Nanobodies are typically derived from heavy chain-only antibodies (without light chains) found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996) and are consequently often referred to as VHH antibodies or VHH sequences. The Camelidae family consists of Old World Camelidae (Camelus bactrianus and Camelus dromedarius) and New World Camelidae (e.g., Lama paccos, Lama glama, Lama guanicoe, and Lama vicugna). Nanobody® and Nanobodies® are registered trademarks of Ablynx NV (Belgium).For a further description of VHHs or nanobodies, see the book "Single domain antibodies", Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, volume 911, in particular the chapter by Vincke and Muyldermans (2012), as well as the non-limiting list of patent applications mentioned as general background art, including: Vrije Universiteit Brussels WO 94 / 04678, WO 95 / 04079, WO 96 / 34103; Unilever WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231, and WO 02 / 48193; Vlaams Instituut voor WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016, and WO 03 / 055527 by Biotechnologie (VIB); WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787, and WO 06 / 122528 by Ablynx NV; see further published patent applications by NV. As known to those skilled in the art, Nanobodies are particularly characterized by the presence of one or more Camelidae "hallmark residues" in one or more framework sequences (according to Kabat numbering), e.g., as described in Table A-3 on page 75 of WO 08 / 020079, which is incorporated herein by reference. It should be noted that the Nanobodies of the invention in their broadest sense are not limited to a particular biological source or a particular method of preparation.(iii) by "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (iv) by "camelizing" a naturally occurring VH domain from any animal species, in particular a mammalian species such as human, or by expressing a nucleic acid encoding such a camelized VH domain; (v) by "camelizing" a "domain antibody" or "Dab" as described in the art or by expressing a nucleic acid encoding such a camelized VH domain; (vi) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (vii) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expressing the nucleic acid so obtained; and / or (8) by any combination of one or more of the foregoing. Further description of Nanobodies, including humanization and / or camelization of Nanobodies, can be found, for example, in WO 08 / 101985 and WO 08 / 142164, and further description herein. A particular class of Nanobody-binding conformational epitopes of natural targets are called Xaperones and are specifically contemplated herein. Xaperone™ is a trademark of VIB and VUB (Belgium). Xaperone™ is a camelid single domain antibody that constrains drug targets into unique, disease-relevant, druggable conformations.

[0071] Within the scope of the present invention, the term "immunoglobulin single variable domain" also encompasses variable domains that have been "humanized" or "camelized", in particular Nanobodies that have been "humanized" or "camelized". For example, both "humanization" and "camelization" can be carried out by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then changing one or more codons of said nucleotide sequence in a manner known per se in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" immunoglobulin single variable domain of the invention, respectively. This nucleic acid can then be expressed in a manner known per se to provide the desired immunoglobulin single variable domain of the invention. Alternatively, the amino acid sequence of the desired humanized or camelized immunoglobulin single variable domain of the invention can be designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using techniques for peptide synthesis known per se. It is also possible to design a nucleotide sequence encoding the desired humanized or camelized immunoglobulin single variable domain of the invention based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesize it de novo using techniques for nucleic acid synthesis known per se, and then express the nucleic acid so obtained in a manner known per se to provide the desired immunoglobulin single variable domain of the invention. Other suitable methods and techniques for obtaining the immunoglobulin single variable domains of the invention and / or nucleic acids encoding them, starting from naturally occurring VH sequences, or preferably VHH sequences, will be clear to the skilled person and may, for example, involve combining one or more parts (such as one or more FR sequences and / or CDR sequences) of one or more naturally occurring VHH sequences, one or more parts (such as one or more FR sequences or CDR sequences) of one or more naturally occurring VHH sequences, and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide a Nanobody of the invention or a nucleotide sequence or nucleic acid encoding it.

[0072] According to certain embodiments of the present invention, a binding agent capable of stabilizing a receptor may bind at an orthosteric or allosteric site. In other specific embodiments, a binding agent capable of stabilizing a receptor may be an active conformation-selective binding agent or a non-active conformation-selective binding agent, either by binding at an orthosteric site or by binding at an allosteric site. Generally, a conformation-selective binding agent that stabilizes the active conformation of a receptor will increase or enhance the affinity of the receptor for an active conformation-selective ligand, such as an agonist, more particularly a full agonist, partial agonist, or biased agonist, compared to the receptor in the absence of the binding agent (or in the presence of a mock binding agent, also referred to as a control binding agent or irrelevant binding agent that is not directed against and / or does not specifically bind to the receptor). Additionally, a binding agent that stabilizes the active conformation of the receptor reduces the affinity of the receptor for non-active conformation-selective ligands, such as inverse agonists, compared to the receptor in the absence of the binding agent (or in the presence of a mock binding agent). In contrast, a binding agent that stabilizes the non-active conformation of the receptor increases the affinity of the receptor for inverse agonists and reduces the affinity of the receptor for agonists, particularly full agonists, partial agonists, or biased agonists, compared to the receptor in the absence of the binding agent (or in the presence of a mock binding agent). The increase or decrease in affinity for the ligand may be measured directly and / or calculated from a decrease or increase, respectively, in EC, IC, K, K, or any other measure of affinity or potency known to those skilled in the art. It is particularly preferred that binding agents that stabilize a particular conformation of the receptor are capable of increasing or decreasing the affinity for the conformation-selective ligand by at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, and more preferably at least 100-fold, even more preferably at least 1000-fold or even more, upon binding to the receptor.It will be appreciated that affinity measurements for conformationally selective ligands that induce / inhibit specific signaling pathways can be performed using any type of ligand, including natural ligands, small molecules, and biological substances; orthosteric ligands and allosteric modulators; single compounds and compound libraries; lead compounds or fragments, etc.

[0073] As used herein, the term "affinity" refers to the degree to which a ligand binds to a target protein (such as a GPCR) such that the equilibrium between the target protein and its bound ligand (as further defined herein) is shifted toward the presence of a complex formed by their binding. Thus, for example, when a GPCR and a ligand are combined at relatively equal concentrations, a high-affinity ligand will bind to an available antigen on the GPCR, shifting the equilibrium toward a higher concentration of the resulting complex. Dissociation constants are commonly used to describe the affinity between a ligand and a target protein. Typically, dissociation constants are between 10 and 10. -5 Preferably, the dissociation constant is less than 10 -6 Less than M, more preferably 10 -7 Most preferably, the dissociation constant is less than 10 -8The affinity between a ligand (including a small molecule ligand) and its target protein is less than M. Another way to describe the affinity between a ligand (including a small molecule ligand) and its target protein is to indirectly assess the potency of the ligand by measuring the association constant (Ka), the inhibition constant (Ki), or the half maximal inhibitory concentration (IC50) or half maximal effective concentration (EC50). Within the scope of the present invention, the ligand may be a binding agent that binds to a conformational epitope of a GPCR, preferably an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH or nanobody. It will be understood that within the scope of the present invention, the term "affinity" is used in the context of a binding agent, particularly an immunoglobulin or immunoglobulin fragment, such as a VHH or nanobody, that binds to a conformational epitope of a target GPCR, as well as in the context of a test compound (as further defined herein) that binds to a target GPCR, particularly an orthosteric or allosteric site of the target GPCR.

[0074] As used herein, the term "specificity" refers to the ability of a protein or other binding agent, in particular an immunoglobulin or immunoglobulin fragment such as a VHH or nanobody, to bind preferentially to one antigen (such as a GPCR) over a different antigen (such as a different GPCR), and does not necessarily imply high affinity.

[0075] The terms "specifically bind" and "specific binding," as used herein, generally refer to the ability of a binding agent, particularly an immunoglobulin such as an antibody, or an immunoglobulin fragment such as a VHH or nanobody, to preferentially bind to a particular antigen present in a homogenous mixture of different antigens. In certain embodiments, a specific binding interaction can distinguish between desired and undesired antigens in a sample by a factor of more than about 10-fold, and in some embodiments, up to 100-fold or more (e.g., about 1000-fold or greater than 10,000-fold). In the context of a range of conformational states of a GPCR, the terms "specifically bind" and "specific binding" particularly refer to the ability of a binding agent (as defined herein) to preferentially recognize and / or bind a particular conformational state of a GPCR compared to alternative conformational states.

[0076] Also, in this specification and the appended claims, when a protein, ligand, compound, binding domain, binding unit, or other chemical entity is said to "bind" to another protein, ligand, compound, binding domain, binding unit, or other chemical entity, or to an epitope or binding site, it is to be understood that such binding is preferably "specific" binding as defined herein. Also, preferably, such binding is "selective binding" as defined herein.

[0077] As used herein, in the context of the present invention, a "conformation-selective binding agent" refers to a binding agent that binds to a target protein (such as a GPCR) in a conformation-selective manner. A binding agent that selectively binds to a particular conformation or conformational state of a protein refers to a binding agent that binds to a protein in a subset of conformations or conformational states with higher affinity than to other possible conformations or conformational states of the protein. Those skilled in the art will recognize that a binding agent that selectively binds to a particular conformation or conformational state of a protein stabilizes or maintains the protein in this particular conformation or conformational state. For example, an active conformation-selective binding agent preferentially binds to GPCRs in an active conformational state and does not bind or binds to a lower extent to GPCRs in an inactive conformational state, and therefore has a higher affinity for the active conformational state. And vice versa. The terms "specifically bind," "selectively bind," "preferentially bind," and their grammatical equivalents are used interchangeably herein. The terms "conformation-specific" or "conformation-selective" are also used interchangeably herein (although it should be noted that the term "conformation-directed" as used herein has a distinct meaning as further defined herein).

[0078] As used herein, the term "stabilize," or grammatically equivalent terms, refers to an increase in the stability of a protein (described herein) or receptor (described herein) in terms of structure (e.g., conformational state) and / or a particular biological activity (e.g., intracellular signaling activity, ligand binding affinity, etc.), as described above. With respect to increased stability to structure and / or biological activity, stabilization can be readily determined by either functional assays for activity (e.g., Ca release, cAMP production or transcriptional activity, β-arrestin recruitment) or ligand binding, or by physical methods such as X-ray crystallography, NMR, or spin labeling, among others. The term "stabilize" also includes increased thermal stability of a receptor under non-physiological conditions induced by denaturing agents or conditions. As used herein, the terms "thermostabilize," "thermostabilization," and "increase the thermal stability of" refer to functional properties of a receptor, not thermodynamic properties, and refer to the resistance of a protein to irreversible denaturation induced by thermal and / or chemical approaches, including, but not limited to, heating, cooling, freezing, chemical denaturants, pH, detergents, salts, additives, proteases, or temperature. Irreversible denaturation results in irreversible unfolding of the protein's functional conformation, loss of biological activity, and aggregation of the denatured protein. With respect to increased stability to heat, stability can be readily determined by measuring ligand binding or by using spectroscopic methods, such as fluorescence, CD, or light scattering, that are sensitive to unfolding at elevated temperatures. Preferably, the binding agent is capable of increasing stability as measured by an increase in the thermal stability of the protein or receptor in its functional conformational state by at least 2°C, at least 5°C, at least 8°C, and more preferably by at least 10°C, 15°C, or 20°C. With respect to increased stability to detergents or chaotropes, typically the protein or receptor is incubated in the presence of a test detergent or chaotropic agent for a predetermined period of time, and stability is determined using, for example, ligand binding or spectroscopy, optionally at elevated temperatures as described above.Alternatively, the binding agent may increase the stability of the functional conformational state of a protein or receptor against pH extremes. With respect to pH extremes, typical test pHs are selected, for example, in the range of 6-8, 5.5-8.5, 5-9, or 4.5-9.5, more particularly, in the range of 4.5-5.5 (low pH) or 8.5-9.5 (high pH). As used herein, the terms "thermostabilize," "thermostabilize," and "increase the thermal stability of" apply to proteins or receptors embedded in lipid particles or lipid layers (e.g., lipid monolayers, lipid bilayers, etc.), as well as proteins or receptors dissolved in detergent.

[0079] Further to the above, the term "stabilization" or "stabilized," with respect to the functional conformational state of a GPCR, refers to maintaining or retaining the GPCR protein in a subset of the possible conformations it might otherwise assume, due to the effect of the interaction of the GPCR with a binding agent according to the present invention. In this context, a binding agent that selectively binds to a particular conformation or conformational state of a protein refers to a binding agent that binds with higher affinity to the protein in a certain conformation or subset of conformational states than to other possible conformations or conformational states of the protein. Those skilled in the art will recognize that a binding agent that specifically or selectively binds to a particular conformation or conformational state of a protein stabilizes this particular conformation or conformational state and its associated activity. Further details are provided further herein.

[0080] As used herein, the terms "compound" or "test compound" or "candidate compound" or "drug candidate compound" describe any naturally occurring or synthetic molecule to be tested in an assay, such as a screening assay or drug discovery assay. Thus, these compounds include organic or inorganic compounds. Compounds include polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) containing about 2 to about 40 amino acids, and larger polypeptides containing about 40 to about 500 amino acids, such as antibodies, antibody fragments, or antibody conjugates. The test compound may also be protein-based. For high-throughput purposes, test compound libraries, such as combinatorial or randomized libraries, that provide sufficient diversity, may be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, fragment-based libraries, and phage display libraries. Further detailed descriptions can be found further herein.

[0081] As used herein, the term "ligand" refers to a molecule that specifically binds to a protein referred to herein, such as a GPCR. A ligand can be, but is not limited to, a polypeptide, lipid, small molecule, antibody, antibody fragment, nucleic acid, or carbohydrate. A ligand can be a synthetic or naturally occurring ligand. Ligands also include "natural ligands," which are endogenous natural ligands for native GPCRs. In the context of the present invention, when a protein is a transmembrane protein such as a GPCR, a ligand can bind to the protein at a ligand-binding site that is exposed to the intracellular environment when the protein is in its native cellular environment (i.e., the ligand can be an "intracellular ligand"), or can bind to the protein at a ligand-binding site that is exposed to the extracellular environment when the protein is in its native cellular environment (i.e., the ligand can be an "extracellular ligand"). Extracellular ligands are often classified based on the manner in which they act to regulate GPCRs (as defined herein), e.g., as agonists, partial agonists, inverse agonists, antagonists, or allosteric modulators. Extracellular ligands can bind at either the orthosteric or allosteric site.

[0082] As described further herein, an intracellular ligand (such as a binding domain or binding unit used in the present invention) may be a "conformation-inducing" ligand (as defined herein), meaning that the ligand is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to the chimeric GPCR (i.e., to the intracellular binding site of the chimeric GPCR). As described further herein, such a conformation-inducing ligand may be capable of inducing and / or stabilizing a complex formed by the GPCR, the conformation-inducing intracellular ligand, and the extracellular ligand (particularly when the extracellular ligand is capable of agonizing the GPCR), in which the GPCR is preferably in a functional, active, and / or druggable state.

[0083] In certain embodiments, a (extracellular or intracellular) ligand may be a "conformation-selective ligand" or "conformation-specific ligand," meaning that such a ligand binds to a protein or GPCR in a conformation-selective manner. As further described herein, a conformation-selective ligand binds with higher affinity to a particular conformation of a protein than to other conformations that the protein can adopt. By way of example, an extracellular ligand that acts as an agonist is an example of an active conformation-selective ligand, and an extracellular ligand that acts as an inverse agonist is an example of a non-active conformation-selective ligand. For clarity, neutral antagonists are not considered conformation-selective ligands because they do not distinguish between different conformations of a GPCR.

[0084] As used herein, "orthosteric ligand" refers to ligands (both natural and synthetic) that bind to the active site of GPCRs and are further classified according to their potency, i.e., their effect on signal transduction through a specific pathway. As used herein, "agonist" refers to a ligand that increases the signal transduction activity of a receptor protein (such as a GPCR) by binding to it. A full agonist allows maximal protein stimulation. A partial agonist cannot elicit full activity even at saturating concentrations. A partial agonist can also function as a "blocker" by preventing the binding of more potent agonists. An "antagonist," also known as a "neutral antagonist," refers to a ligand that binds to a receptor without stimulating any activity. An "antagonist" is also known as a "blocker" because it can prevent the binding of other ligands and thus block agonist-induced activity. Furthermore, an "inverse agonist" refers to an antagonist that, in addition to blocking the agonist effect, reduces the basal or constitutive activity of the receptor below that of the unliganded protein.

[0085] As used herein, a ligand may be a "biased ligand" that has the ability to selectively stimulate a subset of receptor signaling activities, e.g., in the case of GPCRs, selective activation of G-protein or β-arrestin function. Such ligands are known as "biased ligands," "biased agonists," or "functionally selective agonists." More specifically, ligand bias may be incomplete bias (non-absolute selectivity), characterized by ligand stimulation of multiple receptor activities with different relative potencies for different signals, or complete bias, characterized by ligand stimulation of one receptor protein activity without any stimulation of another known receptor protein activity.

[0086] Another type of ligand is known as an allosteric regulator. "Allosteric regulator," or "allosteric modulator," "allosteric ligand," or "effector molecule," as used herein, refers to a ligand that binds to an allosteric site of a GPCR (i.e., a regulatory site that is physically distinct from the active site of the protein). In contrast to orthosteric ligands, allosteric modulators bind to the receptor protein at a different site and are non-competitive, modifying its function even when the endogenous ligand is bound. Allosteric modulators that enhance the activity of a protein are referred to herein as "allosteric activators" or "positive allosteric modulators" (PAMs), while those that decrease the activity of a protein are referred to herein as "allosteric inhibitors" or "negative allosteric modulators" (NAMs).

[0087] As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0088] The term "antibody" is intended to mean an immunoglobulin or any fragment thereof capable of binding to an antigen. The term "antibody" also refers to single-chain antibodies and antibodies with only one binding domain.

[0089] As used herein, the term "complementarity-determining region" or "CDR" in the context of an antibody refers to the variable region of either the heavy (H) or light (L) chain (also abbreviated as VH and VL, respectively) and contains amino acid sequences capable of specifically binding to an antigen target. These CDR regions are responsible for the antibody's basic specificity for a particular antigenic determinant structure. Such regions are also called "hypervariable regions." Although CDRs represent noncontiguous stretches of amino acids within the variable region, the locations of these key amino acid sequences within the variable heavy and light chain regions are found to have similar positions within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all classical antibodies each have three CDR regions (termed LI, L2, L3, H1, H2, and H3) that are noncontiguous with each other for each light (L) and heavy (H) chain. Immunoglobulin single variable domains, and in particular nanobodies, generally comprise a single amino acid chain that can be considered to comprise four "framework sequences or regions" or FRs and three "complementarity-determining regions" or CDRs. Nanobodies have three CDR regions (termed CDR1, CDR2, CDR3), each of which is non-contiguous with the others. As referred to herein, amino acid positions / residues in VHHs, nanobodies or confobodies CDRs are indicated according to the Kabat numbering system, and frameworks and CDRs are defined according to the Abm definition (unless explicitly stated otherwise).

[0090] In general, for purposes of the disclosure herein and in the appended claims, a compound of the invention is considered a "modulator" of a target (and / or signal transduction, pathway(s), mechanism of action, and / or biological, physiological, and / or pharmacological function(s) involving the target) or "modulates" said target when the compound, when present in an appropriate assay or model (i.e., in an appropriate amount or concentration, e.g., a biologically active amount or concentration), alters the appropriate or intended readout of said assay or model (i.e., at least one appropriate value or parameter that can be determined using said assay or model) by at least 0.1%, e.g., at least 1%, at least 10%, and up to 50% or more, compared to the same value or parameter when measured using the same assay or model under essentially the same conditions but in the absence of said compound. Again, said modulation may result in an increase or decrease (i.e., by the percentage given in the preceding sentence) of said value or parameter. The compounds of the present invention are also preferably such compounds that can modulate said target, signal transduction, pathway(s), mechanism of action and / or said biological, physiological and / or pharmacological function in a dose-dependent manner, i.e., within or over at least one concentration range of the compound used in the assay or model.

[0091] There are numerous prior art references that discuss the sequences and structures of GPCRs. Apart from the prior art already cited herein, these references include Mirzadegan and Benko, Biochemistry. 2003 March 18; 42(10):2759-2767; Arakawa et al., Biochimica et Biophysica Acta 1808(2011)1170-1178; Han et al., FEBS Open Bio 5(2015)182-190; Sanchez-Reyes et al., Biophysical Journal 112, 2315-2326, 2017 June 6, 2315; and Kochman, Postepy Hig Med Dosw (Online), 2014 October 31; 68:1225-37. Mirzadegan and Benko present the results of a homology-based multiple sequence analysis performed on 270 family A GPCRs (153 of which were orphan GPCRs with unknown ligands). The results indicate that GPCRs from family A range in length from 290 to 951 amino acid residues, with the majority of receptors ranging from 310 to 470 residues in length. The results also show that GPCRs are characterized by a set of conserved residues distributed among seven helical domains that facilitate multiple alignments between GPCR sequences. These conserved residues (sometimes referred to in the art as "signature residues") are in helix I (Gly and Asn), helix II (Leu and Asp), helix III (Cys and AspArgTyr), helix IV (Trp and Pro), helix V (Pro and Tyr), helix VI (Phe, Trp, and Pro), and helix VII (Asn, Pro, and Tyr of the NPXXY motif). Sanchez-Reyes et al. in Table S1 also list the GPCR signature residues and their degree of conservation within Family A, and these signature residues are located at the following positions:

[0092] [Table 1]

[0093] Most of these signature residues are also shown in red in, for example, Figure 1 of Arakawa et al., which shows a schematic representation of their location within the overall structure of the beta-adrenergic receptor.

[0094] As an alternative to Table A, the positions of the various signature residues may be understood by their relative relationship to specific residues in a selected classical GPCR. Thus, when examining a position in a particular GPCR, one may refer to the relative position in the overall structure of the classical GPCR to determine whether the residue under consideration is present at that position in the particular GPCR. Taking human β2AR (UniProt P07550 (ADRB2_HUMAN), see SEQ ID NO: 17 and FIG. 22) as the base GPCR, the signature residues may be described as follows: Gly at position relative to amino acid 50 of ADRB2_HUMAN; Ans at position relative to amino acid 51 of ADRB2_HUMAN; Leu at position relative to amino acid 75 of ADRB2_HUMAN; Ala at position relative to amino acid 76 of ADRB2_HUMAN; Asp at position relative to amino acid 79 of ADRB2_HUMAN; Cys at position relative to amino acid 106 of ADRB2_HUMAN; Ser at position relative to amino acid 120 of ADRB2_HUMAN; Leu at position relative to amino acid 124 of ADRB2_HUMAN; Asp at position relative to amino acid 130 of ADRB2_HUMAN; Arg at position relative to amino acid 131 of ADRB2_HUMAN; Tyr at position relative to amino acid 132 of ADRB2_HUMAN; Trp at position relative to amino acid 158 of ADRB2_HUMAN; Pro at position relative to amino acid 211 of ADRB2_HUMAN; Tyr at position relative to amino acid 219 of ADRB2_HUMAN; Phe at position relative to amino acid 282 of ADRB2_HUMAN; Cys at position relative to amino acid 285 of ADRB2_HUMAN; Trp at position relative to amino acid 286 of ADRB2_HUMAN; Pro at position relative to amino acid 288 of ADRB2_HUMAN; Asn at position relative to amino acid 322 in ADRB2_HUMAN; Pro at position relative to amino acid 323 of ADRB2_HUMAN; and Tyr at position relative to amino acid 326 of ADRB2_HUMAN.

[0095] As described herein, the present invention aims to provide methodologies for providing assay and screening techniques for desired GPCRs that do not require the production or generation of conformation-specific VHHs against said particular GPCR(s) and thus avoid problems or limitations of prior art methodologies that may be associated with the need to provide the desired GPCR in an isolated, suitably purified form and in a desired conformation for screening and selection purposes, as well as when using native libraries for immunization and display purposes.

[0096] The present invention generally achieves this objective by providing chimeric proteins as described herein and using them in conjunction with binding domains or binding units specific for the intracellular loops (binding sites formed thereby) of the chimeric proteins (as further described herein). Such chimeric GPCRs, in various aspects and embodiments of the chimeric GPCRs described herein, form a first aspect of the invention, and the chimeric proteins of the invention (also referred to herein as "chimeric proteins of the invention" or "chimeric GPCRs of the invention," and these terms are used interchangeably herein), such binding domains or binding units capable of binding to chimeric GPCRs, and their uses are further described herein.

[0097] Generally, as further described herein, the chimeric proteins of the invention have at least one or more portions of amino acid sequence derived from a first GPCR and at least one or more other portions of sequence derived from a second GPCR (different from the first). In particular, the chimeric proteins of the invention have (at least) an extracellular loop derived from the first GPCR and an intracellular loop derived from the second GPCR (different from the first).

[0098] Preferably, the first GPCR and the second GPCR are both naturally occurring GPCRs. Also, when the present invention is used to discover or develop pharmaceutical agents, at least the first GPCR, and preferably in addition the second GPCR, is a GPCR that naturally occurs in the human body (i.e., on the surface of at least one cell present in the human body), particularly a GPCR that naturally occurs in the body (i.e., on the surface of at least one cell present in the body of a subject) to be treated with a compound, ligand, or other therapeutic entity discovered and / or developed using the chimeric proteins and methods described herein, e.g., for therapeutic or prophylactic purposes (as described further herein, one preferred use of the present invention is to generate compounds that can modulate the "first" GPCR from which the ECL is derived, as described herein).

[0099] Although typically and preferably, a chimeric protein of the invention will consist essentially of (and / or consist solely of) a stretch of amino acid residues derived from either the first GPCR or the second GPCR, it is not excluded from the scope of the invention in its broadest sense for the chimeric protein of the invention to also suitably comprise one or more stretches of amino acid residues derived from one or more other GPCRs, one or more stretches of amino acid residues (although usually less preferred) derived from other proteins, and / or one or more stretches of synthetic or semi-synthetic amino acid residues, obtained, for example, by introducing one or more mutations (as defined herein) into a stretch of amino acid residues obtained from the first GPCR or the second (or another) GPCR.

[0100] Chimeric proteins of the present invention generally preferably comprise an N-terminal sequence, a C-terminal sequence, seven transmembrane domains (TM), three extracellular loops (EC or ECL), and three intracellular loops (IC or ICL). More preferably, in chimeric proteins of the present invention, these portions of the total sequence are arranged in the most common configuration for naturally occurring GPCRs, i.e., (from N-terminus to C-terminus) as follows: [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence].

[0101] In particular, in the chimeric proteins of the present invention, at least one (e.g., at least two) of the extracellular loops are derived from a first GPCR and at least one (e.g., at least two) of the intracellular loops are derived from a second GPCR (different from the first).

[0102] Most preferably, in a chimeric protein of the invention, all three (or essentially all three) of the extracellular loops are derived from a first GPCR and all three (or essentially all three) of the intracellular loops are derived from a second GPCR (different from the first). This should be understood to mean that the extracellular loops preferably have no more than two, more preferably no more than one, and most preferably no amino acid difference (as defined herein) with the extracellular loops of the (first) GPCR from which they are derived, and that the intracellular loops preferably have no more than two, more preferably no more than one, and most preferably no amino acid difference (as defined herein) with the intracellular loops of the (second) GPCR from which they are derived.

[0103] The TMs present in the chimeric proteins of the present invention are preferably all derived from essentially the same GPCR. More preferably, the TMs present in the chimeric proteins of the present invention, together with their extracellular loops, form a functional ligand-binding site, particularly a ligand-binding site that is highly similar to and / or closely mimics the binding site for an extracellular ligand of the (first) GPCR from which the extracellular loops are derived. In the present invention, this typically and preferably means that the TM is essentially identical to and / or essentially derived from the first GPCR (except that, as further described herein and depending on how the ICL is provided / inserted into the chimeric GPCR, the TM may contain some amino acid residues at the TM7 position adjacent to the ICL that are the same as and / or derived from the second GPCR). In particular, the portion of the sequence of the chimeric GPCR of the present invention that is derived from the first GPCR may be such that it forms a functional binding site for an extracellular ligand that is essentially identical to and / or closely mimics the extracellular binding site of the first GPCR from which said portion of the chimeric sequence is derived.

[0104] Preferably, each TM in a chimeric GPCR has no more than four, more preferably no more than three, such as no more than two, particularly no more than one, and most preferably no amino acid differences from the amino acid sequence of the corresponding TM present in the naturally occurring GPCR from which it is derived (not taking into account any amino acid residues that are the same as and / or derived from an amino acid residue present in a second GPCR at a position adjacent to the ICL).

[0105] Furthermore, when taking into account amino acid residues that are the same as and / or derived from the second GPCR at a position next to the ICL, each such TM preferably has at least 80%, more preferably at least 85%, such as at least 90%, for example more than 95%, and up to and including 100% sequence identity with the amino acid sequence of the corresponding TM from the naturally-occurring GPCR from which it is derived (again, depending on how many of the optional amino acid residues in each TM are identical to and / or derived from the second GPCR). When any amino acid residues that are the same as and / or derived from the second GPCR at a position next to the ICL are not taken into account, each such TM preferably has at least 90%, more preferably at least 95%, such as at least 98%, and up to and including 100% sequence identity with the amino acid sequence of the corresponding TM from the naturally-occurring GPCR from which it is derived.

[0106] The N-terminal sequence of the chimeric protein of the invention is typically derived from the same GPCR as the first in TM (and typically and preferably is the first GPCR in the practice of the invention, as described herein). The C-terminal sequence is typically derived from the same GPCR as TM (and typically and preferably is the first GPCR in the practice of the invention, as described herein). However, the C-terminal portion can also be derived from a second GPCR, and use of the C-terminal portion of the second GPCR may result in improved expression levels and / or other properties (i.e., compared to the same chimeric GPCR but having the C-terminal sequence from the first GPCR).

[0107] In one aspect of the present invention, the amino acid sequence derived from the first GPCR and the amino acid sequence derived from the second GPCR are all derived from GPCRs belonging to the same class or family of GPCRs (in other words, in the present invention, the first GPCR and the second GPCR preferably belong to the same class or family of GPCRs). Thus, when the standard classification from the IUPHAR database (as of January 2019) is used, the first GPCR and the second GPCR preferably both belong to class A (rhodopsin-like), class B (secretin receptor family), class C (metabotropic glutamate), or class F (frizzled / smoothened). (Because the present invention is primarily directed to applications in vertebrates, particularly humans, GPCR sequences from class D and class E will generally not find utility in the present invention.) When using the "GRAFS" classification of GPCRs from vertebrates, the first GPCR and the second GPCR preferably both belong to the glutamate family, the rhodopsin family, the adhesion family, the Frizzled family, or the secretin family. However, surprisingly, as can be seen from the experimental data presented herein, it is also possible in the present invention to provide and / or use chimeric GPCRs of the present invention in which the EC and TM are essentially from GPCRs from one class or family and the ICL is essentially from another class or family.

[0108] Preferably, the ICL is derived from a GPCR belonging to class A (rhodopsin-like) (classification according to the IUPHAR database as of January 2019). The ECL, TM, C-terminal sequence, and N-terminal sequence are also preferably derived from a GPCR belonging to class A (rhodopsin-like). Thus, more generally, in the present invention, the first GPCR is preferably a GPCR belonging to class A, and the second GPCR is preferably a GPCR belonging to class A. See also the experimental section, where some particularly preferred combinations of ICLs and VHHs specific for said ICLs are used.

[0109] For example, and without limitation, in one aspect, the ICL can be derived from a beta-2-adrenergic receptor and the binding domain can be an ISVD that binds to the ICL of a beta-2-adrenergic receptor, such as one of the ISVDs described in International Application No. 2012 / 007593. International Application No. 2012 / 007593 also provides the sequences and CDRs of certain VHHs that are conformation-driven (as defined herein) for beta-adrenergic receptors, such as CA2780 (SEQ ID NO: 4 in WO 2012 / 007593 and SEQ ID NO: 20 herein), also referred to as "Nb80" and used in the experimental part below.

[0110] In another non-limiting embodiment, the ICL can be derived from an opioid receptor (particularly a mu-opioid receptor), and the binding domain can be an ISVD that binds to the ICL of an opioid receptor (particularly a mu-opioid receptor), such as, for example, one of the ISVDs described in International Application No. 2015 / 121092. International Application No. 2015 / 121092 also provides the sequences and CDRs of certain VHHs that are conformation-driven (as defined herein) for the mu-opioid receptor, such as X8633 (SEQ ID NO: 19 in WO 2014 / 118297, and SEQ ID NO: 21 herein), which is also used in the experimental part below.

[0111] The ICL used is preferably such that it further forms (and is incorporated into the chimeric GPCR of the invention so as to form) a functional binding site, in particular a functional binding site for a binding domain or binding unit used in the methods of the invention (as further described herein, the binding domain or binding unit is most preferably a conformation-induced binding domain or binding unit, in particular a conformation-induced ISVD, e.g. a conformobody). More particularly, the ICL used is one that, together with the remainder of the chimeric GPCR, forms a conformational epitope, i.e., (part of) an epitope or binding site that changes its "shape" (e.g., its geometric shape and / or spatial arrangement) depending on the conformational state of the chimeric GPCR, for example, when the chimeric GPCR undergoes a conformational change, such as from an inactive or less active state to an active, more active and / or functional state, and / or a conformational change that occurs when a first ligand binds to the extracellular binding site of the chimeric GPCR (e.g., a conformational change that is essentially similar to the conformational change that a naturally occurring GPCR may undergo when bound by an agonist).

[0112] Furthermore, the ICL used is preferably such that it further forms (and is incorporated into the chimeric GPCR of the invention so as to form) a functional (intracellular) binding site that mimics the corresponding binding site of the naturally occurring GPCR from which it is derived. In particular, the ICL used may be such that it mimics the G protein binding site of the naturally occurring GPCR from which it is derived. Thus, in a particular embodiment, the chimeric protein of the invention is such that its ICL forms (or forms part of) a functional binding site for a G protein or G protein complex, as further described herein. In many cases, the chimeric protein of the invention is such that its ICL forms (or forms part of) a functional binding site that is both a functional binding site for a G protein or G protein complex and a functional binding site for the specific VHH to which it is raised.

[0113] Thus, in general, a chimeric GPCR of the invention comprises at least two different ligand binding sites, i.e., comprises at least: a first ligand-binding site that most preferably closely mimics or essentially corresponds to the extracellular ligand-binding site of the GPCR from which the extracellular loops (and preferably also the TM) are derived. As is generally known for GPCRs, the ligand-binding site may be formed by one or more ECLs and / or one or more TMs, but does not include an ICL. The ligand-binding site of the chimeric GPCR of the present invention is also generally referred to herein as the "extracellular binding site." As further described herein, and as will be apparent to those skilled in the art, the extracellular binding site of the chimeric GPCR of the present invention typically (and preferably) corresponds to the orthosteric binding site of the first GPCR from which the ECL (and preferably also the TM) is derived (although, as noted herein, the term "extracellular binding site" in its broadest sense as used herein also includes appropriate allosteric binding sites); and a second ligand-binding site comprising at least one (and preferably at least two, e.g., all three) ICLs. As described herein, the ligand-binding site must be such that it can be bound by a protein-binding domain (particularly an ISVD) used in the present invention. Also, preferably, the ligand-binding site comprises and / or mimics a G protein-binding site. This ligand-binding site of the chimeric GPCR of the present invention is also generally referred to herein as the "intracellular binding site."

[0114] However, as is known per se for naturally occurring GPCRs (see, for example, Eglen and Reisine, cited herein), the chimeric GPCRs of the present invention may contain, in addition to a binding site corresponding to the orthosteric binding site of the first GPCR, one or more allosteric binding sites, depending on the ECL and TM present in the chimeric GPCR of the present invention. Preferably, when both the ECL and TM are derived from the first GPCR, the chimeric GPCR of the present invention also contains an allosteric binding site essentially identical to that of the first GPCR. Therefore, it is envisioned that the present invention can also be used to identify, generate, screen, test, and / or develop compounds and ligands directed against allosteric binding sites. As discussed by Eglen and Reisine (cited herein), such allosteric binders may be valuable alternatives to ligands and compounds directed against orthosteric binding sites, particularly for therapeutic applications.

[0115] Thus, the term "extracellular binding site" as used herein and in the claims preferably refers to an orthosteric binding site (i.e., the orthosteric binding site of the GPCR from which the ECL is derived), but it should be understood that the broadest sense of the term "extracellular binding site" also includes allosteric binding sites, particularly allosteric binding sites that project into the extracellular environment (as defined herein) in the GPCR from which the ECL is derived when said GPCR is in its natural cellular environment.

[0116] With respect to the terms "extracellular binding site" and "intracellular binding site," it should also be understood that use of these terms does not mean or imply, respectively, that the chimeric GPCR of the present invention is required to reside within a cellular environment. Instead, these binding sites are referred to by these terms because the extracellular binding site of a chimeric GPCR is generally provided to essentially correspond to and / or closely mimic the extracellular binding site(s) (i.e., at least the orthosteric site and, if present, optionally one or more allosteric sites) of the naturally occurring GPCR from which the ECL (and usually also the TM) is derived, and the intracellular binding site of a chimeric GPCR is generally provided to essentially correspond to and / or closely mimic the intracellular binding site of the naturally occurring GPCR from which the ICL is derived, respectively.

[0117] Thus, in one aspect, the invention relates to a chimeric GPCR, as further described herein, comprising an extracellular binding site, as further described herein, and an intracellular binding site, as further described herein.

[0118] The present invention further relates to a chimeric GPCR comprising an extracellular binding site derived (essentially) from a first GPCR and an intracellular binding site derived (essentially) from a second GPCR (different from the first). The present invention also relates to a composition comprising such a chimeric GPCR, which may be a composition further described herein. The present invention further relates to a composition comprising such a chimeric GPCR and further comprising a binding domain or binding unit capable of specifically binding to the intracellular binding site of said chimeric GPCR. Again, such a composition may be a composition further described herein, wherein the binding domain or binding unit present in said composition is preferably a conformation-induced binding domain or binding unit (as defined herein), more preferably a conformation-induced ISVD (e.g., a conformobody).

[0119] The present invention further relates to a chimeric GPCR comprising an ECL and a TM derived from a first GPCR, wherein the chimeric GPCR comprises a (functional) extracellular binding site that (essentially) corresponds to (and / or closely mimics) the extracellular binding site of the first GPCR, and an ICL derived from a second GPCR (different from the first), wherein the ICL forms (part of) a functional intracellular binding site. The present invention further relates to a composition comprising such a chimeric GPCR and further comprising a binding domain or binding unit capable of specifically binding to the intracellular binding site of the chimeric GPCR. Again, such a composition may be a composition further described herein, wherein the binding domain or binding unit present in the composition is preferably a conformation-induced binding domain or binding unit (as defined herein), more preferably a conformation-induced ISVD (e.g., a conformobody).

[0120] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a chimeric protein comprising an N-terminal sequence, a C-terminal sequence, seven transmembrane domains (TM), three extracellular loops and three intracellular loops; the extracellular loops present in the chimeric protein are (essentially) derived from the first GPCR, the intracellular loop present in the chimeric protein is (essentially) derived from the second GPCR; Chimeric proteins: b) a binding domain or binding unit capable of specifically binding to an intracellular loop (thereby forming a binding site) present in said chimeric protein, preferably a conformation-induced binding domain or binding unit (as defined herein), more preferably a conformation-induced ISVD (e.g., a conformobody); The present invention relates to a composition comprising at least

[0121] In such compositions, the chimeric proteins and binding domains or binding units are preferably as further described herein.

[0122] In one aspect, the binding domain or binding unit may be fused to a chimeric protein essentially as described in International Application WO 2014 / 118297, which describes the fusion of GPCRs and conformobodies and their uses. Thus, in a further aspect, the invention relates to a fusion protein comprising a chimeric GPCR of the invention fused, directly or via a suitable linker or spacer, preferably at its C-terminus, to a binding domain or binding unit as further described herein (the binding domain or binding unit is preferably a conformation-induced binding domain or binding unit, preferably a conformation-induced ISVD).

[0123] In another aspect (also described herein), the invention relates to a fusion protein comprising a chimeric GPCR of the invention fused, either directly or via a suitable linker or spacer, to a binding domain or binding unit that is a first binding member of a binding pair comprising at least a first and a second binding member, wherein the binding pair is capable of producing a detectable signal when said first and second binding members contact or are in proximity to each other.

[0124] In a more general aspect, the invention relates to a fusion protein comprising a chimeric GPCR of the invention and at least one further amino acid sequence, protein or peptide (eg, at least one binding domain or binding unit).

[0125] As further described herein, compositions comprising the chimeric proteins of the invention (and preferably, in addition, the binding domains or binding units described herein) can be cells, cell lines, or suitable fractions or preparations derived from cells or cell lines, such as membrane fractions, cellular fractions containing one or more organelles, or suitable cell lysates (such cells and fractions derived from such cells are also referred to herein as "cellular compositions"). Such compositions can also be liposomes, vesicles, or other suitable liposomal compositions, such as, but not limited to, virus-like lipoparticles, lipid layers (bilayers and monolayers), lipid vesicles, high-density lipoparticles (e.g., nanodiscs), which may contain natural or synthetic lipids or combinations thereof. Typically, the composition is such that the chimeric GPCR is present in the composition in a manner that allows it to adopt the barrel-shaped tertiary structure characteristic of GPCRs. In many cases, this means that the GPCR is suitably associated with (e.g., suitably immobilized in or on) one or more other components of the composition, such as a cell wall, a cell membrane, a fragment of a cell wall or membrane, a wall of a liposome or vesicle, or a lipid bilayer, in a manner that allows the GPCR to adopt the barrel-shaped tertiary structure characteristic of GPCRs. Also, in many cases, the composition is one in which the chimeric GPCR is present in the composition in a manner such that the extracellular binding site of the GPCR is separated from the intracellular binding site by (at least a portion or fragment of) a cell wall, cell membrane, or other layer (such as a lipid bilayer), at least on the size scale of the GPCR.

[0126] As described herein, the chimeric protein of the invention present in the composition preferably has the following overall structure (from N-terminus to C-terminus): [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence].

[0127] Preferably, the chimeric proteins of the present invention are such that their TMs are capable of adopting the barrel-like structure characteristic of naturally occurring GPCRs (7 TMs therein), at least under the conditions applied when the chimeric GPCRs of the present invention are used for assay or screening purposes. Again, reference is made to the prior art cited herein.

[0128] Preferably, in the chimeric protein present in said composition: the extracellular loops present in the chimeric protein form a functional ligand-binding site (optionally together with one or more TMs); The intracellular loops present in the chimeric protein form a functional ligand-binding site, particularly a conformation-dependent functional ligand-binding site (as described herein), to which a binding domain or binding unit can bind.

[0129] Also preferably, in a chimeric protein present in the composition, all of the TMs present in the chimeric protein are derived from the same GPCR (or are essentially derived from the same GPCR, as further described herein). More preferably, the TMs present in the chimeric protein are derived from the same (first) GPCR (or are essentially derived from the same (first) GPCR, as further described herein) from which the extracellular loops are derived.

[0130] Also preferably, an extracellular loop present in the chimeric protein differs from the extracellular loop of the first GPCR from which said extracellular loop is derived by no more than two amino acids, preferably no more than one amino acid, and more preferably zero amino acid differences.

[0131] As described herein, when the present invention is used to identify, select, generate, test or develop ligands or compounds for therapeutic and / or prophylactic use in humans, the portion of the sequence of the chimeric protein of the present invention that is derived from a first GPCR and the portion of the sequence of the chimeric protein of the present invention that is derived from a second GPCR are preferably both derived from GPCRs present in the human body.

[0132] Also preferably, an extracellular loop present in the chimeric protein has no more than two, preferably no more than one, and more preferably no amino acid difference (as defined herein) with the extracellular loop of the first GPCR from which said extracellular loop is derived. Furthermore, preferably, an intracellular loop present in the chimeric protein has no more than two, preferably no more than one, and more preferably no amino acid difference with the intracellular loop of the second GPCR from which said intracellular loop is derived.

[0133] Furthermore, preferably, each of the TMs present in the chimeric protein has at least 80%, more preferably at least 85%, such as at least 90%, for example, more than 95%, up to and including 100% sequence identity (taking into account any amino acid residues derived from the second GPCR that are identical to amino acid residues present in a position adjacent to the ICL, as further described herein) with the amino acid sequence of the corresponding TM from the naturally occurring GPCR from which the TM is derived. Also, preferably, each of the TMs present in the chimeric protein has no more than 7, preferably no more than 5, for example 5, 4, 3, 2, 1, or zero amino acid differences (as defined herein) with the amino acid sequence of the corresponding TM from the naturally occurring GPCR from which the TM is derived (not taking into account any amino acid residues derived from the second GPCR that are identical to amino acid residues present in a position adjacent to the ICL, as further described herein).

[0134] Also preferably, when a chimeric protein has amino acid residues that are the same as and / or derived from a second GPCR located adjacent to an ICL (also referred to herein as "ICL-flanking residues"), the chimeric protein comprises 10 or fewer, preferably 7 or fewer, for example 5 or fewer, for example 5, 4, 3, 2, or 1 such ICL-flanking residues adjacent to each ICL (i.e., immediately adjacent to the first amino acid residue of the associated ICL or the last amino acid residue of the associated ICL, respectively). Also preferably, any such ICL-flanking residues (if present) are the same as or essentially the same as the amino acid residues adjacent to the associated ICL in the second GPCR from which the ICL is derived. Also preferably, any such ICL-flanking residues derived from a second GPCR, when present in a chimeric GPCR, are contiguous with the amino acid sequence of the associated ICL from the second GPCR. Overall, this means, generally, that the ICL and any ICL-flanking residues derived from the second GPCR are arranged N-terminally to C-terminally (i.e., Bold / Underline is meant to have the following structure: [7TM]- [ICL-adjacent residue, if present]-[ICL]-[ICL-adjacent residue, if present] -[7TM]. where the amino acid sequence from the relevant 7TM adjacent (in order) to the ICL-flanking residues is taken from a first GPCR. See, e.g., non-limiting Figures 16A-16C and also Figure 17, which show examples of chimeric GPCRs of the invention.

[0135] Preferably, taken together, each ICL and any ICL-flanking residues have essentially no amino acid differences (as defined herein) with the corresponding portion of the amino acid sequence in the second GPCR from which the associated ICL and ICL-flanking residues are derived. However, in the chimeric proteins of the present invention, each stretch of amino acid residues formed by an ICL and any ICL-flanking residues from a second GPCR may have several amino acid differences (including substitutions, mutations, or deletions) with the corresponding stretch of amino acid residues in the second GPCR from which said portion of sequence is derived, but preferably there are no more than five amino acid differences, e.g., five, four, three, two, or one, for each such stretch of ICL-flanking residues and ICL. Also, as described herein, each ICL by itself preferably has no more than two, more preferably no more than one, and most preferably no amino acid differences (as defined herein) with the intracellular loop of the (second) GPCR from which it is derived.

[0136] It should also be noted that for each ICL, whether any ICL-flanking residues are present, on which side of the ICL such ICL-flanking residues are present (i.e., at the N-terminus, C-terminus, or both), how many ICL-flanking residues are present (if any), and whether the stretch of amino acid residues formed by each ICL and any ICL-flanking residues contains any amino acid differences from the corresponding stretch of amino acid residues in the second GPCR (and if so, how many amino acid differences there are, which amino acids are different, and where in the sequence they are located) can each be selected independently for each ICL.

[0137] Also, although it is usually preferred that any ICL-flanking residues replace the amino acid residue at the corresponding position in the TM to which the relevant ICL is linked, it is also possible that the stretch of amino acid residues formed by each ICL and any ICL-flanking groups adjacent to said ICL are suitably inserted into the sequence of the first GPCR, such that an ICL from the second GPCR replaces the corresponding ICL from the first GPCR, and any ICL-flanking residues from the second GPCR are inserted into the sequence in the TM adjacent to the relevant ICL in the first GPCR, or replace some (but not all) of those amino acid residues. Also, although any amino acid differences present in the portion(s) of the sequence formed by the ICL and its ICL-flanking sequences (if present) in the final sequence of the chimeric GPCR may be derived from the amino acid sequence of the first GPCR (e.g., so that one or more of the ICL-flanking amino acid residues from the second GPCR are replaced by an amino acid residue present at the corresponding position in the amino acid sequence of the first GPCR), it is also possible for the final sequence of the chimeric GPCR to include one or more other amino acid differences at these positions (or an appropriate combination of one or more amino acid differences derived from the first GPCR and one or more other amino acid differences).

[0138] It will also be apparent to those skilled in the art that some of the stretches of amino acid residues in GPCRs adjacent to each ICL contain amino acid residues that are highly conserved at specific positions (see Table A above, which lists some of the so-called "signature residues" within Family A GPCRs).

[0139] It will be apparent to those skilled in the art that such highly conserved amino acid residues at positions close to the ICL are also preferably conserved in the chimeric GPCR of the present invention, particularly when said conserved amino acid residues are present in both the first and second GPCRs. Thus, in one aspect, the chimeric GPCR preferably comprises one or more, for example at least 5, preferably at least 10, and more preferably at least 15, for example 15, 16, 17, 18, 19, 20, or all 21, of the signature residues listed in Table A above, in an appropriate combination.

[0140] Preferably, the chimeric GPCRs of the invention comprise at least the following amino acid residues at the positions indicated: G at position 1.49 and N at position 1.50; L at position 2.46 and A at position 2.47; D at 3.49 and R at 3.50; ·W in 4.50th place; · P at position 5.50 and Y at position 5.58; F at position 6.44, C at position 6.47, and P at position 6.50, and preferably further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9), more preferably at least five (e.g., 5, 6, 7, 8, or 9), additional signature amino acid residues listed in Table A at relevant positions in the sequence.

[0141] Again stated in terms of positions relative to the human β2AR (UniProt P07550 (ADRB2_HUMAN)), preferably the chimeric GPCRs of the invention comprise at least the following amino acid residues at the positions shown: G at position 50 and N at position 51 for ADRB2_HUMAN; L at position 75 and A at position 76 for ADRB2_HUMAN; D at position 130 and R at position 131 for ADRB2_HUMAN; · W at position 158 for ADRB2_HUMAN; P at position 211 and Y at position 219 for ADRB2_HUMAN; F at position 282, C at position 285, and P at position 288 for ADRB2_HUMAN; and preferably further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9), more preferably at least five (e.g., 5, 6, 7, 8, or 9, etc.) additional signature amino acid residues listed in Table A at relatively relevant positions in the sequence.

[0142] To provide the sequence of a chimeric GPCR of the invention, the ICL (and optionally some further ICL-flanking residues) in the amino acid sequence of a first GPCR should be replaced by an ICL (and optionally some further ICL-flanking residues) from a second GPCR. This replacement can be carried out using recombinant DNA techniques known per se. Also, based on the information provided herein, the amino acid sequence of a chimeric GPCR can be designed (e.g., starting from the amino acid sequences of the first and second GPCRs or an alignment of these sequences), and the corresponding chimeric GPCR can then be produced by synthesizing a nucleotide sequence encoding said chimeric GPCR and expressing said nucleotide sequence in a suitable host organism, again using recombinant DNA techniques known per se.

[0143] Regardless of the manner in which the chimeric GPCRs of the invention are provided (i.e. the particular manner in which the ICL and optionally any ICL flanking residues from a first GPCR are replaced with an ICL and optionally any ICL flanking residues from a second GPCR), preferably the chimeric GPCRs of the invention comprise: a) the amino acid residues forming ICL1 in the first GPCR, and optionally one or more further amino acid residues present at positions in the amino acid sequence of the first GPCR between (and including) positions 1.49 and 1.50 (relative to positions 50 and 51 in ADRB2_HUMAN), which are often referred to as GN; and position 2.50 (relative to position 79 in ADRB2_HUMAN), which is often referred to as D, are replaced (as appropriate) by amino acid residues forming ICL1 in the second GPCR, and optionally one or more further amino acid residues present at positions in the amino acid sequence of the second GPCR between (and including) positions 1.49 and 1.50 and 2.50; or b) the amino acid residues forming an ICL2 in the first GPCR, and optionally one or more additional amino acid residues present at positions in the amino acid sequence of the first GPCR between (and including) positions 3.49 and 3.50 (relative to positions 130 and 131 of ADRB2_HUMAN), which are often referred to as DR; and position 4.50 (relative to position 158 of ADRB2_HUMAN), which is often referred to as W, are replaced (as appropriate) by amino acid residues forming an ICL2 in the second GPCR, and optionally one or more additional amino acid residues present at positions in the amino acid sequence of the second GPCR between (and including) positions 3.49 and 3.50 and 4.50; or c) the amino acid residues forming ICL3 in the first GPCR and, optionally, one or more additional amino acid residues at positions in the amino acid sequence of the first GPCR between (and including) position 5.50 (relative to position 211 of ADRB2_HUMAN), which is often referred to as P; and position 6.50 (relative to position 288 of ADRB2_HUMAN), which is often referred to as P, are (appropriately) replaced in the second GPCR by amino acid residues forming ICL3 and, optionally, one or more additional amino acid residues at positions in the amino acid sequence of the second GPCR between (and including) positions 5.50 and 6.50. The chimeric GPCR is preferably a chimeric GPCR to which at least a) and b), at least a) and c), or at least b) and c) apply, and most preferably a chimeric GPCR to which all of a), b), and c) apply.

[0144] Based on the disclosure herein and the alignment and comparison between the amino acid sequence of a first GPCR and the amino acid sequence of a second GPCR, one skilled in the art can, optionally after a more limited degree of trial and error, select one or more ICL-flanking residues in the first GPCR (in addition to the associated ICL) that can be appropriately "replaced" by one or more ICL-flanking residues from the second GPCR. For example, but not limited to, from an alignment between the amino acid sequences of the first GPCR and the second GPCR, one skilled in the art can derive amino acid residues and / or positions that appear to be the same and / or conserved between the amino acid sequences of the first GPCR and the second GPCR, and can use such residues / positions to guide the replacement / insertion of the ICL and any ICL-flanking residues. For example, but not limited to, by searching the GPCR database ( https: / / gpcrdb.org / Based on a comparison of approximately 60 GPCR sequences from ADRB2_HUMAN, the LA motifs at positions 1.52, 1.53 (relative to positions 53 and 54 in ADRB2_HUMAN), 2.46, and 2.47 (relative to positions 75 and 76 in ADRB2_HUMAN), and positions 6.44 and 6.47 (relative to positions 282 and 285 in ADRB2_HUMAN, where positions 6.44 and 6.47, together with the P at position 6.50, may form an FxxCxxP motif) may be conserved between a given first GPCR and a given second GPCR, and such conserved residues may guide the replacement / insertion of the ICL and any ICL-adjacent residues.

[0145] It should also be noted that the stretch of amino acid residues from the second GPCR and the (corresponding) stretch of amino acid residues from the first GPCR that is "replaced" by said stretch of amino acid residues from the second GPCR need not be the same length, particularly with respect to ICL3, which is known to vary in length between different GPCRs and even between GPCRs from the same family.

[0146] It is generally contemplated that the chimeric proteins of the present invention, in combination with an ISVD specific for an ICL present in the chimeric protein (an ISVD as further described herein), may find a variety of uses, particularly in applications where a combination of a GPCR and a conformation-induced binding domain or binding unit (particularly a GPCR and a conformation-induced ISVD) is used, including, but not limited to, the various uses and applications described herein and in WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 175643, WO 2014 / 118297, WO 2014 / 12218283, and WO 2014 / 118297. As further described herein, such applications and uses also include use in the methods and arrangements described in the assignee's co-pending U.S. provisional application filed on April 29, 2019, entitled "Screening methods and assays for use with transmembrane proteins, in particular with GPCRs," and the assignee's co-pending PCT application of the same title, which has the same international filing date as the present application and which incorporates by reference the same priority application as the present application.

[0147] Further applications and uses will be apparent to those skilled in the art based on the disclosure herein.

[0148] In particular, it is envisaged that the chimeric proteins of the invention, in combination with conformation-induced binding domains or binding units that are specific for the ICL present in said chimeric proteins, and cells, cell lines, cellular compositions, vesicles, liposomes, and other compositions that comprise and, where appropriate, express such chimeric proteins (and preferably such binding domains or binding units), will find use and application in a variety of assay techniques and screening methods, particularly for identifying, screening, producing, testing, and developing compounds and ligands that are specific for, directed against, and / or can be used to modulate the GPCR from which the ECL (and, as described herein, generally essentially all TMs) is derived. Accordingly, the chimeric proteins of the invention can be used in such assays and screening methodologies in combination with a conformation-induced binding domain or binding unit specific for an ICL present in the chimeric protein, as a substitute for (i.e., replacing) the (non-chimeric) GPCR (and the ISVD specific for the ICL of such non-chimeric GPCR) used in such methods (e.g., the methods described in WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 175643, WO 2014 / 118297, WO 2014 / 122183 and WO 2014 / 118297). This means that compared to using a naturally occurring GPCR and a conformation-guiding ISVD directed against the ICL of said naturally occurring GPCR, the present invention provides the skilled artisan with an alternative route to providing assay and screening methods for desired naturally occurring GPCRs, which route does not require the generation of a conformation-guiding ISVD against (the ICL of) said naturally occurring GPCR, and therefore avoids any practical problems or technical limitations (as mentioned herein) that may be associated with the need to do so.It is also contemplated that in some cases, replacing the ICL of a desired naturally occurring GPCR with the ICL of another GPCR may result in a chimeric GPCR that is more practical to work with (e.g., in terms of expression, folding, purification, and / or stability) than the original non-chimeric GPCR, particularly under the conditions used in assay and screening techniques.

[0149] Overall, therefore, it is envisaged that the present invention not only provides those skilled in the art with alternative routes towards establishing assay and screening methods involving GPCRs (which may in some respects be more practical or easier to establish or implement than corresponding methods involving the use of corresponding naturally occurring GPCRs), but may also enable the establishment of assay and screening methods for GPCRs that are currently essentially impossible or difficult to achieve using naturally occurring or non-chimeric GPCRs due to potential issues of technical feasibility.

[0150] A binding domain or binding unit for use in the present invention should be capable of binding to (and should be selected to be able to bind to) at least one, preferably at least two, e.g., essentially all three, of the ICLs in a chimeric GPCR of the present invention. In particular, a binding domain or binding unit for use in the present invention should bind, preferably specifically, to an intracellular binding site (as defined herein) of a chimeric GPCR, which may comprise one, two, or essentially all such ICLs.

[0151] Preferably, the binding domain or binding unit is one that, upon binding to the chimeric GPCR (i.e., to the intracellular binding site of the chimeric GPCR), is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR. Such a binding domain or binding unit is also referred to herein as a "conformation-inducing" binding domain or binding unit or a "conformation-stabilizing" binding domain or binding unit (these terms are used interchangeably herein). In particular, such a conformation-inducing binding domain or binding unit may be a binding domain or binding unit that, upon binding to the chimeric GPCR (i.e., to the intracellular binding site of the chimeric GPCR), is capable of stabilizing and / or inducing a druggable conformational state (as defined herein) of the chimeric GPCR.

[0152] Generally, this means that the conformation-induced binding domain or binding unit is specific for at least one functional conformational state of the chimeric GPCR (i.e., compared to at least one other non-functional conformational state of the chimeric GPCR) and / or is specific for at least one active conformational state or states of the chimeric GPCR (i.e., compared to at least one inactive or less active conformational state of the chimeric GPCR). Preferably, the conformation-induced binding domain or binding unit is specific for at least one druggable conformational state of the chimeric GPCR (i.e., compared to at least one other conformational state of the chimeric GPCR that is not or less druggable).

[0153] In particular, the conformation-induced binding domain or binding unit may be such that it preferentially binds to a chimeric GPCR of the present invention (i.e., to an intracellular binding site as defined herein) when the chimeric GPCR of the present invention is bound by an agonist, i.e., it preferentially binds to the conformation(s) that the chimeric GPCR of the present invention adopts when bound by an agonist (i.e., compared to binding to at least one conformation that the chimeric GPCR of the present invention adopts when not bound by an agonist and / or when bound by an inverse agonist and / or antagonist).

[0154] Furthermore, the conformation-induced binding domain or binding unit is preferably a conformation-induced binding domain or binding unit that enhances the affinity of the chimeric GPCR for an agonist, more preferably by at least 2-fold, even more preferably by at least 5-fold, for example by at least 10-fold.

[0155] Furthermore, without being limited to any particular hypothesis or mechanism, the conformation-induced binding domain or binding unit is preferably a conformation-induced binding domain or binding unit that, upon binding to the chimeric GPCR (i.e., to the intracellular binding site as defined herein), is capable of stabilizing and / or inducing the formation of a complex comprising the binding domain or binding unit, the chimeric GPCR, and a compound or ligand that binds to the extracellular binding site (as defined herein) of the chimeric GPCR. In particular, the binding domain or binding unit may be a binding domain or binding unit that, upon binding to the chimeric GPCR (i.e., to the intracellular binding site as defined herein), is capable of stabilizing and / or inducing the formation of a complex comprising the binding domain or binding unit, the chimeric GPCR, and an agonist that binds to the extracellular binding site (as defined herein) of the chimeric GPCR.

[0156] Preferably, the conformation-induced binding domain or binding unit is derived from an immunoglobulin. More preferably, the conformation-induced binding domain or binding unit is an amino acid sequence having an immunoglobulin fold and comprising four framework regions and three complementarity-determining regions. More preferably, the conformation-induced binding domain or binding unit is an immunoglobulin single variable domain, e.g., an ISVD derived from a camelid antibody, such as a VHH or nanobody. The conformation-induced binding domain or binding unit may also be a suitable fragment derived from such an immunoglobulin. ISVDs that are capable of inducing or stabilizing a functional, active, and / or druggable conformational state of a GPCR (and / or are specific for a functional, active, and / or druggable conformational state of a GPCR) are known, for example, from WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 175643, WO 2014 / 118297, WO 2014 / 122183, and WO 2014 / 118297, and such ISVDs may be used in combination with other ISVDs. VDs (also called conformational bodies) may be used in the present invention as conformation-induced binding domains or binding units in combination with chimeric GPCRs comprising an ICL from the GPCR to which the relevant conformational body was generated (see again WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 175643, WO 2014 / 118297, WO 2014 / 122183 and WO 2014 / 118297).

[0157] The binding domain or binding unit can be used as such (i.e., as a separate binding protein, e.g., as a monovalent VHH), or can be part of a larger protein comprising one or more additional amino acid sequences, binding domains, or binding units. For example, but not by way of limitation, as further described herein, when the chimeric proteins and binding domains or binding units of the invention are used in the methods and arrangements described in the co-pending U.S. provisional application referenced below, entitled "Screening methods and assays for use with transmembrane proteins, in particular with GPCRs," filed April 29, 2019, the binding domain or binding unit can form part of a "second fusion protein" used in said methods and arrangements. As further described herein, in such a second fusion protein, the binding domain or binding unit may be linked directly or via a suitable spacer or linker to a binding member that is part of a binding pair capable of generating a detectable signal. A similar arrangement is shown in Figure 3 of the reference Jacobs et al., Int. J. Mol. Sci., 2019, 20, 2597.

[0158] Also, as mentioned herein, in one aspect, the binding domain or binding unit may be fused to the chimeric protein of the invention essentially as described in International Application WO 2014 / 118297.

[0159] The present invention also relates to the use of such binding domains or binding units to induce conformational changes (as further described herein) in the chimeric GPCRs of the invention, and in particular to induce functional, active and / or druggable conformational states in and / or stabilize functional, active and / or druggable conformational states of the chimeric GPCRs of the invention.

[0160] The present invention relates to the use of such binding domains or binding units to induce the formation of a complex comprising said binding domains or binding units and the chimeric GPCR of the present invention and / or to stabilize a complex. Such a complex may further comprise a ligand or compound that binds to the extracellular binding site (as defined herein) of the chimeric GPCR (this ligand or compound may be as further described herein, and may in particular be an agonist). In particular, the present invention also relates to the use of such binding domains or binding units to induce the formation of and / or stabilize a complex in which the chimeric GPCR of the present invention is in a functional, active, druggable conformational state. In a particular aspect, the present invention also relates to the use of such binding domains or binding units to induce the formation of and / or stabilize a complex in which the chimeric GPCR of the present invention is in a ligand-bound (and preferably agonist-bound) conformational state in the chimeric GPCR of the present invention.

[0161] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a chimeric protein comprising an N-terminal sequence, a C-terminal sequence, seven transmembrane domains (TM1 to TM7), three extracellular loops (EC1 to EC3), and three intracellular loops (IC1 to IC3), the extracellular loops present in the chimeric protein are (essentially) derived from the first GPCR, a chimeric protein, wherein the intracellular loop present in the chimeric protein is (essentially) derived from a second GPCR; b) a binding domain or binding unit capable of specifically binding to an intracellular loop present in said chimeric protein (thereby forming a binding site), and optionally c) a ligand or compound that binds to the extracellular binding site (as defined herein) of the chimeric GPCR; The present invention relates to a complex comprising:

[0162] In a further aspect, the present invention relates to such a complex comprising all three of the chimeric GPCR referred to in a), the binding domain or binding unit referred to in b), and the ligand or compound referred to in c).

[0163] In any such complex, the binding domain or binding unit may also be fused to a chimeric protein essentially as described in International Application WO 2014 / 118297, and the present invention also relates to a complex comprising such a fusion protein and (optionally) a ligand or compound that binds to the extracellular binding site (as defined herein) of the chimeric GPCR.

[0164] Preferably, the chimeric GPCR in the complex is in a functional conformational state and / or an active conformational change. In particular, the chimeric GPCR in the complex may be in a druggable conformational change. The functional, active and / or druggable conformational state may also be induced by binding of a binding domain or binding unit referred to in b) to the chimeric GPCR, binding of a ligand or compound referred to in c) to the chimeric GPCR, and / or binding of both the binding domain or binding unit and the compound or ligand to the chimeric GPCR. In one aspect, the functional, active and / or druggable conformational state is a conformational state induced by binding of an agonist to the chimeric GPCR (the agonist is a ligand or compound referred to in c)), optionally accompanied by binding of the binding domain or binding unit referred to in b) to the chimeric GPCR.

[0165] The binding domain or binding unit present in the complex is again preferably a conformation-inducing binding domain or binding unit (as defined herein), i.e. a binding domain or binding unit that is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to the chimeric GPCR (i.e. to the intracellular binding site of the chimeric GPCR). More preferably, the binding domain or binding unit is capable of inducing the formation of a complex formed by the chimeric protein referred to in a), the binding domain or binding unit referred to in b), and the ligand or compound referred to in c), and / or is capable of stabilizing such a complex.

[0166] The ligand or compound present in the complex is preferably a full agonist, partial agonist, inverse agonist or antagonist, more preferably a full agonist or partial agonist. In particular, the ligand or compound may be a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or any suitable fragment thereof.

[0167] In a further aspect, the complex formed by the chimeric GPCR referred to in a), the binding domain or binding unit referred to in b), and (optionally) the ligand or compound referred to in c) is bound and / or immobilized to a suitable solid support. Such a complex can be formed, for example, by first forming a complex of the present invention comprising only the chimeric GPCR referred to in a) and the binding domain or binding unit referred to in b), binding the complex to a solid support, and then contacting the complex with the ligand or compound referred to in c), which may be present in a suitable (preferably liquid, usually aqueous) medium. In a particular aspect, such a complex is formed by performing the following steps (in the order shown): providing a binding domain or binding unit as referred to in point b) above immobilized on a suitable solid support, contacting the immobilized binding domain or binding unit with a chimeric GPCR of the invention (i.e., under conditions such that the immobilized binding domain or binding unit captures the chimeric GPCR, preferably under conditions such that the immobilized binding domain or binding unit captures the chimeric GPCR in a functional and / or active conformation, more preferably in a druggable conformation); and contacting the complex formed by said immobilized binding domain or binding unit and said captured chimeric GPCR with a compound or ligand referred to in c) above.

[0168] The binding domain or binding unit is again preferably a conformation-induced binding domain or binding unit (as defined herein), and again preferably an ISVD (and preferably a conformation-induced ISVD).

[0169] Suitable solid supports and immobilization techniques will be apparent to those skilled in the art and include, for example, beads, columns, slides, chips, or plates, see, for example, WO 2012 / 007593, pages 55-57.

[0170] In another aspect, the present invention relates to a solid support on which is immobilized a complex comprising a chimeric GPCR as referred to in a), a binding domain or binding unit as referred to in b), and optionally a ligand or compound as referred to in c).

[0171] The present invention also relates to the use of a solid support on which a complex comprising a chimeric GPCR as referred to in a) and a binding domain or binding unit as referred to in b) is immobilized. In particular, the present invention relates to identifying, producing, and / or screening (e.g., using standard screening techniques known per se) for ligands or compounds capable of binding (in particular capable of specifically binding as defined herein) to the chimeric GPCR present in the complex; and / or determining (e.g., using standard assay techniques known per se) at least one property of the compound or ligand, such as its ability to bind (and in particular specifically bind as defined herein) to the chimeric GPCR present in said complex and / or its ability to modulate said chimeric GPCR; The present invention relates to the use of such solid supports in

[0172] The present invention also provides a method for determining at least one property of a compound or ligand, comprising at least: providing a complex of a chimeric GPCR of the invention and a binding domain or binding unit described herein; contacting said complex with said compound or ligand; The present invention relates to a method, including:

[0173] The method preferably also includes measuring (a change in) at least one signal or parameter indicative of said at least one property. As described herein, said property may be the ability to bind (particularly the ability to specifically bind) to the chimeric GPCR of the present invention and / or said complex. The at least one property may also be the ability to modulate said chimeric GPCR, for example the ability to act as an agonist of the chimeric GPCR (e.g., as a partial or full agonist), the ability to act as an antagonist of the chimeric GPCR, and / or the ability to act as an inverse agonist of the chimeric GPCR. Again, preferably, said at least one property determined using the chimeric GPCR of the present invention represents essentially the same or essentially similar properties as the naturally occurring GPCR from which the ECL (and preferably, as further described herein, essentially the TM) is derived.

[0174] Again, in the method, the complex may be immobilized on a solid support. Again, the binding domain or binding unit is preferably a conformation-induced binding domain or binding unit (as defined herein), more preferably a conformation-induced ISVD (e.g., a conformobody). Also preferably, in the complex, the chimeric GPCR of the invention is in a functional, active, and / or druggable state.

[0175] It will also be apparent to those skilled in the art, based on the disclosure herein, that when the chimeric GPCRs and binding domains and binding units of the present invention are provided as fusion proteins (e.g., as described in International Application WO 2014 / 118297), the complexes described herein may also be formed by the chimeric GPCR present in the fusion protein, the binding domain or binding unit present in the fusion protein, and, optionally, the compound or ligand referred to in c).

[0176] In another aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to an extracellular binding site (as defined herein) of a GPCR, comprising the steps of: a) providing a chimeric GPCR comprising essentially (at least) the extracellular binding site of said GPCR and comprising the intracellular loops of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to a binding site on the chimeric GPCR that comprises at least one of the intracellular loops; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to the extracellular binding site of the chimeric GPCR; e) assessing whether each of said test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in the presence of said binding domain or binding unit; and optionally f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of the binding domain or binding unit; The present invention relates to a method, including:

[0177] In the above method, the chimeric GPCR and binding domain or binding unit are preferably, again, chimeric GPCRs and binding domains or binding units as further described herein (any preferred or preferred aspects described herein for the chimeric GPCRs of the present invention and / or such binding domains / binding units are also preferred for use in the above method). Again, the chimeric GPCRs provided and used in the above steps are preferably such chimeric GPCRs, and the conditions under which the chimeric GPCR and binding domain or binding unit are used in the above steps are preferably selected so that binding of the test compound to the chimeric GPCR under the conditions used is representative of binding of the test compound(s) to the GPCR. In such a method, the chimeric GPCR and binding domain or binding unit may be present in a suitable cellular composition and / or expressed by a suitable cell or cell line, or may be present in a suitable liposome or vesicle, all as further described herein. The chimeric GPCR or binding domain or binding unit may also be immobilized on a solid support as further described herein. The chimeric GPCR or binding domain or binding unit may suitably be provided and used as a fusion protein, as further described herein and in international application WO 2014 / 118297.

[0178] However, it should be noted that, as described herein and as known per se for naturally occurring GPCRs (see, again, e.g., Eglen and Reisine, cited herein), the chimeric GPCRs of the present invention may comprise, in addition to a binding site corresponding to the orthosteric binding site of a first GPCR, one or more allosteric binding sites corresponding to one or more allosteric binding sites of a first GPCR, depending on the ECL and TM present in the chimeric GPCR of the present invention. Thus, more generally, it is expected that the present invention can be used to identify, generate, screen, test, and / or develop compounds and ligands that act as orthosteric binding agents for a first GPCR, as well as compounds and ligands that act as allosteric binding agents for a first GPCR.

[0179] Thus, in a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to a GPCR, comprising the steps of: a) providing a chimeric GPCR comprising an extracellular loop of said GPCR and a TM of said GPCR (or essentially all of the TM of said GPCR as further described herein), and comprising an intracellular loop of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to a binding site on the chimeric GPCR that comprises at least one of the intracellular loops; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular binding site of the chimeric GPCR; e) assessing whether each of said test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in the presence of said binding domain or binding unit; and optionally f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of said binding domain or binding unit; The present invention relates to a method, including:

[0180] In a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site of a GPCR, comprising the steps of: a) providing a chimeric GPCR comprising the extracellular loops of the GPCR and the TM of the GPCR (or essentially all of the TMs of the GPCR as further described herein), such that the extracellular loops and the TM form a functional extracellular binding site, and comprising an intracellular loop of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to a binding site on the chimeric GPCR that comprises at least one of the intracellular loops; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular binding site of the chimeric GPCR; e) assessing whether each of the test compounds or ligands (and / or which of the test compounds or ligands) binds to the extracellular binding site of the chimeric GPCR in the presence of the binding domain or binding unit; f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of the binding domain or binding unit; The present invention relates to a method, including:

[0181] In a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site of a GPCR, comprising the steps of: a) a cell or cell line, a chimeric GPCR within its cell wall or cell membrane, wherein the chimeric GPCR comprises (at least) the extracellular loops of the GPCR and the chimeric GPCR comprises the intracellular loops of another GPCR, the extracellular loops of the chimeric GPCR protruding into the extracellular environment (as defined herein) and the intracellular loops of the chimeric GPCR protruding into the intracellular environment of the cell or cell line; and a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; providing a cell or cell line comprising: b) contacting the cell or cell line with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of said test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said cell or cell line; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0182] In this method of the present invention, a cell or cell line comprising the chimeric GPCR and the binding domain or binding unit may be prepared, in particular, by maintaining or culturing a cell or cell line capable of expressing the chimeric GPCR and the binding domain or binding unit under conditions such that the cell line expresses the chimeric GPCR (in particular, suitably expresses the chimeric GPCR as defined herein) and also expresses the binding domain or binding unit.

[0183] In a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site of a GPCR, comprising the steps of: a) a vesicle or liposome, a chimeric GPCR within its wall or membrane, wherein the chimeric GPCR comprises (at least) an extracellular loop of the GPCR, and the chimeric GPCR comprises an intracellular loop of another GPCR, the extracellular loop of the chimeric GPCR protruding into the environment outside the vesicle or liposome (as defined herein) and the intracellular loop of the chimeric GPCR protruding into the environment inside the vesicle or liposome, and a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; providing a vesicle or liposome comprising: b) contacting the vesicles or liposomes with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of the test compounds or ligands) binds to the chimeric GPCR present in the vesicles or liposomes; and optionally, d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0184] In another aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to an extracellular binding site (as defined herein) of a GPCR, comprising the steps of: a) providing a chimeric GPCR comprising essentially (at least) the extracellular binding site of said GPCR and comprising the intracellular loops of another GPCR; b) treating said chimeric GPCR with a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, and a test compound or ligand; contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the formation of a complex between the test compound or ligand and the chimeric GPCR; c) assessing whether the test compound or ligand forms a complex with the chimeric GPCR and the binding domain or binding unit; and optionally, d) selecting one or more test compounds or ligands that form a complex with said chimeric GPCR and said binding domain or binding unit; The present invention relates to a method, including:

[0185] In each of the above methods, the chimeric GPCR and binding domain or binding unit are preferably chimeric GPCRs and binding domains or binding units as further described herein (any preferred or preferred aspects described herein for the chimeric GPCRs of the invention and / or such binding domains / binding units are also preferred for use in the above methods). Also, as described herein, chimeric GPCRs and binding domains or binding units may be suitably prepared and used as part of a fusion protein, again essentially as described in WO 2014 / 118297.

[0186] In another aspect, the invention provides a method for identifying a compound capable of binding to a functional conformational state of a GPCR, comprising: a) providing a chimeric GPCR comprising essentially (at least) the extracellular binding site of said GPCR and comprising the intracellular loops of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-inducing binding domain or binding unit (as defined herein) (i.e., a binding domain or binding unit that is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to said chimeric GPCR); c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to the binding site on the chimeric GPCR; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to the extracellular binding site of the chimeric GPCR; e) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in the presence of said binding domain or binding unit; and optionally f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of said binding domain or binding unit; The present invention relates to a method, including:

[0187] In another aspect, the present invention provides a method for identifying a compound capable of binding to an active conformational state of a GPCR, comprising the steps of: a) providing a chimeric GPCR comprising essentially (at least) the extracellular binding site of said GPCR and comprising the intracellular loops of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-inducing binding domain or binding unit (as defined herein) (i.e., a binding domain or binding unit that is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to said chimeric GPCR); c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to the binding site of the chimeric GPCR that comprises at least one of the intracellular loops; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to the extracellular binding site of the chimeric GPCR; e) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in the presence of said binding domain or binding unit; and optionally f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of said binding domain or binding unit; The present invention relates to a method, including:

[0188] In another aspect, the invention provides a method for identifying a compound capable of binding to a functional conformational state of a GPCR, comprising: a) providing a chimeric GPCR comprising an extracellular loop of said GPCR and a TM of said GPCR (or essentially all of the TM of said GPCR as further described herein), and comprising an intracellular loop of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein the binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to the binding site of the chimeric GPCR that comprises at least one of the intracellular loops; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; e) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in the presence of said binding domain or binding unit; and optionally f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of said binding domain or binding unit; The present invention relates to a method, including:

[0189] In another aspect, the present invention provides a method for identifying a compound capable of binding to an active conformational state of a GPCR, comprising the steps of: a) providing a chimeric GPCR comprising an extracellular loop of said GPCR and a TM of said GPCR (or essentially all of the TM of said GPCR as further described herein), and comprising an intracellular loop of another GPCR; b) providing a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein the binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); c) contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the binding domain or binding unit to bind to a binding site on the chimeric GPCR that comprises at least one of the intracellular loops; d) contacting the chimeric GPCR with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; e) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in the presence of said binding domain or binding unit; and optionally f) selecting a test compound or ligand that binds to the chimeric GPCR in the presence of said binding domain or binding unit; The present invention relates to a method, including:

[0190] In another aspect, the invention provides a method for identifying a compound capable of binding to a functional conformational state of a GPCR, comprising: a) a cell or cell line, a chimeric GPCR within its cell wall or cell membrane, wherein the chimeric GPCR comprises (at least) an extracellular loop of the GPCR and the chimeric GPCR comprises an intracellular loop of another GPCR, the extracellular loop of the chimeric GPCR protruding into the extracellular environment (as defined herein) and the intracellular loop of the chimeric GPCR protruding into the intracellular environment of the cell or cell line; and a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; providing a cell or cell line comprising: b) contacting the cell or cell line with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said cell or cell line; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0191] In this method of the invention involving the use of a cell or cell line, the chimeric GPCR and the binding domain or binding unit may particularly be provided by maintaining or culturing a cell or cell line capable of expressing the chimeric GPCR and the binding domain or binding unit under conditions such that the cell line expresses the chimeric GPCR (in particular, suitably expresses the chimeric GPCR as defined herein) and also expresses the binding domain or binding unit.

[0192] In another aspect, the present invention provides a method for identifying a compound capable of binding to an active functional conformational state of a GPCR, comprising: a) a cell or cell line, a chimeric GPCR within its cell wall or cell membrane, wherein the chimeric GPCR comprises (at least) an extracellular loop of the GPCR and the chimeric GPCR comprises an intracellular loop of another GPCR, the extracellular loop of the chimeric GPCR protruding into the extracellular environment (as defined herein) and the intracellular loop of the chimeric GPCR protruding into the intracellular environment of the cell or cell line; and a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; providing a cell or cell line comprising: b) contacting the cell or cell line with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said cell or cell line; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0193] In this method of the present invention, a cell or cell line comprising said chimeric GPCR and said binding domain or binding unit may particularly be prepared by maintaining or culturing a cell or cell line capable of expressing said chimeric GPCR and said binding domain or binding unit under conditions such that said cell line expresses said chimeric GPCR (in particular, suitably expresses said chimeric GPCR as defined herein) and also expresses said binding domain or binding unit.

[0194] In this method of the invention involving the use of a cell or cell line, the chimeric GPCR and the binding domain or binding unit may in particular be prepared by maintaining or culturing a cell or cell line capable of expressing the chimeric GPCR and the binding domain or binding unit under conditions such that the cell line expresses the chimeric GPCR (in particular, suitably expresses the chimeric GPCR as defined herein) and also expresses the binding domain or binding unit.

[0195] In another aspect, the invention provides a method for identifying a compound capable of binding to a functional conformational state of a GPCR, comprising: a) a vesicle or liposome, a chimeric GPCR within its wall or membrane, wherein the chimeric GPCR comprises (at least) an extracellular loop of the GPCR and the chimeric GPCR comprises an intracellular loop of another GPCR, the extracellular loop of the chimeric GPCR protruding into the environment outside the vesicle or liposome (as defined herein) and the intracellular loop of the chimeric GPCR protruding into the environment within the vesicle or liposome, and a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; providing a vesicle or liposome comprising: b) contacting the vesicles or liposomes with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said vesicles or liposomes; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0196] In another aspect, the present invention provides a method for identifying a compound capable of binding to an active conformational state of a GPCR, comprising the steps of: a) a vesicle or liposome, a chimeric GPCR within its wall or membrane, wherein the chimeric GPCR comprises (at least) an extracellular loop of the GPCR and the chimeric GPCR comprises an intracellular loop of another GPCR, the extracellular loop of the chimeric GPCR protruding into the environment outside the vesicle or liposome (as defined herein) and the intracellular loop of the chimeric GPCR protruding into the environment within the vesicle or liposome, and a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; providing a vesicle or liposome comprising: b) contacting the vesicles or liposomes with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said vesicles or liposomes; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0197] In another aspect, the invention provides a method for identifying a compound capable of binding to a functional conformational state of a GPCR, comprising: a) providing a chimeric GPCR comprising essentially (at least) the extracellular binding site of the GPCR and comprising an intracellular loop of another GPCR; b) treating said chimeric GPCR with a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, and a test compound or ligand; contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the formation of a complex between the test compound or ligand and the chimeric GPCR; c) assessing whether the test compound or ligand forms a complex with the chimeric GPCR and the binding domain or binding unit; and optionally, d) selecting one or more test compounds or ligands that form a complex with said chimeric GPCR and said binding domain or binding unit; The present invention relates to a method, including:

[0198] In another aspect, the invention provides a method for identifying a compound capable of binding to a functional conformational state of a GPCR, comprising: a) providing a chimeric GPCR comprising essentially (at least) the extracellular binding site of the GPCR and comprising an intracellular loop of another GPCR; b) treating said chimeric GPCR with a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, and a test compound or ligand; contacting the chimeric GPCR with the binding domain or binding unit under conditions that allow the formation of a complex between the test compound or ligand and the chimeric GPCR; c) assessing whether the test compound or ligand forms a complex with the chimeric GPCR and the binding domain or binding unit; and optionally, d) selecting one or more test compounds or ligands that form a complex with said chimeric GPCR and said binding domain or binding unit; The present invention relates to a method, including:

[0199] Again, in all of these methods, the chimeric GPCR and binding domain or binding unit are preferably, again, as further described herein (any preferred or preferred aspects described herein for the chimeric GPCRs of the present invention and / or such binding domains / binding units are also preferred for use in the methods). Again, the chimeric GPCRs provided and used in the above steps are preferably such chimeric GPCRs, and the conditions under which the chimeric GPCR and binding domain or binding unit are used in the above steps are preferably selected so that binding of the test compound to the chimeric GPCR under the conditions used is representative of binding of the test compound(s) to the GPCR from which the extracellular binding site is derived. In such methods, the chimeric GPCR and binding domain or binding unit may be present in a suitable cellular composition and / or expressed by a suitable cell or cell line, or may be present in a suitable liposome or vesicle, all as further described herein. Again, the chimeric GPCR or binding domain or binding unit may be immobilized on a solid support, as further described herein. The chimeric GPCR or binding domain or binding unit may suitably be provided and used as a fusion protein, as further described herein and in international application WO 2014 / 118297.

[0200] The present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site (as defined herein) of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR that essentially comprises (at least) the extracellular binding site of said GPCR and comprises intracellular loops of another GPCR, and (ii) a binding domain or binding unit that is capable of binding to the binding site of said chimeric GPCR that comprises at least one of said intracellular loops; b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention further relates to a method, including:

[0201] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to an extracellular binding site (as defined herein) of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising an extracellular loop of said GPCR and a TM of said GPCR (or essentially all of the TM of said GPCR as further described herein), and comprising an intracellular loop of another GPCR, and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0202] The present invention also provides a method of forming a complex between a chimeric GPCR, a binding domain or binding unit, and a compound or ligand capable of binding to an extracellular binding site (as defined herein) of said chimeric GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising (at least) an extracellular loop of a first GPCR and an intracellular loop of a second GPCR (different from said first GPCR), and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; The present invention relates to a method, including:

[0203] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to a functional conformation of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising (at least) an extracellular loop of said GPCR and an intracellular loop of a second GPCR (distinct from said first GPCR), and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0204] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to an active conformation of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising (at least) an extracellular loop of said GPCR and an intracellular loop of a second GPCR (distinct from said first GPCR), and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein the binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0205] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to a functional conformation of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising an extracellular binding site (as defined herein) of said GPCR and comprising intracellular loops of another GPCR, and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0206] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to an active conformation of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising an extracellular binding site (as defined herein) of said GPCR and comprising intracellular loops of another GPCR, and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0207] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to a functional conformation of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising an extracellular loop of said GPCR and a TM of said GPCR (or essentially all of the TM of said GPCR as further described herein), and comprising an intracellular loop of another GPCR, and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0208] The present invention also provides a method for identifying and / or producing a compound or ligand capable of binding to an active conformation of a GPCR, comprising the steps of: a) providing a composition comprising (i) a chimeric GPCR comprising the extracellular loops of said GPCR and the TM of said GPCR (or essentially all of the TM of said GPCR as further described herein), and comprising the intracellular loops of another GPCR, and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, wherein said binding domain or binding unit is a conformation-induced binding domain or binding unit (as defined herein); b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the binding domain or binding unit to bind to the binding site of a chimeric GPCR that comprises at least one of the intracellular loops, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR in said composition; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR in said composition; The present invention relates to a method, including:

[0209] Again, in all of these methods, the chimeric GPCRs and binding domains or binding units present in the compositions used are preferably, again, chimeric GPCRs and binding domains or binding units as further described herein (any preferred or preferred aspects described herein for the chimeric GPCRs of the invention and / or such binding domains / binding units are also preferred for use in the methods). Again, the chimeric GPCRs present in the compositions used, and the conditions under which the compositions are used in the above steps, are preferably selected so that binding of the test compound to the chimeric GPCR under the conditions used is representative of binding of the test compound(s) to the GPCR from which the extracellular binding site is derived. Again, the chimeric GPCRs or binding domains or binding units may be immobilized on a solid support, as further described herein. The chimeric GPCRs or binding domains or binding units may suitably be provided and used as fusion proteins, as further described herein and in International Application WO 2014 / 118297.

[0210] Also, in methods in which a composition comprising a chimeric GPCR of the present invention and a binding domain or binding unit capable of binding to an intracellular binding site of the chimeric GPCR is used, the composition may be a cellular composition as described herein, or the composition may be contained in a suitable liposome or vesicle that appropriately contains the chimeric GPCR and the binding domain or binding unit (as described herein).

[0211] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a composition comprising (i) a chimeric GPCR comprising (at least) an extracellular loop of a first GPCR and an intracellular loop of a second GPCR (different from said first GPCR), and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, said binding domain or binding unit preferably being a conformation-induced binding domain or binding unit (as defined herein); a composition comprising (i) a chimeric GPCR comprising an extracellular binding site (as defined herein) of a first GPCR and an intracellular loop of a second GPCR (different from said first GPCR), and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, said binding domain or binding unit preferably being a conformation-induced binding domain or binding unit (as defined herein); a composition comprising (i) a chimeric GPCR comprising an extracellular loop of a first GPCR and a TM of said first GPCR (or essentially all of the TM of said first GPCR, as further described herein), and an intracellular loop of a second GPCR (different from said first GPCR); and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops, said binding domain or binding unit preferably being a conformation-induced binding domain or binding unit (as defined herein). Also relates to.

[0212] The present invention also relates to the use of such compositions, particularly the use of such compositions in the methods described herein.

[0213] The composition may be a cellular composition as described herein, or the composition may be contained in a suitable liposome or vesicle suitably comprising a chimeric GPCR and a binding domain or binding unit (as described herein). Furthermore, the chimeric GPCR and binding domain or binding unit present in the composition used are preferably, again, chimeric GPCRs and binding domains or binding units as further described herein (any preferred or preferred aspects described herein for the chimeric GPCRs of the present invention and / or such binding domains / binding units are also suitable for use in the method). Furthermore, the chimeric GPCR or binding domain or binding unit may, again, be immobilized on a solid support as further described herein. The chimeric GPCR or binding domain or binding unit may suitably be provided and used as a fusion protein, as further described herein and in International Application WO 2014 / 118297.

[0214] As described herein, in some preferred aspects of the invention, the chimeric GPCR of the invention is present in and / or expressed by a suitable cell or cell line.

[0215] Thus, in a further aspect, the present invention relates to a cell or cell line comprising, expressing, and / or capable of expressing a chimeric GPCR of the present invention. Such a cell or cell line is preferably such that the chimeric GPCR is present (i.e., anchored) in the cell membrane or cell wall of the cell or cell line and / or such that the cell or cell line suitably expresses the chimeric GPCR (as defined herein). More preferably, the cell or cell line is such that the chimeric GPCR of the present invention penetrates the cell membrane or cell wall of the cell or cell line, with the extracellular loops protruding into the extracellular environment and the intracellular loops protruding into the intracellular environment of the cell or cell line (and / or is capable of expressing the chimeric GPCR in this manner). In the context of the present application and claims, when one or more ECLs are said to "protrude" into an environment (such as the extracellular environment of a cell or the environment outside a liposome or vesicle), this should be understood to generally mean that the ECLs are exposed to the environment and / or are accessible for binding by ligands, compounds, or other chemical entities present in the environment. In particular, for the chimeric GPCRs of the present invention, this means that the extracellular binding site (as defined herein) of the chimeric protein is accessible for binding by ligands, compounds, or other chemical entities present in the environment. Similarly, when one or more ICLs are said to "protrude" into an environment (e.g., the intracellular environment of a cell or the internal environment of a liposome or vesicle), this should be understood to generally mean that the ICLs are exposed to the environment and / or are accessible for binding by ligands, compounds, or other chemical entities present in the environment.In this context, the phrase "accessible for binding" should generally be understood to mean that a ligand, compound, or other chemical entity present in the relevant environment can bind to a binding pocket or binding site on or within a chimeric GPCR, even if the actual binding site or binding pocket is located deeper within the structure of the chimeric GPCR (even if the actual binding site or binding pocket is located within a portion of the chimera that does not physically protrude beyond the boundary layer itself). See, for example, Chevillard (cited herein), which shows that the binding site of a GPCR for a fragment used in FBDD screening techniques is located deep within the GPCR structure (see, e.g., Figure 2 on page 1120) and is accessible for fragment binding, despite not being present on the surface of the GPCR. See also the teachings regarding GPCR structure, GPCR signaling mechanisms, and GPCR ligand binding sites from some of the other scientific references cited herein.

[0216] A cell or cell line comprising or expressing a chimeric GPCR of the invention preferably further comprises, expresses, and / or is capable of expressing a binding domain or binding unit capable of specifically binding to an intracellular loop (the binding site formed thereby) present in the chimeric protein. Again, such a binding domain or binding unit present in and / or expressed by the cell or cell line is preferably a conformation-induced binding domain or binding unit (as defined herein), more preferably a conformation-induced ISVD (e.g., a conformobody). Furthermore, the cell or cell line preferably comprises or expresses the binding domain or binding unit such that it is capable of binding to the intracellular binding site (as defined herein) of the chimeric GPCR of the invention (as will be apparent to those skilled in the art, this typically means that the cell or cell line comprises or expresses the binding domain or binding unit within its intracellular environment).

[0217] Such cells or cell lines comprising or expressing a chimeric GPCR of the invention (and preferably, in addition, a binding domain or binding unit capable of specifically binding to an intracellular loop present in said chimeric protein) may generally be the cells or cell lines further described herein.

[0218] The present invention also relates to the use of cells or cell lines comprising, expressing and / or capable of expressing the chimeric GPCRs of the invention. In particular, the present invention relates to the use of cells or cell lines comprising, expressing and / or capable of expressing the chimeric GPCRs of the invention, identifying, producing, and / or screening (e.g., using standard screening techniques known per se) for ligands or compounds capable of binding (in particular capable of specifically binding as defined herein) to the chimeric GPCR present in said complex; and / or determining (e.g., using standard assay techniques known per se) at least one property of the compound or ligand, such as its ability to bind (in particular, specifically bind as defined herein) to the chimeric GPCR present in said complex and / or its ability to modulate said chimeric GPCR; In, relating to use.

[0219] For such uses, the cell or cell line comprising, expressing, and / or capable of expressing the chimeric GPCR of the invention can be a cell or cell line as further described herein. Preferably, the cell or cell line used is one that comprises, expresses, or is capable of expressing a binding domain or binding unit that can specifically bind to an intracellular loop (thereby forming a binding site) present in the chimeric protein. Such a binding domain is again preferably a conformation-induced binding domain or binding unit (as defined herein), and again preferably an ISVD (and preferably a conformation-induced ISVD).

[0220] The present invention also provides a method for determining at least one property of a compound or ligand, comprising at least: providing a cell or cell line comprising, expressing or capable of expressing a chimeric GPCR of the invention, and preferably further comprising, expressing or capable of expressing a binding domain or binding unit capable of specifically binding to an intracellular loop present in said chimeric GPCR (the binding site formed thereby), said binding domain or binding unit preferably being a conformation-induced binding domain or binding unit (as defined herein); contacting said cell or cell line with said compound or ligand, wherein said compound or ligand is present in an extracellular environment and said cell or cell line is such that the extracellular binding site (as defined herein) of the chimeric GPCR is available / accessible for binding by the compound or ligand present in the extracellular environment; The present invention relates to a method, including:

[0221] Optionally, the method may comprise maintaining or culturing the cell or cell line under conditions such that the cell or cell line expresses (and in particular suitably expresses as defined herein) a chimeric GPCR of the invention, and in addition expresses the binding domain or binding unit such that the binding domain or binding unit is capable of binding to an ICL of the chimeric GPCR and / or of forming a complex (as described herein) with the chimeric GPCR and optionally the compound or ligand.

[0222] The method preferably also includes measuring (a change in) at least one signal or parameter representative of said at least one property. As described herein, said property can be the ability to bind (and in particular the ability to specifically bind) to a chimeric GPCR of the present invention. The at least one property can also be the ability to modulate said chimeric GPCR, for example the ability to act as an agonist (e.g., as a partial or full agonist) of the chimeric GPCR, the ability to act as an antagonist of the chimeric GPCR, and / or the ability to act as an inverse agonist of the chimeric GPCR. Again, preferably, said at least one property determined using the chimeric GPCR of the present invention represents essentially the same or essentially similar properties as the naturally occurring GPCR from which the ECL (and preferably, as further described herein, essentially further the TM) is derived.

[0223] The present invention also provides a) a chimeric protein comprising an N-terminal sequence, a C-terminal sequence, seven transmembrane domains (TM1 to TM7), three extracellular loops (EC1 to EC3), and three intracellular loops (IC1 to IC3), the extracellular loops present in the chimeric protein are (essentially) derived from the first GPCR, a chimeric protein, wherein the intracellular loop present in the chimeric protein is (essentially) derived from a second GPCR; b) a binding domain or binding unit capable of specifically binding to an intracellular loop present in said chimeric protein (thereby forming a binding site), and optionally c) a ligand or compound that binds to the extracellular binding site (as defined herein) of the chimeric GPCR; A method for forming a complex, comprising: The method comprises: - at least maintaining or culturing a cell or cell line expressing the chimeric protein referred to in a) and the binding domain or binding unit referred to in b) under conditions such that said cell or cell line expresses said chimeric protein and said binding domain, and optionally - also comprising a step of contacting said cells or cell lines with a ligand or compound referred to under c).

[0224] Again, the cell or cell line is preferably such that the extracellular loops of the chimeric GPCR protrude into the extracellular environment (as defined herein) and / or the extracellular binding site of the chimeric GPCR is available / accessible for binding by the ligand or compound referred to in c) when the ligand or compound is present in the extracellular environment. Also, again, the binding domain or binding unit present in and / or expressed by the cell or cell line is preferably a conformation-induced binding domain or binding unit (as defined herein), more preferably a conformation-induced ISVD (e.g., a conformobody). Also preferably, the chimeric GPCR in the complex (once formed) is preferably in a functional, active and / or druggable conformation. In a particular aspect, the ligand or compound referred to in c) is an agonist and the chimeric GPCR in the complex (once formed) is in an agonist-binding conformation.

[0225] As described herein, it is contemplated herein that the chimeric GPCRs of the invention may be fused to a binding domain or binding unit capable of specifically binding to an intracellular loop (the binding site formed thereby) present in the chimeric protein. Such fusions and their uses may be essentially as described in International Application WO 2014 / 118297, entitled "Novel chimeric polypeptides for screening and drug discovery purposes," which describes fusions of GPCRs, particularly GPCRs and conformobodies specific for the intracellular binding site of a GPCR.

[0226] Generally, such fusion proteins comprise a chimeric GPCR of the present invention as described herein fused or linked, optionally via a suitable spacer or linker, to a binding domain or binding unit capable of specifically binding to an intracellular loop (thereby forming a binding site) present in the chimeric protein. The spacer or linker present in such a fusion protein may essentially be a spacer or linker described in International Application WO 2014 / 118297. The chimeric GPCR and binding unit or binding domain present in the fusion protein may essentially be a chimeric GPCR and binding unit or binding domain further described herein. In particular, the binding unit or binding domain is a conformation-induced binding domain or binding unit, more preferably a conformation-induced ISVD (e.g., a conformobody).

[0227] The present invention also relates to the use of such fusion proteins, particularly for drug discovery assays and screening purposes. Such uses may be as further described herein and / or in International Application Publication No. WO 2014 / 118297. For such uses, the chimeric proteins may be expressed in suitable cells or cell lines (essentially as described herein and in International Application Publication No. WO 2014 / 118297), with cells or cell lines expressing such fusion proteins forming a further aspect of the present invention. Also, for such uses, such fusion proteins may be immobilized on a solid support (again, essentially as described herein and in International Application Publication No. WO 2014 / 118297), with the solid support to which such fusion proteins are immobilized forming a further aspect of the present invention.

[0228] Thus, in a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site of a GPCR, comprising the steps of: a) providing a fusion protein comprising (i) a chimeric GPCR that essentially comprises (at least) the extracellular binding site of the GPCR and that comprises the intracellular loops of another GPCR, and (ii) a binding domain or binding unit that is capable of binding to the binding site of the chimeric GPCR that comprises at least one of the intracellular loops; b) contacting the fusion protein with one or more test compounds or ligands under conditions that allow the test compounds to bind to the extracellular binding site of the chimeric GPCR present in the fusion protein; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR present in said fusion protein; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR present in the fusion protein; The present invention relates to a method, including:

[0229] In another aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to a GPCR, comprising the steps of: a) providing a fusion protein comprising (i) a chimeric GPCR comprising the extracellular loops of said GPCR and the TM of said GPCR (or essentially all of the TM of said GPCR, as further described herein), and the intracellular loops of another GPCR, fused or linked to the chimeric GPCR, optionally via a suitable spacer or linker, and (ii) a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops; b) contacting the fusion protein with one or more test compounds or ligands under conditions that allow the test compounds to bind to the extracellular binding site of the chimeric GPCR present in the fusion protein; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to the chimeric GPCR present in said fusion protein; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR present in the fusion protein; The present invention relates to a method, including:

[0230] In methods of the invention in which such fusion proteins are used, the chimeric GPCR and binding domain or binding unit present in the fusion protein are again chimeric GPCR and binding domain or binding unit as further described herein. Again, the conditions under which the chimeric GPCR present in the fusion protein and the fusion protein and binding domain or binding unit are used in the above steps are preferably selected so that binding of the test compound to the chimeric GPCR under the conditions used is representative of binding of the test compound(s) to the GPCR. Also, in such methods, the fusion protein may be present in a suitable cellular composition and / or expressed by a suitable cell or cell line, or may be present in a suitable liposome or vesicle, all as further described herein. Also, the fusion protein may be immobilized on a solid support, again as further described herein.

[0231] Additionally, methods involving the use of such fusion proteins, as well as other methods described herein, may be used to identify and / or generate compounds or ligands that bind to the extracellular binding site of a chimeric GPCR present in the fusion protein and / or compounds or ligands that bind to the allosteric site of the chimeric GPCR.

[0232] In a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site of a GPCR, comprising the steps of: a) providing a cell or cell line containing a fusion protein comprising (i) a chimeric GPCR that essentially comprises (at least) the extracellular binding site of said GPCR and that comprises the intracellular loops of another GPCR, fused or linked to the chimeric GPCR, optionally via a suitable spacer or linker, and (ii) a binding domain or binding unit that is capable of binding to the binding site of said chimeric GPCR that comprises at least one of said intracellular loops; b) contacting the cell or cell line with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said cell or cell line; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0233] In this method of the invention, a cell or cell line comprising the fusion protein can be prepared by maintaining or culturing a cell or cell line capable of expressing the fusion protein, particularly under conditions such that the cell line expresses the fusion protein, preferably such that the chimeric GPCR in the fusion protein is anchored to or incorporated into the cell wall or membrane of the cell or cell line (as generally described herein for the chimeric GPCRs of the invention) and is expressed in an intracellular environment such that the binding domain or binding unit that is part of the fusion protein is capable of binding to the intracellular binding site (as defined herein) of the chimeric protein.

[0234] In a further aspect, the present invention provides a method for identifying and / or producing a compound or ligand capable of binding to the extracellular binding site of a GPCR, comprising the steps of: a) providing a liposome or vesicle containing a fusion protein comprising: (i) a chimeric GPCR that essentially comprises (at least) the extracellular binding site of the GPCR and includes intracellular loops of another GPCR, the chimeric GPCR being fused or linked, optionally via a suitable spacer or linker, to the chimeric GPCR; and (ii) a binding domain or binding unit that is capable of binding to the binding site of the chimeric GPCR that includes at least one of the intracellular loops; b) contacting the cell or cell line with one or more test compounds or ligands under conditions that allow the test compounds to bind to (at least) the extracellular loops of the chimeric GPCR; c) assessing whether each of the test compounds or ligands (and / or which of said test compounds or ligands) binds to said chimeric GPCR present in said cell or cell line; and optionally d) selecting a test compound or ligand that binds to the chimeric GPCR; The present invention relates to a method, including:

[0235] In further aspects, the present invention also relates to compositions comprising such fusion proteins, and to the use of such fusion proteins and such compositions, particularly the use of such fusion proteins and such compositions in the methods described herein. Such compositions may also be cellular compositions as described herein, or may comprise vesicles or liposomes suitably containing such fusion proteins, as further described herein.

[0236] It is also contemplated herein that the chimeric GPCRs of the present invention may find use in the methods and arrangements described in the co-pending U.S. provisional application entitled "Screening methods and assays for use with transmembrane proteins, in particular with GPCRs," filed April 29, 2019 (also referred to herein as the "co-pending application"), which is assigned to Confo Therapeutics NV, the disclosure of which is incorporated herein by reference.

[0237] The co-pending applications generally include at least the following components (all of which are further defined in the co-pending applications): · A boundary layer separating the first environment from the second environment; Translayer proteins; a first ligand for a transmembrane protein present in the first environment (as defined herein); a second ligand for the transmembrane protein present in the second environment (as defined herein); and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; and further describes uses of said arrangements (particularly for assay and screening techniques) and methods of using such arrangements (which again may be particularly assay and screening methods).

[0238] In a particular and preferred embodiment, the chimeric proteins of the present invention are used as transmembrane proteins in the arrangements and methods described in the co-pending applications.

[0239] Generally, this means that such an arrangement comprises, as a transmembrane protein, a chimeric GPCR whose intracellular loops are derived from a first 7TM or GPCR and whose extracellular loops are derived from a second 7TM or GPCR different from the first. The transmembrane domains of such a chimeric protein may be derived from the first or second 7TM or GPCR, and preferably are essentially all derived from the same GPCR, more preferably from the same GPCR as the extracellular loops (although they may include some amino acid residues from the GPCR from which the intracellular loop is derived, depending on the positions chosen for recombinantly deleting the native intracellular loop and inserting the replacement intracellular loop).

[0240] In this aspect of the invention, the resulting chimeric transmembrane protein is still most preferably a transmembrane protein that can be suitably used in the methods and arrangements described in the co-pending applications. Furthermore, the chimeric GPCR of the invention used as a transmembrane protein comprises three intracellular loops and three extracellular loops, the three intracellular loops forming a functional ligand-binding site for a second ligand (which is then selected so as to be capable of binding to the ligand-binding site (9) formed by the intracellular loops). Again, the binding site formed by the three intracellular loops preferably protrudes into the second environment [B] (i.e., the environment inside the cell or liposome, if the method of the invention is carried out in a cell or liposome, respectively), and the three extracellular loops preferably protrude into the first environment [A] (and may form a functional binding site for the first ligand, or the binding site may be located deep within the 7TM structure).

[0241] Therefore, in a further aspect, the present invention relates to an arrangement as described in the co-pending application, wherein the transmembrane protein is a 7TM protein comprising seven transmembrane domains, three intracellular loops, and three extracellular loops (which are linked together in the order known per se for 7TM proteins, i.e., [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence]), wherein the intracellular loops are derived from a first 7TM protein and the extracellular loops are derived from a second 7TM protein different from the first 7TM protein, and the intracellular loops form functional ligand-binding sites. Preferably, the TM domains from the transmembrane protein are essentially derived from the same 7TM protein as the extracellular loops.

[0242] The intracellular loops and 7TM are also such that, taken together, they form a functional ligand-binding site, in particular a functional ligand-binding site to which a (suitable) second ligand (as defined herein) can bind, which again preferably projects into the second environment [B].

[0243] The present invention particularly relates to arrangements described in co-pending applications comprising such chimeric 7TM and a second ligand capable of binding to the ligand binding site formed by said intracellular loop.

[0244] For the remainder, if the second ligand is appropriately selected to be capable of binding to the ligand binding site (9) of the chimeric layer-spanning protein to provide an operable arrangement as described in the co-pending application (and if the chimeric layer-spanning protein itself is operable in such an arrangement), such an arrangement in which the chimeric layer-spanning protein is used can essentially be the arrangement further described herein and in the co-pending application.

[0245] Another aspect of the present invention is a composition or kit of parts as described in the co-pending applications, comprising at least said chimeric transmembrane protein and a ligand capable of binding to an intracellular loop present in said GPCR. Said ligand is preferably a protein, more preferably a protein comprising or consisting essentially of an immunoglobulin single variable domain (e.g. a VHH domain), and in particular may be a conformobody (as described herein).

[0246] Also, when reference is made in the further description and claims herein to such arrangements or any components of such arrangements, it should be understood that such arrangements or component(s) generally (and preferably) are the arrangements or component(s) further described in the co-pending application, and any term not specifically defined herein should generally be understood to have the meaning set forth in the co-pending application.

[0247] Thus, in a further aspect, the present invention provides a method for treating a pulmonary arthritis, comprising administering to a subject a therapeutically effective amount of at least one of the following components (all as further defined herein): · A boundary layer separating the first environment from the second environment; · Translaminar proteins; a first ligand for a transmembrane protein present in the first environment (as defined herein); a second ligand for the transmembrane protein present in the second environment (as defined herein); and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; An arrangement comprising: The present invention relates to an arrangement in which the layer-spanning protein is a chimeric GPCR of the invention as described herein, and the components of the arrangement are arranged (and, where applicable, operably linked and / or associated with each other) with respect to each other in a manner as further described herein and in the co-pending application.

[0248] In certain aspects of such arrangements, the second ligand is a binding domain or binding unit as described herein, i.e., a binding domain or binding unit that can specifically bind to an intracellular loop (and thereby the binding site) present in the chimeric protein.

[0249] As also described in the co-pending applications, the arrangements and methods of the co-pending applications typically (and preferably) involve the use of two fusion proteins: a first fusion protein comprising a layer-spanning protein and a first binding member of a binding pair, and a second fusion protein comprising a second member of the binding pair and a protein capable of binding, directly or indirectly, to the layer-spanning protein (as defined in the co-pending applications). Thus, when a chimeric protein of the invention is used as a layer-spanning protein in such methods and arrangements, the chimeric protein of the invention can be part of such first fusion protein together with the first binding member of the binding pair used in such arrangement.

[0250] Thus, in a further aspect, the present invention relates to fusion proteins comprising the chimeric protein of the invention linked via a suitable linker or spacer to a binding domain, binding unit, or other peptide, protein, or amino acid sequence that is a member of a binding pair as further described herein and in the co-pending applications. The present invention also relates to nucleotide sequences and / or nucleic acids encoding such fusion proteins, as well as to cells, cell lines, or other host cells or organisms that express (and in particular suitably express, as described in the co-pending applications) or are (suitably) capable of expressing such fusion proteins.

[0251] In particular, an arrangement for carrying out the method of the present invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a transmembrane protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (i.e., to form a first fusion protein); a first ligand for a transmembrane protein present in the first environment; and · a second ligand for the transmembrane protein present in the second environment; wherein the layer-spanning protein is a chimeric GPCR of the invention as described herein, and the components of the arrangement are arranged relative to one another (and, where applicable, operably linked and / or associated with one another) in a manner as further described herein. As further described herein and in co-pending applications, the second member of the binding pair may be part of a second fusion protein (different from the first fusion protein comprising the layer-spanning protein and the first binding member of the binding pair), wherein the second fusion protein is a second fusion protein as further described herein.

[0252] In particular, an arrangement for carrying out the method of the present invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., comprising said member of the binding pair such that it is present in the second environment); a second fusion protein present in a second environment, the second fusion protein comprising a protein capable of binding directly or indirectly to the transmembrane protein and the other binding member of said binding pair; and a first ligand for a transmembrane protein present in the first environment; wherein said layer-spanning protein is a chimeric GPCR of the invention as described herein, and wherein the components of the arrangement are arranged relative to each other (and, where applicable, operably linked and / or associated with each other) in a manner further described herein.

[0253] It should be noted that, in the specification and claims, when a ligand, binding domain, binding unit, or other compound or protein is said to be "capable of binding" to another protein or compound, such binding is most preferably a "specific binding" as further defined herein. Also, as further described herein, when a fusion protein is described as "comprising" a first protein, ligand, binding domain, binding member or binding unit, and a second protein, ligand, binding domain, binding member or binding unit (and optionally one or more further proteins, ligands, binding domains, binding members or binding units), it should be understood that in such a fusion protein, such proteins, ligands, binding domains, binding members or binding units are suitably linked to each other, either directly or via a suitable spacer or linker.

[0254] As generally described in the co-pending applications, in the arrangements described therein, a protein (such as a binding domain, binding unit, or ligand) is said to bind "directly or indirectly" to a layer-spanning protein if (i) the protein itself binds (and / or is capable of binding) to the layer-spanning protein (e.g., to an epitope or binding site of the layer-spanning protein, as further described herein); or (ii) the protein binds (and / or is capable of binding) to a ligand or protein that binds (and / or is capable of binding) to the layer-spanning protein; or (iii) the protein binds (and / or is capable of binding) to a protein complex comprising a ligand or protein that binds (and / or is capable of binding) to the layer-spanning protein. In case (i), the protein is said herein to bind "directly" to the layer-spanning protein, and in cases (ii) and (iii), the protein is said herein to bind "indirectly" to the layer-spanning protein. Also, when a protein binds to a protein complex that includes a ligand or protein that binds to a transmembrane protein, the protein may bind to the ligand or protein, or any other part, epitope, or binding site of the complex.

[0255] When the chimeric GPCR of the present invention is used as a layer-spanning protein in the arrangements described in the co-pending applications, the protein that binds to the layer-spanning protein (i.e., the chimeric GPCR of the present invention) may also be selected from (i) a binding domain, binding unit, or other protein that binds (and / or is capable of binding) to an epitope or binding site of the layer-spanning protein, (ii) a binding domain, binding unit, or other protein that binds (and / or is capable of binding) to a ligand or protein that binds (and / or is capable of binding) to said layer-spanning protein, and (iii) a binding domain, binding unit, or other protein that binds (and / or is capable of binding) to a protein complex comprising a ligand or protein that binds (and / or is capable of binding) to said layer-spanning protein. In each such case, such a binding domain, binding unit, or other protein is preferably a binding domain, binding unit, or other protein further described herein.

[0256] In such arrangements comprising a chimeric GPCR of the present invention, when the protein that binds to the transmembrane protein (i.e., the chimeric GPCR of the present invention) is a protein that binds "indirectly" to the chimeric GPCR of the present invention, the protein and second ligand are proteins and second ligands further described in the co-pending applications, and the arrangement typically does not comprise a binding domain or binding unit described herein (i.e., a conformation-induced binding domain or binding unit capable of binding to an intracellular loop of the chimeric GPCR).

[0257] However, in such arrangements comprising a chimeric GPCR of the present invention, when the protein that binds to a transmembrane protein (i.e., a chimeric GPCR of the present invention) is a protein that binds "directly" to the chimeric GPCR of the present invention, the arrangement comprises a binding domain or binding unit as described herein, which binding domain or binding unit functions as a "second ligand." Also, as generally described in the co-pending applications, when the protein binds "directly" to a transmembrane protein, the protein is preferably part of a second fusion protein. Thus, when such arrangements comprising a chimeric GPCR of the present invention also comprise a binding domain or binding unit as described herein as a second ligand, the binding domain or binding unit most preferably also forms part of a second fusion protein.

[0258] Thus, in a further aspect, the present invention relates to fusion proteins comprising the chimeric protein of the invention linked via a suitable linker or spacer to a binding domain, binding unit, or other peptide, protein, or amino acid sequence that is a member of a binding pair as further described herein and in the co-pending applications. The present invention also relates to nucleotide sequences and / or nucleic acids encoding such fusion proteins, as well as to cells, cell lines, or other host cells or organisms that express (and in particular suitably express, as described in the co-pending applications) or are (suitably) capable of expressing) such fusion proteins.

[0259] In a further aspect of the invention, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., comprising said member of the binding pair such that said member is present in the second environment); a second fusion protein present in a second environment, the second fusion protein comprising a transmembrane protein and a protein capable of directly binding (as defined herein) to the other binding member of the binding pair; and a first ligand for a transmembrane protein present in the first environment; wherein the layer-spanning protein is a chimeric GPCR of the invention as described herein, the protein is a binding domain or binding unit capable of binding to a binding site of the chimeric GPCR comprising at least one of the intracellular loops (and preferably is a conformation-induced binding domain as described herein), and the further components of the arrangement are arranged with respect to each other (and, where applicable, are operably linked and / or associated with each other) in a manner as further described herein. In this aspect of the invention, the protein that is capable of directly binding (as defined herein) to the layer-spanning protein (i.e., the chimeric GPCR of the invention) and that is present in the second fusion protein is preferably a binding domain or binding unit, more preferably an immunoglobulin single variable domain. It should also be understood that in this aspect of the invention, the protein that is capable of directly binding (as defined herein) to the layer-spanning protein and that is present in the second fusion protein acts as a second ligand.

[0260] In another aspect of the invention, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., comprising said member of the binding pair such that said member is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a second ligand for the transmembrane protein, which may optionally be part of a protein complex; a second fusion protein present in a second environment, comprising a transmembrane protein and a protein capable of indirectly binding (as defined herein) to the other binding member of said binding pair; and The layer-spanning protein is a chimeric GPCR of the invention as described herein, the components of the arrangement being arranged with respect to one another (and, where applicable, operably linked and / or associated with one another) in a manner as further described herein. In this aspect of the invention, the second ligand may be any suitable ligand (as further described herein), but is most preferably a G protein (or G protein complex), capable of indirectly (as defined herein) binding to the layer-spanning protein, and the protein present in the second fusion protein is preferably a binding domain or binding unit, more preferably an immunoglobulin single variable domain. Also, when the second ligand is a G protein or G protein complex, the chimeric GPCR of the invention is most preferably a chimeric GPCR whose ICL forms (or forms part of) a functional binding site for the G protein. It will also be clear that in this aspect of the invention, the second ligand does not form part of the second fusion protein.

[0261] As described further herein, in practicing the present invention, a first ligand is often added to a further component of an already formed / established arrangement of the present invention as described herein, and it should be noted that, as a result, an arrangement of the present invention in which the first ligand is not present (i.e. before the first ligand is added) forms a further aspect of the present invention (as do methods in which a first ligand is added to an arrangement of the present invention in which said first ligand is not present or not yet present).

[0262] In this specification and claims, the term "second ligand" is used in the methods and arrangements described herein to denote a ligand, binding domain, binding unit or other chemical entity that binds directly to a spanning protein (i.e., a chimeric GPCR of the invention) or is capable of binding directly to a spanning protein (or that forms part of a protein complex that binds directly to a spanning protein or is capable of binding directly to a spanning protein).

[0263] As will be apparent from the further description herein, when the chimeric protein of the invention is used as part of the arrangements described herein and in the co-pending applications, the second ligand can be part of the second fusion protein or can be separate from the second fusion protein. In either case (i.e., whether the second ligand is part of a second fusion protein or not), the second ligand is preferably a second ligand capable of binding to a conformational epitope of the chimeric GPCR (or capable of binding directly to the chimeric GPCR or forming part of a protein complex capable of directly binding to the chimeric GPCR), in particular a second ligand capable of binding to a conformational epitope of a chimeric GPCR that comprises one or more ICLs. More preferably, the second ligand (and / or protein complex comprising the second ligand) is preferably such that it specifically binds to one or more functional, active and / or druggable conformations of the layer-spanning protein (i.e., the chimeric GPCR of the present invention), induces the formation of and / or stabilizes one or more functional, active and / or druggable conformations of the layer-spanning protein (and / or shifts the conformational equilibrium of the layer-spanning protein towards one or more such conformations), and / or induces the formation of and / or stabilizes a complex between the layer-spanning protein, the first ligand and the second ligand.

[0264] When the second ligand is part of a second fusion protein, the second ligand can be any ligand, binding domain, binding unit, peptide, protein, or other chemical entity that can bind directly to the ICL of the chimeric protein of the invention used as a layer-spanning protein and that can be suitably included in the second fusion protein. Preferably, as further described herein, when part of a second fusion protein, the second ligand is a suitable binding domain or binding unit, in particular an immunoglobulin single variable domain (preferably a conformation-inducing ISVD as defined herein). Again, this aspect of the invention, when the chimeric GPCR of the invention is used as a layer-spanning protein together with an immunoglobulin single variable domain specific for the ICL present in the chimeric GPCR (the ISVD is used as the "second ligand" present in the "second fusion protein"), avoids problems or limitations that may be associated with the need to provide the desired GPCR in an isolated, appropriately purified form and in the desired conformation for screening and selection purposes, as well as when a native library is used for immunization and display purposes.

[0265] When the second ligand is separate from the second fusion protein, the second ligand can be any ligand or protein that can bind directly to a spanning protein (i.e., a chimeric GPCR of the present invention) and / or form part of a protein complex that can bind to a spanning protein, but is preferably a G protein or G protein complex (particularly in those aspects of the present invention in which an ICL present in a chimeric GPCR of the present invention forms a functional binding site for a G protein or G protein complex). For example, as further described herein, in such aspects of the present invention, the second ligand can be a naturally occurring G protein; e.g., when an arrangement described herein comprising a chimeric GPCR of the present invention is present in a cell, the G protein can be a G protein that is naturally expressed by the cell in which the chimeric GPCR of the present invention is present or expresses. Such a second ligand can also be a semisynthetic or synthetic analog or derivative of a naturally occurring G protein ligand, or, again, when an arrangement of the present invention is present in a cell, the second ligand can be an orthologue of a G protein that is naturally occurring in said cell. Alternatively, if the second ligand is not part of the second fusion protein, the second fusion protein comprises a binding domain or binding unit capable of indirectly binding (as defined herein) to the layer-spanning protein, i.e., a binding domain or binding unit capable of binding to the second ligand and / or a protein complex comprising the second ligand. Again, as further described herein, such a binding domain or binding unit may in particular be an immunoglobulin single variable domain, for example a camelid-derived ISVD.

[0266] As described further herein, in one aspect of the invention, the arrangements described herein comprising and / or employing the chimeric GPCR of the invention may be present in any suitable cell or cell line, and / or the methods of the invention may be carried out using any suitable cell or cell line that suitably expresses the chimeric GPCR of the invention and / or comprises the chimeric GPCR of the invention in an (operable) arrangement present in said cell or cell line. Such a cell or cell line may again be a cell or cell line as described further herein, and may also express a second fusion protein comprising a binding domain or binding unit capable of binding to the ICl present in the chimeric GPCR.

[0267] In aspects of the invention in which the chimeric GPCR of the invention is used as part of an arrangement as described herein and in co-pending applications, it is most preferred that such cells or cell lines also comprise and / or suitably express (or be capable of suitably expressing) the further components of such arrangement, particularly so as to provide an arrangement that is operable in said cell or cell line. The present invention also relates to cells or cell lines that comprise and / or suitably express or are capable of suitably expressing (as defined herein) a first fusion protein as described herein comprising a chimeric GPCR of the invention. The present invention also relates to cells or cell lines that comprise and / or suitably express or are capable of suitably expressing a second fusion protein as described herein. In yet another aspect, the present invention relates to cells or cell lines that comprise and / or suitably express or are capable of suitably expressing both a first fusion protein as described herein comprising a chimeric GPCR of the invention and a second fusion protein as described herein. In aspects and embodiments in which the second ligand does not form part of a second fusion protein, such cells or cell lines may also comprise or suitably express a suitable second ligand, in particular a G protein or an analogue or derivative thereof, as further described herein.

[0268] Also as described herein, in one aspect of the invention, the arrangements described herein comprising and / or employing the chimeric GPCR of the invention may be present in suitable liposomes or vesicles, and / or the methods of the invention may be carried out using liposomes or vesicles suitably comprising the chimeric GPCR of the invention in an (operable) arrangement as described herein. Such vesicles or liposomes may again be vesicles or liposomes as further described herein, and may also comprise a second fusion protein comprising a binding domain or binding unit capable of binding to the ICl present in the chimeric GPCR.

[0269] In aspects of the invention in which the chimeric GPCR of the invention is used as part of an arrangement as described herein and in co-pending applications, most preferably, such liposomes or vesicles also contain further components of such arrangements, particularly to provide an arrangement that is operable in said liposomes or vesicles. The invention also relates to liposomes or vesicles comprising a first fusion protein as described herein, which first fusion protein comprises a chimeric GPCR of the invention. The invention also relates to liposomes or vesicles comprising a second fusion protein as described herein. In yet another aspect, the invention relates to liposomes or vesicles comprising both a first fusion protein as described herein and a second fusion protein as described herein. In aspects and embodiments in which the second ligand does not form part of the second fusion protein, such liposomes or vesicles may also contain a suitable second ligand (which is also preferably an analogue or derivative of a naturally occurring G protein or a synthetic or semi-synthetic G protein, particularly when the ICL present in the chimeric GPCR of the invention forms a functional G protein binding site).

[0270] Accordingly, the present invention contemplates at least three preferred embodiments of methods and arrangements of the present invention in which the chimeric GPCRs of the present invention are used, depending on whether the second ligand is part of a second fusion protein or not, as further described herein and as illustrated by the accompanying non-limiting figures.

[0271] In a first such preferred embodiment (schematically shown in Figure 1), the second binding member of the binding pair is suitably fused or linked (directly or via a suitable linker or spacer) to a second ligand. According to this preferred embodiment, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a transmembrane protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair; a first ligand for a transmembrane protein present in the first environment; and a second ligand for the layer-spanning protein present in a second environment and suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of said binding pair; wherein said layer-spanning protein is a chimeric GPCR of the invention as described herein, and the components of the arrangement are arranged relative to one another (and, where applicable, operably linked and / or associated with one another) in a manner as further described herein. As further described herein, in this embodiment, the second ligand is preferably a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops (as described herein), more preferably a conformation-induced binding domain or binding unit (as described herein), and may in particular be an ISVD, more particularly a conformation-induced ISVD.

[0272] In particular, as further described herein, such an arrangement may comprise the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; and a second fusion protein comprising a second ligand for the layer-spanning protein and the other binding member of said binding pair, the second fusion protein being present in a second environment; wherein said layer-spanning protein is a chimeric GPCR of the invention as described herein, and the components of the arrangement are arranged relative to one another (and, where applicable, operably linked and / or associated with one another) in a manner as further described herein. As further described herein, in this embodiment, the second ligand present in the second fusion protein is preferably a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops (as described herein), and more preferably (as also described herein) a conformation-induced binding domain or binding unit, and in particular may be an ISVD, more particularly a conformation-induced ISVD.

[0273] In a second such preferred embodiment (shown schematically in Figure 2), the second binding member of the binding pair does not bind directly to the transmembrane protein (i.e., the chimeric GPCR), but instead is suitably fused or linked (directly or via a suitable linker or spacer) to a binding domain or binding unit that binds a second ligand (which can then bind to the chimeric GPCR). According to this preferred embodiment, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a second ligand for the transmembrane protein present in the second environment; and a second fusion protein comprising a binding domain or binding unit present in a second environment and capable of binding to a second ligand, the binding domain or binding unit being suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of said binding pair; wherein said layer-spanning protein is a chimeric GPCR of the invention as described herein, and wherein the components of the arrangement are arranged relative to each other (and, where applicable, operably linked and / or associated with each other) in a manner further described herein.

[0274] It will be apparent to those skilled in the art that in this second embodiment, the binding domain or binding unit present in the second fusion protein binds to the chimeric GPCR "indirectly," i.e., by binding to a second ligand that binds to the chimeric GPCR. Furthermore, said binding domain or binding unit is preferably an immunoglobulin single variable domain, as further described herein. Also, in this embodiment, the second ligand may be any suitable ligand for the chimeric GPCR, as further described herein, but is preferably an analogue or derivative of a naturally occurring G protein or a synthetic or semi-synthetic G protein, as described, particularly where the ICL present in the chimeric GPCR of the invention forms a functional G protein binding site.

[0275] In a third preferred embodiment (shown schematically in Figure 3), the second binding member of the binding pair does not bind directly to the transmembrane protein (i.e. the chimeric GPCR), but instead is suitably fused or linked (directly or via a suitable linker or spacer) to a binding domain or binding unit that binds to a protein complex comprising at least a second ligand for the chimeric GPCR (which protein complex may bind to or be bound by the chimeric GPCR and / or may comprise the chimeric GPCR). According to this preferred embodiment, an arrangement for carrying out the method of the present invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a protein complex comprising a second ligand for at least the transmembrane protein, the protein complex being present in a second environment; and a second fusion protein comprising a binding domain or binding unit present in a second environment and capable of binding to the protein complex, said binding domain or binding unit being suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of said binding pair; wherein said layer-spanning protein is a chimeric GPCR of the invention as described herein, and wherein the components of the arrangement are arranged relative to each other (and, where applicable, operably linked and / or associated with each other) in a manner further described herein.

[0276] It will be apparent to those skilled in the art that in this third embodiment, the binding domain or binding unit present in the second fusion protein binds to the chimeric GPCR "indirectly", i.e., by binding to a protein complex comprising a second ligand, which binding domain or binding unit is preferably an immunoglobulin single variable domain, as further described herein, and which second ligand may be any suitable ligand for what may be part of a protein complex, as further described herein, but is preferably a G protein complex, as described, particularly when the ICL present in the chimeric GPCR of the invention forms a functional G protein binding site.

[0277] More generally, the arrangements described herein that comprise the chimeric GPCRs of the invention typically and preferably comprise at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a second ligand for the transmembrane protein present in the second environment; and a second fusion protein comprising the other binding member of said binding pair (i.e., such that said other member of the binding pair is also present in the second environment); wherein said layer-spanning protein is a chimeric GPCR of the invention as described herein, and the components of the arrangement are arranged relative to one another (and, where applicable, operably linked and / or associated with one another) in a manner as further described herein, in particular In the first preferred embodiment described herein, the second fusion protein comprises the other binding member of said binding pair and a second ligand; In a second preferred embodiment described herein, the second fusion protein comprises a binding domain or binding unit capable of binding to the other binding member of said binding pair and to a second ligand; and In a third preferred embodiment described herein, the second fusion protein comprises a binding domain or binding unit capable of binding to a protein complex comprising the other binding member of said binding pair and at least a second ligand.

[0278] The invention will now be illustrated by the further description herein, the experimental section below, and the accompanying non-limiting figures.

[0279] It will be apparent to those skilled in the art from the drawings and further description herein that several components of the chimeric GPCR-containing arrangements of the invention (e.g., boundary layer, chimeric GPCR, binding pair, optional linker, and first ligand) are present in the various aspects and embodiments of the invention discussed herein. Thus, when a detailed description of any such component (including any preference for any such component) is given herein, it should be understood that such description applies to all aspects and embodiments of the invention in which such component is present or used, unless expressly stated otherwise herein.

[0280] In the methods and arrangements of the present invention, the boundary layer (1) may be any layer (e.g. a wall or membrane) suitable to separate a first environment [A] from a second environment [B] (in a suitable in vitro system or in a suitable in vivo system).

[0281] For example, in a preferred embodiment of the present invention, in which the method of the present invention is carried out in a suitable cell or cell line (as further described herein), the boundary layer (1) is the cell membrane or cell wall of the cell or cell line used in the method of the present invention. In this embodiment, environment [A] is preferably the extracellular environment, and environment [B] is preferably the intracellular environment. Also in this embodiment, the first ligand (3) is preferably present in the extracellular environment, and the second ligand (4) is preferably present in the intracellular environment. Also, the first and second binding members (6) and (7) and the second fusion protein are preferably present in the intracellular environment.

[0282] In another preferred embodiment of the present invention, in which the method of the present invention is carried out in a suitable vesicle or liposome (as further described herein), the boundary layer (1) is the membrane or wall of the vesicle or liposome. In this embodiment, environment [A] is preferably the external environment of the vesicle or liposome, and environment [B] is preferably the internal environment of the vesicle or liposome. Also in this embodiment, the first ligand (3) is preferably present in the external environment of the vesicle or liposome, and the second ligand (4) is preferably present in the internal environment of the vesicle or liposome. The first and second binding members (6) and (7) and the second fusion protein are also preferably present in the internal environment of the vesicle or liposome.

[0283] However, while the present invention in some preferred embodiments is practiced using cells, liposomes or other suitable vesicles, it should be understood that the present invention in its broadest sense is not limited to the use of cells or vesicles, but may be practiced in any other suitable arrangement in which a boundary layer (1) is used to appropriately separate a first environment [A] from a second environment [B]. For example, the boundary layer may be a part or fragment of the cell wall or cell membrane present in a membrane extract, e.g., a membrane extract obtained from whole cells by techniques known per se, such as suitable osmotic and / or mechanical techniques known per se.

[0284] Thus, boundary layer (1) can be any suitable layer, wall, or membrane, particularly a biological wall or membrane (e.g., a cell wall or cell membrane, or a part or fragment thereof), or the wall or membrane of a liposome or other suitable vesicle. In particular, boundary layer (1) can be a suitable lipid bilayer, for example, a phospholipid bilayer. When boundary layer (1) is the wall or membrane of a vesicle or liposome, boundary layer (1) can be unilamellar or multilamellar. Also, as further described herein, when boundary layer (1) is a cell membrane or cell wall, boundary layer (1) is preferably the wall or membrane of a cell or cell line that suitably expresses (as defined herein) layer-spanning protein (2) (i.e., the chimeric GPCR of the present invention), in particular, that suitably expresses the (first) fusion protein described herein comprising layer-spanning protein (2).

[0285] As shown schematically by non-limiting Figures 1, 2 and 3, the boundary layer (1) contains a layer-spanning protein (2) (i.e., a chimeric GPCR of the present invention), which a first binding site (8) for a first ligand (3) (i.e., an extracellular binding site of the chimeric GPCR of the invention, as described herein) protrudes (as defined herein) into a first environment [A] (i.e., such that when a first ligand (3) is present in the first environment [A], the first binding site (8) is accessible for binding by said first ligand); Also, a second binding site (9) for a second ligand (4) (i.e., an intracellular binding site of the chimeric GPCR of the invention, as described herein) projects (as defined herein) into the second environment [B] (i.e., such that when the second ligand (4) is present in the second environment [B], the second binding site (9) is accessible for binding by said second ligand); Thus, it penetrates the boundary layer (1).

[0286] In the methods and arrangements of the present invention, the chimeric GPCR of the present invention (which serves as a layer-spanning protein (2) in said methods and arrangements) penetrates the boundary layer (1) (and / or is prepared and / or arranged in such a manner relative to the boundary layer) such that at least one portion of the amino acid sequence of the chimeric GPCR (in particular at least one ECL, and preferably all ECLs) protrudes (as defined herein) from the boundary layer (1) into a first environment [A], and at least one other portion of the amino acid sequence of the chimeric GPCR (in particular at least one ICL, and preferably all ICLs) protrudes (as defined herein) from the boundary layer (1) into a second environment [B]. In this context, when a portion of the amino acid sequence of a chimeric GPCR of the invention (e.g., the ECL or ICL, respectively) is said to "protrude" from the boundary layer (1) into an environment (i.e., into a first environment [A] or a second environment [B]), this should generally be understood to mean that said portion of the sequence is exposed to said environment and / or is accessible for binding by a ligand, compound, or other chemical present in said environment. Thus, in methods and arrangements using a chimeric GPCR of the invention, at least one portion of the amino acid sequence of the chimeric GPCR (e.g., an epitope or binding site) should be accessible for binding by a ligand, compound, or other chemical present in the first environment (particularly, binding by a first ligand (3)), and at least one other portion of the amino acid sequence of the layer-spanning protein (e.g., another epitope or binding site) should be accessible for binding by a ligand, compound, or other chemical present in the second environment (particularly, binding by a second ligand (4)).In this regard, it should also be noted that the phrase "accessible for binding" should generally be considered to mean that a ligand, compound, or other chemical present in the relevant environment can bind to a binding pocket or binding site on or within a chimeric GPCR of the present invention, even if the actual binding site or binding pocket is located deeper (or deeper) in the structure of the transmembrane protein (even if the actual binding site or binding pocket is located within a portion of the transmembrane protein that does not physically protrude beyond the boundary layer itself). See, for example, the paper by Chevillard (cited herein), which shows that the binding site of a GPCR for a fragment used in the FBDD screening technique is located deep within the GPCR structure (see, e.g., Figure 2 on page 1120), not on the surface of the GPCR, but nevertheless accessible for fragment binding. Theories of GPCR structure, GPCR signaling mechanisms, and GPCR ligand binding sites are also referenced from several other scientific papers cited herein.

[0287] Also, in this specification and claims, when any binding domain, binding unit, epitope, binding site, ligand, protein, or other compound or chemical or other structural entity (e.g., a protein complex) is said to be "present" in an environment (i.e., a first environment [A] or a second environment [B]), this should generally be understood to mean that the binding domain, binding unit, epitope, binding site, ligand, protein, or other compound or chemical or structural entity is exposed to the environment and / or is accessible for binding by another domain, ligand, protein, or compound present in the environment. Thus, for example, a compound or ligand present in the environment may be "free-floating" in the environment (i.e., not bound or immobilized to any other protein or structure), immobilized in the boundary layer, or fused to another protein (which may be immobilized in the boundary layer). Similarly, a binding domain or binding unit present in an environment may be part of a larger protein or structure (e.g., a fusion protein), which may be free-floating in the environment or fixed to a boundary layer or other structure, so long as the binding domain or binding unit is accessible for binding by another domain, ligand, protein, or compound present in the environment. Also, an epitope or binding site present in the environment may be part of a larger protein or structure, which may be free-floating in the environment or fixed to a boundary layer or other structure, so long as the epitope or binding site is accessible for binding by another domain, ligand, protein, or compound present in the environment.

[0288] The one or more portions of the chimeric GPCR of the present invention that protrude into the first environment [A] may be any loop, epitope (linear or conformational), binding site or other portion(s) of the amino acid sequence of the spanning protein; similarly, the one or more portions of the spanning protein that protrude into the second environment [B] may be any loop, epitope (linear or conformational), binding site or other portion(s) of the amino acid sequence of the spanning protein (different from the portion(s) that protrude into the first environment); however, as described herein, preferably at least one ECL (and more preferably all ECLs, particularly extracellular binding sites) of the chimeric GPCR of the present invention protrudes into the first environment [A], and preferably at least one ICL (and more preferably all ICLs, particularly intracellular binding sites) of the chimeric GPCR of the present invention protrudes into the second environment [B].

[0289] In general, the layer-spanning protein (2) (i.e., the chimeric GPCR of the present invention) is usually attached and / or anchored to the boundary layer (1) in a manner known per se, for example, for GPCRs. As further described herein, this can be achieved, for example, by appropriately expressing a nucleotide sequence or nucleic acid (as defined herein) that expresses the first fusion protein in a suitable host cell, such that the chimeric GPCR of the present invention is appropriately anchored to the wall or membrane of said cell. When the method of the present invention is carried out using liposomes or vesicles, this can be achieved by appropriately forming said liposomes or vesicles in the presence of the first fusion protein, such that the chimeric GPCR is appropriately anchored to the wall or membrane of the liposome or vesicle.

[0290] Also, when the methods of the invention are carried out in a cell, the arrangement of the N- and C-termini of the chimeric GPCR of the invention relative to the wall or membrane of the cell used is preferably the same as the arrangement of said termini of the first and second GPCRs from which the ECL and ICL, respectively, are derived (i.e., when said first and second GPCRs are in their natural cellular environment). This also applies when the C-terminus of the chimeric GPCR is derived from the second GPCR instead of the first GPCR.

[0291] When the methods of the present invention are carried out in liposomes or vesicles, the liposomes or vesicles can be liposomes / vesicles in which the chimeric GPCRs of the present invention are arranged in a manner that is essentially the same as the way the first and second GPCRs from which the ECL and ICL, respectively, are arranged relative to the cell wall or membrane in their natural environment (i.e., with the N-terminus and extracellular loop(s) protruding to the outside of the vesicle and the C-terminus and intracellular loop(s) protruding to the inside of the vesicle), and a mixture of vesicles / liposomes in which the proteins are arranged in the opposite manner. Typically, this will not affect the operation of the systems or mechanisms described herein.

[0292] As is known for naturally occurring GPCRs, chimeric GPCRs of the invention should most preferably exist in (i.e., exhibit) two or more conformations (e.g., basal state / conformation, active state / conformation and / or inactive state / conformation, and / or ligand-bound or ligand-free conformation) and / or be capable of undergoing conformational change (particularly a functional conformational change). In particular, chimeric GPCRs of the invention should be capable of exhibiting at least one functional conformation and at least one non-functional conformation (e.g., basal conformation) and / or be capable of undergoing a conformational change from a non-functional conformation to a functional conformation; more particularly, be capable of exhibiting an active (or more active) conformation and an inactive (or less active) conformation and / or be capable of undergoing a conformational change from an inactive (or less active) conformation to an active (or more active) conformation. Furthermore, the chimeric GPCR is preferably capable of exhibiting at least one ligand-bound (particularly agonist-bound) conformation and at least one ligand-free conformation. More specifically, the chimeric GPCR may be capable of exhibiting at least one ligand-bound (particularly agonist-bound) conformation that is an active or functional conformation.

[0293] As described herein, certain classes of functional conformations of (transmembrane) proteins (e.g., certain GPCRs) are referred to / defined as "druggable conformations." Thus, in one particular aspect, a chimeric GPCR can exhibit at least one such druggable conformation (often an active conformation, although the invention is not limited to the use of druggable conformations that are active conformations), and at least one conformation that is not a druggable conformation (often an inactive conformation) and / or a conformation that can undergo a conformational change from a non-druggable conformation to a druggable conformation.

[0294] In particular, the chimeric GPCR preferably undergoes a conformational change upon binding of a ligand (particularly an agonist) to the protein. This conformational change upon ligand binding can be, for example, from an active to an inactive conformation, or from a functional to a non-functional conformation, but is preferably from a non-functional to a functional conformation and / or from an inactive to an active conformation. In certain embodiments, it is a change from a non-druggable conformation to a druggable conformation.

[0295] For example, the conformational change of the chimeric GPCR may be a change from a conformation that is essentially incapable of binding a G protein to a conformation that binds a G protein (or is capable of being bound by a G protein), and in particular, a change from a conformation that is essentially incapable (or is less capable) of binding a conformation-induced binding domain or binding unit to a conformation that binds a conformation-induced binding domain or binding unit (or is more capable of being bound by a conformation-induced binding domain or binding unit).

[0296] As described herein, a ligand capable of inducing a conformational change in a GPCR from a non-functional state to a functional state (e.g., from an inactive state, such as a basal state, to an active state) is also referred to herein as an "agonist" of said GPCR. In particular, an "agonist" of a GPCR may be capable of inducing a conformational change from a conformation that is essentially incapable of binding a G protein to a conformation that binds a G protein.

[0297] In a preferred embodiment, the chimeric GPCR undergoes (or is capable of undergoing) a conformational change (as described herein) when a first ligand (3) binds to the chimeric GPCR; conversely, the first ligand (3) can induce a conformational change in the chimeric GPCR when the first ligand (3) binds to the chimeric GPCR (and / or the present invention can be used to identify such a first ligand). In a more preferred embodiment, the conformational change is a change from an inactive or less active state to a functional or(more) active state, and the first ligand (3) used can induce a conformational change in the chimeric GPCR from an inactive or less active state to a functional or(more) active state when the first ligand binds to the chimeric GPCR. Furthermore, the conformational change upon binding of the first ligand (3) can be a change from a conformation that is essentially incapable of binding a G protein to a conformation that binds a G protein.

[0298] As further described herein, the chimeric GPCR may also be capable of forming a complex with a first and a second ligand. In this regard, it is known that most naturally occurring GPCRs form complexes with an extracellular ligand and a G protein (which is the most common natural intracellular ligand for the GPCR), and that such complexes are stabilized by binding of the G protein to an intracellular conformational epitope of the GPCR. Similarly, in the present invention, the second ligand preferably stabilizes the complex of the chimeric GPCR, the first ligand, and the second ligand. As described herein, for this purpose, the second ligand may be a G protein that associates with the second GPCR (i.e., the GPCR from which the ICL of the chimeric GPCR is derived) in its natural environment (i.e., during GPCR signaling), another naturally occurring G protein that can bind to the chimeric GPCR and stabilize the formation of said complex, or a synthetic or semi-synthetic analog or derivative of a GPCR that can bind to the chimeric GPCR and stabilize the formation of said complex. Also as described herein, the second ligand is preferably a binding domain or binding unit capable of binding to a binding site of said chimeric GPCR comprising at least one of said intracellular loops (as described herein), more preferably a conformation-induced binding domain or binding unit (as described herein), particularly an ISVD, more particularly a conformation-induced ISVD.

[0299] As further described herein and shown diagrammatically in FIGS. 1-3, in the arrangements of the present invention, the layer-spanning protein (2) (i.e., the chimeric GPCR of the present invention) is typically and preferably fused or linked to a first member (6) of a binding pair (6 / 7), either directly or via a suitable spacer or linker (10), to form a first fusion protein. Additionally, the second binding member (7) of the binding pair (6 / 7) is typically and preferably part of a second fusion protein distinct from the first fusion protein, which second fusion protein is also further described herein. The first fusion protein, the second fusion protein (in its various formats as described herein), nucleotide sequences and / or nucleic acids encoding the first or second fusion proteins, and cells, cell lines, or other host cells or organisms that express (particularly suitably expressed as described herein) or are (suitably) capable of expressing) the first and / or second fusion proteins (and preferably both), as well as their various uses as further described herein, form further aspects of the present invention.

[0300] The binding pair (6 / 7) used in the arrangements employing the chimeric GPCRs of the present invention generally comprises at least two separate binding members (6) and (7), also referred to herein as the "first binding member" and the "second binding member," respectively. The binding pair (6 / 7) and its respective members (6) and (7) should be such that the binding pair (6 / 7) is capable of generating a detectable signal when members (6) and (7) contact or are in close proximity to one another. Such a detectable signal can be, for example, a luminescent, fluorescent, or chemiluminescent signal.

[0301] In a particularly preferred aspect, when the methods described herein are carried out in a suitable cell, the first member (6) and the second member (7) of the binding pair (6 / 7) are preferably both polypeptides, proteins, amino acid sequences or other chemical entities obtainable by suitable expression of nucleic acid or nucleotide sequences encoding same, preferably in the cell used in the methods of the invention.

[0302] The first and second binding members may be part of a suitable reporter assay, or may be any other pair of domains or units that are capable of generating a detectable signal when they contact or are in close proximity to each other, such as an enzyme and substrate combination, or binding pairs commonly used in experimental studies of protein-protein interactions. As noted, in order to reduce the level of baseline / background signal, it is preferred that the two members of the binding pair by themselves do not have substantial binding affinity for each other.

[0303] Some preferred, but non-limiting examples of suitable binding pairs are pGFP and Promega's NanoBiT® system, the latter being particularly preferred because the large and small BiTs that make up the NanoBiT® system have, by themselves, low affinity for each other.

[0304] The first binding member (6) can be fused to the chimeric GPCR of the invention in any suitable manner, so long as the resulting first fusion protein allows the first member (6) to contact (or otherwise be in suitable close proximity to) the second member (7) of the binding pair (6 / 7) when the second fusion protein formed by the second ligand (4) and the second member (7) binds to the chimeric GPCR of the invention via the second binding site (9) (i.e., the intracellular binding site as defined herein). Also, preferably, the first binding member (6) is fused or linked to the chimeric GPCR of the invention in a manner that does not substantially affect the conformation and / or conformational changes that the chimeric GPCR of the invention may undergo under the conditions used to practice the methods of the invention.

[0305] Thus, while the present invention does not generally preclude the first binding member (6) from being directly fused or linked to a chimeric GPCR of the present invention, it is generally preferred that the first binding member (6) be fused or linked to the chimeric GPCR via a suitable linker (10). For example, the use of a flexible linker having a total length of between 5 and 50 amino acids, preferably between 10 and 30 amino acids, e.g., about 15-20 amino acids, is generally preferred. Suitable linkers will be apparent to those skilled in the art and include GlySer linkers (e.g., 15GS linkers).

[0306] In the present invention, the first and second binding members of binding pair (6 / 7) will be in the same environment (as defined herein) relative to boundary layer (1) such that they can contact or come into close proximity with each other (as further described herein) and thereby generate a detectable signal. In particular, as shown schematically in Figures 1, 2, and 3, the first and second binding members of binding pair (6 / 7) will be in the same environment (as defined herein) as second binding site (9) (i.e., intracellular binding site, as defined herein) of a chimeric GPCR of the present invention (also relative to boundary layer (1)) such that the first and second binding members of binding pair (6 / 7) can contact when a second fusion protein binds to said binding site directly (as shown in Figure 1) or indirectly (as shown in Figures 2 and 3). To this end, first bindin...

Claims

1. (i) Structure: [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence] wherein the EC and TM form a functional ligand binding site, the EC and TM being derived from a first GPCR and the IC being derived from a second GPCR (different from the first); (ii) an immunoglobulin single variable domain capable of binding to at least one of said ICs derived from said second GPCR.

2. The composition of claim 1 , wherein the first GPCR and the second GPCR both belong to class A.

3. 3. The composition of claim 2, wherein the immunoglobulin single variable domain is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to the chimeric GPCR.

4. The composition of any one of claims 1 to 3, which is a cellular composition.

5. The composition of any one of claims 1 to 4, further comprising a compound or ligand capable of binding to the extracellular binding site of the chimeric GPCR.

6. 1. A method of forming a complex between a chimeric GPCR, an immunoglobulin single variable domain, and a compound or ligand capable of binding to an extracellular binding site of said chimeric GPCR, comprising: a) providing a composition according to any one of claims 1 to 4; b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the immunoglobulin single variable domain to bind to the binding site of the chimeric GPCR comprising at least one of the ICs, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; A method comprising:

7. 1. A method for identifying and / or producing a compound or ligand capable of binding to an extracellular binding site of a GPCR, comprising: a) providing a composition according to any one of claims 1 to 4; b) contacting the composition with one or more test compounds or ligands under conditions that (i) allow the immunoglobulin single variable domain to bind to the binding site of the chimeric GPCR comprising at least one of the ICs, and (ii) allow the test compound to bind to the extracellular binding site of the chimeric GPCR; c) assessing whether each of the test compounds or ligands, and / or which of the test compounds or ligands, binds to the chimeric GPCR in the composition; and optionally, d) selecting said test compound or ligand that binds to said chimeric GPCR in said composition; A method comprising:

8. 1. A method for identifying and / or producing a compound or ligand capable of binding to an active conformation of a GPCR, comprising: a) providing a composition according to any one of claims 1 to 4, wherein the chimeric GPCR comprises the EC and TM of the GPCR, and the immunoglobulin single variable domain is a ligand capable of stabilising and / or inducing an active conformational state of the chimeric GPCR upon binding to the chimeric GPCR; b) contacting said composition with one or more test compounds or ligands under conditions that (i) allow said immunoglobulin single variable domain to bind to said binding site of said chimeric GPCR comprising at least one of said ICs, and (ii) allow said test compound to bind to an extracellular binding site of said chimeric GPCR; c) assessing whether each of the test compounds or ligands, and / or which of the test compounds or ligands, binds to the chimeric GPCR in the composition; and optionally, d) selecting said test compound or ligand that binds to said chimeric GPCR in said composition; A method comprising:

9. 9. The method of claim 7 or 8, wherein the immunoglobulin single variable domain is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to the chimeric GPCR.

10. At least the following components: a boundary layer separating the first environment from the second environment; structure: [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence] wherein the EC and TM form a functional ligand binding site, the EC and TM are derived from a first GPCR and the IC is derived from a second GPCR (different from the first); a ligand for the chimeric GPCR present in the first environment; an immunoglobulin single variable domain present in the second environment and capable of binding to at least one of the ICs derived from the second GPCR; and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; Including, arrangement.

11. 11. The arrangement of claim 10, wherein the chimeric GPCR is fused or linked, either directly or via a suitable spacer or linker, to the first binding member of the binding pair, and the immunoglobulin single variable domain is fused or linked, either directly or via a suitable spacer or linker, to the first binding member of the binding pair.

12. 12. The arrangement of claim 10 or 11, wherein the immunoglobulin single variable domain is capable of stabilizing and / or inducing a functional and / or active conformational state of the chimeric GPCR upon binding to the chimeric GPCR.

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