CELL COMPOSITION, COMPLEX AND IN VITRO METHODS OF IDENTIFICATION OF COMPOUNDS THAT ARE ORTHOSTERIC O

专利摘要:
protein binding domains, complexes, cellular composition, uses of said domains, method of capturing a gpcr in a functional conformational state, kit and method of identifying compounds capable of binding to a functional conformational state of a gpcr. the present invention relates to the field of structural biology and gpcr signaling. in particular, the present invention relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a g protein-coupled receptor (gpcr). more specifically, the present invention provides protein binding domains that are capable of increasing the stability of a functional conformational state of a gpcr, in particular increasing the stability of a gpcr in its active conformational state. The protein binding domains of the present invention can be used as a tool for the structural and functional characterization of g protein-coupled receptors linked to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting gpcrs. further, the invention also encompasses the diagnostic, prognostic and therapeutic utility of these protein-binding domains for gpcr-related diseases.
公开号:BR112013001180B1
申请号:R112013001180-7
申请日:2011-07-18
公开日:2022-01-11
发明作者:Jan Steyaert；Els Pardon；Søren Rasmussen；Juan Fung；Brian Kobilka；Toon Laeremans
申请人:Vrije Universiteit Brussel；Vib Vzw；The Board Of Trustees Of The Leland Stanford Junior University；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention concerns the field of GPCR signaling and structural biology. In particular, the present invention relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a G protein-coupled receptor (GPCR). More specifically, the present invention provides protein binding domains that are capable of increasing the stability of a functional conformational state of a GPCR, increasing the stability of a particular GPCR in its active conformational state. The protein binding domains of the present invention can be used as a tool for structural and functional characterization of G protein-coupled receptors linked to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting GPCRs. Furthermore, the invention also encompasses the diagnostic, prognostic and therapeutic utility of these protein-binding domains for GPCR-related diseases. BACKGROUND
[002] G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in the human genome. They play essential roles in physiological responses to a diverse set of ligands such as biogenic amines, amino acids, peptides, proteins, prostanoids, phospholipids, fatty acids, nucleosides, nucleotides, Ca2+ ions, odorants, bitter and sweet flavors, pheromones and protons ( Heilker et al. 2009). GPCRs are therapeutic targets for a wide range of diseases. GPCRs are characterized by seven transmembrane domains with an extracellular amino terminus and an intracellular carboxyl terminus, and are also called seven transmembrane or heptahelical receptors (Rosenbaum et al. 2009). Rhodopsin, a GPCR that is highly specialized for efficient light detection, has been the paradigm for GPCR signaling and structural biology due to its biochemical stability and natural abundance in bovine retina (Hofmann et al. 2009). In contrast, many GPCRs exhibit complex functional behavior modulating the activity of multiple G protein isoforms as well as G protein-independent signaling pathways (eg, β-arrestin). In some cases, a GPCR may exhibit basal activity for a specific signaling pathway even in the absence of a ligand. Orthosteric ligands that act on a GPCR can have a spectrum of effects on downstream signaling pathways. Full agonists maximally activate the receptor. Partial agonists elicit submaximal stimulation even at saturated concentrations. Reverse agonists inhibit basal activity, while neutral antagonists have no effect on basal activity, but competitively block the binding of other ligands.
[003] The complex behavior of GPCRs for hormones and neurotransmitters can be attributed to their structural plasticity (Kobilka and Deupi, 2007). Evidence from functional and biophysical studies shows that GPCRs can exist in multiple functionally distinct conformational states (Kobilka and Deupi 2007). Although this structural plasticity and dynamic behavior are essential for normal function, they contribute to its biochemical instability and difficulty in obtaining high resolution crystal structures. So far, crystal structures have been reported for human β2AR (Rasmussen et al. 2007; Rosenbaum et al. 2007; Cherezov et al. 2007; Hanson et al. 2008), avian β1AR (Warne et al. 2008), and human adenosine A2 receptor (Jaakola et al. 2008). Although rhodopsin can be crystallized from unaltered protein isolated from native tissue, these other GPCRs required expression in recombinant systems, stabilization of an inactive state by a reverse agonist, and biochemical modifications to stabilize the receptor protein. The first crystal structure of β2AR was stabilized by a selective Fab (Rasmussen et al. 2007). Subsequent structures were obtained from the β2AR and the adenosine A2 receptor with the help of protein engineering: the insertion of T4Lysozyme into the third intracellular loop as originally described for the β2AR (Rosenbaum et al. 2007). Finally, avian β1AR crystals were grown from engineered protein with terminal amino and carboxyl truncations and deletion of the third intracellular loop, as well as 6 amino acid substitutions that enhanced the thermostability of the purified protein (Warne et al. 2008).
[004] Obtaining structures from an active state of a GPCR is more difficult because this state is relatively unstable. Fluorescence lifetime studies show that β2AR is structurally heterogeneous in the presence of saturated concentrations of a full agonist (Ghanouni et al. 2001). This structural heterogeneity is incompatible with crystal formation. Stabilization of the active state of β2AR requires the presence of its cognate G protein Gs, the stimulating protein for adenylyl cyclase (Yao et al. 2009). So far, the only active-state structure of the GPCR is that of opsin, the ligand-free form of rhodopsin (Park et al. 2008). These crystals were grown at acidic pH (5.5) where opsin was shown to be structurally similar to photoactivated rhodopsin (meta-rhodopsin II) at physiological pH by FTIR spectroscopy. Although β2AR also shows higher basal activity at reduced pH, it is biochemically unstable (Ghanouni et al. 2000).
[005] Unraveling the structures of different functional conformational states of GPCRs in the complex with various natural and synthetic ligands and proteins is valuable both for understanding the mechanisms of GPCR signal transduction as well as for structure-based drug discovery efforts. The development of new direct tools for high resolution structure analysis of the individual conformers of GPCRs is therefore necessary. SUMMARY OF THE INVENTION
[006] A first aspect of the invention pertains to a protein binding domain capable of specifically binding to a functional conformational state of a GPCR.
[007] According to a preferred embodiment, said protein-binding domain is capable of stabilizing a functional conformational state of a GPCR upon binding. Preferably, said protein-binding domain is capable of inducing a functional conformational state in a GPCR upon binding.
[008] According to another preferred embodiment, said functional conformational state of a GPCR is selected from the group consisting of a basal conformational state, either an active conformational state or an inactive conformational state. Preferably, said functional conformational state of a GPCR is an active conformational state.
[009] According to another preferred embodiment, said protein-binding domain is capable of specifically binding to an agonist-bound GPCR and/or enhancing the affinity of a GPCR for an agonist.
[0010] According to another preferred embodiment, said protein-binding domain is capable of increasing the thermostability of a functional conformational state of a GPCR upon binding.
[0011] In a specific embodiment, said protein binding domain is capable of specifically binding to a conformational epitope of said functional conformational state of a GPCR. Preferably, said conformational epitope is an intracellular epitope. More preferably, said conformational epitope is comprised of a binding site for a downstream signaling protein. Most preferably, said conformational epitope is comprised of the G protein binding site.
[0012] Preferably, the protein binding domain of the present invention comprises an amino acid sequence comprising 4 framework regions and 3 complementary determining regions, or any suitable fragment thereof. More preferably, said protein binding domain is derived from a camelid antibody. Most preferably, said protein-binding domain comprises a nanobody sequence, or any suitable fragment thereof. For example, said nanobody comprises a sequence selected from the group consisting of SEQ ID NOs: 1-29, or any suitable fragment thereof.
[0013] According to another preferred embodiment, said GPCR is a mammalian protein, or a plant protein, or a microbial protein, or a viral protein, or an insect protein. Said mammalian protein may be a human protein. In particular, said GPCR is chosen from the group comprising a GPCR from the family of Glutamate GPCRs, a GPCR from the family of Rhodopsin GPCRs, a GPCR from the family of Adhesion GPCRs, a GPCR from the family of Frizzled/Taste2 GPCRs, and a GPCR from Secretin family of GPCRs. More specifically, said GPCR is an adrenergic receptor, such as an α-adrenergic receptor or an α-adrenergic receptor, or wherein said GPCR is a muscarinic receptor, such as an M1 muscarinic receptor or an M2 muscarinic receptor, or a M3 muscarinic receptor, or an M4 muscarinic receptor, or an M5 muscarinic receptor, or wherein said GPCR is an angiotensin receptor, such as an angiotensin II type 1 receptor or an angiotensin II type 2 receptor.
[0014] A second aspect of the invention pertains to a complex comprising (i) a protein-binding domain according to the invention, (ii) a GPCR in a functional conformational state, and (iii) optionally, a receptor ligand . Said receptor ligand may be selected from the group comprising a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or any suitable fragment thereof. Said complex may be in a solubilized form or immobilized on a solid support. In particular, said complex is crystalline. The invention further encompasses a crystalline form of a complex comprising (i) a protein-binding domain, (ii) a GPCR in a functional conformational state, and (iii) optionally, a receptor ligand, wherein said crystalline form is obtained by using a protein-binding domain according to the invention.
[0015] A third aspect of the invention relates to a cellular composition comprising a protein-binding domain according to the invention and/or a complex according to the invention. Preferably, the protein-binding domain comprised in the cellular composition is capable of stabilizing and/or inducing a functional conformational state of a GPCR upon binding of said protein-binding domain.
[0016] A fourth aspect of the invention concerns the use of a protein-binding domain according to the invention or a complex according to the invention or a cellular composition according to the invention to stabilize and/or induce a conformational state functionality of a GPCR.
[0017] According to a preferred embodiment, said protein-binding domain or said cell complex or said composition can be used to crystallize and/or resolve the structure of a GPCR into a functional conformational state.
[0018] The invention also encompasses a method of determining a crystal structure of a GPCR in a functional conformational state, said method comprising the steps of: (i) providing a protein binding domain according to the invention, a GPCR target, and optionally a receptor ligand, and (ii) form a complex of the protein binding domain, the GPCR, and optionally the receptor ligand, and (iii) crystallize said complex of step (ii) to form a crystal ,
[0019] in which the crystal structure is determined from a GPCR in a functional conformational state.
[0020] The above method of determining a crystal structure of a GPCR can complex comprise the step of obtaining the atomic coordinates of the crystal.
[0021] According to another preferred embodiment, said protein-binding domain or said cell complex or said composition can be used to capture a GPCR in a functional conformational state, optionally with a receptor ligand or with one or more downstream signaling proteins.
[0022] The invention thus also encompasses a method of capturing a GPCR in a functional conformational state, said method comprising the steps of: (i) providing a protein binding domain according to the invention and a target GPCR, and (ii) form a complex of the protein-binding domain and the GPCR,
[0023] in which a GPCR is captured in a functional conformational state.
[0024] Further, the invention also encompasses a method of capturing a GPCR in a functional conformational state, said method comprising the steps of: (i) applying a solution containing a GPCR in a plurality of conformational states to a solid support having an immobilized protein-binding domain according to the invention, and (ii) forming a complex of the protein-binding domain and the GPCR, and (iii) removing loosely bound or unbound molecules,
[0025] in which a GPCR is captured in a functional conformational state.
[0026] The above methods of capturing a GPCR in a functional conformational state may comprise the step of purifying the complex.
[0027] According to another preferred embodiment, the invention also relates to the use of the protein-binding domain, or the complex, or the cellular composition, according to the invention, in screening and/or program identification for compounds or conformation-specific (drug) ligands of a GPCR.
[0028] The invention thus also encompasses a method of identifying compounds capable of binding to a functional conformational state of a GPCR, said method comprising the steps of: (i) providing a GPCR and a protein binding domain in accordance with the invention, and (ii) providing a test compound, and (iii) evaluating whether the test compound binds to the functional conformational state of the GPCR, and (iv) selecting a compound that binds to the functional conformational state of the GPCR.
[0029] Preferably, the method described above for identifying other compounds comprises the step of forming a complex comprising the protein-binding domain and the GPCR in a functional conformational state, in accordance with the invention. Said complex may complex comprise a receptor ligand which may be selected from the group comprising a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or any suitable fragment thereof. same. Preferably, said receptor ligand is a full agonist, or a polarized agonist, or a reverse agonist, or antagonist. Preferably, said protein-binding domain and/or said complex is provided essentially in purified form. Alternatively, said protein-binding domain and/or said complex is provided in a solubilized form. Alternatively, said protein-binding domain and/or said complex is immobilized on a solid support. Alternatively, said protein-binding domain and/or said complex are provided in a cellular composition.
[0030] According to another preferred embodiment, the test compound used in the method described above to identify compounds is selected from the group comprising a polypeptide, a peptide, a small molecule, a natural product, a peptidemimetic, a nucleic acid, a lipid, lipopeptide, a carbohydrate, an antibody or any fragment derived therefrom, such as Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising a VL or VH domain, a heavy chain antibody (hcAb), a single domain antibody (sdAb), a minibody, the variable domain derived from camelid heavy chain antibodies (VHH or nanobody), the variable domain of novel shark antibody-derived antigen receptors (VNAR), a protein scaffold including an alphabody, protein A, protein G, projected ankyrin repeat domains (DARPins), fibronectin type III repeats, ankyrin allins, quinotines, engineered CH2 domains (nanoantibodies).
[0031] Preferably, said test compounds are labeled. In addition, a library of test compounds can be used. Furthermore, the method described above for identifying compounds may be a high throughput screening method.
[0032] According to another specific embodiment, the protein-binding domain or the cellular complex or composition, all according to the invention, can be used to diagnose or predict a GPCR-related disease, such as cancer, autoimmune disease , infectious disease, neurological disease, cardiovascular disease.
[0033] A fifth aspect of the invention pertains to a pharmaceutical composition comprising a therapeutically effective amount of a protein-binding domain according to the invention and at least one of a pharmaceutically acceptable carrier, adjuvant or diluents.
[0034] A sixth aspect of the invention relates to the use of a protein-binding domain according to the invention or a pharmaceutical composition according to the invention to modulate GPCR signaling activity, more specifically, to block mediated signaling. by G protein.
[0035] The protein-binding domain or pharmaceutical composition according to the invention can also be used in the treatment of a GPCR-related disease, such as cancer, autoimmune disease, infectious disease, neurological disease, cardiovascular disease.
[0036] A seventh aspect of the invention relates to a kit comprising a protein-binding domain according to the invention or a complex according to the invention or a cellular composition according to the invention.
[0037] Other applications and uses of the amino acid and polypeptide sequences of the invention will become apparent to the skilled person from the further disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGURE 1. β2AR-SPECIFIC NANOBODIES BIND AND STABILIZE AN ACTIVE RECEPTOR STATE.
[0039] (a) Representative size exclusion chromatography (SEC) trace for Nb80. Purified β2AR (20 μM) bound to an agonist (β2AR agonist) was incubated with and without 40 μM Nb80 (black and blue, respectively) for 2 h at room temperature before analyzing by FPLC. In the presence of Nb80, the elution peak of the β2AR agonist increases in UV absorbance (280 nm) and elutes at an earlier volume than the β2AR agonist alone, with a simultaneous decrease in the Nb80 elution peak (green), suggesting the formation of the β2AR-Nb80 agonist complex. Incubation of β2AR (20 μM) bound to a reverse agonist with Nb80 (red) resulted in a smaller shift and smaller increase in UV absorbance when compared to the β2AR-Nb80 agonist complex.
[0040] (b) Dose-response competition binding experiments on Sf9 insect cell membranes expressing β2AR. Seven nanobodies that bound β2AR by SEC were individually incubated for 90 min at room temperature with β2AR expression membranes. All seven nanobodies increased the affinity of β2AR for (-)-isoproterenol (Table 3). Nb80 (blue) was selected as the lead nanobody. Data represent the mean ± s.e. of two independent experiments performed in triplicate.
[0041] (c) A fluorescence-based functional assay using purified receptor labeled with monobromobimane (mBBr) shows that 1 μM Nb80 (blue) stabilizes a more active state of β2AR (bound to the full agonist (-)-isoproterenol) when compared to the receptor in the absence of Nb80 (black). The active state is characterized by a quenching of mBBr fluorescence and a red shift in mBBr fluorescence (Yao et al., 2009).
[0042] FIGURE 2. REPRESENTATIVE DOT BLOTS SHOWING NANOBODY SPECIFICITY FOR THE TERTIARY STRUCTURE OF β2AR.
[0043] (a) Equal amounts of native or purified SDS-denatured β2AR bound to an agonist (top and middle, respectively), or native β2AR bound to a reverse agonist (bottom) were labeled in triplicate on nitrocellulose strips. Strips were blocked with 5% non-fat dry milk in PBS (pH 7.4) with 0.05% Tween-20 and then incubated with 1 mg/ml of the indicated nanobodies diluted in blocking buffer. Binding of the nanobodies was detected by a primary mouse anti-histidine antibody (a-6His), followed by incubation with IR-800 labeled secondary goat anti-mouse antibody. M1 antibody, which recognizes the linear FLAG epitope, was tagged with Alexa-688 and directly detected β2AR. The dot-shaped markings were scanned and imaged using the Odyssey Infrared Imaging System (Li-cor Biosciences). Labels detecting nanobodies were processed separately from labels detecting β2AR by M1 since two different channels (800 nm versus 700 nm, respectively) were used for imaging; therefore, labels cannot be directly compared and quantified (ie, comparison of nanobody binding versus M1 binding is only qualitative in nature).
[0044] (b) Representative Dot Blots showing nanobodies with reduced binding to natively folded β2AR.
[0045] FIGURE 3. SELECTIVE CONNECTION OF NANOBODIES TO THE ACTIVE STATE OF THE RECEPTOR
[0046] Purified β2AR (20 μM) bound to an agonist was incubated with and without 40 μM nanobodies (black and blue, respectively) for 2 h at room temperature before analyzing by size exclusion chromatography. Samples of β2AR (20 μM) bound to a reverse agonist in the presence of nanobodies (red) were also analyzed. In the presence of several nanobodies (Nb72, Nb65, Nb71, Nb69, Nb67 and Nb84), the elution peak of the β2AR agonist increases in UV absorbance (280 nm) and elutes in an earlier volume (black line) than the β2AR agonist. β2AR alone (blue), with a simultaneous decrease in Nb80 elution peak (green), suggesting formation of the β2AR-Nb80 agonist complex. The formation of a β2AR-reverse agonist-Nb80 complex is not observed (red line).
[0047] FIGURE 4. SELECTIVE CONNECTION OF NANOBODIES TO THE ACTIVE STATE OF THE RECEPTOR
[0048] Purified β2AR (20 μM) bound to an agonist was incubated with and without 40 μM nanobodies (black and blue, respectively) for 2 h at room temperature before analyzing by size exclusion chromatography.
[0049] FIGURE 5. FLUORESCENCE EMISSION SPECTRUM SHOWING CONFORMATIONAL CHANGES INDUCED BY MONOBROMOBIMAN-LABELED B2AR NANOBODY
[0050] Nanobodies that increase agonist binding affinity for the β2AR stabilize an active state of the receptor. A fluorescence-based functional assay using purified receptor labeled with monobromobimane (mBBr) shows that 1 μM of nanobodies 65, 67, 69, 71, 72 and 84 (red) stabilizes a more active state of β2AR (bound to the full agonist isoproterenol) when compared to the receptor in the absence of nanobodies (black). This active state is characterized by a quenching of mBBr fluorescence and a red shift in mBBr fluorescence (Yao et al., 2009).
[0051] FIGURE 6. EFFECT OF Nb80 ON THE STRUCTURE AND FUNCTION OF β2AR
[0052] (a) The drawing illustrates the movement of the environmentally sensitive bimanne probe attached to Cys2656.27 at the cytoplasmic end of TM6 from a more buried hydrophobic environment to a more polar position exposed to solvent during receptor activation which results in a decrease in fluorescence seen in Figure 6b-c.
[0053] (b)-(c) Fluorescence emission spectra showing conformational changes induced by monobromobiman-labeled β2AR ligand reconstituted in high density lipoprotein particles (mBB-β2AR/HDL) in the absence (solid black line) or presence of the full agonist isoproterenol (ISO, green dotted line), reverse agonist ICI-118.551 (ICI, black dotted line), Gs heterotrimer (red solid line), nanobody-80 (Nb80, blue solid lines), and Gs combinations with ISO (red spaced dotted line), Nb80 with ISO (blue spaced dotted line), and Nb80 with ICI (blue dotted line).
[0054] (d)-(f) Ligand binding curves for ISO competing against [3H]-dihydroalprenolol ([3H]-DHA) for d, β2AR/HDL reconstituted with Gs heterotrimer in the absence or presence of GTPYS, and, β2AR/HDL in the absence and presence of Nb80, ef, β2AR-T4L/HDL in the absence and presence of Nb80. Error bars represent standard errors.
[0055] FIGURE 7. PACKAGING OF AGONIST-β2AR-T4L-Nb80 COMPLEX IN CRYSTALS FORMED IN LIPID CUBIC PHASE.
[0056] Three different views of the structure of β2AR indicated in orange, Nb80 in blue, and agonist in green. T4 lysozyme (T4L) could not be modeled due to weak electron density. Its position is likely indicated by the light blue circle with black dotted lines connected to the intracellular ends of TM5 and TM6 where it is fused in the β2AR-T4L construct. PyMOL (http://www.pymol.org) was used for the preparation of all figures.
[0057] FIGURE 8. COMPARISON OF CRYSTAL STRUCTURES STABILIZED WITH REVERSE AGONIST AND Nb80 AGONIST OF β2AR.
[0058] The structure of the carazolol reverse agonist linked to β2AR-T4L (β2AR-Cz) is shown in blue with carazolol in yellow. The structure of agonist-bound β2AR-T4L and stabilized with Nb80 (β2AR-Nb80) is shown in orange with the agonist in green. These two structures were aligned using Pymol's alignment function. (a) Side view of the overlapping structures showing significant structural changes in the intracellular and G-protein facing part of the receptors. (b) The side view following 90 degrees of rotation on the vertical axis. (c) Comparison of extracellular ligand binding domains showing modest structural changes.
[0059] FIGURE 9. INTRACELLULAR DOMAIN STABILIZED WITH Nb80 COMPARED WITH INACTIVE STRUCTURES OF β2AR AND OPSIN.
[0060] (a) Side view of β2AR (orange) with light blue (CDR1) and blue (CDR3) Nb80 CDRs interacting with the receptor. (b) Closer view focusing on CDRs 1 and 3 introducing the β2AR. The side chains at TM3, 5, 6, and 7 within 4 Å of the CDRs. The larger CDR3 penetrates 13 Â into the receiver. (c) Interaction of CDR1 and CDR3 seen from the intracellular side. (d) Agonist-bound β2AR-T4L stabilized with Nb80 (β2AR-Nb80) is overlapped with the inactive carazolol-linked structure of β2AR-T4L (β2AR-Cz). The ion lock interaction between Asp3.49 and Arg3.50 of the DRY motif in TM3 is broken in the β2AR-Nb80 structure. The intracellular end of TM6 is moved out and away from the receptor nucleus. The arrow indicates an 11.4 Â change in distance between the α-carbon of Glu6.30 in the β2AR-Cz and β2AR-Nb80 structures. The intracellular ends of TM3 and TM7 move to the nucleus by 4 and 2.5 Â respectively, while TM5 moves out by 6 Â. (e) The structure of β2AR-Nb80 superimposed with the opsin structure crystallized with the C-terminal peptide of Gt (transducin).
[0061] FIGURE 10. INTRACELLULAR DOMAIN STABILIZED WITH β2AR Nb80 COMPARED TO OPSIN STRUCTURES.
[0062] (a) Interactions between β2AR and Nb80. (b) Interactions between opsin and the carboxyl-terminal peptide of transducin.
[0063] FIGURE 11. REARRANGEMENT OF TRANSMEMBRANE SEGMENT PACKAGING INTERACTIONS UNDER AGONIST BINDING
[0064] (a) Conditioning interactions that stabilize the inactive state are observed between Pro211 in TM5, Ile121 in TM3, Phe282 in TM6 and Asn316 in TM5. (b) Inward movement of TM5 upon agonist binding disrupts the packaging of Ile121 and Pro211 resulting in a rearrangement of interactions between Ile121 and Phe282. These changes contribute to a rotational movement in and out of TM6 and an inward movement of TM7.
[0065] FIGURE 12. DIFFERENT NANOBODY AMINO ACID SEQUENCES ELEVATED AGAINST β2AR. The sequences were aligned using standard software tools. CDRs were defined according to the IMGT numbering (Lefranc et al., 2003).
[0066] FIGURE 13. EFFECT OF NB80 ON THE THERMAL STABILITY OF THE β2AR RECEPTOR. Comparison of melting curves of detergent-solubilized β2AR (DDM) bound to agonist (isoproterenol) in the presence and absence of Nb80. The evident melting temperature for β2AR without Nb80 is 12.0°C. The evident melting temperature for β2AR with Nb80 is 24°C.
[0067] FIGURE 14. EFFECT OF Nb80 ON TEMPERATURE-INDUCED AGGREGATION OF THE β2AR RECEPTOR.
[0068] A. Detergent-solubilized β2AR (DDM) was heated for 10 minutes at 50°C in the presence of Nb80 or isoproterenol and receptor aggregation was analyzed by SEC. B. Temperature dependence of the isoproterenol-bound receptor in the absence of Nb80.
[0069] FIGURE 15. Nb80 HAS SMALL EFFECT ON B2AR BINDING TO REVERSE AGONIST ICI-118.551
[0070] β2AR or β2AR-T4L were reconstituted in HDL particles and agonist competition binding experiments were performed in the absence or presence of Nb80. Ligand binding curves for the reverse agonist ICI-118551 competing against [3H]-dihydroalprenolol ([3H]-DHA) for a, β2AR/HDL in the absence and presence of Nb80, eb, β2AR-T4L/HDL in the absence and presence of Nb80.
[0071] FIGURE 16. Nb80 INCREASES β2AR AFFINITY FOR AGONISTS BUT NOT FOR ANTAGONISTS.
[0072] Competitive ligand binding experiments were performed on membranes derived from commercial insect cells containing complete β2AR in the absence or presence of Nb80. Dose shift curves of radioligand dependent on the presence of Nb80 and an irrelevant Nanobody (Irr Nb) for two representative agonists (isoproterenol, procaterol) and two representative antagonists (ICI-118,551 and carvedilol).
[0073] FIGURE 17: SEQUENCE ALIGNMENT OF HUMAN β1AR AND HUMAN β2AR.
[0074] The β2-adrenoreceptor amino acids that interact with Nb80 at the β2AR-Nb80 interface are underlined.
[0075] FIGURE 18: Nb80 SELECTIVELY TURNS ON THE ACTIVE CONFORMATION OF THE HUMAN β1AR RECEPTOR
[0076] Ligand binding curves for agonists and reverse agonists competing against [3H]-dihydroalprenolol ([3H]-DHA). A) Isoproterenol agonist (ISO) binding to β2AR in the presence and absence of Nb80. B) ICI-118.551 reverse agonist (ICI) binding to β2AR in the presence and absence of Nb80. C) Isoproterenol agonist (ISO) binding to β1AR in the presence and absence of Nb80. D) CGP20712A reverse agonist (CPG) binding to β1AR in the presence and absence of Nb80. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[0077] The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but solely by the claims. Any reference marks in the claims should not be interpreted as limiting the scope. The drawings described are schematic only and are non-limiting. In drawings, the size of some of the elements may be exaggerated and drawn without scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun, for example "a" or "the", it includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and others in the description and claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in sequences other than those described or illustrated herein.
[0078] Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In general, the nomenclatures used in association and the techniques of molecular and cell biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification unless otherwise indicated. See, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements 2002)
[0079] The term "protein binding domain" generally refers to any naturally occurring or non-naturally occurring molecule thereof that is capable of binding a protein or peptide using specific intermolecular interactions. A variety of molecules can function as protein binding domains, including, but not limited to, proteinaceous molecules (protein, peptide, in protein form or containing protein), nucleic acid molecules (nucleic acid, in nucleic acid form, containing nucleic acid), and carbohydrate molecules (carbohydrate, in carbohydrate form, containing carbohydrate). A more detailed description can still be found in the descriptive report.
[0080] 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, chemical amino acids or biochemically modified or derivatized, and polypeptides having modified peptide backbones.
[0081] As used herein, the terms "multiprotein complex" or "protein complex" or simply "complex" refer to a group of two or more associated polypeptide chains. Proteins in a protein complex are linked through non-covalent protein-protein interactions. The "quaternary structure" is the structural arrangement of the associated proteins folded into the protein complex. A "multimeric complex" refers to a protein complex as defined herein which may complex comprise a non-proteinaceous molecule.
[0082] As used herein, the terms "nucleic acid molecule", "polynucleotide", "poly(nucleic acid)", "nucleic acid" are used interchangeably and refer to a polymeric form of the nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogues thereof. Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence , control regions, RNA isolated from any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
[0083] As used herein, the terms "ligand" or "receptor ligand" mean a molecule that specifically binds to a GPCR, either intracellularly or extracellularly. A ligand can be, without the intent of being limiting, a protein, a (poly)peptide, a lipid, a small molecule, a protein scaffold, a nucleic acid, an ion, a carbohydrate, an antibody or an antibody fragment. , such as a nanobody (all as defined here). A binder can be either synthetic or naturally occurring. A linker also includes a "native linker" which is a linker that is an endogenous, natural linker to a native GPCR. A "modulator" is a ligand that increases or decreases the signaling activity of a GPCR (i.e., through an intracellular response) when it comes into contact, e.g., binds to a GPCR that is expressed in a cell. This term includes agonists, full agonists, partial agonists, reverse agonists, and antagonists, of which a more detailed description can be found further in the specification.
[0084] The term "conformation" or "conformational state" of a protein generally refers to the range of structures that a protein can adopt at any given time. One of skill in the art will recognize that determinants of conformation or conformational state include the primary structure of a protein as reflected in a protein's amino acid sequence (including modified amino acids) and the environment that surrounds the protein. The conformation or conformational state of a protein also concerns structural features such as secondary structures of the protein (e.g. α helix, β sheet, among others), tertiary structure (e.g. the three-dimensional folding of a polypeptide chain), and quaternary structure (eg, interactions of a polypeptide chain with other protein subunits). Post-translational and other modifications to a polypeptide chain such as linker binding, phosphorylation, sulfation, glycosylation, or hydrophobic group attachments, among others, can influence the conformation of a protein. In addition, environmental factors such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and cofactors, among others, can affect protein conformation. The conformational state of a protein can be determined by any functional assay for activity or binding to another molecule or by physical methods such as X-ray crystallography, NMR, or spin labeling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993. A "specific conformational state" is any subset of the range of conformations or conformational states that a protein can adopt.
[0085] A "functional conformation" or a "functional conformational state", as used herein, refers to the fact that proteins have different conformational states having a dynamic range of activity, ranging in particular from no activity to maximum activity. It should be clear that "a functional conformational state" is meant to encompass any conformational state of a GPCR, having some activity, including no activity; and it is not meant to encompass the denatured states of proteins.
[0086] As used herein, the terms "complementarity determining region" or "CDR" within the context of antibodies refer to variable regions of H (heavy) or L (light) chains (also abbreviated as VH and VL, respectively) and contains the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the antibody's basic specificity to a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions". The CDRs represent non-contiguous stretches of amino acids within the variable regions but, irrespective of the species, the positional locations of these critical amino acid sequences within the heavy and light variable chain regions were observed to have similar locations within the variable chain amino acid sequences. The variable heavy and light chains of all canonical antibodies each have 3 CDR regions, each not contiguous with the others (labeled L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy ( H). Nanobodies, in particular, generally comprise a single amino acid chain which can be considered to comprise 4 "framework sequences or regions" or FR and 3 "complementary determining regions" or CDRs. Nanobodies have 3 CDR regions, each not contiguous with the others (named CDR1, CDR2, CDR3). The delineation of the FR and CDR sequences is based on the unique IMGT numbering system for V domains and V-like domains (Lefranc et al. 2003).
[0087] An "epitope", as used herein, refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation that is unique to the epitope. In general, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, it consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.
[0088] A "conformational epitope", as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded three-dimensional conformation of the polypeptide. In general, a conformational epitope consists of amino acids that are discontinuous in the linear sequence that enter together in the folded structure of the protein. However, a conformational epitope can also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded three-dimensional conformation of the polypeptide (and not present in a denatured state). In multiprotein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together in the folding of the different folded polypeptides and their association into a single quaternary structure. Similarly, conformational epitopes herein may also consist of a linear sequence of amino acids of one or more polypeptides that come together and adopt a conformation that is unique to the quaternary structure.
[0089] The term "specificity", as used herein, refers to the ability of a protein-binding domain, in particular an immunoglobulin or an immunoglobulin fragment, such as a nanobody, to preferentially bind to an antigen, versus a different antigen, and does not necessarily imply high affinity.
[0090] The term "affinity", as used herein, refers to the degree to which a protein-binding domain, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a nanobody, binds to an antigen to shift the balance of antigen and protein-binding domain to the presence of a complex formed by their binding. Thus, for example, where an antigen and antibody (fragment) are combined in relatively equal concentration, a high-affinity antibody (fragment) will bind to the available antigen to shift the equilibrium to high concentration of the resulting complex. The dissociation constant is commonly used to describe the affinity between the protein-binding domain and the antigenic target. Typically, the dissociation constant is less than 10-5M. Preferably, the dissociation constant is less than 10-6M, more preferably, less than 10-7M. Most preferably, the dissociation constant is less than 10 -8 M
[0091] The terms "specifically binding" and "specific binding", as used herein, generally refer to the ability of a domain to bind to protein, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a nanobody, to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (eg, more than about 1000 or 10,000-fold). Within the context of the spectrum of conformational states of GPCRs, the terms particularly refer to the ability of a protein-binding domain (as defined herein) to preferentially recognize and/or bind to a particular conformational state of a GPCR when compared to another conformational state. For example, an active state-selective protein-binding domain 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 thus will have a higher affinity for said active conformational state. The terms "specifically binds", "selectively binds", "preferably binds", and grammatical equivalents thereof, are used interchangeably herein. The terms "specific conformational" or "selective conformational" are also used interchangeably herein.
[0092] An "antigen", as used herein, means a molecule capable of eliciting an immune response in an animal. Within the context of the GPCR conformational state spectrum, said molecule comprises a conformational epitope of a GPCR in a particular conformational state that is not formed or less accessible in another conformational state of said GPCR.
[0093] A "deletion" is defined herein as a change in amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent when compared to an amino acid sequence or nucleotide sequence of a polypeptide or parent nucleic acid. Within the context of a protein, a deletion may involve 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 a fragment thereof may contain more than one deletion. Within the context of a GPCR, a deletion can also be a loop deletion, or an N and/or C-terminal deletion.
[0094] An "insertion" or "addition" is that change in an amino acid or nucleotide sequence that has resulted in the addition of one or more amino acid or nucleotide residues, respectively, when compared to an amino acid or nucleotide sequence of a parent protein. "Insertion" in general refers to adding to one or more amino acid residues within an amino acid sequence a polypeptide, while "addition" may be an insertion or may refer to amino acid residues added to an N-terminus or C, or both terms. Within the context of a protein or a fragment thereof, an insertion or addition is usually 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 insert.
[0095] A "substitution", as used herein, is the result of replacing one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, when compared to an amino acid sequence or nucleotide sequence of a parent protein or a fragment of the parent protein. same. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions that have substantially no effect on the activity of the protein. By conservative substitutions are intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gln; be, thr; lys, arg; cys, met; and phe, tyr, trp.
[0096] "Crystal" or "crystal structure", as used herein, refers to a solid material whose constituent atoms, molecules, or ions are arranged in an ordered repeating pattern that spans all three spatial dimensions. The process of forming a crystal structure of a fluid or materials dissolved in the fluid is often referred to as "crystallization" or "crystallogenesis". Protein crystals are almost always grown in solution. The most common approach is to gradually decrease the solubility of your component molecules. Crystal growth in solution is characterized by two steps: nucleation of a microscopic crystallite (possibly having only 100 molecules), followed by growth of that crystallite, ideally to a diffraction quality crystal.
[0097] "X-ray crystallography", as used herein, is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays impinges on a crystal and scatters in many specific directions. From the angles and intensities of these scattered beams, a crystallographer can produce a three-dimensional picture of the electron density within the crystal. From this electron density, the average positions of atoms in the crystal can be determined, as well as their chemical bonds, their disorder, and various other information.
[0098] The term "atomic coordinates", as used herein, refers to a set of three-dimensional coordinates for atoms within a molecular structure. In one embodiment, atomic coordinates are obtained using X-ray crystallography in accordance with methods well known to those of ordinary skill in the art of biophysics. Briefly described, X-ray diffraction patterns can be obtained through X-rays diffracting out of a crystal. The diffraction data is used to calculate an electron density map of the unit cell comprising the crystal; said maps are used to establish the positions of atoms (ie atomic coordinates) within the unit cell. Those skilled in the art understand that a set of structure coordinates determined by X-ray crystallography contains standard errors. In other embodiments, atomic coordinates can be obtained using other experimental biophysical structure determination methods that may include electron diffraction (also known as electron crystallography) and nuclear magnetic resonance (NMR) methods. In still other embodiments, atomic coordinates can be obtained using molecular modeling tools that can be based on one or more ab initio protein folding, energy minimization, and homology-based modeling algorithms. These techniques are well known to persons of ordinary skill in biophysical and bioinformatics techniques.
[0099] "Solving the structure" as used herein refers to determining the arrangement of atoms or the atomic coordinates of a protein, and is often done by a biophysical method, such as X-ray crystallography.
[00100] The terms "compound" or "test compound" or "candidate compound" or "drug candidate compound" as used herein describe any molecule, naturally occurring or synthetic, that is tested in an assay, such as a drug discovery screening or trial. As such, these compounds comprise organic or inorganic compounds. Compounds include polynucleotides, lipids or hormone analogues that are characterized by low molecular weights. Other biopolymeric organic test compounds include small peptides or peptide-shaped molecules (peptide mimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies, antibody fragments, or antibody conjugates. Test compounds can also be protein scaffolds. For high throughput purposes, libraries of test compounds can be used, such as combinatorial or randomized libraries that provide a sufficient range of diversity. 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, and the like. A more detailed description can still be found in the descriptive report.
[00101] As used herein, the terms "determining", "measuring", "evaluating", "monitoring" and "assaying" are used interchangeably and include both quantitative and qualitative determinations.
[00102] The term "biologically active", with respect to a GPCR, refers to a GPCR having a biochemical function (e.g., a binding function, a signal transduction function, or an ability to change conformation as result of ligand binding) of a naturally occurring GPCR.
[00103] The terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount", as used herein, mean the amount necessary to achieve the desired result or results.
[00104] The term "pharmaceutically acceptable", as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the compound without causing any undesirable biological effects or interacting in any way. in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. DETAILED DESCRIPTION
[00105] Structural information on GPCRs will provide insight into the structural, functional, and biochemical changes involved in signal transfer from the receptor to intracellular interacting proteins (G-proteins, β-arrestins, etc.) and delineate ways to interfere with these interactions pharmacologically. relevant. Efforts to obtain and crystallize GPCRs are therefore of great importance. However, this is a particularly difficult endeavor due to the biochemical challenges of working with GPCRs and the inherent instability of these complexes in detergent solutions. Also, the intrinsic conformational flexibility of GPCRs complicates high resolution structure analysis of GPCRs alone because increasing diffraction quality crystals require stable, conformationally homogeneous proteins (Kobilka et al. 2007). The present invention provides new experimental and analytical tools to capture or "freeze" the functional conformational states of a GPCR of interest, in particular its active conformational state, allowing structural and functional analysis of the GPCR, including high resolution structural analysis and many applications. derived therefrom.
[00106] A first aspect of the present invention pertains to a protein binding domain that is capable of specifically binding to a functional conformational state of a GPCR.
[00107] The protein binding domain of the present invention can be any naturally occurring or non-naturally occurring molecule thereof (as defined above) that is capable of specifically binding to a functional conformational state of a target GPCR. In a preferred embodiment, the protein binding domains as described herein are protein scaffolds. The term "protein scaffold" generally refers to hinge units that form structures, particularly protein or peptide structures comprising structures for the attachment of another molecule, e.g. a protein (See, e.g., Skerra, J. , 2000, for review). A protein-binding domain can be derived from a naturally occurring molecule, for example from components of the innate or adaptive immune system, or it can be engineered completely artificially. A protein-binding domain can be immunoglobulin-based or based on domains present in proteins, including, but limited to, microbial proteins, protease inhibitors, toxins, fibronectin, lipocalins, single-chain anti-parallel coiled-coil proteins, or protein-binding proteins. repeat reason. Examples of protein binding domains that are known in the art include, but are not limited to: antibodies, heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies, the variable domain derived from heavy chain antibodies of camelid (VHH or nanobodies), the variable domain of shark antibody-derived novel antigen receptors (VNAR), alphabodies, protein A, protein G, projected ankyrin repeat domains (DARPins), fibronectin type III repeats, anticalines, quinotines, engineered CH2 domains (nanoantibodies), peptides and proteins, lipopeptides (eg pepducins), DNA, and RNA (see, for example, Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., 1997; Binz et al., 1997; Binz et al. al., 2004; Koide et al., 1998; Dimitrov, 2009; Nygren et al. 2008; WO2010066740). Often, when generating a particular type of protein-binding domain using selection methods, combinatorial libraries comprising a consensus or framework sequence containing the potential randomized interaction residues are used to screen for binding to a molecule of interest, such as a protein.
[00108] "G protein-coupled receptors", or "GPCRs", as used herein, are polypeptides that share a common structural motif, having seven regions of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which traverses the membrane. Each span is identified by the number, i.e. transmembrane-1 (TM1), transmembrane-2 (TM2), etc. Transmembrane helices are joined by amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the outer, or "extracellular," side of the membrane. cell membrane, referred to as "extracellular" regions 1, 2 and 3 (EC1, EC2 and EC3), respectively. The transmembrane helices are also joined by amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the inner, or "intracellular," side of the membrane. cell membrane, referred to as "intracellular" regions 1, 2 and 3 (IC1, IC2 and IC3), respectively. The "carboxy" ("C") terminus of the receptor is in the intracellular space inside the cell, and the "amino" ("N") terminus of the receptor is in the extracellular space outside the cell. Any of these regions are readily identifiable by analyzing the primary amino acid sequence of a GPCR.
[00109] Structure and classification of GPCRs are generally well known in the art and further discussion of GPCRs can be found in Probst et al. 1992; Marchese et al. 1994; Lagerstrom & Schioth, 2008; Rosenbaum et al. 2009; and the following books: Jurgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled Receptors published by Wiley-Liss (1st edition; October 15, 1999); Kevin R. Lynch (Ed) Identification and Expression of G Protein-Coupled Receptors published by John Wiley & Sons (March 1998) and Tatsuya Haga (Ed), G Protein-Coupled Receptors, published by CRC Press (September 24, 1999) ); and Steve Watson (Ed) G-Protein Linked Receptor Factsbook, published by Academic Press (1st edition; 1994).
[00110] GPCRs can be grouped based on sequence homology into several distinct families. Although all GPCRs have a similar architecture of seven transmembrane helices, the different families within this receptor class do not show any sequence homology to each other, thus suggesting that the similarity of their transmembrane domain structure could define common functional requirements. A comprehensive view of the GPCR repertoire was made possible when the first drawing of the human genome became available. Fredriksson and colleagues divided 802 human GPCRs into families based on phylogenetic criteria. This showed that most human GPCRs can be found in five main families, named Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin (Fredriksson et al., 2003).
[00111] In a preferred embodiment of the invention, the protein binding domain is directed against or is capable of specifically binding a functional conformational state of a GPCR, wherein said GPCR is chosen from the group comprising a GPCR of the family of Glutamate GPCRs, a GPCR of the Rhodopsin family of GPCRs, a GPCR of the Adhesion GPCR family, a GPCR of the Frizzled/Taste2 family of GPCRs, and a GPCR of the Secretin family of GPCRs. Preferably, the GPCR is a mammalian protein, or a plant protein, or a microbial protein, or a viral protein, or an insect protein. Even more preferably, the GPCR is a human protein.
[00112] Members of the Rhodopsin family (corresponding to Class A (Kolakowski, 1994) or Class 1 (Foord et al (2005) in older classification systems) only have small extracellular loops and ligand interaction occurs with residues within This is by far the largest group (>90% of GPCRs) and contains receptors for odorants, small molecules such as catecholamines and amines, (neuro)peptides and glycoprotein hormones. Rhodopsin, a representative of this family, is the first GPCR for which the structure was resolved (Palczewski et al., 2000) β2AR, the first receptor interacting with a diffusible ligand for which the structure was resolved (Rosenbaum et al., 2007) also belongs to this family. In Phylogenetic analysis, class B or Class 2 GPCR receptors (Foord et al, 2005) have recently been subdivided into two families: adhesion and secretin (Fredriksson et al., 2003). by a relatively long amino terminal extracellular domain involved in ligand binding. Little is known about the orientation of the transmembrane domains, but it is probably quite different from that of rhodopsin. Ligands for these GPCRs are hormones such as glucagon, secretin, gonadotropin releasing hormone and parathyroid hormone. The Glutamate family of receptors (Class C or Class 3 receptors) also have a large extracellular domain that functions as a "flycatcher" as it can open and close with the agonist bound inside. Members of the family are metabotropic glutamate, Ca2+-sensitive receptors, and Y-aminobutyric acid (GABA)-B.
[00113] GPCRs include, without limitation, serotonin olfactory receptors, glycoprotein hormone receptors, chemokine receptors, adenosine receptors, biogenic amine receptors, melanocortin receptors, neuropeptide receptors, chemotactic receptors, somatostatin receptors, opiate receptors , melatonin receptors, calcitonin receptors, PTH/PTHrP receptors, glucagon receptors, secretin receptors, latrotoxin receptors, metabotropic glutamate receptors, calcium receptors, GABA-B receptors, pheromone receptors, protease-activated receptors , the rhodopsins and seven other G protein-coupled transmembrane segment receptors. GPCRs also include these GPCR receptors associated with each other as homomeric or heteromeric dimers or as higher-order oligomers. The amino acid sequences (and the nucleotide sequences of the cDNAs encoding them) of the GPCRs are readily available, for example, by reference to GenBank (http://www.ncbi.nlm.nih.gov/entrez).
[00114] According to a preferred embodiment, the GPCR is chosen from the group comprising adrenergic receptors, preferably a-adrenergic receptors, such as a1-adrenergic receptors and a2-adrenergic receptors, and β-adrenergic receptors, such as such as β-adrenergic receptors, α2-adrenergic receptors and β3-adrenergic receptors; or from the group comprising muscarinic receptors, preferably M1 muscarinic receptors, M2 muscarinic receptors, M3 muscarinic receptors, M4 muscarinic receptors and M5 muscarinic receptors; or from the group of angiotensin receptors, preferably angiotensin II receptor type 1, angiotensin II receptor type 2 and other atypical angiotensin II receptors; all of these are well known in the art.
[00115] A GPCR, as used herein, may be any naturally occurring or non-naturally occurring (ie, man-altered) polypeptide. The term "naturally occurring" in reference to a GPCR means a GPCR that is produced naturally (for example, without limitation, by a mammal, more specifically by a human, or by a virus, or by a plant, or by an insect, among others). Such GPCRs are found in nature. The term "non-naturally occurring" in reference to a GPCR means a GPCR that is not naturally occurring. Wild-type GPCRs that have been made constitutively active through mutation, and variants of naturally occurring GPCRs are examples of non-naturally occurring GPCRs. Non-naturally occurring GPCRs may have an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to a naturally occurring GPCR. Taking the e2-adrenergic receptor as a particular non-limiting example of a GPCR within the scope of the present invention, it should be clear from the above that in addition to the human e2-adrenergic receptor (e.g. the sequence described by Genbank accession number NP_000015) , the mouse e2-adrenergic receptor (e.g., as described by Genbank accession number NM 007420) or another mammalian e2-adrenergic receptor may also be employed. Furthermore, the term is intended to encompass polymorphic wild-type variants and certain other active variants of the e2-adrenergic receptor of a particular species. For example, a "human e2-adrenergic receptor" has an amino acid sequence that is at least 95% identical (e.g., at least 95% or at least 98% identical) to the naturally occurring "human β2-adrenoreceptor" of number Genbank access code NP_000015. Furthermore, it will be appreciated that the present invention also targets GPCRs with a loop deletion, or an N and/or C-terminal deletion, or a substitution, or an insertion or addition with respect to their amino acid or nucleotide sequence, or any combination of them (as defined above, and see also the Example section). It is further expected that the protein binding domains according to the invention in general will be able to bind all analogues, variants, mutants, alleles, naturally occurring or synthetic, of said GPCR.
[00116] Various methods can be used to determine specific binding between the protein binding domain and a target GPCR, including, for example, enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance assays, phage display , and others, which are common practice in the art eg discussed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual. Third edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. It will be appreciated that for this purpose often a unique tag or tag will be used, such as a peptide tag, a nucleic acid tag, a chemical tag, a fluorescent tag, or a radiofrequency tag, as described further herein.
[00117] It should be clear that GPCRs are conformationally complex membrane proteins that exhibit spectrum functional behavior in response to natural and synthetic ligands. Defining the pathway from agonist binding to protein activation will require a combination of the crystal structures of the different conformational states of the receptor under investigation in complex with various natural or synthetic ligands (including structures of active agonist-bound states and GPCR- G protein), which will provide snapshots along the activation pathway.
[00118] Thus, in a preferred embodiment, the protein-binding domain is capable of stabilizing, or otherwise enhancing the stability of a particular functional conformational state of a GPCR. Preferably, the protein-binding domain is capable of inducing the formation of a functional conformational state in a GPCR upon binding of said GPCR. Said functional conformational state of said GPCR may be a basal conformational state, or an active conformational state or an inactive conformational state. Preferably, the protein-binding domain is capable of stabilizing a GPCR in its active conformational state and/or is capable of forcing the GPCR to adopt its active conformational state upon binding.
[00119] The words "inducing" or "forcing" or "locking" or "capturing" or "fixing" or "freezing" with respect to a functional conformational state of a GPCR (as defined herein), as used herein, refer to whether to retain or contain a GPCR in a subset of the possible conformations it might otherwise assume, due to the effects of the GPCR's interaction with the protein-binding domain according to the invention. Consequently, a protein that is "conformationally captured" or "conformational fixed" or "conformationally locked" or "conformationally frozen", as used herein, is one that is retained in a subset of the possible conformations it might otherwise assume, due to the effects of the interaction of the GPCR with the protein-binding domain according to the invention. Within this context, a protein-binding domain that specifically or selectively binds in a specific conformation or conformational state of a protein refers to a protein-binding domain that binds with a higher affinity to a protein in a subset of conformations or conformational states than to other conformations or conformational states that the protein can assume. One of skill in the art will recognize that protein binding domains that specifically or selectively bind to a specific conformation or conformational state of a protein will stabilize that specific conformation or conformational state.
[00120] The term "a functional conformational state", as used herein, refers to the fact that proteins, in particular membrane proteins such as GPCRs, have many different conformational states having a dynamic range of activity, varying in particular from no activity to maximum activity (reviewed in Kobilka and Deupi, 2007). It should be clear that "a functional conformational state" is not meant to encompass the denatured states of proteins. The functional versatility of GPCRs is inherently coupled with the flexibility of these proteins resulting in such a spectrum of conformations. The conformational energy landscape is intrinsically coupled to such factors as the presence of bound ligands (effector molecules, agonists, antagonists, reverse agonists,...), the lipid environment or the binding of interacting proteins. For example, a "basal conformational state" can be defined as a low energy state of the receptor in the absence of a ligand (as defined above, e.g., effector molecules, agonists, antagonists, reverse agonists). The probability that a protein will transition to another conformational state is a function of the energy difference between the two states and the height of the energy barrier between the two states. In the case of a receptor protein, such as a GPCR, the binding energy of the ligand can be used to change the energy barrier between the two states, or to change the relative energy levels between the two states, or both. Changing the energy barrier would have an effect on the rate of transition between the two states, while changing the energy levels would have an effect on the equilibrium distribution of receptors in the two states. Binding of an agonist or polarized agonist would lower the energy barrier and/or reduce the energy of the more active conformational state relative to the inactive conformational state. A reverse agonist would increase the energy barrier and/or reduce the energy of the "inactive state conformation" relative to the "active conformation". Coupling the receptor to its G protein could further alter the energy landscape. The activities of integral membrane proteins, including GPCRs, are also affected by the structures of the lipid molecules that surround them in the membrane. Membrane proteins are not rigid entities, and they deform to ensure good hydrophobic pairing to the surrounding lipid bilayer. An important parameter is the hydrophobic thickness of the lipid bilayer, defined by the lengths of the fatty lipid acyl chains. Also, the structure of the lipid head group region is likely to be important in defining the structures of those parts of a membrane protein that are located in the lipid head group region. Among other lipids, palmitoylation and binding of cholesterol to GPCRs may also play a structural role within monomeric receptors and contribute to the formation/stabilization of receptor oligomers (Lee 2004; Chini and Parenti 2009).
[00121] "Receptor ligands", or simply "ligands", as defined above, can be "orthosteric" ligands (both natural and synthetic), meaning that they bind to the active site of the receptor and are further classified according to their effectiveness or in other words with the effect they have on receptor signaling through a specific pathway. As used herein, an "agonist" refers to a ligand which, by binding to a receptor, increases the signaling activity of the receptor. Full agonists are capable of maximal receptor stimulation; partial agonists cannot elicit full activity even at saturated concentrations. Partial agonists can also function as "blockers" preventing the binding of more robust agonists. An "antagonist" refers to a ligand that binds to a receptor without stimulating any activity. An "antagonist" is also known as a "blocker" because of its ability to prevent binding of other ligands and therefore block agonist-induced activity. Further, a "reverse agonist" refers to an antagonist that, in addition to blocking the effects of the agonist, reduces the basal or constitutive activity of receptors below that of the unbound receptor.
[00122] The canonical view of how GPCRs regulate cell physiology is that binding ligands (such as hormones, neurotransmitters, or sensory stimuli) stabilize an active conformational state of the receptor, thus allowing interactions with heterotrimeric G proteins. In addition to interacting with G proteins, agonist-bound GPCRs associate with GPCR kinases (GRKs), leading to receptor phosphorylation. A common result of GPCR phosphorylation by GRKs is a decrease in GPCR interactions with G proteins and an increase in GPCR interactions with arrestins, which sterically still interdict G protein signaling, resulting in receptor desensitization. As β-arrestins turn off G protein signals, they can simultaneously initiate a second parallel set of signal cascades, such as the MAPK pathway. GPCRs also associate with several proteins outside the general GPCR interacting protein families (G proteins, GRKs, arrestins, and other receptors). These selective GPCR pairs can mediate GPCR signaling, organize GPCR signaling through G proteins, direct GPCR traffic, dock GPCRs in particular subcellular areas, and/or influence the pharmacology of GPCRs (Ritter and Hall 2009). In this regard, ligands as used herein may also be "partial ligands" with the ability to selectively stimulate a subset of a receptor's signaling activities, for example, selective G protein activation or β-arrestin function. Such ligands are known as "polarized ligands", "polarized agonists" or "functionally selective agonists". More particularly, ligand polarization may be imperfect polarization characterized by ligand stimulation of multiple receptor activities with different relative efficiencies by different signals (non-absolute selectivity) or it may be perfect polarization characterized by ligand stimulation of one activity. receptor without any stimulation of other known receptor activity.
[00123] The signaling activities of GPCRs (and thus their conformational behavior) may also be affected by the binding of another type of ligand known as allosteric regulators. "Allosteric regulators" or otherwise "allosteric modulators", "allosteric ligands" or "effector molecules" bind to an allosteric site on a GPCR (i.e., a regulatory site physically distinct from the protein's active site). In contrast to orthosteric ligands, allosteric modulators are non-competitive because they bind receptors at a different site and modify receptor function even if the endogenous ligand is also binding. Because of this, allosteric modulators are not limited to simply turning a receptor on or off, the way most drugs are. Otherwise, they act more like a dimming switch, offering control over the amount of on or off, while allowing the body to retain its natural control when initiating receptor activation. Allosteric regulators that enhance protein activity are referred to herein as "allosteric activators" or "positive allosteric modulators", while those that decrease protein activity are referred to herein as "allosteric inhibitors" or otherwise "negative allosteric modulators".
[00124] Preferably, the protein-binding domain of the present invention is capable of specifically binding to an agonist-bound GPCR and/or enhancing the affinity of a GPCR for an agonist. It is preferred that the protein binding domain is capable of increasing affinity for the agonist at least two-fold, at least five-fold, and more preferably at least ten-fold upon receptor binding as measured by a decrease in EC50, IC50, Kd , or any other measure of affinity or potency known to one of skill in the art.
[00125] It will be appreciated that having increased stability with respect to the structure and/or a particular biological activity of a GPCR may also signal the stability of other denaturing or denaturing conditions including heat, a detergent, a chaotropic agent and a extreme pH. Consequently, in another embodiment, the protein binding domain according to the invention is capable of increasing the stability of a functional conformational state of a GPCR under non-physiological conditions induced by dilution, concentration, buffer composition, heat, cooling, freezing, detergent, chaotropic agent, pH. In contrast to water-soluble proteins, thermodynamic studies of membrane protein folding and stability have proven to be extremely challenging, and complicated by the difficulty of finding conditions for reversible folding. Unfolding of helical membrane proteins induced by most methods, such as thermal and chemical approaches, is irreversible as reviewed by Stanley and Fleming (2008). The terms "thermostabilize", "thermostabilization", "increase the thermostability of", as used herein, therefore, refer to the functional rather than thermodynamic properties of a GPCR and the resistance of the protein to irreversible denaturation induced by thermal and/or thermal approaches. or chemicals including, but not limited to, heat, denaturing coolants, cold, chemical agent, pH, detergents, salts, additives, proteases or temperature. Irreversible denaturation leads to irreversible unfolding of functional protein conformations, loss of biological activity and aggregation of the denatured protein. The terms "(thermo)stabilize", "(thermo)stabilization", "increase the (thermo)stability of", as used herein, apply to GPCRs embedded in lipid particles or lipid layers (e.g., lipid monolayers). lipid, lipid bilayers, and others) and for GPCRs that have been solubilized in detergent.
[00126] Preferably, the protein binding domain according to the invention is capable of increasing the thermostability of a functional conformational state of a GPCR, preferably an active conformational state of a GPCR. Regarding increased heat stability, this can be easily determined by measuring ligand binding or using spectroscopic methods such as fluorescence, CD or light diffraction which are sensitive to unfolding at elevated temperatures (see also Example section). It is preferred that the protein-binding domain is capable of increasing stability as measured by an increase in the thermal stability of a GPCR in a functional conformational state at at least 2°C, at least 5°C, at least 8°C , and more preferably at least 10°C or 15°C or 20°C. According to another preferred embodiment, the protein-binding domain is capable of increasing the thermal stability of a functional conformation of a GPCR in complex with a ligand such as, but not restricted to, an agonist, a reverse agonist, antagonist and/or a modulator or an inhibitor of the GPCR or the GPCR-dependent signaling pathway. According to another preferred embodiment, the protein binding domain according to the invention is capable of enhancing the stability in the presence of a detergent or a chaotrope of a functional conformational state of a GPCR. Preferably, the protein-binding domain is capable of enhancing stability to thermally or chemically induced denaturation of the active conformational state of a GPCR. With respect to increased stability to heat, a detergent or a chaotrope, typically the GPCR is incubated for a defined time in the presence of a test detergent or a test chaotropic agent and stability is determined using, for example, binding of binder or a spectroscopic method, optionally at elevated temperatures as discussed above. In accordance with yet another preferred embodiment, the protein-binding domain according to the invention is capable of enhancing the extreme pH stability of a functional conformational state of a GPCR. Preferably, the protein-binding domain is capable of enhancing the extreme pH stability of the active conformational state of a GPCR. Regarding a pH extreme, a typical test pH would 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, plus specifically in the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).
[00127] The protein binding domains according to the invention may in general be directed against any desired GPCR, and may be directed in particular against any conformational epitope of any GPCR, preferably a functional conformational state of any GPCR, more preferably a active conformational state of a GPCR (all as defined above). More particularly, said conformational epitope may be part of an intracellular or extracellular region, or an intramembranous region, or a domain or loop structure of any desired GPCR. According to particular embodiments, the protein binding domains may be directed against any suitable extracellular region, domain, loop or other extracellular conformational epitope of a GPCR, but are preferably directed against one of the extracellular parts of the transmembrane domains or more preferably against one of the extracellular loops connecting the transmembrane domains. Alternatively, the protein-binding domains may be directed against any suitable intracellular region, domain, loop or other intracellular conformational epitope of a GPCR, but are preferably directed against one of the intracellular parts of the transmembrane domains or more preferably against one of the intracellular loops that link the transmembrane domains. A protein-binding domain that specifically binds to a "three-dimensional" epitope or "conformational" epitope specifically binds to a tertiary (i.e., three-dimensional) structure of a folded protein, and binds at very low affinity ( that is, by a factor of at least 2, 5, 10, 50, or 100) to the linear (i.e., uncoiled, denatured) form of the protein. It is further expected that the protein binding domains of the invention in general will bind all analogues, variants, mutants, naturally occurring or synthetic alleles of said GPCR.
[00128] In a specific embodiment, the protein binding domain according to the invention is capable of specifically binding to an intracellular conformational epitope of a GPCR. Preferably, the protein-binding domain is capable of specifically binding a conformational epitope that is comprised of, located or overlapped with the G protein binding site of a GPCR. More preferably, said protein binding domains may occupy the G protein binding site of a functional conformational state of a GPCR, more preferably of an active conformational state of a GPCR. Most preferably, said protein-binding domains show G-protein-like behavior. The term "G-protein-like behavior" as used herein refers to the property of protein binding domains to bind agonist-bound receptor preferentially versus, for example, reverse agonist-bound receptor. Protein binding domains showing G-protein-like behavior also enhance the receptor's affinity for agonists which are attributed to the cooperative interaction between the agonist-bound receptor and G protein (see also the Example section).
[00129] In a preferred embodiment, the protein-binding domain is derived from an innate or adaptive immune system. Preferably, said protein-binding domain is derived from an immunoglobulin. Preferably, the protein-binding domain according to the invention is an antibody or a derivative thereof. The term "antibody" (Ab) generally refers to a polypeptide encoded by an immunoglobulin gene, or functional fragments thereof, which specifically binds and recognizes an antigen, and is known to the person skilled in the art. A conventional immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" (about 25 kDa) and a "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. The term "antibody" is meant to include whole antibodies, including whole single chain antibodies, and antigen-binding fragments. In some embodiments, the antigen-binding fragments can be antibody antigen-binding fragments that include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (dsFv), and fragments comprising or consisting of a VL or VH domain, and any combination thereof or any other functional portion of an immunoglobulin peptide capable of binding the target antigen. The term "antibodies" is also intended to include heavy chain antibodies, or functional fragments thereof, such as single domain antibodies, more specifically, nanobodies, as defined further herein.
[00130] Preferably, said protein binding domain comprises an amino acid sequence comprising 4 framework regions and 3 complementary determining regions, preferably in a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 sequence, or any fragment of the same (which will then usually contain at least some of the amino acid residues that form at least one of the complementary determining regions). Protein binding domains comprising 4 FRs and 3 CDRs are known to the person skilled in the art and are described, as a non-limiting example, in Wesolowski et al. (2009)
[00131] Preferably, the protein binding domain according to the invention is derived from a camelid antibody. More preferably, the protein-binding domain according to the invention comprises an amino acid sequence of a nanobody, or any suitable fragment thereof. More specifically, the protein-binding domain is a nanobody or any suitable fragment thereof. A "nanobody" (Nb), as used herein, refers to the smaller antigen-binding fragment or single variable domain ("VHH") derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain-only antibodies seen in camelids (Hamers-Casterman et al. 1993; Desmyter et al. 1996). In the "camelid" family, immunoglobulins devoid of light polypeptide chains are found. "Camelid" comprises old world camelid (Camelus bactrianus and Camelus dromedarius) and new world camelid (eg Lama paccos, Lama glama, Lama guanicoe and Lama vicugna). Said single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody. Nanobody™, Nanobodies™ and Nanoclone™ are registered trademarks of Ablynx NV (Belgium). The small size and unique biophysical properties of Nbs outperform conventional antibody fragments for recognizing unusual or hidden epitopes and for binding to cavities or active sites on protein targets. Furthermore, Nbs can be designed as bispecific and bivalent antibodies or bound to reporter molecules (Conrath et al. 2001). Nbs are stable and rigid single domain proteins that can be easily manufactured and survive the gastrointestinal system. Therefore, Nbs can be used in many applications including drug discovery and therapy (Saerens et al. 2008) but also as a versatile and valuable tool for purification, functional study and crystallization of proteins (Conrath et al. 2009).
[00132] The nanobodies according to the invention generally comprise a single amino acid chain which can be considered to comprise 4 "framework sequences" or FR and 3 "complementary determination regions" or CDRs (as defined above). Non-limiting examples of nanobodies of the invention are described in further detail here. It should be clear that the framework regions of nanobodies may also contribute to the binding of their antigens (Desmyter et al 2002; Korotkov et al. 2009).
[00133] Non-limiting examples of nanobodies according to the invention include, but are not limited to, nanobodies as defined by SEQ ID NOs: 1-29 (see Fig. 12, table 1). The delineation of the CDR sequences is based on the IMGT unique numbering system for V domains and V-shaped domains (Lefranc et al. 2003). In a specific embodiment, the above nanobodies may comprise at least one of the complementary determining regions (CDRs) with an amino acid sequence selected from SEQ ID NOs: 30-70 (see Fig. 12; Table 2). More specifically, the above nanobodies can be selected from the group comprising SEQ ID NOs: 1-29, or a functional fragment thereof. A "functional fragment" or a "suitable fragment", as used herein, may for example comprise one of the CDR loops. Preferably, said functional fragment comprises CDR3. More specifically, said nanobodies consist of any one of SEQ ID NOs: 1-29 and said functional fragment of said nanobodies consists of any one of SEQ ID NOs: 30-70. In yet another embodiment, a nucleic acid sequence encoding any of the above nanobodies or functional fragments also forms part of the present invention. Furthermore, the present invention also targets expression vectors comprising nucleic acid sequences encoding any of the nanobodies or functional fragments above them, as well as host cells expressing such expression vectors. Suitable expression systems include constitutive and inducible expression systems in bacteria or yeast, viral expression systems such as baculovirus, Semliki Forest virus and lentivirus, or transient transfection into insect or mammalian cells. Suitable host cells include E. coli, Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and others. Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 and others. Cloning, expression and/or purification of the nanobodies can be done according to techniques known to the person skilled in the art.
[00134] It should be noted that the term nanobodies as used herein in its broadest sense is not limited to a specific biological source or a specific method of preparation. For example, nanobodies of the invention can generally be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by "camelizing" a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as a human, or by expressing a nucleic acid encoding such a camelized VH domain ; (5) by "camelizing" a "domain antibody" or "Dab" as described in the art, or by expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques to prepare the proteins, polypeptides or other amino acid sequences known per se; (7) preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.
[00135] A preferred class of nanobodies corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against a functional conformational state of a GPCR. While pure or synthetic nanobody libraries (for examples of such libraries, see WO9937681, WO0043507, WO0190190, WO03025020 and WO03035694) may contain conformational binders against a GPCR in a functional conformational state, a preferred embodiment of this invention includes immunizing a Camelidae with a GPCR in a functional conformational state, optionally linked to a receptor ligand, to expose the animal's immune system with conformational epitopes that are unique to the GPCR (e.g., agonist-bound GPCR to raise antibodies directed against a GPCR in its active conformational state). Thus, as further described herein, such VHH sequences can preferably be generated or suitably obtained by immunizing a Camelid species with a GPCR, preferably a GPCR in a functional conformational state, more preferably an active conformational state (i.e., to raise a immune response and/or heavy chain antibodies directed against said GPCR), obtaining a suitable biological sample from said Camelid (such as a blood sample, or any B cell sample), and generating VHH sequences directed against said GPCR , from said sample. Such techniques will be clear to the skilled person. Yet another technique for obtaining the desired VHH sequences involves immunizing a transgenic mammal that is capable of adequately expressing heavy chain antibodies (i.e., to raise an immune response and/or heavy chain antibodies directed against a GPCR in a functional conformational state). ), obtain a suitable biological sample from said transgenic mammal (such as a blood sample, or any B cell sample), and then generate VHH sequences directed against said GPCR from said sample, using any suitable technique known per se. if. For example, for this purpose, mice expressing heavy chain antibody and the other methods and techniques described in WO02085945 and WO04049794 can be used.
[00136] Accordingly, the invention encompasses methods of generating protein-binding domains in accordance with the invention. As a non-limiting example, a method is provided of generating nanobodies that specifically bind a conformational epitope of a functional conformational state of a GPCR, comprising: (i) immunizing an animal with a GPCR, and (ii) screening for nanobodies that specifically bind to a conformational epitope of a functional conformational state of said GPCR.
[00137] Preferably, the immunization of an animal will be with a GPCR in the presence of a receptor ligand, wherein said ligand induces a particular functional conformational state of said GPCR. For example, nanobodies can be generated that are specifically binding to a conformational epitope of an active conformational state of a GPCR by immunizing an animal with a GPCR in the presence of an agonist that induces the formation of an active conformational state of said GPCR. (see also the Example section).
[00138] For immunization of an animal with a GPCR, the GPCR can be produced and purified using conventional methods which may employ expression of a recombinant form of the GPCR in a host cell, and purification of the GPCR using affinity chromatography and/or methods antibody based. In particular embodiments, the baculovirus/Sf-9 system may be employed for expression, although other expression systems (e.g., bacterial, yeast, or mammalian cell systems) may also be used. Exemplary methods for expressing and purifying GCPRs are described, for example, in Kobilka (1995), Eroglu et al (2002), Chelikani et al (2006) and the book "Identification and Expression of G Protein-Coupled Receptors" (Kevin R Lynch (Ed.), 1998), among many others. A GPCR can also be reconstituted into phospholipid vesicles. Likewise, methods to reconstitute an active GPCR into phospholipid vesicles are known, and are described in: Luca et al (2003), Mansoor et al (2006), Niu et al. (2005), Shimada et al. (2002), and Eroglu et al. (2003), among others. In certain cases, the GPCR and phospholipids can be reconstituted at high density (eg, 1 mg of receptor per mg of phospholipid). In particular embodiments, phospholipid vesicles can be tested to confirm that the GPCR is active. In many cases, a GPCR can be present in the phospholipid vesicle in both orientations (in the normal orientation, and in the "upside-down" orientation where the intracellular loops are on the outside of the vesicle). Other methods of immunization with a GPCR include, without limitation, the use of whole cells expressing a GPCR, vaccination with a nucleic acid sequence encoding a GPCR (e.g., DNA vaccination), immunization with viruses or viruses as particles expressing a GPCR. GPCR, among others.
[00139] Any suitable animal, for example a warm-blooded animal, in particular a mammal such as a rabbit, mouse, rat, camel, sheep, cow, shark, or pig or a bird such as a chicken or turkey, may be immunized using any of the techniques well known in the art suitable for generating an immune response.
[00140] Screening for nanobody, as a non-limiting example, specifically binding to a conformational epitope of a functional conformational state of said GPCR can be, for example, performed by screening a set, collection or library of cells expressing the heavy chain antibodies on their surface (e.g. B cells obtained from a suitably immunized Camellid), or bacteriophages that display a genIII and nanobody fusion on their surface, by sorting a library (pure or immune) of VHH sequences or nanobody sequences, or by sorting a library (pure or immune) of nucleic acid sequences encoding the VHH sequences or the nanobody sequences which can all be performed in a manner known per se, and whose method may optionally complex comprise one or more other suitable steps, such as , for example and without limitation, an affinity maturation step, a step of expressing the desired amino acid sequence, a step of screening for binding and/or activity against the desired antigen (in this case, the GPCR), a step of determining the desired amino acid sequence or nucleotide sequence, a step of introducing one or more humanized substitutions, a step of formatting into a format multivalent and/or multispecific, a step of screening for desired biological and/or physiological properties (i.e., using a suitable assay known in the art), and/or any combination of one or more of such steps, in any suitable order.
[00141] A particularly preferred class of protein-binding domains of the invention comprises nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but which has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular framework sequences) by one or more of the amino acid residues occurring at the corresponding position(s) in a domain of VH of a conventional 4-chain antibody from a human. This can be performed in a manner known per se, which will be clear to the skilled person, based on the further description herein and the prior art on humanization. Again, it should be noted that such humanized nanobodies of the invention can be obtained in any suitable manner known per se (i.e., as indicated under points (1) - (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material. Humanized nanobodies may have several advantages, such as reduced immunogenicity compared to corresponding naturally occurring VHH domains. Such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring VHH with amino acid residues that occur at the same position in a human VH domain, such as a human VH3 domain. Humanization substitutions should be selected such that the resulting humanized nanobodies still retain the favorable properties of nanobodies as defined herein. The skilled person may select suitable humanization substitutions or combinations of humanization substitutions that optimize or achieve a desired or suitable balance between the favorable properties provided on the one hand by the humanization substitutions and, on the other hand, by the favorable properties of the VHH domains of naturally occurring.
[00142] Another particularly preferred class of protein-binding domains of the invention comprises nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but has been "camelized", i.e., replacing one or more more amino acid residues in the amino acid sequence of a naturally occurring VH domain of a conventional 4-chain antibody per one or more of the amino acid residues occurring at the corresponding position(s) in a VHH domain of a heavy chain antibody. Such "camelization" substitutions are preferably inserted at the amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called conspicuous residues of Camelidae, as defined herein (see, for example, WO9404678). Preferably, the VH sequence that is used as a starting material or starting point to generate or design the camelized nanobodies is preferably a VH sequence from a mammal, more preferably the VH sequence from a human, such as a sequence of VH3. However, it should be noted that such camelized nanobodies of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) - (8) above) and thus are not strictly limited to the polypeptides that have been obtained. using a polypeptide comprising a naturally occurring VH domain as a starting material.
[00143] 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 altering it in a manner known per se , one or more codons in said nucleotide sequence in such a way that the novel nucleotide sequence encodes a "humanized" or "camelized" nanobody of the invention, respectively. This nucleic acid can then be expressed in a manner known per se to provide the desired nanobody of the invention. Alternatively, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized nanobodies of the invention, respectively, can be designed and then de novo synthesized using techniques for the synthesis of peptide known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized nanobody of the invention, respectively, can be designed and then synthesized. again using techniques for the synthesis of nucleic acid known per se after which the nucleic acid thus obtained can be expressed in a manner known per se, to provide the desired nanobody of the invention. Other suitable methods and techniques for obtaining the nanobodies of the invention and/or nucleic acids encoding the same 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 more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more sequences of FR or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, to provide a nanobody of the invention or a nucleotide or nucleic acid sequence encoding the same.
[00144] It is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs") of the protein binding domains according to the invention, preferably for nanobodies , and in particular analogs of the nanobodies of SEQ ID NOs: 1-29 (see Table 1, Fig. 12). Thus, according to one embodiment of the invention, the term "nanobodies of the invention" in its broadest sense also encompasses such analogues. In general, in such analogs, one or more amino acid residues may have been substituted, deleted and/or added, compared to the nanobodies of the invention as defined herein. Such substitutions, insertions, deletions or additions may be made in one or more of the framework regions and/or in one or more of the CDRs, and in particular analogues of the CDRs of the nanobodies of SEQ ID NOs: 1-29, said CDRs corresponding with SEQ ID NOs: 30-70 (see Table 2, Fig. 12). Analogs, as used herein, are sequences in which each or any framework region and each or any complementary determining region shows at least 80% identity, preferably at least 85% identity, more preferably 90% identity, even more preferably 95% identity with the corresponding region in the reference sequence (i.e. FR1_analog versus FR1_reference, CDR1_analog versus CDR1_reference, FR2_analog versus FR2_reference, CDR2_analog versus CDR2_reference, FR3_analog versus FR3_reference, CDR3_analog versus CDR3_reference, FR4_analog FR4_reference, FR3_analog versus FR3_reference, CDR3_analog versus CDR3_reference, FR4_analog BLASTp alignment (Altschul et al; 1987; definitions of FR and CDR according to the IMGT unique numbering system for V domains and V-shaped domains (Lefranc et al. 2003)).
[00145] By way of non-limiting examples, a substitution can, for example, be a conservative substitution (as described herein) and/or an amino acid residue can be substituted for another amino acid residue that naturally occurs at the same position in another domain of VHH. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improves the properties of the nanobody of the invention or that at least does not greatly diminish the desired properties or the balance or combination of the desired properties of the nanobody of the invention (ie, to the extent that the nanobody is no longer suitable for its intended use) are included within the scope of the invention. A person of ordinary skill will be able to determine and select suitable substitutions, deletions, insertions, additions, or suitable combinations thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may, for example, involve introducing a number limited number of possible substitutions and to determine their influence on the properties of the nanobodies thus obtained.
[00146] For example, and depending on the host organism used to express the protein-binding domain of the invention, preferably the nanobody, such deletions and/or substitutions can be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the skill of the person skilled in the art. Alternatively, substitutions or insertions can be designed to introduce one or more sites for attachment of functional groups (as described herein), for example, to allow site-specific pegylation.
[00147] Examples of modifications, as well as examples of amino acid residues within the protein-binding domain sequence, preferably the nanobody sequence that can be modified (i.e., either in the protein backbone, but preferably in a side chain ), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person. For example, such a modification may involve the introduction (e.g. covalently or otherwise suitable) of one or more groups, residues or functional moieties in or on the nanobody of the invention, and in particular one or more groups, residues or functional moieties that impart one or more desired properties or functionalities to the nanobody of the invention. Examples of such functional groups and of techniques for introducing the same will be clear to the skilled person, and may in general comprise all the functional groups and techniques mentioned in the background of the general art cited above as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv and single domain antibodies) for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may, for example, be linked directly (eg covalently) to a nanobody of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the widely used techniques to increase the half-life and/or reduce the immunogenicity of pharmaceutical proteins comprises binding a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). In general, any suitable form of pegylation can be used, such as pegylation used in the art for antibodies and antibody fragments (including but not limited to domain (single) and ScFv antibodies); reference is made for example to Chapman, Nat. Biotechnol., 54, 531-545 (2002 ); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO04060965. Various reagents for pegylation of proteins are also commercially available, for example, from Nektar Therapeutics, USA. Preferably, site-directed pegylation is used, in particular via a cysteine residue (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG can be attached to a naturally occurring cysteine residue in a nanobody of the invention, a nanobody of the invention can be modified to suitably introduce one or more cysteine residues for PEG binding, or an amino acid sequence comprising one or more residues of cysteine to PEG binding may be fused to the N-terminus and/or C-terminus of a nanobody of the invention, all using protein engineering techniques known per se to the skilled person. Preferably, for the nanobodies and proteins of the invention, a PEG is used having a molecular weight greater than 5000, such as greater than 10,000 and less than 200,000, such as less than 100,000, for example in the range of 20,000-80,000. Another, usually less preferred modification comprises N-linked glycosylation or O-linked, usually as part of co-translational and/or post-translational modification, depending on the host cell used to express the nanobody or polypeptide of the invention. Another technique for increasing the half-life of a nanobody may comprise engineering bifunctional nanobodies (e.g., a nanobody against the target GPCR and one against a serum protein such as albumin) or fusions of the nanobodies with peptides (e.g., a peptide against a whey protein such as albumin).
[00148] Yet another modification may comprise the introduction of one or more detectable tags or other signal generating groups or moieties, depending on the intended use of the tagged protein binding domain, in particular the nanobody. Labels and techniques suitable for binding, using and detecting the same will be clear to the skilled person, and, for example, include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, and fluorescamine and fluorescent metals such as Eu or other lanthanide series metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogues), radioisotopes, metals, metal chelates or metal cations or other metals or metal cations which are particularly suited for use in in vivo, in vitro or in situ diagnostics and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase genase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and, for example, include halves that can be detected using NMR or ESR spectroscopy. Such nanobodies and labeled polypeptides of the invention can, for example, be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as diagnostic and in vivo imaging purposes, depending on the choice of specific label. As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metal cations referred to above. Suitable chelating groups include, for example, without limitation, diethyl enettriaminopentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is a part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group can be used to link the nanobody of the invention to another protein, polypeptide or chemical compound that is linked to the other half of the binding pair, i.e. through binding pair formation. For example, a nanobody of the invention can be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated nanobody can be used as a reporter, for example in a diagnostic system where a detectable signal producing agent is conjugated to avidin or streptavidin. Such binding pairs can also be used for example to link the nanobody of the invention to a carrier, including carriers suitable for pharmaceutical purposes. A non-limiting example is the liposomal formulations described by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs can also be used to bind a therapeutically active agent to the nanobody of the invention.
[00149] In a particular embodiment, the nanobody of the invention is bivalent and formed by ligation, chemically or through recombinant DNA techniques, together with two monovalent single domains of heavy chains. In another particular embodiment, the nanobody of the invention is bispecific and formed by linking two heavy chain variable domains, each with a different specificity. Similarly, polypeptides comprising multivalent or multispecific nanobodies are included herein as non-limiting examples. Preferably, a monovalent nanobody of the invention is such that it will bind to a part, region, domain, extracellular loop or other extracellular epitope of a functional conformational state of a GPCR, more preferably an active conformational state of a GPCR, with a lower affinity. than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Alternatively, a monovalent nanobody of the invention is such that it will bind to a part, region, domain, intracellular loop or other intracellular epitope of a functional conformational state of a GPCR, more preferably an active conformational state of a GPCR with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Also, in accordance with this aspect, any multivalent or multispecific nanobodies of the invention (as defined herein) may also be suitably targeted against two or more different parts, regions, domains, extracellular or intracellular loops or other extracellular or intracellular epitopes on the same antigen, for example against two different extracellular or intracellular loops or against two different extracellular or intracellular parts of the transmembrane domains. Such multivalent or multispecific nanobodies of the invention may also have (or be engineered and/or selected for) increased avidity and/or improved selectivity for the desired GPCR, and/or for any other desired property or combination of desired properties that can be obtained by the use of such multivalent or multispecific nanobodies. In a particular embodiment, such multivalent or multispecific nanobodies of the invention may also have (or be engineered and/or selected for) improved efficacy in modulating the signaling activity of a GPCR (see also further herein). It will be appreciated that the multivalent or multispecific nanobodies according to the invention may additionally be suitably targeted to a different antigen, such as, but not limited to, a ligand that interacts with a GPCR or one or more downstream signaling proteins.
[00150] A second aspect of the present invention pertains to a complex comprising (i) a protein binding domain according to the invention, (ii) a GPCR in a functional conformational state, and optionally, (iii) a ligand of receiver. A "receptor ligand" or a "ligand", as defined herein, can be a small compound, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or any suitable fragment. derived therefrom, and others. Preferably, the ligand is of the agonist class and the receptor is in an active conformational state. The ligand can also be a reverse agonist, antagonist, or a polarized ligand. The ligands can be orthosteric or allosteric. Ligands also include allosteric modulators, enhancers, enhancers, negative allosteric modulators, and inhibitors. They may have biological activity by themselves or they may modulate activity only in the presence of another ligand. Neubig et al (2003) describe many of the ligand classes.
[00151] Preferably, the invention provides a complex wherein the protein-binding domain is linked to the GPCR; more preferably, the protein binding domain is linked to the GPCR, wherein said GPCR is linked to a receptor ligand. To further illustrate this, and not intended to be limiting, a stable ternary complex containing a nanobody, a GPCR and an agonist can be purified by affinity chromatography or gel filtration from a mixture containing (1) a nanobody that is specific for the active conformation of that GPCR, (2) the detergent-solubilized receptor, and (3) an agonist.
[00152] In another preferred embodiment, the complex is crystalline. Thus, a crystal of the complex is also provided, as well as methods of making said crystal being described in more detail below. Preferably, a crystalline form of a complex comprising (i) a protein-binding domain according to the invention, (ii) a GPCR in a functional conformational state, more preferably an active conformational state, and optionally, (iii) a ligand receptor, is envisaged, wherein said crystalline form is obtained by using a protein-binding domain according to the invention.
[00153] In yet another preferred embodiment, the complex according to the invention is in a solubilized form, for example after aqueous solubilization with a detergent. In an alternative preferred embodiment, the complex according to the invention is immobilized on a solid support. Non-limiting examples of solid supports as well as methods and techniques for immobilization are further described in the detailed description. In yet another embodiment, the complex according to the invention is in a "cellular composition", including an organism, a tissue, a cell, a cell lineage, and a membrane composition derived from said organism, tissue, cell or cell lineage. . Said membrane composition is also intended to include any liposomal composition which may comprise natural or synthetic lipids or a combination thereof. Examples of membrane or liposomal compositions include, but are not limited to, organelles, membrane preparations, Virus-Like Lipparticles, lipid layers (bilayers and monolayers), lipid vesicles, high density lipoparticles (e.g. nanodisks), and others. It will be appreciated that a cellular composition, or a membrane or liposome-like composition, may comprise natural or synthetic lipids.
[00154] Accordingly, a third aspect of the invention pertains to a cellular composition, including a membrane or liposomal composition derived therefrom (all as defined above) comprising a protein-binding domain and/or a complex according to the invention. Preferably, the cellular composition providing and/or expressing the protein-binding domain is capable of stabilizing and/or inducing a functional conformational state of a GPCR upon binding of said protein-binding domain. It will be understood that it is essential to retain sufficient functionality of the respective proteins, for which methods and techniques are available and known to the person skilled in the art. It will also be appreciated that said cellular composition can provide and/or express a target GPCR endogenously or exogenously.
[00155] Preparations of GPCRs formed from membrane fragments or membrane-detergent extracts are reviewed in detail in Cooper (2004), incorporated herein by reference. Non-limiting examples of solubilized receptor preparations are further provided in the Example section.
[00156] Transfection of target cells (eg, mammalian cells) can be performed following the principles outlined by Sambrook and Russell (Molecular Cloning, A Laboratory Manual, 3rd Edition, Volume 3, Chapter 16, Section 16.1-16.54). Furthermore, viral transduction can also be performed using reagents such as adenoviral vectors. Selection of the appropriate viral vector system, regulatory regions and host cell is common knowledge within the skill level usual in the art. The resulting transfected cells are maintained in culture or frozen for later use according to standard practices. Preferably the cells are eukaryotic cells, for example yeast cells, or cultured cell lines, for example mammalian cell lines, preferably human cell lines expressing a GPCR of interest. Preferred cell lines functionally expressing the protein binding domain and/or the GPCR include insect cells (e.g., SF-9), human cell lines (e.g., HEK293), rodent cell lines (e.g., CHO -K1), camelid cell lines (Dubca).
[00157] Protein binding domains or complexes or cellular composition as described above can be used in a variety of contexts and applications which will be described in more detail below. CRYSTALLIZATION AND SOLVING THE STRUCTURE OF A GPCR
[00158] Crystallization of membrane proteins including GPCRs remains a formidable challenge. While expression and purification methods are appearing to allow for the generation of milligram amounts, achieving stability with these molecules is perhaps the most difficult hurdle to overcome. Purification requires a release of the GPCR from the lipid bilayer through detergent solubilization, a process during which the hydrophobic surfaces of the protein are coated with surfactant monomers to form a protein-detergent complex (PDC). However, the detergent belt formed around the protein is a poor replacement for the lipid bilayer, much of the lateral pressure exerted on the protein by the surrounding lipids is lost. Thus, solubilization of membrane proteins often results in destabilization, unfolding, and subsequent aggregation. GPCRs other than rhodopsin typically have poor stability in detergents and are prone to aggregation and proteolysis. Efforts to crystallize GPCRs have been thwarted by other intrinsic features of the integral membrane proteins. The seven hydrophobic transmembrane helices of GPCRs make surfaces poor for crystal contacts, and the extracellular and intracellular domains are often relatively short and/or poorly structured. In addition to rhodopsin (an atypical GPCR in terms of natural abundance and stability), the first crystals of GPCRs were obtained from β2AR bound to a Fab fragment that recognized an epitope composed of the amino- and carboxyl-terminal ends of the third intracellular loop connecting TMs 5 and 6 (Rasmussen, 2007). In the second approach, the third intracellular loop was replaced by T4 lysozyme (β2AR-T4L; Rosenbaum, 2007). Finally, the remarkable versatility of GPCRs as signaling molecules can be attributed to their flexible and dynamic three-dimensional structure. Unfortunately, such dynamic behavior is particularly challenging for high resolution structure analysis. Developing diffraction quality crystals requires stable, conformationally homogeneous protein. As such, diffraction quality crystals from a native unbound GPCR are difficult to obtain, and even when this goal is achieved, the crystal structure will represent only one of many native conformations. Many of these problems in the invention can be solved by using protein binding domains, in particular nanobodies, as tools to stabilize, purify and crystallize the specific conformational states of GPCRs for high resolution structure analysis.
[00159] It is therefore one of the goals of the invention to use protein binding domains as tools to stabilize GPCR proteins and further use these protein binding domains as co-crystallization aids for GPCRs, or in other words to facilitate the crystallogenesis of GPCRs , preferably in a functional conformational state.
[00160] Accordingly, a fourth aspect of the invention pertains to the use of a protein-binding domain according to the invention, or in specific embodiments, a complex comprising the protein-binding domain or a cellular composition that provides the protein-binding domain. protein binding, to stabilize a GPCR in a functional conformational state, in particular an active conformational state; and/or induce the formation of a particular functional (preferably active) conformational state within a GPCR. It will be appreciated that such a protein-binding domain is a very useful tool for purifying, crystallizing and/or solving the structure of a GPCR in a functional conformational state, in particular in its active conformational state. As clearly outlined above, it should be clear that the protein binding domains according to the invention that will be used to purify, stabilize, crystallize and/or resolve the structure of a GPCR can be targeted against any desired GPCR and can specifically bind or recognizing a conformational epitope of a functional conformational state, preferably an active conformational state, of any desired GPCR. In particular, said conformational epitope may be part of an intracellular or extracellular region, or an intramembrane region, or a domain or loop structure of any desired GPCR.
[00161] First, the protein binding domains according to the invention can increase the thermostability of detergent-solubilized receptors, stabilized in a particular conformational state, protecting them from proteolytic degradation and/or aggregation and facilitating purification and concentration of homogeneous samples from the correctly folded receiver. Persons of ordinary skill in the art will recognize that such samples are the preferred starting point for generating diffraction crystals.
[00162] Crystallization of the purified receptor is another major bottleneck in the process of determining macromolecular structure through X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth usually occurs spontaneously in a supersaturated solution as a result of homogeneous nucleation. Proteins can be crystallized in a typical sparse matrix screening experiment in which precipitants, additives, and protein concentration are extensively sampled, and supersaturation conditions suitable for nucleation and crystal growth can be identified for a particular protein. The approach to sparse matrix sorting is to generate structural variation in the protein itself, for example by adding ligands that bind to the protein, or by making different mutations, preferably at surface residues of the target protein, or by trying to crystallize orthologs from different species of the target protein ( Chang 1998). An unexpected finding of the present invention is the utility of protein binding domains such as nanobodies that specifically bind to a GPCR to introduce a degree of structural variation upon binding while preserving the overall folding of the GPCR. Different nanobodies will generate different quaternary structures that provide distinct new interfaces for crystal lattice formation that results in multiple crystal shapes while preserving the overall folding of the GPCR.
[00163] Because crystallization involves an unfavorable loss of conformational entropy in the molecule to be assembled in the crystal lattice, methods that reduce the conformational entropy of the target while still in solution should enhance the probability of crystallization by decreasing the entropic lattice penalty of the formation of a target. trellis. The 'surface entropy reduction approach' has proven to be highly effective (Derewenda 2004). Likewise, binding pairs such as ions, small molecule ligands, and peptides can reduce conformational heterogeneity by binding and stabilizing a subset of conformational states of a protein. Although such binding pairs are effective, not all proteins have a known binding pair, and even when a binding pair is known, its affinity, solubility, and chemical stability may not be compatible with crystallization experiments. Therefore, it was surprisingly found that the protein binding domains of the present invention, in particular the nanobodies, can be used as tools to increase the probability of obtaining well-regulated crystals by minimizing conformational heterogeneity in the target GPCR by binding to a particular conformation of the receptor.
[00164] Crystallization of GPCRs for high resolution structural studies are particularly difficult because of the amphipathic surface of these membrane proteins. Embedded in the membrane bilayer, protein contact sites with phospholipid acyl chains are hydrophobic, while polar surfaces are exposed to lipid polar head groups and aqueous phases. To obtain well-ordered three-dimensional crystals - a precondition for high resolution X-ray structural analysis - GPCRs are solubilized with the aid of detergents and purified as protein-detergent complexes. The detergent micelle covers the hydrophobic surface of the membrane protein in a belt-like fashion (Hunte and Michel 2002; Ostermeier et al. 1995). The GPCR-detergent complexes form three-dimensional crystals in which the contacts between adjacent protein molecules are made by the polar surfaces of the protein that protrude from the detergent micelle (Day et al. 2007). Obviously, the detergent micelle requires space in the crystal lattice. Although attractive interactions between micelles could stabilize crystal packing (Rasmussen et al. 2007; Dunn et al. 1997), these interactions do not lead to rigid crystal contacts. Because many membrane proteins, including GPCRs, contain relatively small or highly flexible hydrophilic domains, one strategy to increase the likelihood of acquiring well-regulated crystals is to increase the protein's polar surface and/or reduce its flexibility. Thus, the nanobodies of the present invention can be used to increase the polar surfaces of the protein, supplementing the amount of protein surface that can facilitate the primary contacts between the molecules in the crystal lattice. Nanobodies of the present invention can also reduce the flexibility of their extracellular regions to grow well-regulated crystals. Nanobodies are especially suitable for this purpose because they are composed of a single rigid globular domain and are devoid of flexible linker regions unlike conventional antibodies or derived fragments such as Fab's.
[00165] In another embodiment of the invention, the complex comprising the protein binding domain according to the invention and the target GPCR in a functional conformational state, preferably an active conformational state, can be crystallized using any of a variety of methods. crystallization methods for membrane proteins, many of which are reviewed in Caffrey (2003). In general terms, the methods are lipid-based methods that include adding lipid to the GPCR-nanobody complex prior to crystallization. Such methods have previously been used to crystallize other membrane proteins. Many of these methods, including the lipid cubic phase crystallization method and the bicell crystallization method, exploit the spontaneous self-assembly properties of lipids and detergent such as vesicles (vesicle fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic phase method). Methods of crystallization of lipid cubic phases are described, for example, in: Landau et al. 1996; Gouaux 1998; Rummel et al. 1998; Nollert et al. 2004 whose publications are incorporated by reference for disclosure of those methods. Bicell crystallization methods are described, for example, in: Faham et al. 2005; Faham et al. 2002 whose publications are incorporated by reference for disclosure of those methods.
[00166] According to another embodiment, the invention further encompasses the use of a protein-binding domain as described above to solve a structure of a GPCR. The structure of a protein, in particular a GPCR, includes the primary, secondary, tertiary and, if applicable, quaternary structure of said protein. "Structure solving" as used herein refers to determining the arrangement of atoms or the atomic coordinates of a protein, and is often done by a biophysical method, such as X-ray crystallography.
[00167] In X-ray crystallography, the diffraction data when correctly assembled gives the 3D Fourier transform amplitude of the electron density of the molecule in the unit cell. If the phases are known, the electron density can simply be obtained by Fourier synthesis. For a protein complex, the success of deriving molecular substitution (MR) phase information alone is questionable when the fraction of proteins with a known structure (the research models) is low (less than 50% amino acid content) and/or when the crystals exhibit limited diffraction quality. Although the combination of multiple isomorphic substitution (MIR) and MR phasing has proven successful for protein complexes (eg, Ostermeier et al. 1995; Li et al. 1997; Hunte et al. 2000), the requirement to produce a good heavy atom derivative is almost always problematic. Over the past decade, classical MIR approaches have largely been replaced by the use of anomalous scatter data using selenomethionine (SeMet) incorporation (MAD or SAD) (Hendrickson 1991). In fact, anomalous experimental data using edge energies of Se generally provide higher or less polarized phase information compared to model-based MIR or MR phasing data. One embodiment of this invention concerns the use of nanobodies for phasing GPCR-nanobody complexes by MR or MAD. Nanobodies generally express robustly and are suitable for SeMet incorporation. Staging a GPCR-nanobody Complex by introducing all SeMet sites alone into the nanobody avoids the need to incorporate SeMet sites into the GPCR.
[00168] In many cases, obtaining a diffraction quality crystal is the main barrier to solving its atomic resolution structure. Thus, according to specific embodiments, protein binding domains as described above, in particular nanobodies, can be used to improve the diffraction quality of crystals so that the GPCR protein crystal structure can be solved. .
[00169] There is great interest in structural information to help guide GPCR drug discovery. For the GPCRs whose structures have now been solved, these modeling efforts have been shown to be imprecise to the level required by in silico drug designers. With the inactive-state receptor structures of β2AR, β1AR and A2A, pharmaceutical chemists now have experimental data to guide the development of ligands for various active therapeutic targets. However, the value of these high resolution structures for in silico screening may be limited. Recent molecular docking studies using the crystal structure of β2AR as a model have identified six novel β2AR ligands that bind with affinities ranging from 9 nM to 4 μM; however, each compound exhibited reverse agonist activity. In addition to crystallizing more GPCRs, methods to acquire the structures of receptors bound to different classes of ligands including agonists, antagonists, allosteric regulators and/or G proteins must be developed. For example, agonist-bound receptor crystals can provide three-dimensional representations of the active states of GPCRs. These structures will help clarify the conformational changes that link ligand binding and G protein interaction sites, and lead to more precise mechanistic hypotheses and eventually new therapeutics. Given the conformational flexibility inherent in ligand-activated GPCRs and the greater heterogeneity displayed by agonist-bound receptors, stabilizing such a state is not easy. Such efforts may benefit from stabilizing the agonist-bound receptor conformation by adding protein-binding domains that are specific for an active conformational state of the receptor. Especially suitable are nanobodies showing G-protein-like behavior and exhibiting cooperative properties with respect to agonist binding, as provided in the present invention (see Example section).
[00170] Accordingly, the present invention also provides a method of determining a crystal structure of a GPCR in a functional conformational state, said method comprising the steps of: (i) providing a protein binding domain in accordance with the invention, a target GPCR, and optionally a receptor ligand, and (ii) form a complex of the protein binding domain, the GPCR, and optionally the receptor ligand, and (iii) crystallize said complex of step (ii) to form a crystal
[00171] wherein the crystal structure is determined from a GPCR in a functional conformational state, preferably the active conformational state.
[00172] Said crystal structure determination can be done by a biophysical method such as X-ray crystallography. The method can further comprise a step of obtaining the atomic coordinates of the crystal (as defined above). CAPTURE AND/OR PURIFICATION OF A GPCR IN A FUNCTIONAL CONFORMATIONAL STATE
[00173] In yet another embodiment, the invention provides a method for capturing and/or purifying a GPCR in a functional conformational state, preferably an active conformational state, making use of any of the protein binding domains described above, or complexes or cellular compositions comprising such protein-binding domains. Capture and/or purification of a GPCR, in a functional conformational state, preferably an active conformational state, according to the invention, will allow subsequent crystallization, ligand characterization and compound screening, immunizations, among others. In practice, such methods and techniques include, without limitation, affinity-based methods such as affinity chromatography, affinity purification, immunoprecipitation, protein detection, immunochemistry, surface display, among others, and are all well known to those skilled in the art. technique.
[00174] Thus, in a particular embodiment, the invention concerns the use of a protein-binding domain according to the invention, or a complex or a cellular composition comprising the same as previously described herein, to capture a GPCR in a functional conformational state, preferably capturing a GPCR in its active conformational state. Optionally, but not necessarily, capturing a GPCR in its functional conformational state as described above may include capturing a GPCR in a functional conformational state in complex with a receptor ligand or one or more downstream interacting proteins.
[00175] Accordingly, the invention also provides a method of capturing a GPCR in a functional conformational state, said method comprising the steps of: (i) providing a protein binding domain according to the invention and a target GPCR, and, (ii) form a complex of the protein binding domain and the GPCR,
[00176] wherein a GPCR is captured in a functional conformational state, preferably an active conformational state.
[00177] More specifically, the invention also aims at a method of capturing a GPCR in a functional conformational state, said method comprising the steps of: (i) applying a solution containing a GPCR in a plurality of conformational states to a solid support that has an immobilized protein-binding domain in accordance with the invention, and (ii) forms a complex of the protein-binding domain and the GPCR, and (iii) loosely removes bound or unbound molecules,
[00178] wherein a GPCR is captured in a functional conformational state, preferably an active conformational state.
[00179] It will be appreciated that any of the methods as described above may further comprise the step of purifying the protein-binding domain complex and the GPCR into its functional conformational state. THERAPEUTIC AND DIAGNOSTIC APPLICATIONS
[00180] Traditionally, small molecules are used for drug development directed against GPCRs, not only because pharmaceutical companies have historical reasons for working with them, but more importantly because of the structural constraints of Family 1 GPCRs having the binding site of linker within the cleaved transmembrane (Nat Rev Drug Discov. (2004)). The state of GPCR research in 2004. Nature Reviews Drug Discovery GPCR Questionnaire Participants 3(7):575, 577-626). For this reason it has proved difficult or impossible to generate monoclonal antibodies against this target class. The protein-binding domains of the present invention, in particular the nanobodies, can solve this particular problem through their intrinsic property of binding via CDR loops extending into the wells.
[00181] Accordingly, a fifth aspect of the invention pertains to a pharmaceutical composition comprising a therapeutically effective amount of a protein-binding domain according to the invention and at least one of a pharmaceutically acceptable carrier, adjuvant or diluents.
[00182] A 'vehicle', or 'adjuvant', in particular a pharmaceutically acceptable carrier of' or 'pharmaceutically acceptable adjuvant' is any suitable excipient, diluent, vehicle and/or adjuvant which, in itself, does not induce the production of antibodies harmful to the individual receiving the composition nor provide protection. Thus, pharmaceutically acceptable carriers are inherently non-toxic and non-therapeutic, and they are known to the person skilled in the art. Suitable carriers or adjuvants typically comprise one or more of the compounds included in the following non-exhaustive list: large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Carriers or adjuvants may be, as a non-limiting example, Ringer's solution, dextrose solution or Hank's solution. Nonaqueous solutions such as fixed oils and ethyl oleate can also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.
[00183] Administration of a protein-binding domain according to the invention or a pharmaceutical composition thereof may be via oral, inhaled or parenteral administration. In particular embodiments, the protein-binding domain is released via intrathecal or intracerebroventricular administration. The active compound can be administered alone or preferably formulated as a pharmaceutical composition. An amount effective to treat a certain disease or disorder expressing the antigen recognized by the protein-binding domain depends on usual factors such as the nature and severity of the disorder being treated and the weight of the mammal. However, a unit dose will normally be in the range of 0.1 mg to 1 g, e.g. 0.1 to 500 mg, e.g. 0.1 to 50 mg, or 0.1 to 2 mg of binding domain to the protein or a pharmaceutical composition thereof. Unit doses will normally be administered once a month, once a week, biweekly, once or more than once a day, for example 2, 3, or 4 times a day, more usually 1 to 3 times a day. It is greatly preferred that the protein-binding domain or a pharmaceutical composition thereof is administered in the form of a unit dose composition, such as an oral, parenteral, unit dose, or inhaled composition. Such compositions are prepared by mixing and suitably adapted for oral, inhaled or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and non-injectable solutions or suppositories. or aerosols. Usually tablets and capsules are presented for oral administration in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tableting agents, lubricants, disintegrants, coloring agents, flavors, and wetting agents. The tablets can be coated according to methods well known in the art. Fillers suitable for use include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycollate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate. These solid oral compositions can be prepared by conventional mixing, filling, tableting or other methods. Repeated mixing operations can be used to distribute the active agent throughout those compositions employing large amounts of fillers. Such operations are, of course, conventional in the art. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or they can be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate , or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerin, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents. Oral formulations also include conventional continuous release formulations, such as tablets or granules having an enteric coating. Preferably, the inhalation compositions for administration to the respiratory tract are presented as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the active compound particles suitably have diameters of less than 50 microns, preferably less than 10 microns, for example between 1 and 5 microns, such as between 2 and 5 microns. A favored inhaled dose will be in the range of 0.05 to 2 mg, for example 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2 mg. For parenteral administration, fluid unit dose forms are prepared containing a compound of the present invention and a sterile vehicle. The active compound, depending on the vehicle and concentration, can either be suspended or dissolved. Parenteral solutions are normally prepared by dissolving the compound in a vehicle and sterilizing the filter before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle. To enhance stability, the composition can be frozen after being filled into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle rather than being dissolved and sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound. Where appropriate, small amounts of bronchodilators, for example, sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylphrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included. As is common practice, the compositions will usually be accompanied by written or printed directions for use in the medical treatment concerned.
[00184] Release of protein binding domains, in particular nanobodies, into cells can be performed as described for peptides, polypeptides and proteins. If the antigen is extracellular or an extracellular domain, the protein-binding domain can exert its function by binding this domain, without the need for intracellular release. The protein binding domains of the present invention as described herein can target intracellular conformational epitopes of GPCRs of interest. Using these protein binding domains as effective and safe therapeutics within a cell, intracellular delivery can be enhanced by protein transduction or delivery systems known in the art. Protein transduction domains (PTDs) have attracted considerable interest in the drug delivery field for their ability to translocate across biological membranes. PTDs are relatively short sequences (1-35 amino acids) that confer this translocation activity evident to proteins and other macromolecular cargo to which they are conjugated, complexed, or fused (Sawant and Torchilin 2010). HIV-derived TAT peptide (YGRKKRRQRRR), for example, has been widely used for intracellular delivery of various agents ranging from small molecules to proteins, peptides, range of pharmaceutical nanovehicles and imaging agents. Alternatively, receptor-mediated endocytic mechanisms can also be used for intracellular drug delivery. For example, the transferrin receptor-mediated internalization pathway is an efficient cellular uptake pathway that has been exploited for site-specific release of drugs and proteins (Qian et al. 2002). This is achieved either chemically by conjugating transferrin to therapeutic drugs or proteins or genetically by infusing therapeutic peptides or proteins into the transferrin structure. Naturally existing proteins (such as the iron-binding protein transferrin) are very useful in this area of drug targeting as these proteins are biodegradable, non-toxic, and non-immunogenic. Furthermore, they can achieve site-specific targeting due to the high amounts of their receptors present on the cell surface. Still other delivery systems include, without intending to be limiting, polymer-based and liposome-based delivery systems.
[00185] The effectiveness of the protein binding domains of the invention, and compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved.
[00186] A sixth aspect of the invention pertains to the use of the protein binding domain or pharmaceutical composition as described above to modulate GPCR signaling activity.
[00187] The protein binding domains of the invention as described herein can bind to the GPCR to activate or enhance receptor signaling; or alternatively to decrease or inhibit receptor signaling. The protein binding domains of the invention can also bind to the GPCR in such a way that they block the constitutive activity of the GPCR. The protein binding domains of the invention may also bind to the GPCR in such a way that they mediate allosteric modulation (e.g. bind to the GPCR at an allosteric site). In this way, the protein binding domains of the invention can modulate receptor function by binding to different regions on the receptor (e.g., at allosteric sites). Reference is made, for example, to George et al. (2002), Kenakin (2002) and Rios et al. (2001). The protein binding domains of the invention may also bind GPCR in such a way that they prolong the duration of GPCR-mediated signaling or that they enhance receptor signaling by increasing receptor-ligand affinity. Furthermore, the protein-binding domains of the invention may also bind GPCR in such a way that they inhibit or enhance the set of functional GPCR homomers or heteromers.
[00188] In a particular embodiment, the protein binding domain or pharmaceutical composition as described above blocks G protein-mediated signaling.
[00189] In another embodiment, the invention also relates to the protein-binding domain or pharmaceutical composition as described above for use in the treatment of a GPCR-related disease, such as cancer, autoimmune disease, infectious disease, neurological disease, disease cardiovascular.
[00190] Certain protein binding domains described above may have therapeutic utility and be administered to a subject having a condition to treat the subject for the condition. Therapeutic utility for a protein-binding domain can be determined by the GPCR to which the protein-binding domain binds where signaling through that GPCR is linked to the condition. In certain cases, the GPCR can be activated in the condition by binding to a ligand. In other embodiments, the GPCR can be mutated to make it constitutively active, for example. A protein binding domain in question can be employed for the treatment of a GPCR-mediated condition such as schizophrenia, migraine headache, reflux, asthma, bronchospasm, prostatic hypertrophy, ulcers, epilepsy, angina, allergy, rhinitis, cancer , for example, prostate cancer, glaucoma and stroke. Other conditions related to exemplary GPCRs in the Mendelian Inheritance in Man Online Database found on the NCBI World Wide Web site. Thus, a particular embodiment of the present invention also provides for the use of a protein-binding domain or a pharmaceutical composition for the treatment of a GPCR-related disease or disorder.
[00191] In a more specific embodiment, the protein-binding domain may bind to the e2-adrenergic receptor in which case it may be employed in the treatment of a condition requiring relaxation of the smooth muscle of the uterus or vascular system. Such a protein-binding domain can thus be used for the prevention or alleviation of preterm birth in pregnancy, or in the treatment of chronic or acute asthma, urticaria, psoriasis, rhinitis, allergic conjunctivitis, actinitis, hay fever, or mastocytosis, whose conditions have been linked to the β2-adrenoreceptor. In these embodiments, the protein-binding domain can be employed as co-therapeutic agents for use in combination with other drug substances such as anti-inflammatory, bronchodilator or antihistamine drug substances, particularly in the treatment of obstructive or inflammatory diseases of the airways. such as those mentioned above, for example as enhancers of the therapeutic activity of such drugs or as a means of reducing the required dosage or potential side effects of such drugs. A protein binding domain in question can be mixed with the other drug substance in a fixed pharmaceutical composition or it can be administered separately, before, simultaneously with or after the other drug substance. Broadly speaking, these protocols involve administering to an individual suffering from a GPCR-related disease or disorder an effective amount of a protein-binding domain that modulates a GPCR to modulate the GPCR in the host and treating the individual for the disorder.
[00192] In some embodiments where a reduction in the activity of a certain GPCR is desired, one or more compounds that decrease the activity of the GPCR may be administered, whereas when an increase in the activity of a certain GPCR is desired, one or more compounds that increase the activity of GPCR activity can be administered.
[00193] A variety of individuals are treatable according to the methods in question. In general, such individuals are mammals, where this term is widely used to describe organisms that are within the class of mammalia, including orders of carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates. (eg humans, chimpanzees and monkeys). In many embodiments, the individuals will be human beings. Methods of treatment in question are typically performed on individuals with such disorders or on individuals with a desire to avoid such disorders.
[00194] According to yet another embodiment, the protein-binding domain or complex according to the invention may also be useful for the diagnosis or prognosis of a GPCR-related disease, such as cancer, autoimmune disease, infectious disease, neurological disease, cardiovascular disease. IDENTIFICATION OF COMPOUNDS SELECTIVELY AIMING A GPCR IN A FUNCTIONAL CONFORMATIONAL STATE
[00195] In the process of compound screening, lead compound optimization, and drug discovery, there is a requirement for faster, more effective, less expensive, and especially information-rich screening assays that provide simultaneous information about multiple compound characteristics and its effects on various cellular pathways (i.e. drug efficacy, specificity, toxicity and metabolism). Thus, there is a need for rapid and inexpensive screening of large numbers of compounds to identify novel specific binders of a GPCR of interest, preferably conformation-specific binders, that could be potential new drug candidates. The present invention solves this problem by providing protein binding domains that stabilize and/or lock a GPCR into a functional conformational state, preferably an active conformational state, which can then be used as immunogens or selection reagents to screen in a variety of contexts. . A major advantage of the protein binding domains according to the invention is that a GPCR can be maintained in a stabilized functional conformation, preferably in an active state conformation. For example, library compounds that selectively bind this active conformation of the receptor have a higher tendency to behave as agonists because orthosteric or allosteric stabilization of the active conformation of the GPCR elicits biological responses. Another advantage is that the protein-binding domain increases the thermostability of the active conformation of the GPCR, thereby protecting the GPCR against irreversible or thermal denaturation induced by the non-native conditions used in compound screening and drug discovery, without the need to rely on the mutant GPCRs with increased stability. Another main advantage of the conformation-selective protein-binding domains according to the invention is that they allow to quickly and reliably screen and differentiate between receptor agonists, reverse agonists, antagonists and/or modulators as well as inhibitors of GPCRs and GPCR-dependent pathways. , thus increasing the likelihood of identifying a ligand with the desired pharmacological properties.
[00196] To illustrate this further, it is a well-established concept that most GPCRs exhibit higher agonist binding affinity when complexed with the G protein. This is attributed to the cooperative interaction between the agonist-coupled receptor and the G protein ( Delean et al. 1980). The protein binding domains of the present invention, in particular the nanobodies, are capable of stabilizing an active conformational state of a GPCR (and thereby showing G-protein-like behavior) and destabilizing inactive conformational states, thereby increasing affinity. of the GPCR by agonists and decreasing the affinity for reverse agonists or antagonists. It follows that protein-binding domains, such as nanobodies, according to the invention that recognize the active functional conformation of the GPCR can be used efficiently in a high-throughput screening assay for agonists because they increase the receptor's affinity for agonists, with compared to reverse agonists or antagonists. Conversely, protein binding domains, in particular nanobodies that stabilize the inactive state conformation of a GPCR will increase affinity for a reverse agonist or antagonist and decrease affinity for an agonist. Such protein binding domains can be used, for example, to screen for reverse agonists. Thus, protein binding domains, particularly nanobodies, which recognize particular functional conformational states thereby modulating affinities for agonists and reverse agonists in a reciprocal manner are also part of the present invention.
[00197] Thus, in a preferred embodiment, the present invention encompasses the use of protein-binding domains, or complexes, or cellular compositions, all as described above, in screening and/or pairwise identification programs. conformation-specific binding of a GPCR, which could ultimately lead to potential new drug candidates.
[00198] According to one embodiment, the invention aims at a method of identifying compounds capable of selectively binding to a functional conformational state of a GPCR, said method comprising the steps of: (i) providing a GPCR and a domain of binding protein capable of specifically binding to a functional conformational state of said GPCR according to the invention, and (ii) providing a test compound, and (iii) evaluating whether the test compound binds to the functional conformational state of the GPCR, and (iv) ) select a compound that selectively binds to the functional conformational state of the GPCR.
[00199] Preferably, the above method further comprises a step of forming a complex comprising the protein binding domain and the GPCR in a functional conformational state, more preferably in an active conformational state.
[00200] Thus, the invention also aims at a method of identifying compounds capable of selectively binding to a functional conformational state of a GPCR, said method comprising the steps of: (i) providing a complex comprising a binding domain protein according to the invention and a GPCR in a functional conformational state, and (ii) provide a test compound, and (iii) assess whether the test compound binds to the functional conformational state of the GPCR, and (iv) select a compound that binds to the functional conformational state of the GPCR.
[00201] Preferably, the protein binding domain as used in any of the above methods is capable of stabilizing and/or inducing a functional conformational state of a GPCR upon binding. Preferably, said functional conformational state of a GPCR is selected from the group consisting of a basal conformational state, either an active conformational state or an inactive conformational state (as defined above). Most preferably, said functional conformational state of a GPCR is an active conformational state.
[00202] In another preferred embodiment, the protein binding domain as used in any of the above screening methods comprises an amino acid sequence comprising 4 framework regions and 3 complementary determining regions, or any suitable fragment thereof. Preferably, said protein binding domain is derived from a camelid antibody. More preferably, said protein-binding domain comprises a nanobody sequence, or any suitable fragment thereof. In particular, said nanobodies comprise a sequence selected from the group consisting of SEQ ID NOs: 1-29, or any suitable fragment thereof.
[00203] Other preferences for protein binding domains and/or complexes are as defined above with respect to the first and second aspects of the invention.
[00204] In a preferred embodiment, the protein binding domain, the GPCR or the complex comprising the protein binding domain and the GPCR, as used in any of the above screening methods, are provided as whole cells, or cell extracts (organelles) such as membrane extracts or fractions thereof, or may be incorporated into lipid layers or vesicles (comprising natural and/or synthetic lipids), high-density lipoparticles, or any nanoparticles, such as nanodisks, or are supplied as VLPs , so that sufficient functionality of the respective proteins is maintained. Preparations of GPCRs formed from membrane fragments or membrane-detergent extracts are reviewed in detail in Cooper (2004), incorporated herein by reference. Alternatively, GPCRs and/or the complex may also be solubilized in detergents. Non-limiting examples of solubilized receptor preparations are further provided in the Example section.
[00205] Often, high-throughput screening of GPCR targets for conformation-specific binding pairs will be preferred. This will be facilitated by immobilizing a protein-binding domain according to the invention, a GPCR in a functional conformational state, or a complex comprising them, on a suitable solid surface or support which may otherwise be arranged multiplexed. Non-limiting examples of suitable solid supports include beads, columns, slides, slices or plates.
[00206] More particularly, solid supports can be particulate (e.g. beads or granules, generally used in extraction columns) or sheet-like (e.g. membranes or filters, glass or plastic slides, test plates microtiter tube, dipstick, capillary supply devices or the like) which may be flat, pleated, or hollow fibers or tubes. The following matrices are given as examples and are not exhaustive, such examples could include silica (porous amorphous silica), i.e. the FLASH series of cartridges containing 60A irregular silica (3263 µm or 35-70 µm) supplied by Biotage (a division of Dyax Corp.), agarose or polyacrylamide supports, for example the Sepharose product range provided by Amersham Pharmacia Biotech, or the Affi-gel supports provided by Bio-Rad. In addition, there are macroporous polymers such as the pressure stable Affi-Prep supports as supplied by Bio-Rad. Other supports that could be used include; dextran, collagen, polystyrene, methacrylate, calcium alginate, controlled pore glass, aluminum, titanium and porous ceramics. Alternatively, the solid surface may comprise part of a sensor-dependent mass, for example a plasmon surface resonance detector. Other examples of commercially available supports are discussed in, for example, Protein Immobilisation, R.F. Taylor ed., Marcel Dekker, Inc., New York, (1991).
[00207] Immobilization can be non-covalent or covalent. In particular, immobilization or non-covalent adsorption on a solid surface of the protein-binding domain, the GPCR or the complex comprising said protein-binding domain and said GPCR, according to the invention may occur by means of a coating of surface with either an antibody, or streptavidin or avidin, or a metal ion, recognizing a molecular marker attached to the protein-binding domain or the GPCR, in accordance with standard techniques known to the skilled person (e.g., biotin marker , Histidine tag, etc.).
[00208] In particular, the protein-binding domain, the GPCR or the complex comprising said protein-binding domain and said GPCR, according to the invention, can be attached to a solid surface by covalent cross-linking using chemical conventional couplings. A solid surface may naturally comprise suitable crosslinkable residues for covalent bonding or may be coated or derivatized to introduce suitable crosslinkable groups according to methods well known in the art. In a particular embodiment, sufficient functionality of the immobilized protein is maintained following direct covalent coupling to the desired matrix via a reactive moiety that does not contain a chemical spacer arm. Additional examples and more detailed information on methods of immobilizing antibody (fragments) on solid supports are discussed in Jung et al. (2008); similarly, membrane receptor immobilization methods are reviewed in Cooper (2004); both incorporated herein by reference.
[00209] Advances in molecular biology, particularly through loco-directed mutagenesis, allow the mutation of specific amino acid residues in a protein sequence. Mutation of a particular amino acid (in a protein with a known or deduced structure) to a lysine or cysteine (or other desired amino acid) can provide a specific site for covalent coupling, for example. It is also possible to re-engineer a specific protein to alter the distribution of available surface amino acids involved in chemical coupling (Kallwass et al, 1993), in effect controlling the orientation of the coupled protein. A similar approach can be applied to the protein binding domains according to the invention, as well as to conformationally stabilized GPCRs, whether or not comprised in a complex, providing a means of targeted immobilization thus without the addition of other peptide tails or domains containing natural or unnatural amino acids. In the case of an antibody or an antibody fragment, such as a nanobody, the introduction of mutations in the framework region, minimizing disruption to the antigen binding activity of the antibody (fragment) is preferred.
[00210] Conveniently, the immobilized proteins can be used in immunoadsorption processes such as immunoassays, for example ELISA, or immunoaffinity purification processes by contacting the immobilized proteins according to the invention with a test sample according to conventional standard methods. in the technique. Alternatively, and particularly for high throughput purposes, the immobilized proteins can be arrayed or otherwise multiplexed. Preferably, the immobilized proteins according to the invention are used for the screening and selection of compounds that specifically bind to a GPCR in a functional conformational state, in particular a GPCR in an active conformational state.
[00211] It will be appreciated that either the protein-binding domain or the GPCR in a functional conformational state, or said complex comprising said protein-binding domain and said GPCR can be immobilized, depending on the type of application or the type sorting that needs to be done. Also, the choice of the GPCR stabilizing protein binding domain (targeting a particular conformational epitope of the GPCR), will determine the orientation of the GPCR and hence the desired outcome of the identification of compounds, e.g. compounds that specifically bind to extracellular moieties. , intramembrane parts or intracellular parts of said conformationally stabilized GPCR.
[00212] In an alternative embodiment, the test compound (or a library of test compounds) may be immobilized on a solid surface, such as a slice surface, while the protein-binding domain, GPCR or complex is supplied, for example, in a detergent solution or in a membrane-like preparation.
[00213] Most preferably, neither the protein binding domain, nor the GPCR, nor the test compound are immobilized, for example, in solution phage display screening protocols, or radioligand binding assays.
[00214] Various methods can be used to determine binding between the stabilized GPCR and a test compound, including, for example, enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance assays, slice-based assays, immunocytofluorescence , yeast two-hybrid technology and phage display, which are common practice in the art, for example in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Other methods of detecting binding between a test compound and a GPCR include ultrafiltration with ion spray mass spectroscopy/HPLC or other (bio)physical and analytical methods. Fluorescence Resonance Energy Transfer (FRET) methods, for example, well known to those skilled in the art, can also be used. It will be appreciated that a bound test compound can be detected using a single tag or tag associated with the compound, such as a peptide tag, a nucleic acid tag, a chemical tag, a fluorescent tag, or a radio frequency tag, as described further here.
[00215] In addition to establishing binding to a GPCR in a functional conformational state, it will also be desirable to determine the functional effect of a compound on the GPCR. In particular, protein binding domains or cellular complexes or compositions as described herein can be used to screen for compounds that modulate (increase or decrease) the biological activity of the GPCR. The desired modulation in biological activity will depend on the GPCR of choice. The compounds to be tested can be any small chemical compound, or a macromolecule, such as a protein, sugar, nucleic acid, or lipid. Typically, test compounds will be small chemical compounds, peptides, antibodies or fragments thereof. It will be appreciated that in some circumstances high throughput screening of test compounds is preferred and that methods as described above may be used as a "library" screening method, a term well known to those skilled in the art. Thus, the test compound may be a library of test compounds. In particular, high throughput screening assays for therapeutic compounds such as agonists, antagonists or reverse agonists and/or modulators form part of the invention. For high throughput purposes, compound libraries such as allosteric compound libraries, peptide libraries, antibody libraries, fragment-based libraries, synthetic compound libraries, natural compound libraries, phage display libraries and others can be used. used. Methodologies for preparing and screening such libraries are known in the art.
[00216] In a preferred embodiment, high-throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic ligands. Such "combinatorial libraries" or "compound libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that exhibit a desired characteristic activity. A "compost library" is usually a collection of stored chemical agents used ultimately in high throughput sorting. A "combinatorial library" is a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis, combining various "building blocks" such as reagents. Preparation and screening of combinatorial libraries are well known to those of skill in the art. Compounds so identified can serve as conventional "lead compounds" or can be used as potential or current therapeutics. Thus, in another embodiment, the screening methods as described above complex comprise modifying a test compound that has been shown to bind to a GPCR in a functional conformational state, preferably an active conformational state, and determining whether the modified test compound binds to the GPCR. GPCR when residing in the particular conformation.
[00217] In a particular embodiment, the test compound is provided as a biological sample. In particular, the sample may be any suitable sample taken from an individual. For example, the sample may be a sample of body fluid such as blood, serum, plasma, spinal fluid. Alternatively, the sample is tissue or cell extract.
[00218] The compounds can bind to the target GPCR which results in the modulation (activation or inhibition) of the biological function of the GPCR, in particular downstream receptor signaling. This modulation of GPCR signaling can occur ortho or allosterically. Compounds can bind to the target GPCR to activate or enhance receptor signaling; or alternatively to decrease or inhibit receptor signaling. The compounds can also bind to the target GPCR in such a way that they block the constitutive activity of the GPCR. The compounds may also bind to the target complex in such a way that they mediate allosteric modulation (eg bind to the GPCR at an allosteric site). In this way, compounds can modulate receptor function by binding to different regions on the GPCR (eg, at allosteric sites). Reference is made, for example, to George et al. (2002), Kenakin (2002) and Rios et al. (2001). Compounds of the invention may also bind to the target GPCR in such a way that they prolong the duration of GPCR-mediated signaling or that they enhance receptor signaling by increasing receptor-ligand affinity. Furthermore, the compounds may also bind to the target complex in such a way that they inhibit or enhance the assembly of functional GPCR homomers or heteromers.
[00219] In one embodiment, it is determined whether the compound alters the binding of the GPCR to a ligand on the receptor (as defined herein). Binding of a GPCR to its ligand can be assayed using standard ligand binding methods known in the art as described herein. For example, a linker may be radiolabeled or fluorescently labeled. The assay can be performed on whole cells or on membranes obtained from the cells or receptor solubilized aqueous with a detergent. The compound will be characterized by its ability to alter the binding of the labeled ligand (see also the Example section). The compound may decrease binding between GPCR and its ligand, or may increase binding between GPCR and its ligand, for example by a factor of at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold , 30 times, 50 times, 100 times.
[00220] Thus, in accordance with more specific embodiments, the complex as used in any of the above screening methods additionally comprises a receptor ligand. Preferably, the receptor ligand is chosen from the group comprising a small molecule, a polypeptide, an antibody or any fragment derived therefrom, a natural product, and the like. More preferably, the receptor ligand is a full agonist, or a polarized agonist, or a reverse agonist, or antagonist, as described above.
[00221] According to a specific embodiment, the protein binding domains of the present invention, particularly the nanobodies, may also be useful for leader identification and the design of peptidomimetics. Using a biologically relevant peptide or protein structure as a starting point for leader identification represents one of the most powerful approaches in modern drug discovery. Peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect. Peptidomimetics are designed to avoid some of the problems associated with a natural peptide: for example stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, can often be substantially improved. By way of non-limiting example, the nanobodies of the present invention can bind with the long CDR loops deep in the receptor nucleus to exert a biological effect. These peptides and their concomitant structures in the nanobody-GPCR complex can serve as starting points for leader identification and the design of peptidemimetics.
[00222] Consequently, protein binding domains, in particular the nanobodies of the present invention, may be useful in screening assays. Screening assays for drug discovery can be solid-phase or solution-phase assays, for example, a binding assay, such as radioligand binding assays. In high-throughput assays, up to several thousand different modulators or ligands can be screened in a single day in 96, 384, or 1536-well formats. For example, each well of a microtiter plate can be used to refer to a separate assay against a selected potential modulator, or, if concentration or incubation time effects are observed, every 5-10 wells can test one modulator only. Thus, a single standard microtiter plate can assay about 96 modulators. It is possible to rehearse many boards per day; Assay screens for up to about 6,000, 20,000, 50,000 or more different compounds are possible today.
[00223] In addition, protein binding domains, such as the nanobodies of the present invention, may also be useful in cell-based assays. Cell-based assays are also critical to assess the mechanism of action of novel biological targets and the biological activity of chemical compounds. Current cell-based assays for GPCRs include measurements of pathway activation (Ca2+ release, cAMP generation or transcriptional activity); protein traffic measurements by labeling the GPCRs and downstream elements with GFP; and direct measurements of interactions between proteins using Forster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), or yeast two-hybrid approaches. Introducing the protein binding domains, in particular the nanobodies of the present invention, within the cell to the relevant compartment of the cell (intracellularly or extracellularly) by any means well known and commonly used in the art, can lead to assays based on new or improved cells.
[00224] In particular, there is a need to "deorphanize" those GPCRs for which a naturally activated ligand has not been identified. Stabilization of GPCRs in a functional conformational state using the protein binding domains according to the invention allows for screening approaches that can be used to identify ligands of "orphaned" GPCRs where the natural ligand is unknown. Orphan GPCR ligands can be identified from biological samples such as blood or tissue extract or from libraries of ligands. For example, several approaches to "deorphanize" have been adopted including array-screening against families of known ligands.
[00225] The effectiveness of compounds and/or compositions comprising the same can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved.
[00226] Accordingly, in a specific embodiment, a solid support to which a protein-binding domain according to the invention is immobilized and/or a complex comprising a protein-binding domain and a GPCR in a functional conformational state in accordance with with the invention is provided for use in any of the above screening methods.
[00227] In one embodiment, the test compound as used in any of the above screening methods is selected from the group comprising a polypeptide, a peptide, a small molecule, a natural product, a peptidemimetic, a nucleic acid, a lipid, lipopeptide, a carbohydrate, an antibody or any fragment derived therefrom, such as Fab, Fab' and F(ab')2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising a VL or VH domain, a heavy chain antibody (hcAb), a single domain antibody (sdAb), a minibody, the variable domain derived from camelid heavy chain antibodies (VHH or nanobody), the variable domain of shark antibody-derived novel antigen receptors (VNAR), a protein scaffold including an alphabody, protein A, protein G, projected ankyrin repeat domains (DARPins), fibronectin type III repeats, anticalines, quinotines, domains of engineered CH2 (nanoantibodies), as defined above.
[00228] The test compound may optionally be covalently or non-covalently linked to a detectable label. Suitable detectable markings and techniques for binding, using and detecting the same will be clear to the skilled person, and include, but are not limited to, any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads (e.g. Dynabeads), fluorescent dyes (e.g. all Alexa Fluor dyes, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein and others), radiolabels (e.g. 3H, 125I, 35S, 14C , or 32P), enzymes (e.g. horseradish peroxidase, alkaline phosphatase), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g. polystyrene, polypropylene, latex, etc.). Means of detecting such markings are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination. Enzyme labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected simply by viewing the colored label. Other suitable detectable labels were described earlier within the context of the first aspect of the invention relating to a protein binding domain.
[00229] In a preferred embodiment, the test compound is an antibody or any fragment derived therefrom, as described above, including a nanobody. For example, and not intended to be limiting, the test compound may be an antibody (as defined herein in its broadest sense) that has been raised against a complex comprising a protein-binding domain according to the invention and a GPCR ( including variants as described above) in a functional conformational state, preferably in an active conformational state. Methods for raising antibodies in vivo are known in the art. Preferably, immunization of an animal will be done in a similar manner as described previously herein (immunization with GPCR in the presence of receptor ligand; see also Example section) with a GPCR in the presence of a state-stabilizing protein binding domain. functional conformational, more preferably an active state stabilization protein binding domain. The invention also relates to methods for selecting antibodies specific for a GPCR in a functional conformational state, preferably an active conformational state, involving screening of expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria, yeast, filamentous fungi , ribosome or ribosomal subunits or other display systems in a complex containing a GPCR and a protein-binding domain that stabilizes a functional conformational state of the GPCR.
[00230] A seventh aspect of the invention relates to a kit comprising a protein-binding domain according to the invention or a complex according to the invention or a cellular composition according to the invention. The kit may further comprise a combination of reagents such as buffers, molecular markers, vector constructs, reference sample material, as well as suitable solid supports, and the like. Such a kit may be useful for any of the applications of the present invention as described herein. For example, the kit may comprise (a library of) test compounds useful for compound screening applications.
[00231] Finally, a last aspect of the invention is the use of any protein-binding domain according to the invention to isolate amino acid sequences that are responsible for specific binding to a conformational epitope of a functional conformational state of a GPCR, in particular an active conformational state of a GPCR and to construct artificial protein binding domains based on said amino acid sequences. It will be appreciated that in the protein-binding domains according to the invention, framework regions and complementary determining regions are known, and the study of protein-binding domain derivatives binding to the same conformational epitope of a functional conformational state of a GPCR, in particular an active conformational state of a GPCR, will make it possible to deduce the essential amino acids involved in the binding of said conformational epitope. This knowledge can be used to construct a minimal protein-binding domain and create derivatives thereof, which can be done ordinarily by techniques known to those skilled in the art.
[00232] The following examples are intended to promote a better understanding of the present invention. While the present invention is described herein with reference to the illustrated embodiments, it should be understood that the invention is not limited thereto. Those having ordinary skill in the technique and access to the teachings herein will recognize additional modifications and modalities within its scope. Therefore, the present invention is limited only by the claims appended hereto. EXAMPLES 1 PROTEIN BINDING DOMAINS STABILIZING FUNCTIONAL CONFORMATIONAL STATES OF Human β2AR EXAMPLE 1. IMMUNIZATION, LIBRARY BUILDING AND INITIAL SCREENING
[00233] To obtain mature nanobodies in vivo against β2AR, a llama (Llama glama) was immunized with recombinant β2AR truncated at Gly365 (β2AR-365) to exclude an immune response to carboxyl termination. β2AR-365 was expressed in insect cells and the antigen was previously reconstituted as described (Day et al, 2007). After six-weekly administrations of the reconstituted truncated agonist-bound receptor, lymphocytes were isolated from the blood of the immunized llama and a phage library prepared and sorted as described in Materials and Methods for the Examples (see below). Two screens identified conformational nanobodies that recognize the native β2AR, but not the denatured receptor. EXAMPLE 2. SELECTION OF CONFORMATIONAL SPECIFIC NANOBODIES BY ELISA
[00234] In a first screening we compared the binding of nanobodies on native and thermodenatured β2AR antigen in an ELISA. For each nanobody, one well was coated with phospholipid vesicles containing agonist-bound β2AR-365 (0.1 μg protein/well). Then, this plate was incubated at 80°C for 2 h. Then, another well of the same plate was coated with phospholipid vesicles containing agonist-bound β2AR-365 (0.1 μg protein/well) without heating. All nanobodies were able to selectively bind the native receptor, but not the thermoinactivated receptor, indicating that 16 binders recognize conformational epitopes. EXAMPLE 3. SELECTION OF SPECIFIC CONFORMATIONAL NANOBODIES THROUGH DOT SPOT
[00235] In an upcoming screening we compared the specificity of the nanobodies for a native agonist-bound β2AR receptor, versus a native reverse agonist-bound receptor, versus an SDS-denatured receptor by spot blot analysis. Screening identified 16 different conformational nanobodies that recognize β2AR-365 bound to the native agonist, but not the reverse agonist, or the thermodenatured receptor (Fig. 2 (dot spots)). EXAMPLE 4. SELECTION OF NANOBODIES WITH G PROTEIN SIMILAR BEHAVIOR
[00236] Initial screening identified 16 clones that recognized β2AR bound to the native agonist but not the thermodenatured receptor. Our next goal was to identify nanobodies that had G protein-like behavior. β2AR preferentially couples to Gs and agonist binding enhances G protein interactions. Furthermore, in the presence of Gs, β2AR binds to the agonist with higher affinity. Therefore, we observed (1) the effect of the agonist on the binding of the nanobodies to β2AR using size exclusion chromatography, (2) the effect of the nanobodies on the binding affinity of the β2AR agonist on membranes, and (3) the effect of the nanobodies on the alterations conformational patterns of β2AR as monitored by the environmentally sensitive monobromine bimane (mBBr) fluorophore. EXAMPLE 5. NANOBODIES WITH G-PROTEIN-LIKE BEHAVIOR SPECIFICALLY BIND TO PURIFIED AGONIST BOUND RECEPTOR.
[00237] Purified nanobodies were incubated with detergent-solubilized purified β2AR receptor in the presence of an agonist or a reverse agonist that stabilizes an inactive conformation. The mixture was then analyzed by Size Exclusion Chromatography (SEC) which separates the protein based on size. Seven of the nanobodies bound purified β2AR and migrated as a complex into SEC only in the presence of the agonist and not a reverse agonist (an example is shown in Fig. 1A, Fig. 3 and Fig. 4). The remaining nanobodies did not bind with high enough affinity to displace β2AR mobility in SEC. EXAMPLE 6. NANOBODIES WITH G-PROTEIN-LIKE BEHAVIOR STRENGTHEN β2AR AFFINITY FOR AGONISTS.
[00238] Many GPCRs exhibit higher agonist binding affinity when complexed with the G protein. This is attributed to the cooperative interaction between the agonist-occupied receptor and the G protein. Our SEC experiments provide evidence that 7 nanobodies bind preferentially to agonist-occupied β2AR and can stabilize an active state, thus in a similar manner to G proteins. Agonist competition binding experiments were performed in the presence and absence of these 7 nanobodies. The affinity of β2AR for the agonist (isoproterenol) was enhanced 2-30 fold in the presence of nanobodies 65, 67, 69, 71, 72, 80 and 84 (Fig. 1B and Table 1). In contrast, Nb80 does not increase the affinity of β2AR or β2AR-T4L for the reverse agonist ICI-118,551 (ICI) (Figure 15). EXAMPLE 7. Nb80 AND G PROTEIN INDUCE SIMILAR CONFORMATIONAL CHANGES IN THE CYTOPLASMIC DOMAIN OF TM6.
[00239] The recent crystal structure of opsin as well as biophysical studies on rhodopsin (Park et al. 2008) and β2AR (Yao et al. 2009) show that the cytoplasmic end of transmembrane segment 6 (TM6) undergoes conformational changes under agonist binding that are required for the coupling of G proteins. To investigate the effect of nanobodies on the movement of the cytoplasmic domain of TM6 we C265-labeled purified β2AR with monobromobimane (mBB-β2AR). We previously showed that both agonist binding and G protein coupling induced changes in mBB-β2AR fluorescence consistent with an outward movement of TM6 (Yao et al. 2008) as seen in the crystal structure of opsin (Park et al. . 2008). In mBB-β2AR, the addition of the agonist together with the G protein results in greater fluorescent changes than either the agonist or the G protein alone. This is consistent with the cooperative interactions observed in agonist competition binding assays (Yao et al. 2009). Similarly, relatively small changes in fluorescence were observed in mBB-β2AR with the addition of the agonist isoproterenol alone or Nb80 alone; however, greater changes were observed when both agonist and nanobody were added together (Fig. 1C). Comparable results were observed for nanobodies 65, 67, 69, 71, 72 and 84 (Fig. 5). EXAMPLE 8. CHARACTERIZATION OF THE ACTIVE STATE STABILIZED WITH β2AR NANOBODY
[00240] The effect of Nb80 with Gs on β2AR structure and agonist binding affinity was then compared. β2AR was labeled on the cytoplasmic end of TM6 to C265 with monobromobimane and reconstituted into HDL particles. TM6 moves relative to TM3 and TM5 upon agonist activation (Fig. 6A), and we have previously shown that the environment around bimane covalently bound to C265 changes with both agonist binding and G protein coupling, resulting in in a decrease in bimane intensity and a red shift in Àmax (Yao et al. 2009). The change in bimanne fluorescence is compatible with TM6 movements similar to those seen in rhodopsin by DEER spectroscopy and in the low pH opsin structure. As shown in Fig. 6B, the catecholamine agonist, isoproterenol, and Gs both stabilize an active-like conformation, but the effect of Gs is greater in the presence of isoproterenol, consistent with cooperative interactions of the agonist and Gs in the β2AR structure. Nb80 alone has an effect on bimanne fluorescence and β2AR unbound β2AR is similar to that of Gs (Fig. 6C). This effect was not observed in β2AR bound to the reverse agonist ICI-118,551. The effect of Nb80 was enhanced in the presence of 10 μM isoproterenol. These results show that Nb80 does not recognize the inactive conformation of β2AR, but efficiently binds to agonist-occupied β2AR and produces a change in bimanne fluorescence that is indistinguishable from that observed in the presence of Gs and isoprotererenol.
[00241] Figures 6D and 6E show the effect of Gs and Nb80 on agonist affinity for β2AR. β2AR was reconstituted in HDL particles and agonist competition binding experiments were performed in the absence or presence of Nb80 and Gs. In the absence of any protein, isoproterenol has an inhibition constant (Ki) of 107 nM. In the presence of Gs two affinity states are observed, because not all of the β2AR is coupled to Gs. In the Gs-coupled state, the affinity of isoproterenol increases by 100-fold (Ki = 1.07 nM) (Fig 6D and Table 4). Similarly, in the presence of Nb80 the affinity of isoproterenol increases by 95-fold (Ki = 1.13 nM) (Fig. 6E and Table 4). These binding data suggest that Nb80 stabilizes a conformation in WT β2AR that is very similar to that stabilized by Gs, such that the energetic coupling of agonist and Gs binding is closely mimicked by Nb80.
[00242] The high resolution structure of the inactive state of β2AR was obtained with a β2AR-T4L fusion protein. We have previously shown that β2AR-T4L has a higher affinity for isoproterenol than β2AR of WT (Rosenbaum et al. 2007). Nevertheless, in the presence of Nb80 the affinity increased by 60-fold, resulting in an affinity (Ki = 0.56 nM) comparable to that of Nb80-bound WT β2AR (Fig 6F and Table 4). However we could not study G protein coupling in β2AR-T4L due to steric hindrance by T4L, the results show that T4L does not prevent Nb80 binding, and Ki values almost identical for agonist binding to wild-type and β2AR β2AR. β2AR-T4L in the presence of Nb80 suggests that Nb80 stabilizes a similar conformation in these two proteins. EXAMPLE 9. NANOBODIES FACILITATE THE CRYSTALLIZATION OF AGONIST BOUND β2AR.
[00243] β2AR was crystallized originally bound to the reverse agonist carazolol using two different approaches. The first crystals were obtained from β2AR bound to a Fab fragment that recognized an epitope composed of the terminal amino and carboxyl ends of the third intracellular loop connecting TMs 5 and 6 (Rasmussen et al. 2007). In the second approach, the third intracellular loop was replaced by T4 lysozyme (β2AR-T4L) (Rosenbaum et al. 2007). Efforts to crystallize the complex of β2AR-Fab and β2AR-T4L bound to different agonists failed to produce crystals of sufficient quality for structure determination.
[00244] An attempt was therefore made to crystallize the agonist-bound β2AR and β2AR-T4L in the complex with Nb80. While crystals of both complexes were obtained in lipid bicells and lipid cubic phase (LCP), high resolution diffraction was obtained from β2AR-T4L-Nb80 crystals grown in LCP. These crystals were grown at pH 8.0 between 3944% PEG400, 100 mM Tris, 4% DMSO, and 1% 1,2,3-heptanetriol. EXAMPLE 10. Nb80 CONTRIBUTES TO THE CONDITIONING OF β2AR IN A CRYSTAL truss.
[00245] High resolution diffraction was obtained from β2AR-T4L-Nb80 crystals grown in LCP. These crystals were grown at pH 8.0 between 3944% PEG400, 100 mM Tris, 4% DMSO, and 1% 1,2,3-heptanetriol.
[00246] A dataset fused at 3.5 Â was obtained from 23 crystals (Table 5). The structure was solved by molecular substitution using the structure of β2AR linked to carazolol and a nanobody as research models. Figure 7 shows the packaging of the β2AR-T4L-Nb80 complex in the crystal lattice. Nb80 binds to the cytoplasmic end of the β2AR, with the third loop of the complementarity determining region (CDR) projecting into the receptor nucleus. The β2AR-nanobody complexes are arranged with the lipid bilayer approximately parallel to the bc plane of the crystal. Double symmetry related nanobody molecules interact along a geometric axis a to generate a lattice tightly packed in this direction. Within the bilayer, receptor molecules interact in an antiparallel arrangement with TM1,2,3 and 4 of a β2AR molecule packaging against TM4 and 5 of the adjacent molecule. Contacts are also made between the helix 8 and TM5 of the parallel lattice neighbor along the geometric axis b, and between the extracellular portion of TM1 and the cytoplasmic end of TM6 of a third antiparallel neighbor. Packing is weaker along the c axis which may be in part due to non-specific interactions of T4L with neighboring receptor and/or nanobody molecules. There is no electron density interpretable by T4L, but given the visible ends of TM5 and TM6 the position of T4L is highly constrained. Presumably T4L adopts several orientations with respect to the receiver, and perhaps a range of internal conformations due to its hinge motion (Zhang et al. 1995), which has moved out of its average density. However, T4L likely contributes to the crystal structure since we could not produce native β2AR-nanobody complex crystals under these conditions, although it is possible that the flexible loop connecting TM5 and TM6 in the native receptor prevents lattice formation. EXAMPLE 11. STRUCTURE OF AN ACTIVE STATE STABILIZED WITH β2AR NANOBODY.
[00247] Figure 8 compares the inactive β2AR structure (from the carazolol-bound β2AR-T4L structure) with the active state of β2AR. The greatest differences are found on the cytoplasmic face of the receptor, with outward displacement of TM5 and TM6 and an inward movement of TM7 and TM3 in the β2AR-T4L-Nb80 complex with respect to the inactive structure (Fig 8A, B). There are relatively small changes on the extracellular surface (Fig. 8C). The second intracellular loop (ICL2) between TM3 and TM4 adopts a two-turn alpha helix, similar to that observed in the turkey β1AR structure (Warne et al. 2008). The absence of this helix in the inactive β2AR structure may reflect crystal lattice contacts involving ICL2.
[00248] Figure 9A-C shows in greater detail the interaction of Nb80 with the cytoplasmic side of β2AR. A sequence of eight amino acids CDR 3 penetrates a hydrophobic pocket formed by amino acids from segments of TM 3, 5, 6 and 7. A sequence of four amino acids CDR1 provides additional stabilizing interactions with the cytoplasmic ends of segments of TM 5 and 6. Figure 9 D compares the cytoplasmic surface of the active and inactive conformations of β2AR. CDR3 occupies a position similar to the carboxyl-terminal peptide of transducin in opsin (Scheerer et al. 2008) (Fig. 10). Most interactions between Nb80 and β2AR are mediated by hydrophobic contacts.
[00249] When comparing active and inactive structures, the greatest change is seen in TM6, with a helix movement of 11.4 Â in Glu2686.30 (part of the ion lock) (superscripts in this form indicate Ballesteros-Weinstein numeration for conserved GPCR residues (Ballesteros and Weinstein 1995) (Fig. 9D) This large change is accomplished by a small right rotation of TM6 in the loop preceding the conserved Pro2886. proline and repackaging of Phe2826.44 (see below), which swings the helix out.
[00250] The changes in active β2AR-T4L-Nb80 relative to inactive carazolol-bound β2AR-T4L is remarkably similar to those observed between rhodopsin and opsin (Scheerer et al. 2008; Park et al. 2008) (Fig. 9E). , Fig. 10). The salt bridge in the ionic lock between highly conserved Arg1313.50 and Asp/Glu1303.49 is broken. In opsin, Arg1353,50 interacts with Tyr2235,58 on TM5 and a main-chain carbonyl of the transducin peptide. Arg1313,50 of β2AR also interacts with a carbonyl backbone of CDR3 of Nb80. However, Nb80 prevents an interaction between Arg1313,50 and Tyr2195,58, although tyrosine occupies a similar position in opsin and the active conformation of β2AR-T4L-Nb80. As in opsin, Tyr3267,53 of the highly conserved NPxxY sequence passes into the space occupied by TM6 in the inactive state. In β2AR-T4L bound to inactive carazolol we observed a network of hydrogen-bonding interactions involving highly conserved amino acids in TMs 1, 2, 6 and 7 and various water molecules (Rosenbaum et al. 2007). Although the resolution of β2AR-T4L-Nb80 is inadequate to detect water, it is clear that the structural changes we observed would substantially alter this network.
[00251] In contrast to the relatively large changes seen in the cytoplasmic domains of β2AR-T4L-Nb80, the changes in the agonist binding pocket are quite subtle. Trp6,48 is highly conserved in Family A GPCRs, and its rotameric state has been proposed to play a role in GPCR (alternate rotamer switch) activation (Shi et al. 2002). We did not observe any change in the side chain rotamer of Trp2866.48 in TM6, which lies close to the base of the ligand binding pocket although its position shifts slightly due to the rearrangement of residues close to Ile1213,40 and Phe2826,44. Although there is spectroscopic evidence for changes in the Trp6,48 environment upon rhodopsin activation (Ahuja et al. 2009), a rotamer change is not observed in the low pH rhodopsin and opsin crystal structures. Furthermore, recent histamine receptor mutagenesis experiments demonstrate that Trp6,48 is not required for activation of the 5HT4 receptor through serotonin (Pellissier et al. 2009).
[00252] It is interesting to speculate how the small changes around the agonist binding pocket are coupled to the much larger structural changes in the cytoplasmic regions of TMs 5, 6 and 7 that facilitate the binding of Nb80 and Gs. A potential conformational binding is shown in Figure 11. Agonist interactions can stabilize an active receptor conformation that includes an inward movement of 2.1 Å of TM5 at position 2075.46 and an inward movement of 1.4 Å of Pro2115, 50 conserved with respect to the inactive structure. In the inactive state, the relative positions of TM5, TM3, TM 6 and TM7 are stabilized through interactions between Pro2115.50, Ile1213.40, Phe2826.44 and Asn3187.45. The position of Pro2115,50 observed in the active state is incompatible with this network of interactions, and Ile1213,40 and Phe2826,44 are repositioned, with a rotation of TM6 around Phe2826,44 leading to an outward movement of the cytoplasmic end of TM6.
[00253] Although some of the structural changes observed in the cytoplasmic domains of the β2AR-T4L-Nb80 complex arise from specific interactions with Nb80, the fact that Nb80 and Gs induce or stabilize similar structural changes in β2AR, as determined by fluorescence spectroscopy and by Agonist binding affinity suggests that Nb80 and Gs recognize similar agonist-stabilized conformations. The observation that the cytoplasmic domains of rhodopsin and β2AR undergo similar structural changes upon activation provides further support that agonist-bound β2AR-T4L-Nb80 represents an active conformation and is consistent with a conserved mechanism of G protein activation.
[00254] However, the mechanism by which agonists induce or stabilize these conformational changes likely differs for different ligands and for different GPCRs. The conformational equilibria of rhodopsin and β2AR differ, as shown by the fact that rhodopsin can adopt a fully active conformation in the absence of a G protein whereas β2AR cannot. Thus, activation energetics and conformational sampling may differ between different GPCRs, which likely gives rise to the variety of ligand efficiencies exhibited by these receptors. An agonist need only disrupt a fundamental intramolecular interaction necessary to stabilize the inactive state, as constitutive receptor activity can result from single amino acid mutations of different regions of GPCRs (Parnot et al. 2002). Thus, disruption of these stabilizing interactions by agonists or mutations lowers the energy barrier separating inactive and active states and increases the probability with which a receptor can interact with a G protein.
[00255] In conclusion, these above results demonstrate the ability to generate nanobodies that recognize and stabilize an agonist-bound state of a GPCR. In the case of β2AR, this state stabilized by the nanobody is functionally similar to the state stabilized by the G proteins. Finally, the nanobodies facilitated the formation of diffraction quality crystals. This approach is now applied to other GPCRs and other membrane proteins. EXAMPLE 12. Nb80 STABILIZES ACTIVE STATE CONFORMATION OF ADRENERGIC RECEPTOR FAMILY MEMBERS
[00256] Active state stabilization nanobodies that are cross-reactive with related receptors can be used as a tool to stabilize a conformational state of those related receptors. To demonstrate this principle, we analyzed whether Nb80 selectively binds to the active conformation of the human β1AR receptor. β1AR and β2AR are closely related adrenergic receptors. Using the PISA server (Krissinel & Henrick, 2007) and based on the crystal structure of the Nb80-β2AR complex, 30 AA residues of β2AR were identified to interact with Nb80 at the β2AR-Nb80 interface. An amino acid sequence alignment (Figure 17) indicates that 28 of these 30 residues involved in the Nb80 interaction are conserved between β1AR and β2AR. The remaining interface residues are lysine in β2AR and have been replaced by arginines in β1AR corresponding to the conserved substitutions (shown in gray boxes in Figure 17). It appears that both receptors share a binding site very similar to Nb80. Based on this analysis, we also measured the effect of Nb80 on the affinity of β1AR for the agonist isoproterenol and the reverse agonist CGP20712A (Figure 18). As for β2AR, Nb80 also induced an increased affinity of isoproterenol for β1AR (Figure 18, panels A and C). Nb80 does not alter the affinity for the antagonist CGP20712A (Figure 18, panel D), demonstrating the selective stabilization of the active state conformation of β1AR by Nb80. No such effect of Nb80 was observed at unrelated receptors such as the dopamine D1 receptor (data not shown). EXAMPLE 13. β2AR ACTIVE STATE STABILIZATION NANOBODIES ARE EXCELLENT TOOLS FOR IMPROVED AGONIST SCREENING.
[00257] Many GPCRs exhibit higher agonist binding affinity when complexed with G protein. This is attributed to the cooperative interaction between the agonist-occupied receptor and the G protein. Nanobodies with G protein-like behavior likely enhance the affinity of β2AR for agonists (see example 6). This behavior may have important implications for the discovery of new agonists. For example, Nbs with G-protein-like behavior may increase the evident affinity of GPCRs for agonists compared to antagonists. This can cause a bias towards agonists between repeats when a compound library is screened against such a GPCR-Nb complex. To assess the applicability of this approach, we analyzed the effect of the β2AR Nb80 active state stabilization nanobody on the interaction of β2AR with various well-known β2AR ligands in competition radioligand binding experiments (see Materials and Methods). Ten agonists and five antagonists were tested: (-) isoproterenol HCl (agonist), (-)-alprenolol (antagonist), salbutamol (polar agonist), ICI118551 (reverse agonist), carvedilol (antagonist), CGP12177A (antagonist), xinafoate salmeterol (full agonist), terbutaline hemisulfate salt (polar agonist), dobutamine hydrochloride (polar agonist), metaproterenol hemisulfate salt (agonist), procaterol hydrochloride (agonist), ritodrine hydrochloride (agonist), fenoterol (full agonist), formoterol fumarate dihydrate (agonist) and timolol maleate salt (antagonist) all purchased through Sigma Aldrich.
[00258] Competition ligand binding experiments were performed in the presence and absence of 500 nM Nb80 on commercial membranes containing whole human β2AR. 3H-Dihydroalprenolol (DHA) was used as the competing radioligand. Representative examples of competition ligand binding experiments are shown in Figure 16. For all compounds, IC50 values in the presence of excess Nb80 were obtained and compared to IC50 obtained in the presence of an irrelevant nanobody (negative control). (Table 6). Consistent with examples 6, 8 & 12, we observed an increase in potency for all agonists when β2AR is complexed with Nb80. Such an effect is not observed for antagonists or reverse agonists. EXAMPLE 14. LIBRARY SCREENING OF COMPOUNDS USING A β2AR ACTIVE STATE STABILIZATION NANOBODY.
[00259] Trapping the GPCR in a particular conformation has an advantage in using a non-conformationally stabilized target (representing a repertoire of conformations) in a screening assay with the goal of identifying compounds against that particular conformation, the so-called 'drugable target conformation'. '. Conformational selective Nb80 allows the use of wild-type receptors that minimize the potential risk of artificial conformational changes, therefore, as a result of site-directed mutagenesis.
[00260] To demonstrate whether Nb80 facilitates the identification of ligands that selectively bind β2AR in its active conformation, a fragment library consisting of about 1500 distinct low molecular weight (< 300 Da) compounds is screened for agonists using a competition radioligand binding. For this assay, membranes containing full-length β2AR are pre-incubated with Nb80 or an irrelevant Nb (negative control) and added to 96-well plates containing the library compounds and 2nM 3H-dihydroalprenolol (DHA) radioligand. (see materials and methods). Library compounds that significantly displace the radioligand in the sample containing β2AR in the complex with Nb80 when compared to the sample containing irrelevant nanobody are compounds that preferentially bind to the active conformation of the receptor. Library compounds that selectively bind the active conformation of the receptor have a high tendency to behave as agonists because orthosteric or allosteric stabilization of the active conformation of the GPCR elicits biological responses. Selected library compounds must be further screened for agonism by measuring for example G protein coupling, downstream signaling events, physiological yield. EXAMPLE 15: THERMOSTABILIZATION OF THE β2AR RECEPTOR WITH NANOBODIES.
[00261] Structural and functional studies on integral membrane proteins have long been hampered by their instability in detergent. Although expression and purification methods are emerging that allow the generation of milligram amounts, achieving stability with these molecules is perhaps the most difficult hurdle to overcome. Purification necessitates a release of the membrane protein from the lipid bilayer by detergent solubilization, a process during which the hydrophobic surfaces of the protein are coated with surfactant monomers to form a protein-detergent complex. However, the detergent belt formed around the protein is a poor replacement for the lipid bilayer. Thus, solubilization of membrane proteins often results in destabilization, unfolding, and subsequent aggregation. Thermostabilization of membrane proteins can be achieved through loco-directed mutagenesis (Zhou & Bowie, 2000; Magnini et al., 2008). Here we show that binding of conformationally selective nanobodies represents an innovative alternative for the thermostabilization of detergent-solubilized GPCRs.
[00262] The effect of conformationally selective nanobodies on thermostability and subsequent aggregation of the β2AR receptor was analyzed using a fluorescent thermal stability assay (Fig 13) and size exclusion chromatography (Fig 14). For these experiments, recombinant β2AR was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, 100 mM NaCl, 20 mM Hepes pH 7.5 and protease inhibitors, and purified by M1 FLAG affinity chromatography ( see Materials and Methods for the Examples).
[00263] The fluorescent thermal stability assay makes use of the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) to measure the chemical reactivity of native cysteines embedded in the protein interior as a sensor for the overall paste state integrity of membrane proteins (Alexandrov et al., 2008). For the fluorescent thermal stability assay, receptor solubilized in detergent (in 0.1% dodecylmaltoside, 100 mM NaCl and 20 mM Hepes pH 7.5) was pre-incubated with isoproterenol (10 μM) in the presence or absence of a 2:1 molar excess of Nb80 for 1h at RT. The samples were then mixed with CPM fluorophore and incubated for 2 min at a temperature ranging from 10°C to 94°C and the fluorescence emission was collected. The experiments were performed in triplicate and resulted in casting curves that resemble the stability profiles obtained for the APJ of GPCR (Alexandrov et al., 2008). Unfolding transitions could be described by a simple two-state model, including the native (folded) state and the denatured state, representing the lower and upper plateaus of the casting curves. Comparison of the melting curve of the agonist-bound receptor with the melting curve of the agonist-bound receptor in the Nb80 complex indicates that the nanobody stabilizes the active conformation of β2AR by increasing the melting temperature (Tm) of the agonist-bound receptor by 12° Ç.
[00264] The effect of Nb80 on the thermal unfolding and aggregation of the agonist-bound receptor was also analyzed by size exclusion chromatography (SEC). For this experiment, receptor solubilized in dodecylmaltoside was pre-incubated with isoproterenol (10 μM) in the presence or absence of a 2:1 molar excess of Nb80 for 45 minutes at RT. The samples were then incubated for 10 minutes at increasing temperatures and subsequently analyzed by SEC (Fig 14). Comparison of the different chromatograms indicates that Nb80 protects the agonist-bound receptor against temperature-induced aggregation. PROTEIN BINDING DOMAINS STABILIZING FUNCTIONAL CONFORMATIONAL STATES OF RAT ANGIOTENSIN II TYPE 1A RECEPTOR (AT1AR) EXAMPLE 16. IMMUNIZATION, LIBRARY BUILDING AND INITIAL SCREENING
[00265] To obtain mature nanobodies in vivo against mouse AT1aR, two llamas (Llama glama) were immunized with a fusion of recombinant AT1aR-T4lysozyme (T4L) truncated after Lys318 excluded a carboxyl-terminus immune response. AT1aR was expressed in insect cells (Shluka et al. 2006) and the antigen was previously reconstituted in lipid vesicles as described (Day et al., 2007). One llama was immunized with angiotensin-bound receptor (non-polarized agonist), one llama was immunized with β-arrestin polarized ligand TRV023 (Violin et al. 2010) bound to the receptor. After six weekly administrations of the reconstituted truncated agonist-bound receptor, lymphocytes were isolated from the blood of the immunized llama and a phage library prepared and sorted as described in Materials and Methods for the Examples. Solid phase ELISAs identified nanobodies that recognize the AT1a receptor. EXAMPLE 17. SELECTION OF CONFORMATIONAL SPECIFIC NANOBODIES BY ELISA
[00266] In a first screening we compared the binding of purified nanobodies to native and thermodenatured mouse AT1aR antigen in an ELISA. For each nanobody, a well is coated with receptor. Then, this plate is incubated at 80°C for 2 h. Then another empty well of the same plate is coated with receptor without heating. Nanobodies that are able to selectively bind the native receptor but not the thermoinactive receptor recognize conformational epitopes. EXAMPLE 18. SELECTION OF NANOBODY SPECIFIC TO THE CONFORMATIONAL STATE THROUGH SPOT STAIN
[00267] In a next screen we compared the binding of those nanobodies that bind conformational epitopes to an agonist-bound AT1aR receptor versus an antagonist-bound receptor by spot blot analysis. This screen identifies nanobodies that selectively recognize a conformation of an agonist-bound receptor (active state) versus antagonist-bound (inactive state). EXAMPLE 19. SCREENING FOR AT1AR NANOBODY SELECTIVELY STABILIZING AN ACTIVE RECEPTOR CONFORMATION.
[00268] In addition to the binding assay for AT1aR specificity (ELISA), the purified nanobodies are evaluated in a radioligand competition experiment similar to the radioligand assays described in Examples 6,12 & 13. Nanobodies that increase AT1aR affinity to an agonist are considered to stabilize an active conformation of the receptor. PROTEIN BINDING DOMAINS STABILIZING FUNCTIONAL CONFORMATIONAL STATES OF RAT M3 MUSCARINIC RECEPTOR EXAMPLE 20. IMMUNIZATION, LIBRARY BUILDING AND INITIAL SCREENING.
[00269] To obtain mature nanobodies in vivo against mouse M3R, a llama (Llama glama) was immunized with a recombinant M3R-T4lysozyme (M3R-T4L) fusion truncated at N and C-terminal sites to exclude an immune response at the termini. . In M3R-T4L, the 3rd intracellular loop was replaced by T4lysozyme. M3R-T4L was expressed in insect cells and the antigen was previously reconstituted as described (Day et al, 2007). After six weekly administrations of the receptor bound to the reconstituted antagonist (tiotropium), lymphocytes were isolated from the blood of the immunized llama and a phage library prepared and sorted as described in Materials and Methods for the Examples. EXAMPLE 21. SELECTION OF CONFORMATIONAL SPECIFIC NANOBODIES BY ELISA
[00270] In a first screening we compared the binding of purified nanobodies to native and thermodenatured mouse M3R antigen in an ELISA. For each nanobody, a well is coated with receptor. Then, this plate is incubated at 80°C for 2 h. Then another empty well of the same plate is coated with receptor without heating. Nanobodies that are able to selectively bind the native receptor but not the thermoinactive receptor recognize conformational epitopes. EXAMPLE 22. SELECTION OF NANOBODY SPECIFIC TO THE CONFORMATIONAL STATE THROUGH SPOT STAIN
[00271] In a next screen we compared the binding of those nanobodies that bind conformational epitopes to an agonist-bound M3 receptor versus an antagonist-bound receptor by spot blot analysis. This screen identifies nanobodies that selectively recognize a conformation of an agonist-bound receptor (active state) versus antagonist-bound (inactive state). EXAMPLE 23. SCREENING FOR M3R NANOBODIES SELECTIVELY STABILIZING AN ACTIVE RECEPTOR CONFORMATION.
[00272] In addition to the binding assay for M3R specificity (ELISA), the purified nanobodies are evaluated in a radioligand competition experiment similar to the radioligand assays described in Examples 6,12 & 13. Nanobodies that increase M3R affinity to an agonist are considered to stabilize an active conformation of the receptor. MATERIALS AND METHODS FOR THE EXAMPLES Preparation of β2AR
[00273] β2AR truncated after amino acid 365 (β2AR-365) having an amino terminal Flag epitope tag was expressed in Sf9 insect cells and previously purified by M1 antibody and alprenolol sequential affinity chromatography as described (Kobilka 1995). Purified β2AR-365 was immobilized on a Flag column (Sigma) and equilibrated with 10 column volumes of a mixture of 5 mg/mL DOPC (Avanti Polar Lipids) and 0.5 mg/mL Lipid A (Sigma) in 1% (w/v) octylglucoside (Anatrace), 100 mM NaCl, 20 mM Hepes pH 7.5, 2 mM CaCl 2 and 1 µM agonist (eg isoproterenol). The β2AR was then eluted in the same buffer containing EDTA. The eluted β2AR concentration was adjusted to 5 mg/mL. This usually involved diluting the protein with the same buffer, but occasionally required concentrating the protein up to twice with an Amicon ultrafiltration cell (100 kDa pore). The protein was then dialyzed against phosphate buffered saline containing 1 µM agonist at 4°C to remove the detergent. Reconstituted protein was stored at -80°C prior to use for immunization. AT1aR preparation
[00274] AT1aR with T4lysozyme fusion in the third loop was truncated after amino acid 318 (AT1aR-318). This construct has an amino-terminal Flag epitope tag and a 10-histidine C-terminal tag. AT1aR-318 was expressed in Tni insect cells and solubilized in 20 mM Hepes, pH 7.4, 1M NaCl and 0.5% MNG for 2 h at room temperature. The receptor was purified by sequential Ni-NTA and FLAG-M1 antibody affinity chromatography. Purified AT1aR was reconstituted in a mixture of 5 mg/mL DOPC (Avanti Polar Lipids) and 0.5 mg/mL Lipid A (Sigma) in 1% (w/v) octylglucoside (Anatrace), 100 mM NaCl , 20 mM Hepes pH 7.5, and 100 µM agonist (e.g. angiotensin II). The concentration of the eluted At1aR was adjusted to 1-2 mg/mL. The protein was then dialyzed against phosphate buffered saline containing 100 µM agonist at 4°C to remove the detergent. Reconstituted protein was stored at -80°C prior to use for immunization. M3 receiver preparation
[00275] M3 muscarinic receptor with an amino-terminal FLAG epitope tag and carboxy-terminal hexa-histidine tag were expressed in Sf9 insect cells in the presence of 1 µM atropine (Vasudevan et al. 1995). Receptor had intracellular loop 3 deleted (M3RΔi3) or replaced with T4 lysozyme (M3R-T4L). Cells were centrifuged and then lysed by osmotic shock, and the protein was solubilized in 1% dodecylmaltoside, 0.1% cholesterol hemisuccinate, 750 mM sodium chloride, 20 mM HEPES pH 7.5. Solubilized receptor was then purified by nickel affinity chromatography followed by FLAG affinity chromatography. The purified protein was then separated by size exclusion chromatography to select the monomeric receptor which was reconstituted as described (Day et al, 2007). Nanobody selection against β2AR
[00276] A single llama received six weekly administrations of the reconstituted truncated β2AR. Lymphocytes were isolated from the blood of the immunized llama and total RNA was prepared from these cells. The nanobody repertoire coding sequences were amplified by a TA-PCR and cloned into the pMES4 phage display vector (Genbank GQ907248) (Conrath et al. 2001). Specific β2AR-phage were enriched by in vitro selection on 96-well Maxisorp (Nunc) plates coated with reconstituted β2AR-365 receptor. Antigen-bound phage were recovered from antigen-coated wells with triethylamine pH 11 and neutralized with Tris-HCl pH 7 by the addition of freshly grown TG1 E. coli cells. After two cycles of bio-panning, 96 individual colonies were randomly chosen and the nanobodies produced as a soluble His-tagged protein in the periplasm of TG1 cells. Solid phase ELISA identified 16 different conformational nanobodies that recognize β2AR-365 bound to the native agonist but not to the thermodenatured receptor. Selection of nanobodies against AT1aR
[00277] A single llama received six weekly administrations of reconstituted AT1aR-318 bound to its angiotensin agonist. Another llama alone received six weekly administrations of AT1aR-318 linked to a polarized agonist TRV023. After immunization, lymphocytes from each llama were separately isolated from the blood. Total RNA was prepared from these cells. From each sample, the nanobody repertoire coding sequences were amplified by TA-PCR and cloned separately (Conrath et al. 2001) into the pMESy4 phage display vector to generate two independent libraries. pMESy4 is a derivative of pMES4 (Genbank GQ907248) carrying a C-terminal His6tag followed by the EPEA amino acids (De Genst et al. 2010). J. Mol. Biol 402: 326-343.
[00278] AT1aR-specific phage was enriched by in vitro selection on 96-well Maxisorp (Nunc) plates coated with reconstituted truncated AT1aR receptor (a variant of the recombinant receptor lacking the T4L insert) bound to angiotensin or TRV023, respectively. Antigen-bound phage were recovered from antigen-coated wells by trypsin digestion. After two cycles of bio-panning, 92 individual colonies (46 on AT1aR-angiotensin and 46 on AT1aR-TRV023) were randomly chosen and nanobodies were produced as a soluble HisIS-EPEA-tagged protein in the periplasm of TG1 cells. Selection of nanobodies against M3R
[00279] A single llama received six weekly administrations of reconstituted truncated M3R-T4L bound to the antagonist tiotropium. After immunization, the lymphocytes of this llama were isolated from the blood and total RNA was prepared from these cells. The nanobody repertoire coding sequences were amplified by TA-PCR and cloned (Conrath et al. 2001) into the phage display vector pMESy4 vector.
[00280] To enrich the M3R-specific phages, different in vitro selection strategies were followed using different formats of the antigen in the presence of the carbachol agonist or the quinuclidinyl benzylate (QNB) antagonist. Antigen formats include virus-like particles (VLPs) carrying the mouse M3RΔi3 receptor (i.e., the M3 receptor with a deletion in the 3rd intracellular loop), human CHO cell membranes containing M3R (Perkin Elmer), M3R-T4L reconstituted recombinant, or reconstituted recombinant M3RΔi3. Optionally, VLPs carrying the mouse M3RΔi3 receptor or human M3R membranes were captured by wheat germ agglutinin coated in Maxisorp (Nunc) 96-well plates. Antigen-bound phage were recovered from antigen-coated wells by a trypsin digest. Alternatively, phage selected on agonist-bound antigen is eluted using an excess of antagonist or vice versa.
[00281] After two cycles of bio-panning, 180 colonies were randomly chosen and nanobodies were produced as a soluble His-EPEA-tagged protein in the periplasm of TG1 cells. A comparative solid phase ELISA on the M3R-T4L receptor versus the AT1aR-T4L receptor resulted in 66 M3R-specific nanobodies. Purification of nanobodies for biochemical characterization
[00282] His-tagged or His-EPEA-tagged nanobodies were expressed in WK6 E. coli cells. Periplasmic extracts were subjected to metal affinity chromatography immobilized on nickel (II) sulfate fast-flow sepharose (GE Healthcare). IMAC protein fractions were dialyzed overnight in 100 mM MES pH 6.5, 100 mM NaCl buffer. Nanobodies subjected to dialysis were further purified by cation exchange chromatography (ÃKTA FPLC with Mono S 10/100 GL column). Size exclusion chromatography
[00283] Agonist-selective nanobodies were identified by size exclusion chromatography following incubation of 20 μm of agonist-bound β2AR-365N or reverse agonist (carazolol) for 1 h at RT in the absence or presence of 40 μM nanobody. Chromatography was performed in 0.1% DDM, 20 mM HEPES pH 7.5, 100 mM NaCl in the presence of 1 µM of the respective binders using an ÃKTA FPLC with Superdex 200 10/300 GL column.
[00284] Ligand binding at truncated β2AR receptor in insect cell membrane preparations
[00285] Competition binding experiments were performed on β2AR-365 expressed on Sf9 insect cell membranes in the absence or presence of 1 μM nanobodies for 90 min at RT in binding buffer (75 mM Tris, pH 7, 5, 12.5 mM MgCl2, 1 mM EDTA, 0.05% BSA, and 10 μM GTPYS) containing 0.5 nM [3H]-dihydroalprenolol and (-)-isoproterenol in varying concentrations from 1011 M to 10-4 M. Bound radioligand was separated from unbound on Whatman GF/B filters using Brandel collector. Data are the mean ± S.E. of two independent experiments performed in triplicate. Bimane fluorescence.
[00286] Purified β2AR was reacted with 1:1 equivalent of monobromobimane (mBBr, Invitrogen) in 100 mM NaCl, 20 mM HEPES, pH 7.5, 0.1% dodecyl maltoside and incubated overnight in ice in the dark. The fluorophore-labeled receptor was purified right before use by gel filtration on a desalting column equilibrated with the same buffer. Fluorescence spectroscopy experiments were performed on a FluoroMax-3 Spex spectrofluorometer (Jobin Yvon Inc, NJ) with photon counting mode using an excitation and emission bandpass of 4 nm. All experiments were performed at 25°C. For emission scans, excitation was set at 370 nm and emission was measured from 430-530 nm with an integration time of 1 s/nm. To determine the effect of nanobodies and ligands, three individual labeled protein samples were incubated with 1 μM nanobodies or 10 μM Isoproterenol or both. Emission spectra of the samples were taken after 1 hour incubation. Fluorescence intensity was corrected for background fluorescence from buffer and ligands in all experiments. Data are the mean ± S.E. of two independent experiments performed in triplicate. Preparation of β2AR-T4L and nanobody-80 for crystallography
[00287] β2AR-T4L was expressed in β2AR-T4L baculovirus-infected cultures of insect Sf-9 cells, and solubilized according to previously described methods (Kobilka 1995). Functional protein was obtained by FLAG affinity chromatography of M1 (Sigma) before and following alprenolol-Sepharose chromatography (Kobilka 1995). In the second M1 chromatography step, receptor-bound alprenolol was exchanged for a high-affinity agonist and dodecylmaltoside was exchanged for the MNG-3 amphiphile for increased receptor stability (Chae and Gellman, unpublished). Agonist-bound, detergent-exchanged β2AR-T4L was eluted in 10 mM HEPES pH 7.5, 100 mM NaCl, 0.02% MNG-3, and 10 μM agonist followed by removal of N-linked glycosylation. through treatment with PNGaseF (NEB). Protein was concentrated to ~50 mg/mL with a 100 kDa molecular weight cut-off Vivaspin concentrator (Vivascience).
[00288] Nanobody-80 (Nb80) carrying a C-terminal His6 tag was expressed in the periplasm of E. coli strain WK6 following induction with IPGT. The 0.6 L cultures were grown at OD600 = 0.7 at 37°C in TB media containing 0.1% glucose, 2 mM MgCl2, and 50 μg/mL ampicillin. Induced cultures were grown overnight at 28°C. Cells were harvested by centrifugation and lysed in ice-cold buffer (50 mM Tris pH 8.0, 12.5 mM EDTA, and 0.125 M sucrose). then centrifuged to remove cell debris. Nb80 was purified by nickel affinity chromatography, dialyzed together with buffer (10 mM HEPES pH 7.5, 100 mM NaCl), and concentrated by spin to ~120 mg/mL. Crystallization
[00289] Agonist-bound β2AR-T4L and nanobody (e.g. Nb80) were mixed in a 1:1.2 molar ratio, incubated for 2 hours at RT before mixing with liquefied mono-olein (M7765, Sigma) containing 10% cholesterol ( C8667, Sigma) in a protein to lipid ratio of 1:1.5 (w/w) using the double syringe mixing method developed by Martin Caffrey (Caffrey and Cherezov 2009). Initial crystallization leaders were identified using in-house screens and optimized in 24-well glass sandwich plates using 50 nL of manually released protein:lipid drops coated with 0.8 μL of precipitant solution in each well and sealed with a slide. of glass cover. Crystals for data collection were grown at 20°C by hanging drip vapor diffusion using 0.8 μL of reservoir solution (36 to 44% PEG 400, 100 mM Tris pH 8.0, 4% DMSO , 1% 1,2,3-heptanetriol) diluted 2-4 times in Milli-Q water. The crystals grew to full size within 7 to 10 days. The crystals were flash frozen and stored in liquid nitrogen with reservoir solution as a cryoprotectant.
[00290] Microcrystallography data collection and processing
[00291] Diffraction data were measured in-line from the Advanced Photon Source 23-ID beam, using a 10 μm diameter beam. Low dose 1.0° rotation images were used to locate and center the crystals for data collection. Data were measured in 1.0° frame with exposure times typically 5-10 s with a 5x attenuated beam. Only 5-10° of the data could be measured before significant radiation damage occurred. The data were integrated and scaled with the HKL2000 package (Otwinowski 1997). Structure and refinement solution
[00292] The molecular substitution phases were obtained with the Phaser program (McCoy 2007). Research models were 1) the high resolution carazolol-bound β2AR structure, PDB id 2RH1, but with T4L and all water, ligand and lipid molecules removed) and a nanobody (PDB id 3DWT, water molecules removed) as research models. The rotation and translation function Z scores were 8.7 and 9.0 after placing the β2AR model, and the subsequently placed nanobody model had rotation and translation function Z scores of 3.5 and 11.5. The model was refined in Phenix (Afonine 2005) and Buster (Blanc 2004), using a group B factor model with a B for the main chain and a B for the side chain atoms. Refinement statistics are given in Table 5. Despite the strong anisotropy (Table 5), the electron density was clear for the placement of the side chains.
[00293] Ligand binding on truncated β2AR receptor reconstituted on HDL particles.
[00294] The effect of Nb80 and Gs on receptor affinity for agonists was compared in competition binding experiments. β2AR and β2AR-T4L (both truncated at position 365) previously purified as described (Rosenbaum et al. 2007; Rasmussen et al. 2007) were reconstituted into high-density lipoprotein (HDL) particles followed by reconstitution of Gs into HDL particles. HDL containing β2AR according to previously published methods (Whorton et al. 2007). 0.6 nM [3H]-dihydroalprenolol (3H-DHA) was used as a radioligand and (-)-isoproterenol (ISO) in concentrations ranging from 10-12 to 10-4 M as a competitor. Nb80 was used at 1 μM. GTPYS was used at 10 μM. TBS (50 mM Tris pH 7.4, 150 mM NaCl) containing 0.1% BSA was used as a binding buffer. Bound 3 H-DHA was separated from unbound on a Brandel collector bypassing a Whatman GF/B filter (pre-swollen in TBS with 0.3% polyethyleneimine) and washed in cold TBS. Radioligand binding was measured on a Beckman LS6000 scintillation counter. DHA ligand binding affinity (Kd) was determined from saturation binding curves using GraphPad Prism software. ISO binding affinities (Ki values, tabulated in Table 4) were determined from the IC50 values using the equation Ki = IC50 / (1 + [L] / Kd)
[00295] Ligand binding at the full-length β2AR receptor in insect cell membrane extracts for improved agonist identification.
[00296] Radioligand competition binding experiments were performed on membrane extracts essentially as described by Seifert et al. (Seifert et al. 1998. Eur. J. Biochem. 255:369-382). Ten μg of homogenized membrane extracts from insect cells containing human β2AR (Perkin Elmer, cat nr 6110106400UA) were incubated with Nb80 or an unrelated nanobody (negative control; Irr Nb) for 1 h at 37°C in incubation buffer ( 75 mM Tris-HCl, 12.5 mM MgCl 2 , 1 mM EDTA and 0.2% w/v BSA) in 24-well plates (Corning Costar). Nanobodies were applied at a final concentration of 500 nM, corresponding to a 3000-fold excess of Nanobodies versus the adrenergic receptor. Subsequently, an appropriate dilution series of the ligand under investigation was added to the nanobody-bound membrane extracts along with 2 nM 3H-DHA radioligand (Perkin Elmer nr cat NET720001MC; specific activity 104.4 Ci/mmol). The total volume per well was adjusted with incubation buffer to 500 μL and the reaction mixture was further incubated for another hour at 37°C in a water bath. After harvesting the membrane extracts with a cell harvester (Inotech) onto glass fiber filters (Whatmann GF/B filter paper), the filters were washed with ice-cold wash buffer (50 mM Tris-HCl pH 7 ,4) and air-dried filter parts were transferred to scintillation tubes containing 3.5 ml of Optiphase 'Hisafe 2' liquid scintillation (Perkin Elmer). Radioactivity was measured in an LKB Wallace scintillation counter after 1 hour incubation at room temperature. Compound library screening.
[00297] A compound library was screened for agonists using a competition radioligand binding assay. For this purpose, 10 μg of home-prepared membrane layers of HEK293T cells expressing human β2AR (expression level ~10 pmol/mg membrane protein) were pre-incubated with Nb80 or an unrelated nanobody (negative control) for 1h at 30°C in incubation buffer (50 mM Hepes pH 7.4, 1 mM CaCl 2 , 5 mM MgCl 2 , 100 mM NaCl and 0.5% w/v BSA). Nanobodies are applied at a final concentration of 500 nM, roughly corresponding to a 3000-fold excess of Nanobody versus β2AR. Subsequently, Nanobody-loaded membranes are added to 96-well plates containing library compounds and 2 nM 3H-dihydroalprenolol (DHA) radioligand. The total volume per well is adjusted with incubation buffer to 100 μL and the reaction mixture is further incubated for another hour at 30°C. Subsequently, membrane-bound radioligand is collected using a 96-well GF/B filter plate of glass fiber (Perkin Elmer) pre-swollen in 0.3% polyethyleneimine. Filter plates are washed with ice-cold wash buffer (50 mM Tris-HCl pH 7.4), and dried for 30 minutes at 50°C. After adding 25 μL of scintillation fluid (MicroScint™-O, Perkin Elmer), radioactivity (cpm) is measured on a Wallac MicroBeta TriLux scintillation counter.
[00298] Fluorescence spectroscopy of bimanne in β2AR reconstituted in HDL particles.
[00299] To compare the effects on receptor conformation of Gs and Nb80 binding, purified β2AR was labeled with the environmentally sensitive monobromo-bimane (Invitrogen) fluorescent probe on cysteine 265 located at the cytoplasmic end of TM6, and reconstituted into TM6 particles. HDL (mBB-β2AR/HDL). Prior to obtaining fluorescence emission spectra, 10 nM mBB-β2AR/HDL incubated 30 min at RT in buffer (20 mM HEPES pH 7.5, 100 mM NaCl) in the absence or presence of 10 μM ISO, 1 μM ICI-118.551 reverse agonist (ICI), 300 nM Gs heterotrimer, or 300 nM Nb80, or in combinations of ISO with Gs, ISO with Nb80, and ICI with Nb80. Fluorescence spectroscopy was performed on a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) with photon counting mode, using an excitation and emission bandpass of 5 nm. Excitation was set at 370 nm and emission was collected from 415 to 535 nm in 1 nm increments with an integration time of 0.3 s/nm. Fluorescence intensity was corrected for background fluorescence of buffer and ligands. TABLE 1. LIST OF β2AR-SPECIFIC NANOBODIES

TABLE 2. β2AR-SPECIFIC NANOBODY CDRS



TABLE 3. TOTAL BINDING PROPERTIES OF THE MEMBRANE AGONIST EXPRESSING β2AR IN THE PRESENCE AND ABSENCE OF NANOBODY.
[00300] Competition binding of [3H]-DHA was performed on membranes of Sf9 insect cells expressing β2AR, in the presence or absence of 1 μM nanobodies. Data represent the mean ± s of two independent experiments performed in triplicate. The IC50 values used for calculations of Ki values were obtained from the average of the pIC50 values determined by nonlinear regression analysis using Prism (GraphPad Software, San Diego, CA) and the range s of pIC50 ± s.
TABLE 4. PHARMACOLOGICAL CHARACTERIZATION OF β2AR RECONSTITUTED IN HDL PARTICLES IN COMPLEX WITH Gs AND Nb80
TABLE 5. X-RAY DATA COLLECTION AND β2AR-Nb80 COMPLEX REFINING STATISTICS A. DATA COLLECTION STATISTICS
B. REFINEMENT STATISTICS

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权利要求:
Claims (27)
[0001]
1. Cellular composition, characterized in that it comprises a complex, said complex comprising (i) a nanobody capable of specifically binding to an intracellular conformational epitope of a GPCR and capable, after binding, of stabilizing an active conformational state of the GPCR said GPCR, wherein said nanobody comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 29; and (ii) a GPCR in an active conformational state.
[0002]
2. Cellular composition according to claim 1, characterized in that said complex further comprises a receptor ligand, preferably wherein said receptor ligand is a full agonist, or partial agonist, or an inverse agonist or an antagonist. .
[0003]
3. Cellular composition according to claim 2, characterized in that said receptor ligand is a full agonist or a partial agonist.
[0004]
4. Cellular composition according to claim 2 or 3, characterized in that said receptor ligand is chosen from the group comprising a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or any suitable fragment thereof.
[0005]
5. Cellular composition according to any one of claims 1 to 4, characterized in that said nanobody is specifically binding to a conformational epitope that is comprised in a binding site for a signaling protein downstream of said GPCR.
[0006]
6. Cellular composition according to any one of claims 1 to 5, characterized in that said nanobody is specifically binding to a conformational epitope that is comprised of, located in or overlaps with the G protein binding site of said GPCR.
[0007]
7. Cellular composition according to any one of claims 1 to 6, characterized in that said nanobody occupies the G protein binding site of an active conformational state of said GPCR.
[0008]
8. Cellular composition according to any one of claims 1 to 7, characterized in that said nanobody increases the affinity of the GPCR for an agonist.
[0009]
A cellular composition according to any one of claims 1 to 8, characterized in that said nanobody increases the affinity of the GPCR for an agonist at least twice, preferably at least five times, more preferably at least ten times, after connection to the GPCR.
[0010]
10. Cellular composition according to any one of claims 1 to 9, characterized in that it is a tissue, a cell, a cell lineage, a membrane composition or a liposomal composition.
[0011]
11. Cell composition according to claim 10, characterized in that it is a cell or a cell lineage.
[0012]
12. Cellular composition according to claim 10, characterized in that it is a membrane composition or a liposomal composition.
[0013]
13. In vitro method of identifying compounds that are orthosteric or allosteric ligands of a GPCR and that are capable of binding to an active conformational state of said GPCR, characterized in that it comprises the steps of: (a) providing a GPCR and a nanobody capable of specifically binding a conformational epitope of said GPCR, wherein said nanobody comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 29; (b) forming a complex comprising said nanobody and said GPCR, wherein said GPCR is in an active conformational state; and (c) providing a test compound, and (d) evaluating whether the test compound binds to the active conformational state of the GPCR; and (e) selecting a compound that binds to the active conformational state of the GPCR; preferably wherein said nanobody and/or said complex is immobilized on a solid support.
[0014]
14. In vitro method of capturing a GPCR in an active conformational state, characterized in that it comprises the steps of: a. applying a solution containing a GPCR in a plurality of conformational states to a solid support that has an immobilized nanobody that is capable of specifically binding to a conformational epitope of said GPCR and that is capable, after binding, of stabilizing an active conformational state of the said GPCR, wherein said nanobody comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 29, and b. form a complex of the nanobody and the GPCR, wherein said GPCR is in an active conformational state, and c. remove weakly bound or unbound molecules.
[0015]
15. In vitro method according to claim 13 or 14, characterized in that said nanobody is capable of increasing the stability of an active conformational state of a GPCR after binding.
[0016]
16. In vitro method according to any one of claims 13 to 15, characterized in that said nanobody is capable of inducing an active conformational state in a GPCR after binding.
[0017]
17. In vitro method according to any one of claims 13 to 16, characterized in that said nanobody is capable of specifically binding a GPCR bound to an agonist and/or increasing the affinity of a GPCR for an agonist.
[0018]
18. In vitro method according to any one of claims 13 to 17, characterized in that said nanobody is directed against an intracellular region, domain, loop or suitable intracellular conformational epitope of a GPCR.
[0019]
19. In vitro method according to any one of claims 13 to 18, characterized in that said nanobody is capable of specifically binding to a conformational epitope that is comprised, located or overlaps with the G protein binding site of a GPCR.
[0020]
20. Complex, characterized in that it comprises a nanobody that is capable of specifically binding to a conformational epitope of a GPCR and capable, after binding, of stabilizing an active conformational state of said GPCR, and a GPCR in an active conformational state , wherein said nanobody binding comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 29.
[0021]
21. Complex according to claim 20, characterized in that said complex further comprises a receptor ligand, preferably wherein said receptor ligand is a full agonist, or a partial agonist, or an inverse agonist or a antagonist, more preferably wherein said receptor ligand is a full agonist or a partial agonist.
[0022]
22. Complex according to claim 21, characterized in that said receptor ligand is chosen from the group comprising a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or any suitable fragment thereof.
[0023]
23. Complex according to any one of claims 20 to 22, characterized in that said nanobody is capable of increasing the stability of an active conformational state of said GPCR after binding.
[0024]
24. Complex according to any one of claims 20 to 23, characterized in that said nanobody is capable of specifically binding a GPCR bound to an agonist and/or increasing the affinity of a GPCR for an agonist.
[0025]
25. Complex according to any one of claims 20 to 24, characterized in that said nanobody is directed against an intracellular region, domain, loop or suitable intracellular conformational epitope of said GPCR.
[0026]
26. Complex according to any one of claims 20 to 25, characterized in that said nanobody is capable of specifically binding to a conformational epitope that is comprised in, located in or overlaps with the G protein binding site of said GPCR
[0027]
27. Complex according to any one of claims 20 to 26, characterized in that said complex is immobilized on a solid support.

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法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

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2020-08-04| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-09-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-06| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 18/07/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US39978110P| true| 2010-07-16|2010-07-16|

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US61/399,781|2010-07-16|

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GB1014715.5|2010-09-06|

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GBGB1014715.5A|GB201014715D0|2010-09-06|2010-09-06|Nanobodies stabilizing functional conformational states of GPCRS|

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PCT/EP2011/062287|WO2012007593A1|2010-07-16|2011-07-18|Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof|

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