Screening methods and assays for transmembrane proteins, in particular gpcr

Abstract

The invention provides a method and a device for screening membrane proteins. The device comprises a first fusion protein comprising a membrane protein present in a boundary layer (e.g., the wall of a cell, liposome, or vesicle) and fused to one member of a binding pair, and a second fusion protein comprising an intracellular ligand fused to the membrane protein of the other member of the binding pair, wherein the combination pair is capable of generating a detectable signal.

Description

[0001] This application is a divisional application of the application with an international application date of April 28, 2020, international application number PCT / EP2020 / 061803, application number 202080046276.7 entering the Chinese national phase, and invention name “Screening methods and assays for transmembrane proteins, especially GPCRs”.

[0002] The present invention relates to methods and tools that can be used in assays, screening, and drug discovery and development efforts.

[0003] In particular, the present invention relates to methods and tools that can be used for screening and assay techniques involving the use of membrane proteins (i.e. as targets to be screened, e.g., finding candidate compounds for said targets), and for the discovery, production, optimization and / or development of therapeutic, prophylactic and diagnostic agents for membrane proteins (i.e., specific for membrane proteins). The present invention further relates to methods for preparing tools that can be used for screening and assay techniques.

[0004] Advantageously, the methods and tools of the present invention can be used for screening and assay techniques involving the use of membrane proteins that can present / exist in a variety of conformations (such as, but not limited to, active and inactive conformations), as well as for the discovery, generation, optimization and / or development of therapeutic, preventive and diagnostic agents for such membrane proteins. Such membrane proteins include, but are not limited to, transmembrane proteins, such as GPCRs and other cell surface receptors.

[0005] In a particularly preferred but non-limiting aspect, the methods and tools of the invention can be used for screening and assay techniques and for discovering, producing, optimizing and / or developing therapeutic, preventive and diagnostic agents for such membrane proteins, the screening and assay techniques involving the use of membrane proteins that can undergo conformational changes (again, for example but not limited to, from an inactive conformation to an active conformation) in response to a ligand that binds to the protein. Likewise, such membrane proteins can be cell surface receptors, such as GPCRs.

[0006] The present invention generally provides methods that can be used to perform 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 "hits" against a target). The present invention also provides devices that can be used in the methods, i.e., as a system or device (set-up) for performing the assay or screening. The device includes the elements described herein. The elements can also be provided or established as a kit of parts, and such a kit of parts forms another aspect of the present invention. The present invention also provides components for identifying and creating such a device and methods for assembling such a device.

[0007] The methods and devices described herein can generally be used to test one or more properties of (known) compounds or ligands (i.e., those properties that can be determined using the methods described herein) and / or for identifying compounds or ligands having such one or more 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, including but not limited to small molecules, small peptides, biomolecules, or other chemical entities, and examples of such compounds will be clear to those skilled in the art based on the further disclosure herein. In addition, 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 development work (e.g., using the well-known techniques of so-called "hits-to-leads" chemistry), and such further work may also involve using the methods of the present invention (e.g., as a functional assay or an assay for quality control purposes).

[0008] Compounds identified using the methods and techniques of the invention (i.e., "hits"), as well as any compounds generated or developed using such hits as a starting point, are also collectively referred to herein as "compounds of the invention" and form further aspects of the invention. The skilled person will appreciate that such compounds may be, for example, so-called "hits," "leads," "development candidates," "preclinical compounds," "clinical candidates," or commercial compounds or products, depending on their stage of development and the specific terminology used by the company or entity developing and / or commercializing them.

[0009] 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 labeled or radioactively labeled) and can therefore also be applied to membrane proteins for which no antagonists are available or known. In addition, as further described herein, the methods and assays of the present invention can allow the identification and / or characterization of allosteric agonists (positive and negative), antagonists and / or inverse agonists (depending on the specific target and assay used).

[0010] Other features, aspects, embodiments, utilities and advantages of the invention will become apparent from the further description herein.

[0011] Membrane proteins (e.g., cell surface receptors, including GPCRs) and assays and screening techniques for membrane proteins are well known in the art. It is estimated that more than half of modern drugs target membrane proteins and about one-third of modern drugs target GPCRs. Reference is made to standard manuals and other prior art cited herein.

[0012] As is well known in the field of protein dynamics, most proteins are not static objects whose function is determined solely by their primary, secondary, tertiary and (when the protein comprises two or more polypeptide chains) quaternary structures, but are typically flexible structures that can undergo transitions (also called "conformational changes") between different conformational states, such that the protein can exist in equilibrium between these different states. Some of these states may be functional and / or active, while others may be basal states (which may or may not exhibit some level of constitutive activity), substantially inactive states, and / or states that are less active than more functional or active states. In addition, the geometries of different epitopes, binding sites (including ligand binding sites), and / or catalytic sites that may be present in or on the protein may differ between these different conformations, such that, for example, in some conformational states, the binding site may be unavailable / inaccessible for ligand binding, and / or such that the affinity of the interaction between the binding site and the associated ligand is reduced compared to the more active conformational states.

[0013] It is also known that for some protein / ligand combinations, binding of a ligand to a protein may change conformation (e.g., from an inactive / less active conformation to an active / more active conformation) and / or shift the equilibrium from an inactive / less active conformation to an active / more active conformation. Binding of a ligand to one binding site of a protein may also make another binding site on the protein more accessible to its associated ligand, and / or may result in an increase in the affinity of the other binding site for the ligand, and / or shift the equilibrium from a conformation in which the other binding site has a lower affinity for the ligand to a conformation in which the other binding site has a better affinity for the ligand. For example, for some transmembrane proteins such as GPCRs, binding of an extracellular ligand to an extracellular binding site on a protein may increase the affinity of an intracellular binding site for the intracellular ligand (e.g., increase the affinity of the interaction between a G-protein and a G protein binding site on a GPCR), and vice versa. This change in binding affinity for the intercellular ligand following binding of the extracellular ligand, and subsequent binding of the intracellular ligand to the intracellular binding site, may be part of the way in which the protein transduces the extracellular signal.

[0014] Generally, as further described herein, it can be said that for a receptor protein that can undergo conformational changes, an "agonist" of the receptor shifts the conformational equilibrium from an inactive state (or one or more less active states) to an active state (or one or more more active states), while an "inverse agonist" of the receptor will do the opposite.

[0015] It is also possible that a protein forms a complex with two ligands, these ligands bind to two different binding sites on the protein, and the interaction between the protein and each ligand is stabilized by the binding of the other ligand (in other words, the complex is stabilized by the binding of both ligands). Again, in this case, the binding of one or both ligands may also shift the conformational equilibrium of the protein to (the formation and / or stabilization of) the complex. See, for example, WO 2012 / 007593 cited below.

[0016] Given that the perceived "overall" state of such a protein is largely governed by the (statistical) distribution of the protein across its various possible conformations, and therefore by the equilibrium that exists between these conformational states, it should be understood that in this specification or claims, when a protein is referred to as undergoing a conformational change to a certain conformation (i.e. from one or more other conformations), this will include mechanisms or circumstances where the conformational equilibrium of the protein shifts to said conformation (i.e. under the specific conditions used, e.g. conditions used for a screening or related assay). Similarly, when a ligand is referred to as inducing a conformational change of a protein to a certain conformation (i.e. from one or more other conformations), this includes a mechanism or circumstances where the binding of the ligand shifts the conformational equilibrium of the protein to said conformation (i.e. under the specific conditions used, e.g. conditions used for a screening or related assay).

[0017] However, it should also be noted that while any one of the mechanisms described herein (or any combination thereof) may be involved in the practice of the present invention at any given time (depending also, for example, on the particular protein and / or ligand to which the present invention is applied), the present invention is in its broadest sense and is not limited to any particular mechanism, explanation or hypothesis, so long as the application of the present invention to a particular target or protein results in the technical effects outlined herein.

[0018] One of the challenges of screening for compounds against membrane proteins that exist in multiple conformations is that if the protein is expressed or used in isolation from its native environment, the correct conformation of the protein may be lost (if it is feasible or possible to express the protein and ensure that it is correctly folded outside the cellular environment). In addition, it may be challenging to ensure that the protein is in its desired conformation (usually a functional conformation, such as its active conformation) under the conditions used for screening. It may also be necessary or beneficial to achieve a shift in the conformational equilibrium of the protein to a conformational state that is more suitable for screening or assay purposes (e.g., an active state or a state in which the relevant binding site is more accessible and / or has a more optimal geometry for assay or screening purposes). As further described herein, this conformation is also referred to as a "drugable" conformation, and according to a preferred aspect of the present invention, means (as further described herein) are applied to ensure that the protein is in this druggable conformation and / or to ensure that the conformational equilibrium of the protein is shifted to a more druggable conformation when performing the method of the present invention.

[0019] For example, WO2012 / 007593, WO2012 / 007594, WO2012 / 175643, WO2014 / 118297, WO2014 / 122183 and WO2014 / 118297 relate to protein binding domains that can be used to stabilize a specific conformational state of a GPCR for determining its structure and for drug screening and discovery purposes. In these references, the use of VHH domains can stabilize a GPCR in a desired conformation, in particular a (more) druggable conformation, such as a functional state and / or an active state, such as a conformation that occurs when an activating ligand (agonist) binds to the extracellular side of the GPCR, thereby activating the GPCR to activate the heterotrimeric G protein. For example, reference is also made to 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 and other references cited therein. VHH domains that can be used to stabilize a desired conformation of a membrane protein such as a GPCR are also referred to herein as ConfoBody. TM is a registered trademark of Confo Therapeutics, Ghent, Belgium].

[0020] Some specific but non-limiting examples of ConfoBodys that are capable of binding to an intracellular epitope of a GPCR and that can be used to stabilize a GPCR in a desired conformation (and that can also be used in the present invention) are the VHHs designated 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 (see, e.g., WO2012 / 007593, Tables 1 and 2 and SEQ ID NO:1 to 29); VHHs designated CA5669, Nb9-1, Nb9-8, XA8633 and CA4910 (see, e.g., WO2014 / 118297, Tables 1 and 2 and SEQ ID NO:15, 16, 17, 19 and 20); VHHs designated 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 (see, e.g., WO2014 / 122183, Tables 1 and 2 and SEQ ID NO:1-19); and VHHs designated XA8639, VHHs of XA8635, XA8727 and XA9644 (see, e.g., WO2015 / 121092, Tables 2 and 3 and SEQ ID NOs: 2 to 6 and 74).

[0021] Some specific but non-limiting examples of VHHs capable of binding to G protein are CA4435, CA4433, CA4436, CA4437, CA4440 and CA4441 (see, e.g., WO2012 / 175643007593, Tables 2 and 3 and SEQ ID NOs: 1 to 6).

[0022] As further described herein, the present invention generally provides improved screening methods and assay technologies that can be used to discover and develop (e.g., identify, generate, test and optimize) compounds that are directed against membrane proteins (i.e., are specific for and / or intended to target one or more membrane proteins, e.g., for therapeutic, prophylactic and / or diagnostic purposes). Preferably, such compounds are specific for (i.e., are selective for) a particular membrane protein as compared to other (closely related) membrane proteins.

[0023] Compounds identified and / or developed using the methods of the invention can be used to modulate membrane proteins (as defined herein), their signal transduction and / or biological functions, pathways and / or mechanisms in which the membrane proteins or their signal transduction are involved. For example, the invention can be used to discover and develop compounds that are agonists, antagonists, inverse agonists, inhibitors or modulators (e.g., positive and negative allosteric modulators) of the membrane proteins and / or signal transduction, pathways and / or physiological and / or biological mechanisms involved in the membrane proteins.

[0024] The present invention can be used to discover and develop compounds against membrane proteins that are integral membrane proteins or peripheral membrane proteins in their natural environment. The present invention can be particularly useful for discovering and developing compounds against transmembrane proteins, as further described herein. In a specific but non-limiting aspect, compounds discovered and / or developed using the present invention will be directed against receptors, particularly cell surface receptors.

[0025] As further described herein, a transmembrane protein may in particular be a membrane protein that passes through the membrane multiple times, such as a 7TM or a GPCR. [In this regard, it should be noted that, generally within the field, the terms "7TM receptor" and "7TM" are often used interchangeably with "GPCR", although according to the IUPHAR database, there are some 7TM receptors that do not signal through G proteins. For the purposes of this specification and claims, the terms "GPCR" and "7TM" are used interchangeably herein to include all transmembrane proteins (especially transmembrane receptors) having 7 transmembrane domains, regardless of their intracellular signaling cascades or signal transduction mechanisms, although it should be understood throughout the specification and claims that 7TMs that signal through G proteins are a preferred aspect of the present invention].

[0026] Typically, compounds discovered and / or developed using the present invention will be directed against membrane proteins that are expressed and / or exposed on the surface of a cell when in its natural environment, and in particular will be directed against such membrane proteins that are expressed by or on cells present in a subject to be treated with a compound that has been discovered or developed using the methods and techniques of the present invention.

[0027] The present invention can be used to find and / or develop any kind of compound suitable for its intended use, which is generally used as a therapeutic agent, diagnostic agent or preventive agent. Therefore, these compounds can be small molecules, peptides, biomolecules or other chemical entities. Examples of suitable biomolecules can include, for example, antibodies and antibody fragments (e.g., Fab, VH, VL and VHH domains) and compounds based on antibody fragments (e.g., ScFv and diabodies and other compounds or constructs comprising one or more VH, VL and / or VHH domains), compounds based on other protein scaffolds, such as Alphabodies and avimer-based scaffolds, PDZ domains, protein A domains (e.g., Affibodies), ankyrin repeats (e.g., DARPins), fibronectins (e.g., Adnectins) and lipocalins (e.g., Anticalins) and binding moieties based on DNA or RNA, including but not limited to DNA or RNA aptamers. Reference is made to further description herein, and to, 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.

[0028] The methods and techniques of the invention can, for example, be used to screen libraries of such compounds to identify one or more "hits" that are specific for a membrane protein (particularly a desired conformation of a membrane protein, and / or a conformation capable of inducing a desired conformation of a membrane protein, such as ligand binding, particularly agonist binding), and / or used as an assay as part of a strategy to improve the affinity and / or potency of compounds directed to membrane proteins and / or otherwise improve the (pharmacological and / or other properties) of such compounds (e.g., in the case of small molecules, as part of a "hit to lead" campaign).

[0029] The methods and techniques of the present invention can also be used for the purpose of so-called "fragment-based drug discovery" or "FBDD" (also called "fragment-based lead discovery" or "FBLD"). For example, see Lamoree and Hubbard, Essays in Biochemistry (2017) 61, 453–464, and standard manuals such as 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.

[0030] The present invention will be described herein with respect to specific embodiments and with reference to certain non-limiting examples and drawings. Any reference numerals in the claims should not be interpreted as limiting the scope. The drawings described are only schematic and non-restrictive. In the drawings, the sizes of some elements may be enlarged and not drawn to scale for the purpose of illustration. When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. If an indefinite article or definite article is used when referring to a singular noun, such as "a" or "an", "the", the plural form of the noun is included unless otherwise expressly stated. In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe order or time sequence. It should be understood that the terms used in this way are interchangeable where appropriate, and the embodiments of the present invention described herein can be operated in other orders different from those described or illustrated herein.

[0031] Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and techniques associated with molecular and cell biology, structural biology, biophysics, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2DED., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide a general dictionary of many terms used in the present disclosure to the skilled person. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); up, Biomolecular crystallography: principles, Practice and Applications to Structural Biology, 1st ed., GarlandScience, Taylor & Francis Group, LLC, an informa Business, NY (2009); Limbird, Cell Surface Receptors, 3rd ed., Springer (2004).

[0032] As used herein, the terms "polypeptide", "protein", "peptide" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids (chemically or biochemically modified or derivatized amino acids), as well as polypeptides with modified peptide backbones. Throughout the application, the standard single-letter symbols for amino acids will be used. Generally, the term "amino acid" will refer to "proteinogenic amino acids", i.e., those amino acids naturally occurring in proteins. Most particularly, the amino acids are in the L isomeric form, but D amino acids are also contemplated.

[0033] As used herein, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. 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 may be linear or circular.

[0034] Any peptide, polypeptide, nucleic acid, compound, etc. disclosed herein may be "isolated" or "purified". "Isolated" is used herein to indicate that the material referred to is (i) separated from one or more substances with which it exists in nature (e.g., separated from at least some cellular material, separated from other polypeptides, separated from its native sequence context), and / or (ii) produced by a process involving artificiality, such as recombinant DNA technology, protein engineering, chemical synthesis, etc.; and / or (iii) has a sequence, structure or chemical composition not found in nature. "Isolated" is intended to include compounds within a sample that is substantially enriched in the compound of interest and / or in which the compound of interest is partially or substantially purified. As used herein, "purified" means that the material referred to is removed from its natural environment and is at least 60%, at least 75%, or at least 90% free of other components with which it is naturally associated, also referred to as "substantially pure".

[0035] As used herein, the term "sequence identity" refers to the extent to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over the comparison window.

[0036] Therefore, "sequence identity percentage" is calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) appears in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity. Determining the sequence identity percentage can be done manually or by using a computer program available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0037] "Similarity" refers to the percentage of amino acids that are identical or constitute conservative substitutions. Sequence comparison programs such as GAP (Deveraux et al. 1984) can be used to determine similarity. In this way, sequences with similar or substantially different lengths than those cited herein can be compared by inserting gaps into the alignment, such as by determining such gaps through the comparison algorithm used by GAP. As used herein, "conservative substitutions" are amino acid substitutions with other amino acids having similar biochemical properties (e.g., aliphatic, aromatic, positively charged, etc.) in the side chain, and are well known to technicians. Non-conservative substitutions are amino acid substitutions with other amino acids whose side chains do not have similar biochemical properties (e.g., replacing hydrophobic residues with polar residues). Conservative substitutions typically produce sequences that are no longer identical but still highly similar. Conservative substitutions mean combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

[0038] A "deletion" is defined herein as a change in an amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are deleted compared to the amino acid sequence or nucleotide sequence of a parent polypeptide or nucleic acid. In the context of a protein, a deletion may involve a 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 more than one deletion. In the context of a GPCR, a deletion may also be a loop deletion, or an N- and / or C-terminal deletion. As is clear to the skilled person, an N- and / or C-terminal deletion of a GPCR is also referred to as a truncation of the GPCR amino acid sequence or a truncated GPCR.

[0039] An "insertion" or "addition" is a change in an amino acid or nucleotide sequence that results in the addition of one or more amino acid or nucleotide residues, respectively, compared to the amino acid sequence or nucleotide sequence of the parent protein. An "insertion" generally refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, while an "addition" may be an insertion or refers to an amino acid residue added at the N- or C-terminus or both termini. In the context of a protein or fragment thereof, an insertion or addition is generally about 1, about 3, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

[0040] As used herein, "substitution" is produced by replacing one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid sequence or nucleotide sequence of a parent protein or fragment thereof. It should be understood that a protein or fragment thereof may have conservative amino acid substitutions that have substantially no effect on protein activity. Conservative substitutions mean combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

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

[0042] The term "orthologs" when used to refer 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. It should be understood that two sequences are orthologs to each other when they are derived from a common ancestral sequence by linear descent and / or are closely related in terms of their sequence and their biological function. Orthologs typically have a high degree of sequence identity, but may not (and usually do not) share 100% sequence identity.

[0043] The term "recombinant" when used in reference to a cell, nucleic acid, protein or vector, means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses nucleic acids or polypeptides not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, over-expressed or not expressed at all.

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

[0045] As used herein, the term "operably linked" refers to a regulatory sequence that is adjacent to a target gene to control the connection of the target gene, as well as a regulatory sequence that acts in trans or remotely to control the target gene. For example, when a DNA sequence is connected to a promoter downstream of the transcription start site of the promoter, the DNA sequence is operably linked to the promoter and allows transcription extension to proceed through the DNA sequence. If the DNA of the signal sequence is expressed as a preprotein that participates in polypeptide transport, the DNA of the signal sequence is operably linked to the DNA encoding the polypeptide. The connection of the DNA sequence to the regulatory sequence is usually achieved by connecting at a suitable restriction site or adapter or a joint inserted in its place using restriction endonucleases known to those skilled in the art.

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

[0047] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule connected thereto. The vector may be of any suitable type, including but not limited to bacteriophage, virus, plasmid, phagemid, cosmid, bacmid or even artificial chromosome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors having a replication origin that works in the host cell). Other vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some preferred vectors are capable of directing the expression of certain target genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, etc., as required and according to specific host organisms (e.g., bacterial cells, yeast cells). Typically, the recombinant vector according to the present invention comprises at least one "chimeric gene" or "expression cassette." The expression cassette is usually a DNA construct, preferably including (5' to 3' in the transcription direction): a promoter region, a polynucleotide sequence of the present invention operably linked to a transcription initiation region, a homologue, a variant or a fragment thereof, and a termination sequence including a termination signal of an RNA polymerase and a polyadenylation signal. It should be understood that all these regions should be able to operate in biological cells to be transformed, such as prokaryotes or eukaryotic cells. The promoter region (which preferably includes an RNA polymerase binding site) and a polyadenylation signal comprising a transcription initiation region may be natural to the biological cells to be transformed, or may be derived from an alternative source, wherein the region is functional in the biological cell.

[0048] As used herein, the term "host cell" is intended to refer to a cell that has been introduced into a recombinant vector. It should be understood that these terms refer not only to a specific test cell, but also to the offspring of this cell. Because some modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from the parental cell, but are still included in the scope of the term "host cell" used herein. The host cell can be an isolated cell or cell line grown in culture, or can be a cell present in a living tissue or an organism. In particular, the host cell is derived from bacteria or fungi, but can also be derived from plants or mammals. The wording "host cell", "recombinant host cell", "expression host cell", "expression host system", "expression system" is intended to have the same meaning and is used interchangeably in this article.

[0049] "G-protein coupled receptors" or "GPCRs" are polypeptides that share a common structural motif, having an extracellular amino terminus (N-terminus), an intracellular carboxyl terminus (C-terminus), and seven hydrophobic transmembrane spans (seven regions of 22 to 24 hydrophobic amino acids forming seven alpha helices), each spanning the membrane. Each span is identified by a number, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are connected by amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the outside or "extracellular" side of the cell membrane, referred to as "extracellular" regions 1, 2, and 3 (EC1, EC2, and EC3), respectively. The transmembrane helices are also connected by amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the inside or "intracellular" side of the cell membrane, referred to as "intracellular" regions 1, 2, and 3 (IC1, IC2, and IC3), respectively. The "carboxyl" ("C") terminus of the receptor is located in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor is located in the extracellular space outside the cell.GPCR structure and classification are generally well known in the art, and further discussion of GPCRs can be found in: Cvicek et al., PLoS Comput Biol. 2016 Mar 30;12(3):e1004805. doi:10.1371 / journal.pcbi.1004805; Ventakakrishnan, Current Opinion in Structural Biology, 2014, 27:129-137; Isberg, Trends Pharmacol. Sci., 2015 Jan, 22-13, Probst, DNA Cell Biol. 1992 11:1-20; Marchese et al. Genomics 23: 609-618, 1994; and the following books: Jurgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled Receptors, 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).

[0050] The International Union of Basic and Clinical Pharmacology (IUPHAR) maintains a database of receptors (including GPCRs) and their known endogenous ligands and signaling mechanisms (http: / / www.guidetopharmacology.org / targets.jsp). According to the database, as of January 2019, approximately 800 GPCRs have been identified in humans, of which approximately half have sensory functions (e.g., smell, taste, light perception, and pheromone signaling), and approximately half mediate signaling associated with ligands that range in size from small molecules to peptides to 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 (rhodopsins), Class B (secretin receptor family), Class C (metabotropic glutamate), Class D (fungal mating pheromone receptors, not found in vertebrates), Class E (cyclic AMP receptors, also not found in vertebrates), and Class F (frizzled / smooth). The IUPHAR database also mentions an alternative classification scheme called "GRAFS", which divides vertebrate GPCRs into five classes (overlapping with the AF nomenclature), as follows: the glutamate family (overlapping with the above-mentioned "class C"), which includes, among others, metabotropic glutamate receptors, calcium-sensing receptors, and GABAB receptors; the rhodopsin family (overlapping with the above-mentioned "class A"), which includes receptors for various small molecules, neurotransmitters, peptides, and hormones, as well as olfactory receptors, visual pigments, type 2 taste receptors, and five pheromone receptors (V1 receptors); the adhesion family G PCR (phylogenetically related to class B receptors); the frizzled family, which consists of 10 frizzled proteins (FZD (1-10)) and smoothened (SMO); and the secretin family, which are receptors for peptide ligands / hormones with 27-141 amino acid residues, including glucagon, glucagon-like peptides (GLP-1, GLP-2), glucose-dependent insulinotropic polypeptide (GIP), secretin, vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone releasing hormone (GHRH). In this specification and the appended claims, unless otherwise expressly stated, the A to F type classification will be used. Further reference is made to Cvicek et al. cited herein.

[0051] With respect to a GPCR, the term "biologically active" refers to a GPCR that has the biochemical function of a naturally occurring GPCR (eg, binding function, signal transduction function, or the ability to change conformation due to ligand binding).

[0052] In general, the term "naturally occurring" with respect to a GPCR refers to a GPCR that is naturally produced (e.g., by a wild-type mammal, such as a human). Such a GPCR is found in nature. The term "non-naturally occurring" with respect to a GPCR refers to a non-naturally occurring GPCR. Variants of naturally occurring GPCRs and naturally occurring transmembrane receptors that are constitutively active by mutation (e.g., epitope-tagged GPCRs and GPCRs lacking their native N-termini) are examples of non-naturally occurring GPCRs. Non-naturally occurring versions of naturally occurring GPCRs are typically activated by the same ligands as naturally occurring GPCRs. Non-limiting examples of naturally occurring or non-naturally occurring GPCRs in the context of the present invention are further provided herein.

[0053] As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope may contain 3 amino acids in a spatial conformation that is unique to the epitope. Typically, an epitope is composed of at least 4, 5, 6, or 7 such amino acids, and more typically, at least 8, 9, or 10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art, including, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a "conformational epitope" refers to an epitope comprising amino acids in a spatial conformation that is unique to the folded 3-dimensional conformation of a polypeptide. Typically, a conformational epitope is composed of discontinuous amino acids in a linear sequence that are aggregated together in the folded structure of a protein. However, a conformational epitope may also be composed of a linear amino acid sequence that adopts a conformation that is unique to the folded 3-dimensional conformation of a polypeptide (and does not exist in a denatured state).

[0054] The term "conformation" or "conformational state" of a protein generally refers to the spatial arrangement, structure or range of structures that a protein may adopt at any time. Those skilled in the art will recognize that the determinants of conformation or conformational state include the primary structure of the protein as reflected in the amino acid sequence of the protein (including modified amino acids) and the surrounding environment of the protein. The conformation or conformational state of a protein also involves structural features such as protein secondary structure (e.g., α-helix, β-sheet, etc.), tertiary structure (e.g., three-dimensional folding of a polypeptide chain), and quaternary structure (e.g., interaction of a polypeptide chain with other protein subunits). Post-translational and other modifications of polypeptide chains, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, can affect the conformation of a protein. In addition, environmental factors, such as pH, salt concentration, ionic strength, and osmotic pressure of the surrounding solution, as well as interactions with other proteins and cofactors, can affect protein conformation. The conformational state of a protein can be determined by functional assays of activity or binding to another molecule or by physical methods such as X-ray crystallography, NMR, or spin labeling. For a general discussion of protein conformation and conformational states, reference may be made to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, WH Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, WH Freeman and Company, 1993.

[0055] As used herein, "functional conformation" or "functional conformational state" refers to the fact that a protein possesses different conformational states with a dynamic range of activity, particularly from inactive to maximal activity. It should be understood that a "functional conformational state" refers to any conformational state of a protein with any activity (including inactivity), and does not include a denatured state of a protein. Non-limiting examples of functional conformations include an active conformation, an inactive conformation, or a basal conformation (as further defined herein). As described above, a specific class of functional conformations is defined as a "druggable conformation" and generally refers to a therapeutically relevant conformational state of a protein. For example, reference is made to Johnson and Karanicolas, PLoS Comput Biol 9(3): e1002951.doi:10.1371 / journal.pcbi.1002951 and, for example, WO2014 / 122183, which describes that the agonist binding active conformation of the muscarinic acetylcholine receptor M2 corresponds to a druggable conformation of the receptor associated with pain and gliobastoma, and describes VHHs capable of stabilizing the druggable conformation for assay and screening purposes. It will therefore be understood that druggability is limited to a specific conformation depending on the therapeutic indication. Further details are provided herein.

[0056] For proteins that act as receptors, as used herein, the term "active conformation" more specifically refers to a conformation or a spectrum of receptor conformations that allow signal transduction to an intracellular effector system (e.g., G protein-dependent signaling and / or G protein-independent signaling (e.g., β-arrestin signaling). Thus, the "active conformation" encompasses a range of ligand-specific conformations, including agonist-specific active state conformations, partial agonist-specific active state conformations, or biased agonist-specific active state conformations, such that they induce cooperative binding of intracellular effector proteins.

[0057] In addition to the above, for GPCRs, the terms "active conformation" and "active form" used herein refer to GPCRs that are folded in a certain way to have (functional) activity. The GPCR can be placed in an active conformation using an activating ligand (agonist) of the receptor, and this conformational change generally enables the receptor to activate a heterotrimeric G protein. For example, a GPCR in an active conformation binds to a heterotrimeric G protein and catalyzes nucleotide exchange of the G protein to activate a downstream signal transduction pathway. The activated GPCR binds to an inactive GDP-bound heterotrimeric G protein and causes the G protein to release its GDP, so that GTP can bind. This process produces a transient "nucleotide-free" state that enables GTP to bind. Once GTP binds, the receptor and G protein dissociate, allowing the GTP-bound G protein to activate downstream signal transduction pathways, such as adenylate cyclase, ion channels, RAS / MAPK, etc. The terms "inactive conformation" and "inactive form" refer to GPCRs that are folded in a certain way so as to be inactive. The GPCR can be placed in an inactive conformation using an inverse agonist of the receptor. For example, a GPCR in an inactive conformation does not bind to a heterotrimeric G protein with high affinity. The terms "active conformation" and "inactive conformation" will be further described herein. As used herein, the term "basal conformation" refers to a GPCR folded in such a way that it exhibits activity (also referred to as basal activity or constitutive activity) for a specific signal transduction pathway even in the absence of an agonist. Inverse agonists can inhibit this basal activity. Therefore, in the absence of a ligand or accessory protein, the basal conformation of a GPCR corresponds to a stable conformation or a prominent structural species.

[0058] Similarly, for proteins that act as receptors, the term "inactive conformation" as used herein refers to a spectrum of receptor conformations that do not allow or block signal transduction to intracellular effector systems. Thus, "inactive conformations" encompass a range of ligand-specific conformations, including inverse agonist-specific inactive state conformations, thereby preventing cooperative binding of intracellular effector proteins. It should be understood that the binding site of the ligand is not critical for obtaining an active or inactive conformation. Thus, orthosteric ligands as well as allosteric modulators are equally capable of stabilizing receptors in active or inactive conformations.

[0059] As used herein, the term "binding agent" refers to all or part of a proteinaceous (proteinaceous, protein-like or protein-containing) molecule that can bind to a membrane protein using specific intermolecular interactions. In a specific embodiment, the term "binding agent" is not intended to include naturally occurring binding partners of the relevant membrane protein, such as G proteins, inhibitory proteins, endogenous ligands; or variants or derivatives (including fragments) thereof. More specifically, the term "binding agent" refers to a polypeptide, more specifically a protein domain. A suitable protein domain is an element of the overall protein structure that is self-stable and folds independently of the rest of the protein chain and is generally referred to as a "binding domain". The length of such a binding domain varies from about 25 amino acids to 500 amino acids and more. Many binding domains can be classified as folded and are recognizable, identifiable, 3-D structures. Some folds are so common in many different proteins that they are given specific names. Non-limiting examples are binding domains selected from the group consisting of 3 or 4 helical bundles, armadillo repeat domains, leucine-rich repeat domains, PDZ domains, SUMO or SUMO-like domains, cadherin domains, immunoglobulin-like domains, phosphotyrosine binding domains, pleckstrin homology domains, src homology 2 domains, etc. Thus, the binding domain may be derived from a naturally occurring molecule, e.g., from a component of the innate or adaptive immune system, or it may be completely artificially designed.

[0060] Typically, the binding domain may be immunoglobulin based or it may be based on a domain present in a protein including, but not limited to, a microprotein, a protease inhibitor, a toxin, a fibronectin, a lipocalin, a single-chain antiparallel coiled-coil protein, or a repeat motif protein. Specific examples of binding domains known in the art include, but are not limited to, antibodies, heavy chain antibodies (hcAbs), single domain antibodies (sdAbs), minibodies, variable domains derived from camelid heavy chain antibodies (VHHs or nanobodies), variable domains of new antigen receptors derived from shark antibodies (VNAs), alphabodies, protein A, protein G, designed ankyrin repeat domains (DARPins), fibronectin type III repeats, anticalins, knottins, engineered CH2 domains (nanobodies), engineered SH3 domains, affibodies, peptides and proteins, 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; WO2010066740). Typically, when selection methods are used to generate a particular type of binding domain, a combinatorial library containing a consensus or framework sequence containing randomized potential interacting residues is screened for binding to a molecule of interest (e.g., a protein).

[0061] According to a preferred embodiment, it is particularly envisioned that the binding agent of the present invention is derived from the innate or adaptive immune system. Preferably, the binding agent is derived from an immunoglobulin. Preferably, the binding agent according to the present invention is derived from an antibody or an antibody fragment. The term "antibody" (Ab) generally refers to a polypeptide encoded by an immunoglobulin gene or a functional fragment thereof, which specifically binds to and recognizes an antigen, and is known to those skilled in the art. Antibodies are intended to include conventional four-chain immunoglobulins, which comprise two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 kDa). Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. The term "antibody" is intended to include complete antibodies, including single-chain complete antibodies and antigen binding fragments. In some embodiments, the antigen-binding fragment can be an antigen-binding antibody fragment, including but not limited to Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), and fragments comprising or consisting of a VL or VH domain, and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen. The term "antibody" is also intended to include heavy chain antibodies or fragments thereof, including immunoglobulin single variable domains, as further defined herein.

[0062] The term "immunoglobulin single variable domain" or "ISVD" defines molecules in which the antigen binding site is present on and formed by a single immunoglobulin domain (unlike conventional immunoglobulins or fragments thereof, in which typically two immunoglobulin variable domains interact to form an antigen binding site). However, it should be clear that the term "immunoglobulin single variable domain" does encompass fragments of conventional immunoglobulins in which the antigen binding site is formed by a single variable domain. Preferably, the binding agent within the scope of the present invention is an immunoglobulin single variable domain.

[0063] Typically, an immunoglobulin single variable domain is an amino acid sequence comprising four framework regions (FR1 to FR4) and three complementary determining regions (CDR1 to CDR3), preferably according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1), or any suitable fragment thereof (which typically contains at least some of the amino acid residues that form at least one complementary determining region). 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, as long as it is able to form a single antigen binding unit. Therefore, according to a preferred embodiment, the binding agent is an immunoglobulin single variable domain, which is a light chain variable domain sequence (e.g., a VL domain sequence) or a heavy chain variable domain sequence (e.g., a VH domain sequence); more specifically, 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. The immunoglobulin single variable domain can be a domain antibody, or a single domain antibody, or a "dAB" or dAb, or a nanobody (as defined herein), or another immunoglobulin single variable domain, or any suitable fragment of any of them. For a general description of single domain antibodies, reference is made to the following book: "Single domain antibodies", Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, Vol. 911. An immunoglobulin single variable domain generally comprises a single amino acid chain, which can be considered to comprise four "framework sequences" or FRs and three "complementarity determining regions" or CDRs (as defined above). It should be clear that the framework regions of an immunoglobulin single variable domain may also contribute to its antigen binding (Desmyter et al. 2002; Korotkov et al. 2009).

[0064] As further described herein, the total number of amino acid residues in a VHH, Nanobody or ConfoBody may be in the range of 110-120, preferably 112-115, most preferably 113. However, it should be noted that parts, fragments, analogs or derivatives of VHH or Nanobodies (as further described herein) are not particularly limited in their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.

[0065] In the present application, the amino acid residues / positions in the immunoglobulin heavy chain variable domain will be indicated by the numbering according to Kabat ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195 (see, for example, the publication of this publication). Figure 2 ) is applied to the VHH domains of camelids. For example, reference is also made to the international application WO2108 / 134235 Figure 1 , which gives a table listing some amino acid positions in VHH and their numbers according to some alternative numbering systems (such as Aho and IMGT. Note: Unless explicitly stated otherwise, for this description and claims, the Kabat numbering is decisive for the amino acid residues / positions in VHH, Nanobodies or ConfoBody; other numbering systems are for reference only).

[0066] Regarding CDRs, as is well known in the art, there are a variety of conventions to define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the position of the structural loop regions). For example, reference is made to the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, even though reference may also be made to the CDRs according to Kabat, the CDRs are most preferably defined based on the Abm definition (which is based on Oxford Molecular's AbM antibody modeling software) because this is considered to be the optimal compromise between the Kabat and Chothia definitions. Reference is again made to the website http: / / www.bioinf.org.uk / abs / .

[0067] Therefore, in this specification and claims, unless otherwise explicitly stated herein, all CDRs or VHHs, Nanobodies or ConfoBody are defined according to the Abm rule.

[0068] It should be noted that immunoglobulin single variable domains as binding agents are not limited to a specific biological source or a specific preparation method in its broadest sense. The term "immunoglobulin single variable domain" or "ISVD" covers variable domains of different origins, including mouse, rat, rabbit, donkey, human, shark, camelid variable domains. According to a specific embodiment, the immunoglobulin single variable domain is derived from shark antibodies (so-called immunoglobulin new antigen receptors or IgNARs), more specifically from naturally occurring heavy chain shark antibodies (without light chains), and is referred to 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 (without light chains), and is referred to as VHH domain sequences or nanobodies.

[0069] According to a particularly preferred embodiment, the binding agent of the present invention is an immunoglobulin single variable domain, which is a nanobody (as further defined herein, and including but not limited to VHH). As used herein, the term "nanobody" (Nb) is a single domain antigen binding fragment. It particularly refers to a single variable domain derived from a naturally occurring heavy chain antibody and is known to those skilled in the art. Nanobodies are generally derived from only heavy chain antibodies (without light chains) found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996), and are therefore generally referred to as VHH antibodies or VHH sequences. Camelids include Old World camelids (Camelusbactrianus and Camelus dromedarius) and New World camelids (e.g., Lamapaccos, Lama glama, Lama guanicoe and Lama vicugna). Nanobody® and Nanobodies® are registered trademarks of Ablynx NV (Belgium).For a further description of VHHs or Nanobodies, reference is made to the book "Single Domain Antibodies", Methods in Molecular Biology, edited by Saerens and Muyldermans, 2012, Volume 911, in particular the chapter by Vincke and Muyldermans (2012), and to a non-limiting list of patent applications mentioned as general background art, including: WO 94 / 04678, WO 95 / 04079, WO 96 / 34103 of Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231 and WO 02 / 48193 of Unilever; 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 to Ablynx NV, and further published patent applications to Ablynx N.V. As known to the skilled person, Nanobodies are particularly characterized by the presence of one or more Camelidae "hallmark residues" in one or more framework sequences (according to Kabat numbering), as for example described in Table A-3 on page 75 of WO 08 / 020079 (incorporated herein by reference). It should be noted that the Nanobodies of the invention in their broadest sense are not limited to a specific biological source or a specific method of preparation.For example, Nanobodies can generally be obtained by: (i) isolating the VHH domain of a naturally occurring heavy chain antibody; (ii) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (iii) "humanizing" a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain; (iv) "camelizing" a naturally occurring VH domain from any animal species, in particular from a mammalian species, such as from a human, or expressing a nucleic acid encoding such a camelized VH domain; (v) "camelizing" a "domain antibody" or "Dab" as described in the art, or expressing a nucleic acid encoding such a camelized VH domain; (vi) using 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 by using nucleic acid synthesis techniques known per se, and then expressing the nucleic acid thus obtained; and / or (8) any combination of one or more of the above. Further description of nanobodies, including humanization and / or camelization of nanobodies, can be found in, for example, WO08 / 101985 and WO08 / 142164, as well as further description herein. A specific class of nanobodies that bind conformational epitopes of natural targets is called Xaperones, and is specifically contemplated here. Xaperone™ is a trademark of VIB and VUB (Belgium). Xaperone™ is a camelid single domain antibody that can restrict drug targets to unique, disease-related druggable conformations.

[0070] Within the scope of the present invention, the term "immunoglobulin single variable domain" also encompasses "humanized" or "camelized" variable domains, in particular "humanized" or "camelized" Nanobodies. For example, both "humanization" and "camelization" can be performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then changing one or more codons in the nucleotide sequence in a manner known per se, wherein the changed codons are in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" immunoglobulin single variable domain, respectively, of the present invention. The nucleic acid can then be expressed in a manner known per se to provide the desired immunoglobulin single variable domain of the present invention. Alternatively, the amino acid sequence of the desired humanized or camelized immunoglobulin single variable domain of the present invention can be designed, respectively, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using techniques for peptide synthesis known per se. Furthermore, 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 invention can be designed, respectively, and then synthesized de novo using techniques for nucleic acid synthesis known per se, and the nucleic acid thus obtained can then be expressed in a manner known per se, to provide the desired immunoglobulin single variable domain of the invention. Other suitable methods and techniques for obtaining the immunoglobulin single variable domains of the invention and / or nucleic acids encoding the same starting from naturally occurring VH sequences, or preferably VHH sequences, will be clear to the skilled person and may, for example, comprise combining one or more parts of one or more naturally occurring VH sequences (e.g. one or more FR sequences and / or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (e.g. one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner, to provide the Nanobodies of the invention or nucleotide sequences or nucleic acids encoding the same.

[0071] According to a specific embodiment of the present invention, the binding agent that can stabilize the receptor can be combined at the orthosteric site or the allosteric site. In other specific embodiments, the binding agent that can stabilize the receptor can be an active conformation selective binding agent or an inactive conformation selective binding agent, by binding at the orthosteric site or the allosteric site. Generally, compared with the receptor in the absence of a binding agent (or in the presence of a non-specific and / or non-specific binding receptor analog binding agent (also referred to as a control binding agent or an irrelevant binding agent)), the conformation selective binding agent that stabilizes the active conformation of the receptor will increase or enhance the affinity of the receptor to an active conformation selective ligand (e.g., an agonist, more specifically, a full agonist, a partial agonist, or a biased agonist). In addition, compared with the receptor in the absence of a binding agent (or the presence of a simulated binding agent), the binding agent that stabilizes the active conformation of the receptor will reduce the affinity of the receptor to an inactive conformation selective ligand (e.g., an inverse agonist). In contrast, a binding agent that stabilizes an inactive conformation of a receptor will enhance the affinity of the receptor for an inverse agonist and reduce the affinity of the receptor for an agonist (particularly a full agonist, a partial agonist, or a biased agonist) compared to a receptor in the absence of a binding agent (or in the presence of a mimetic binding agent). An increase or decrease in affinity for a ligand can be directly measured and / or calculated by and / or from a decrease or increase in EC50, IC50, Kd, ​​K, or any other measure of affinity or potency known to those skilled in the art, respectively. It is particularly preferred that, upon binding to a receptor, a binding agent that stabilizes a specific conformation of a receptor is able to increase or decrease the affinity for a conformationally 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. It should be understood that affinity measurements of conformationally selective ligands that trigger / inhibit specific signaling pathways can be performed with: any type of ligand, including natural ligands, small molecules, and biologics; orthosteric ligands and allosteric modulators; single compounds and compound libraries; lead compounds or fragments; and the like.

[0072] As used herein, the term "affinity" refers to the degree to which a ligand (as further defined herein) binds to a target protein thereby shifting the equilibrium of the target protein and the ligand toward the presence of a complex formed by their binding. Thus, for example, when a GPCR and a ligand are combined in relatively equal concentrations, a high affinity ligand will bind to the available antigen on the GPCR, thereby shifting the equilibrium toward a high concentration of the resulting complex. A dissociation constant is often used to describe the affinity between a ligand and a target protein. Typically, a 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 -8M. Other ways of describing the affinity between a ligand (including a small molecule ligand) and its target protein are the binding constant (Ka), the inhibition constant (Ki) (also called the inhibitory constant), or indirectly assessing the potency of the ligand by measuring the half maximal inhibitory concentration (IC50) or the half maximal effective concentration (EC50). Within the scope of the present invention, the ligand may be a binding agent, preferably an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH or a nanobody, that binds to a conformational epitope on a GPCR. It should be understood that within the scope of the present invention, the term "affinity" is used in the context of a binding agent, particularly an immunoglobulin or an immunoglobulin fragment, such as a VHH or a nanobody, that binds to a conformational epitope of a target GPCR, and in the context of a test compound (as further defined herein) that binds to a target GPCR, more particularly to an orthosteric or allosteric site of a target GPCR.

[0073] 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 a nanobody, to preferentially bind one antigen over a different antigen, and does not necessarily mean high affinity.

[0074] As used herein, the term "specifically bind" or "specific binding" generally refers to the ability of a binding agent, particularly an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH or a nanobody, to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens. In certain embodiments, the specific binding interaction will discriminate between desired and undesired antigens in a sample, in some embodiments by more than about 10 to 100 times or more (e.g., more than about 1000 times or 10,000 times). In the context of the spectrum of conformational states of a GPCR, the term particularly refers to the ability of a binding agent (as defined herein) to preferentially recognize and / or bind to a specific conformational state of a GPCR compared to another conformational state.

[0075] Furthermore, it should be understood that in this specification and the appended claims, where a protein, ligand, compound, binding domain, binding unit or other chemical entity is referred to as "binding" another protein, ligand, compound, binding domain, binding unit or other chemical entity or epitope or binding site, such binding is preferably "specific" binding as defined herein. Furthermore, preferably, such binding is "selective binding" as defined herein.

[0076] As used herein, the term "conformational selective binder" in the context of the present invention refers to a binder that binds to a target protein in a conformationally selective manner. A binder that selectively binds to a specific conformation or conformational state of a protein refers to a binder that binds to a protein in a subset of conformations or conformational states with a higher affinity than other conformations or conformational states that the protein may assume. One skilled in the art will recognize that a binder that selectively binds to a specific conformation or conformational state of a protein will stabilize or retain the protein in that specific conformation or conformational state. For example, an active conformation selective binder will preferentially bind to a GPCR in an active conformational state, and will not or to a lesser extent bind to a GPCR in an inactive conformational state, and will therefore have a higher affinity for the active conformational state; or vice versa. The terms "specific binding", "selective binding", "preferential binding" and their grammatical equivalents are used interchangeably herein. The terms "conformational specificity" or "conformational selectivity" are also used interchangeably herein.

[0077] As used herein, the term "stable" or grammatically equivalent terms as defined above refers to the increased stability of a protein (as described herein) or a receptor (also as described herein) in terms of structure (e.g., conformational state) and / or specific biological activity (e.g., intracellular signaling activity, ligand binding affinity, etc.). Regarding increased stability in terms of structure and / or biological activity, this can be easily determined by functional assays of activity (e.g., Ca2+ release, cAMP generation or transcriptional activity, β-arrestin recruitment, etc.) or ligand binding, or by physical methods such as X-ray crystallography, NMR, or spin labeling. The term "stable" also includes an increase in the thermal stability of a receptor under non-physiological conditions induced by a denaturant or denaturing condition. As used herein, the term "thermostabilize, thermostabilizing", "increase in thermal stability..." refers to the functionality of a receptor rather than thermodynamic properties and the resistance of a protein to irreversible denaturation induced by heat and / or chemical methods (including but not limited to heating, cooling, freezing, chemical denaturants, pH, detergents, salts, additives, proteases, or temperature). Irreversible denaturation leads to irreversible unfolding of the functional conformation of the protein, loss of biological activity and aggregation of denatured proteins. Regarding increased thermal stability, this can be easily determined by measuring ligand binding or by using spectroscopic methods (such as fluorescence, CD or light scattering), which are sensitive to unfolding when the temperature rises. Preferably, the binding agent is able to increase stability, as measured by the increase in the thermal stability of the protein or receptor in the functional conformational state at at least 2°C, at least 5°C, at least 8°C, more preferably at least 10°C or 15°C or 20°C. Regarding increasing stability to detergents or chaotropes, proteins or receptors are usually incubated for a specified time in the presence of a test detergent or a test chaotropic agent, and stability is optionally determined at an elevated temperature as described above using, for example, ligand binding or spectroscopic methods. Otherwise, the binding agent is able to increase the stability of the functional conformational state of a protein or receptor to extreme pH. Regarding extreme pH, a typical test pH will be selected, for example, in the range 6 to 8, the range 5.5 to 8.5, the range 5 to 9, the range 4.5 to 9.5, more specifically the range 4.5 to 5.5 (low pH) or the range 8.5 to 9.5 (high pH). As used herein, the terms "(thermo)stabilize, (thermo)stabilizing", "increasing the (thermo)stability of..." apply to proteins or receptors embedded in lipid particles or lipid layers (e.g., lipid monolayers, lipid bilayers, etc.), and proteins or receptors that have been dissolved in detergents.

[0078] In addition to the above, with respect to the functional conformational states of a GPCR, the term "stabilizing" or "stabilized" refers to retaining or keeping the GPCR protein in a subset of possible conformations that may be assumed as a result of the effect of the interaction of the GPCR with the binding agent according to the present invention. In this context, a binding agent that selectively binds to a specific conformation or conformational state of a protein refers to a binding agent that binds to the protein in a subset of conformations or conformational states with a higher affinity than other conformations or conformational states that the protein may assume. Those skilled in the art will recognize that a binding agent that specifically or selectively binds to a specific conformation or conformational state of a protein will stabilize that specific conformation or conformational state and its associated activity. More details are provided further herein.

[0079] As used herein, the term "compound" or "test compound" or "candidate compound" or "drug candidate compound" describes any naturally occurring or synthetic molecule tested in an assay (e.g., a screening assay or a drug discovery assay). Thus, these compounds include organic or inorganic compounds. These compounds include polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptide mimetics) containing about 2 to about 40 amino acids, and larger polypeptides containing about 40 to about 500 amino acids, such as antibodies, antibody fragments, or antibody conjugates. The test compound can also be a protein scaffold. For high throughput purposes, test compound libraries can be used, such as combinatorial or random libraries that provide a sufficient diversity range. 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, etc. A more detailed description can be found further in the specification.

[0080] As used herein, the term "ligand" refers to a molecule that specifically binds to a protein (e.g., GPCR) as referred to herein. A ligand may be, but is not limited to, a polypeptide, a lipid, a small molecule, an antibody, an antibody fragment, a nucleic acid, a carbohydrate. A ligand may be synthetic or naturally occurring. A ligand also includes a "natural ligand," which is a ligand that is an endogenous natural ligand of a natural GPCR. In the context of the present invention, when a protein is a transmembrane protein such as a GPCR, when the protein is in its natural cell environment, the ligand may bind to the protein at a ligand binding site exposed to the intracellular environment (i.e., the ligand may be an "intracellular ligand"), or when the protein is in its natural cell environment, the ligand may bind to the protein at a ligand binding site exposed to the extracellular environment (i.e., the ligand may be an "extracellular ligand"). A ligand may be an agonist, a partial agonist, an inverse agonist, an antagonist, an allosteric modulator, and may bind at an orthosteric site or an allosteric site. In certain embodiments, the ligand may be a "conformation selective ligand" or a "conformation specific ligand," meaning that such ligand binds to a protein or GPCR in a conformation selective manner. As further described herein, a conformation selective ligand binds to a specific conformation of a protein with higher affinity than to other conformations that the protein may adopt. For purposes of illustration, an agonist is an example of an active conformation selective ligand, while an inverse agonist is an example of an inactive conformation selective ligand. For clarity, a neutral antagonist is not considered a conformation selective ligand because a neutral antagonist does not discriminate between different conformations of a GPCR.

[0081] As used herein, "orthosteric ligands" refer to ligands (natural and synthetic) that bind to the active site of a GPCR and are further classified according to their efficacy or in other words their effect on signal transduction through a specific pathway. As used herein, "agonists" refer to ligands that increase the signaling activity of a receptor by binding to a receptor protein. Full agonists are able to stimulate the protein to the greatest extent; partial agonists are unable to induce full activity even at saturating concentrations. Partial agonists can also act as "blockers" by preventing the binding of more robust agonists. "Antagonists", also known as "neutral antagonists", refer to ligands that bind to a receptor without stimulating any activity. "Antagonists" are also called "blockers" because they are able to prevent the binding of other ligands, thereby blocking agonist-induced activity. In addition, "inverse agonists" refer to an antagonist that, in addition to blocking the action of agonists, reduces the basal or constitutive activity of a receptor to below the activity of an unliganded protein.

[0082] As used herein, a ligand may also be a "biased ligand" that has the ability to selectively stimulate a subset of receptor signaling activities, such as selectively activating G-protein or β-arrestin function in the case of a GPCR. Such ligands are referred to as "biased ligands," "biased agonists," or "functionally selective agonists." More specifically, ligand bias may be imperfect bias, characterized by ligand stimulation of multiple receptor activities with different relative efficacies for different signals (non-absolute selectivity), or may be perfect bias, characterized by ligand stimulation of one receptor protein activity without any stimulation of another known receptor protein activity.

[0083] Another type of ligand is called an allosteric modulator. As used herein, an "allosteric modulator" or other "allosteric modulators," "allosteric ligands," or "effector molecules" refers to a ligand that binds at an allosteric site of a GPCR (i.e., a regulatory site that is physically different from the active site of the protein). In contrast to orthosteric ligands, allosteric modulators are non-competitive because they bind to receptor proteins at different sites and alter their function, even if endogenous ligands are also binding. Allosteric modulators that enhance protein activity are referred to herein as "allosteric activators" or "positive allosteric modulators" (PAMs), while allosteric modulators that reduce protein activity are referred to herein as "allosteric inhibitors" or other "negative allosteric modulators" (NAMs).

[0084] As used herein, the terms "determine," "measure," "evaluate," "measure," and "assay" are used interchangeably and include both quantitative and qualitative determination.

[0085] The term "antibody" is intended to mean an immunoglobulin or any fragment thereof that is capable of binding to an antigen. The term "antibody" also refers to single-chain antibodies and antibodies with only one binding domain.

[0086] As used herein, the term "complementarity determining region" or "CDR" in the context of antibodies refers to the variable region of H (heavy) or L (light) chain (also abbreviated as VH and VL, respectively) and comprises an amino acid sequence that can specifically bind to an antigen target. These CDR regions explain the basic specificity of antibodies to specific antigenic determinant structures. Such regions are also referred to as "hypervariable regions". CDR represents non-adjacent amino acid segments in the variable region, but regardless of species, it has been found that the positions of these key amino acid sequences in the variable heavy and light chain regions have similar positions in the amino acid sequences of the variable chains. The variable heavy and light chains of all classical antibodies have 3 CDR regions, and for the corresponding light (L) and heavy (H) chains, each region is not adjacent to other regions (referred to as L1, L2, L3, H1, H2, H3). Immunoglobulin single variable domains, particularly nanobodies, typically comprise a single amino acid chain that can be considered to comprise 4 "framework sequences or regions" or FRs and 3 "complementarity determining regions" or CDRs. Nanobodies have 3 CDR regions, each region is not adjacent to the other regions (called CDR1, CDR2, CDR3). As described herein, to indicate the amino acid position / residue CDR in a VHH, Nanobody or ConfoBody, the Kabat numbering system will be followed, and the framework and CDR are defined based on the Abm definition (unless otherwise explicitly stated).

[0087] In general, for the purposes of this disclosure and the claims thereto, a compound of the invention will be considered a "modulator" of a target, or "modulates" a target (and / or signaling, pathway, mechanism of action, and / or the biological, physiological, and / or pharmacological function involved in the target) when the presence of the compound in a suitable assay or model (i.e., in a suitable amount or concentration, such as a biologically active amount or concentration) changes the appropriate or expected reading of the assay or model (i.e., at least one suitable value or parameter that can be determined using the assay or model) by at least 0.1%, such as at least 1%, such as at least 10%, and up to 50% or more, compared to the same value or parameter measured using the same assay or model under essentially the same conditions but in the absence of the compound. Again, the modulation may result in an increase or decrease in the value or parameter (i.e., an increase or decrease by the percentage given in the preceding sentence). In addition, the compounds of the invention are preferably capable of modulating the target, signaling, pathway, 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.

[0088] The method of the present invention is generally carried out with an apparatus comprising at least the following elements (each as further defined herein):

[0089] a boundary layer separating the first environment and the second environment;

[0090] translamin;

[0091] a first ligand for a translaminar protein present in a first environment (as defined herein);

[0092] a second ligand for the translaminar protein present in a second environment (as defined herein); and

[0093] 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;

[0094] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein.

[0095] In particular, in the devices of the invention and as further described herein, the first binding member of the binding pair may be part of a "first fusion protein" (as further described herein) and the second member of the binding pair may be part of a "second fusion protein" (also as further described herein, and different from the first fusion protein), and such first fusion protein, such second fusion protein (in its various forms as described herein), nucleotide sequences and / or nucleic acids encoding thereof, and cells, cell lines or other host cells or host organisms expressing (and in particular suitably expressing, as described herein) or capable of (suitably) expressing the first and / or second fusion protein (and preferably both) form further aspects of the invention.

[0096] In particular, the apparatus for executing the method of the present invention may include at least the following elements:

[0097] - a boundary layer separating the first environment and the second environment;

[0098] - 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;

[0099] - a translaminar protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (ie forming a first fusion protein);

[0100] - a first ligand for the translaminar protein present in a first environment; and

[0101] - a second ligand for the translaminar protein present in a second environment;

[0102] The elements are arranged relative to each other (and, where applicable, operably connected and / or associated with each other) in a manner further described herein. In particular, the second member of the binding pair can be part of a second fusion protein (which is different from a first fusion protein comprising a spanning protein and a first binding member of the binding pair), the second fusion protein being further described herein.

[0103] More specifically, the apparatus for executing the method of the present invention may include at least the following elements:

[0104] - a boundary layer separating the first environment and the second environment;

[0105] - 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;

[0106] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0107] - a second fusion protein comprising a protein that can bind directly or indirectly to the translaminar protein and to the other binding member of the binding pair, the second fusion protein being present in a second environment; and

[0108] - a first ligand for a translaminar protein present in a first environment;

[0109] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein.

[0110] It should be noted that in this specification and claims, when it is said that a ligand, binding domain, binding unit or other compound or protein is "capable of binding" to another protein or compound, such binding is most preferably "specific binding" as further defined herein. In addition, 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 other proteins, ligands, binding domains, binding members or binding units), it should be understood that in such a fusion protein, such proteins, ligands, binding domains, binding members or binding units are appropriately connected to each other directly or through suitable spacers or linkers.

[0111] For the purposes of this specification and claims, a protein (e.g., a binding domain, binding unit, or ligand) is said to bind "directly or indirectly" to a translaminar protein if: (i) the protein itself binds (and / or is capable of binding) to a translaminar protein (e.g., binds to an epitope or binding site on a translaminar protein, as further described herein); or if (ii) the protein binds (and / or is capable of binding) to a ligand or protein, wherein the ligand or protein binds (and / or is capable of binding) to the translaminar protein; or if (iii) the protein binds (and / or is capable of binding) to a protein complex, wherein the protein complex comprises a ligand or protein that binds (and / or is capable of binding) to the translaminar protein. In the case of (i), the protein is referred to herein as "directly" binding to a translaminar protein, while in the cases of (ii) and (iii), the protein is referred to herein as "indirectly" binding to a translaminar protein. Furthermore, when a protein binds to a protein complex comprising a ligand or protein that binds to a translaminar protein, the protein may bind to the ligand or protein or to any other part, epitope, or binding site of the complex.

[0112] Thus, in one aspect of the invention, the protein that binds to a translaminar protein is selected from (i) a binding domain, a binding unit or other protein that binds (and / or is capable of binding) an epitope or binding site on a translaminar protein; (ii) a binding domain, a binding unit or other protein that binds (and / or is capable of binding) a ligand or protein that binds (and / or is capable of binding) to the translaminar protein; and (iii) a binding domain, a binding unit or other protein that binds (and / or is capable of binding) to a protein complex that contains a ligand or protein that binds (and / or is capable of binding) to the translaminar protein. In each such case, such a binding domain, binding unit or other protein is preferably as further described herein.

[0113] In particular, the protein that directly or indirectly binds to a translaminar protein may be selected from (i) an ISVD that binds (and / or is capable of binding) an epitope or binding site on a translaminar protein; (ii) an ISVD that binds (and / or is capable of binding) a ligand or protein that binds (and / or is capable of binding) to the translaminar protein; (iii) an ISVD that binds (and / or is capable of binding) a protein complex that contains a ligand or protein that binds (and / or is capable of binding) to the translaminar protein. Again, in each such case, such an ISVD is preferably as further described herein.

[0114] In a further aspect of the present invention, an apparatus for performing the method of the present invention may include at least the following elements:

[0115] - a boundary layer separating the first environment and the second environment;

[0116] - 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;

[0117] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0118] - a second fusion protein comprising a protein that can directly bind to the translaminar protein (as defined herein) and to the other binding member of said binding pair, the second fusion protein being present in a second environment; and

[0119] - a first ligand for a translaminar protein present in a first environment;

[0120] The elements are arranged relative to each other (and where applicable are operably connected and / or associated with each other) in a manner further described herein. In this aspect of the invention, the protein that can directly bind (as defined herein) to a spanning protein and is present in the second fusion protein is preferably a binding domain or binding unit, and is more preferably an immunoglobulin single variable domain. It should also be understood that in this aspect of the invention, the protein that can directly bind (as defined herein) to a spanning protein and is present in the second fusion protein acts as a second ligand.

[0121] In another aspect of the present invention, an apparatus for performing the method of the present invention may include at least the following elements:

[0122] - a boundary layer separating the first environment and the second environment;

[0123] - 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;

[0124] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0125] - a first ligand for a translaminar protein present in a first environment;

[0126] - a second ligand for the translaminin, which may optionally be part of a protein complex;

[0127] a second fusion protein comprising a protein that can indirectly bind (as defined herein) to a translaminar protein and to the other binding member of said binding pair, the second fusion protein being present in a second environment;

[0128] The elements are arranged relative to each other (and where applicable are operably connected and / or associated with each other) in a manner further described herein. In this aspect of the invention, the second ligand may be any suitable ligand (as further described herein), and the protein capable of indirectly binding (as defined herein) to the translaminar protein and present in the second fusion protein is preferably a binding domain or binding unit, and more preferably an immunoglobulin single variable domain. It will also be clear that in this aspect of the invention, the second ligand does not form part of the second fusion protein.

[0129] It should be noted that, as further described herein, in the practice of the present invention, the first ligand will typically be added to further elements of an already formed / established device of the present invention as described herein, and thus the device of the present invention in which the first ligand is not present (i.e., prior to the addition of the first ligand) forms other aspects of the present invention (such as a method of adding a first ligand to a device of the present invention in which the first ligand is not present or does not yet exist).

[0130] In this specification and claims, the term "second ligand" is used to refer to a ligand, binding domain, binding unit or other chemical entity that directly binds to a translaminar protein or is capable of directly binding to a translaminar protein (or forms part of a protein complex that directly binds to a translaminar protein or is capable of directly binding to a translaminar protein) in the methods and devices described herein.

[0131] It will be clear from the further description herein that the second ligand may be part of the second fusion protein, or it may be separate from the second fusion protein. In either case (i.e., whether or not the second ligand is part of the second fusion protein), the second ligand is preferably capable of binding to a conformational epitope on the translaminar protein (or it may form part of a protein complex that directly binds to the translaminar protein or is capable of directly binding to the translaminar protein). More preferably, the second ligand (and / or a protein complex comprising the second ligand) is preferably one that specifically binds to one or more functional, active and / or druggable conformations of the translaminar protein, it induces the formation and / or stabilization of one or more functional, active and / or druggable conformations of the translaminar protein (and / or shifts the conformational equilibrium of the translaminar protein to one or more such conformations); and / or it induces the formation and / or stabilization of a complex of the translaminar protein, the first ligand and the second ligand.

[0132] When the second ligand is part of the second fusion protein, it can be any ligand, binding domain, binding unit, peptide, protein or other chemical entity that can directly bind to the cross-layer protein and can be appropriately contained in the second fusion protein. Preferably, as further described herein, when it is part of the second fusion protein, the second ligand will be a suitable binding domain or binding unit, and in particular an immunoglobulin single variable domain.

[0133] When the second ligand is separate from the second fusion protein, it can be any ligand or protein that can bind directly to the translaminar protein and / or can form part of a protein complex that can bind to the translaminar protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of the translaminar protein (e.g., a naturally occurring G-protein, such as a G-protein that occurs naturally in the cell or cell line used), a semisynthetic 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). In addition, when the second ligand is not part of the second fusion protein, the second fusion protein will comprise a binding domain or binding unit that can bind indirectly (as defined herein) to the translaminar protein, i.e., a binding domain or binding unit that can bind to the second ligand and / or bind to a protein complex comprising the second ligand. Likewise, as also further described herein, such a binding domain or binding unit may in particular be an immunoglobulin single variable domain, such as a Camelidae-derived ISVD (or it may comprise one or more such immunoglobulin single variable domains, such as two or three such immunoglobulin single variable domains, which may be the same or different, as further described herein).

[0134] As further described herein, in one aspect of the invention, the device of the invention may be present in a suitable cell or cell line and / or the method of the invention may be performed using a suitable cell or cell line, which cell or cell line suitably contains (is operable with) the device of the invention.

[0135] Thus, as further described herein, the present invention also relates to a cell or cell line, which suitably comprises the device of the present invention and / or suitably expresses (as defined herein) or is capable of suitably expressing an element of the device of the present invention so as to provide a device of the present invention (especially a device of the present invention operable in said cell or cell line). The present invention also relates to a cell or cell line, which comprises and / or suitably expresses (as defined herein) or is capable of suitably expressing a first fusion protein as described herein. The present invention also relates to a cell or cell line, which comprises and / or suitably expresses or is capable of suitably expressing a second fusion protein as described herein. On the other hand, the present invention relates to a cell or cell line, which comprises and / or suitably expresses or is capable of suitably expressing a first fusion protein as described herein and a second fusion protein as described herein. In aspects and embodiments in which the second ligand does not form part of the second fusion protein, such a cell or cell line may also comprise or suitably express a suitable second ligand.

[0136] As also described herein, in one aspect of the invention, the device of the invention may be present in a suitable liposome or vesicle and / or the method of the invention may be performed using a liposome or vesicle suitably containing (operably containing) the device of the invention.

[0137] Therefore, as further described herein, the present invention also relates to liposomes or vesicles that suitably comprise (elements of) the device of the present invention, in particular so as to provide a device of the present invention operable in said liposomes or vesicles. The present invention also relates to liposomes or vesicles comprising a first fusion protein as described herein. The present invention also relates to liposomes or vesicles of cells or cell lines comprising a second fusion protein as described herein. In yet another aspect, the present invention relates to liposomes or vesicles comprising a first fusion protein as described herein and a second fusion protein as described herein. In aspects and embodiments in which the second ligand does not form part of the second fusion protein, such liposomes or vesicles may also contain a suitable second ligand.

[0138] Thus, as further described herein and as will be illustrated by the accompanying non-limiting figures, and depending on whether the second ligand is part of a second fusion protein, the present invention contemplates at least three preferred embodiments of the methods and devices of the present invention.

[0139] In the first such preferred embodiment ( Figure 1 In the embodiment shown schematically in Figure 2, the second binding member of the binding pair will be appropriately fused or connected (directly or through a suitable linker or spacer) to the second ligand. According to this preferred embodiment, the device for performing the method of the present invention may therefore include at least the following elements:

[0140] - a boundary layer separating the first environment and the second environment;

[0141] - 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;

[0142] - a translaminar protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair;

[0143] - a first ligand for the translaminar protein present in a first environment; and

[0144] - a second ligand of the translaminar protein present in a second environment and suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of the binding pair;

[0145] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein.

[0146] In particular, as further described herein, such an apparatus may include the following elements:

[0147] - a boundary layer separating the first environment from the second environment;

[0148] - 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;

[0149] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0150] - a first ligand for the translaminar protein present in a first environment; and

[0151] - a second fusion protein comprising a second ligand for the layer-spanning protein and the other binding member of the binding pair, the second fusion protein being present in a second environment;

[0152] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein.

[0153] It will be clear to the skilled person that in this first embodiment, the "second ligand" will be a binding domain, binding unit or other protein that directly binds (and / or is capable of binding) to an epitope or binding site on a translaminar protein. Again, the binding domain or binding unit is preferably an immunoglobulin single variable domain as further described herein.

[0154] In a second such preferred embodiment (schematically shown in Figure 2 The second binding member of the binding pair will be suitably fused or linked (directly or via a suitable linker or spacer) to a binding domain or binding unit that does not directly bind to the translaminar protein but binds to a second ligand (which in turn is capable of binding to the translaminar protein). According to this preferred embodiment, the device for performing the method of the invention may therefore comprise at least the following elements:

[0155] - a boundary layer separating the first environment and the second environment;

[0156] - 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;

[0157] a first fusion protein comprising a spanning protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in a second environment);

[0158] - a first ligand for a translaminar protein present in a first environment;

[0159] - a second ligand for the translaminar protein present in a second environment; and

[0160] a second fusion protein, which is present in a second environment and comprises a binding domain or binding unit capable of binding a second ligand, which binding domain or binding unit is suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of the binding pair;

[0161] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein.

[0162] It will be clear to the skilled person that in this second embodiment, the binding domain or binding unit present in the second fusion protein will bind to the cross-layer protein "indirectly", i.e., by binding to a second ligand that is bound to the cross-layer protein. Likewise, the binding domain or binding unit is preferably the following (and / or preferably consists essentially of the following): an immunoglobulin single variable domain as further described herein. The binding domain or binding unit may also comprise or consist essentially of the following: two or more immunoglobulin single variable domains (e.g., two or three immunoglobulin single variable domains), each of which is capable of (specifically) binding to a second ligand (i.e., the same epitope or binding site on the second ligand or a different epitope / binding site on the second ligand), and they may be the same or different (as further described herein), and they are suitably connected or fused to each other and to the other binding member 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, but not limited to, such a binding domain or binding unit may comprise two or three copies of ConfoBody CA4437 (SEQ ID NO: 4 in WO2012 / 75643 and SEQ ID No: 2 herein), which are suitably linked or fused to each other and to the other binding member of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for use in the present invention. Furthermore, in this embodiment, the second ligand may be any suitable ligand of a translaminar protein as further described herein. Again, such a "multivalent" binding domain comprising two or more ISVDs should most preferably be such that its binding to the second ligand does not substantially interfere with the ability of the second ligand to bind to the translaminar protein and / or to form (or promote the formation of) a complex between the second ligand, the translaminar protein and the first ligand.

[0163] In a third preferred embodiment (schematically shown in Figure 3In the embodiment of the present invention, the second binding member of the binding pair will be suitably fused or linked (directly or via a suitable linker or spacer) to the binding domain or binding unit, wherein the binding domain or binding unit does not directly bind to the translaminar protein, but binds to a protein complex comprising at least a second ligand of the translaminar protein (the protein complex may bind to the translaminar protein or be bound by the translaminar protein and / or comprise the translaminar protein). According to this preferred embodiment, the device for performing the method of the present invention may therefore comprise at least the following elements:

[0164] - a boundary layer separating the first environment and the second environment;

[0165] - 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;

[0166] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0167] - a first ligand for a translaminar protein present in a first environment;

[0168] - a protein complex comprising at least a second ligand of the translaminar protein, the protein complex being present in a second environment; and

[0169] a second fusion protein, which is present in a second environment and comprises a binding domain or a binding unit capable of binding to the protein complex, which binding domain or binding unit is suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of the binding pair;

[0170] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein.

[0171] It is clear to the skilled person that in this third embodiment, the binding domain or binding unit present in the second fusion protein will bind to the spanning protein "indirectly", i.e. by binding to a protein complex comprising the second ligand. Again, the binding domain or binding unit is preferably an immunoglobulin single variable domain as further described herein, and the second ligand may be any suitable ligand of the spanning protein, which ligand may be part of a protein complex as further described herein.

[0172] In addition, the binding domain or binding unit in the second fusion protein may comprise two or more immunoglobulin single variable domains, each domain being capable of binding to a different epitope, part, domain or subunit on / on the protein complex, such as two different epitopes on the G protein complex. For example, but not limited to, when the protein complex is a heterotrimeric G protein, the binding domain or binding unit may comprise two or three different ISVDs, wherein each ISVD is capable of (specifically) binding to a different subunit of the G-protein (wherein preferably, at least one of the ISVDs is capable of specifically binding to the G-α subunit present in the heterotrimeric G-protein). Specific but non-limiting examples of such binding domains or binding units may, for example, include ConfoBody CA4435 (SEQ ID NO: 1 in WO2012 / 75643 and SEQ ID NO: 1 herein) and CA4437 (SEQ ID NO: 4 in WO2012 / 75643 and SEQ ID NO: 2 herein), which are suitably linked or fused to each other and to the other binding member of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for use in the present invention.

[0173] The use of such "multivalent" binding domains or binding units (ie comprising two or more ISVDs) in the second fusion protein may also lead to improved sensitivity in the assays described herein compared to the use of the corresponding ISVD in a monovalent format (ie comprising only one of said ISVDs).

[0174] More generally, an apparatus for carrying out the method of the present invention in its various aspects and embodiments will typically and preferably include at least the following elements:

[0175] - a boundary layer separating the first environment and the second environment;

[0176] - 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;

[0177] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0178] - a first ligand for a translaminar protein present in a first environment;

[0179] - a second ligand for the translaminar protein present in a second environment; and

[0180] - a second fusion protein comprising the other binding member of the binding pair (i.e. the other member of the binding pair is also present in the second environment);

[0181] The elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in the manner further described herein. In particular:

[0182] - in the first preferred embodiment described herein, the second fusion protein will comprise the other binding member of the binding pair and the second ligand;

[0183] - in a second preferred embodiment described herein, the second fusion protein will comprise the other binding member of the binding pair and a binding domain or binding unit capable of binding a second ligand; and

[0184] - In a third preferred embodiment described herein, the second fusion protein will comprise the other binding member of the binding pair and a binding domain or binding unit capable of binding to a protein complex comprising at least the second ligand. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] The invention will now be illustrated by the further description herein, the following experimental section and the accompanying non-limiting drawings. In the drawings:

[0186] a) Figure 1 A first device of the invention is schematically shown, wherein the second ligand (in Figure 1 (4)) to form a second fusion protein (in Figure 1 In the embodiment shown, it is formed from a second ligand (4), a linker (11) and a second member (7) of a binding pair (6 / 7) and binds directly (as defined herein) to a translaminar protein (2). Figure 1 In the setup shown:

[0187] - The boundary layer is represented by (1);

[0188] - The first environment is denoted as [A];

[0189] - The second environment is denoted as [B];

[0190] - Translaminin is represented as (2);

[0191] - the first ligand is represented by (3);

[0192] - a first binding site on the translaminar protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is denoted (8);

[0193] - The second ligand is represented by (4);

[0194] - a second binding site on the translaminar protein (2) that is exposed to a second environment [B] and to which a second ligand (4) can bind is represented by (9);

[0195] - a binding pair capable of generating a detectable signal is denoted (6 / 7) and consists of a first binding member (6) linked to a translaminar protein (2) (directly or via a linker or spacer (10)) and a second binding member (7) linked to a second ligand (4) (directly or via a linker or spacer (11));

[0196] - the first fusion protein comprises a translaminar protein (2) fused directly or via a linker (10) to a first binding member (6);

[0197] - a second fusion protein comprising a second ligand (4) fused directly or via a linker (11) to a second binding member (7); and

[0198] - the first and second fusion proteins are arranged relative to each other and to the boundary layer (1) in such a way that when the second ligand (4) binds to the translaminar protein (2) (i.e. directly via the binding site (9)), the first binding member (6) and the second binding member (7) are able to contact or come into close proximity with each other (or otherwise appropriately associate to produce a detectable signal (with Figure 1 Indicated by the flashing symbol in the figure).

[0199] b) Figure 2 A second device of the invention is schematically shown, wherein the second ligand (in Figure 2 denoted as (4)) and the second fusion protein (in Figure 2 In the embodiment shown, the binding domain (5), the linker (11) and the second member (7) of the binding pair (6 / 7) are separated, and the binding domain (5) present in the second fusion protein indirectly binds (as defined herein, and Figure 2 In the case of , through the second ligand (4)) to the translaminar protein (2). Figure 2 In the setup shown:

[0200] - The boundary layer is represented by (1);

[0201] - The first environment is denoted as [A];

[0202] - The second environment is denoted as [B];

[0203] - Translaminin is represented as (2);

[0204] - the first ligand is represented by (3);

[0205] - a first binding site on the translaminar protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is denoted (8);

[0206] - The second ligand is represented by (4);

[0207] - a second binding site on the translaminar protein (2) that is exposed to a second environment [B] and to which a second ligand (4) can bind is represented by (9);

[0208] - a binding domain or binding unit capable of binding a second ligand (4) is denoted (5);

[0209] - a binding pair capable of generating a detectable signal is denoted (6 / 7) and consists of a first binding member (6) linked to a translaminar protein (2) (directly or via a linker or spacer (10)) and a second binding member (7) linked to a binding domain or binding unit (5) (directly or via a linker or spacer (11));

[0210] - the first fusion protein comprises a translaminar protein (2) fused directly or via a linker (10) to a first binding member (6);

[0211] - a second fusion protein comprising a binding domain (5) fused directly or via a linker (11) to a second binding member (7); and

[0212] - the first and second fusion proteins are arranged relative to each other and to the boundary layer (1) in such a way that when the binding domain (5) binds to the translaminar protein (2) (i.e. indirectly via binding to the second ligand (4) (which in turn binds to the translaminar protein (2) via the binding site (9))), the first binding member (6) and the second binding member (7) are able to contact or be in close proximity to each other (or otherwise suitably associate) to produce a detectable signal (expressed as Figure 2 Indicated by the flashing symbol in the figure).

[0213] c) Figure 3 A third device of the invention is schematically shown, wherein the second ligand (in Figure 3 4)) is separated from the second fusion protein and forms part of a protein complex (12) consisting of a second ligand (4) and one or more other proteins (in Figure 3 In the case of, for illustrative purposes, the complex is illustrated as comprising a second ligand (4) and two other proteins (4a) and (4b), see also Figure 3 ). Figure 3 In the embodiment shown, the second ligand (4) is again combined with the second fusion protein (which is in Figure 3 In the embodiment shown, the binding domain (5), linker (11) and second member (7) of the binding pair (6 / 7) are separated, and the binding domain (5) present in the second fusion protein indirectly binds (as defined herein and Figure 3 In the case of , through a protein complex (12)) to the translaminar protein (2). Figure 3In the setup shown:

[0214] - The boundary layer is represented by (1);

[0215] - The first environment is denoted as [A];

[0216] - The second environment is denoted as [B];

[0217] - Translaminin is represented as (2);

[0218] - the first ligand is represented by (3);

[0219] - a first binding site on the translaminar protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is denoted (8);

[0220] - The second ligand is represented by (4) and forms a complex with one or more other proteins (12) (for illustrative purposes, Figure 3 In the example, complex (12) is represented as a complex comprising three proteins / subunits, namely the second ligand (4) and two other subunits (4a) and (4b), see also Figure 3 illustrations in );

[0221] - a second binding site on the translaminar protein (2) that is exposed to a second environment [B] and to which the complex (12) (4) can bind is represented by (9);

[0222] - a binding domain or binding unit capable of binding to the complex (12) is denoted as (5);

[0223] - a binding pair capable of generating a detectable signal is denoted (6 / 7) and consists of a first binding member (6) linked to a translaminar protein (2) (directly or via a linker or spacer (10)) and a second binding member (7) linked to a binding domain or binding unit (5) (directly or via a linker or spacer (11));

[0224] - the first fusion protein comprises a translaminar protein (2) fused directly or via a linker (10) to a first binding member (6);

[0225] - a second fusion protein comprising a binding domain (5) fused directly or via a linker (11) to a second binding member (7); and

[0226] - the first and second fusion proteins are arranged relative to each other and to the boundary layer (1) in such a way that when the binding domain (5) binds to the translaminar protein (2) (i.e. indirectly via binding to the complex (12) which in turn binds to the translaminar protein (2) via the binding site (9))), the first binding member (6) and the second binding member (7) are able to contact or be in close proximity to each other (or otherwise suitably associate) to produce a detectable signal (expressed as Figure 3 Indicated by the flashing symbol in the figure).

[0227] d) Figure 4 is a graph showing a dose response curve for NDP-α-MSH obtained using the MC4R screening assay of CA4437 described in Example 1;

[0228] e) Figures 5A to 5C is a graph showing dose response curves for the indicated compounds obtained using the GLP-1R screening assay for CA4437 described in Example 2;

[0229] f) Figure 6 is a graph showing the dose response curve for GLP-1(7-36)amide obtained using the GLP-1R screening assay of CA4435 described in Example 2;

[0230] g) Figure 7 A and 7B are graphs showing assay results obtained using the β-2-AR screening assay for CA4437 described in Example 3;

[0231] h) Figure 8 Figures A to 8E show the use of CA4435 ( Figure 8 A, 8B, 8D), CA4437( Figure 8 C) and CA4435-35GS-CA4437 fusion ( Figure 8 E) the assay results obtained by the β-2-AR screening assay;

[0232] i) Fig. 9 and 10 is a graph showing dose response curves for the indicated compounds obtained using the MOR screening assay described in Example 4;

[0233] j) Fig.11 is a graph showing assay results obtained using the M2R screening assay described in Example 5;

[0234] k) Fig.12 is a graph showing assay results obtained using the β-2AR screening assay described in Example 6;

[0235] l) Fig.13is a graph showing dose response curves for the indicated compounds obtained using the AT1R screening assay described in Example 7;

[0236] m) Fig.14 is a graph showing assay results obtained using the AT1R screening assay described in Example 7;

[0237] n) Fig.15 shows the results of the compound library screening performed in Example 8;

[0238] o) Fig.16 is a graph showing dose response curves for the indicated compounds obtained using the recombinant MC4R screening assay described in Example 9;

[0239] p) Fig.17 is a graph showing assay results obtained using the recombinant MC4R screening assay described in Example 9;

[0240] q) Figures 18 to 22 is a graph showing dose response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 10;

[0241] r) Fig.23A A and B are graphs showing assay results obtained using the two recombinant APJ receptor screening assays described in Example 11. Fig.23A show the results obtained using a recombinant apelin receptor with an ICL of the μ-opioid receptor (MOR), and Fig. 23B Results obtained with recombinant apelin receptors with ICLs from the β-2AR receptor are shown;

[0242] s) Figures 24 to 27 The results of the compound library screening performed in Example 12 are shown.

[0243] t) Fig.28 is a graph of the screening results obtained for the collection of 78 compound fragments in Example 14 when tested using two assays of the invention (both using the β-2AR-LgBiT fusion, but one assay using the CA2780-SmBiT fusion and one assay using the CA4435-35GS-CA4437-LgBiT fusion). Fig.28 In FIG, the x-axis represents the results obtained in the assay using the CA4435-35GS-CA4437-LgBiT fusion, the y-axis represents the results obtained in the assay using the CA2780-SmBiT fusion, and each point represents the result obtained in both assays for one of the 78 compounds tested.

[0244] u) Fig.29A and29B is a graph of the screening results obtained in Example 15 for a collection of compound fragments when tested using a radioligand assay and a corresponding assay of the present invention. Fig.29A In A and B, the x-axis represents the results obtained using the assay of the present invention, the y-axis represents the results obtained in this assay using the radioligand assay, and each point represents the results obtained for a compound when tested in both the radioligand assay and the assay of the present invention.

[0245] v) Fig. 30A and 30B is a graph showing the test results of the compounds mentioned in Example 16 and Table 3 in the GloSensor cAMP assay of β-2AR (100 μM and 200 μM, respectively);

[0246] w) Fig.31A and 31B is a graph showing a comparison of the results obtained in Example 17 when a cell-based assay of the present invention is compared to a comparable membrane-based assay of the present invention;

[0247] x) Fig.32A to C show dose response curves for apelin obtained using different VHHs (Example 18).

[0248] y) Fig.33 Shown are dose response curves for iperoxo production against the M2 receptor using the assay of the present invention in Example 19 in the presence and absence of LY2119620, an allosteric modulator of the M2 receptor.

[0249] z) Fig.34 is a graph obtained in Example 20, comparing the results from the OX2 assay of the present invention (using recombinant OX2 fusions) and the OX2 IP-One assay, wherein the x-axis represents the data obtained in the assay of the present invention, the y-axis represents the data obtained in the IP-One assay, and each point represents the results for a single compound.

[0250] aa) Fig.35A and 35B is a graph obtained when using the assay of the present invention to screen a large library of compounds against the recombinant OX2 receptor in Example 21. Fig.35A Results obtained when the compounds were tested at 30 µM are shown, and Fig.35B Results obtained when compounds were tested at 200 µM are shown, where the x-axis represents the ratio of the signal obtained for the test compound ("sample") to the signal given by the carrier solvent ("blank"), and each point represents the result obtained for a single compound.

[0251] Based on the drawings and further description herein, it will be clear to those skilled in the art that some elements of the device of the present invention (e.g., boundary layer, spanning protein, binding pair, any linker and first ligand) will be present in various aspects and embodiments of the present invention, as contemplated herein. Therefore, when a detailed description of any such element is given herein (including any preference for any such element), it should be understood that such description applies to all aspects and embodiments of the present invention in which such element is present or used, unless otherwise expressly stated herein.

[0252] In the methods and devices of the present invention, the boundary layer (1) may be any layer suitable for separating (in a suitable in vitro system or in a suitable in vivo system) a first environment [A] from a second environment [B].

[0253] For example, in a preferred aspect of the invention, wherein the method of the invention is performed in a suitable cell or cell line (as further described herein), the boundary layer (1) is a cell membrane or cell wall of the cell or cell line used in the method of the invention. In this regard, environment [A] is preferably the extracellular environment, and environment [B] is preferably the intracellular environment. Moreover, in this regard, the first ligand (3) is preferably present in the extracellular environment, and the second ligand (4) is preferably present in the intracellular environment. In addition, the first and second binding members (6) and (7) and the second fusion protein are preferably also present in the intracellular environment,

[0254] In another preferred aspect of the invention, wherein the method of the invention is carried out in a suitable vesicle or liposome (as further described herein), the boundary layer (1) is a membrane or wall of the vesicle or liposome. In this regard, environment [A] is preferably an environment outside the vesicle or liposome, and environment [B] is preferably an environment inside the vesicle or liposome. In addition, in this regard, the first ligand (3) is preferably present in an environment outside the vesicle or liposome, and the second ligand (4) is preferably present in an environment inside the vesicle or liposome. In addition, the first and second binding members (6) and (7) and the second fusion protein are preferably also present in an environment inside the vesicle or liposome.

[0255] However, it should be understood that, although the present invention in some preferred aspects is carried out using cells, liposomes or other suitable vesicles, the present invention in its broadest sense is not limited to the use of cells or vesicles, but may be carried out in any other suitable device in which a boundary layer (1) is used to appropriately separate the first environment [A] from the second environment [B]. For example, the boundary layer may also be a part or fragment of a cell wall or cell membrane present in a membrane extract (e.g. a membrane extract obtained from whole cells by techniques known per se, such as appropriate osmotic pressure and / or mechanical techniques known per se).

[0256] Thus, the boundary layer (1) may be any suitable layer, wall or membrane, in particular 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) may be a suitable lipid bilayer, such as a phospholipid bilayer. When the boundary layer (1) is a wall or membrane of a vesicle or liposome, it may be monolayer or multilayer. Furthermore, as further described herein, when the boundary layer (1) is a cell membrane or cell wall, it is preferably the wall or membrane of a cell or cell line, wherein the cell or cell line suitably expresses (as defined herein) a translaminar protein (2) and in particular suitably expresses a (first) fusion protein as described herein comprising a translaminar protein (2).

[0257] If non-restrictive Figure 1 , 2 As schematically shown in Figures 3 and 4, the boundary layer (1) comprises a translaminar protein (2) which spans the boundary layer (1) such that:

[0258] - the first binding site (8) of the first ligand (3) extends (as defined herein) into the first environment [A] (i.e. such that the first binding site (8) is accessible for binding by the first ligand (3) when the first ligand is present in the first environment [A]);

[0259] And also makes

[0260] - the second binding site (9) of the second ligand (4) extends (as defined herein) into the second environment [B] (i.e. such that the second binding site (9) is accessible for binding of the second ligand (4) when the second ligand is present in the second environment [B]).

[0261] In the present specification and claims, the term "spanning protein" is used to denote a protein used (e.g. screened) in the methods and apparatus of the present invention. In the methods and apparatus of the present invention, the spanning protein (2) is such (and / or provided and / or arranged in a manner relative to the boundary layer) that it spans the boundary layer (1) such that at least a portion of the amino acid sequence of the spanning protein (2) extends (as defined herein) from the boundary layer (1) into the first environment [A], and such that at least one other portion of the amino acid sequence of the spanning protein (2) extends (as defined herein) from the boundary layer (1) into the second environment [B]. In this case, when a portion of the amino acid sequence of the spanning protein (2) is said to "extend" from the boundary layer (1) into the environment (i.e. into the first environment [A] or the second environment [B]), this is generally understood to mean that said portion of the sequence is exposed to said environment and / or is accessible for binding by a ligand, compound or other chemical entity present in said environment. Thus, in the methods and devices of the present invention, at least a portion of the amino acid sequence of the translamin protein (e.g. an epitope or binding site) should be accessible for binding of a ligand, compound or other chemical entity present in the first environment (in particular binding of the first ligand (3)), and at least one other portion of the amino acid sequence of the translamin protein (e.g. another epitope or binding site) should be accessible for binding of a ligand, compound or other chemical entity present in the second environment (and in particular binding of the second ligand (4)). In this regard, it should also be noted that the wording "accessible for binding" should generally be understood to mean that a ligand, compound or other chemical entity present in the relevant environment can bind to a binding pocket or binding site on or in the translamin protein, even if the actual binding site or binding pocket is located deep (deeper) in the structure of the translamin protein (even such that the actual binding site or binding pocket is located in a portion of the translamin protein that does not itself physically extend beyond the boundary layer). For example, reference is made to the publication by Chevillard (cited herein), which shows that the binding site on a GPCR for a fragment used in the FBDD screening technique may be located deep in the GPCR structure (see, for example, p. 1120). Figure 2 ) rather than being located on the surface of the GPCR, but still accessible for fragment binding. Reference is also made to some other scientific references cited herein for teachings on GPCR structure, GPCR signaling mechanism, and GPCR ligand binding site,

[0262] Furthermore, in the present specification and claims, when any binding domain, binding unit, epitope, binding site, ligand, protein or other compound or chemical or other structural entity (e.g., protein complex) is referred to as being "present" in an environment (i.e., in a first environment [A] or a second environment [B]), this should generally be understood to mean that the binding domain, binding unit, epitope, binding site, ligand, protein or other compound or chemical or structural entity is exposed to the environment and / or is accessible for binding to another domain, ligand, protein or compound present in the environment. Thus, for example, a compound or ligand present in an environment may be "free floating" in the environment (i.e., not bound or anchored to any other protein or structure) or may be anchored to a boundary layer or fused to another protein (which other protein may be anchored to a boundary layer). Similarly, a binding domain or binding unit present in an environment may be part of a larger protein or structure (e.g., a fusion protein) that may be free floating in the environment or anchored to a boundary layer or another structure, as long as the binding domain or binding unit is accessible for binding to another domain, ligand, protein or compound present in the environment. Furthermore, an epitope or binding site present in the environment may be part of a larger protein or structure, which may again be free floating in the environment or anchored to a boundary layer or another structure, as long as the epitope or binding site is accessible for binding by another domain, ligand, protein or compound present in the environment.

[0263] The portion or portions of the translamin protein (2) extending into the first environment [A] may be any loop, epitope (linear or conformational), binding site or other portion of the amino acid sequence of the translamin protein, and similarly, the portion or portions of the translamin protein extending into the second environment [B] may also be any loop, epitope (linear or conformational), binding site or other portion of the amino acid sequence of the translamin protein (but different from the portion extending into the first environment).

[0264] In a preferred aspect of the invention, the translaminar protein (2) comprises at least two different / distinct ligand binding sites, wherein at least one first binding site extends (as defined herein) into a first environment [A] (in particular making it accessible for binding of a first ligand (3)), and wherein at least one second binding site extends (as defined herein) into a second environment [B] (in particular making it accessible for binding of a second ligand (4)).

[0265] Typically, the translaminar protein (2) will typically be attached to and / or anchored in the boundary layer (1), for example in a manner known per se for (trans)membrane proteins (which in their natural environment are anchored in the cell wall or cell membrane). As further described herein, this can be achieved, for example, by suitably expressing a nucleotide sequence or nucleic acid expressing a first fusion protein (as defined herein) in a suitable host cell, such that the translaminar protein (2) becomes suitably anchored in the wall or membrane of the cell. When liposomes or vesicles are used to carry out the method of the invention, this can be achieved by suitably forming the liposomes or vesicles in the presence of the first fusion protein, such that the translaminar protein (2) becomes suitably anchored in the wall or membrane of the liposomes or vesicles.

[0266] The translaminar protein (2) may comprise one or more domains (especially one or more transmembrane domains) and is typically and preferably a transmembrane protein, such as a (transmembrane) receptor.

[0267] When the translayer protein (2) is a transmembrane protein, it can be a bitopic membrane protein (i.e., a transmembrane protein that passes through the membrane once) or a polytopic membrane protein (i.e., a transmembrane protein that passes through the membrane twice or more). Therefore, the translayer protein (2) can be any known or newly discovered transmembrane protein (or its synthetic or recombinant analogue) with known or unknown biological functions and known or unknown ligands (for example, the translayer protein (2) can be a so-called "orphan" GPCR).

[0268] The translaminar protein (2) may be an α-helical protein or a β-barrel protein and, depending on the position of the protein's N-terminus and C-terminus relative to the boundary layer, may be a type I, II, III or IV transmembrane protein. Preferably, the translaminar protein is a protein having an amino terminus outside the cell and a carboxyl terminus inside the cell in its natural cellular environment, although the invention in its broadest sense is not limited thereto.

[0269] Furthermore, when the method of the invention is performed in a cell, the N-terminus and C-terminus of the protein are preferably arranged relative to the wall or membrane of the cell used as they are in the protein's natural cellular environment.

[0270] When the method of the present invention is carried out in liposomes or vesicles, the liposomes or vesicles can be the following: a mixture of liposomes / vesicles in which the arrangement of the protein is substantially the same as that of the protein in its natural environment relative to the cell wall or cell membrane (i.e., the N-terminus and the extracellular loop extend to the outside of the vesicle, and the C-terminus and the intracellular loop extend to the inside of the vesicle); and vesicles / liposomes in which the protein is arranged in the opposite manner. Generally, this does not affect the performance of the system or arrangement described herein.

[0271] As further described herein, typically and preferably, the translaminar protein (2) will be a protein that exists (i.e. can assume) two or more conformations (e.g. a basal state / conformation, an active state / conformation and / or an inactive state / conformation, and / or a ligand-bound or ligand-free conformation), and / or a protein that can undergo conformational changes (particularly functional conformational changes). In particular, the translaminar protein (2) may be a protein that can assume at least one functional conformation and at least one non-functional conformation (e.g. a basal conformation) and / or can undergo a conformational change from a non-functional conformation to a functional conformation; and more particularly, may be a protein that can assume 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 translaminar protein (2) may also be a protein that can assume at least one ligand-bound (particularly agonist-bound) conformation and at least one ligand-free conformation. More particularly, the translaminar protein (2) may be a protein that can assume at least one ligand-binding (particularly agonist-binding) conformation, which is an active conformation or a functional conformation.

[0272] As described herein, a particular class of functional conformations of (transmembrane) proteins (e.g. certain GPCRs) is referred to / defined as a "druggable conformation". Thus, in a specific aspect, a transmembrane protein (2) may be a protein that can exhibit at least one such druggable conformation (which is typically an active conformation, although the invention is not limited to use with druggable conformations that are active conformations) and at least one conformation that is a non-druggable conformation (typically an inactive conformation) and / or a transmembrane protein that can undergo a conformational change from a non-druggable conformation to a druggable conformation.

[0273] In particular, the translaminar protein (2) may be a protein that undergoes a conformational change after a ligand (particularly an agonist) binds to the protein. This conformational change after binding to the ligand may 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 preferably a change from a non-functional conformation to a functional conformation and / or from an inactive conformation to an active conformation. In a particular aspect, it is a change from a non-drugable conformation to a druggable conformation.

[0274] For example, when the translaminin (2) is a receptor such as a cell surface receptor (or a synthetic analog thereof), it may be a protein that undergoes a conformational change when a natural or synthetic (extracellular) ligand for the receptor binds to the receptor.

[0275] When the translaminar protein (2) is a GPCR, the conformational change may be, in a preferred but non-limiting aspect, a change from a conformation that is substantially unable to bind to a G-protein to a conformation that binds to a G-protein (or is capable of being bound by a G protein).

[0276] As described herein, a ligand that can cause a conformational change of a translamin (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 translamin. When the translamin (2) is a GPCR, the "agonist" is particularly capable of causing a conformational change from a conformation that is substantially unable to bind to a G protein to a conformation that binds to a G protein.

[0277] In a preferred aspect of the present invention, the translamin (2) is a protein that undergoes (or is capable of undergoing) a conformational change (as described herein) when a first ligand (3) binds thereto, and conversely, the first ligand (3) is such that, when it binds to the translamin (2), it can cause a conformational change in the translamin (2) (and / or the present invention is used to identify such a first ligand). Similarly, in a more preferred aspect, the conformational change is from an inactive or less active state to a functional or (higher) active state, and the first ligand (3) used is such that, when it binds to the translamin (2), it can cause a conformational change in the translamin from an inactive or less active state to a functional or (higher) active state. In addition, when the translamin (2) is a GPCR, in a preferred but non-limiting aspect, the conformational change after ligand binding can be a change from a conformation that is substantially incapable of binding to a G protein to a conformation that binds to a G protein.

[0278] As further described herein, the translaminar protein may be a protein that can form a complex with the first and second ligands. In particular, the translaminar protein may be a protein that can form a complex with an intracellular ligand and an extracellular ligand in its natural 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 (which is the most common natural intracellular ligand of a GPCR), and that this 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 is preferably a complex (formation) of the translaminar protein, the first ligand and the second ligand that stabilizes the translaminar protein. For example, for this purpose, and as further described herein, when the translaminar protein (2) is a GPCR, the second ligand may be a G-protein associated with the GPCR in its natural environment (i.e., signal transduction by the GPCR), another naturally occurring G protein that can bind to the GPCR and stabilize the formation of the above-mentioned complex, or a synthetic or semi-synthetic analog or derivative of the GPCR that can bind to the GPCR and stabilize the formation of the above-mentioned complex. As also mentioned herein, the second ligand may be a ConfoBody, ie an immunoglobulin single variable domain (eg a VHH or a Nanobody) that has been designed / generated to stabilize the formation of a complex of the ConfoBody, the spanning protein and the first ligand.

[0279] In a preferred but non-limiting aspect of the invention, the translaminar protein (2) will be a "seven transmembrane protein", in particular a 7TM that is a receptor (e.g., a cell surface receptor). In a particularly preferred aspect, the translaminar protein (2) may be a 7TM that signals through 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 to include all transmembrane proteins having a 7-transmembrane domain, regardless of their intracellular signaling cascade or signal transduction mechanism, although it should be understood that throughout the specification and claims, 7 TMs that signal through G proteins are a preferred aspect of the invention].

[0280] The translaminar protein (2) may be a naturally occurring protein or receptor or a synthetic or semisynthetic analog of a naturally occurring protein or receptor (again, obtained by protein chemistry or recombinant DNA techniques as generally described herein). Such a synthetic analog may be, for example, an analog of a naturally occurring transmembrane protein in which 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) have been inserted, deleted and / or replaced by other amino acid residues or stretches of amino acid residues (e.g., stretches or loops substantially corresponding to other (preferably structurally related) membrane proteins) compared to the sequence of the naturally occurring protein (in other words, comprising one or more "amino acid differences" as defined herein compared to the native sequence). Typically, the native sequence of the naturally occurring protein used will be obtained from the species to be treated with the compounds of the invention or from an animal (preferably a mammal) to be used for animal model purposes for testing the compounds of the invention.

[0281] It will be clear to those skilled in the art that such synthetic analogs can be obtained using standard techniques of protein chemistry known per se and / or standard techniques of protein chemistry and / or recombinant DNA techniques. For example, when the present invention is carried out in a cell as described herein, a synthetic analog can be obtained by suitably expressing a DNA sequence (or other suitable nucleotide sequence) encoding the synthetic analog in the cell.

[0282] In addition, as is known in the art, 7TM and other transmembrane proteins typically comprise one or more intracellular loops and one or more extracellular loops. Similarly, the translaminar proteins used in the device of the present invention may comprise one or more loops extending (as defined herein) into the first environment and one or more loops extending (as defined herein) into the second environment. For example, when the method of the present invention is performed in a cell, the translaminar proteins used in the device of the present invention may comprise one or more loops extending into the intracellular environment and one or more loops extending into the extracellular environment. Similarly, when the method of the present invention is performed in a vesicle or liposome, the translaminar proteins used in the device of the present invention may comprise one or more loops extending into the internal environment of the liposome or vesicle and one or more loops extending into the external environment of the liposome or vesicle. In each case, the loops extending into the first environment are most preferably such (and arranged such) that they can form a functional ligand binding site (and in particular a functional binding site for the first ligand), and / or they can form a functional ligand binding site (and in particular a functional binding site for the first ligand) when the spanning protein assumes a suitable conformation; and the loops extending into the second environment are most preferably such (and arranged such) that they can form a functional ligand binding site (and in particular a functional binding site for the second ligand), and / or they can form a functional ligand binding site (and in particular a functional binding site for the second ligand) when the spanning protein assumes a suitable conformation (e.g., after the first ligand binds to the spanning protein).

[0283] In one specific but non-limiting aspect, loops of a translamin protein that extend out into one environment will correspond substantially to extracellular loops of the translamin protein, and loops of a translamin protein that extend out into the other environment will correspond substantially to intracellular loops (again, preferably in each case such that the extracellular loop will form a functional ligand binding site and such that the intracellular loop will form another functional ligand binding site). Preferably, loops of a translamin protein that extend out into a first environment [A] will correspond substantially to extracellular loops of the translamin protein, and loops of a translamin protein that extend out into a second environment [B] will correspond substantially to intracellular loops of the translamin protein, particularly when the second environment [B] is the environment inside a cell or liposome (again, preferably such that the extracellular loop will form a functional ligand binding site extending out into the first environment, and such that the intracellular loop will form a different functional ligand binding site extending into the second environment).

[0284] For example, when the translaminar protein is a transmembrane protein (e.g., 7TM), the translaminar protein may comprise one or more extracellular loops of a transmembrane protein (particularly one or more extracellular loops of 7TM) and one or more intracellular loops of a transmembrane protein (particularly one or more intracellular loops of 7TM), more particularly such that the extracellular loops form or can form a functional ligand binding site and such that the intracellular loops form or can form a different functional ligand binding site. Similarly, the ligand binding site formed by the extracellular loop will preferably extend out (as defined herein) to one environment, and the ligand binding site formed by the intracellular loop will preferably extend out (as defined herein) to other environments. In particular, when the method of the present invention is performed in a cell or liposome, the extracellular loop will extend out to the environment outside the cell or liposome, and the intracellular loop will extend out to the environment inside the cell or liposome. Furthermore, preferably, the intracellular loops are such (and are arranged such) that they form or can form a functional ligand binding site for a second ligand (or in other words: in the present invention, the ligand binding site of the second ligand is preferably composed of and / or comprises one or more intracellular loops of a transmembrane protein. It is also possible that the ligand binding site of the first ligand is composed of and / or comprises one or more extracellular loops, but as further described herein, the actual binding / docking site of the first ligand may also be located deeper in the transmembrane protein structure).

[0285] For example, when the translaminin is 7TM, the translaminin may comprise three intracellular loops (i.e., three intracellular loops from 7TM) and three extracellular loops (i.e., three extracellular loops from 7TM), wherein the three intracellular loops form or can form a functional ligand binding site, wherein the three extracellular loops form or can form different functional ligand binding sites. Again, preferably, the functional ligand binding site formed by the three intracellular loops extends out into one environment (and preferably the second environment [B]), and the functional ligand binding site formed by the three extracellular loops extends out into the other environment (and preferably the second environment [A]). In addition, the three intracellular loops preferably form a functional binding site for a second ligand (and the three extracellular loops may form a functional binding site for a first ligand or the binding site may be located deeper in the 7TM structure). Most preferably, the three intracellular loops will form a binding site for the second ligand that extends out into the second environment [B] (i.e., the environment inside the cell or liposome, respectively, when the method of the invention is performed in a cell or liposome), and the three extracellular loops will extend out into the first environment [A] (and may form a functional binding site for the first ligand, or the binding site may be located deeper in the 7TM structure).

[0286] In one aspect of the invention, the intracellular loops and the extracellular loops of the transmembrane protein are derived from or are substantially derived from the same transmembrane protein (i.e., are identical or substantially identical to those present in the native transmembrane protein). In this aspect of the invention, the transmembrane protein may have an amino acid sequence that is identical or substantially identical to a native transmembrane protein (which is to be used as a target in the screening or assay methods of the invention).

[0287] In another aspect of the invention, the intracellular loop and the extracellular loop of the translamin can be derived from different transmembrane proteins. In particular, in this aspect of the invention, the intracellular loop and the extracellular loop can be derived from different but related transmembrane proteins, for example from two different but related 7TMs, such as two GPCRs. In particular, in this aspect of the invention, the intracellular loop can be derived from a first 7TM or GPCR, and the extracellular loop can be derived from a second 7TM or GPCR that is different from the first 7TM or GPCR. The transmembrane domain of such a chimeric protein can be derived from a first or second 7TM or GPCR, and preferably both are substantially derived from the same GPCR, more preferably from the same GPCR as the extracellular loop (but may contain some amino acid residues from the GPCR from which the intracellular loop is derived, depending on the position selected for the recombinant deletion of the native intracellular loop and the insertion of the replacement intracellular loop).

[0288] In this aspect of the invention, the resulting chimeric translaminar protein should most preferably still be suitable for use in the methods and apparatus of the invention. Likewise, again, in the case of 7TM, the translaminar protein will comprise three intracellular loops and three extracellular loops, the three intracellular loops forming a functional ligand binding site for a second ligand (and the second ligand is then selected such that it can bind to the ligand binding site formed by the intracellular loops (9)). Likewise, the binding site formed by the three intracellular loops will preferably extend out into the second environment [B] (i.e., the environment inside a cell or liposome, respectively, when the methods of the invention are performed in a cell or liposome, respectively), and the three extracellular loops will preferably extend out into the first environment [A] (and may form a functional binding site for the first ligand, or the binding site may be located deeper in the 7TM structure).

[0289] Thus, in another aspect, the invention relates to a device as further described herein, wherein the translaminar protein is a 7TM comprising 7 transmembrane domains, 3 intracellular loops and 3 extracellular loops (which are connected to each other and in an order known as 7TM itself), i.e. [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-C-terminal sequence), wherein the intracellular loops are derived from a first 7TM and the extracellular loops are derived from a second 7TM different from the first 7TM, wherein the intracellular loops form a functional ligand binding site. Preferably, the TM domains from the translaminar protein are substantially derived from the same 7TM as the extracellular loops.

[0290] Furthermore, the intracellular loop and the 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. The ligand binding site again preferably extends out into the second environment [B].

[0291] In one specific aspect, the chimeric translaminar protein comprises an intracellular loop derived from a beta-2-adrenergic receptor. In another specific aspect, the chimeric translaminar protein comprises an intracellular loop derived from a μ-opioid receptor. For some non-limiting examples of such chimeric receptors, reference is also made to the assignee's publication entitled "Chimeric proteins and methods to screen for compounds and ligands that bind to GPCRs."

[0292] This application is a co-pending PCT application entitled "Binding to GPCRs)" which has the same international filing date and cites the same priority application as the present application.

[0293] The present invention particularly relates to devices comprising such chimeric 7TM and a second ligand capable of binding to the ligand binding site formed by the intracellular loop.

[0294] For the remainder, it is a prerequisite that the second ligand is appropriately selected so that it is capable of binding to the ligand binding site (9) on the chimeric translaminar protein, thereby providing an operable device of the invention (and it is a prerequisite that the chimeric translaminar protein itself is operable in the device of the invention), wherein such devices of the invention using the chimeric translaminar protein can be substantially as further described herein.

[0295] Furthermore, such chimeric translayer proteins, nucleotide sequences and nucleic acids encoding the same, and cells, cell lines or host organisms comprising such nucleotide sequences or nucleic acids and / or which can express such chimeric translayer proteins form further aspects of the present invention, as do further uses of such chimeric translayer proteins, nucleotide sequences, nucleic acids, cells, cell lines and host organisms.

[0296] Another aspect of the invention is a composition or kit of parts comprising at least the chimeric translayer protein and a ligand that can bind to an intracellular loop present in the GPCR. The ligand is preferably a protein, more preferably a protein comprising or essentially consisting of an immunoglobulin single variable domain (e.g., a VHH domain), and in particular may be a ConfoBody (as described herein).

[0297] As described above, the chimeric translaminar protein is preferably a 7TM / GPCR. In addition, in a specific aspect, the chimeric translaminar protein comprises an intracellular loop derived from a beta-2-adrenergic receptor. In another specific aspect, the chimeric translaminar protein comprises an intracellular loop derived from a μ-opioid receptor.

[0298] As further described herein, and as Figures 1 to 3 As shown schematically, in the device of the present invention, the cross-layer protein (2) is usually and preferably fused or connected to the first member (6) of the binding pair (6 / 7) directly or via a suitable spacer or linker (10) to form a first fusion protein. In addition, the second binding member (7) of the binding pair (6 / 7) is usually and preferably part of a second fusion protein different from the first fusion protein, and the second fusion protein is also as further described herein. The first fusion protein, the second fusion protein (in its various forms as described herein), the nucleotide sequence and / or nucleic acid encoding the first or second fusion protein, and the cells, cell lines or other host cells or host organisms that express (and particularly suitably express, as described herein) or can (suitably) express the first and / or second fusion protein (and preferably both), and the various uses of the above substances as further described herein, form further aspects of the present invention.

[0299] The binding pair (6 / 7) used in the device of the present invention generally comprises at least two separate binding members (6) and (7), which are also referred to herein as "first binding member" and "second binding member", respectively. The binding pair (6 / 7) and each of its members (6) and (7) should be such that when the members (6) and (7) are in contact with each other or in close proximity, the binding pair (6 / 7) is able to produce a detectable signal. This detectable signal can be, for example, a luminescent signal, a fluorescent signal or a chemiluminescent signal, based on a reporter gene, or based on a DNA connection. Some specific but non-limiting examples of technologies (including binding pairs and their associated detectable signals) are technologies based on protein complementation, such as the NanoBit™ system, the NanoLuc™ system, the hGLuc system (Remy and Michnick, Nature Methods, 2006, 977), BiFC (bimolecular fluorescence complementation) and DHFR-PCA (dihydrofolate reductase protein fragment complementation assay); technologies based on direct interactions, such as BRET (bioluminescence resonance energy transfer), FRET (fluorescence / Foster resonance energy transfer) and BioID (approach-dependent biotin recognition); systems based on reporter genes (e.g., KISS / kinase substrate sensors) or proximity ligation assays (Weilbrecht et al., Expert Review of Proteomics, 7:3, 401-409). Technologies based on protein complementation and luminescent, fluorescent or chemiluminescent signals (e.g., NanoLuc™ or NanoBit™) are generally preferred.

[0300] In a particularly preferred aspect, when the method of the present invention is performed in a suitable cell, the first member (6) and the second member (7) of the binding pair (6 / 7) are preferably both polypeptides, proteins, amino acid sequences or other chemical entities obtainable by appropriately expressing (preferably in a cell used in the method of the present invention) a nucleic acid or nucleotide sequence encoding the same.

[0301] The first and second binding members may also be part of a suitable reporter gene assay, may be a combination of an enzyme and a substrate, or any other domain or unit pair that can generate a detectable signal when they are in contact or close proximity to each other, such as binding pairs commonly used in experimental studies of protein-protein interactions. As described above, in order to reduce the level of baseline / background signal, it is preferred that the two members of the binding pair themselves do not have substantial binding affinity for each other.

[0302] Some preferred but non-limiting examples of suitable binding pairs are pGFP and the NanoBiT® system from Promega. The latter is particularly preferred because the large BiT and small BiT that make up the NanoBiT® system themselves have low affinity for each other.

[0303] The first binding member (6) may be fused to the layer-spanning protein (2) in any suitable manner, provided that the resulting first fusion protein is such that, when the second fusion protein formed by the second ligand (4) and the second member (7) binds to the layer-spanning protein (2) via the second binding site (9), it 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). Furthermore, preferably, the first binding member (6) is fused or linked to the layer-spanning protein (2) in a manner that does not substantially affect the conformation and / or conformational changes that the layer-spanning protein (2) can undergo under the conditions used to carry out the method of the invention.

[0304] Thus, generally, although the present invention does not exclude the direct fusion or connection of the first binding member (6) to the translaminar protein (2), it is generally preferred that the first binding member (6) is fused or connected to the translaminar protein (2) via a suitable linker (10). It is generally preferred to use a flexible linker, for example having a total of 5 to 50 amino acids, preferably 10 to 30 amino acids, for example about 15 to 20 amino acids. Suitable linkers are clear to the skilled person and include GlySer linkers (e.g. 15GS linkers).

[0305] In the present invention, the first and second binding members of the binding pair (6 / 7) will be present in the same environment (as defined herein) relative to the boundary layer (1), such that they can contact or be in close proximity to each other (in a manner further described herein) and can generate a detectable signal when doing so. In particular, if Figure 1 , 2 3, the first and second binding members of the binding pair (6 / 7) will be present in the same environment (as defined herein) as the second binding site (9) on the translaminar protein (2) (again, relative to the boundary layer (1)) so as to allow for the second fusion protein to bind directly (e.g. Figure 1 as shown) or indirectly (as Figure 2 and 3 When the first binding member (as shown) binds to the binding site, the first and second binding members of the binding pair (6 / 7) come into contact. To this end, the first binding member (6) will typically be attached directly or via a linker (10) to an amino acid residue / position in / on the translaminar protein (2) that is exposed to the same environment as the second binding site (9). As further described herein, the environment (at Figures 1 to 3 The environment (denoted as environment [B]) may be, for example, the intracellular environment (when the method of the invention is performed in a cell) or the environment within a vesicle or liposome.

[0306] In a preferred aspect of the invention, the first binding member (6) will be fused, either directly or via a linker (10), to one end of the primary amino acid sequence of the translaminar protein (2). This may be the N-terminus or the C-terminus of the translaminar protein (2), again as long as in the final device of the invention the first binding member (6) is located on the same side of the boundary layer (1) as the second binding site (9). Thus, in aspects of the invention carried out in cells as further described herein, and when 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 (typically the C-terminus in the case of 7TM).

[0307] The first fusion protein can be provided and produced using suitable protein chemistry techniques and / or recombinant DNA techniques known per se. Based on the further disclosure of this article and the standard manuals and other scientific references mentioned herein, these techniques will be clear to the technician. When the method of the present invention is carried out in a cell (as further described herein), the first fusion protein is preferably provided by suitably expressing a nucleotide sequence and / or nucleic acid encoding the first fusion protein in the cell. This can be carried out using the appropriate techniques of recombinant DNA techniques known per se again, and the cell that suitably expresses or (can suitably express) the first fusion protein forms another aspect of the present invention.

[0308] As further described herein, in the device of the present invention, the second member (7) of the binding pair (6 / 7) will typically and preferably also form part of a fusion protein, which will typically comprise the 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 ligand, protein, binding domain or binding unit is such that it can bind directly (as defined herein) or indirectly (as defined herein) to the translaminar protein (2). To this end, as further described herein, the ligand, protein, binding domain or binding unit may for example be a second ligand (resulting in Figure 1 A device of the invention of the type schematically shown in FIG. 1 , wherein (4) is a second ligand), is a binding domain or binding unit that can bind to a second ligand (resulting in Figure 2 A device of the invention of the type schematically shown in , wherein (4) is a second ligand and (5) is a binding domain or binding unit that binds to the second ligand), or a binding domain or binding unit that can bind to a protein complex that can bind to a translaminar protein (such as Figure 3 As shown schematically, (4) is the second ligand, (12) is the protein complex comprising the second ligand, and (5) is the binding domain or binding unit that binds to the protein complex).

[0309] In the second fusion protein, the second binding member (7) is most preferably linked to the other ligand, protein, binding domain or binding unit in a suitable manner that allows the second binding member (7) to contact (or otherwise be in suitable close proximity to) the first member (6) of the binding pair (6 / 7) when the second fusion protein binds directly or indirectly to the second binding site (9) on the translaminar protein (2). To this end, 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 a suitable linker (11), which is preferably a flexible linker, for example having a total of 5 to 50 amino acids, preferably 10 to 30 amino acids, and generally preferably about 15 to 20 amino acids. Suitable linkers will be clear to the skilled person and include GlySer linkers (e.g. 15GS linkers).

[0310] As described herein, the second ligand can be any ligand, protein, binding domain or binding unit that is capable of binding to the translaminar protein, i.e., via the binding site (9) (when the second ligand is part of a second fusion protein, it should most preferably also be appropriately comprised in the second fusion protein).

[0311] Typically, in the present invention (and regardless of whether the binding site is directly or indirectly bound by the second fusion protein used in the device of the present invention), the binding site (9) may be a conformational epitope on the spanning protein (2). More particularly, the binding site (9) may be a conformational epitope on the spanning protein (2) that changes its "shape" (i.e., the spatial arrangement of domains, loops and / or amino acid residues forming the epitope) when the spanning 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 the first ligand binds to the spanning protein.

[0312] Preferably, the binding site (9) and the second ligand are such that the affinity of the interaction between the binding site (9) and the second ligand (4) changes when the binding site (9) changes its shape because the translamin (2) undergoes a conformational shape. In particular, the binding site (9) and the second ligand may be such that the affinity of the interaction between the binding site (9) and the second ligand (4) increases when the translamin (2) undergoes a conformational change from an inactive or less active state to an active, more active, functional and / or druggable state and / or undergoes a conformational change that occurs when the first ligand (3) (particularly a first ligand (3) that is an agonist with respect to the translamin (2)) binds to the translamin (2).

[0313] In particular, the second ligand (4) and its interaction with the binding site (9) can enable the second ligand (4) to bind to the binding site (9) with a higher affinity when the translaminar protein (2) is in an active, highly active and / or functional state and / or enable the second ligand (4) to bind to the binding site (9) with a higher affinity when the first ligand (3) (in particular, the first ligand (3) acts as an agonist with respect to the translaminar protein (2)) binds to the translaminar protein (2). For example, the second ligand (4) and its interaction with the binding site (9) may be such that the affinity of the second ligand (4) for the translamin (2) increases by 10-fold, for example 100-fold or more when the translamin (2) undergoes such a conformational change, for example from an affinity in the micromolar range (i.e., greater than 1000 nM) when the translamin (2) is in an inactive, less active or ligand-free conformation, to an affinity in the nanomolar range (i.e., less than 1000 nM, for example, less than 100 nM) when the translamin (2) is in a functional, active or more active and / or ligand-bound conformation. For example, in the case of a GPCR, it is known that when a ligand (particularly an agonist) binds to the extracellular binding site of the GPCR, the affinity of the interaction between the G protein and the G protein binding site increases. Furthermore, WO2012 / 007593, WO2012 / 007594, WO2012 / 75643, WO 2014 / 118297, WO2014 / 122183 and WO2014 / 118297 describe VHH domains (ConfoBody) having a higher affinity for a GPCR when the GPCR is in a functional, active or more active and / or ligand-bound conformation than when the ConfoBody is in an inactive, less active or ligand-free conformation (e.g. in the nanomolar range for a functional, active or ligand-bound conformation and in the micromolar range for an inactive or ligand-free conformation).

[0314] The second ligand itself may also undergo a conformational change when it binds to the translaminar protein (2). In embodiments where the second fusion protein indirectly binds to the translaminar protein (2), this may also mean that the binding domain or binding unit (5) in the second fusion protein that binds to the second ligand (4) may be such that it has a higher affinity for the conformation adopted by the second ligand (4) when it is bound to the translaminar protein (2) than for the conformation adopted by the second ligand (4) when it is not bound to the translaminar protein (2). For example, it is known that G proteins undergo conformational changes when they bind to GPCRs, and it may be that the VHH domain present in the second fusion protein has a higher affinity for the GPCR-bound conformation of the G protein than for the GPCR-unbound conformation.

[0315] In a preferred aspect, the binding site (9) is a binding site on the translamin (2) that, when the translamin is in its natural environment, serves as a binding site for a natural ligand of the translamin. More specifically, the binding site (9) can be a binding site on the translamin (2) that, when the translamin is in its natural environment, serves as an intracellular binding site for a natural intracellular ligand of the translamin. For example, when the translamin (2) is a receptor, the binding site (9) can be a binding site on the translamin (2) that, when the translamin is in its natural environment, serves as an intracellular binding site for one or more intracellular ligands of the translamin (2) involved in signal transduction.

[0316] In a specific aspect, when the translaminar protein (2) is a GPCR, the binding site (9) can be a binding site of a G-protein (and / or a G-protein complex). As further described herein, in this case, the second ligand can be a natural, synthetic or recombinant protein or can be another ligand that can bind to a G protein binding site on a GPCR.

[0317] The second ligand (4) is typically a protein or a ligand of a protein. In aspects of the invention carried out in suitable cells or cell lines, the second ligand (4) may be a protein native to the cell or cell line used, or may be a suitable (recombinant) protein expressed in the cell or cell line used. For example, when the second ligand (4) is not part of the second fusion protein, it may be a ligand of the translamin (2) naturally present in the cell or cell line (e.g., when the translamin (2)) is a GPCR, the second ligand (4) may be a G protein naturally expressed by the cell or cell line used). Alternatively, the second ligand may be a protein recombinantly expressed in the cell or cell line used, for example when the cell or cell line does not naturally express a suitable ligand for the translamin (2) or when it is desired to use a ligand different from the ligand naturally expressed by the cell or cell line (e.g., when it is desired to use an analog, derivative or ortholog of a naturally expressed ligand, in which case the natural expression of the naturally expressed ligand may also be temporarily or constitutively inhibited or knocked out in the cell or cell line used). When the second ligand (4) forms part of a second fusion protein, the second ligand is typically recombinantly expressed as part of the second fusion protein.

[0318] As further described herein, the second ligand (4) can be part of the second fusion protein or it can be separate from the second fusion protein. In either case (i.e., whether or not the second ligand is part of the second fusion protein), the second ligand is preferably capable of binding to a conformational epitope on the translamin (or such that it is part of a protein complex that directly binds to the translamin or is capable of directly binding to the translamin). More preferably, the second ligand (and / or the protein complex comprising the second ligand) is preferably such that it specifically binds to one or more functional, active and / or druggable conformations of the translamin, such that it induces the formation and / or stabilization of one or more functional, active and / or druggable conformations of the translamin (and / or shifts the conformational equilibrium of the translamin to one or more such conformations); and / or such that it induces the formation and / or stabilization of a complex of the translamin, the first ligand and the second ligand.

[0319] When the second ligand is part of the second fusion protein, it can be any ligand, binding domain, binding unit, peptide, protein or other chemical entity that can directly bind to the cross-layer protein and can be appropriately contained in the second fusion protein. Preferably, as further described herein, when it is part of the second fusion protein, the second ligand will be a suitable binding domain or binding unit, and in particular an immunoglobulin single variable domain.

[0320] When the second ligand is separate from the second fusion protein, it may be any ligand or protein that can bind directly to the translaminin and / or can form part of a protein complex that can bind to the translaminin. For example, such a second ligand may be a naturally occurring ligand of the translaminin, a semisynthetic or synthetic analog or derivative of such a naturally occurring ligand, or an ortholog of such a naturally occurring ligand, as further described herein. Furthermore, when the second ligand is not part of the second fusion protein, the second fusion protein will comprise a binding domain or binding unit that can bind indirectly (as defined herein) to the translaminin, i.e., a binding domain or binding unit that can bind to the second ligand and / or to a protein complex comprising the second ligand. Likewise, such a binding domain or binding unit may in particular be an immunoglobulin single variable domain, such as an ISVD derived from a camelid, as further described herein. As also mentioned herein, such a binding domain or binding unit may comprise two or more (e.g. two or three) ISVDs (suitably fused or linked, optionally via a suitable linker or spacer), which ISVDs may be identical or different, and (when identical) they will generally bind to the same binding site or epitope on the second ligand, or (when different) they may bind to the same or different epitopes or binding sites on the second ligand (and, when the second ligand is a protein complex, such as a G protein complex, may bind to the same or different subunits of the protein complex).

[0321] It will also be clear to the skilled person that when the second ligand does not form part of the second fusion protein, the binding domain or binding unit present in the second fusion protein and that can bind to the second ligand should not substantially interfere with the binding of the second ligand to the translamin. For example, it preferably binds to a binding site or epitope on the second ligand that is different from the binding site on the second protein that binds to the translamin (and preferably also sufficiently removed from the binding site on the second protein that binds to the translamin to avoid any major steric hindrance).

[0322] When the second ligand (4) is a naturally occurring ligand of the translaminar protein (2), it may be, for example, a ligand that participates in a signaling pathway or signal transduction in which the translaminar protein (2) is involved. For example, when the second ligand (4) is a receptor, the second ligand (4) may be a naturally occurring ligand of the receptor, in particular a naturally occurring intracellular ligand of the receptor, such as an intracellular ligand that binds to an intracellular binding site on the receptor when the extracellular ligand binds to the extracellular binding site on the receptor, or when the receptor has a certain degree of constitutive activity, an intracellular ligand that binds to the intracellular binding site of the receptor as part of a pathway that provides said constitutive activity. Suitable examples of such natural ligands will be clear to the skilled person based on the disclosure herein, and will generally depend on the translaminar protein (2) used. For example, when the translaminar protein (2) is a 7TM or a GPCR, the second ligand (4) can be a G protein (preferred), including but not limited to a naturally occurring G protein (e.g., a G protein naturally present in the cell or cell line used) or a synthetic or semisynthetic analog or derivative (including a chimeric G protein) of a naturally occurring G-protein, all as further described herein.

[0323] As further described herein, particularly in aspects and embodiments of the invention performed 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 proteins. For example, when the translaminar 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 the G protein and optionally one or more other proteins. A preferred but non-limiting example of such a complex is a G-protein trimer comprising a G-α subunit, a G-β subunit and a G-γ subunit. The complex may also comprise the translaminar protein itself (e.g. a GPCR and a G-protein or a GPCR and a G-protein trimer). It will be clear to the skilled person that when the second ligand forms part of such a complex, it is generally preferred that the second ligand does not form part of the second fusion protein. Instead, the second fusion protein will comprise a binding domain or binding unit that can bind to the second ligand or the complex. For example, where the second ligand forms part of a G protein complex, the binding domain or binding unit in the second fusion protein can be a VHH domain that binds to the complex (e.g., to a subunit within the complex or to an interface between two or more of the subunits). As described herein, an example of such a VHH domain is the VHH referred to as "CA4435" (SEQ ID NO: 1 in WO2012 / 75643 and SEQ ID NO: 1 herein).

[0324] The second ligand (4) may also be a synthetic or semi-synthetic analog or derivative of such a naturally occurring ligand, for example an analog or derivative having a primary amino acid sequence that differs from the primary amino acid sequence of the corresponding natural ligand by deletion, insertion and / or substitution of a limited number of amino acid residues or amino acid residue fragments. Such analogs or derivatives may again be provided using suitable techniques of recombinant DNA technology known per se, which again in one aspect may involve expressing a nucleotide sequence or nucleic acid encoding the analog or derivative (preferably as part of the entire second fusion protein, also including the second binding member (7) and any linker (11), if present) in a suitable host or host cell. For example, when the translaminar protein (2) is 7TM or GPCR, the second ligand (4) may be an analog or derivative of the G protein (preferably), which may also have one or more amino acid differences (as defined herein) from the natural sequence, provided that the analog or derivative still has sufficient affinity for the translaminar protein (2) to allow the analog or derivative to be suitably used in the method of the invention.

[0325] For example, in a specific embodiment, an analog or derivative of such 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 amino acid residue stretches) are replaced by (substantially) one or more amino acid residues (and / or one or more amino acid residue stretches) occurring at the same or corresponding position in another naturally occurring G protein.

[0326] When the G protein is a heterotrimeric protein, such replacement of one or more amino acid residues (and / or one or more amino acid residue stretches) may be present or performed in any one, two or all three subunits of the G-α, G-β and / or G-γ subunits, and may particularly be in the G-α subunit.

[0327] For example, it is well known that there are multiple genes in humans, each gene encoding a different G-α subunit, and G-α has multiple isoforms that can be divided into different functional subfamilies (e.g., see Flock et al., Nature, 2015, 524(7564), 173-179; and Nehme et al., PLoS One, 2017, 12(4)), and the analogs or derivatives of the naturally occurring G-α subunit used in the present invention can be obtained by replacing one or more amino acid residues (and / or one or more amino acid residue stretches) in (the amino acid sequence of) a naturally occurring G-α subunit with one or more amino acids (and / or one or more amino acid residue stretches) present at (substantially) the same or corresponding positions in another naturally occurring α subunit (which may belong to the same subfamily or a different subfamily as the original subunit). Some specific but non-limiting examples are naturally occurring Gαs subunits 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 occurring at (substantially) the same or corresponding positions of the Gαi subunit, or naturally occurring Gαs subunits 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 occurring at (substantially) the same or corresponding positions of the Gαq subunit. Typically, but not exclusively, such substituted / replaced amino acids or stretches of amino acids will be present at or near the C-terminus of the α-subunit.

[0328] Some specific but non-limiting examples of such "chimeric" G-proteins and their design can also be found in the scientific literature. Reference is made, for example, again to the above-mentioned publications by Flock et al. and Nehme et al.

[0329] The second ligand (4) may also be another type of ligand that has been generated to bind to the binding site (9) on the layer-spanning protein (2), and preferably in the manner further described herein.

[0330] In a particularly preferred aspect, when the method of the present invention is carried out in a suitable cell, the second ligand (4) is preferably a polypeptide, protein, amino acid sequence or other chemical entity that can be obtained by appropriately expressing (preferably in the cell used in the method of the present invention) a nucleic acid or nucleotide sequence encoding it.

[0331] As described herein, and regardless of whether it is a naturally occurring ligand of the translamin (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 as described above), or another ligand (e.g. a ConfoBody as further described herein), the second ligand (4) generally enables binding, in particular specific binding, to an epitope on the translamin (2), in particular the binding site (9). In particular, the second ligand (4) may enable it to bind, in particular specifically bind, to an epitope (which would be an intracellular epitope if the translamin (2) were in its natural cellular environment).

[0332] As described herein, the epitope (i.e., binding site (9) may be a linear epitope or a conformational epitope, and is preferably a conformational epitope (as described herein). For example, when the translaminar protein (2) is a GPCR, the epitope may comprise one or more amino acid residues and / or amino acid residue stretches on at least one intracellular loop of the GPCR, and in particular may be a conformational epitope formed by or comprising one or more amino acid residues and / or amino acid residue stretches on at least two different intracellular loops of the GPCR.

[0333] The epitope of the second ligand (4) may in particular be a (partial) epitope on the translamin (2) which is involved in the signal transduction mediated by the translamin (2). For example, the second ligand may bind to an epitope on the translamin (2) which is located within the binding site of the downstream signal transduction protein. For example, when the translamin (2) is a GPCR, the second ligand (4) may be a binding domain or binding unit which is capable of specifically binding to a conformational epitope contained in, located in or overlapping the G protein binding site of the GPCR.

[0334] When the translaminar protein (2) used is a protein that can present / exist in two or more conformations (e.g. a basal state / conformation, an active state / conformation and / or an inactive state / conformation) and / or can undergo conformational changes (in particular functional conformational changes), the second ligand (4) is preferably such that it is able to bind, in particular specifically bind, to a functional conformational state of the translaminar protein (2).

[0335] In particularly preferred aspects, the second ligand (4) is such that, after binding to the translamin (2), it is capable of stabilizing and / or inducing a functional and / or active conformational state of the translamin (2) (and / or is capable of shifting the conformational equilibrium of the translamin (2) from an inactive or less active state to a more active state), is capable of causing the translamin to enter a more druggable conformation (and / or is capable of shifting the conformational equilibrium of the translamin (2) from a less druggable conformation to a more druggable conformation), is capable of changing the conformation of (the relevant binding pocket on) the protein to make it more amenable or accessible for binding to the first ligand (3) or generally increasing the affinity of the interaction between the first ligand (3) and / or is capable of shifting the conformational equilibrium of the protein toward such a conformation), and / or is capable of inducing and / or stabilizing the formation of a complex comprising the second ligand, the translamin and the first ligand (and / or is capable of shifting the conformational equilibrium of the translamin (2) toward the formation of such a complex), or any combination thereof.

[0336] Thus, when the translaminar protein (2) is a GPCR, the second ligand (4) may be such that it can bind, in particular stabilize and / or induce a functional conformational state of the GPCR, more preferably an active conformational state of the GPCR. The second ligand (4) if preferably also is such that it preferentially / specifically binds to the protein or GPCR when it is bound to an agonist (e.g. it is bound by the first ligand (3) which is an agonist of the protein or GPCR) compared to a conformational state in which the protein or GPCR is not bound by any first ligand (3) or is not bound by a ligand (3) which is an inverse agonist; and / or is such that it increases the affinity of the protein or GPCR for at least one compound or ligand which is an agonist of the protein or GPCR (i.e. at least two-fold, in particular at least five-fold, more preferably at least ten-fold).

[0337] As described above, a class of preferred compounds for use as a second ligand in the present invention (particularly when the second ligand is comprised in a second fusion protein) is generally described in WO2012 / 007593, WO2012 / 007594, WO2012 / 75643, WO2014 / 118297, WO2014 / 122183 and WO2014 / 118297, and comprises a VHH domain (Confobody) capable of stabilizing the GPCR in a desired conformation.

[0338] In addition, WO2012 / 75643 discloses a number of VHH domains that can bind to GPCRs indirectly, i.e., by binding to a G protein or a G protein complex. Some preferred but non-limiting examples thereof are the VHH referred to as "CA4435" (SEQ ID NO: 1 in WO2012 / 75643 and SEQ ID NO: 1 herein), which can bind to a G protein complex, and the VHH referred to as "CA4437" (SEQ ID NO: 4 in WO2012 / 75643 and SEQ ID NO: 2 herein), which can bind to a G protein. Such VHH domains can be appropriately included in a second fusion to provide a second fusion protein that can bind to a GPCR indirectly by binding to a G protein or a G protein complex.

[0339] Therefore, in a preferred aspect of the invention, the second fusion protein comprises at least one such VHH or ConfoBody and a second binding member (7).

[0340] Typically, in the present invention, when the second fusion protein binds directly or indirectly (as defined herein) to the translaminar protein (2), the first binding member (6) and the second binding member (7) will be in close proximity to each other. In particular, when the second ligand (4) present in the second fusion protein binds directly to the translaminar protein (2), or when the binding domain or binding unit (5) present in the second fusion protein binds indirectly to the translaminar protein, i.e. when the binding domain or binding unit (5) binds to the second ligand (4), or when Figure 3 In the case of the embodiment shown, bound to the protein complex (12), the first and second binding members will be in close proximity to each other, the second ligand (4) or the protein complex (12) in turn binding to or being bound by the translaminar protein (2). It will be clear to the person skilled in the art that preferably the first and second binding members themselves should not have a high affinity for each other, so that their association (and the concomitant generation of a detectable signal) is mainly driven by the first and second binding members being in close proximity to each other, because the second ligand is bound (directly or indirectly) to the translaminar protein, and is not substantially or only to a minor extent driven by affinity between the first and second ligands (the NanoBiT system from Promega is an example of such a suitable binding pair). However, it should also be noted that any such affinity between the first and second ligands will generally provide a baseline for the detectable signal, which baseline should not substantially interfere with the assays of the invention, as the readout of the assay focuses primarily on any changes in the detectable signal, such as when the first ligand is added to a device of the invention that does not already contain the first ligand (more generally, it should also be noted that for certain uses of the methods and devices of the invention, it may be preferable to have a certain level of baseline signal, as the readout may also include a decrease in signal compared to the baseline).

[0341] Thus, in general, 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 spanning protein (2). This in turn will depend on the binding interaction between the second ligand (4) and the spanning protein (especially between the second ligand and one or more specific conformations that the spanning protein can assume, such as functional, active and / or druggable conformations) and / or any change in said binding interaction (especially any change in said binding interaction that is the result of a conformational change of the spanning protein and / or a shift in the conformational equilibrium of the spanning protein, such as due to binding of the first ligand to the spanning protein and / or the formation of a complex between the first ligand, the spanning protein and the second ligand).

[0342] Included herein are methods of identifying and creating the various components of the above-described devices and compositions, as well as methods of assembling such devices and compositions.Such methods may be combined with and form part of any assays and methods for measuring or determining one or more properties of the first ligand.

[0343] By way of non-limiting example, a method as described herein for determining one or more properties of a first ligand may include one or more of the following steps, which are intended to determine that a second ligand: binds to a spanning protein, specifically binds to a spanning protein, specifically binds to a domain of a spanning protein located in a second environment, is a conformationally selective binder of a spanning protein, stabilizes the conformation of a spanning protein, stabilizes an inactive conformation of a spanning protein, stabilizes a functional, active, and / or druggable conformation of a spanning protein, and / or stabilizes a complex of a spanning protein and a first ligand.

[0344] Based on this and the further disclosure herein, it will be clear to the skilled person that the methods and devices of the present invention can be used to measure or determine one or more properties of a first ligand (particularly properties of the first ligand that are related to, influence and / or determine the interaction between the first ligand and the translaminar protein), one or more properties of a second ligand (particularly properties of the second ligand that are related to, influence and / or determine the interaction between the second ligand and the translaminar protein), and / or one or more properties of any binding domain or binding unit present in the second fusion protein (particularly properties of the binding domain or binding unit that are related to, influence and / or determine the interaction between the binding domain or binding unit and the translaminar protein when the binding domain or binding unit directly binds to the translaminar protein; or, properties of the binding domain or binding unit that are related to, influence and / or determine the interaction between the binding domain or binding unit and the second ligand and / or a protein complex comprising the same).

[0345] More specifically, with respect to the first ligand, the methods and devices of the present invention can be used to measure or determine the ability of the first ligand to bind to a translaminar protein to cause a conformational change in the translaminar protein and / or to cause a conformational equilibrium shift in the translaminar protein. For example, as further described herein, the methods and devices of the present invention can be used to measure or determine the ability of a given first ligand to act as an agonist, antagonist, inverse agonist, inhibitor, or regulator (e.g., allosteric) modulator of a translaminar protein and / or to screen or identify small molecules, proteins, or other compounds or chemical entities that are or can act as agonists, antagonists, inverse agonists, inhibitors, or regulators (e.g., allosteric) modulators of a translaminar protein. In this regard, it will be clear to the skilled person based on the disclosure herein that when the methods and devices of the present invention are used for such purposes (i.e. for purposes relating to a first ligand), typically (and preferably) the other elements used in the devices of the present invention (e.g. a second ligand and / or any binding domains or binding units present in a second fusion protein) will be selected such that they have known properties (i.e. their properties relevant to their use in the methods and devices of the present invention are known and / or have been characterized) and / or such that they have been validated for use in the methods and devices of the present invention.

[0346] The assay of the invention may also be performed in the presence of a compound with a known effect on the translaminar protein (e.g., in the presence of a known agonist, antagonist, inverse agonist, inhibitor or modulator (e.g., an allosteric modulator) of the translaminar protein) at a concentration at which the "known" compound is known to have an effect on the translaminar protein. The known compound will then typically be present in the same environment as the first ligand (i.e., the ligand whose properties are determined using the assay of the invention). For example, in the method of the invention, in which the first ligand is added to the device of the invention in which the first ligand is not already present, the known compound may be added at substantially the same time as the first ligand, may be added separately before the first ligand is added, or may be added after the first ligand is added, and the read-out from the assay may vary depending on the order in which the first ligand and the known compound are added, and the time between the time of adding the first ligand and the time of adding the known compound when the first ligand and the known compound are not added at substantially the same time (or vice versa). It is also possible that by varying the order and / or timing of adding the first ligand and the known compound, different properties of the first ligand may be determined and / or different first ligands having these properties may be identified.

[0347] For example, but not limited to, the assay of the invention can be performed in the presence of a known agonist of a translaminar protein (i.e., in the same environment as the first ligand). In this setting, the assay of the invention can, for example, be used to determine whether and how the first ligand can counteract the agonist effect of a known compound, for example, because it acts as an antagonist (thus, in this setting, the assay of the invention can be used to identify and / or characterize potential antagonists of translaminar proteins). The setting in which a known agonist is present can also be used, for example, to identify and / or characterize a first ligand that can act as an allosteric modulator that increases or decreases the effect of the agonist and / or can act as an inverse agonist of a translaminar protein. Competition assays can also be performed between the first ligand and a known compound.

[0348] The methods and devices of the present invention can be used to measure or determine the ability of a second ligand to bind to a translamin, in particular to bind and / or stabilize a specific conformation of a translamin (e.g., a functional, active, or druggable conformation) and / or to stabilize and / or induce the formation of a complex between a first ligand, a second ligand, and a translamin. For example, as further described herein, the methods and devices of the present invention can be used to measure or determine the ability of a given VHH to act as a ConfoBody of a translamin, or to identify, optimize, or validate a VHH that can act as a ConfoBody. To this end, typically, the VHH or candidate VHH will be present in a second fusion protein (i.e., as a second ligand) and will directly bind to a translamin or test its ability to directly bind to a translamin or a more specific conformation of one or more of the translamin. Also as further described herein, the methods and devices of the present invention can also be used to measure or determine the ability of an analog, derivative, or ortholog of a natural ligand of a translamin as a ligand of a translamin (e.g., to test an analog, derivative, or ortholog of a naturally occurring G protein as a ligand of a related GPCR). In this case, typically, the second ligand will not be present in the second fusion protein (although a second fusion protein comprising the analog, derivative or ortholog may also be used as the second ligand), but the second fusion protein will comprise a binding domain or binding unit (e.g., VHH) that can bind to the second ligand (or a complex comprising it). In this regard, it will be clear to the skilled person based on the disclosure herein that when the methods and devices of the present invention are used for such purposes (i.e., for purposes with respect to the second ligand), typically (and preferably) the other elements used in the devices of the present invention (e.g., the first ligand and / or any binding domain or binding unit present in the second fusion protein) will be selected so that they have known properties (i.e., their properties relevant to their use in the methods and devices of the present invention are known and / or have been characterized) and / or so that they have been validated for use in the methods and devices of the present invention.

[0349] The methods and devices 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 devices 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 VHHs that can indirectly bind to a translaminar protein (which can then, for example, be used as a binding domain or binding unit in a device of the present invention as described herein or for any other suitable purpose). Such methods and devices 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 VHHs (again, such VHHs can be used as binding domains or binding units in a device of the present invention as described herein or for any other suitable purpose). In this regard, it will be clear to the skilled person based on the disclosure herein that when the methods and devices of the present invention are to be used for such a purpose (i.e. for the purpose of indirectly binding to a binding domain or binding unit of a translaminar protein), then typically (and preferably) the other elements used in the devices of the present invention (e.g. the first ligand and the second ligand) will be selected so that they have known properties (i.e. their properties relevant to their use in the methods and devices of the present invention are known and / or have been characterized) and / or so that they have been validated for use in the methods and devices of the present invention.

[0350] In the present invention, generally speaking, the detectable signal will preferably be generated in response to a conformational change of a translaminar protein and / or a shift in the conformational equilibrium of a translaminar protein, and more preferably in proportion thereto. As further described herein, but again without limitation to any particular mechanism or explanation, the conformational change of a translaminar protein and / or the shift in the conformational equilibrium of the translaminar protein may in turn be caused by a first ligand that binds to the translaminar protein (or otherwise causes a conformational change of the translaminar protein) and / or by forming a complex of a first ligand, a translaminar protein, and a second ligand (the second ligand may, for example, stabilize the complex or otherwise induce or promote the formation of the complex). Therefore, more generally, in the present invention, a detectable signal (or any change therein, as further described herein) is generated in response to the presence of a first ligand in a first environment and / or in response to a first ligand that binds to a translaminar protein (or otherwise causes a conformational change of a translaminar protein and / or a shift in the conformational equilibrium of a translaminar protein).

[0351] In addition, typically and particularly when the methods and devices of the present invention are used to test, optimize and / or validate a first ligand and / or identify a small molecule, protein, ligand or other chemical entity that can act as an agonist, antagonist, inverse agonist, inhibitor or modulator (e.g., allosteric) modulator of a translaminar protein, the detectable signal (or any change therein, as further described herein) will be proportional to the amount and / or concentration of the first ligand present in the first environment (and / or to which the translaminar protein is exposed) and / or the affinity of the first ligand for the translaminar protein (e.g., compared to other ligands tested).

[0352] Therefore, based on the description herein, it will be clear to the skilled person that in one aspect of the invention, the methods and apparatus described herein will be used to detect the presence of a first ligand in a first environment and / or determine the amount and / or concentration of a first ligand in a first environment. The methods and apparatus described herein can also be used to measure the amount of signal generated when different concentrations of the first ligand are present in the first environment, for example to establish a relationship between the amount / concentration of the first ligand in the first environment and (the level and / or change in) the detectable signal. The methods and apparatus described herein can also be used to determine the affinity of the first ligand for the translaminar protein, for example by comparing the signal generated by one or more known concentrations of the first ligand in the first environment with the signal generated by other ligands with known affinity for the translaminar protein in the same device.

[0353] As further described herein, the methods and apparatus of the invention can also be used to determine whether a given (first) ligand is an agonist, antagonist, inverse agonist, inhibitor or modulator (eg, allosteric) modulator of a translamin protein.

[0354] It will also be clear to the skilled person that when using the methods and devices of the invention to determine one or more characteristics of a first ligand, the device of the invention will generally first be set up or otherwise established without the presence of the first ligand, the device will then be contacted with the ligand (e.g. by adding the ligand to the first environment), after which the detectable signal (or any change therein) resulting from the presence of the first ligand will be measured (and optionally compared to the signal in the absence of the first ligand and / or with one or more reference values). Thus, the devices described herein in the absence of the first ligand (e.g. before the first ligand is added) form a further aspect of the invention.

[0355] Another aspect of the invention is a method for providing a device of the invention as described herein, the method comprising the step of adding a first ligand to a device of the invention (as described herein), the device (as described herein) not (already) comprising the first ligand. The device so obtained can then be used to measure or otherwise determine at least one property of the first ligand, and in particular a property of the first ligand that can be measured or otherwise determined using the device of the invention.

[0356] It will be clear to a person skilled in the art based on the disclosure herein that a device of the invention without the presence of a first ligand (i.e. a device of the invention that does not yet comprise a first ligand) will include at least the following elements:

[0357] - a boundary layer separating the first environment and the second environment;

[0358] - translaminins;

[0359] - ligands for the translaminar proteins present in the second environment; and

[0360] - 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;

[0361] The elements are arranged relative to each other (and where applicable are operably connected and / or associated with each other) in the manner further described herein (i.e. essentially in the same manner as described for the device of the invention comprising the first ligand).

[0362] In particular, a device of the invention in the absence of the first ligand (ie a device of the invention which does not yet comprise the first ligand) will comprise at least the following elements:

[0363] - a boundary layer separating the first environment and the second environment;

[0364] - 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;

[0365] - a translaminar protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (ie forming a first fusion protein); and

[0366] - a second ligand for the translaminar protein present in a second environment;

[0367] The elements are arranged relative to each other (and where applicable operably connected and / or associated with each other) in a manner further described herein (i.e. substantially in the same manner as described for the device of the invention comprising the first ligand). In particular, the second member of the binding pair may be part of a second fusion protein (which is different from the first fusion protein comprising the spanning protein and the first binding member of the binding pair), the second fusion protein being further described herein.

[0368] More particularly, a device of the invention in the absence of the first ligand (i.e. a device of the invention that does not yet comprise the first ligand) will comprise at least the following elements:

[0369] - a boundary layer separating the first environment and the second environment;

[0370] - 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;

[0371] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0372] - a second fusion protein comprising a protein that can bind directly or indirectly to the translaminar protein and to the other binding member of the binding pair, the second fusion protein being present in a second environment;

[0373] The elements are arranged relative to each other (and where applicable are operably connected and / or associated with each other) in the manner further described herein (i.e. essentially in the same manner as described for the device of the invention comprising the first ligand).

[0374] Further aspects, embodiments and preferences of the device of the invention without the first ligand are as described herein for the device of the invention with the first ligand but in the subsequent absence of the first ligand.

[0375] Typically, once the first ligand is added as part of the methods described herein, any such device of the invention in which the first ligand is not present will become a corresponding device of the invention with the first ligand. Thus, another aspect of the invention is a method of providing a device of the invention as described herein, the method comprising the step of adding the first ligand to a device of the invention (as described herein) that does not (already) contain the first ligand. The device so obtained can then be used to measure or otherwise determine at least one property of the first ligand, and in particular a property of the first ligand that can be measured or otherwise determined using the device of the invention.

[0376] The present invention also relates to a method for measuring or otherwise determining at least one property of a compound or a ligand, the method comprising at least the following steps:

[0377] - adding said compound or ligand as a first ligand to a device of the invention which does not yet comprise a first ligand; and

[0378] - measuring or otherwise determining at least one property of said compound or ligand, wherein said property is a property that can be measured or otherwise determined using said device.

[0379] In this aspect of the invention, the property is preferably a property that represents the ability of the compound or ligand to bind to and / or modulate a translaminar protein (eg affinity).

[0380] The present invention also relates to a method of measuring or otherwise determining the ability of a compound or ligand to alter a detectable signal generated by a binding pair present in a device of the present invention as further described herein, the method comprising at least the steps of:

[0381] - adding said compound or ligand as a first ligand to a device of the invention which does not yet comprise a first ligand; and

[0382] - determining whether adding said compound or ligand results in a change in a detectable signal produced by a binding pair used in said device, and optionally measuring said change in said detectable signal.

[0383] Therefore, in another aspect, the present invention relates to a method comprising at least the following steps:

[0384] a) Provide an apparatus comprising at least the following components:

[0385] - a boundary layer separating the first environment and the second environment;

[0386] - translaminins;

[0387] - ligands for the translaminar proteins present in the second environment; and

[0388] - 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;

[0389] Therein, the elements are arranged relative to each other (and where applicable operatively connected and / or associated with each other) in a manner further described herein.

[0390] as well as;

[0391] b) adding a first ligand to the first environment.

[0392] The method preferably further comprises the following steps:

[0393] c) measuring a signal produced by the binding pair and / or measuring a change in a signal produced by the binding pair.

[0394] Therefore, in a more specific aspect, the present invention relates to a method comprising at least the following steps:

[0395] a) Provide an apparatus comprising at least the following components:

[0396] - a boundary layer separating the first environment and the second environment;

[0397] - 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;

[0398] - a translaminar protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (ie forming a first fusion protein); and

[0399] - a second ligand for the translaminar protein present in a second environment;

[0400] wherein the elements are arranged relative to each other (and where applicable operably connected and / or associated with each other) in the manner further described herein;

[0401] as well as;

[0402] b) adding a first ligand to the first environment.

[0403] The method preferably further comprises the following steps:

[0404] c) measuring a signal produced by the binding pair and / or measuring a change in a signal produced by the binding pair.

[0405] In another specific aspect, the present invention relates to a method comprising at least the following steps:

[0406] a) Provide an apparatus comprising at least the following components:

[0407] - a boundary layer separating the first environment and the second environment;

[0408] - 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;

[0409] - a first fusion protein comprising a translaminar protein and one of the binding members of said binding pair (i.e. such that said member of the binding pair is present in a second environment);

[0410] - a second fusion protein comprising a protein that can bind directly or indirectly to the translaminar protein and to the other binding member of the binding pair, the second fusion protein being present in a second environment;

[0411] wherein the elements are arranged relative to each other (and where applicable operably connected and / or associated with each other) in the manner further described herein;

[0412] as well as;

[0413] b) adding a first ligand to the first environment.

[0414] The method preferably further comprises the following steps:

[0415] c) measuring a signal produced by the binding pair and / or measuring a change in a signal produced by the binding pair.

[0416] As further described herein, in this aspect of the invention, the first ligand may be any desired and / or suitable compound or ligand, including but not limited to small molecules, small peptides, biomolecules or other chemical entities. It is also clear to those skilled in the art that the method according to this aspect (and other methods of the invention) can be used to measure or otherwise determine at least one property of a compound or ligand added to a device as a first ligand, and in particular to measure or otherwise determine the ability of the compound or ligand to cause a detectable signal change produced by a binding pair, the ability of the compound or ligand to bind to a translaminar protein, the ability of the compound or ligand to cause a conformational change in a translaminar protein, and / or the ability of the compound or ligand to modulate (as defined herein) a translaminar protein and / or a signal transduction pathway and / or a biological mechanism in which a translaminar protein is involved. In particular, the method can be used to determine whether such a compound or ligand is or can be used as an agonist, antagonist, inverse agonist, inhibitor or modulator (e.g., an allosteric modulator) of a translaminar protein, and / or a signal transduction pathway and / or a biological mechanism in which a translaminar protein is involved. In addition, the methods and devices of the present invention can be used to identify and / or screen compounds or ligands that have the ability to cause a change in a detectable signal produced by a binding pair, the ability to bind to a translamin, the ability to cause a conformational change in a translamin, the ability to modulate a translamin and a signaling pathway and / or biological mechanism in which the translamin is involved, and / or the ability to act as an agonist, antagonist, inverse agonist, inhibitor and / or modulator (e.g., an allosteric modulator) of a translamin, and such uses of the methods and devices described herein form further aspects of the present invention.

[0417] It should also be noted that, in another aspect, the methods and devices of the present invention can also be used to measure or otherwise determine at least one property of a second ligand, such as the ability of a second ligand to bind to a translaminar protein, the ability of a second ligand to bind to and / or stabilize a particular conformation (e.g., an active and / or druggable conformation) of a translaminar protein, and / or the ability of a second ligand to stabilize a complex of a translaminar protein, a first ligand, and a second ligand. Typically, in this aspect of the invention, one or more first ligands with a known ability to bind to and / or modulate a translaminar protein will be used to determine whether a device of the present invention comprising a (candidate) second ligand will produce a detectable signal when the first ligand is added to the device (e.g., at one or more known concentrations).

[0418] For example, this aspect of the invention can be used to identify or optimize binding domains or binding units (e.g., ISVDs) that can bind directly (as defined herein) to a translaminar protein, and in particular binding domains or binding units that are specific and / or selective for the translaminar protein conformation that occurs when the first ligand used binds to the translaminar protein. The binding domains or binding units so identified, optimized, and / or validated can, for example, be used in the device of the invention (i.e., as part of a second fusion protein) and / or for inducing or stabilizing a specific conformation of the translaminar protein (e.g., for screening or crystallization purposes, as described in the prior art for conformation-specific ligands for GPCRs cited herein). Thus, for example, this aspect of the invention can be used to identify, optimize, and / or validate an ISVD for use as a ConfoBody, which can then be used for the purposes described herein and / or for uses known per se for conformation-specific ISVDs. Reference is again made to the other prior art cited herein.

[0419] Typically, in this aspect of the invention, the binding domain, binding unit, ligand or other protein to be tested or verified to bind (directly) to the translaminar protein will be part of the second fusion protein. Therefore, the invention further relates to a method for measuring or otherwise determining at least one property of a binding domain, binding unit, ligand or other protein, the method comprising at least the following steps:

[0420] - providing a device of the invention which does not yet comprise the first ligand, wherein the second fusion protein comprises said binding domain, binding unit, ligand or other protein;

[0421] - adding a first ligand to the device; and

[0422] - determining whether adding said first ligand results in a change in a detectable signal produced by a binding pair used in said device, and optionally measuring said change in said detectable signal.

[0423] It will be clear to a person skilled in the art based on the disclosure herein that the at least one characteristic of the binding domain, binding unit, ligand or other protein will be, in particular, the ability of the binding domain, binding unit, ligand or other protein to bind to a translaminar protein (especially the conformation of the translaminar protein when the first ligand used is bound to the translaminar protein), the ability of the binding domain, binding unit, ligand or other protein to stabilize the conformation of the translaminar protein when the first ligand used is bound to the translaminar protein, and / or the ability of the binding domain, binding unit, ligand or other protein to promote or induce the formation of a complex of the first ligand used, the translaminar 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.

[0424] In another aspect of the invention, the device described herein is again used to measure or otherwise determine at least one property of a second ligand and / or identify, optimize and / or validate a candidate second ligand, but in this regard, the second fusion protein will not comprise the second ligand to be tested or the candidate second ligand, but rather a binding domain or binding unit known to bind to the second ligand to be tested or the candidate second ligand. In other words, in this aspect, the second fusion protein will comprise a binding domain or binding unit that can bind to the translaminar protein indirectly (as defined herein), i.e., through the second ligand to be tested or the candidate second ligand or through a protein complex comprising it, provided that the second ligand or complex is capable of binding to the translaminar protein (and in particular, to the conformation of the translaminar protein produced when the first ligand binds to the translaminar protein). As with the aforementioned aspect, this aspect can also be used to identify, optimize and / or validate (candidate) ligands of the translaminar protein, such as ligands that are synthetic or semi-synthetic analogs or derivatives of naturally occurring ligands of the translaminar protein. For example, when the translaminar protein is a GPCR, this aspect of the invention can be used to identify, optimize and / or validate analogs or derivatives of a G protein that is a natural ligand for the GPCR, or to determine whether an ortholog of a naive G protein for the GPCR of interest is able to bind to the GPCR and / or stabilize a complex of the GPCR and a first ligand used.

[0425] Therefore, the present invention further relates to a method for measuring or otherwise determining at least one property of a ligand or other protein, the method comprising at least the following steps:

[0426] - providing a device according to the invention which does not yet comprise a first ligand, wherein said ligand or other protein is present and / or serves as a second ligand, and wherein the second fusion protein comprises a binding domain or binding unit which can bind to said ligand or other protein and / or to a protein complex comprising said ligand or other protein;

[0427] - adding a first ligand to the device; and

[0428] - determining whether adding said first ligand results in a change in a detectable signal produced by a binding pair used in said device, and optionally measuring said change in said detectable signal.

[0429] As described herein, in a particular aspect of the invention, the method of the invention is performed using a suitable cell or cell line, wherein all elements of the device of the invention are suitably present and arranged so as to provide an operable device of the invention. Such a cell or cell line will suitably comprise the translamin (2) in its cell wall or cell membrane, i.e. such that the translamin (2) is present in and spans the cell wall or cell membrane of the cell, such that at least a portion of the amino acid sequence of the translamin extends out (as defined herein) into the extracellular environment, and at least one of the other portions of the amino acid sequence of the translamin extends out (as defined herein) into the intracellular environment. Furthermore, preferably and as further described herein, the translamin will form part of a first fusion protein as described herein and the device will also comprise a second fusion protein as described herein. More preferably, the extracellular environment will be 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 will be the "second environment" (i.e. the environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0430] Therefore, in another aspect, the invention relates to a method or device as described herein, wherein the boundary layer (2) is the wall or membrane of a cell.

[0431] As also described herein, when the method of the invention is performed in a cell or a suitable cell line, the cell or cell line used is preferably such that it suitably expresses one or more, and preferably all, of the following elements of the device of the invention:

[0432] - a first fusion protein comprising a translaminar protein (2) and a first binding member (6);

[0433] - a second fusion protein comprising a second binding member (7) and a protein that can bind directly or indirectly (as defined herein) to the translaminar protein (2);

[0434] and / or

[0435] - When the second fusion protein indirectly binds to the translaminar protein (2), the second ligand (4) and / or the protein constituting the protein complex (12).

[0436] In the context of a cell or cell line expressing one or more elements of the device of the invention, and more generally in the context of the present specification and claims, the term "suitably expresses" means that the cell or cell line expresses or is capable of expressing (i.e. under conditions useful for carrying out the method of the invention) a nucleotide sequence or nucleic acid encoding the element, such that when such element is expressed it is capable of being used as an operable part of the device of the invention. For example, with respect to a translamin (2), this means that the translamin is expressed as part of a first fusion protein such that the expressed translamin (2) is suitably anchored or otherwise incorporated into the cell wall or cell membrane of a cell such that the translamin spans the cell wall or cell membrane, wherein at least a portion of the amino acid sequence of the translamin extends out (as defined herein) into the extracellular environment and at least one other portion of the amino acid sequence of the translamin extends out (as defined herein) into the intracellular environment. With respect to the first and second fusion proteins, "suitably expressed" means that the first and second fusion proteins are expressed (and most preferably expressed in an intracellular environment) such that when the second fusion protein binds directly or indirectly to the translaminar protein (2) in a manner further described herein, the first and second binding members of the binding pair (6 / 7) can contact or be in close proximity to each other.

[0437] Any suitable expression of each such element of the apparatus of the invention may be transient or constitutive, so long as all required elements of the apparatus of the invention are suitably and operatively present in sufficient amounts at the point in time when the cells are used to perform the methods of the invention.

[0438] In one aspect of the invention, in the case of an embodiment of the invention wherein the second fusion protein binds indirectly to the translaminar protein (i.e. wherein the second ligand (4) is not part of the second fusion protein), the cell or cell line used is preferably such that it naturally expresses the second ligand (4) and / or the proteins constituting the protein complex (12). For example, but not limited to, in this aspect of the invention, when the translaminar protein (2) is a GPCR, the second ligand (4) may be a G protein naturally expressed by the cell or cell line used and / or the protein complex (12) may be a protein trimer comprising a G-α subunit, a G-β subunit and a G-γ subunit G naturally expressed by the cell or cell line used. More generally, in these aspects of the invention, the cell or cell line used may be a cell or cell line that naturally expresses one or more natural ligands (and in particular intracellular ligands) of the translaminar protein (2) and / or a cell or cell line that naturally expresses one or more ligands that can be used as the second ligand of the translaminar protein (2), depending on the translaminar protein (2) being used or screened.

[0439] The cell or cell line may be any cell or cell line suitable for use with the methods and devices of the invention, including but not limited to mammalian cells and insect cells. Some preferred but non-limiting examples are human cell lines, such as HEK 293T.

[0440] Suitable techniques for transiently or stably expressing the desired protein in such cells or cell lines so that the translaminar protein (2) is appropriately anchored to the cell wall or membrane of the cells will be clear to the skilled person and for example include techniques involving the use of a suitable transfection reagent, such as X-tremeGENE™ or polyethyleneimine (PEI) from Sigma Aldrich.

[0441] When the invention is performed using cells or cell lines which suitably express one or more elements of the device of the invention, the method of the invention generally further comprises the step of culturing or maintaining said cells under conditions such that said cells or cell lines suitably express said elements.

[0442] Thus, in another aspect, the present invention relates to a cell or cell line comprising a fusion protein comprising a translaminar protein (as described herein) fused directly or via a suitable linker to a binding domain or binding unit which is the first binding member of a binding pair comprising at least said binding domain or binding unit as the first binding member and another binding domain or binding unit as the second binding member, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when in contact with each other or in close proximity to each other. The present invention also relates to a cell or cell line expressing or capable of expressing (i.e. under suitable conditions) such a fusion protein.

[0443] Such a cell or cell line may be as further described herein, and preferably is such that it expresses or is capable of expressing the fusion protein in such a way that the translaminin is incorporated into and spans the cell wall or cell membrane of the cell or cell line, more preferably such that at least a portion of the amino acid sequence of the translaminin extends out (as defined herein) into the extracellular environment, and at least one other portion of the amino acid sequence of the translaminin extends out (as defined herein) into the intracellular environment. More preferably, in the 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 is such that it expresses or is capable of expressing the fusion protein such that upon such expression, the first binding member is present in the intracellular environment of the cell (as defined herein).

[0444] Furthermore, the translamin protein present in the fusion protein is preferably as further described herein, and more preferably has at least two ligand binding sites, one of which extends out (as defined herein) into the extracellular environment and one of which extends out (as defined herein) into the intracellular environment. Furthermore, as described herein, the translamin protein is preferably such that it is capable of undergoing a conformational change from one of its conformations to another conformation (and in particular, a conformational change from a substantially inactive or less active conformation to an active or more active conformation) upon binding of a ligand to a ligand binding site on the translamin protein and in particular upon binding of a ligand present in the extracellular environment to a ligand binding site on the translamin protein present in the extracellular environment (as defined herein). As further described herein, the translaminin is preferably further such that it can be stabilized in a functional and / or active (or more active) conformation (and in particular in a druggable conformation and / or a ligand-bound conformation, and more particularly in an agonist-bound conformation) by binding of a suitable ligand, binding domain or binding unit (e.g., a ConfoBody as described herein or a natural ligand of the translaminin, e.g., a natural intracellular ligand) to an intracellular binding site on the translaminin (which can be a binding site on the translaminin that is an intracellular binding site when the translaminin is in its natural environment, and / or when the translaminin is present in a cell or cell line used in the present invention, it can be a binding site on the translaminin that is an intracellular binding site, and preferably both are present). In particular, as also described herein, the translaminin may be able to form a complex when a first ligand binds to an extracellular binding site and a second ligand binds to an intracellular binding site. More specifically, as described herein, the translaminar protein may be capable of forming a complex, wherein the translaminar protein is in a functional or active conformation, which is induced by a first ligand that binds to an extracellular binding site, wherein the active or functional conformation is stabilized by the binding of a second ligand to an intracellular binding site, which second ligand is capable of stabilizing the functional, active or ligand-bound conformation and / or the complex. In a preferred but non-limiting aspect, the translaminar protein is a transmembrane protein, particularly a 7TM. In addition, the members of the binding pair and any linkers used may be as further described herein.

[0445] In another aspect, the invention relates to a cell or cell line comprising a fusion protein comprising a protein that can (directly or indirectly, as described herein) bind to a translaminar protein (as described herein), fused directly or via a suitable linker to a binding domain or binding unit that is the first binding member of a binding pair, the binding pair comprising at least a first binding member and the binding domain or binding unit as the second binding member, wherein the first and second binding members of the binding pair are such that they are capable of generating a detectable signal when in contact with each other or in close proximity to each other. The invention also relates to a cell or cell line that expresses or is capable of expressing (i.e. under suitable conditions) such a fusion protein.

[0446] The protein present in the fusion protein and that can bind to the translaminar protein is preferably as further described herein for the protein that can be present in the second fusion protein. In addition, the members of the binding pair and any linkers used can be as further described herein. As also described herein, the protein can bind to the translaminar protein directly (as described herein) or indirectly (as described herein). Again, in this regard, the translaminar protein that the protein can bind to is preferably also as further described herein, and can be in particular a transmembrane protein, more in particular a 7TM.

[0447] As described herein, when the protein present in the fusion protein directly binds to the spanning protein, it is preferably such that it specifically binds to one or more functional, active and / or druggable conformations of the spanning protein, such that it induces the formation and / or stabilization of one or more functional, active and / or druggable conformations of the spanning protein (and / or shifts the conformational equilibrium of the spanning protein to one or more such conformations); and / or such that it induces the formation and / or stabilization of a complex of the protein, the spanning protein and another ligand of the spanning protein (all as further described herein). In addition, when the protein present in the fusion protein directly binds to the spanning protein, the protein is preferably such that it can bind to an intracellular binding site on the spanning protein. The intracellular binding site on the spanning protein can be a binding site on the spanning protein that is an intracellular binding site when the spanning protein is in its native environment, and / or a binding site on the spanning protein that is an intracellular binding site when the spanning protein is present in the cells or cell lines used in the present invention (and preferably both).

[0448] Furthermore, when the protein present in the fusion protein directly binds to the translaminar 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).

[0449] As also described herein, when the protein present in the fusion protein is indirectly bound to the translaminar protein, it is preferably made available to bind to a ligand that can bind to the translaminar protein. When the second ligand does not form part of the second fusion protein, the ligand may be as described herein for "second ligand". Again, the ligand preferably allows it to specifically bind to one or more functional, active and / or druggable conformations of the translaminar protein, so that it induces the formation and / or stabilization of one or more functional, active and / or druggable conformations of the translaminar protein (and / or shifts the conformational equilibrium of the translaminar protein to one or more such conformations); and / or allows it to induce the formation and / or stabilization of a complex of the ligand, the translaminar protein and another ligand of the translaminar protein (all as further described herein). In addition, the ligand preferably allows it to bind to an intracellular binding site on the translaminar protein. The intracellular binding site on the translamin can be a binding site on the translamin that is an intracellular binding site when the translamin is in its native environment, and / or a binding site on the translamin that is an intracellular binding site when the translamin is present in the cells or cell lines used in the present invention (and preferably both). In addition, as described herein, the ligand can also be part of a protein complex that can bind to the translamin (i.e., to the intracellular binding site on the translamin), in which case the protein present in the fusion protein can also bind to the protein complex.

[0450] In addition, when the protein present in the fusion protein indirectly binds to the translaminar protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain. In addition, in a preferred aspect, when the protein present in the fusion protein indirectly binds to the translaminar protein, and the translaminar protein is a GPCR, the ligand bound to the GPCR is a G protein, and the protein present in the fusion protein is capable of specifically binding to the G-protein or G-protein complex, such as a G-protein trimer comprising a G-α subunit, a G-β subunit, and a G-γ subunit. In addition, the G protein may be native to the cell or cell line used, or may be a suitable analog or derivative of a native G protein (as described herein and recombinantly expressed in the cell or cell line) or a suitable ortholog of a G protein native to the cell or cell line used (again, recombinantly expressed in the cell or cell line used).

[0451] Regardless of whether the protein present in the fusion protein binds directly or indirectly to the translaminar protein, the cell or cell line is preferably such that it expresses or is capable of expressing the fusion protein in its intracellular environment. Another aspect of the invention relates to such a cell or cell line comprising such a fusion protein in its intracellular environment.

[0452] In another aspect, the present invention relates to a cell or cell line comprising a first fusion protein and a second fusion protein, wherein:

[0453] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0454] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0455] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair.

[0456] The invention also relates to cells or cell lines expressing or capable of expressing (ie under suitable conditions) such a first and a second fusion protein.

[0457] The present invention particularly relates to a cell or cell line comprising a first fusion protein and a second fusion protein, wherein:

[0458] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0459] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0460] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0461] - When the second fusion protein binds (directly or indirectly, as described herein) to a spanning protein forming part of the first fusion protein, the first and second binding members of the binding pair may be in contact or in close proximity with each other.

[0462] The present invention also relates to a cell or cell line comprising the first fusion protein and the second fusion protein, wherein:

[0463] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0464] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0465] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0466] - The first and second binding members of the binding pair are present in the intracellular environment of a cell (as defined herein).

[0467] The present invention also relates to a cell or cell line comprising the first fusion protein and the second fusion protein, wherein:

[0468] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0469] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0470] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0471] - said cell or cell line is capable of producing a detectable signal (and in particular a detectable signal produced by the first and second binding members of a binding pair) when the second fusion protein binds (directly or indirectly, as described herein) to a translaminar protein forming part of the first fusion protein.

[0472] The present invention also relates to a cell or cell line comprising the first fusion protein and the second fusion protein, wherein:

[0473] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0474] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0475] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0476] - said cell or cell line produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or a change in such a signal produced by the first and second binding members of a binding pair) when a ligand for the translamin present in the extracellular environment binds to the translamin.

[0477] In a particular aspect, the invention relates to a cell or cell line comprising a first fusion protein and a second fusion protein, wherein:

[0478] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0479] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0480] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0481] - said cell or cell line produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or a change in such a signal produced by the first and second binding members of a binding pair) when an agonist of the translamin protein present in the extracellular environment binds to the translamin protein.

[0482] Again, such cells or cell lines comprising or expressing such first and second fusion proteins can be as further described herein, and preferably are such that they express or are capable of expressing the first fusion protein in a manner such that the translaminin is incorporated into and spans the cell wall or cell membrane of the cell or cell line, more preferably such that at least a portion of the amino acid sequence of the translaminin extends out (as defined herein) into the extracellular environment and at least one other portion of the amino acid sequence of the translaminin extends out (as defined herein) into the intracellular environment.

[0483] The cells or cell lines are also preferably such that they express or are capable of expressing the first and second fusion proteins, such that upon such expression, when the second fusion protein binds (directly or indirectly, as described herein) to a translaminar protein forming part of the first fusion protein, the first and second binding members of the binding pair may be in contact or in close proximity with each other. It is clear to the skilled person that this generally means that such cells or cell lines will express the first and second fusion proteins in such a way that upon such expression, the first and second binding members of the binding pair will be present in the same environment relative to the cell wall or cell membrane (as defined herein). Preferably, the cells or cell lines are such that they express or are capable of expressing the first and second fusion proteins, such that upon such expression, the first and second binding members of the binding pair will both be present in the intracellular environment of the cell (as defined herein). This also generally means that the cells or cell lines are preferably such that they express or are capable of expressing the second fusion protein in their intracellular environment.

[0484] Again, in aspects of the invention involving expression or being capable of expressing such first and second fusion proteins, the spanning proteins, proteins that can directly or indirectly bind to the spanning proteins, members of the binding pairs and any linkers used can be as further described herein.

[0485] In a further aspect, the present invention also relates to methods, and in particular to assays or screening methods using the cells or cell lines described herein. As further described herein, such assays and screening methods can be particularly useful for identifying compounds and other chemical entities that bind (and in particular specifically bind) to a translaminar protein, can modulate a translaminar protein, and / or modulate signaling, signaling pathways, and / or biological or physiological activities involving a translaminar protein, its signaling, and / or its signaling pathways. Thus, the cells and cell lines described herein can be used in methods for identifying compounds or other chemical entities that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) modulators of a translaminar protein.

[0486] The present invention also relates to the use of the cells or cell lines described herein, in particular in assay and screening methods and techniques. For the methods and uses of the device of the present invention, these methods and uses may also be further described here again, and will generally also include the step of culturing or maintaining the cells under conditions that allow the cells or cell lines to appropriately express the desired fusion protein or protein.

[0487] Again, in all these aspects, such cells, cell lines and uses thereof are preferably as further described herein.

[0488] In another aspect of the invention, the method of the invention is carried out using suitable liposomes or vesicles, wherein all elements of the device of the invention are suitably present and arranged to provide an operable device of the invention. Such liposomes or vesicles will suitably contain the translaminar protein (2) in their wall or membrane, i.e., such that the translaminar protein (2) is present in and spans the wall or membrane of the liposome or vesicle, such that at least a portion of the amino acid sequence of the translaminar protein extends (as defined herein) to the environment outside the liposome or vesicle, and at least one other portion of the amino acid sequence of the translaminar protein extends (as defined herein) to the environment inside the liposome or vesicle. Furthermore, preferably and as further described herein, in the aspect of the invention carried out in liposomes or vesicles, the environment outside the liposome or vesicle will be 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 will be the "second environment" (i.e., the environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0489] Thus, in another aspect, the invention relates to a method or device as described herein, wherein the boundary layer (2) is the wall or membrane of a liposome or other (suitable) vesicle.

[0490] As also described herein, when the method of the invention is carried out in a liposome or vesicle, the liposome or vesicle is preferably such that it suitably contains (i.e. in a manner that provides an operable device of the invention) the following elements of the device of the invention:

[0491] - a first fusion protein comprising a translaminar protein (2) and a first binding member (6);

[0492] - a second fusion protein comprising a second binding member (7) and a protein that can bind directly or indirectly (as defined herein) to the translaminar protein (2);

[0493] and / or

[0494] - When the second fusion protein indirectly binds to the translaminar protein (2), the second ligand (4) and / or the protein constituting the protein complex (12).

[0495] Liposomes or vesicles containing the elements can generally be provided by forming liposomes or vesicles in the presence of the relevant elements of the device of the present invention, so that the elements are suitably incorporated into the liposomes or vesicles. This can generally be carried out by methods and techniques known per se for forming liposomes or vesicles, preferably in a suitable aqueous buffer or another suitable aqueous medium. Such methods may also include the following steps: liposomes or vesicles in which the elements of the desired device of the present invention are suitably and operably included, and vesicles or liposomes that do not contain all the desired elements of the device and / or in which the elements do not form an operable device of the present invention are separated. The device elements incorporated into the liposomes or vesicles can be provided in a manner known per se, for example by recombinantly expressing a suitable host cell or host organism, and then isolating and purifying the expressed elements obtained thereby.

[0496] Generally, in aspects of the invention carried out in liposomes or vesicles, wherein the second ligand does not form part of the second fusion protein, a sufficient amount of the second ligand should also be provided and suitably contained within the vesicle or liposome.

[0497] The liposome or vesicle may be any liposome or vesicle suitable for use in the methods and devices of the 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 liposome and vesicle may also be liposomes or vesicles comprising and / or based on (e.g., reconstituted from) one or more membrane fractions obtained from cells expressing the desired elements of the device of the invention.

[0498] Thus, in another aspect, the present invention relates to a liposome or vesicle comprising a fusion protein comprising a translaminar protein (as described herein) fused directly or via a suitable linker to a binding domain or binding unit which is the first binding member of a binding pair comprising at least said binding domain or binding unit as the first binding member and another binding domain or binding unit as the second binding member, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when in contact with each other or in close proximity to each other. The present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating such a fusion protein into a liposome or vesicle and / or forming a liposome or vesicle in the presence of said fusion protein.

[0499] As further described herein, the liposome or vesicle is preferably such that the translaminar protein is anchored or otherwise suitably incorporated into and spans the wall or membrane of the liposome or vesicle, more preferably such that at least a portion of the amino acid sequence of the translaminar protein extends (as defined herein) into the environment outside the liposome or vesicle, and at least one of the other portions of the amino acid sequence of the translaminar protein extends (as defined herein) into the environment inside the liposome or vesicle. More preferably, the first binding member of the binding pair is present in the environment inside the liposome or vesicle (as defined herein),

[0500] Furthermore, the translaminar protein present in the fusion protein is preferably as further described herein, and more preferably has at least two ligand binding sites, one of which extends (as defined herein) into the environment outside the liposome or vesicle, and one of which extends (as defined herein) into the environment inside the liposome or vesicle. Furthermore, as described herein, the translaminar protein is preferably such that it is capable of undergoing a conformational change from one of its conformations to another conformation (and in particular, a conformational change from a substantially inactive or less active conformation to an active or more active conformation) upon binding of a ligand to a ligand binding site on the translaminar protein and in particular upon binding of a ligand present in the environment outside the liposome or vesicle to a ligand binding site on the translaminar protein present in the environment outside the liposome or vesicle (as defined herein). As further described herein, the laminar protein is preferably further such that it can be stabilized in a functional and / or active (or more active) conformation (and in particular in a druggable conformation and / or a ligand-bound conformation, and more particularly in an agonist-bound conformation) by binding of a suitable ligand, binding domain or binding unit (e.g., a ConfoBody as described herein or a natural ligand of the laminar protein) to an intracellular binding site on the laminar protein (which can be a binding site on the laminar protein that is an intracellular binding site when the laminar protein is in its natural environment, and / or a binding site on the laminar protein that is present in the liposome or vesicle internal environment when the laminar protein is present in the liposome or vesicle used in the present invention, and preferably both). In particular, as also described herein, the laminar protein may be able to form a complex when a first ligand binds to a binding site that is present in the liposome or vesicle external environment (as defined herein) and a second ligand binds to a binding site that is present in the liposome or vesicle internal environment (as defined herein). More specifically, as described herein, the translaminar protein may be capable of forming a complex wherein the translaminar protein is in a functional or active conformation induced by the binding of a first ligand to a binding site present in the liposome or vesicle external environment (as defined herein), wherein the active or functional conformation is stabilized by the binding of a second ligand to a binding site present in the liposome or vesicle internal environment (as defined herein), the second ligand being capable of stabilizing the functional, active or ligand-bound conformation and / or the complex. In a preferred but non-limiting aspect, the translaminar protein is a transmembrane protein, in particular a 7TM. Furthermore, the members of the binding pair and any linkers used may be as further described herein.

[0501] In another aspect, the present invention relates to a liposome or vesicle comprising a fusion protein, the fusion protein comprising a protein that can (directly or indirectly, as described herein) bind to a translaminar protein (as described herein), the protein being fused directly or via a suitable linker to a binding domain or binding unit that is the first binding member of a binding pair, the binding pair comprising at least a first binding member and the binding domain or binding unit as a second binding member, wherein the first and second binding members of the binding pair are such that they are capable of generating a detectable signal when in contact with each other or in close proximity to each other. The present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating such fusion proteins into liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion proteins.

[0502] The protein present in the fusion protein and that can bind to the translaminar protein is preferably as further described herein for the protein that can be present in the second fusion protein. In addition, the members of the binding pair and any linkers used can be as further described herein. As also described herein, the protein can bind to the translaminar protein directly (as described herein) or indirectly (as described herein). Again, in this regard, the translaminar protein that the protein can bind to is preferably also as further described herein, and can be in particular a transmembrane protein, more in particular a 7TM.

[0503] As described herein, when the protein present in the fusion protein binds directly to the translaminin, it is preferably such that it specifically binds to one or more functional, active and / or druggable conformations of the translaminin, such that it induces the formation and / or stabilization of one or more functional, active and / or druggable conformations of the translaminin (and / or shifts the conformational equilibrium of the translaminin toward one or more such conformations); and / or such that it induces the formation and / or stabilization of a complex of the protein, the translaminin and another ligand of the translaminin (all as further described herein). In addition, when the protein present in the fusion protein binds directly to the translaminin, the protein is preferably such that it can bind to the following binding sites: a binding site on the translaminin that is an intracellular binding site when the translaminin is in its native state, and / or a binding site on the translaminin that is present in the internal environment of the liposome or vesicle (as defined herein) when the translaminin is present in a liposome or vesicle (when the liposome or vesicle is used in the present invention), (and preferably both).

[0504] Furthermore, when the protein present in the fusion protein directly binds to the translaminar 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).

[0505] As also described herein, when the protein present in the fusion protein is indirectly bound to the translaminar protein, it is preferably such that it can bind to a ligand that can bind to the translaminar protein. When the second ligand does not form part of the second fusion protein, the ligand may be as described herein for "second ligand". Again, the ligand preferably allows it to specifically bind to one or more functional, active and / or druggable conformations of the translaminar protein, such that it induces the formation and / or stabilization of one or more functional, active and / or druggable conformations of the translaminar protein (and / or shifts the conformational equilibrium of the translaminar protein to one or more such conformations); and / or allows it to induce the formation and / or stabilization of a complex of the ligand, the translaminar protein and another ligand of the translaminar protein (all as further described herein). Furthermore, the ligand is preferably such that it can bind to a binding site on the translaminar protein that is an intracellular binding site when the translaminar protein is in its native state, and / or a binding site on the translaminar protein that is present in the internal environment of the liposome or vesicle (as defined herein) when the translaminar protein is present in a liposome or vesicle (when the liposome or vesicle is used in the present invention), (and preferably both). Furthermore, as described herein, the ligand may also be part of a protein complex that can bind to the translaminar protein, in which case the protein present in the fusion protein may also bind to the protein complex.

[0506] In addition, when the protein present in the fusion protein indirectly binds to the translaminar protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain. In addition, in a preferred aspect, when the protein present in the fusion protein indirectly binds to the translaminar protein, and the translaminar protein is a GPCR, the ligand bound to the GPCR is a G protein, and the protein present in the fusion protein can specifically bind to the G-protein or G-protein complex, such as a G-protein trimer comprising a G-α subunit, a G-β subunit and a G-γ subunit.

[0507] Regardless of whether the protein present in the fusion protein is directly or indirectly bound to the translaminar protein, the fusion protein is preferably present in the environment inside the liposome or vesicle (as defined herein). In addition, 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.

[0508] In another aspect, the present invention relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein, wherein:

[0509] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0510] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0511] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair.

[0512] The present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming the liposomes or vesicles in the presence of the fusion protein.

[0513] The present invention particularly relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0514] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0515] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0516] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0517] - When the second fusion protein binds to the spanning protein forming part of the first fusion protein (directly or indirectly, as described herein), the first and second binding members of the binding pair may be in contact or in close proximity with each other.

[0518] Again, the present invention also relates to a method for providing such liposomes or vesicles, which method comprises at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming the liposomes or vesicles in the presence of the fusion protein.

[0519] The present invention also relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein, wherein:

[0520] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0521] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0522] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0523] - The first and second binding members of the binding pair are present in the environment inside a liposome or vesicle (as defined herein).

[0524] Again, the present invention also relates to a method for providing such liposomes or vesicles, which method comprises at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming the liposomes or vesicles in the presence of the fusion protein.

[0525] The present invention further relates to a liposome or vesicle comprising the first fusion protein and the second fusion protein, wherein:

[0526] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0527] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0528] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0529] - the liposome or vesicle is capable of generating a detectable signal (and in particular a detectable signal generated by the first and second binding members of a binding pair) when the second fusion protein binds (directly or indirectly, as described herein) to a translaminar protein forming part of the first fusion protein.

[0530] Again, the present invention also relates to a method for providing such liposomes or vesicles, which method comprises at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming the liposomes or vesicles in the presence of the fusion protein.

[0531] The present invention further relates to a liposome or vesicle comprising the first fusion protein and the second fusion protein, wherein:

[0532] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0533] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0534] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0535] - When a ligand for the translamellar protein present in the environment outside the liposome or vesicle binds to the translamellar protein, the liposome or vesicle produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or a change in such a signal produced by the first and second binding members of a binding pair).

[0536] Again, the present invention also relates to a method for providing such liposomes or vesicles, which method comprises at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming the liposomes or vesicles in the presence of the fusion protein.

[0537] In a particular aspect, the invention relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein, wherein:

[0538] - said first fusion protein comprises a binding domain or a binding unit as a first binding member of a binding pair, and said second fusion protein comprises a binding domain or a binding unit as a second binding member of said binding pair, wherein said first and second binding members of said binding pair are such that they are capable of generating a detectable signal when they are in contact with each other or in close proximity to each other; and

[0539] - said first fusion protein comprises a translaminar protein (as described herein) fused directly or via a suitable linker to said first binding member of a binding pair; and

[0540] - the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translaminar protein, which protein is fused directly or via a suitable linker to the second binding member of the binding pair; and

[0541] - When an agonist of the translamellar protein present in the environment outside the liposome or vesicle binds to the translamellar protein, the liposome or vesicle produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or a change in such a signal produced by the first and second binding members of a binding pair).

[0542] Again, the present invention also relates to a method for providing such liposomes or vesicles, which method comprises at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming the liposomes or vesicles in the presence of the fusion protein.

[0543] Such liposomes or vesicles comprising such first and second fusion proteins may be as further described herein, and preferably such that they have the translaminar protein appropriately anchored or otherwise incorporated into and spanning the wall or membrane of the liposome or vesicle, more preferably such that at least a portion of the amino acid sequence of the translaminar protein extends (as defined herein) into the environment outside the liposome or vesicle, and at least one other portion of the amino acid sequence of the translaminar protein extends (as defined herein) into the environment inside the liposome or vesicle.

[0544] - When the second fusion protein binds to the translaminar protein forming part of the first fusion protein (directly or indirectly, as described herein), the liposome or vesicle is also preferably such that the first and second binding members of the binding pair are in contact or in close proximity with each other. It will be clear to the skilled person that this generally means that the first and second binding members of the binding pair will be present in the same environment (as defined herein) relative to the wall or membrane of the liposome or vesicle. Preferably, the liposome or vesicle is such that both the first and second binding members of the binding pair are present in the environment inside the liposome or vesicle (as defined herein).

[0545] Again, in aspects of the invention involving liposomes or vesicles comprising such first and second fusion proteins, the translaminar proteins, proteins that can directly or indirectly bind to the translaminar proteins, members of the binding pairs and any linkers used can be as further described herein.

[0546] In a further aspect, the present invention also relates to methods, and in particular to assay methods or screening methods using the liposomes or vesicles described herein. As further described herein, such assays and screening methods can be particularly useful for identifying compounds and other chemical entities that bind (and in particular specifically bind) to a translaminar protein, can modulate a translaminar protein, and / or modulate signaling, signaling pathways, and / or biological or physiological activities involving a translaminar protein, its signaling, and / or its signaling pathways. Thus, the liposomes or vesicles described herein can be used in methods for identifying compounds or other chemical entities that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) modulators of a translaminar protein.

[0547] The present invention also relates to the use of the liposomes or vesicles described herein, in particular in assay and screening methods and techniques. Such methods and uses may again be as further described herein for the methods and uses of the device of the present invention.

[0548] Likewise, in all these aspects, such liposomes or vesicles and their uses are preferably as further described herein.

[0549] It will be clear to the skilled person that the compounds discovered, developed, produced and / or optimized using the methods and techniques described herein can be used for any suitable or desired purpose. The purpose is generally related to the target for which the compound is screened / generated, the signal transduction, pathway and / or mechanism of action associated with the target, and / or the biological, physiological and / or pharmacological functions involved in the target, pathway, signal transduction and / or mechanism of action. Typically and preferably, the compound of the present invention will be such, and / or will be selected so that it can regulate the target, signal transduction, pathway, mechanism of action and / or the biological, physiological and / or pharmacological function in a desired or expected manner. As described herein, the regulation can take any desired or expected form, including but not limited to the up-regulation and down-regulation of the target, signal transduction, pathway, mechanism of action and / or the biological, physiological and / or pharmacological function. Therefore, the compound of the present invention can be used, for example, as an agonist, antagonist, inverse agonist, inhibitor or another type of regulator (e.g., allosteric regulator) of the target and / or its signal transduction, pathway, mechanism of action and / or the biological, physiological and / or pharmacological function. All of this can be determined using appropriate in vitro, cellular and / or in vivo assays (e.g., appropriate efficacy or potency assays) and / or appropriate animal models, depending on the specific target, signaling, pathway, mechanism of action and / or the biological, physiological and / or pharmacological function involved. Suitable assays and models will be clear to the skilled person.

[0550] Typically, when a compound of the invention is an agonist (or antagonist) of a target, it will also be an agonist (or antagonist) of the signaling, pathway, mechanism of action and / or the biological, physiological and / or pharmacological function in which the target is involved. However, as will be clear to the skilled person, it is also possible (and not excluded from the scope of the invention) that the compound of the invention is an agonist (or antagonist) of the target or its signaling, for example but not limited to any kind of hypothesis or explanation, but the action as an agonist (or antagonist) of the target or its signaling results in an action as an antagonist (or antagonist) in terms of the biological, physiological and / or pharmacological function in which the target or signaling is involved.

[0551] In one aspect of the practice of the invention, the devices and methods described herein will be used to test whether a compound or ligand present in an environment [A] (e.g., in the extracellular environment if the invention is practiced in a cell, or in the environment outside of a liposome or vesicle if the invention is practiced in a liposome or vesicle) is capable of producing a detectable signal when it is contacted with a device of the invention (i.e., in a manner that allows the compound or ligand to bind to a binding site (8) on a translaminar protein (2)). Similarly, when the methods and devices of the invention are used to screen a panel, series or library of compounds or ligands, the methods and devices of the invention will be used to determine which compounds or ligands from the panel, series or library produce a detectable signal (i.e., a "hits").

[0552] Typically, in the present invention, the detectable signal will be measured by measuring the signal generated (or can be) generated by the binding pair (6 / 7) (i.e., the signal generated when the first member (6) and the second member (7) are in contact with each other, close to each other, or otherwise associated with each other to generate a detectable signal). It should be noted that, in the present invention, the change in the signal is usually measured, and such change is also included in the term "generating a detectable signal" as used herein.

[0553] The change may be an increase in the signal compared to a basal level (which basal level may also be below the detection limit of the device used to measure the signal, in which case a signal will be detected in the presence of the ligand of the compound where essentially no signal was measured before, and this is also included in the term "increase in the signal"...

Claims

1. A device comprising at least the following elements: - a boundary layer separating the first environment and the second environment; - translaminins; - a first ligand for a translaminar protein present in a first environment; - a second ligand for the translaminar protein present in a second environment; and - a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; in: The translaminar protein is a GPCR; The second ligand is a G protein or a G protein complex; The first binding member of the binding pair is part of a first fusion protein comprising the first binding member, wherein the first binding member is fused or linked to the GPCR directly or via a suitable linker or spacer; The second binding member of the binding pair is part of a second fusion protein comprising the second binding member, wherein the second binding member is fused or linked directly or via a suitable linker or spacer to an immunoglobulin single variable domain capable of binding to a G protein or a G protein complex.

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

3. The device of claim 1, wherein the boundary layer is a wall or membrane of a liposome or vesicle, and wherein the first environment is the environment outside the liposome or vesicle and the second environment is the environment inside the liposome or vesicle.

4. A device comprising at least the following elements: - a boundary layer separating the first environment and the second environment; - translaminins; - a second ligand for the translaminar protein present in a second environment; and - a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; in: The translaminar protein is a GPCR; The second ligand is a G protein or a G protein complex; The first binding member of the binding pair is part of a first fusion protein comprising the first binding member, wherein the first binding member is fused or linked to the GPCR directly or via a suitable linker or spacer; The second binding member of the binding pair is part of a second fusion protein comprising the second binding member, wherein the second binding member is fused or linked directly or via a suitable linker or spacer to an immunoglobulin single variable domain capable of binding to a G protein or a G protein complex.

5. The device of claim 4, wherein the boundary layer is a cell wall or a cell membrane of a cell, and wherein the first environment is an extracellular environment and the second environment is an intracellular environment.

6. The device of claim 4, wherein the boundary layer is a wall or membrane of a liposome or vesicle, and wherein the first environment is the environment outside the liposome or vesicle and the second environment is the environment inside the liposome or vesicle.

7. A method comprising the following steps: a) providing a device according to any one of claims 4 to 6; and b) adding a first ligand to a first environment of said device.

8. The method according to claim 7, further comprising the steps of: c) measuring a signal produced by the binding pair and / or measuring a change in a signal produced by the binding pair.

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