Screening methods and assays for use with transmembrane proteins, particularly those associated with GPCRs

Abstract

The present invention provides methods and arrangements for screening membrane proteins, the arrangements comprising a first fusion protein comprising a membrane protein present in a boundary layer, such as the wall of a cell, liposome or vesicle, fused to one member of a binding pair, and a second fusion protein comprising an intracellular ligand of said membrane protein fused to another member of the binding pair, the binding pair being capable of generating a detectable signal.

Description

Technical Field

[0001] The present invention relates to methods and tools that can be used for assays, screening, and attempts at drug discovery and development.

[0002] In particular, the present invention relates to screening and assay techniques involving the use of membrane proteins (i.e., as targets to be screened, e.g., to discover candidate compounds that act on said targets), and to methods and tools that can be used in attempts to discover, generate, optimize, and / or develop therapeutic, prophylactic, and diagnostic agents directed to (i.e., having specificity for) membrane proteins. The present invention further relates to methods for making tools that can be used in screening and assay techniques.

[0003] Advantageously, the methods and tools of the present invention can be used in screening and assay techniques involving the use of membrane proteins that can assume or are present in multiple conformations (e.g., but not limited to, active and inactive conformations), and in attempts to discover, generate, optimize, and / or develop therapeutic, prophylactic, and diagnostic agents directed to such membrane proteins. Such membrane proteins include, but are not limited to, transmembrane proteins such as GPCRs and other cell surface receptors.

[0004] In a particularly preferred but non-limiting aspect, the methods and tools of the present invention can be used in screening and assay techniques involving the use of membrane proteins that can undergo a conformational change (again, e.g., but not limited to, a conformational change from an inactive conformation to an active conformation) in response to the binding of a ligand to said protein, and in attempts to discover, generate, optimize, and / or develop therapeutic, prophylactic, and diagnostic agents directed to such membrane proteins. Again, such membrane proteins can be cell surface receptors such as GPCRs.

[0005] The present invention generally provides a method that can be used for performing an assay (i.e., for a given compound or ligand) or for screening purposes (i.e., for screening a group, series or library of compounds or ligands to identify a “hit” against a target). The present invention also provides an arrangement that can be used in said method, i.e., as a system or setup for performing said assay or screening. Said arrangement includes the components described herein. Said components can also be provided or established as a kit of parts, and such a kit of parts forms a further aspect of the present invention. The present invention also provides a method for identifying and fabricating elements of such an arrangement, as well as a method for assembling such an arrangement.

[0006] The methods and arrangements described herein can generally be used for testing a (known) compound or ligand for one or more of its properties (i.e., those properties that can be determined using the methods described herein), and / or for identifying a compound or ligand having such desired properties (i.e., from a group, series or library of compounds or ligands). These compounds or ligands can be any desired and / or suitable compounds or ligands, such as, 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. Also, compounds identified using the methods of the present invention (i.e., “hits” from such screening) can be used as a starting point for further drug discovery and drug development efforts (e.g., using well-known techniques of so-called “hits-to-leads” chemistry), and such further efforts can also involve the use of the methods of the present invention (e.g., as a functional assay, or an assay used for quality control purposes).

[0007] 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 also collectively referred to herein as "compounds of the present invention" and form further aspects of the present invention. Such compounds can be, for example, so-called "hits", "leads", "development candidates", "pre-clinical compounds", "clinical candidates", or commercial compounds or products, depending on their stage of development and the specific terms used by the company or entity developing and / or commercializing them, which will be apparent to those skilled in the art.

[0008] Advantageously, compared to conventional radioligand assays or functional assays, the methods and assays of the present invention do not require the use of labeled antagonists (e.g., fluorescently or radioactively labeled antagonists), and thus can also be applied to membrane proteins for which antagonists are not available or are not known. Also, as further described herein, the methods and assays of the present invention can (depending on the specific target and assay used) identify and / or characterize allosteric agonists (both positive and negative), antagonists and / or inverse agonists.

[0009] Other features, aspects, embodiments, uses and advantages of the present invention will become apparent from the further description herein.

Background Art

[0010] Membrane proteins (such as cell surface receptors including GPCRs), as well as assay and screening techniques for membrane proteins, 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. See standard handbooks and further prior art cited herein.

[0011] As is well known in the field of protein dynamics, most proteins are not static objects whose function is determined only by their primary, secondary, tertiary, and quaternary structures (when the protein contains two or more polypeptide chains), but rather are flexible structures that can transition between different conformational states such that the protein can exist in equilibrium between these different states (also called "conformational changes"). Some of these states may be functional and / or active, while other states may be basal states (which may or may not exhibit a certain level of constitutive activity), essentially inactive states, and / or states that are less active relative to a relatively high functional or active state. The geometric shape of different epitopes, binding sites (including ligand-binding sites), and / or catalytic sites that may be present within or on the protein can 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 the interaction with the ligand(s) associated with the binding site may be reduced compared to a more active conformational state.

[0012] For some protein / ligand combinations, it is also known that ligand binding to a protein can change its conformation (e.g., from an inactive / low-activity conformation to an active / higher-activity conformation) and / or shift the equilibrium from an inactive / low-activity conformation to an active / higher-activity conformation. Binding of a ligand to one binding site on a protein can make another binding site on the protein more accessible to its associated ligand(s), and / or result in an increase in the affinity of the other binding site(s) for said ligand(s), and / or shift the equilibrium from a conformation with lower affinity of the other binding site(s) for said ligand(s) to a conformation with higher affinity of the other binding site(s) for said ligand(s). For example, for some transmembrane proteins such as GPCRs, binding of an extracellular ligand to the extracellular binding site of the protein can increase the affinity of the intracellular binding site for an intracellular ligand (e.g., the affinity for the interaction between a G protein and the G-protein binding site of the GPCR), and vice versa. This change in the binding affinity for an intracellular ligand after binding of an extracellular ligand, and subsequent binding of the intracellular ligand to the intracellular binding site, is part of the means by which the protein transmits extracellular signals.

[0013] Generally, as further described herein, for receptor proteins that can undergo conformational changes, an “agonist” of the receptor can shift the conformational equilibrium from an inactive state (or one or more low-activity states) to an active state (or one or more higher-activity states), while an “antagonist” of the receptor can do the opposite.

[0014] A protein may form a complex with two ligands that bind to two different binding sites of the protein, and the interaction between the protein and each of the ligands 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, the binding of one or both of the ligands may shift the protein's conformational equilibrium towards the formation and / or stabilization of this complex. See, for example, International Publication No. WO 2012 / 007593, cited below.

[0015] Considering that the recognized "overall" state of such a protein is largely governed by the (statistical) distribution of the protein over its various possible conformations, and thus by the equilibrium existing between these conformational states, when, in this specification or in the claims, a protein 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 the mechanism or situation by which the protein's conformational equilibrium shifts towards the said conformation (i.e., under the particular conditions used, such as those used for screening or related assays). Similarly, when a ligand is said to induce a conformational change of a protein to a particular conformation (i.e., from one or more other conformations), this includes the mechanism or situation by which the binding of the ligand shifts the protein's conformational equilibrium towards the said conformation (i.e., under the particular conditions used, such as those used for screening or related assays).

[0016] However, while any one of the mechanisms (or any combination thereof) described herein may be involved in the practice of the invention at any given time (e.g., depending also on the particular protein and / or ligand(s) to which the invention is applied), it should also be noted that the invention in its broadest sense is not limited to any particular mechanism, explanation, or hypothesis, so long as the application of the invention to a particular target or protein provides the technical effect(s) outlined herein.

[0017] One problem in screening for compounds directed to a membrane protein that exists in multiple conformations is that the correct conformation of the protein can be lost when the protein is expressed or used in isolation from its native environment (even when it is feasible or possible to express the protein and ensure proper folding outside of its cellular environment). Also, it can be difficult to ensure that the protein is in its desired conformation (often a functional conformation such as its active conformation) under the conditions used for screening. Further, there may be a need to achieve or an advantage in achieving a shift in the protein's conformational equilibrium to a conformation state (such as an active state or a state where the relevant binding site has a more accessible and / or more favorable geometry for assay or screening purposes) that is more suitable for screening or assay purposes. As further described herein, such conformations are also referred to as "drugable" conformations, and according to a preferred embodiment of the invention, means are applied (as further described herein) to ensure that the protein is in such a drugable conformation and / or to ensure that the protein's conformational equilibrium is shifted to a more drugable conformation when the methods of the invention are carried out.

[0018] For example, International Publication Nos. 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 for the purpose of determining the structure of GPCRs and for drug screening and discovery to stabilize specific conformational states of GPCRs. In these references, for example, in the conformation that occurs when an activating ligand (agonist) binds to the extracellular side of a GPCR such that the GPCR can activate a heterotrimeric G protein, a VHH domain that can stabilize the GPCR in a desired conformation, particularly a (more) drugable conformation, such as a functional state and / or an active state, is used. 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; and Rasmussen et al., Nature, 2011, 469(7329):175-180, as well as additional references cited herein. The VHH domain that can be used to stabilize a desired conformation of a membrane protein such as a GPCR is also referred to herein as a Confobody [Confobody (trademark) is a registered trademark of Confo Therapeutics, Ghent, Belgium].

[0019] Non-limiting examples of some of the conformational antibodies that can bind to intracellular epitopes of GPCRs and can be used (and can also be used in the present invention) to stabilize GPCRs in a desired conformation are CA2764, CA3431, CA3413, CA2780, CA2765, CA2761, CA3475, CA2770, CA3472, CA3420, CA3433, CA3434, CA3484, CA2760, CA2773, CA3477, CA2774, CA2768, CA3424, CA2767, CA2786, CA3422, CA2763, CA2772, CA2771, CA2769, CA2782, CA2783 and CA2784 VHHs (see, for example, WO 2012 / 007593, Tables 1 and 2 and SEQ ID NOs: 1-29); CA5669, Nb9-1, Nb9-8, XA8633 and CA4910 VHHs (see, for example, Tables 1 and 2 of WO 2014 / 118297 and SEQ ID NOs: 15, 16, 17, 19, and 20); 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-E1, Nb_A2, Nb_B4, Nb_D3, Nb_D1 and Nb_H1 VHHs (see, for example, Tables 1 and 2 of WO 2014 / 122183 and SEQ ID NOs: 1-19), and XA8639, XA8635, XA8727 and XA9644 VHHs (see, for example, WO 2015 / 121092, Tables 2 and 3 and SEQ ID NOs: 2-6 and 74).

[0020] Non-limiting examples of some of the VHHs that can bind to G proteins are CA4435, CA4433, CA4436, CA4437, CA4440 and CA4441 (see, for example, Tables 2 and 3 of WO 2012 / 175643007593 and SEQ ID NOs: 1-6). SUMMARY OF THE INVENTION

[0021] As further described herein, the present invention generally provides improved screening methods and assay techniques that can be used to discover and develop compounds (i.e., intended to have specificity for and / or target one or more membrane proteins, e.g., for therapeutic, prophylactic, and / or diagnostic purposes) for membrane proteins (e.g., for identification, generation, testing, and optimization). Preferably, such compounds are specific (i.e., selective) for one particular membrane protein as compared to other (closely related) membrane proteins.

[0022] Compounds identified and / or developed using the methods of the present invention can be used to modulate (as defined herein) membrane proteins, their signaling, and / or biological functions, pathways, and / or mechanisms in which the membrane proteins or their signaling are 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 positive and negative allosteric modulators) of the membrane proteins and / or signaling, pathways, and / or physiological and / or biological mechanisms in which the membrane proteins are involved.

[0023] The present invention can be used to discover and develop compounds for membrane proteins that are integral membrane proteins or surface membrane proteins in their native environment. The present invention can be used, in particular, to discover and develop compounds for transmembrane proteins, as further described herein. In one specific but non-limiting aspect, the compounds discovered and / or developed using the present invention are directed to receptors, particularly cell surface receptors. As further described herein, the transmembrane protein can in particular be a membrane protein having a plurality of passes through the membrane, such as a 7TM or GPCR. [In this regard, it should generally be noted that in the art, the terms "7TM receptor" and "7TM" are often used interchangeably with "GPCR". However, according to the IUPHAR database, there are some 7TM receptors that do not signal via G proteins. For the purposes of this specification and the claims, the terms "GPCR" and "7TM" are used interchangeably herein and include all transmembrane proteins having seven transmembrane domains, particularly transmembrane receptors, regardless of their intracellular signaling cascade or signaling mechanism. However, it should be understood that throughout this specification and the claims, 7TMs that signal through G proteins are a preferred embodiment of the present invention.]

[0024] Typically, the compounds discovered and / or developed using the present invention target membrane proteins that are expressed and / or exposed on the surface of cells when in their natural environment, and in particular target membrane proteins expressed by or in cells present within the body of a subject being treated with a compound 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 kind of compound suitable for its intended use, which is often an use 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 antibody fragments (such as ScFv and diabodies, and other compounds or constructs containing one or more VH, VL, and / or VHH domains), compounds based on alphabodies (trademark) and avimers, scaffolds based on PDZ domains, protein A domains (such as Affibodies (trademark)), ankyrin repeats (such as DARPins (trademark)), fibronectin (such as Adnectins (trademark)) and lipocalins (such as Anticalins (trademark)) and other protein scaffolds, and 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, Vol. 23:1257; and Ulrich et al., Comb Chem High Throughput Screen, 2006, 9(8):619-32.

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

[0027] The methods and techniques of the present invention can also be used for the purposes of so-called "fragment-based drug discovery" or "FBDD" (also known as "fragment-based lead discovery" or "FBLD"). See, for example, the 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 Description of the Drawings

[0028]

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[0029] The present invention is described herein with reference to specific embodiments and with reference to specific non-limiting examples and drawings. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and non-limiting. In the drawings, the sizes of some of the elements may be exaggerated and may not be drawn to scale for illustrative purposes. When the term "comprising" is used in this specification and the claims, other elements or steps are not excluded. When an indefinite or definite article is used in reference to a singular noun (e.g., "a" or "an", "the"), the plural form of that noun is included unless specifically stated otherwise. Also, the terms first, second, third, etc. in this specification and the claims are used to distinguish similar elements and are not necessarily used to describe an order or time sequence. The terms so used are interchangeable under appropriate circumstances, and it should be understood that the embodiments of the invention described herein may operate in an order 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 as commonly understood by those of ordinary skill in the art. In general, nomenclature used in connection with, and the laboratory procedures and techniques of, molecular and cell biology, structural biology, biophysics, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly employed in the art. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Methods for protein purification, and enzymatic reactions and assays, can be performed according to commonly used procedures in the art. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. 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 one of skill with a general dictionary of many of the terms used in this disclosure. The methods and techniques of the present invention, unless otherwise indicated, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements up to 2002); up, Biomolecular crystallography: principles, Practice and Applications to Structural Biology, 1st ed., Garland Science, Taylor & Francis Group, LLC, an informa Business, N.Y. (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 a polymeric form of amino acids of any length, including coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Throughout the application, the standard one-letter notation for amino acids is used. Typically, the term "amino acid" refers to "proteinogenic amino acids", i.e., amino acids that are naturally present in proteins. In particular, the amino acids are in the L-isomeric 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 either deoxyribonucleotides or ribonucleotides or analogs thereof of any length. A polynucleotide can have any three-dimensional structure and can 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 in this specification may be "isolated" or "purified". As used herein, "isolated" means that the substance being referred to is (i) separated from one or more substances in which it naturally occurs (e.g., separated from at least some cellular substances, separated from other polypeptides, or separated from its natural sequence context), and / or (ii) produced by a process involving human hand such as recombinant DNA technology, protein engineering, chemical synthesis, etc., and / or (iii) used to indicate having a sequence, structure, or chemical composition not found in nature. "Isolated" is intended to include a compound in a sample in which the compound of interest is substantially concentrated and / or the compound of interest is partially or substantially purified. As used herein, "purified" indicates that the substance being referred to is removed from its natural environment and contains no less than 60%, no less than 75%, or no less than 90% of other components naturally associated therewith, and is 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 the 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, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences, counting the number of positions that match, dividing the number of positions that match by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The determination of 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 analysis 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 the sequences cited herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. As used herein, "conservative substitution" is a substitution of an amino acid with another amino acid having similar biochemical properties of the side chain (e.g., aliphatic, aromatic, or positively charged), which is well known to those skilled in the art. A non-conservative substitution is a substitution of an amino acid with another amino acid having dissimilar biochemical properties of the side chain (e.g., substitution of a hydrophobic residue with a polar residue). Typically, conservative substitutions result in sequences that are no longer identical but are still very similar. Conservative substitutions are intended to include combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp, etc.

[0037] "Deletion" is defined herein as a change in either an amino acid sequence or a nucleotide sequence in which one or more amino acid residues or nucleotide residues are absent as compared to the amino acid sequence or nucleotide sequence of the 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 those skilled in the art, an N-terminal and / or C-terminal deletion of a GPCR is also referred to as a truncated or truncated GPCR of the amino acid sequence of the GPCR.

[0038] "Insertion" or "addition" refers to a change in an amino acid sequence or nucleotide sequence, respectively, in which one or more amino acid or nucleotide residues are added as compared to the amino acid sequence or nucleotide sequence of the parent protein. "Insertion" generally refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, and "addition" can refer to an insertion or the addition of amino acid residues at the N-terminus or C-terminus, or both termini. In the context of a protein or a 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 a fragment thereof may contain two or more insertions.

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

[0040] The term "amino acid difference" refers to the total number of amino acid residues in a sequence that have changed (i.e., by substitution, insertion, and / or deletion) as compared to the starting sequence or a reference sequence. The number of amino acid differences between a sequence and a reference sequence can typically be determined by aligning and comparing these sequences, for example.

[0041] The term "ortholog" when used with respect to an amino acid or nucleotide / nucleic acid sequence from a given species refers to the same amino acid or nucleotide / nucleic acid sequence from a different species. Two sequences should be understood to be orthologs of each other if they are derived from a common ancestral sequence through a direct lineage and / or are otherwise closely related in terms of both their sequences and their biological functions. Orthologs typically have a high degree of sequence identity, but do not necessarily (and often do not) share 100% sequence identity.

[0042] The term "recombinant" when used with respect 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 by a change in 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 not found in the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise abnormally expressed, suppressed, overexpressed or not expressed at all.

[0043] 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.

[0044] As used herein, the term "operably linked" refers to a linkage in which a regulatory sequence is contiguous with a target gene for controlling the target gene, and a linkage in which a regulatory sequence acts in trans or at a certain distance for controlling the target gene. For example, a DNA sequence is operably linked to a promoter when it is ligated downstream of the promoter with respect to the transcription start site of the promoter, allowing transcription elongation to proceed through the DNA sequence. The DNA of a signal sequence is operably linked to the DNA encoding a polypeptide when it is expressed as a preprotein involved in the transport of the polypeptide. The linkage between a DNA sequence and a regulatory sequence is typically achieved by ligation at an appropriate restriction site using restriction endonucleases known to those skilled in the art or alternatively by an adapter or linker inserted.

[0045] As used herein, the term "regulatory sequence" is also referred to as a "control sequence" and refers to a polynucleotide sequence necessary for affecting the expression of a coding sequence to which it is operably linked. A regulatory sequence is a sequence that controls transcription, post-transcriptional events, and translation of a nucleic acid sequence. Examples of regulatory sequences include appropriate transcription start sequences, termination sequences, promoter sequences, 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.

[0046] 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 is linked. A vector can be any suitable type of vector including, but not limited to, phage, virus, plasmid, phagemid, cosmid, bacmid, or even artificial chromosome. A particular vector can be capable of autonomous replication within the host cell into which it is introduced (e.g., a vector having an origin of replication that functions within the host cell). Other vectors can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicated with the host genome. Further, a particular preferred vector can direct the expression of a particular target gene. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Suitable vectors can have regulatory sequences such as promoters, enhancers, terminator sequences, etc., as desired and depending on the particular host organism (e.g., bacterial cell, yeast cell). Typically, the recombinant vector according to the invention comprises at least one "chimeric gene" or "expression cassette". An expression cassette is generally a DNA construct, preferably a promoter region (5' to 3' in the transcription direction), a polynucleotide sequence of the invention or a homolog, variant or fragment thereof operably linked to a transcription start region, and a termination sequence comprising a termination signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be operable in a living cell such as a prokaryotic or eukaryotic cell to be transformed. The promoter region comprising a transcription start region preferably containing an RNA polymerase binding site, and the polyadenylation signal can be specific to the living cell to be transformed or can be derived from another source where the region is functional in the living cell.

[0047] 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 term is intended to refer not only to a particular target cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations either as a result of mutations or environmental influences, such progeny may not actually be identical to the parental 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 a cell present in a biological tissue or organism. In particular, a host cell is a cell derived from bacteria or fungi, but may also be a cell derived from plants or mammals. 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.

[0048] The "G-protein coupled receptor" or "GPCR" is a polypeptide having a common structural motif, with an extracellular amino terminus (N-terminus), an intracellular carboxy terminus (C-terminus), and seven hydrophobic transmembrane regions of 22 to 24 hydrophobic amino acids each forming seven α-helices that traverse 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, each of which is referred to as extracellular region 1, 2, and 3 (EC1, EC2, and EC3) on the outer, or "extracellular", side of the cell membrane. Also, the transmembrane helices are connected by regions of amino acids between transmembrane-1 and transmembrane-2, between transmembrane-3 and transmembrane-4, and between transmembrane-5 and transmembrane-6, each of which is referred to as intracellular region 1, 2, and 3 (IC1, IC2, and IC3) on the inner, or "intracellular", side of the cell membrane. The "carboxy" ("C") terminus of the receptor is in the intracellular space inside 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. March 30, 2016; 12(3):e1004805. doi:10.1371 / journal.pcbi.1004805; Ventakakrishnan, Current Opinion in Structural Biology, 2014, 27:129-137; Isberg, Trends Pharmacol. Sci., January 2015, 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, published by Wiley Liss (1st Edition; 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 Edition; 1994).

[0049] The International Union of Basic and Clinical Pharmacology (IUPHAR) maintains a database of receptors (including GPCRs), 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, about half of which have sensory functions (e.g., olfaction, taste, photoreception, and pheromone signaling), and about half mediate signaling related to ligands ranging in size from small molecules to peptides and large proteins. The IUPHAR database as of January 2019 describes two systems for classifying GPCRs, one of which is based on six classes of GPCRs as follows: class A (rhodopsin-like), class B (secretin receptor family), class C (metabotropic glutamate), class D (fungal mating pheromone receptor, not found in vertebrates), class E (cyclic AMP receptor, also not found in vertebrates), and class F (frizzled / Smoothened).The IUPHAR database also refers to an alternative classification scheme known as "GRAFS" that divides vertebrate GPCRs into five classes (overlapping with the A-F nomenclature) as follows: the Glutamate family (overlapping with "Class C" above), including in particular metabotropic glutamate receptors, calcium-sensing receptors and GABAB receptors; the Rhodopsin family (overlapping with "Class A" above), including 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); the Adhesion family of GPCRs (phylogenetically related to Class B receptors); the Frizzled family, consisting of ten Frizzled proteins (FZD(1-10)) and Smoothened (SMO); and the Secretin family, which are receptors for peptide ligands / hormones having 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, unless expressly stated otherwise, the A-F type classification is used. Further, see Cvicek et al. cited herein.

[0050] The term "biologically active" with respect to a GPCR refers to a GPCR having the biochemical function of a naturally occurring GPCR (e.g., binding function, signal transduction function, or the ability to change conformation as a result of ligand binding).

[0051] In general, the term "naturally occurring" with respect to a GPCR means a GPCR that is produced naturally (e.g., by a wild-type mammal such as a human). Such GPCRs are found in nature. "Not naturally occurring" with respect to a GPCR means a GPCR that is not a naturally occurring one. Naturally occurring GPCRs that are constitutively activated by mutation, and variants of naturally occurring transmembrane receptors, such as epitope-tagged GPCRs, and GPCRs lacking the native N-terminus, are examples of GPCRs that are not naturally occurring. Non-naturally occurring variants of naturally occurring GPCRs are often activated by the same ligands as the naturally occurring GPCRs. Non-limiting examples of naturally occurring GPCRs or non-naturally occurring GPCRs in the context of the present invention are further provided herein.

[0052] 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 4, 5, 6, or 7 such amino acids, and more usually, at least 8, 9, or 10 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, "conformational epitope" refers to an epitope that comprises amino acids in a spatial conformation unique to the folded three-dimensional conformation of the polypeptide. Generally, conformational epitopes consist of amino acids that are discontinuous in the linear sequence and that come together in the folded structure of the protein. However, a conformational epitope may consist of a linear sequence of amino acids that assume a conformation unique to the folded three-dimensional conformation of the polypeptide (and that does not exist in the variant state).

[0053] The term "conformation" or "conformational state" of a protein generally refers to the spatial arrangement, structure, or range of structures that a protein can assume at any given time. One of ordinary skill in the art will recognize that the determinants of conformation or conformational state include the primary structure of the protein as reflected in its amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein is also related to structural features such as protein secondary structure (e.g., in particular, α - helices, β - sheets), tertiary structure (e.g., the three - dimensional folding of a polypeptide chain), and quaternary structure (e.g., the interaction of polypeptide chains with other protein subunits). In particular, post - translational modifications and other modifications of the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or the binding of hydrophobic groups, can affect the conformation of the protein. Furthermore, environmental factors, such as in particular the pH, salt concentration, ionic strength, and osmotic pressure of the surrounding solution, as well as the interaction with other proteins and cofactors, can influence the conformation of the 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, in particular, X - ray crystallography, NMR, or spin labeling. For general considerations of protein conformation and conformational state, 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.

[0054] As used herein, "functional conformation" or "functional conformation state" refers to the fact that a protein has a range of conformational states with dynamic range of activity, particularly having various conformational states from inactive to maximum activity. It will be apparent that the "functional conformation state" is intended to include any conformational state of a protein having any activity, including inactivity, and is not intended to include the misfolded state of the protein. Non-limiting examples of functional conformations include active conformations, inactive conformations, or basal conformations (further defined herein). As described above, a particular class of functional conformations is defined as "drugable conformations" and generally refers to the therapeutically relevant conformational state(s) of a protein. See, for example, Johnson and Karanicolas, PLoS Comput Biol 9(3):e1002951.doi:10.1371 / journal.pcbi.1002951. Also see, for example, International Publication No. WO 2014 / 122183, which describes that the agonist-binding active conformation of the muscarinic acetylcholine receptor M2 corresponds to the drugable conformation of this receptor associated with pain and glioblastoma, and describes VHHs capable of stabilizing said drugable conformation for assay and screening purposes. Thus, it will be understood that drugability is limited to specific conformations depending on the therapeutic indication. Further details are provided herein.

[0055] As used herein, with respect to a protein that is a receptor, the term "active conformation" more particularly refers to a conformation or series of receptor conformations that enable signaling 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 series of ligand-specific conformations, including agonist-specific active-state conformations, partial agonist-specific active-state conformations, or biased agonist-specific active-state conformations, which result in the cooperative binding of intracellular effector proteins.

[0056] In addition to the above, with respect to GPCRs, as used herein, the terms “active conformation” and “active form” refer to a GPCR that is folded in a manner such that it is (functionally) active. A GPCR can be placed in an active conformation using an activating ligand (agonist) of the receptor, and such a conformational change generally enables 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 of the G protein to activate downstream signaling pathways. An activated GPCR binds to an inactive GDP-bound heterotrimeric G protein and causes the G protein to release GDP so that GTP can bind. This process results in a transient “nucleotide-free” state that enables GTP binding. When GTP binds, the receptor and G protein dissociate, and the GTP-bound G protein is able to activate downstream signaling pathways such as adenylyl cyclase, ion channels, RAS / MAPK. The terms “inactive conformation” and “inactive form” refer to a GPCR that is folded in a manner such 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 to a heterotrimeric G protein with high affinity. The terms “active conformation” and “inactive conformation” are further described herein. As used herein, the term “basal conformation” refers to a GPCR that is folded in a manner that exhibits activity (also called basal or constitutive activity) directed towards a particular signaling pathway even in the absence of an agonist. 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.

[0057] Similarly, with respect to a protein that is a receptor, the term "inactive conformation" as used herein refers to a series of receptor conformations that do not permit or block signal transduction to an intracellular effector system. Thus, "inactive conformation" encompasses a series of ligand-specific conformations that includes the inverse agonist-specific inactive state conformation, which as a result impedes the cooperative binding of intracellular effector proteins. It will be understood that the binding site of the ligand is not critical for obtaining the active or inactive conformation. Thus, orthosteric ligands as well as allosteric modulators can similarly stabilize the receptor in the active or inactive conformation.

[0058] As used herein, the term "binding agent" means all or part of a proteinaceous (protein, protein-like, or protein-containing) molecule that is capable of binding using specific intermolecular interactions to a membrane protein. In certain embodiments, the term "binding agent" 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 particularly, the term "binding agent" refers to a polypeptide, and in particular, a protein domain. A suitable protein domain is an element of the overall protein structure that is self-stabilizing and folds independently of the rest of the protein chain, and is often referred to as a "binding domain". Such binding domains vary in length from about 25 amino acids up to and more than 500 amino acids. Many binding domains can be classified into foldings, which are recognizable and distinguishable three-dimensional structures. Some foldings are common to many different proteins and have been given special names. Non-limiting examples include, inter alia, binding domains selected from 3- or 4-helix bundles, armadillo repeat domains, leucine-rich repeat domains, PDZ domains, SUMO or SUMO-like domains, cadherin domains, immunoglobulin-like domains, phosphotyrosine-binding domains, plekstrin homology domains, src homology 2 domains. Thus, a binding domain may be derived from a naturally occurring molecule, such as a component of the innate or adaptive immune system, or may be entirely artificially designed.

[0059] Generally, the binding domain can be immunoglobulin-based or based on domains present in proteins such as, but not limited to, microbial proteins, protease inhibitors, toxins, fibronectin, lipocalin, single-chain antiparallel coiled-coil proteins or repetitive motif proteins. Specific examples of binding domains known in the art include, but are not limited to, antibodies, heavy-chain antibodies (hcAb), single-domain antibodies (sdAb), minibodies, variable domains derived from camelid heavy-chain antibodies (VHH or Nanobody), variable domains of novel antigen receptors derived from shark antibodies (VNA), 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, lipopeptides (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 generating a particular type of binding domain using a selection method, combinatorial libraries containing consensus or framework sequences that include randomized potential interaction residues are often used to screen for binding to a molecule of interest, e.g., a protein.

[0060] According to a preferred embodiment, it is particularly contemplated that the binder of the present invention is derived from the innate or adaptive immune system. Preferably, said binder is derived from an immunoglobulin. Preferably, the binder 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. An antibody is intended to include a conventional four-chain immunoglobulin comprising a pair of two identical polypeptide chains, each having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 kDa). Typically, in a conventional immunoglobulin, 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 includes, consists of, or consists of any combination of Fab, Fab', and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-bonded Fv (dsFv), and fragments containing or consisting of either the VL or VH domain, and any other functional portion of an immunoglobulin peptide capable of binding to a target antigen, but is not limited thereto. The term "antibody" is also intended to include heavy-chain antibodies or fragments thereof that include an immunoglobulin single variable domain, as further defined herein.

[0061] The term "immunoglobulin single variable domain" or "ISVD" defines a molecule in which the antigen-binding site resides in a single immunoglobulin domain (which is different from a conventional immunoglobulin or fragment thereof, in which typically two immunoglobulin variable domains interact to form the antigen-binding site). However, it will be apparent 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, the binder within the scope of the present invention is an immunoglobulin single variable domain.

[0062] Generally, an immunoglobulin single variable domain comprises four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1 to CDR3), preferably an amino acid sequence or any suitable fragment thereof (usually comprising at least a part of the amino acid residues forming at least one of the complementarity determining regions), according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1). ISVDs comprising four FRs and three CDRs are known to those skilled in the art and are described, as non-limiting examples, 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, insofar as they can form a single antigen-binding unit. Thus, according to a preferred embodiment, the binder is an immunoglobulin single variable domain that is a light chain variable domain sequence (e.g., VL domain sequence) or a heavy chain variable domain sequence (e.g., VH domain sequence), and more particularly, the immunoglobulin single variable domain is a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. An immunoglobulin single variable domain can be a domain antibody, or single domain antibody, or "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, Volume 911. An immunoglobulin single variable domain generally comprises a single amino acid chain that can be considered to include four "framework sequences" or FRs (as defined above herein) and three "complementarity determining regions", or CDRs.It is apparent that the framework regions of immunoglobulin single variable domains can also contribute to their antigen binding (Desmyter et al., 2002; Korotkov et al., 2009).

[0063] 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, most preferably 113. It should be noted, however, that parts, fragments, analogues or derivatives of a VHH or Nanobody (as further described herein) are not particularly limited with respect to their length and / or size, so long as such parts, fragments, analogues or derivatives meet the further requirements outlined herein and are suitable for the purposes described herein.

[0064] 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 Riechmann and Muyldermans, J. Immunol. Methods, 2000 Jun. 23; 240(1-2):185-195. HH As applied to domains (see e.g. FIG. 2 of this document), they are depicted using the numbering according to Kabat ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). See also, e.g., FIG. 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 is definitive with regard to amino acid residues / positions in VHHs, nanobodies or conformos, and other numbering systems are presented for reference only).

[0065] Regarding CDRs, as is well known in the art, there are multiple rules for defining and describing the CDRs of VH or VHH fragments. For example, there are the Kabat definition (based on sequence variability and the most commonly used), the Chothia definition (based on the positions of structural loop regions), etc. For example, see the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and the claims, the CDRs according to Kabat may also be referred to, but the CDRs are most preferably defined based on the Abm definition (based on Oxford Molecular's AbM antibody modeling software), because this Abm definition is considered to be the optimal compromise between the Kabat definition and the Chothia definition. Again, see the website http: / / www.bioinf.org.uk / abs / .

[0066] Accordingly, in this specification and the claims, unless explicitly defined otherwise herein, all CDRs or VHHs, nanobodies or conformatibodies are defined according to the Abm rules.

[0067] It should be noted that the immunoglobulin single variable domain as a binder in the broadest sense is not limited to a specific biological source or a specific preparation method. The term "immunoglobulin single variable domain" or "ISVD" encompasses variable domains of various origins, including mouse, rat, rabbit, camel, human, shark, camelid variable domains. According to certain embodiments, the immunoglobulin single variable domain is derived from shark antibodies (so-called immunoglobulin new antigen receptors or IgNARs), and more specifically, from natural heavy-chain shark antibodies that do not have a light chain and are known as VNAR domain sequences. Preferably, the immunoglobulin single variable domain is derived from camelid antibodies. More preferably, the immunoglobulin single variable domain is derived from naturally occurring heavy-chain camelid antibodies that do not have a light chain and are known as VHH domain sequences or nanobodies.

[0068] According to a particularly preferred embodiment, the binder of the present invention is an immunoglobulin single variable domain that is a nanobody (further defined herein and including, but not limited to, VHH). As used herein, the term "nanobody" (Nb) is a single domain antigen-binding fragment. In particular, it refers to a single variable domain derived from naturally occurring heavy chain antibodies, which is known to those skilled in the art. Nanobodies are usually derived from heavy chain-only antibodies (without light chains) found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996) and as a result are often referred to as VHH antibodies or VHH sequences. Camelids consist of Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., Lama paccos, Lama glama, Lama guanicoe, and Lama vicugna). Nanobody® and Nanobodies® are registered trademarks of Ablynx NV (Belgium).For further description of VHH or nanobody, 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), and the following non-limiting list of patent applications mentioned as general background art: International Publication No. WO94 / 04678, International Publication No. WO95 / 04079, International Publication No. WO96 / 34103 of Vrije Universiteit Brussel; International Publication No. WO94 / 25591, International Publication No. WO99 / 37681, International Publication No. WO00 / 40968, International Publication No. WO00 / 43507, International Publication No. WO00 / 65057, International Publication No. WO01 / 40310, International Publication No. WO01 / 44301, EP1134231, and International Publication No. WO02 / 48193 of Unilever; International Publication No. WO97 / 49805, International Publication No. WO01 / 21817, International Publication No. WO03 / 035694, International Publication No. WO03 / 054016, and International Publication No. WO03 / 055527 of Vlaams Instituut voor Biotechnologie (VIB); International Publication No. WO04 / 041867, International Publication No. WO04 / 041862, International Publication No. WO04 / 041865, International Publication No. WO04 / 041863, International Publication No. WO04 / 062551, International Publication No. WO05 / 044858, International Publication No. WO06 / 40153, International Publication No. WO06 / 079372, International Publication No. WO06 / 122786, International Publication No. WO06 / 122787 and International Publication No. WO06 / 122528 of Ablynx N.V., and further published patent applications of Ablynx N.V. It should be noted that, as is known to those skilled in the art, nanobodies are particularly characterized by the presence of one or more "characteristic residues" of camelids in one or more framework sequences (according to Kabat numbering), as described on page 75, Table A-3 of International Publication No. WO08 / 020079, which is incorporated herein by reference. It should also be noted that the nanobodies of the present invention in the broadest sense are not limited to a specific biological source or a specific preparation method.For example, nanobodies can generally be obtained by (i) isolation of the VHH domain of naturally occurring heavy-chain antibodies, (ii) expression of a nucleotide sequence encoding a naturally occurring VHH domain, (iii) "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain, (iv) "camelization" of a naturally occurring VH domain from any animal species, particularly mammalian species such as humans, or by expression of a nucleic acid encoding such a camelized VH domain, (v) "camelization" of a "domain antibody" or "Dab" as described in the art or by expression of a nucleic acid encoding such a camelized VH domain; (vi) use of synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (vii) preparing a nucleic acid encoding a nanobody using techniques known per se for nucleic acid synthesis and then expressing the nucleic acid thus obtained; and / or (viii) 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, as well as in further description herein. A particular class of nanobody-binding conformational epitopes of natural targets is called Xaperone and is specifically contemplated herein. Xaperone™ is a trademark of VIB and VUB (Belgium). Xaperone™ is a camelid single-domain antibody that binds a drug target in a disease-specific, drugable conformation.

[0069] Within the scope of the present invention, the term "immunoglobulin single variable domain" also encompasses variable domains that are "humanized" or "camelized", in particular nanobodies that are "humanized" or "camelized". For example, both "humanization" and "camelization" can be carried out, respectively, by preparing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, and then changing one or more codons of said nucleotide sequence in a manner such that the new nucleotide sequence encodes, respectively, the "humanized" or "camelized" immunoglobulin single variable domain of the present invention, by methods known per se. This nucleic acid can then be expressed, by methods known per se, to provide the desired immunoglobulin single variable domain of the present invention. Alternatively, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized immunoglobulin single variable domain of the present invention can be designed, and then newly synthesized using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized immunoglobulin single variable domain of the present invention can be designed, and then newly synthesized using techniques for nucleic acid synthesis known per se, and thereafter the nucleic acid thus obtained can be expressed, by methods known per se, to provide the desired immunoglobulin single variable domain of the present invention. Other suitable methods and techniques for obtaining the immunoglobulin single variable domain of the present invention and / or the nucleic acid encoding it, starting from a naturally occurring VH sequence or preferably a VHH sequence, will be apparent to those skilled in the art, and include, for example, combining one or more parts (such as one or more FR sequences and / or CDR sequences) of one or more naturally occurring VH 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 the nanobody of the present invention or the nucleotide sequence or nucleic acid encoding it.

[0070] According to certain embodiments of the present invention, a binder capable of stabilizing a receptor can bind at an orthosteric site or an allosteric site. In other certain embodiments, a binder capable of stabilizing a receptor can be an active conformation selective binder or an inactive conformation selective binder by either binding at an orthosteric site or binding at an allosteric site. Generally, a conformation selective binder that stabilizes the active conformation of a receptor increases or enhances the affinity of the receptor for an active conformation selective ligand, such as an agonist, more particularly, a full agonist, a partial agonist, or a biased agonist, compared to the receptor in the absence of the binder (or in the presence of a mock binder, which is also referred to as a control binder or an irrelevant binder that is not directed to and / or does not specifically bind to the receptor). Also, a binder that stabilizes the active conformation of a receptor decreases the affinity of the receptor for an inactive conformation selective ligand, such as an inverse agonist, compared to the receptor in the absence of the binder (or in the presence of a mock binder). In contrast, a binder that stabilizes the inactive conformation of a receptor enhances the affinity of the receptor for an inverse agonist and decreases the affinity of the receptor for an agonist, particularly a full agonist, a partial agonist, or a biased agonist, compared to the receptor in the absence of the binder (or in the presence of a mock binder). The increase or decrease in affinity for a ligand may be measured directly and / or may be calculated from a decrease or increase in, respectively, EC50, IC50, Kd, K, or any other measure of affinity or potency known to those skilled in the art. A binder that stabilizes a particular conformation of a receptor is particularly preferably capable of increasing or decreasing the affinity for a 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 more upon binding to the receptor.It will be appreciated that measurements of affinity for a conformational selective ligand that induces / inhibits a particular signaling pathway can be performed using any type of ligand, including natural ligands, small molecules, and biological substances; orthosteric ligands as well as allosteric modulators; single compounds as well as compound libraries; lead compounds or fragments, etc.

[0071] As used herein, the term "affinity" refers to the degree to which a ligand binds to a target protein such that the equilibrium of the target protein and the ligand that binds thereto (further defined herein) shifts towards the existence of the complex formed by their binding. Thus, for example, when a GPCR and a ligand are combined at relatively equal concentrations, a high-affinity ligand binds to the available antigen of the GPCR and shifts the equilibrium towards the side where the resulting complex is at a high concentration. Dissociation constants are commonly used to describe the affinity between a ligand and a target protein. Typically, the dissociation constant is less than 10 -5 M. Preferably, the dissociation constant is less than 10 -6 M, more preferably less than 10 -7 M. Most preferably, the dissociation constant is less than 10 -8It is less than M. Other ways to describe the affinity between a ligand (including small molecule ligands) and its target protein are to indirectly evaluate the potency of the ligand by measuring the association constant (Ka), inhibition constant (Ki), or half maximal inhibitory concentration (IC50) or half maximal effective concentration (EC50). Within the scope of the present invention, the ligand can be an 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. Within the scope of the present invention, the term "affinity" is used in the context of an agent that binds to a conformational epitope of a target GPCR, particularly an immunoglobulin or immunoglobulin fragment, such as a VHH or nanobody, and also in the context of a test compound that binds to a target GPCR, particularly the orthosteric or allosteric site of the target GPCR (further defined herein).

[0072] As used herein, the term "specificity" refers to the ability of a protein or other binding agent, particularly an immunoglobulin or immunoglobulin fragment such as a VHH or nanobody, to bind preferentially to one antigen over different antigens, and does not necessarily imply high affinity.

[0073] As used herein, the terms "specifically binds" and "specific binding" generally refer to the ability of a binder, particularly an immunoglobulin such as an antibody, or an immunoglobulin fragment such as a VHH or nanobody, to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens. In certain embodiments, the specific binding interaction can distinguish a desired antigen from an undesired antigen in a sample, in some embodiments, by a factor of greater than about 10-fold to up to 100-fold or more (e.g., greater than about 1000-fold or greater than 10,000-fold). In the context of a series of conformational states of a GPCR, the terms "specifically binds" and "specific binding" particularly refer to the ability of a binder (as defined herein) to preferentially recognize and / or bind to a specific conformational state of a GPCR as compared to another conformational state.

[0074] 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 an epitope or binding site, it should be understood that such binding is preferably "specific" binding as defined herein. Also preferably, such binding is "selective binding" as defined herein.

[0075] As used herein, in the context of the present invention, a "conformation-selective binder" refers to a binder that binds to a target protein in a conformation-selective manner. A binder that selectively binds to a particular conformation or conformational state of a protein refers to a binder that binds to the protein in a subset of conformations or conformational states with a higher affinity than other possible conformations or conformational states of the protein. One of ordinary skill in the art will recognize that a binder that selectively binds to a particular conformation or conformational state of a protein stabilizes or holds the protein in this particular conformation or conformational state. For example, an active conformation-selective binder preferentially binds to a GPCR in the active conformation state and does not bind or binds to a lesser extent to a GPCR in the inactive conformation state, and thus has a higher affinity for the active conformation state. And vice versa. The terms "specifically binds," "selectively binds," "preferentially binds," and grammatically equivalent terms thereof are used interchangeably herein. The terms "conformation-specific" or "conformation-selective" are also used interchangeably herein.

[0076] As used herein, the term "stabilization" or a grammatically equivalent term means, as described above, an increase in the stability of a protein (described herein) or a 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.). With respect to increased stability to structure and / or biological activity, stabilization can be readily determined by any of a functional assay for activity (e.g., Ca2+ release, cAMP production or transcriptional activity, β-arrestin recruitment) or ligand binding, or by physical methods such as, inter alia, X-ray crystallography, NMR, or spin labeling. The term "stabilize" also includes increased thermal stability of a receptor under non-physiological conditions induced by a denaturing agent or denaturing conditions. As used herein, the terms "thermostabilize", "thermostabilization", "increase the thermal stability of ~" refer to the functional properties, not the thermodynamic properties, of a receptor 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 denaturing agents, pH, surfactants, salts, additives, proteases, or temperature. Irreversible denaturation results in irreversible unfolding of the functional conformation of the protein, 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 spectroscopy such as fluorescence, CD or light scattering sensitive to unfolding at elevated temperatures. A binder preferably is capable of increasing the stability as measured by an increase in the thermal stability of a protein or receptor in a functional conformational state of at least 2 °C, at least 5 °C, at least 8 °C, and more preferably at least 10 °C, or 15 °C, or 20 °C. With respect to increased stability to a surfactant or chaotrope, typically, a protein or receptor is incubated for a predetermined time in the presence of a test surfactant or test chaotropic agent and stability is determined, optionally at an elevated temperature as described above, using, for example, ligand binding or spectroscopy.Alternatively, the binder can increase the stability of the functional conformational state of a protein or receptor against extreme pH. For extreme pH, typical test pHs are selected, for example, in the range of 6 - 8, 5.5 - 8.5, 5 - 9, 4.5 - 9.5, and more particularly in the range of 4.5 - 5.5 (low pH) or 8.5 - 9.5 (high pH). As used herein, the terms "(thermo)stabilize", "(thermo)stabilization", "increase the (thermo)stability of ~" are applied 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 surfactants.

[0077] In addition to the above, with respect to the functional conformational state of a GPCR, the terms "stabilize" or "stabilized" refer to maintaining or holding the GPCR protein in a subset of possible conformations that might otherwise be expected, due to the effect of the interaction of the GPCR with the binder according to the present invention. In this context, a binder that selectively binds to a particular conformation or conformational state of a protein refers to a binder that binds to the protein in a particular conformation or subset of conformational states with a higher affinity than other possible conformations or conformational states of the protein that might be expected. One of ordinary skill in the art will recognize that a binder that specifically or selectively binds to a particular conformation or conformational state of a protein will stabilize this particular conformation or conformational state and its associated activity. Further details are provided herein.

[0078] As used herein, the terms "compound", "test compound", "candidate compound", or "drug candidate compound" describe any natural or synthetic molecule that is 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 peptidomimetic molecules (peptide mimetics) containing from about 2 to about 40 amino acids, and larger polypeptides containing from about 40 to about 500 amino acids, such as antibodies, antibody fragments or antibody conjugates, etc. A test compound may be a protein backbone. For high-throughput purposes, a test compound library such as a combinatorial library or a randomized library that provides a sufficient range of 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, phage display libraries. Further details can be found elsewhere herein.

[0079] As used herein, the term "ligand" means 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, carbohydrate. A ligand can be a synthetic ligand or a naturally occurring ligand. Ligands include "natural ligands" that are endogenous natural ligands for native GPCRs. In the context of the present invention, when the protein is a transmembrane protein such as a GPCR, the ligand can bind to the protein at a ligand binding site that is exposed to the intracellular environment when the protein is in the 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 the native cellular environment (i.e., the ligand can be an "extracellular ligand"). A ligand can be an agonist, partial agonist, inverse agonist, antagonist, allosteric modulator, and can bind at either an orthosteric site or an allosteric site. In certain embodiments, a ligand can be a "conformation-selective ligand" or a "conformation-specific ligand", which means 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 the protein can adopt. By way of example, an agonist is an example of an active conformation-selective ligand and an inverse agonist is an example of an inactive conformation-selective ligand. For clarity, neutral antagonists are not considered as conformation-selective ligands because they do not distinguish between different conformations of a GPCR.

[0080] As used herein, an "orthosteric ligand" refers to ligands (both natural and synthetic) that bind to the active site of a GPCR and are further classified according to their efficacy, or rather their effect on signaling through a particular pathway. As used herein, an "agonist" refers to a ligand that, by binding to a receptor protein, increases the signaling activity of the receptor. A full agonist allows for maximal protein stimulation. A partial agonist cannot elicit full activity even at saturating concentrations. A partial agonist can also function as an "antagonist" by preventing the binding of a more potent agonist. An "antagonist", also referred to as a "neutral antagonist", refers to a ligand that binds to a receptor without stimulating any activity. An "antagonist" is also known as an "antagonist" because it can prevent the binding of other ligands and thus block agonist-induced activity. Further, 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.

[0081] A ligand used herein may also be a "biased ligand" that has the ability to selectively stimulate a subset of the receptor's signaling activity, such as in the case of a GPCR, the 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 an incomplete bias (non-absolute selectivity) characterized by ligand stimulation of multiple receptor activities with different relative potencies for different signals, or it may be a complete bias characterized by ligand stimulation of one receptor protein activity without any stimulation of another known receptor protein activity.

[0082] Another type of ligand is known as an allosteric regulator. An “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 physically distinct from the active site of the protein). In contrast to an orthosteric ligand, an allosteric regulator binds to the receptor protein at a different site and is non-competitive because it modifies the function even when an endogenous ligand is bound. An allosteric regulator that enhances the activity of a protein is referred to herein as an “allosteric activator” or “positive allosteric modulator” (PAM), and one that decreases the activity of a protein is referred to herein as an “allosteric inhibitor” or “negative allosteric modulator” (NAM).

[0083] As used herein, the terms “determine,” “measure,” “evaluate,” “assay” are used interchangeably and include both quantitative and qualitative determinations.

[0084] 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 having only one binding domain.

[0085] As used herein, the term "complementary determining region" or "CDR" in the context of an antibody refers to the variable region of either the H (heavy) or L (light) chain (abbreviated VH and VL, respectively), and includes amino acid sequences capable of specifically binding to an antigen target. These CDR regions are responsible for the basic specificity of the antibody for a particular antigen determinant structure. Such regions are also referred to as "hypervariable regions". CDRs represent non - contiguous stretches of amino acids within the variable region, and although species - independent, the positions where these important amino acid sequences are placed within the variable heavy and light chain regions have been found to have similar positions within the amino acid sequence of the variable chain. All classical antibody variable heavy and light chains each have three CDR regions (designated L1, L2, L3, H1, H2, H3), which are non - contiguous with each other with respect to each light (L) and heavy (H) chain, respectively. An immunoglobulin single variable domain, particularly a nanobody, can generally be considered to comprise a single amino acid chain that can potentially contain four "framework sequences or regions" or FRs and three "complementary determining regions" or CDRs. Nanobodies have three CDR regions (designated CDR1, CDR2, CDR3) that are non - contiguous with each other. As referred to herein, the amino acid positions / residues CDR in VHH, nanobodies or conformatibodies are shown according to the Kabat numbering system, and the frameworks and CDRs are defined based on the Abm definition (unless otherwise explicitly indicated).

[0086] In general, for the purposes of the disclosure of this specification and the appended claims, a compound of the invention is a "modulator" of a target (and / or signaling, pathway(s), mechanism of action and / or biological, physiological and / or pharmacological function in which the target is involved) or is considered to "modulate" the target when the compound, when present in a suitable assay or model (i.e., in a suitable amount or concentration, such as a biologically active amount or concentration), causes a suitable or intended readout of the assay or model (i.e., at least one suitable value or parameter that can be determined using the assay or model) to vary by at least 0.1%, such as at least 1%, at least 10%, and up to 50% or more, compared to the same value or parameter measured in the same assay or model under essentially the same conditions but in the absence of the compound. Again, the modulation can result in an increase or decrease of the value or parameter (i.e., by the percentages given above). Also, a compound of the invention is preferably a compound that can modulate the target, signaling, pathway(s), mechanism of action and / or the 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.

[0087] The method of the invention generally comprises at least the following components (all further defined herein): · A boundary layer separating a first environment and a second environment; · A transmembrane protein; · A first ligand for the 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 capable of generating a detectable signal, comprising at least a first binding member and a second binding member in an arrangement, The components are arranged relative to each other in the manner further described herein (and, where applicable, are operably linked to each other and / or associated with each other), and are carried out in the arrangement.

[0088] In particular, in the arrangement of the invention, and as further described herein, the first binding member of a binding pair may be part of a "first fusion protein" (further described herein), and the second member of the binding pair may be part of a "second fusion protein" (further described herein and different from the first fusion protein). Such first fusion proteins, such second fusion proteins in the various formats described herein, the nucleotide sequences and / or nucleic acids encoding them, and cells, cell lines or other host cells or host organisms capable of expressing either the first protein and / or the second protein (in particular, appropriately expressing as described herein) or capable of (appropriately) expressing also form further aspects of the invention.

[0089] In particular, an arrangement for carrying out the method of the invention comprises at least the following components: · a boundary layer separating a first environment from a second environment; · a binding pair comprising at least a first binding member and a second binding member, which binding pair is capable of generating a detectable signal; · a transmembrane protein appropriately fused or linked (i.e., so as to form the first fusion protein) (either directly or via a suitable linker or spacer) to one of the binding members of the binding pair; · a first ligand for the transmembrane protein present in the first environment; and · a second ligand for the transmembrane protein present in the second environment; can include, and the components are arranged relative to one another in a manner further described herein (operably linked and / or associated with one another where applicable). In particular, the second member of the binding pair may be part of a second fusion protein (different from the first fusion protein that includes the transmembrane protein and the first binding member of the binding pair), where the second fusion protein is the second fusion protein further described herein.

[0090] More particularly, an arrangement for performing the method of the present invention includes at least the following components: · A boundary layer separating a first environment and a second environment; · A binding pair including at least a first binding member and a second binding member, the binding pair capable of generating a detectable signal; · A first fusion protein including a transmembrane protein and one of the binding members of the binding pair (i.e., including the member of the binding pair such that it is present in the second environment); · A second fusion protein including a protein capable of binding directly or indirectly to the transmembrane protein and the other binding member of the binding pair, the second fusion protein being present in the second environment; and · A first ligand for the transmembrane protein present in the first environment can include, and the components are arranged relative to one another in a manner further described herein (operably linked and / or associated with one another where applicable).

[0091] In this specification and the claims, when a ligand, binding domain, binding unit, or other compound or protein is said to "be able to bind" to another protein or compound, it should be noted that such binding is most preferably "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 additional proteins, ligands, binding domains, binding members or binding units), in such a fusion protein, such proteins, ligands, binding domains, binding members or binding units are understood to be appropriately linked to each other, either directly or via a suitable spacer or linker.

[0092] For the purposes of this specification and the claims, a protein (such as a binding domain, binding unit, or ligand) binds “directly or indirectly” to a transmembrane protein if (i) the protein itself binds (and / or is capable of binding) to the transmembrane protein (e.g., to an epitope or binding site of the transmembrane 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 transmembrane protein; or (iii) the protein binds (and / or is capable of binding) to a protein complex that includes a ligand or protein that binds (and / or is capable of binding) to the transmembrane protein. In case (i), the protein is said in this specification to bind “directly” to the transmembrane protein, and in cases (ii) and (iii), the protein is said in this specification to bind “indirectly” to the transmembrane protein. Also, if a protein binds to a protein complex that includes a ligand or protein that binds to the transmembrane protein, the protein may bind to the ligand or protein, or to any other part, epitope, or binding site of the complex.

[0093] Thus, in one aspect of the present invention, the protein that binds to the transmembrane protein is selected from (i) a binding domain, binding unit, or other protein that binds to (and / or is capable of binding to) an epitope or binding site of the transmembrane protein, (ii) a binding domain, binding unit, or other protein that binds to (and / or is capable of binding to) a ligand or protein that binds to (and / or is capable of binding to) the transmembrane protein, and (iii) a binding domain, binding unit, or other protein that binds to (and / or is capable of binding to) a protein complex comprising a ligand or protein that binds to (and / or is capable of binding to) the transmembrane 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.

[0094] In particular, the protein that binds directly or indirectly to the transmembrane protein can be selected from (i) an ISVD that binds to (and / or is capable of binding to) an epitope or binding site of the transmembrane protein, (ii) an ISVD that binds to (and / or is capable of binding to) a ligand or protein that binds to (and / or is capable of binding to) the transmembrane protein, and (iii) an ISVD that binds to (and / or is capable of binding to) a protein complex comprising a ligand or protein that binds to (and / or is capable of binding to) the transmembrane protein. Again, in each such case, such an ISVD is preferably an ISVD further described herein.

[0095] In a further aspect of the present invention, an arrangement for carrying out the method of the present invention comprises at least the following components: · A boundary layer separating the first environment and 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 the binding pair (i.e., comprising the member of the binding pair such that it is present in the second environment); ·A second fusion protein present in a second environment, comprising a transmembrane protein and a protein that can directly bind to the other binding member of the binding pair (as defined herein); and ·A first ligand for the transmembrane protein present in the first environment which can be included, and the components are arranged relative to one another in a manner further described herein (operatively linked and / or associated with one another where applicable). In this aspect of the invention, the protein that can directly bind to the transmembrane protein (as defined herein) and the protein present in the second fusion protein are preferably a binding domain or binding unit, more preferably an immunoglobulin single variable domain. Also, in this aspect of the invention, it should be understood that the protein that can directly bind to the transmembrane protein (as defined herein) and the protein present in the second fusion protein can act as a second ligand.

[0096] In another aspect of the invention, an arrangement for performing the method of the invention comprises at least the following components: ·A boundary layer separating the first environment and the second environment; ·A binding pair comprising at least a first binding member and a second binding member, which can generate a detectable signal; ·A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., comprising the member of the binding pair such that it is present in the second environment); ·A first ligand for the transmembrane protein present in the first environment, ·Optionally, a second ligand for the transmembrane protein, which may 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 the binding pair; Can comprise components that are arranged relative to each other in a manner further described herein (operatively linked and / or associated with each other where applicable). In this aspect of the invention, the second ligand can be any suitable ligand (further described herein) that can bind indirectly (as defined herein) to the transmembrane 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. It will also be apparent in this aspect of the invention that the second ligand does not form part of the second fusion protein.

[0097] As further described herein, in the practice of the invention, the first ligand is often added to further components of an already formed / established arrangement of the invention as described herein, such that it should be noted that the arrangement of the invention in the absence of the first ligand (i.e., prior to addition of the first ligand) forms a further aspect of the invention (and the same applies to the manner in which the first ligand is added to an arrangement of the invention in which the first ligand is absent or not yet present).

[0098] As used herein and in the claims, the term "second ligand" refers to a ligand, binding domain, binding unit or other chemical entity that binds directly or is capable of binding directly to the transmembrane protein (or forms part of a protein complex that binds directly or is capable of binding directly to the transmembrane protein) in the methods and arrangements described herein.

[0099] As will be apparent from further description herein, the second ligand can be part of the second fusion protein or can be separated from the second fusion protein. In either case (i.e., regardless of whether the second ligand is part of the second fusion protein), the second ligand is preferably such that it can bind to a conformational epitope of the transmembrane protein (or can form part of a protein complex that binds directly to or can bind directly to the transmembrane protein). More preferably, the second ligand (and / or the protein complex comprising the second ligand) preferably specifically binds to one or more functional, active and / or druggable conformations of the transmembrane protein, induces the formation of one or more functional, active and / or druggable conformations of the transmembrane protein and / or stabilizes such conformations (and / or shifts the conformational equilibrium of the transmembrane protein to one or more such conformations), and / or induces the formation of a complex of the transmembrane protein with the first ligand and the second ligand and / or stabilizes such complex, and is the second ligand.

[0100] When the second ligand is part of the second fusion protein, the second ligand can bind directly to the transmembrane protein and can be any ligand, binding domain, binding unit, peptide, protein, or other chemical entity that can be appropriately included in the second fusion protein. Preferably, as further described herein, when part of the second fusion protein, the second ligand is an appropriate binding domain or binding unit, and in particular, an immunoglobulin single variable domain.

[0101] If the second ligand is separated from the second fusion protein, the second ligand can be any ligand or protein that can directly bind to the transmembrane protein and / or form part of a protein complex that can bind to the transmembrane protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of the transmembrane protein (e.g., a naturally occurring G protein, e.g., a G protein that is naturally present in the cell or cell line being used), a semi-synthetic or synthetic analog or derivative of such a naturally occurring ligand or an ortholog of such a naturally occurring ligand (e.g., a "chimeric" G protein as described herein). Also, if the second ligand is not part of the second fusion protein, the second fusion protein includes a binding domain or binding unit that can bind indirectly (as defined herein) to the transmembrane protein, i.e., a binding domain or binding unit that can bind 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 can in particular be an immunoglobulin single variable domain, e.g., a camelid-derived ISVD. (Or, as further described herein, can include one or more such immunoglobulin single variable domains, which can be the same or different, e.g., two or three such immunoglobulin single variable domains).

[0102] As further described herein, in one aspect of the invention, the arrangement of the invention can be present in a suitable cell or cell line, and / or the method of the invention can be carried out using a suitable cell or cell line that appropriately contains the (operable) arrangement of the invention.

[0103] Accordingly, as further described herein, the invention also relates to cells or cell lines that suitably comprise the arrangement of the invention and / or that suitably express or are capable of suitably expressing the components of the arrangement of the invention (as defined herein), particularly an arrangement of the invention that is operable in a cell or cell line. The invention also relates to cells or cell lines that contain and / or suitably express or are capable of suitably expressing the first fusion protein described herein. The invention also relates to cells or cell lines that contain and / or suitably express or are capable of suitably expressing the second fusion protein described herein. In yet another aspect, the invention relates to cells or cell lines that contain and / or suitably express or are capable of suitably expressing both the first fusion protein described herein and the second fusion protein described herein. In embodiments and aspects where the second ligand does not form part of the second fusion protein, such cells or cell lines may also contain or suitably express a suitable second ligand.

[0104] Also as described herein, in one aspect of the invention, the arrangement of the invention can be present in suitable liposomes or vesicles and / or the method of the invention can be carried out using liposomes or vesicles that suitably contain the (operable) arrangement of the invention.

[0105] Accordingly, as further described herein, the invention also relates to liposomes or vesicles appropriately containing the arrangement (or components thereof) of the invention, such as to provide an arrangement of the invention that is operable particularly in liposomes or vesicles. The invention also relates to liposomes or vesicles containing the first fusion protein described herein. The invention also relates to liposomes or vesicles containing the second fusion protein described herein. In yet another aspect, the invention relates to liposomes or vesicles containing both the first fusion protein described herein and the second fusion protein described herein. In embodiments and forms where the second ligand does not form part of the second fusion protein, such liposomes or vesicles may also contain a suitable second ligand.

[0106] Accordingly, as further described herein and as shown by the accompanying non-limiting drawings, depending on whether the second ligand is or is not part of the second fusion protein, the invention contemplates at least three preferred embodiments of the methods and arrangements of the invention.

[0107] In such a preferred first embodiment (schematically shown in FIG. 1), the second binding member of the binding pair is appropriately fused or linked to the second ligand (either directly or via a suitable linker or spacer). According to this preferred embodiment, the 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, which binding pair is capable of generating a detectable signal; · A transmembrane protein appropriately fused or linked (either directly or via a suitable linker or spacer) to one of the binding members of the binding pair; · A first ligand for the transmembrane protein present in the first environment; and · A second ligand for a transmembrane protein that is present in a second environment and is appropriately fused or linked (directly or via a suitable linker or spacer) to the other binding member of the binding pair; It may include, and the components are arranged relative to each other in the manner further described herein (operatively linked and / or associated with each other where applicable).

[0108] In particular, as further described herein, such an arrangement is the following components: · A boundary layer separating the first environment and the second environment; · A binding pair consisting of at least a first binding member and a second binding member, which can generate a detectable signal; · A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., such that the 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 transmembrane protein and the other binding member of the binding pair, which is present in the second environment; It may include, and the components are arranged relative to each other in the manner further described herein (operatively linked and / or associated with each other where applicable).

[0109] In this first embodiment, it will be apparent to those skilled in the art that the "second ligand" is a binding domain, binding unit or other protein that binds directly (and / or is capable of binding) to an epitope or binding site of the transmembrane protein. Again, the binding domain or binding unit is preferably an immunoglobulin single variable domain further described herein.

[0110] In a second such preferred embodiment (schematically shown in FIG. 2), the second binding member of the binding pair does not bind directly to the transmembrane protein, but instead is suitably fused or linked (either directly or via a suitable linker or spacer) to a binding domain or binding unit that binds to a second ligand (which can then bind to the transmembrane protein). 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, which can generate a detectable signal; · A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., such that said member of the binding pair is present in the second environment); · A first ligand for the transmembrane protein present in the first environment; · A second ligand for the transmembrane protein present in the second environment; and · A second fusion protein present in the second environment and comprising a binding domain or binding unit that can bind to the second ligand and is suitably fused or linked (either directly or via a suitable linker or spacer) to the other binding member of the binding pair; and may include, and the components are arranged relative to each other in the manner further described herein (operatively linked and / or associated with each other where applicable).

[0111] In this second embodiment, it will be apparent to those skilled in the art that the binding domain or binding unit present in the second fusion protein binds to the transmembrane protein "indirectly", i.e., by binding to a second ligand that binds to the transmembrane protein. Also, as further described herein, the binding domain or binding unit is preferably an immunoglobulin single variable domain (and / or preferably consists essentially thereof). The binding domain or binding unit can also comprise or consist essentially of two or more immunoglobulin single variable domains (e.g., two or three immunoglobulin single variable domains), each of which is capable of binding (specifically) to a second ligand (i.e., the same epitope or binding site of the second ligand, or a different epitope / binding site of the second ligand), which may be the same or different (as further described herein), and are appropriately linked or fused to each other and to the other binding members of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for use in the present invention. For example, without limitation, such a binding domain or binding unit may comprise two or three copies of the affibody CA4437 (SEQ ID NO: 4 in WO 2012 / 75643 and SEQ ID NO: 2 herein), which are appropriately linked or fused to each other and to the other binding members of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for use in the present invention. Also, in this embodiment, the second ligand can be any suitable ligand of the transmembrane protein further described herein. Again, such "multivalent" binding domains comprising two or more ISVDs are most preferably multivalent binding domains such that binding to the second ligand does not substantially interfere with the ability of the second ligand to bind to the transmembrane protein and / or the ability of the second ligand to form (or promote the formation of) a complex of the transmembrane protein, the second ligand, and the first ligand.

[0112] In a third preferred embodiment (schematically shown in Figure 3), the second binding member of the binding pair does not bind directly to the transmembrane protein, but instead binds (either directly or via a suitable linker or spacer) to a binding domain or binding unit that binds to a protein complex that includes at least a second ligand for the transmembrane protein, which protein complex may bind to the transmembrane protein or be bound by the transmembrane protein and / or may include the transmembrane protein. 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, which binding pair is capable of generating a detectable signal; · A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., such that said member of the binding pair is present in the second environment); · A first ligand for the transmembrane protein present in the first environment; · A protein complex comprising at least a second ligand for the transmembrane protein, which protein complex is present in the second environment; and · A second fusion protein present in the second environment and comprising a binding domain or binding unit capable of binding to the protein complex, which binding domain or binding unit is suitably fused or linked (either directly or via a suitable linker or spacer) to the other binding member of the binding pair; and the components are arranged relative to one another in the manner further described herein (and are operably linked and / or associated with one another, where applicable).

[0113] In this third embodiment, it will be apparent to those skilled in the art that the binding domain or binding unit present in the second fusion protein binds to the transmembrane protein "indirectly", i.e., by binding to a protein complex containing a second ligand. Also, as further described herein, the binding domain or binding unit is preferably an immunoglobulin single variable domain, and the second ligand can be any suitable ligand for the transmembrane protein that can be part of a protein complex as further described herein.

[0114] Also, the binding domain or binding unit in the second fusion protein can comprise two or more immunoglobulin single variable domains, each of which is capable of binding to a different epitope, moiety, domain or subunit of the protein complex, for example, capable of binding to two different epitopes of a G protein complex. For example, but not limited to, where the protein complex is a heterotrimeric G protein, the binding domain or binding unit can comprise two or three different ISVDs, each ISVD being capable of (specifically) binding to a different subunit of the G protein (preferably, at least one of the ISVDs is capable of specifically binding to the G alpha subunit present in the heterotrimeric G protein). Specific but non-limiting examples of such binding domains or binding units can include, for example, Conformatibody CA4435 (SEQ ID NO: 1 in WO 2012 / 75643 and SEQ ID NO: 1 herein) and Conformatibody CA4437 (SEQ ID NO: 4 in WO 2012 / 75643 and SEQ ID NO: 2 herein), which are suitably linked or fused (optionally via a suitable linker or spacer) to each other and to the other binding members of the binding pair to form a second fusion protein suitable for use in the present invention.

[0115] The use of such "multivalent" (i.e., containing two or more ISVDs) binding domains or binding units in the second fusion protein can result in improved sensitivity in the assays described herein compared to the use of the corresponding ISVD(s) in a monovalent format (i.e., containing only one of said ISVDs).

[0116] More generally, the arrangements for performing the methods of the invention in various aspects and embodiments typically and preferably include at least the following components: · A boundary layer separating a first environment and a second environment; · A binding pair capable of generating a detectable signal, consisting of at least a first binding member and a second binding member; · A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., such that said member of the binding pair is present in the second environment); · A first ligand for the 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 the binding pair (i.e., such that said other member of the binding pair is also present in the second environment) and at least include, The components are arranged relative to each other in the manner further described herein (and, where applicable, are operably linked and / or associated with each other). In particular, · In the first preferred embodiment described herein, the second fusion protein comprises the other binding member of the binding pair and the second ligand; · In the second preferred embodiment described herein, the second fusion protein comprises the other binding member of the binding pair and a binding domain or binding unit capable of binding to the second ligand; and · In the 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 the binding pair and at least a second ligand.

[0117] The present invention is now illustrated by further description herein, the following experimental section, and the accompanying non-limiting drawings.

[0118] From the drawings and further description herein, it will be apparent to those skilled in the art that some of the components of the arrangements of the present invention (e.g., the boundary layer, the transmembrane protein, the binding pair, any linker, and the first ligand) are present in the various aspects and embodiments of the present invention contemplated herein. Thus, when a detailed description of any such component (including any preference for any such component) is given herein, it is to be understood that such description applies to all aspects and embodiments of the present invention in which such component is present or used, unless otherwise expressly stated herein.

[0119] In the methods and arrangements of the present invention, the boundary layer (1) can be any layer (e.g., a wall or a membrane) suitable for separating a first environment [A] from a second environment [B] (in a suitable in vitro system or a suitable in vivo system).

[0120] For example, in a preferred aspect of the present invention where the method of the present invention is practiced 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 aspect, environment [A] is preferably the extracellular environment and environment [B] is preferably the intracellular environment. Also, in this aspect, 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 also preferably present in the intracellular environment.

[0121] (As further described herein) In another preferred embodiment of the invention, where the method of the invention is carried out with suitable vesicles or liposomes, the boundary layer (1) is the membrane or wall of the vesicle or liposome. In this embodiment, the environment [A] is preferably the environment outside the vesicle or liposome, and the environment [B] is preferably the environment inside the vesicle or liposome. Also, in this embodiment, the first ligand (3) is preferably present in the environment outside the vesicle or liposome, and the second ligand (4) is preferably present in the environment inside the vesicle or liposome. Also, the first and second binding members (6) and (7) and the second fusion protein are also preferably present in the environment inside the vesicle or liposome.

[0122] However, although the invention in some preferred embodiments is carried out using cells, liposomes or other suitable vesicles, the invention in its broadest sense is not limited to the use of cells or vesicles, and it should be understood that it can be carried out in any other suitable arrangement where a boundary layer (1) is used to appropriately separate the first environment [A] from the second environment [B]. For example, the boundary layer can be a cell wall or a part or fragment of a cell membrane present in a membrane extract obtained from whole cells by techniques known per se, such as suitable osmotic and / or mechanical techniques known per se.

[0123] Thus, the 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, the boundary layer (1) can be a suitable lipid bilayer, e.g., a phospholipid bilayer. When the boundary layer (1) is the wall or membrane of a vesicle or liposome, the boundary layer (1) can be unilamellar or multilamellar. Also, as further described herein, when the boundary layer (1) is a cell membrane or cell wall, the boundary layer (1) is preferably the wall or membrane of a cell or cell line that appropriately expresses the transmembrane protein (2) (as defined herein), particularly the (first) fusion protein described herein that contains the transmembrane protein (2).

[0124] As schematically shown by non-limiting FIGS. 1, 2 and 3, the boundary layer (1) contains a transmembrane protein (2), and the transmembrane protein (1) has: · a first binding site (8) for a first ligand (3) that protrudes into a first environment [A] (as defined herein) (i.e., when the first ligand (3) is present in the first environment [A], the first binding site (8) is accessible for binding by said first ligand); and, as such, · a second binding site (9) for a second ligand (4) that protrudes into a second environment [B] (as defined herein) (i.e., 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); and passes through the boundary layer (1).

[0125] In this specification and the claims, the term "transmembrane protein" is used to denote a protein that is used (e.g., screened) in the methods and arrangements of the present invention. In the methods and arrangements of the present invention, the transmembrane protein (2) is such that at least one part of the amino acid sequence of the transmembrane protein (2) protrudes from the boundary layer (1) into a first environment [A] (as defined herein), and at least one other part of the amino acid sequence of the transmembrane protein (2) protrudes from the boundary layer (1) into a second environment [B] (as defined herein), and the transmembrane protein (2) penetrates (and / or is provided and / or arranged in such a manner with respect to the boundary layer) through the boundary layer (1). In this context, when a part of the amino acid sequence of the transmembrane protein (2) is said to "protrude" from the boundary layer (1) into an environment (i.e., into the first environment [A] or the second environment [B]), this is generally to be understood to mean that the said part of the sequence is exposed to the said environment and / or is accessible for binding by a ligand, compound or other chemical substance present in the said environment. Thus, in the methods and arrangements of the present invention, at least one part (e.g., an epitope or binding site) of the amino acid sequence of the transmembrane protein should be accessible for binding by a ligand, compound or other chemical entity present in the first environment (in particular, binding by the first ligand (3)), and at least one other part (e.g., another epitope or binding site) of the amino acid sequence of the transmembrane protein should be accessible for binding by a ligand, compound or other chemical entity present in the second environment (in particular, binding by the second ligand (4)). In this regard, it should also be noted that the expression "accessible for binding" generally means that a ligand, compound or other chemical substance present in the relevant environment can bind to a binding pocket or binding site on or within the transmembrane protein, even if the actual binding site or binding pocket is deep (deeper) within the structure of the transmembrane protein (even if the actual binding site or binding pocket is located within a part of the transmembrane protein that does not physically protrude beyond the boundary layer itself).For example, refer to the paper by Chevillard (cited herein) which shows that the binding site of GPCR for fragments used in FBDD screening technology is deep within the GPCR structure (see, e.g., Figure 2 on page 1120), not on the surface of the GPCR, but is nevertheless accessible for binding of the fragment. The teachings of GPCR structures, GPCR signaling mechanisms and GPCR ligand binding sites are also referenced from several other chemical papers cited herein.

[0126] Also, in this specification and the claims, when any binding domain, binding unit, epitope, binding site, ligand, protein or other compound or chemical or other structural substance (e.g., protein complex) is said to "be present" in an environment (i.e., the first environment [A] or the second environment [B]), this is generally to be understood as meaning that the binding domain, binding unit, epitope, binding site, ligand, protein or other compound or chemical or structural substance 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 an environment may be "floating" in the environment (i.e., not bound or immobilized to any other protein or structure), immobilized to a boundary layer, or fused to another protein (the other protein may be immobilized to a boundary layer). Similarly, a binding domain or binding unit present in an environment may be part of a large protein or structure (e.g., a fusion protein), and as long as the binding domain or binding unit is accessible for binding by another domain, ligand, protein or compound present in the environment, the large structure may be floating in the environment or immobilized to a boundary layer or another structure. Also, an epitope or binding site present in an environment may be part of a large protein or structure, and as long as the epitope or binding site is accessible for binding by another domain, ligand, protein or compound present in the environment, the large protein or structure may also be floating in the environment or immobilized to a boundary layer or another structure.

[0127] One or more portions of the transmembrane protein (2) protruding into the first environment [A] are any loop, epitope (linear or conformational), binding site or other portion(s) of the amino acid sequence of the transmembrane protein, and likewise one or more portions of the transmembrane protein protruding into the second environment [B] can also be any loop, epitope (linear or conformational), binding site or other portion(s) of the amino acid sequence of the transmembrane protein (provided that they are different from the portion(s) protruding into the first environment).

[0128] In a preferred embodiment of the invention, the transmembrane protein (2) comprises at least two different / distinct ligand binding sites, at least the first binding site of which protrudes into the first environment [A] (in particular, so as to be accessible for binding by the first ligand (3)) (as defined herein), and at least the second binding site protrudes into the second environment [B] (in particular, so as to be accessible for binding by the second ligand (4)) (as defined herein).

[0129] Generally, the transmembrane protein (2) is bound and / or immobilized to the boundary layer (1) in a manner known per se for membrane (trans-) proteins that are usually fixed to the cell wall or cell membrane in their natural environment, for example. As further described herein, this can be achieved, for example, by appropriately expressing a nucleotide sequence or nucleic acid that expresses a first fusion protein in a suitable host cell such that the transmembrane protein (2) is appropriately fixed to the wall or membrane of said cell (as defined herein). When the method of the 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 transmembrane protein (2) is appropriately fixed to the wall or membrane of the liposomes or vesicles.

[0130] The transmembrane protein (2) can include one or more domains (especially one or more transmembrane domains) and is usually, preferably, a transmembrane protein, such as a (transmembrane) receptor.

[0131] When the transmembrane protein (2) is a transmembrane protein, the transmembrane protein (2) can be a bitopic membrane protein (i.e., a transmembrane protein having one passage through the membrane) or a polytopic membrane protein (i.e., a transmembrane protein having two or more passages through the membrane). Thus, the transmembrane protein (2) can be any known or newly discovered transmembrane protein (or its synthetic or recombinant analog) having a known or unknown biological function and having a known or unknown ligand (for example, the transmembrane protein (2) can be a so-called "orphan" GPCR).

[0132] The transmembrane protein (2) can be an alpha-helix protein or a beta-barrel protein and can be a type I, type II, type III, or type IV transmembrane protein depending on the position of the N-terminal and C-terminal boundary layers of the protein. Preferably, the transmembrane protein is a protein having its amino terminus outside the cell and its carboxy terminus inside the cell in its native cellular environment, but the invention in its broadest sense is not limited thereto.

[0133] Also, when the method of the present invention is carried out in a cell, the arrangement of the N-terminal and C-terminal of the protein with respect to the wall or membrane of the cell used is preferably the same as the arrangement of said termini in the native cellular environment of the protein.

[0134] When the method of the present invention is carried out with liposomes or vesicles, the liposomes or vesicles are arranged in a manner that is essentially the same as the way the protein is arranged with respect to the cell wall or cell membrane in its natural environment (i.e., by the N-terminus and extracellular loop(s) protruding outside the vesicle and the C-terminus and intracellular loop(s) protruding inside the vesicle), and can be a mixture of liposomes / vesicles and vesicles / liposomes in which the protein is arranged in the reverse. Usually, this will not affect the implementation of the systems or mechanisms described herein.

[0135] As further described herein, generally and preferably, the transmembrane protein (2) is a protein that exists in 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) (i.e., can take on two or more of its conformations), and / or is a protein that can undergo a conformational change (particularly a functional conformational change). In particular, the transmembrane protein (2) can exhibit at least one functional conformation and at least one non-functional conformation (e.g., basal conformation), and / or can undergo a conformational change from a non-functional conformation to a functional conformation; more specifically, it can exhibit an active (or more active) conformation and an inactive (or less active) conformation, and / or can undergo a conformational change from an inactive (or less active) conformation to an active (or more active) conformation. The transmembrane protein (2) can also be a protein that can take on at least one ligand-bound (particularly agonist-bound) conformation and at least one ligand-free conformation. In particular, the transmembrane protein (2) can be a protein that can take on at least one ligand-bound (particularly agonist-bound) conformation that is an active or functional conformation.

[0136] As described herein, certain classes of (transmembrane) protein functional conformations (e.g., certain GPCRs) are referred to / defined as "drugable conformations". Thus, in certain embodiments, the transmembrane protein (2) can be a protein that exhibits at least one such drugable conformation (which is often the active conformation, although the invention is not limited to the use of drugable conformations that are active conformations), and at least one conformation that is not a drugable conformation (which is often an inactive conformation), and / or can be a transmembrane protein that undergoes a conformational change from a non-drugable conformation to a drugable conformation.

[0137] In particular, the transmembrane protein (2) can be a protein that undergoes a conformational change upon binding of a ligand (especially an agonist) to the protein. This conformational change upon ligand binding can be, for example, a conformational change from an active conformation to an inactive conformation, or from a functional conformation to a non-functional conformation, but is preferably a change from a non-functional conformation to a functional conformation and / or from an inactive conformation to an active conformation. In certain embodiments, it is a change from a non-drugable conformation to a drugable conformation.

[0138] For example, when the transmembrane protein (2) is a receptor such as a cell surface receptor (or a synthetic analog thereof), the transmembrane protein (2) can be a protein that undergoes a conformational change when a natural or synthetic (extracellular) ligand of the receptor binds to the receptor.

[0139] When the transmembrane protein (2) is a GPCR, in a preferred but non-limiting embodiment, the conformational change can be from a conformation in which it is essentially impossible to bind a G protein to a conformation that can bind (or can be bound by) a G protein.

[0140] As mentioned herein, a ligand capable of inducing a conformational change of the transmembrane protein (2) 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 the transmembrane protein (2). In particular, when the transmembrane protein (2) is a GPCR, the “agonist” may be capable of inducing a conformational change from a conformation in which it is essentially impossible to bind a G protein to a conformation that can bind a G protein.

[0141] In a preferred embodiment of the present invention, the transmembrane protein (2) is a protein that undergoes (or is capable of undergoing) a conformational change (described herein) when the first ligand (3) binds to the transmembrane protein (2). Conversely, the first ligand (3) is a first ligand capable of causing a conformational change in the transmembrane protein (2) when the first ligand (3) binds to the transmembrane protein (2) (and / or the present invention is used to identify such a first ligand). In a more preferred embodiment, the conformational change is a change from an inactive or low-activity state to a functional or (more) active state, and the first ligand (3) used can cause a conformational change of the transmembrane protein from an inactive or low-activity state to a functional or (more) active state when the first ligand binds to the transmembrane protein (2). Also, when the transmembrane protein (2) is a GPCR, the conformational change upon ligand binding can, in a preferred but non-limiting embodiment, be a change from a conformation in which it is essentially impossible to bind a G protein to a conformation that can bind a G protein.

[0142] Also, as further described herein, the transmembrane protein can be a protein capable of forming a complex with a first ligand and a second ligand. In particular, the transmembrane protein can be a protein capable of forming a complex with an intracellular ligand and an extracellular ligand in its native environment. For example, from the references cited herein, it is known that most GPCRs form a complex with an extracellular ligand and a G protein (the most common native intracellular ligand of GPCRs), and that such a complex is stabilized by the 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 (formation) of the transmembrane protein, the first ligand, and the second ligand. For example, for this purpose, and as further described herein, when the transmembrane protein (2) is a GPCR, the second ligand is a G protein that associates with the GPCR in its native environment (i.e., has signal transduction by the GPCR), another naturally occurring G protein capable of binding to the GPCR and stabilizing the formation of the aforementioned complex, or a synthetic or semi-synthetic analog or derivative of the GPCR capable of binding to the GPCR and stabilizing the formation of the aforementioned complex. As also mentioned herein, the second ligand can be a conformatibody, i.e., an immunoglobulin single variable domain (e.g., VHH or nanobody) designed / produced to stabilize the formation of a complex between the conformatibody, the transmembrane protein, and the first ligand.

[0143] In a preferred but non-limiting embodiment of the present invention, the transmembrane protein (2) is a "seven-transmembrane protein", in particular a 7TM which is a receptor (such as a cell surface receptor). In a particularly preferred embodiment, the transmembrane protein (2) can be a 7TM that signals via a G protein. Such 7TMs are also known in the art as GPCRs. [As described above, the terms "GPCR" and "7TM" are used interchangeably herein and include all transmembrane proteins having a seven-membrane-spanning domain, regardless of the intracellular signaling cascade or signaling mechanism. However, it should be understood that throughout this specification and the claims, 7TMs that signal via a G protein are a preferred embodiment of the present invention.]

[0144] The transmembrane protein (2) can be a naturally occurring protein or receptor, or a synthetic or semi-synthetic analog thereof (again, obtained through protein chemistry or recombinant DNA techniques as generally described herein) of a naturally occurring protein or receptor. Such synthetic analogs can, for example, have one or more amino acid residues or stretches of amino acid residues (including one or more loops or portions thereof, and / or one or more domains and / or portions thereof) inserted, deleted, and / or substituted by other amino acid residues or stretches of amino acid residues (e.g., by an essentially corresponding stretch or loop of amino acids of another (preferably structurally related) membrane protein) compared to the sequence of a naturally occurring protein (in other words, include one or more "amino acid differences" as defined herein), and can be analogs of naturally occurring transmembrane proteins. In many cases, the native sequence of the naturally occurring protein used is obtained from the species to be treated with the compounds of the present invention or from the animal (preferably a mammal) used for the purpose of an animal model for testing the compounds of the present invention.

[0145] As will be apparent to those skilled in the art, such synthetic analogs can be obtained using standard techniques of protein chemistry and / or recombinant DNA techniques that are known per se. For example, when the invention is practiced in the cells described herein, the synthetic analog can be obtained by appropriately expressing in the cells a DNA sequence (or other suitable nucleotide sequence) encoding the synthetic analog.

[0146] Also, as is well known in the art, 7TM and other transmembrane proteins typically include one or more intracellular loops and one or more extracellular loops. Similarly, the transmembrane proteins used in the arrangements of the present invention can include one or more loops that project into a first environment (as defined herein) and one or more loops that project into a second environment (as defined herein). For example, when the method of the present invention is practiced in a cell, the transmembrane proteins used in the arrangements of the present invention can include one or more loops that project into the intracellular environment and one or more loops that project into the extracellular environment. Similarly, when the method of the present invention is practiced in a vesicle or liposome, the transmembrane proteins used in the arrangements of the present invention can include one or more loops that project into the environment inside the liposome or vesicle and one or more loops that project into the environment outside the liposome or vesicle. In each case, the loops that project into the first environment are most preferably loops that can form a functional ligand binding site (particularly the functional binding site for the first ligand) and / or can form it (and are so arranged) when the transmembrane protein adopts an appropriate conformation, and the loops that project into the second environment are most preferably loops that can form a functional ligand binding site (particularly the functional binding site for the second ligand) and / or can form it (and are so arranged) when the transmembrane protein adopts an appropriate conformation (e.g., upon binding of the first ligand to the transmembrane protein).

[0147] In one specific but non-limiting embodiment, the loops of the transmembrane protein protruding into one environment essentially correspond to the extracellular loops of the transmembrane protein, and the loops of the transmembrane protein protruding into another environment essentially correspond to the intracellular loops (again, in each case, preferably, the extracellular loop forms a functional ligand-binding site and the intracellular loop forms a different functional ligand-binding site). Preferably, the loops of the transmembrane protein protruding into the first environment [A] essentially correspond to the extracellular loops of the transmembrane protein, and the loops of the transmembrane protein protruding into the second environment [B] essentially correspond to the intracellular loops of the transmembrane protein, especially when the second environment [B] is the environment inside a cell or a liposome (again, preferably, the extracellular loop forms a functional ligand-binding site protruding into the first environment and the intracellular loop forms a different functional ligand-binding site protruding into the second environment).

[0148] For example, when the transmembrane protein is a transmembrane protein (such as 7TM), the transmembrane protein can include one or more extracellular loops of the transmembrane protein (particularly, one or more extracellular loops of 7TM) and one or more intracellular loops of the transmembrane protein (particularly, one or more intracellular loops of 7TM), and in particular, a transmembrane protein such that the extracellular loop forms or can form a functional ligand binding site and the intracellular loop forms or can form a different functional ligand binding site. Again, the ligand binding site formed by the extracellular loop preferably protrudes into one environment (as defined herein), and the ligand binding site formed by the intracellular loop preferably protrudes into the other environment (as defined herein). In particular, when the method of the present invention is carried out in a cell or a liposome, the extracellular loop protrudes into the external environment of the cell or liposome, and the intracellular loop protrudes into the internal environment of the cell or liposome. Also preferably, the intracellular loop is an intracellular loop such that it forms or can form a functional ligand binding site for a second ligand (and is so arranged) (in other words, in the present invention, the ligand binding site for the second ligand preferably consists of and / or includes one or more intracellular loops of the transmembrane protein. Also, the ligand binding site for the first ligand may consist of and / or include one or more extracellular loops, but as further described herein, the actual binding / docking site for the first ligand may be deeply located within the structure of the transmembrane protein).

[0149] For example, when the transmembrane protein is 7TM, the transmembrane protein can include three intracellular loops (i.e., three intracellular loops from 7TM) and three extracellular loops (i.e., three extracellular loops from 7TM), the three intracellular loops can form or be capable of forming a functional ligand binding site, and the three extracellular loops can form or be capable of forming different functional ligand binding sites. Again, preferably, the functional ligand binding site formed by the three intracellular loops projects into one environment (preferably, the second environment [B]), and the functional ligand binding site formed by the three extracellular loops projects into another environment (preferably, the second environment [A]). Also, the three intracellular loops preferably form a functional binding site for the second ligand (and the three extracellular loops can form a functional binding site for the first ligand, or the binding site can be deeply located within the structure of 7TM). Most preferably, the three intracellular loops form a binding site for the second ligand that projects into the second environment [B] (i.e., the environment inside the cell or liposome when the method of the present invention is carried out in the cell or liposome, respectively), and the three extracellular loops project into the first environment [A] (and can form a functional binding site for the first ligand, or the binding site can be deeply located within the structure of 7TM).

[0150] In one aspect of the present invention, the intracellular and extracellular loops of the transmembrane protein are derived from or essentially derived from the same transmembrane protein (i.e., are the same as or essentially the same as the intracellular and extracellular loops present in a native transmembrane protein). In this aspect of the present invention, the transmembrane protein can have the same or essentially the same amino acid sequence as a native transmembrane protein that is used as a target in the screening or assay method of the present invention.

[0151] In another aspect of the invention, the intracellular and extracellular loops of the transmembrane protein may be derived from different transmembrane proteins. In particular, in this aspect of the invention, the intracellular and extracellular loops may be derived from different but related transmembrane proteins, such as different but related two 7TMs, such as two GPCRs. In particular, in this aspect of the invention, the intracellular loop may be derived from a first 7TM or GPCR, and the extracellular loop may be derived from a second 7TM or GPCR different from the first 7TM or GPCR. The transmembrane domains of such chimeric proteins may be derived from the first or second 7TM or GPCR, preferably, are essentially all derived from the same GPCR, more preferably, are derived from the same GPCR as the extracellular loop (however, depending on the position selected for recombinant deletion of the native intracellular loop and insertion of a replacement intracellular loop, may contain some amino acid residues from the GPCR from which the intracellular loop is derived).

[0152] In this aspect of the invention, it is most preferred that the resulting chimeric transmembrane protein is still a transmembrane protein that can be appropriately used in the methods and arrangements of the invention. Also, again, in the case of 7TM, the transmembrane protein contains three intracellular loops and three extracellular loops, and the three intracellular loops form a functional ligand binding site for a second ligand (the second ligand is then selected to be able to bind to the ligand binding site (9) formed by the intracellular loop). Again, the binding site formed by the three intracellular loops preferably protrudes into a second environment [B] (i.e., the internal environment of the cell or liposome when the method of the invention is carried out in the 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 structure of the 7TM).

[0153] Accordingly, in a further aspect, the invention relates to an arrangement as further described herein, wherein the transmembrane protein is a 7TM comprising seven transmembrane domains, three intracellular loops, and three extracellular loops (which are linked to each other in the order known per se for 7TMs, i.e., [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence]), the intracellular loops are derived from a first 7TM, the extracellular loops are derived from a second 7TM different from the first 7TM, and the intracellular loops form a functional ligand binding site. Preferably, the TM domains from said transmembrane protein are essentially from the same 7TM as the extracellular loops.

[0154] Also, the intracellular loops and 7TM as a whole are such that 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. Preferably, the ligand binding site again extends into a second environment [B].

[0155] In a specific embodiment, such a chimeric transmembrane protein comprises an intracellular loop derived from the beta-2 adrenergic receptor. In another specific embodiment, such a chimeric transmembrane protein comprises an intracellular loop derived from the mu-opioid receptor. For some non-limiting examples of such chimeric receptors, reference is also made to the co-pending PCT application by the assignee entitled "Chimeric proteins and Methods to screen for compounds and ligands binding to GPCRs" having the same international filing date as this application and relying on the same priority application.

[0156] The present invention relates in particular to an arrangement comprising such a chimeric 7TM and a second ligand capable of binding to the ligand binding site formed by said intracellular loop.

[0157] For the rest, when the second ligand is appropriately selected such that it can bind to the ligand binding site (9) of the chimeric transmembrane protein in order to provide a workable arrangement of the present invention (and when the chimeric transmembrane protein itself is workable in the arrangement of the present invention), such an arrangement of the present invention in which the chimeric transmembrane protein is used can essentially be the arrangement further described herein.

[0158] Also, such chimeric transmembrane proteins, nucleotide sequences and nucleic acids encoding them, and cells, cell lines or host organisms capable of containing such nucleotide sequences or nucleic acids and / or expressing such chimeric transmembrane proteins form further aspects of the present invention, as well as further uses of such chimeric transmembrane proteins, nucleotide sequences, nucleic acids, cells, cell lines and host organisms.

[0159] Another aspect of the present invention is a composition or parts kit comprising at least said chimeric transmembrane protein and a ligand capable of binding to the intracellular loop present in said GPCR. The ligand is preferably a protein, more preferably a protein comprising or consisting essentially of an immunoglobulin single variable domain (e.g., VHH domain), and in particular can be a conformatibody (described herein).

[0160] As described above, the chimeric transmembrane protein is preferably a 7TM / GPCR. Also, in one particular aspect, the chimeric transmembrane protein comprises an intracellular loop derived from the beta-2 adrenergic receptor. In another specific aspect, the chimeric transmembrane protein comprises an intracellular loop derived from the mu-opioid receptor.

[0161] As further described herein and schematically shown in FIGS. 1-3, in the arrangement of the present invention, the transmembrane protein (2) is usually and preferably fused or linked, directly or via a suitable spacer or linker (10), to the first member (6) of the binding pair (6 / 7) so as to form a first fusion protein. Also, the second binding member (7) of the binding pair (6 / 7) is usually and preferably part of a second fusion protein that is different from the first fusion protein, and the second fusion protein is also further described herein. The first fusion protein, the second fusion protein (in its various formats as described herein), the nucleotide sequence and / or nucleic acid encoding the first or second fusion protein, and cells, cell lines or other host cells or host organisms that express (particularly appropriately expressed as described herein) or are capable of expressing (appropriately) the first and / or second fusion protein (and preferably both), and their various uses as further described herein form further aspects of the present invention.

[0162] The binding pair (6 / 7) used in the arrangement of the present invention generally comprises at least two separate binding members (6) and (7), which are 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) can generate a detectable signal when members (6) and (7) are in contact with or very close to each other. Such a detectable signal may be, for example, a luminescence signal, a fluorescence signal, or a chemiluminescence signal, may be based on a reporter gene, or may be based on DNA ligation. Some specific but non-limiting examples of the technology (including the binding pair and the detectable signals associated therewith) include technologies based on protein complementarity, such as the NanoBit™ system, the NanoLuc™ system, the hGLuc system (Remy and Michnick, Nature Methods, 2006, 977), BiFC (bimolecular fluorescence complementarity), and DHFR-PCA (dihydrofolate reductase protein-fragment complementation assay); technologies based on direct interaction, such as BRET (bioluminescence resonance energy transfer), FRET (fluorescence / Foerster resonance energy transfer), and BioID (proximity-dependent biotin identification); reporter gene-based systems (such as the KISS / kinase substrate sensor) or proximity ligation assays (Weilbrecht et al., Expert Review of Proteomics, 7:3, 401-409). Usually, technologies based on protein complementarity and on luminescence signals, fluorescence signals or chemiluminescence signals (such as NanoLuc™ or NanoBit™) are preferred.

[0163] In a particularly preferred embodiment, when the method of the present invention is 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 substances that can be obtained by appropriately expressing the nucleic acid or nucleotide sequence encoding it in the cell used in the method of the present invention.

[0164] The first and second binding members may be part of a suitable reporter assay and can be any other pair of domains or units that generate a detectable signal when they contact or are in very close proximity to each other, such as an enzyme - substrate combination or a binding pair commonly used in experimental studies of protein - protein interactions. As described, in order to reduce the level of the baseline / background signal, the two members of the binding pair preferably do not have substantial binding affinity for each other by themselves.

[0165] Some preferred but non - limiting examples of suitable binding pairs are pGFP and Promega's NanoBiT® system. The latter is particularly preferred because the Large BiT and small BiT that make up the NanoBiT® system do not have affinity for each other by themselves.

[0166] The first binding member (6) is such that when the second fusion protein formed by the second ligand (4) and the second member (7) binds to the transmembrane protein (2) via the second binding site (9), 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). Also, preferably, the first binding member (6) is fused or linked to the transmembrane protein (2) in a manner that does not substantially affect the conformation and / or conformational changes that the transmembrane protein (2) can undergo under the conditions used to carry out the method of the present invention.

[0167] Thus, in general, although it is not excluded in the present invention that the first binding member (6) is directly fused or linked to the transmembrane protein (2), generally, the first binding member (6) is preferably fused or linked to the transmembrane protein (2) via a suitable linker (10). For example, the use of a flexible linker having between 5 and 50 amino acids in total, preferably between 10 and 30 amino acids, such as about 15 - 20 amino acids, is usually preferred. Suitable linkers will be apparent to those skilled in the art and include GlySer linkers (such as 15GS linker).

[0168] In the present invention, the first and second binding members of the binding pair (6 / 7) are (in the manner further described herein) in the same environment with respect to the boundary layer (1) (as defined herein) such that they can contact each other or be in very close proximity and thereby generate a detectable signal. In particular, as schematically shown in FIGS. 1, 2 and 3, when the second fusion protein binds directly (shown in FIG. 1) or indirectly (shown in FIGS. 2 and 3) to the binding site, the first and second binding members of the binding pair (6 / 7) will be in the same environment with respect to the second binding site (9) of the transmembrane protein (2) (and also with respect to the boundary layer (1)) such that they can contact each other. For this reason, the first binding member (6) will generally be attached directly or via a linker (10) to an amino acid residue / position within / on the transmembrane protein (2) that is exposed to the same environment as the second binding site (9). As further described herein, said environment (shown as environment [B] in FIGS. 1 - 3) can be, for example, an intracellular environment (when the method of the present invention is carried out in cells) or the environment inside vesicles or liposomes.

[0169] In a preferred embodiment of the invention, the first binding member (6) will be fused, directly or via a linker (10), to one end of the primary amino acid sequence of the transmembrane protein (2). This may be the N-terminus or C-terminus of the transmembrane protein (2) as long as, in the final arrangement of the invention, the first binding member (6) is on the same side of the boundary layer (1) as the second binding site (9). Thus, in embodiments of the invention that are carried out in cells as further described herein where the second binding site (9) is exposed to the intracellular environment, the first member (6) may be fused to the end of the primary amino acid sequence that terminates in the intracellular environment (which would usually be the C-terminus in the case of 7TM).

[0170] The first fusion protein may be prepared and produced using appropriate techniques of protein chemistry and / or recombinant DNA technology known per se. Such techniques will be apparent to the person skilled in the art based on the further disclosure herein as well as standard manuals and other scientific literature referred to herein. If the method of the invention is carried out in cells (as further described herein), the first fusion protein is preferably provided in said cells by appropriately expressing the nucleotide sequence and / or nucleic acid encoding the first fusion protein. This can also be carried out using appropriate techniques of recombinant DNA technology known per se, and cells that appropriately express (or are capable of appropriately expressing) the first fusion protein form a further aspect of the invention.

[0171] As further described herein, in the arrangements of the invention, the second member (7) of the binding pair (6 / 7) typically and preferably also forms part of a fusion protein which generally comprises said second binding unit fused or linked, directly or via a suitable spacer or linker (11), to another ligand, protein, binding domain or binding unit which can bind directly (as defined herein) or indirectly (as defined herein) to the transmembrane protein (2). For this purpose, as further described herein, said ligand, protein, binding domain or binding unit may be, for example, a second ligand (4 which is a second ligand, giving rise to an arrangement of the invention of the type schematically shown in Figure 1), a binding domain or binding unit which can bind to the second ligand (4 which is a second ligand and 5 which is a binding domain or binding unit which can bind to the second ligand, giving rise to an arrangement of the invention of the type schematically shown in Figure 2), or a binding domain or binding unit which can bind to a protein complex which can bind to the transmembrane protein (4 which is a second ligand, 12 which is said protein complex comprising the second ligand, and 5 which is a binding domain or binding unit which can bind to the protein complex, as schematically shown in Figure 3).

[0172] In the second fusion protein, when the second fusion protein binds directly or indirectly to the second binding site (9) of the transmembrane protein (2), the second binding member (7) enables the second binding member (7) to contact (or, if not, appropriately approach very closely) the first member (6) of the binding pair (6 / 7) in an appropriate manner. Most preferably, the second binding member (7) is linked to the other ligand, protein, binding domain or binding unit. For this purpose, the second binding member (7) may be directly fused or linked to the other ligand, protein, binding domain or binding unit, but preferably they are linked via an appropriate linker (11), which preferably has a flexible linker having, for example, between 5 and 50 amino acids in total, preferably between 10 and 30 amino acids, for example about 15 to 20 amino acids. Appropriate linkers will be apparent to those skilled in the art and include GlySer linkers (e.g., 15GS linker).

[0173] As described herein, the second ligand can be any ligand, protein, binding domain or binding unit and can bind to the transmembrane protein, i.e., via the binding site (9) (when the second ligand is part of the second fusion protein, the second ligand should most preferably also be appropriately included in the second fusion protein).

[0174] Generally, in the present invention (regardless of whether the binding site is directly or indirectly bound by a second fusion protein used in the arrangement of the present invention), the binding site (9) can be a conformational epitope of the transmembrane protein (2). More specifically, the binding site (9) undergoes a conformational change when the transmembrane protein (2) undergoes a conformational change, such as a conformational change from an inactive or less active state to an active, more active, and / or functional state and / or when a first ligand binds to the transmembrane protein, and its "shape" (i.e., the spatial arrangement of domains, loops, and / or amino acid residues that form the epitope) changes. It can be a conformational epitope of the transmembrane protein (2).

[0175] Preferably, when the binding site (9) changes its shape for the transmembrane protein (2) to receive a conformational shape, the binding site (9) and the second ligand change the affinity of the interaction between the binding site (9) and the second ligand (4). In particular, when the transmembrane protein (2) undergoes a conformational change from an inactive or less active state to an active, more active, functional, and / or drug - creatable state and / or when a first ligand (3) (especially a first ligand (3) that acts as an agonist for the transmembrane protein) binds to the transmembrane protein (2) and undergoes a conformational change, the binding site (9) and the second ligand can increase the affinity of the interaction between the binding site (9) and the second ligand (4).

[0176] In particular, its interaction with the second ligand (4) and the binding site (9) may be such that the second ligand (4) binds to the binding site (9) with high affinity when the transmembrane protein (2) is in an active, more active and / or functional state, and / or when the first ligand (3) (in particular, the first ligand (3) acting as an agonist with respect to the transmembrane protein (2)) binds to the transmembrane protein (2), the second ligand (4) may bind to the binding site (9) with high affinity. For example, its interaction with the second ligand (4) and the binding site (9) may range from an affinity in the micromolar range (i.e., higher than 1000 nM), for example, when the transmembrane protein is in an inactive, low-activity or ligand-free conformation, to the nanomolar range (i.e., lower than 1000 nM, for example, lower than 100 nM) when the transmembrane protein (2) is in a functional, active or more active and / or ligand-bound conformation. When the transmembrane protein (2) undergoes such a conformational change, the affinity of the second ligand (4) for the transmembrane protein (2) can increase by 10-fold, for example, 100-fold or more. For example, in the case of GPCRs, it is known that the affinity of the interaction between the G protein and the G protein binding site increases when a ligand (in particular, an agonist) binds to the extracellular binding site of the GPCR. Also, WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 75643, WO 2014 / 118297, WO 2014 / 122183 and WO 2014 / 118297 describe VHH domains (ConfoBodies) that have a high affinity for transmembrane proteins (e.g., in the nanomolar range for functional, active or ligand-bound conformations versus the micromolar range for inactive or ligand-free conformations) compared to when the transmembrane protein is in an inactive, low-activity or ligand-free conformation.

[0177] The second ligand itself may also undergo a conformational change when binding to the transmembrane protein (2). In embodiments where the second fusion protein binds indirectly to the transmembrane protein (2), this means that the binding domain or binding unit (5) of the second fusion protein that binds to the second ligand (4) has a higher affinity for the conformation adopted when the second ligand (4) is bound to the transmembrane protein (2), compared to the conformation adopted when the second ligand (4) is not bound to the transmembrane protein (2). For example, G proteins are known to undergo conformational changes when binding to GPCRs, and the VHH domain present in the second fusion protein may have a higher affinity for the GPCR-binding conformation of the G protein compared to the unbound conformation of the GPCR.

[0178] In a preferred embodiment, the binding site (9) is a binding site of the transmembrane protein (2) that functions as the binding site for the natural ligand of the transmembrane protein when the transmembrane protein (2) is in its native environment. More specifically, the binding site (9) can be a binding site of the transmembrane protein (2) that functions as the intracellular binding site for the natural intracellular ligand of the transmembrane protein when the transmembrane protein (2) is in its native environment. For example, when the transmembrane protein (2) is a receptor, the binding site (9) can be a binding site of the transmembrane protein (2) that functions as the intracellular binding site for one or more intracellular ligands of the transmembrane protein (2) involved in signal transduction when the transmembrane protein (2) is in its native environment.

[0179] In certain embodiments, when the transmembrane protein (2) is a GPCR, the binding site (9) can be a binding site for the G protein (and / or G protein complex). As further described herein, in such cases, the second ligand can be a natural, synthetic, or recombinant protein, or other ligand that can bind to the G protein-binding site of the GPCR.

[0180] If the second ligand is not a conformation-inducing binding domain or binding unit, the second ligand (4) will typically be a protein or proteinaceous ligand. In embodiments of the invention carried out in a suitable cell or cell line, the second ligand (4) may be a protein native to the cell or cell line used, or a suitable (recombinant) protein expressed in the cell or cell line used. For example, if the second ligand (4) is not part of a second fusion protein, the second ligand (4) can be a ligand for the transmembrane protein (2) that is naturally present in said cell or cell line (for example, if the transmembrane protein (2) is a GPCR, the second ligand (4) can be a G protein naturally expressed by the cell or cell line used). Alternatively, for example, if said cell or cell line does not naturally express a suitable ligand for the transmembrane protein (2), or if it is desired to use a ligand different from the ligand(s) naturally expressed by said cell or cell line (for example, if it is desired to use an analog, derivative or ortholog of the naturally expressed ligand, in which case the natural expression of the naturally expressed ligand may also be temporarily or constitutively suppressed or knocked out in the cell or cell line used), the second ligand may be a protein recombinantly expressed in the cell or cell line used. If the second ligand (4) forms part of a second fusion protein, the second ligand will typically be recombinantly expressed as part of the second fusion protein.

[0181] As further described herein, the second ligand (4) can be either part of the second fusion protein or separable from the second fusion protein. In either case (i.e., regardless of whether the second ligand is part of the second fusion protein or not), the second ligand can preferably bind to a conformational epitope of the transmembrane protein (or is directly bound to the transmembrane protein or is part of a protein complex that can directly bind to the transmembrane protein). More preferably, the second ligand (and / or the protein complex comprising the second ligand) preferably specifically binds to one or more functional, active and / or drugable conformations of the transmembrane protein, induces and / or stabilizes the formation of one or more functional, active and / or drugable conformations of the transmembrane protein (and / or shifts the conformational equilibrium of the transmembrane protein with respect to one or more such conformations); and / or induces and / or stabilizes the formation of a complex of the transmembrane protein, the first ligand and the second ligand.

[0182] When the second ligand is part of the second fusion protein, the second ligand can be any ligand, binding domain, binding unit, peptide, protein or other chemical substance that can directly bind to the transmembrane protein and can suitably be included in the second fusion protein. Preferably, as further described herein, when the second ligand is part of the second fusion protein, the second ligand is a suitable binding domain or binding unit, particularly an immunoglobulin single variable domain.

[0183] When the second ligand is separated from the second fusion protein, the second ligand can be any ligand or protein that can bind directly to the transmembrane protein and / or can form part of a protein complex that can bind to the transmembrane protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of the transmembrane protein, a semi-synthetic or synthetic analog or derivative of such a naturally occurring ligand, or an ortholog of such a naturally occurring ligand. Also, when the second ligand is not part of the second fusion protein, the second fusion protein will include a binding domain or binding unit that can bind indirectly (as defined herein) to the transmembrane protein, i.e., a binding domain or binding unit that can bind to the second ligand and / or a protein complex comprising the second ligand. Also, as further described herein, such a binding domain or binding unit can in particular be an immunoglobulin single variable domain, such as the camelid-derived ISVD. As also mentioned herein, such a binding domain or binding unit can be two or more (e.g., two or three) identical or different ISVDs (appropriately, ISVDs that are fused or linked via an optional suitable linker or spacer), which, when identical, generally bind to the same binding site or epitope of the second ligand, and when different, can bind to the same or different epitopes or binding sites of the second ligand (also, when the second ligand is a protein complex such as a G protein complex, can bind to the same or different subunits of said protein complex), and can include an ISVD.

[0184] If the second ligand does not form part of the second fusion protein, it will also be apparent to those skilled in the art that the binding domain or binding unit present in the second fusion protein and capable of binding to the second ligand should not substantially interfere with the binding of the second ligand to the transmembrane protein. For example, the second ligand preferably binds to a binding site or epitope of the second ligand that is different from the binding site of the second protein that binds to the transmembrane protein (preferably sufficiently removed from the binding site of the second protein that binds to the transmembrane protein so as to avoid any major steric hindrance).

[0185] If the second ligand (4) is a naturally occurring ligand of the transmembrane protein (2), the second ligand may be, for example, a ligand involved in the signal transduction pathway or signal transduction in which the transmembrane protein (2) is included. For example, if the second ligand (4) is a receptor, the second ligand (4) can be a naturally occurring ligand of the receptor, particularly a naturally occurring intracellular ligand of the receptor. For example, when an extracellular ligand binds to the extracellular binding site of the receptor, or when the receptor has a certain degree of constitutive activity and binds to the intracellular binding site of the receptor as part of the pathway providing said constitutive activity, it can be an intracellular ligand that binds to the intracellular binding site of the receptor. Suitable examples of such natural ligands will be apparent to those skilled in the art based on the disclosure herein and generally depend on the transmembrane protein (2) used. For example, if the transmembrane protein (2) is a 7TM or GPCR, the second ligand (4) can be (preferably) a G protein, for example, but not limited to, naturally occurring G proteins (e.g., G proteins naturally present in the cells or cell lines used) further described herein, or synthetic or semi-synthetic analogs or derivatives of naturally occurring G proteins (such as chimeric G proteins).

[0186] As further described herein, in particular in embodiments and implementations of the invention carried out using cells or cell lines, the second ligand (4) may also be part of a complex comprising the second ligand (4) and optionally one or more additional proteins. For example, if the transmembrane protein is a GPCR and the second ligand is a G protein or an analog or derivative of a G protein, the second ligand may be part of a complex formed by said G protein and optionally one or more additional proteins. One preferred but non-limiting example of such a complex is a G protein trimer comprising a G alpha subunit, a G beta subunit and a G gamma subunit. The complex may also include the transmembrane protein itself (e.g., a GPCR and a G protein or a GPCR and a G protein trimer). When the second ligand forms part of such a complex, it will generally be apparent to those skilled in the art that it is preferably not part of a second fusion protein. Instead, the second fusion protein will include a binding domain or binding unit that can bind to the second ligand or said complex. For example, if the second ligand forms part of a G protein complex, the binding domain or binding unit of the second fusion protein may be a VHH domain that binds to said complex, e.g., to a subunit within said complex or to the interface between two or more of said subunits. As described herein, an example of such a VHH domain is the VHH called "CA4435" (SEQ ID NO: 1 of WO 2012 / 75643 and SEQ ID NO: 1 herein).

[0187] The second ligand (4) may be a synthetic or semi-synthetic analogue or derivative of such a naturally occurring ligand, for example an analogue or derivative having a primary amino acid sequence different from that of the corresponding natural ligand by deletion, insertion and / or substitution of a limited number of amino acid residues or stretches of amino acid residues. Such analogues or derivatives may also be provided using appropriate techniques of recombinant DNA technology known per se, and in one embodiment may include (preferably, when present, as part of the whole of the second fusion protein including the second binding member (7) and any linker (11)) expression in a suitable host or host cell of a nucleotide sequence or nucleic acid encoding the analogue or derivative. For example, the transmembrane protein (2) is a 7TM or GPCR, and the second ligand (4) may be an analogue or derivative of a (preferred) G protein provided that the analogue or derivative still has sufficient affinity for the transmembrane protein (2) such that the analogue or derivative is suitably used in the method of the present invention, and may also have one or more amino acid differences from the natural sequence (as defined herein).

[0188] For example, in one particular embodiment, such an analogue or derivative of a naturally occurring G protein may be a naturally occurring G protein in which one or more amino acid residues (and / or one or more stretches of amino acid residues) have been replaced by one or more amino acid residues (and / or one or more stretches of amino acid residues) that are (essentially) in the same or corresponding position(s) in another naturally occurring G protein.

[0189] Where the G-protein is a heterotrimeric protein, such substitutions of one or more amino acid residues (and / or one or more stretches of amino acid residues) may be present or effected in any one, any two, or all three of the G-alpha, G-beta and / or G-gamma subunits, and in particular may be substitutions in the G-alpha subunit.

[0190] For example, in humans, it is well known that there are multiple genes encoding different G-alpha subunits, each of which can be grouped into different functional subfamilies and there are multiple G-alpha isoforms (see, for example, Flock et al., Nature, 2015, 524(7564), 173-179; and Nehme et al., PLoS One, 2017, 12(4)). Such analogs or derivatives of naturally occurring G-alpha subunits that can be used in the present invention are obtained by replacing one or more amino acid residues (and / or one or more stretches of amino acid residues) within the naturally occurring G-alpha subunit (amino acid sequence) with one or more amino acid residues (and / or one or more stretches of amino acid residues) at (essentially) the same or corresponding positions (s) within another naturally occurring alpha subunit (which may belong to the same or a different subfamily as the original subunit). Some specific but non-limiting examples are those in which one or more amino acid residues and / or one or more stretches of amino acid residues are replaced with one or more amino acid residues and / or one or more stretches of amino acid residues at (essentially) the same or corresponding positions (s) within the Gα i subunit, or those in which one or more amino acid residues and / or one or more stretches of amino acid residues are replaced with one or more amino acid residues and / or one or more stretches of amino acid residues at (essentially) the same or corresponding positions (s) within the Gα s subunit, or those in which one or more amino acid residues and / or one or more stretches of amino acid residues are replaced with one or more amino acid residues and / or one or more stretches of amino acid residues at (essentially) the same or corresponding positions (s) within the Gα q subunit, or those in which one or more amino acid residues and / or one or more stretches of amino acid residues are replaced with one or more amino acid residues and / or one or more stretches of amino acid residues at (essentially) the same or corresponding positions (s) within the Gα s subunit. In many cases, although not exclusively, such replaced / substituted amino acids or stretches of amino acids are present at or near the C-terminus of the alpha subunit.

[0191] Such "chimeric" G proteins and some specific but non-limiting examples of their design can also be found in the scientific literature. For example, again, see the references to Flock et al. and Nehme et al. cited above.

[0192] The second ligand (4) may also be another type of ligand generated to bind to the binding site (9) of the transmembrane protein (2), preferably binding in a manner further described herein.

[0193] In a particular preferred embodiment, when the methods herein are carried out in suitable cells, the second ligand (4) is preferably a polypeptide, protein, amino acid sequence or other chemical entity, preferably a polypeptide, protein, amino acid sequence or other chemical entity that can be obtained by appropriately expressing the nucleic acid or nucleotide sequence encoding it in the cells used in the methods of the invention.

[0194] As referred to herein, whether the transmembrane protein (2) (e.g., a naturally occurring G protein), a synthetic or semi-synthetic analog or derivative of such a naturally occurring ligand (e.g., a chimeric G protein described herein), or another type of ligand (e.g., a conformatibody further described herein), the second ligand (4) is generally a second ligand capable of binding, particularly specifically binding, to an epitope of the transmembrane protein (2), particularly the binding site (9). In particular, the second ligand (4) can be a second ligand capable of binding, particularly specifically binding, to an epitope that would be an intracellular epitope when the transmembrane protein (2) is in its native cellular environment.

[0195] As mentioned in this specification, the epitope (i.e., the binding site (9)) can be a linear epitope or a conformational epitope, preferably a conformational epitope (as described herein). For example, when the transmembrane protein (2) is a GPCR, the epitope may include one or more amino acid residues and / or a stretch of amino acid residues in at least one intracellular loop of the GPCR, and in particular, a conformational epitope formed by and / or including one or more amino acid residues and / or a stretch of amino acid residues in at least two different intracellular loops of the GPCR.

[0196] The epitope of the second ligand (4) can in particular be (part of) the epitope of the transmembrane protein (2) involved in signal transduction mediated by the transmembrane protein (2). For example, the second ligand can bind to an epitope on the transmembrane protein (2) that is within the binding site of a downstream signal transduction protein. For example, when the transmembrane protein (2) is a GPCR, the second ligand (4) can be a binding domain or binding unit that can specifically bind to a conformational epitope contained within, located at, or overlapping with the G protein binding site of the GPCR.

[0197] If the transmembrane protein (2) used can adopt two or more conformations (such as a basal state / conformation, an active state / conformation, and / or an inactive state / conformation, etc.) and / or is a protein that can undergo a conformational change (in particular a functional conformational change), the second ligand (4) is preferably a protein that can bind, in particular specifically bind, to the functional conformational state of the transmembrane protein (2).

[0198] In certain preferred embodiments, the second ligand (4), upon binding to the transmembrane protein (2), stabilizes and / or induces the functional and / or active conformational state of the transmembrane protein (2) (and / or shifts the conformational equilibrium of the transmembrane protein (2) from an inactive or less active state(s) to a more active state), makes the transmembrane protein adopt a more drug - discovery - promising conformation (and / or shifts the conformational equilibrium of the transmembrane protein (2) from a less drug - discovery - promising conformation(s) to a more drug - discovery - promising conformation(s)), changes the conformation of the protein (and the associated binding pocket) to be more suitable or accessible for the binding of the first ligand (3), or overall increases the affinity of the interaction with the first ligand (3) (and / or shifts the conformational equilibrium of the protein to such a conformation), and / or induces and / or stabilizes the formation of a complex comprising the second ligand, the transmembrane protein, and the first ligand (and / or shifts the conformational equilibrium of the transmembrane protein to the formation of such a complex), or is a second ligand such that any combination thereof is possible.

[0199] Thus, when the transmembrane protein (2) is a GPCR, the second ligand (4) can be a second ligand that binds to the functional conformational state of the GPCR, more preferably the active conformational state of the GPCR, and can in particular stabilize and / or induce it. Also, the second ligand (4) is preferably a conformational state in which the protein or GPCR is not bound by any first ligand (3) or is bound by a ligand (3) acting as an inverse agonist, compared to when the protein or GPCR is bound by an agonist (e.g., bound by a first ligand (3) acting as an agonist of the protein or GPCR). It is a second ligand that preferentially / specifically binds to the protein or GPCR when it is bound by an agonist, and / or increases the affinity of the protein or GPCR for at least one compound or ligand acting as an agonist of the protein or GPCR (i.e., increases by at least 2-fold, particularly at least 5-fold, and more preferably at least 10-fold).

[0200] As mentioned, one preferred class of compounds for use in the present invention as the second ligand (particularly when the second ligand is included in a second fusion protein) is generally described in WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 75643, WO 2014 / 118297, WO 2014 / 122183 and WO 2014 / 118297, and includes VHH domains (conformatibodies) capable of stabilizing the GPCR in the desired conformation.

[0201] WO 2012 / 75643 also discloses a number of VHH domains that can bind indirectly to a GPCR by binding to a G protein or G protein complex. Some preferred but non-limiting examples of these are the VHH called "CA4435" (SEQ ID NO: 1 of WO 2012 / 75643 and SEQ ID NO: 1 herein) that can bind to a G protein complex, and the VHH called "CA4437" (SEQ ID NO: 4 of WO 2012 / 75643 and SEQ ID NO: 2 herein) that can bind to a G protein. Such VHH domains can be appropriately included in a second fusion protein to provide a second fusion protein that can bind indirectly to a GPCR by binding to a G protein or G protein complex.

[0202] Accordingly, in one preferred embodiment of the present invention, the second fusion protein comprises at least one such VHH or conformatibody and a second binding member (7).

[0203] Generally, in the present invention, the first binding member (6) and the second binding member (7) will be in very close proximity to each other when the second fusion protein binds directly or indirectly (both as defined herein) to the transmembrane protein (2). In particular, the first and second binding members are such that when the second ligand (4) present in the second fusion protein binds directly to the transmembrane protein (2), or when the binding domain or binding unit (5) present in the second fusion protein binds indirectly to the transmembrane protein, i.e., when the binding domain or binding unit (5) binds to the second ligand (4), or in the case of the embodiment shown in FIG. 3, to the protein complex (12), they are in very close proximity to each other, and the second ligand (4) or the protein complex (12) then binds to or is bound by the transmembrane protein (2). Preferably, the first and second binding members should not have a high affinity for each other per se, so that their association (resulting in the generation of an associated detectable signal) is mainly due to the second ligand binding (directly or indirectly) to the transmembrane protein and is essentially independent of, or only to a low extent, the affinity between the first ligand and the second ligand (an example of such a suitable binding pair by the NanoBiT system from Promega), and it will be apparent to those skilled in the art that it is mainly driven by the first and second binding members coming into proximity to each other. However, any such affinity between the first ligand and the second ligand generally provides a baseline for the detectable signal, which should not substantially interfere with the assay of the present invention as the readout of this assay mainly looks at any change in the detectable signal upon addition of the first ligand to an arrangement of the present invention that does not contain the first ligand (more generally, for some uses of the methods and arrangements of the present invention, it should also be noted that it may be preferable to have some level of baseline signal when the readout may also include a decrease in signal compared to the baseline).

[0204] Thus, generally in the present invention, the detectable signal (or any change in said signal) generated by the first and second binding members will be proportional to the amount of the second fusion protein that is directly or indirectly bound to the transmembrane protein (2). This will then be due to the binding interaction between the second ligand (4) and the transmembrane protein (in particular, one or more specific conformations that the second ligand and the transmembrane protein can assume, such as functional, active and / or druggable conformations), and / or any change to said binding interaction (in particular, as a result of a conformational change of the transmembrane protein and / or a shift in the conformational equilibrium of the transmembrane protein, for example, any change to said binding interaction due to the binding of the first ligand to the transmembrane protein and / or the formation of a complex between the first ligand, the transmembrane protein and the second ligand).

[0205] Methods for identifying and making the various elements of the above-described arrangements and compositions, and methods for assembling such arrangements and compositions are included herein. Such methods can be combined with, and can form part of, any assay and method for measuring or determining one or more properties of the first ligand.

[0206] By way of non-limiting example, methods for determining one or more properties of the first ligand described herein may include one or more steps directed to determining whether the second ligand binds to the transmembrane protein, binds specifically to the transmembrane protein, binds specifically to a domain of the transmembrane protein located in a second environment, is a conformational selective binder of the transmembrane protein, stabilizes the conformation of the transmembrane protein, stabilizes the inactive conformation of the transmembrane protein, stabilizes the functional, active, and / or druggable conformation of the transmembrane protein, and / or stabilizes the complex between the transmembrane protein and the first ligand.

[0207] Based on this and further disclosure of the present invention, the methods and arrangements of the present invention are directed to one or more properties of the first ligand (in particular, properties of the first ligand that affect and / or determine the interaction between the first ligand and the transmembrane protein), one or more properties of the second ligand (in particular, properties of the second ligand that affect and / or determine the interaction between the second ligand and the transmembrane protein), and / or one or more properties of any binding domain or binding unit present in the second fusion protein (in particular, when the binding domain or binding unit binds directly to the transmembrane protein, properties of the binding domain or binding unit that affect, and / or determine the interaction between the binding domain or binding unit and the transmembrane protein; or when the binding domain or binding unit binds to the second ligand and / or a protein complex comprising the same, properties of the binding domain or binding unit that affect and / or determine the interaction between the binding domain or binding unit and the second ligand or the complex), it will be apparent to those skilled in the art that they can be used to measure or determine such properties.

[0208] More specifically, with respect to the first ligand, the methods and arrangements of the present invention can be used to measure or determine the ability of the first ligand to bind to a transmembrane protein, affect a conformational change of the transmembrane protein, and / or affect a change in the conformational equilibrium of the transmembrane protein. For example, as further described herein, the methods and arrangements of the present invention measure or determine the ability of a given first ligand to act as an agonist, antagonist, inverse agonist, inhibitor, or modulator (e.g., allosteric) modulator of a transmembrane protein, and / or screen for or identify small molecules, proteins, or other compounds or chemical substances that act or are capable of acting as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) modulators of a transmembrane protein. In this regard, when the methods and arrangements of the present invention are used for such purposes (i.e., for purposes related to the first ligand), then typically (and preferably) other components used in the arrangements of the present invention (e.g., the second ligand present in the second fusion protein and / or any binding domain or binding unit) will have properties that are known (i.e., their properties related to their use in the methods and arrangements of the present invention are known and / or characterized), and / or will be selected such that their use in the methods and arrangements of the present invention has already been established, which will be apparent to those skilled in the art based on the disclosure herein.

[0209] The assays of the present invention can also be performed in the presence of a compound having a known effect on the transmembrane protein (e.g., in the presence of a known agonist, antagonist, inverse agonist, inhibitor or modulator (e.g., allosteric modulator) of the transmembrane protein) at a concentration at which it is known that the "known" compound has its effect on the transmembrane protein. The known compound is usually present in the same environment as the first ligand (i.e., the ligand whose properties are determined using the assay of the present invention). For example, in the method of the present invention in which the first ligand is added to the arrangement of the present invention in which the first ligand is not yet present, the known compound may be added essentially simultaneously with the first ligand, may be added separately before the addition of the first ligand, or may be added after the first ligand can be added. The readout from the assay can vary depending on the order in which the first ligand and the known compound are added, and, if the first ligand and the known compound are not added essentially simultaneously, the time between the moment the first ligand is added and the moment the known compound is added (or vice versa). By varying the order and / or timing of addition of the first ligand and the known compound, it is also possible to determine different properties of the first ligand and / or to identify different first ligands having such properties.

[0210] For example, but not limited to, the assays of the present invention can be performed in the presence of a known agonist of the transmembrane protein (i.e., in the same environment as the first ligand). In this setting, the assays of the present invention can be used, for example, to determine whether and how the first ligand can counteract the agonist effect of a known compound, since the first ligand acts as an antagonist in this example. (Thus, in this setting, the assays of the present invention may be used to identify and / or characterize potential antagonists and agonists of the transmembrane protein.) The setting by the presence of a known agonist can also be used, for example, to determine whether an allosteric modulator that increases or decreases the effect of the agonist can be used and / or to identify and / or characterize a first ligand that can act as an inverse agonist of the transmembrane protein. It may also be possible to perform a competitive assay between the first ligand and a known compound.

[0211] The methods and arrangements of the present invention can be used to measure or determine the ability of a second ligand to bind to a transmembrane protein, in particular its ability to bind to and / or stabilize a specific conformation of the transmembrane protein (such as a functional, active or drugable conformation), and / or its ability to stabilize and / or induce the formation of a complex between a first ligand, a second ligand and a transmembrane protein. For example, as further described herein, the methods and arrangements of the present invention can be used to measure or determine the ability of a given VHH to act as a conformational body of a transmembrane protein or to identify, optimize or validate a VHH that can act as a conformational body. For this purpose, usually, the VHH or candidate VHH is present in a second fusion protein (i.e., as a second ligand) and is tested for its ability to bind directly to the transmembrane protein or to a specific conformation of the transmembrane protein or one or more of the transmembrane proteins. As further described herein, the methods and arrangements of the present invention can also be used to measure or determine the ability of an analog, derivative or ortholog of a native ligand of a transmembrane protein that acts as a ligand of the transmembrane protein (for example, to test an analog, derivative or ortholog of a naturally occurring G protein that acts as a ligand of a related GPCR). In this case, usually, the second ligand is not present in the second fusion protein (although in some cases a second fusion protein containing the analog, derivative or ortholog as the second ligand may be used), but instead, the second fusion protein contains a binding domain or binding unit (such as a VHH) that can bind to the second ligand (or a complex containing it).In this regard, when the methods and arrangements of the present invention are used for such purposes (i.e., for purposes regarding the second ligand), it will be apparent to those skilled in the art based on the disclosure herein that the other components used in the arrangements of the present invention (e.g., the first ligand and / or any binding domain or binding unit present in the second fusion protein) will typically (and preferably) be selected such that they have known properties (i.e., their properties relevant to their use in the methods and arrangements of the present invention are known and / or characterized) and / or such that they have already been verified for use in the methods and arrangements of the present invention.

[0212] The methods and arrangements of the present invention can also be used to measure or determine the ability of a binding domain or binding unit present in a second fusion protein to bind to a given second ligand and / or a protein complex comprising the second ligand. For example, as further described herein, the methods and arrangements of the present invention can be used to measure or determine the ability of a given VHH to bind to a G protein and / or to identify, optimize or validate such a VHH that can bind indirectly to a transmembrane protein (whereupon the transmembrane protein can then be used, for example, as a binding domain or binding unit in the arrangements of the present invention as described herein or for any other suitable purpose). Such methods and arrangements of the present invention can also be used to measure or determine the ability of a given VHH to bind to a protein complex comprising a G protein and / or to identify, optimize or validate such a VHH (again, such a VHH can be used as a binding domain or binding unit in the arrangements of the present invention as described herein or for any other suitable purpose). In this regard, it will be apparent to those skilled in the art based on the disclosure herein that when the methods and arrangements of the present invention are used for such purposes (i.e., for purposes related to a binding domain or binding unit that binds indirectly to a transmembrane protein), usually (and preferably) other components used in the arrangements of the present invention (such as the first ligand and the second ligand) will be selected such that they have known properties (i.e., their properties relevant to their use in the methods and arrangements of the present invention are known and / or characterized) and / or such that they have already been validated for use in the methods and arrangements of the present invention.

[0213] In the present invention, generally, a detectable signal is preferably generated in response to, and more preferably in proportion to, a conformational change in the transmembrane protein and / or a shift in the conformational equilibrium of the transmembrane protein. As further described herein, without being limited to any particular mechanism or explanation, the conformational change and / or shift in the conformational equilibrium of the transmembrane protein is then caused by the binding of a first ligand to the transmembrane protein (or otherwise causing a conformational change in the transmembrane protein) and / or by the formation of a complex of the first ligand, transmembrane protein, and second ligand, where the second ligand can, for example, stabilize the complex or otherwise induce or facilitate the formation of the complex. Thus, more generally, in the present invention, a detectable signal (or any change thereof, as further described herein) will be generated in response to the presence of the first ligand in a first environment and / or in response to the binding of the first ligand to the transmembrane protein (or otherwise causing a conformational change in the transmembrane protein and / or a shift in the conformational equilibrium of the transmembrane protein).

[0214] Also, typically, particularly when using the methods and arrangements of the present invention to test, optimize, and / or validate a first ligand and / or to identify small molecules, proteins, ligands, or other chemical substances that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) of the transmembrane protein, the detectable signal (or any change thereof, as further described herein) will be proportional to the amount and / or concentration of the first ligand present in the first environment (and / or the environment to which the transmembrane protein is exposed) and / or to the affinity of the transmembrane protein for the first ligand (e.g., as compared to other ligands tested).

[0215] Accordingly, based on the description herein, in one aspect of the invention, it will be apparent to those skilled in the art that the methods and arrangements described herein can be used to detect the presence of a first ligand in a first environment and / or to determine the amount and / or concentration of the first ligand in the first environment. The methods and arrangements described herein can also be used, for example, to measure the amount of signal that occurs when different concentrations of the first ligand are present in the first environment, e.g., to establish the relationship between the amount / concentration of the first ligand in the first environment and the detectable signal (level and / or change). The methods and arrangements described herein can also be used, for example, to determine the affinity of the first ligand for the transmembrane protein by comparing the signal generated by one or more known concentrations of the first ligand in the first environment with the signal generated by the same arrangement with a known concentration of another ligand that has a known affinity for the transmembrane protein.

[0216] As further described herein, the methods and arrangements of the invention can also be used to determine whether a given (first) ligand is an agonist, antagonist, inverse agonist, inhibitor or modulator, (e.g., allosteric) modulator of the transmembrane protein.

[0217] When the methods and arrangements of the invention are used to determine one or more characteristics of the first ligand, the arrangement of the invention is typically first set up or otherwise established such that the first ligand is absent, and then the arrangement is contacted with the ligand (e.g., by adding the ligand to the first environment), and then the detectable signal (or any change thereof) resulting from the presence of the first ligand is measured (optionally compared with the signal without the presence of the first ligand and / or one or more reference values). Accordingly, the arrangements described herein in which the first ligand is absent (e.g., before the first ligand is added) form a further aspect of the invention.

[0218] Another aspect of the invention is a method of providing an arrangement of the invention described herein, the method comprising the step of adding a first ligand to an arrangement of the invention (as described herein) that does not (yet) contain the first ligand. Thus, the resulting arrangement can be used to measure at least one property of the first ligand, particularly a property of the first ligand that can be measured or otherwise determined using the arrangement of the invention, or otherwise determined.

[0219] As will be apparent to those skilled in the art based on the disclosure herein, an arrangement of the invention in the absence of the first ligand (i.e., an arrangement of the invention that does not yet contain the first ligand) comprises at least the following components: · A boundary layer separating a first environment and a second environment; · A transmembrane protein; · A ligand for the transmembrane protein present in the second environment; and · A binding pair comprising at least a first binding member and a second binding member that can generate a detectable signal; The components are arranged relative to each other (and, where applicable, operably linked and / or associated with each other) in a manner further described herein (i.e., in substantially the same manner as described for an arrangement of the invention that contains the first ligand).

[0220] In particular, an arrangement of the invention in the absence of the first ligand (i.e., an arrangement of the invention that does not yet contain the first ligand) comprises at least the following components: · A boundary layer separating a first environment and a second environment; · A binding pair comprising at least a first binding member and a second binding member that can generate a detectable signal; · A transmembrane protein (i.e., forming a first fusion protein) appropriately fused or linked (either directly or via a suitable linker or spacer) to one of the binding members of the binding pair; and · A second ligand for a transmembrane protein present in the second environment; comprising components arranged relative to each other (and, where applicable, operably linked and / or associated with each other) in a manner further described herein (i.e., in substantially the same manner as described for the arrangements of the invention that include a first ligand). In particular, the second member of the binding pair may be part of a second fusion protein (different from the first fusion protein that includes the transmembrane protein and the first binding member of the binding pair), and the second fusion protein is further described herein.

[0221] More particularly, an arrangement of the invention in the absence of a first ligand (i.e., an arrangement of the invention that does not yet include a first ligand) includes at least the following components: · A boundary layer separating the first environment and the second environment; · A binding pair comprising at least a first binding member and a second binding member, the binding pair capable of generating a detectable signal; · A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., the member of the binding pair is present in the second environment); · A second fusion protein comprising a transmembrane protein and a protein capable of binding directly or indirectly to the other binding member of the binding pair, the second fusion protein being present in the second environment; comprising components arranged relative to each other (and, where applicable, operably linked and / or associated with each other) in a manner further described herein (i.e., in substantially the same manner as described for the arrangements of the invention that include a first ligand).

[0222] Other aspects, embodiments and references of arrangements of the invention that do not include a first ligand are those other aspects, embodiments and references of arrangements of the invention that include a first ligand as described herein but in the absence of a first ligand.

[0223] Generally, any such arrangement of the present invention in the absence of the first ligand will, once the first ligand is added as part of the method described herein, become the corresponding arrangement of the present invention that includes the first ligand. Accordingly, another aspect of the present invention is a method of preparing an arrangement of the present invention as described herein, the method comprising the step of adding a first ligand to an arrangement of the present invention (as described herein) that does not (yet) contain the first ligand. Accordingly, the resulting arrangement may be used to measure or otherwise determine at least one property of the first ligand, particularly a property of the first ligand that can be measured or otherwise determined using the arrangement of the present invention.

[0224] The present invention also relates to a method of measuring or otherwise determining at least one property of a compound or ligand, the method comprising at least: · adding the compound or ligand as a first ligand to an arrangement of the present invention that does not yet contain the first ligand; and · measuring or otherwise determining at least one property of the compound or ligand, wherein the property is a property that can be measured or otherwise determined using the arrangement. The method includes the steps of.

[0225] In this aspect of the present invention, the property is preferably a property typified by the ability (e.g., affinity) of a compound or ligand to bind to and / or modulate a transmembrane protein.

[0226] The present invention also relates to a method of measuring or otherwise determining the ability of a compound or ligand to change a detectable signal generated by a binding pair present in an arrangement of the present invention as further described herein, the method comprising at least: · adding the compound or ligand as a first ligand to an arrangement of the present invention that does not yet contain the first ligand; and · A step of determining whether adding the compound or ligand results in a change in a detectable signal generated by a binding pair used in the arrangement, and optionally a step of measuring the change in the detectable signal including the step of

[0227] Thus, in another aspect, the present invention is at least: a) The following components: · A boundary layer separating a first environment and a second environment; · A transmembrane protein; · A ligand for the transmembrane protein present in the second environment; and · A binding pair consisting of at least a first binding member and a second binding member, which can generate a detectable signal; including at least, and arranging the components in a manner further described herein (operatively connected and / or associated with each other where applicable) to provide an arrangement; and; b) A step of adding a first ligand to the first environment relates to a method including the step of The method preferably further includes: c) A step of measuring the signal generated by the binding pair and / or a step of measuring the change in the signal generated by the binding pair including the step of

[0228] In a more specific aspect, the present invention is at least: a) The following components: · A boundary layer separating a first environment and a second environment; · A binding pair consisting of at least a first binding member and a second binding member, which can generate a detectable signal; ·A transmembrane protein appropriately fused or linked (either directly or via a suitable linker or spacer) to one of the binding members of the binding pair (i.e., to form a first fusion protein); and ·A second ligand for the transmembrane protein present in the second environment; Providing an arrangement comprising at least the above components, wherein the components are arranged relative to each other in a manner further described herein (and operably linked and / or associated with each other where applicable); and; b) Adding a first ligand to the first environment relates to a method comprising the steps of: The method preferably further comprises: c) Measuring the signal generated by the binding pair and / or measuring a change in the signal generated by the binding pair in further steps.

[0229] In another particular aspect, the invention relates to an arrangement comprising at least: a) The following components: ·A boundary layer separating the first environment from the second environment; ·A binding pair comprising at least a first binding member and a second binding member, which can generate a detectable signal; ·A first fusion protein comprising a transmembrane protein and one of the binding members of the binding pair (i.e., said member of the binding pair is present in the second environment); ·A second fusion protein comprising a transmembrane protein and a protein capable of binding directly or indirectly to the other binding member of the binding pair, the second fusion protein being present in the second environment; Providing an arrangement comprising at least the above components, wherein the components are arranged relative to each other in a manner further described herein (and operably linked and / or associated with each other where applicable); and; b) Adding a first ligand to the first environment Regarding a method including the steps of, The method preferably: c) Measuring the signal generated by the coupling pair and / or measuring the change in the signal generated by the coupling pair further includes the step of.

[0230] As further described herein, in this aspect of the invention, the first ligand can be any desired and / or suitable compound or ligand, including but not limited to small molecules, low molecular weight peptides, biomolecules or other chemical substances. The methods described in this aspect (and other methods of the invention) are used to measure or otherwise determine at least one property of the compound or ligand added to the arrangement as the first ligand, particularly the ability of the compound or ligand to produce a detectable signal change generated by a binding pair, the ability of the compound or ligand to bind to a transmembrane protein, the ability of the compound or ligand to affect conformational changes in the transmembrane protein, and / or the ability of the compound or ligand to modulate (as defined herein) the transmembrane protein and / or the signal transduction pathway(s) and / or biological mechanism(s) in which the transmembrane protein is included. It will be apparent to those skilled in the art that the method can be used to determine whether such a compound or ligand can act as an agonist, antagonist, inverse agonist, inhibitor or modulator (e.g., allosteric modulator) of the transmembrane protein and / or the signal transduction pathway(s) and / or biological mechanism(s) in which the transmembrane protein is included. Also, the methods and arrangements of the invention can be used to identify and / or screen for compounds or ligands having the ability to produce a detectable signal change generated by a binding pair, the ability to bind to a transmembrane protein, the ability to affect conformational changes in the transmembrane protein, the ability to modulate the transmembrane protein and / or the signal transduction pathway(s) and / or biological mechanism(s) in which the transmembrane protein is included, and / or the ability to act as an agonist, antagonist, inverse agonist, inhibitor and / or modulator (e.g., allosteric modulator) of the transmembrane protein, and such use of the methods and arrangements described herein forms a further aspect of the invention.

[0231] In a further aspect, it should be noted that the methods and arrangements of the present invention can also be used to measure or otherwise determine at least one property of the second ligand, for example, the ability of the second ligand to bind to the transmembrane protein, the ability of the second ligand to bind to and / or stabilize a particular conformation of the transmembrane protein (such as an active and / or drugable conformation), and / or the ability of the second ligand to stabilize a complex of the transmembrane protein with the first ligand and the second ligand. Usually, in this aspect of the invention, one or more first ligands having a known ability to bind to and / or modulate the transmembrane protein are used to determine whether the arrangement of the invention, which includes the (candidate) second ligand, produces a detectable signal when the first ligand is added to the arrangement (e.g., at one or more known concentrations).

[0232] For example, this aspect of the invention can be used to identify or optimize a binding domain or binding unit (e.g., an ISVD) that can bind directly to a transmembrane protein (as defined herein), particularly a binding domain or binding unit that is specific and / or selective for the conformation of the transmembrane protein that occurs when the first ligand used binds to the transmembrane protein. A binding domain or binding unit thus identified, optimized, and / or validated can be used, for example, in an arrangement of the invention (i.e., as part of a second fusion protein), and / or to induce or stabilize a specific conformation of the transmembrane protein (e.g., for screening or crystallization purposes as described in the prior art cited herein for conformation-specific ligands of GPCRs). Thus, for example, this aspect of the invention can be used to identify, optimize, and / or validate an ISVD for use as a conformational body, which can then be used for the purposes described herein and / or for uses known per se for conformation-specific ISVDs. Again, see further prior art cited herein.

[0233] Generally, in this aspect of the invention, a binding domain, binding unit, ligand, or other protein that is tested or validated for (direct) binding to a transmembrane protein is part of a second fusion protein. Thus, the invention provides a method of measuring or otherwise determining at least one property of a binding domain, binding unit, ligand, or other protein, comprising at least: · providing an arrangement of the invention that does not yet contain a first ligand, wherein the second fusion protein comprises the binding domain, binding unit, ligand, or other protein; · adding a first ligand to the arrangement; · A method comprising determining whether adding the first ligand results in a change in a detectable signal generated by a binding pair used in the arrangement, and optionally measuring the change in the detectable signal.

[0234] As will be apparent to those skilled in the art based on the disclosure herein, the at least one property of the binding domain, binding unit, ligand or other protein may in particular be the ability of the binding domain, binding unit, ligand or other protein to bind to the transmembrane protein (in particular to the putative conformation of the transmembrane protein when the first ligand used binds to the transmembrane protein), the ability of the binding domain, binding unit, ligand or other protein to stabilize the putative conformation of the transmembrane protein when the first ligand used binds to the transmembrane protein, and / or the ability of the binding domain, binding unit, ligand or other protein to promote or induce the formation of a complex between the first ligand used, the transmembrane protein and the binding domain, binding unit, ligand or other protein, and / or the ability of the binding domain, binding unit, ligand or other protein to stabilize such a complex.

[0235] In another aspect of the invention, the arrangements described herein are again used to measure or determine at least one property of a second ligand and / or to identify, optimize, and / or validate a candidate second ligand. In this aspect, however, the second fusion protein does not include the second ligand being tested or the candidate second ligand. Instead, it includes a binding domain or binding unit known to bind to the second ligand being tested or the candidate second ligand. In other words, in this aspect, the second fusion protein can bind indirectly to the transmembrane protein (as defined herein), i.e., via the second ligand being tested or the candidate second ligand, or via a protein complex containing it, provided that the second ligand or complex is capable of binding to the transmembrane protein (particularly to the conformation of the transmembrane protein that occurs when the first ligand binds to the transmembrane protein). Similar to the previous aspect, this aspect can also be used to identify, optimize, and / or validate (candidate) ligands of the transmembrane protein, such as synthetic or semi-synthetic analogs or derivatives of the naturally occurring ligand of the transmembrane protein. For example, if the transmembrane protein is a GPCR, this aspect of the invention can be used to identify, optimize, and / or validate analogs or derivatives of the G protein that is the natural ligand of the GPCR, or to determine whether an ortholog of the natural G protein of a related GPCR can bind to the GPCR, and / or to stabilize the complex of the GPCR and the first ligand used.

[0236] Accordingly, the present invention is a method of measuring or otherwise determining at least one property of a ligand or other protein, comprising at least ·Preparing an arrangement of the invention that does not yet contain the first ligand, wherein the ligand or other protein is present and / or used as a second ligand, and the second fusion protein comprises a binding domain or binding unit capable of binding to the ligand or other protein and / or a protein complex comprising the ligand or other protein, preparing the arrangement; ·Adding a first ligand to the arrangement; ·Determining whether adding the first ligand results in a change in a detectable signal generated by a binding pair used in the arrangement, and optionally measuring the change in the detectable signal, relating to a method.

[0237] As described herein, in a particular embodiment of the invention, the method of the invention is carried out using a suitable cell or cell line in which all components of the arrangement of the invention are appropriately present and arranged to provide a workable arrangement of the invention. Such a cell or cell line would appropriately contain a transmembrane protein (2) in its cell wall or cell membrane such that at least a portion of the amino acid sequence of the transmembrane protein protrudes into the extracellular environment (as defined herein) and at least one other portion of the amino acid sequence of the transmembrane protein protrudes into the intracellular environment (as defined herein). Also, preferably, and as further described herein, the transmembrane protein (2) forms part of the first fusion protein as described herein, and the arrangement will also contain the second fusion protein as described herein. More preferably, the extracellular environment is the "first environment" (i.e., the environment in which the first ligand (3) is present or to which the first ligand (3) is added), and the intracellular environment is the "second environment" (i.e., the environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0238] Accordingly, in a further aspect, the invention relates to the method or arrangement described herein, wherein the boundary layer (2) is a cell wall or membrane.

[0239] Also as described herein, when the method of the invention is carried out with a cell or a suitable cell line, the cell or cell line used preferably comprises one or more, and preferably all, of the following components of all arrangements of the invention: · a first fusion protein comprising a transmembrane protein (2) and a first binding member (6); · a second fusion protein comprising a second binding member (7) and a protein that can bind directly or indirectly (as defined herein) to the transmembrane protein (2); and / or · when the second fusion protein binds directly to the transmembrane protein (2), a second ligand (4) and / or a protein that form a protein complex (12) are appropriately expressed.

[0240] In the context of a cell or cell line that expresses one or more components of the arrangement of the present invention, and more generally in the context of the present description and claims, the term "appropriately expresses" means that when such a component is expressed, the cell or cell line expresses, or is capable of expressing, the nucleotide sequence or nucleic acid encoding said component (i.e., under the conditions used to carry out the method of the present invention) such that it can function as an operative part of the arrangement of the present invention. For example, with respect to the transmembrane protein (2), this means that the transmembrane protein is expressed as part of a first fusion protein such that the expressed transmembrane protein (2) is appropriately anchored or otherwise incorporated into the cell wall or cell membrane of the cell so that at least a part of the amino acid sequence of the transmembrane protein protruding into the extracellular environment (as defined herein) and at least one other part of the amino acid sequence of the transmembrane protein protruding into the intracellular environment (as defined herein) cause the transmembrane protein (2) to penetrate the cell wall or cell membrane. With respect to the first and second fusion proteins, "appropriately expresses" means that the first and second fusion proteins are expressed such that when the second fusion protein binds directly or indirectly to the transmembrane protein (2), the first binding member and the second binding member of the binding pair (6 / 7) can contact or be in very close proximity to each other (most preferably, expressed in the intracellular environment).

[0241] When a cell is used to carry out the method of the present invention, any appropriate expression of each component of the arrangement of the present invention can be transient or constitutive expression, provided that all the necessary components of the arrangement of the present invention are present appropriately and operably in sufficient amounts at the appropriate time.

[0242] In one aspect of the present invention, in embodiments of the present invention where the second fusion protein binds indirectly to the transmembrane protein (i.e., the second ligand (4) is not part of the second fusion protein), the cells or cell lines used preferably naturally express the second ligand (4) and / or the protein that constitutes the protein complex (12). For example, but not limited to, in this aspect of the present invention, the transmembrane protein (2) may be a GPCR, the second ligand (4) may be a G protein naturally expressed by the cells or cell lines used, and / or the protein complex (12) may be a G protein trimer containing a G alpha subunit, a G beta subunit, and a G gamma subunit naturally expressed by the cells or cell lines used. More generally, in these aspects of the present invention, the cells or cell lines used naturally express one or more natural ligands (especially intracellular ligands) of the transmembrane protein (2) and / or one or more ligands that can function as the second ligand of the transmembrane protein (2), depending on the transmembrane protein (2) being used or screened.

[0243] The cells or cell lines can be any cells or cell lines suitable for use in the methods and arrangements of the present invention, including but not limited to mammalian cells and insect cells. Some preferred, but not limited, examples are human cell lines such as HEK293T.

[0244] Appropriate techniques for transiently or stably expressing the desired protein in such cells or cell lines so that the transmembrane protein (2) is properly immobilized on the cell wall or cell membrane of the cells will be apparent to those skilled in the art and include techniques involving the use of appropriate transfection reagents such as SigmaAldrich's X-tremeGENE™ or polyethyleneimine (PEI).

[0245] When the method of the present invention is carried out using a cell or cell line that appropriately expresses one or more components of the arrangement of the present invention, the method of the present invention will generally also include the step of culturing or maintaining the cells under conditions such that the cells or cell line appropriately express the components.

[0246] Accordingly, in another aspect, the present invention relates to a cell or cell line comprising a fusion protein, said fusion protein comprising a transmembrane protein (described herein) fused directly or via a suitable linker to a binding domain or binding unit that is a first binding member of a binding pair, said binding pair comprising at least said binding domain or binding unit as a first binding member and a further binding domain or binding unit as a second binding member, wherein said first and second binding members of said binding pair are capable of generating a detectable signal when they contact each other or are in very close proximity to each other. The present invention also relates to a cell or cell line that expresses or is capable of expressing such a fusion protein (i.e., under appropriate conditions).

[0247] Such a cell or cell line may be further described herein, and preferably, the transmembrane protein is incorporated into the cell wall or cell membrane of the cell or cell line, penetrates the cell wall or cell membrane, and more preferably, at least a portion of the amino acid sequence of the transmembrane protein protrudes into the extracellular environment (as defined herein), and at least one other portion of the amino acid sequence of the transmembrane protein protrudes into the intracellular environment (as defined herein) in such a manner that the fusion protein is expressed or is capable of being expressed. More preferably, in said cell or cell line, the first binding member of the binding pair is present in the intracellular environment of the cell (as defined herein) and / or the cell or cell line expresses or is capable of expressing the fusion protein, and upon such expression, the first binding member is present in the intracellular environment of the cell (as defined herein).

[0248] In addition, the transmembrane protein present in the fusion protein is preferably a transmembrane protein further described herein, more preferably having at least two ligand-binding sites, one protruding into the extracellular environment (as defined herein) and one protruding into the intracellular environment (as defined herein). Furthermore, as described herein, the transmembrane protein preferably undergoes a conformational change (in particular, a conformational change from an essentially inactive or less active conformation to an active or more active conformation) upon binding of a ligand to the ligand-binding site of the transmembrane protein, particularly upon binding of a ligand present in the extracellular environment to the ligand-binding site of the transmembrane protein present in the extracellular environment (as defined herein). As further described herein, the transmembrane protein is preferably further stabilized in a functional and / or active (or more active) conformation (in particular, a drugable conformation and / or a ligand-binding conformation, more particularly an agonist-binding conformation) by binding of an appropriate ligand, binding domain or binding unit (e.g., a conformer or the natural ligand of the transmembrane protein described herein, e.g., a natural intracellular ligand) to the intracellular binding site of the transmembrane protein (which may be the binding site of the transmembrane protein that is the intracellular binding site when the transmembrane protein is in its natural environment and / or may be the binding site of the transmembrane protein that is the intracellular binding site when the transmembrane protein is present in the cell or cell line used in the present invention, preferably both). In particular, as also described herein, the transmembrane protein may be capable of forming a complex when a first ligand binds to the extracellular binding site and a second ligand binds to the intracellular binding site.More specifically, as described herein, the transmembrane protein is in a functional or active conformation induced by the binding of a first ligand to an extracellular binding site, the active or functional conformation being stabilized by the binding of a second ligand to an intracellular binding site, and the second ligand being capable of stabilizing the functional, active or ligand-binding conformation and / or the complex, and may be capable of forming a complex. In a preferred but non-limiting embodiment, the transmembrane protein is a membrane-spanning protein, particularly a 7TM. Also, the members of the binding pair and any linker used can be the members and linkers further described herein.

[0249] In another aspect, the invention relates to a cell or cell line comprising a fusion protein, the fusion protein comprising a protein capable of binding (either directly or indirectly as described herein) to a transmembrane protein (as described herein), the protein being fused, either directly or via a suitable linker, to a binding domain or binding unit that is a binding member of a binding pair, the binding pair comprising the binding domain or binding unit as at least a first binding member and a second binding member, and the first and second binding members of the binding pair being capable of generating a detectable signal when they come into contact with each other or are in very close proximity to each other. The invention also relates to a cell or cell line that expresses or is capable of expressing such a fusion protein (i.e., under appropriate conditions).

[0250] The protein that is present in the fusion protein and can bind to the transmembrane protein is preferably a protein that can be present in a second fusion protein, as further described herein. Also, the members of the binding pair and any linker used can be further described herein. Also, as described herein, the protein can bind directly (as described herein) or indirectly (as described herein) to the transmembrane protein. Again, in this aspect, the transmembrane protein to which the protein can bind is preferably a transmembrane protein further described herein, particularly a membrane-spanning protein, and more particularly a 7TM.

[0251] As described herein, when the protein present in the fusion protein binds directly to the transmembrane protein, it specifically binds to one or more functional, active and / or drugable conformations of the transmembrane protein, inducing and / or stabilizing the formation of one or more functional, active and / or drugable conformations of the transmembrane protein (and / or shifting the conformational equilibrium of the transmembrane protein with respect to one or more such conformations); and / or inducing and / or stabilizing the formation of a complex of the protein, the transmembrane protein, and a further ligand of the transmembrane protein (as further described herein in all cases). Also, when the protein present in the fusion protein binds directly to the transmembrane protein, the protein can preferably bind to an intracellular binding site of the transmembrane protein. The intracellular binding site of the transmembrane protein can be a binding site of the transmembrane protein that is an intracellular binding site when the transmembrane protein is in its native environment, and / or a binding site of the transmembrane protein that is an intracellular binding site when the transmembrane protein is present in the cells or cell lines used in the present invention (and preferably both).

[0252] In addition, when the protein present in the fusion protein binds directly to the transmembrane protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain, in particular a ConfoBody (described herein).

[0253] Also, as described herein, when the protein present in the fusion protein binds indirectly to the transmembrane protein, it preferably binds to a ligand that can bind to the transmembrane protein. The ligand can be a "second ligand" as described herein when the second ligand does not form part of a second fusion protein. Also, the ligand preferably binds specifically to one or more functional, active and / or druggable conformations of the transmembrane protein, inducing the formation of one or more functional, active and / or druggable conformations of the transmembrane protein (and / or shifting the conformational equilibrium of the transmembrane protein with respect to one or more such conformations); and / or inducing and / or stabilizing the formation of a complex of the ligand, the transmembrane protein, and a further ligand of the transmembrane protein (as further described herein). Also, the ligand is preferably a ligand capable of binding to an intracellular binding site of the transmembrane protein. The intracellular binding site of the transmembrane protein can be the binding site of the transmembrane protein that is the intracellular binding site when the transmembrane protein is in its native environment and / or the binding site of the transmembrane protein that is the intracellular binding site when the transmembrane protein is present in the cells or cell lines used in the present invention (and, preferably, both). Also, as described herein, the ligand is also part of a protein complex capable of binding to the transmembrane protein (i.e., the intracellular binding site of the transmembrane protein), in which case the protein present in the fusion protein can also bind to the protein complex.

[0254] In addition, when the protein present in the fusion protein binds indirectly to the transmembrane protein, it is preferably a binding domain or binding unit derived from a VHH domain or a VHH domain. Also, in a preferred embodiment, when the protein present in the fusion protein binds indirectly to the transmembrane protein and the transmembrane protein is a GPCR, the ligand that binds to the GPCR is a G protein, and the protein present in the fusion protein can specifically bind to the G protein or a G protein complex such as a G protein trimer containing a G-alpha subunit, a G-beta subunit, and a G-gamma subunit. Further, the G protein may be native to the cell or cell line used, a suitable analog or derivative of the native G protein (described herein and recombinantly expressed in the cell or cell line), or a suitable ortholog of the G protein native to the cell or cell line used (and also recombinantly expressed in the cell or cell line used).

[0255] Regardless of whether the protein present in the fusion protein binds directly or indirectly to the transmembrane protein, the cell or cell line preferably expresses or is capable of expressing the fusion protein in the intracellular environment. Another aspect of the present invention relates to such a cell or cell line containing such a fusion protein in its intracellular environment.

[0256] In another aspect, the present invention relates to a cell or cell line containing a first fusion protein and a second fusion protein: · The first fusion protein contains a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein contains a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair can generate a detectable signal when they come into contact with each other or are very close to each other; · The first fusion protein includes a transmembrane protein that is fused to the first binding member of the binding pair either directly (as described herein) or via a suitable linker; · The second fusion protein includes a protein that can bind to the transmembrane protein either directly or indirectly (as described herein), and that protein is fused to the second binding member of the binding pair either directly or via a suitable linker.

[0257] The present invention also relates to cells or cell lines that express or are capable of expressing such first and second fusion proteins (i.e., under appropriate conditions).

[0258] The present invention particularly relates to cells or cell lines comprising a first fusion protein and a second fusion protein: · The first fusion protein includes a binding domain or binding unit that is the first binding member of the binding pair, and the second fusion protein includes a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair can generate a detectable signal when they contact each other or are very close to each other; · The first fusion protein includes a transmembrane protein that is fused to the first binding member of the binding pair either directly (as described herein) or via a suitable linker; · The second fusion protein includes a protein that can bind to the transmembrane protein either directly or indirectly (as described herein), and that protein is fused to the second binding member of the binding pair either directly or via a suitable linker; · When the second fusion protein binds to a transmembrane protein that forms part of the first fusion protein either directly or indirectly (as described herein), the first and second binding members of the binding pair can contact or be very close to each other.

[0259] The present invention also relates to a cell or cell line comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact or are in very close proximity to each other; · The first fusion protein comprises a transmembrane protein that is fused, directly or via a suitable linker as described herein, to the first binding member of the binding pair; · The second fusion protein comprises a protein that can bind, directly or indirectly as described herein, to the transmembrane protein, and the protein is fused, directly or via a suitable linker, to the second binding member of the binding pair; · The first and second binding members of the binding pair are present in the intracellular environment of the cell as described herein.

[0260] The present invention further relates to a cell or cell line comprising a first fusion protein and a second fusion protein, wherein · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first binding member and the second binding member of the binding pair are capable of generating a detectable signal when they contact or are in proximity to each other; · The first fusion protein comprises a transmembrane protein as described herein that is fused, directly or via a suitable linker, to the first binding member of the binding pair; · The second fusion protein comprises a protein that can bind, directly or indirectly as described herein, to the transmembrane protein, and the protein is fused, directly or via a suitable linker, to the second binding member of the binding pair; · When the second fusion protein binds (either directly or indirectly as described herein) to a transmembrane protein that forms part of the first fusion protein, said cell or cell line relates to a cell or cell line that can generate a detectable signal (in particular a detectable signal generated by the first binding member and the second binding member of a binding pair).

[0261] The present invention further relates to a cell or cell line comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair can generate a detectable signal when they contact each other or are very close to each other; · The first fusion protein comprises a transmembrane protein fused (as described herein) directly or via a suitable linker to the first binding member of the binding pair; · The second fusion protein comprises a protein that can bind (either directly or indirectly as described herein) to the transmembrane protein, and the protein is fused directly or via a suitable linker to the second binding member of the binding pair; · When a ligand of the transmembrane protein present in the extracellular environment binds to the transmembrane protein, said cell or cell line produces a detectable signal and / or a change in a detectable signal (in particular a detectable signal generated by the first and second binding members of a binding pair and / or a change in such a signal).

[0262] In certain embodiments, the present invention is a cell or cell line comprising a first fusion protein and a second fusion protein, wherein · The first fusion protein contains a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein contains a binding domain or binding unit that is the second binding member of the binding pair, and the first binding member and the second binding member of the binding pair can generate a detectable signal when they contact or are close to each other; · The first fusion protein contains a transmembrane protein (described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; · The second fusion protein contains a protein that can bind (either directly or indirectly as described herein) to the transmembrane protein, and the protein is fused directly or via a suitable linker to the second binding member of the binding pair; · Relates to a cell or cell line in which, when an agonist of the transmembrane protein present in the extracellular environment binds to the transmembrane protein, the cell or cell line produces a detectable signal and / or a change in a detectable signal (in particular, a detectable signal generated by the first binding member and the second binding member of the binding pair and / or a change in such a signal).

[0263] Also, such cells or cell lines containing or expressing such first and second fusion proteins are further described herein, and preferably they express or can express the first fusion protein in such a manner that the transmembrane protein is incorporated into the cell wall or cell membrane of the cell or cell line, penetrates the cell wall or cell membrane, and more preferably at least a portion of the amino acid sequence of the transmembrane protein protrudes into the extracellular environment (as defined herein) and at least one other portion of the amino acid sequence of the transmembrane protein protrudes into the intracellular environment (as defined herein).

[0264] The cell or cell line preferably expresses or is capable of expressing the first and second fusion proteins, and when such expression occurs, if the second fusion protein binds (either directly or indirectly as described herein) to a transmembrane protein that forms part of the first fusion protein, the first and second binding members of the binding pair can be in contact with or very close to each other. This generally means that such a cell or cell line expresses the first and second fusion proteins in such a manner that, when such expression occurs, the first and second binding members of the binding pair will be in the same environment with respect to the cell wall or membrane (as defined herein). Preferably, the cell or cell line expresses or is capable of expressing the first and second fusion proteins, and when such expression occurs, both the first and second binding members of the binding pair are present in the intracellular environment of the cell (as defined herein). This also generally means that the cell or cell line preferably expresses or is capable of expressing the second fusion protein in its intracellular environment.

[0265] Also, in aspects of the invention relating to cells or cell lines that express or are capable of expressing such first and second fusion proteins, the transmembrane protein, the protein that can bind directly or indirectly to the transmembrane protein, the members of the binding pair, and any linker used can all be further described herein.

[0266] In a further aspect, the invention also relates to methods, in particular assay or screening methods, comprising the use of the cells or cell lines described herein. As further described herein, such assay and screening methods can be used, in particular, to identify compounds and other chemical substances that bind (in particular specifically bind) to a transmembrane protein, can modulate a transmembrane protein, and / or modulate a signal transduction, a signal transduction pathway and / or a biological and / or physiological activity (ies) in which the transmembrane protein, its signal transduction and / or its signal transduction pathway are involved. Thus, the cells and cell lines described herein can be used in methods for identifying compounds or other chemical substances that can act as agonists, antagonists, inverse agonists, inhibitors or modulators (e.g., allosteric) of a transmembrane protein.

[0267] The invention also relates to the use of the cells or cell lines described herein in assays, and in screening methods and techniques in particular. Such methods and uses may also be further described herein with respect to the methods and uses of the arrangements of the invention and will generally also include the step of culturing or maintaining the cells under conditions in which the cells or cell lines appropriately express the desired one or more fusion proteins.

[0268] Also, in all these aspects, such cells, cell lines and their uses are preferably further described herein.

[0269] In another aspect of the invention, the method of the invention is carried out using suitable liposomes or vesicles, and all components of the arrangement of the invention are appropriately present and arranged to provide a workable arrangement of the invention. Such liposomes or vesicles appropriately contain a transmembrane protein (2) in their wall or membrane, i.e., the transmembrane protein (2) is present and penetrates the wall or membrane of the liposome or vesicle, and at least one part of the amino acid sequence of the transmembrane protein protrudes into the environment outside the liposome or vesicle (as defined herein), and at least one other part of the amino acid sequence of the transmembrane protein protrudes into the environment inside the liposome or vesicle (as defined herein). Also, preferably, and as further described herein, in the aspect of the invention carried out with liposomes or vesicles, the environment outside the liposome or vesicle is the "first environment" (i.e., the environment in which the first ligand (3) is present or to which the first ligand (3) is added), and the environment inside the liposome or vesicle is the "second environment" (i.e., the environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0270] Accordingly, in a further aspect, the invention relates to the method or arrangement described herein, wherein the boundary layer (2) is the wall or membrane of a liposome or other (suitable) vesicle.

[0271] Also, as described herein, when the method of the invention is carried out with liposomes or vesicles, the liposomes or vesicles preferably comprise the following components of the arrangement of the invention: · A first fusion protein comprising a transmembrane protein (2) and a first binding member (6); · A second fusion protein comprising a second binding member (7) and a protein capable of binding directly or indirectly (as defined herein) to the transmembrane protein (2); and / or · When the second fusion protein binds indirectly to the transmembrane protein (2), a second ligand (4) and / or a protein constituting the protein complex (12) contain it appropriately (i.e., in a manner that provides a workable arrangement of the present invention).

[0272] Liposomes or vesicles containing said components can generally be provided by forming the liposomes or vesicles in the presence of the relevant components of the arrangement of the present invention such that the components are appropriately incorporated into the liposomes or vesicles. This can generally be carried out, preferably, by methods and techniques known per se for forming liposomes or vesicles in a suitable aqueous buffer or another suitable aqueous medium. Such methods may also include the step of separating liposomes or vesicles containing the components of the desired arrangement of the present invention in an appropriate and workable manner from vesicles or liposomes that do not contain all the necessary components of the arrangement and / or whose components do not form a workable arrangement of the present invention. The components of the arrangement incorporated into the liposomes or vesicles can be provided, for example, by methods known per se by isolating and purifying the resulting expressed components after recombinant expression in a suitable host cell or host organism.

[0273] Generally, in embodiments of the present invention carried out with liposomes or vesicles, a second ligand does not form part of a second fusion protein, a sufficient amount of the second ligand is also provided, and it should be appropriately included in the vesicles or liposomes.

[0274] The liposomes or vesicles can be any liposomes or vesicles suitable for use in the methods and arrangements of the present invention, including but not limited to liposomes based on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The liposomes and vesicles can also be liposomes or vesicles containing and / or based on (e.g., reconstituted from) one or more membrane fractions obtained from cells expressing the desired component(s) of the arrangement of the present invention.

[0275] Thus, in another aspect, the present invention relates to liposomes or vesicles comprising a fusion protein, wherein the fusion protein comprises a transmembrane protein fused directly or via a suitable linker to a binding domain or binding unit that is the first binding member of a binding pair (as described herein), said binding pair comprising at least said binding domain or binding unit as the first binding member and a further binding domain or binding unit as the second binding member, and said first and second binding members of said binding pair being capable of generating a detectable signal when they contact each other or are in very close proximity to each other. The present invention also relates to a method of preparing such liposomes or vesicles, said method comprising at least the step of incorporating such a fusion protein into the liposomes or vesicles and / or the step of forming liposomes or vesicles in the presence of said fusion protein.

[0276] As further described herein, the liposomes or vesicles preferably have the transmembrane protein either immobilized to the wall or membrane of the liposomes or vesicles or otherwise appropriately incorporated and spanning the wall or membrane, more preferably with at least a portion of the amino acid sequence of the transmembrane protein protruding into the environment outside the liposomes or vesicles (as defined herein) and at least one other portion of the amino acid sequence of the transmembrane protein protruding into the environment inside the liposomes or vesicles (as defined herein). More preferably, the first binding member of the binding pair is present in the environment inside the liposomes or vesicles (as defined herein).

[0277] In addition, the transmembrane protein present in the fusion protein is preferably a transmembrane protein further described herein, more preferably having at least two ligand-binding sites, one protruding into the environment outside the liposome or vesicle (as defined herein) and one protruding into the environment inside the liposome or vesicle (as defined herein). Further, as described herein, the transmembrane protein preferably undergoes a conformational change (in particular, a conformational change from an essentially inactive or less active conformation to an active or more active conformation) upon binding of a ligand to the ligand-binding site of the transmembrane protein, particularly upon binding of a ligand present in the environment outside the liposome or vesicle (as defined herein) to the ligand-binding site of the transmembrane protein present in the environment outside the liposome or vesicle. As further described herein, the transmembrane protein preferably further has a functional and / or active (or more active) conformation (in particular, a drugable conformation and / or a ligand-binding conformation, more particularly an agonist-binding conformation) stabilized by the binding of an appropriate ligand, binding domain or binding unit (e.g., a conformatibody or the natural ligand of the transmembrane protein described herein) to the intracellular binding site of the transmembrane protein (which can be the binding site of the transmembrane protein that is the intracellular binding site when the transmembrane protein is in its natural environment and / or the binding site of the transmembrane protein present in the environment inside the liposome or vesicle when the transmembrane protein is present in the liposome or vesicle used in the present invention, preferably both). In particular, as also described herein, the transmembrane protein may be capable of forming a complex when a first ligand binds to a binding site present in the environment outside the liposome or vesicle (as defined herein) and a second ligand binds to a binding site present in the environment inside the liposome or vesicle (as defined herein).More particularly, as described herein, the transmembrane protein is in a functional or active conformation induced by binding of a first ligand to a binding site present in the environment outside the liposome or vesicle (as defined herein), and the active or functional conformation is stabilized by binding of a second ligand to a binding site present in the environment inside the liposome or vesicle (as defined herein), and the second ligand can form a complex that can stabilize the functional, active or ligand-bound conformation and / or the complex. In a preferred but non-limiting embodiment, the transmembrane protein is a membrane-spanning protein, particularly a 7TM. Also, the members of the binding pair and any linker used can be the members and linkers further described herein.

[0278] In another aspect, the invention relates to a liposome or vesicle comprising a fusion protein, the fusion protein comprising a protein that can bind (either directly or indirectly as described herein) to a transmembrane protein (as described herein), the protein being fused directly or via a suitable linker to a binding domain or binding unit that is a binding member of a binding pair, the binding pair comprising at least a first binding member and a second binding member as the binding domain or binding unit, and the first and second binding members of the binding pair being capable of generating a detectable signal when they contact or are in very close proximity to each other. The invention also relates to a method of preparing such liposomes or vesicles, the method comprising at least the step of incorporating such a fusion protein into the liposome or vesicle and / or the step of forming the liposome or vesicle in the presence of the fusion protein.

[0279] The protein that is present in the fusion protein and can bind to the transmembrane protein is preferably a protein that can be present in a second fusion protein, as further described herein. Also, the members of the binding pair and any linker used can be further described herein. Also, as described herein, the protein can bind to the transmembrane protein either directly (as described herein) or indirectly (as described herein). Again, in this aspect, the transmembrane protein to which the protein can bind is preferably a transmembrane protein further described herein, particularly a membrane-spanning protein, and more particularly a 7TM.

[0280] As described herein, when the protein present in the fusion protein binds directly to the transmembrane protein, the protein specifically binds to one or more functional, active, and / or drugable conformations of the transmembrane protein, induces and / or stabilizes the formation of one or more functional, active, and / or drugable conformations of the transmembrane protein (and / or shifts the conformational equilibrium of the transmembrane protein with respect to one or more such conformations); and / or preferably induces and / or stabilizes the formation of a complex of the protein, the transmembrane protein, and a further ligand of the transmembrane protein (as further described herein). Also, when the protein present in the fusion protein binds directly to the transmembrane protein, the protein is preferably a binding site of the transmembrane protein that is an intracellular binding site when the transmembrane protein is in its native environment, and / or a binding site of the transmembrane protein that is present in the inner environment of the liposome or vesicle (as defined herein) when the transmembrane protein is present in the liposome or vesicle when liposomes or vesicles are used in the present invention (and preferably to both).

[0281] Also, when the protein present in the fusion protein binds directly to the transmembrane protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain, in particular a ConfoBody (as described herein).

[0282] Also, as described herein, when the protein present in the fusion protein binds indirectly to the transmembrane protein, it preferably binds to a ligand that can bind to the transmembrane protein. The ligand can be a "second ligand" as described herein when the second ligand does not form part of the second fusion protein. Also, the ligand preferably binds specifically to one or more functional, active and / or druggable conformations of the transmembrane protein, inducing and / or stabilizing the formation of one or more functional, active and / or druggable conformations of the transmembrane protein (and / or shifting the conformational equilibrium of the transmembrane protein with respect to one or more such conformations); and / or inducing and / or stabilizing the formation of a complex of the ligand, the transmembrane protein, and a further ligand of the transmembrane protein (as further described herein). Also, the ligand preferably binds to the binding site of the transmembrane protein that is the intracellular binding site when the transmembrane protein is in its natural environment, and / or to the binding site of the transmembrane protein that is present inside the liposome or vesicle when the liposome or vesicle is used in the present invention and the transmembrane protein is present in the liposome or vesicle (as defined herein) (and preferably to both). Also, as described herein, the ligand is also part of a protein complex that can bind to the transmembrane protein, in which case the protein present in the fusion protein can also bind to the protein complex.

[0283] In addition, when the protein present in the fusion protein binds indirectly to the transmembrane protein, it is preferably a binding domain or binding unit derived from the VHH domain or VHH domain. Also, in a preferred embodiment, when the protein present in the fusion protein binds indirectly to the transmembrane protein, the transmembrane protein is a GPCR, the ligand binding to the GPCR is a G protein, and the protein present in the fusion protein can specifically bind to a G protein complex such as the G protein trimer including the G protein or G alpha subunit, G beta subunit, and G gamma subunit.

[0284] Regardless of whether the protein present in the fusion protein binds directly or indirectly to the transmembrane protein, the fusion protein is preferably present in the environment inside the liposome or vesicle (as defined herein). Also, when the second ligand does not form part of the fusion protein, the environment inside the liposome or vesicle will also contain an appropriate amount of the second ligand.

[0285] In another aspect, the present invention relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair can generate a detectable signal when they contact each other or are in very close proximity to each other; · The first fusion protein comprises a transmembrane protein fused to the first binding member of the binding pair directly or via an appropriate linker (as described herein); · The second fusion protein comprises a protein that can bind to the transmembrane protein (directly or indirectly as described herein), and the protein is fused to the second binding member of the binding pair directly or via an appropriate linker.

[0286] The present invention also relates to a method for preparing such liposomes or vesicles, the method comprising at least the step of incorporating such a fusion protein into the liposomes or vesicles and / or the step of forming liposomes or vesicles in the presence of said fusion protein.

[0287] The present invention particularly relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they come into contact with each other or are very close to each other; · The first fusion protein comprises a transmembrane protein that is fused, either directly (as described herein) or via a suitable linker, to the first binding member of the binding pair; · The second fusion protein comprises a protein that can bind (either directly or indirectly, as described herein) to the transmembrane protein of the present invention, and the protein is fused, either directly or via a suitable linker, to the second binding member of the binding pair; · When the second fusion protein binds (either directly or indirectly, as described herein) to a transmembrane protein that forms part of the first fusion protein, the first and second binding members of the binding pair can come into contact with each other or be very close to each other.

[0288] The present invention also relates to a method for preparing such liposomes or vesicles, the method comprising at least the step of incorporating the fusion protein into the liposomes or vesicles and / or the step of forming liposomes or vesicles in the presence of the fusion protein.

[0289] The present invention also relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or come very close to each other; · The first fusion protein comprises a transmembrane protein that is fused, either directly (as described herein) or via a suitable linker, to the first binding member of the binding pair; · The second fusion protein comprises a protein that can bind to the transmembrane protein (either directly or indirectly as described herein), and that protein is fused, either directly or via a suitable linker, to the second binding member of the binding pair; · The first and second binding members of the binding pair are present in the inner environment of the liposome or vesicle (as defined herein).

[0290] The present invention also relates to a method of preparing such liposomes or vesicles, the method comprising at least the step of incorporating the fusion protein into the liposome or vesicle and / or the step of forming the liposome or vesicle in the presence of the fusion protein.

[0291] The present invention further relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or come very close to each other; · The first fusion protein comprises a transmembrane protein that is fused, directly or via a suitable linker (as described herein), to the first binding member of a binding pair; · The second fusion protein comprises a protein that can bind (directly or indirectly, as described herein) to the transmembrane protein, and the protein is fused, directly or via a suitable linker, to the second binding member of the binding pair; · When the second fusion protein binds (directly or indirectly, as described herein) to a transmembrane protein that forms part of the first fusion protein, the liposome or vesicle can generate a detectable signal (in particular, a detectable signal generated by the first and second binding members of the binding pair).

[0292] The invention also relates to a method of preparing such liposomes or vesicles, the method comprising at least the step of incorporating the fusion protein into the liposomes or vesicles and / or the step of forming liposomes or vesicles in the presence of the fusion protein.

[0293] The invention further relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein: · The first fusion protein comprises a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein comprises a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair can generate a detectable signal when they contact each other or are in very close proximity to each other; · The first fusion protein comprises a transmembrane protein that is fused, directly or via a suitable linker (as described herein), to the first binding member of a binding pair; · The second fusion protein includes a protein that can bind to the transmembrane protein (either directly or indirectly as described herein), and that protein is fused to the second binding member of the binding pair, either directly or via a suitable linker; · When a ligand for a transmembrane protein present in the environment outside the liposome or vesicle binds to the transmembrane protein, the liposome or vesicle produces a detectable signal and / or a change in a detectable signal (in particular, a detectable signal produced by the first and second binding members of the binding pair and / or a change in such a signal).

[0294] The invention also relates to a method of preparing such liposomes or vesicles, the method including at least the step of incorporating the fusion protein into the liposomes or vesicles and / or the step of forming liposomes or vesicles in the presence of the fusion protein.

[0295] In certain embodiments, the invention relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein: · The first fusion protein includes a binding domain or binding unit that is the first binding member of a binding pair, the second fusion protein includes a binding domain or binding unit that is the second binding member of the binding pair, and the first and second binding members of the binding pair can produce a detectable signal when they contact each other or are in very close proximity to each other; · The first fusion protein includes a transmembrane protein that is fused, either directly or via a suitable linker as described herein, to the first binding member of the binding pair; · The second fusion protein includes a protein that can bind to the transmembrane protein (either directly or indirectly as described herein), and that protein is fused to the second binding member of the binding pair, either directly or via a suitable linker; · When an agonist for a transmembrane protein present in the environment outside the liposome or vesicle binds to the transmembrane protein, the liposome or vesicle produces a detectable signal and / or a change in a detectable signal (in particular, a detectable signal generated by the first and second binding members of the binding pair and / or a change in such a signal).

[0296] The present invention also relates to a method of preparing such liposomes or vesicles, the method comprising at least the step of incorporating the fusion protein into the liposome or vesicle and / or the step of forming the liposome or vesicle in the presence of the fusion protein.

[0297] Such liposomes or vesicles containing such first and second fusion proteins may be as further described herein, and preferably they are appropriately immobilized on or otherwise incorporated into the wall or membrane of the liposome or vesicle, have a transmembrane protein that penetrates the wall or membrane, and more preferably at least a part of the amino acid sequence of the transmembrane protein protrudes into the environment outside the liposome or vesicle (as defined herein), and at least one other part of the amino acid sequence of the transmembrane protein protrudes into the environment inside the liposome or vesicle (as defined herein).

[0298] The liposome or vesicle also preferably allows the first and second binding members of the binding pair to come into contact with or very close to each other when the second fusion protein binds to the transmembrane protein forming part of the first fusion protein (either directly or indirectly as described herein). It will be apparent to those skilled in the art that this generally means that the first and second binding members of the binding pair are present in the same environment with respect to the wall or membrane of the liposome or vesicle (as defined herein). Preferably, the liposome or vesicle will be such that the first and second binding members of the binding pair are both present in the environment inside the liposome or vesicle (as defined herein).

[0299] Also, in aspects of the invention relating to liposomes or vesicles containing such first and second fusion proteins, the transmembrane protein, the protein that can bind directly or indirectly to the transmembrane protein, the members of the binding pair, and any linker used can all be further described herein.

[0300] In a further aspect, the invention also relates to methods, particularly assay or screening methods, that involve the use of the liposomes or vesicles described herein....

Claims

1. At least the following components: A boundary layer separating a first environment and a second environment; A transmembrane protein; A first ligand for the transmembrane protein present in the first environment; A second ligand for the transmembrane protein present in the second environment; and A binding pair consisting of at least a first binding member and a second binding member, which can generate a detectable signal comprising wherein the transmembrane protein is a GPCR, the second ligand is an immunoglobulin single variable domain, the first binding member of the binding pair is part of a first fusion protein in which the first binding member is fused or linked to the GPCR directly or via a suitable linker or spacer, an arrangement, wherein the second binding member of the binding pair is part of a second fusion protein in which the second binding member is fused or linked to the immunoglobulin single variable domain directly or via a suitable linker or spacer.

2. The immunoglobulin single variable domain according to claim 1, wherein the second ligand specifically binds to one or more functional, active and / or drug - creatable conformations of the GPCR, or induces the formation of one or more functional, active and / or drug - creatable conformations of the GPCR and / or stabilizes the conformation, and / or induces the formation of a complex of the GPCR, the first ligand and the immunoglobulin single variable domain and / or stabilizes the complex.

3. The arrangement according to claim 1 or 2, wherein the boundary layer is a cell wall or cell membrane of a cell, the first environment is an extracellular environment, and the second environment is an intracellular environment.

4. The arrangement according to claim 1 or 2, wherein the boundary layer is a wall or membrane of a liposome or vesicle, the first environment is an environment outside the liposome or vesicle, and the second environment is an environment inside the liposome or vesicle.

5. At least the following components: A boundary layer separating a first environment and a second environment; A transmembrane protein; A second ligand for the transmembrane protein present in the second environment; and A binding pair consisting of at least a first binding member and a second binding member, which can generate a detectable signal comprising wherein the transmembrane protein is a GPCR wherein the second ligand is an immunoglobulin single variable domain wherein the first binding member of the binding pair is part of a first fusion protein comprising the first binding member fused or linked to the GPCR directly or via a suitable linker or spacer wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member fused or linked to the immunoglobulin single variable domain directly or via a suitable linker or spacer, an arrangement **Claim 6** The arrangement according to claim 5, wherein the boundary layer is a cell wall or cell membrane of a cell, the first environment is an extracellular environment, and the second environment is an intracellular environment **Claim 7** The arrangement according to claim 5, wherein the boundary layer is a wall or membrane of a liposome or vesicle, the first environment is an environment outside the liposome or vesicle, and the second environment is an environment inside the liposome or vesicle **Claim 8** a) preparing the arrangement according to any one of claims 5 to 7; and b) adding a first ligand to the first environment of the arrangement A method comprising **Claim 9** The method according to claim 8, wherein the second ligand specifically binds to one or more functional, active and / or drug - creatable conformations of the GPCR, or induces the formation of one or more functional, active and / or drug - creatable conformations of the GPCR and / or stabilizes the conformation, and / or induces the formation of a complex of the GPCR, the first ligand and the immunoglobulin single variable domain and / or stabilizes the complex, and is an immunoglobulin single variable domain **Claim 10** c) measuring the signal generated by the binding pair and / or measuring the change in the signal generated by the binding pair The method according to claim 8 or 9, further comprising

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