WO2019155041A1 - Gβγ COMPLEX ANTIBODIES AND USES THEREOF - Google Patents

Abstract

The present invention relates to antibodies or antibody fragments specifically binding to Gβγ dimers and uses thereof. More specifically, immunoglobulin single variable domain antibodies causing a GPCR-independent shift in the equilibrium of heterotrimeric G proteins towards dissociated Gα and antibody-bound Gβγ subunits were identified. The invention discloses antibodies and active antibody fragments inhibiting Gβγ signaling, without affecting Gα, and outcompeting other Gβγ-regulatory proteins/ effectors, thereby targeting an epitope of Gβ overlapping with the Gα binding site. The invention further relates to methods and uses of said antibodies or active antibody fragments in structural analysis, as a tool, to select for GPCR signaling via Gβγ targeting independent of Gα, and to said antibodies or active antibody fragments for use as a medicament or as a diagnostic.

Description

G y complex antibodies and uses thereof

FIELD OF THE INVENTION

The present invention relates to antibodies or antibody fragments specifically binding to qbg dimers and uses thereof. More specifically, immunoglobulin single variable domain antibodies causing a GPCR- independent shift in the equilibrium of heterotrimeric G proteins towards dissociated Ga and antibody- bound qbg subunits were identified. The invention discloses antibodies and active antibody fragments selectively inhibiting qbg signaling, without affecting Ga, and outcompeting other ΰbg-^uIqIqGg proteins/ effectors, thereby targeting an epitope of qb overlapping with the Ga binding site. The invention further relates to methods and uses of said antibodies or active antibody fragments in structural analysis, as a tool, to select for GPCR signaling via qbg targeting independent of Ga, and to said antibodies or active antibody fragments for use as a medicament or as a diagnostic.

BACKGROUND

G protein-coupled receptors (GPCRs) comprise the most abundant family of cell membrane receptors and share a common mechanism of signal transduction. GPCRs respond to a wide variety of extracellular signals, including photons, ions, lipids, small molecules, peptides, and proteins. G protein-coupled receptors (GPCRs) function by translating extracellular stimuli across the plasma membrane into intracellular signaling events^{1 5}. The latter are accomplished by a ligand-induced conformational change in the GPCR that activates a downstream heterotrimeric G protein⁶·⁷. Heterotrimeric G proteins, consisting of three subunits, Ga, qb and Gy, undergo a Ga-GDP/GTP exchange that leads to dissociation of the qbg dimer from the heterotrimer^{8 10}. The Ga-GTP and qbg dimer each regulate a variety of downstream pathways to control various aspects of human physiology. Dysregulated ΰbg-e^hqI^ is a central element of various neurological and cancer-related anomalies. However, qbg also serves as a negative regulator of Ga that is essential for G protein inactivation, and thus has the potential for numerous side effects when targeted therapeutically. Notably, heterotrimeric G proteins maintain an equilibrium between their heterotrimeric and dissociated states by undergoing spontaneous GPCR-independent association/dissociation^{11 13}.

While GPCRs serve as the largest class of drug-targeted membrane proteins, producing the majority of FDA-approved drugs available on the market, they have been mostly targeted therapeutically with either small molecule or peptide modulators¹⁴. These modulators can be classified as either agonists, antagonists, or inverse agonists, depending on their ability to either stabilize the activated state of receptors, inhibit agonist competitively, or reduce the basal spontaneous coupling to G proteins, respectively¹⁵. However, drug development for GPCR signaling pathways has been hampered by difficulties in identifying molecules with suitable selectivity¹⁸. Related GPCRs share a promiscuous ligand binding site for lipophilic ligands¹⁹, which allows several off-target effects when pursued therapeutically. Additionally, lipophilic ligands can penetrate the central nervous system (CNS), and thereby interact with undesirable neuronal receptor targets. Therefore, functional monoclonal antibodies (mAbs) are currently being used to target less well-conserved allosteric sites of GPCRs to selectively modulate their signaling²⁰. Although mAbs and their fragments have played an instrumental role in selectively targeting GPCR signaling pathways, their commercial success is limited due to their high costs of production and treatment, especially when their efficacy in prolonging survival is similar to that of less expensive alternatives. Antibody alternatives are now known as versatile tools for exploring the determinants of GPCR recognition. For instance, Nanobodies^® (Nbs) derived from the variable region of camelid heavy chain are endowed with favorable characteristics in terms of size, solubility, affinity and ease of production²¹. In addition to their use in (GPCR) structural studies^{22 25}·²⁷, therapeutic Nbs are being discovered that target GPCR signaling²⁶. Also i-bodies, derived from the l-set immunoglobulin superfamily, that selectively block CXCR4 b-arrestin recruitment, were reported¹¹⁴’¹¹⁷. With the exception of b-arrestin specific antibody fragments, all small-molecule-, antibody- and Nanobody-based approaches target GPCR-mediated signaling at the GPCR level²⁶·²⁸, and thereby are GPCR-specific and cannot be used generically. Additionally, these approaches activate both Ga-GTP- and Gbg-mediated signaling pathways that lead to the activation of undesired cellular signaling events.

The liberated qbg dimer is a very efficient signal transducer^{29 44} and can dysregulate various cellular functions leading to numerous side effects associated with dysregulated ion channels, phosphoinositide- 3-kinase (PI3K), adenylyl cyclase and mitogen-activated protein kinase (MAPK) pathways. These diverse qbg functions make it an attractive target for the treatment of many medical conditions³³·⁴⁵. However, the ability of qbg to play essential roles in various cellular functions, including the formation of heterotrimeric G proteins, necessitates highly-specific qbg inhibitors that modulate its function while keeping Go mediated signaling intact.

SUMMARY OF THE INVENTION

The present invention is based on the finding that an antibody or more specifically a Nanobody family specifically binds to the qbg dimer and shifts the association/dissociation equilibrium of the heterotrimeric visual G protein (Gt) towards dissociated Gat and Nb-bound Qb1g1 subunits. Differential hydrogen/ deuterium exchange and crystallography studies suggest a competition between the Nb and other ΰbg- regulatory proteins for a common binding site on the qbg dimer. Interestingly, binding was shown for qb subtypes 1-4, showing its broad applicability to various cell types. Additionally, said Nanobodies respond to all combinations of b- and g-subtypes and compete with other ΰbg-^uIqIqGg proteins for a common binding site on the qbg dimer. Moreover, the Nb-binding suppresses the activation of both the ΰbg- regulated G-protein-gated inward rectifier potassium channels and the Gbg-mediated phosphoinositide- 3-kinase and mitogen-activated protein kinase pathways. Despite its inhibitory effect on Gbg-mediated signaling, surprisingly the Nb-binding to qbg has no effect on Ga-GTP-mediated signaling events, i.e. it has no effect on Ga_q and Gas-mediated signaling events in the environment of a living cell. In conclusion, said Nbs represent a versatile tool to achieve selective modulation of GPCR-signaling, opening new avenues for G Y-signaling modulation to treat various excitatory neurological conditions and cancer progression.

In a first aspect, the invention relates to an antibody or an active antibody fragment specifically binding the ΰbg complex at an interface overlapping the Ga binding site. In one embodiment, said antibody or active antibody fragment binds the ΰbg complex epitope conserved among the ΰbi-₄ subtypes, comprising the amino acid acids 80-99 and 1 1 1-1 18 of said ΰb subtypes b1-4. In another embodiment, said antibodies or active antibody fragments promote dissociation of Ga-GDP from the ΰbg complex, upon GPCR activation.

In some embodiments, the antibody or active antibody fragment of the invention has an affinity for the ΰbg complex corresponding to a KD between 5 nM and 50 nM. An embodiment relates to a KD above about 5nM, in order not to affect Ga signaling, and a KD below about 50 nM, in order to outcompete effector proteins of ΰbg such as PDC and GRK2. In a specific embodiment, said antibody or active antibody fragment is able to inhibit ΰbg signaling. In another specific embodiment, said antibody or active antibody fragment is able to selectively inhibit ΰbg signaling, without affecting Ga signaling.

In a particular embodiment, the antibody or active antibody fragment is an immunoglobulin single variable domain (ISVD) comprising the amino acid sequence that comprises 4 framework regions (FR) and 3 complementary determining regions (CDR) according to the formula FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4. More specifically, said ISVD comprises a CDR3 with SEQ ID NO: 1 , or an amino acid sequence with at least 80 % identity thereof. Or even more specifically, the ISVD comprises SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a homologue with at least 80 % amino acid identity thereof, or a humanized variant of any one thereof. Most preferably, said ISVD comprises SEQ ID NO: 2, corresponding to Nb5 for specific binding and inhibition of ΰbg complex activity.

Another embodiment relates to said antibody or active antibody fragment of the invention bound to the ΰbg complex inhibiting GIRK channel activation in neuronal cells. Another embodiment relates to said antibody or active antibody fragment of the invention that blocks activation of PI3K-AKT and/or activation of MAP ERK.

In another embodiment, the antibody or active antibody fragment further comprises a detection agent, such as a tag or a label, or comprises a functional moiety, such as a blood-brain-barrier (BBB) crossing moiety, or comprises a cell penetrant carrier, or any combination thereof.

Another aspect of the invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or active antibody fragment of the present invention. One embodiment comprises an expression cassette comprising said nucleic acid molecule, and another embodiment comprises a vector comprising said expression cassette or nucleic acid molecule. In a particular embodiment, said nucleic acid sequence, expression cassette or vector encodes the antibody or active antibody fragment, as an intrabody.

A third aspect of the invention relates to a solid substance, which can be a surface, resin or support for instance, comprising the antibody or active antibody fragment of the invention. Further aspects of the invention relate to the use of the antibody or active antibody fragment, or the use of said solid substance or support comprising the antibody or active antibody fragment, for affinity chromatography, affinity purification, immunoprecipitation, in-vivo imaging, protein detection, immunochemistry, surface-display, FRET-type applications or for structural analysis.

In one embodiment the use of said antibody or active antibody fragment, said nucleic acid molecule, expression cassette or vector, or the solid substance of the invention, as a tool to distinguish in a system the GPCR activated Ga from qbg signaling.

In another embodiment, said antibody or active antibody fragment, said nucleic acid molecule, said expression cassette, said vector, or said solid substance are of use as a medicament or as a diagnostic. More specifically, said antibody or active antibody fragment, said nucleic acid molecule, said expression cassette, and/or said vector may be applied for use as a medicament or therapeutic, and said antibody or active antibody fragment, said nucleic acid molecule, said expression cassette, said vector, and/or said solid substance as solid surface or resin may be applicable for use as a diagnostic or provided in a kit. In particular embodiments, the invention comprises a kit comprising means for qbg complex binding without affecting Ga, said kit comprising the antibody or active antibody fragment, said nucleic acid molecule, said expression cassette, said vector, or said solid substance of the invention.

In another aspect of the invention, a host cell is disclosed comprising the antibody or active antibody fragment, the nucleic acid molecule, the expression cassette or the vector of the invention. In a specific embodiment, said host cell comprises the intrabody comprising the antibody or active antibody fragment, or comprises the intrabody encoded by the nucleic acid molecule of the invention.

A final aspect relates to a method for identifying or producing a compound that modulates G protein signaling, comprising the steps of: a) providing the host cell of the invention, and transfecting said cell with a GPCR of interest, b) adding a test compound to said cell, and c) evaluating the effect of said test compound on G protein signaling in said cell as compared to a cell line without the test compound.

DESCRIPTION OF THE FIGURES

The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Figure 1. Nanobody-mediated shift in heterotrimeric G_t equilibrium.

(a) Effect of Nb5 on the heterotrimeric state of Gt in solution. Lane 1 shows the dissociation of heterotrimeric Gt in the presence of light-activated Rh (Rh*), GTP and Nb5. ΰbigi was later co-purified with Nb5 by immobilized-Ni²⁺ affinity chromatography and Gat emerged as an unbound protein in the flowthrough (lane 2). Note the decrease in Gyi Coomassie staining in the presence of 300 mM imidazole during protein elution. Similar results were obtained from the reaction mixture in the absence of either Rh* alone (lanes 3 and 4) or both Rh* and GTP (lanes 5 and 6). These findings suggest that Nb5 alone can trap ΰbigi after its spontaneous dissociation from Gat, and thereby shifts the dynamic equilibrium of heterotrimeric Gt towards its dissociated subunits. However, Nb17 did not affect the heterotrimeric configuration of Gt (lanes 7 and 8, compare lanes marked with asterisks). Additionally, non-specific interactions between ΰb-igi and Ni²⁺-NTA resin were not seen (lanes 9 and 10). The eluate and flowthrough obtained from affinity chromatography purification are denoted as Έ” and“FT”, respectively.

(b) Proteins in typical eluates obtained after immobilized-Ni²⁺ affinity chromatography were analyzed by SDS-PAGE (lane 1 ) and immunoblotting with antibodies specific to ΰb-i , His-tag, and Gy-i, respectively (lanes 2-4).

(c) Effect of Nb5 on the heterotrimeric state equilibrium of Gt in ROS membranes. Light activation of ROS membranes in the presence of GTP released Gat in the high salt wash (lane 1 ). Similar Gat release was obtained with Nb5 with or without GTP during the high salt wash (lanes 2 and 3). Treatment of ROS membranes with Nb17 had no effect on Gat release (lane 4, compare lanes marked with asterisks). Untreated ROS membranes showed no Gat release during the high salt wash (lane 5).

(d) Gt activation by Rh* was monitored by an increase in the intrinsic tryptophan fluorescence of Gat in the presence of GTPyS. The initial Gt activation rate of Rh* was reduced upon pre-treatment of heterotrimeric Gt with Nb5 (greencyan) as compared to either untreated (red) or Nb17-treated Gt (purple).

(e) Quantification of the initial Gt activation rates of Rh*with and without Nanobody pre-treatment.

(f) The decrease in Gt activation rates after Nb5 treatment were explained by Rh single turnover experiments that exhibited an exponential increase in Gt activation rates with increasing concentrations of Gt.

Figure 2. Kinetic profiling and hydrogen-deuterium exchange (HDX) of the GPiyi-Nb5 complex.

Binding of ΰb-igi to immobilized Nb5 in SPR equilibrium binding experiments. Association of the ΰb-igi dimer with Nb5 was investigated by single-cycle kinetics (a) and affinity based analysis (b). Resonance signals are indicated in response units (RU). The determined dissociation constants (KD) and kinetic parameters (Kon and Koff) are shown as insets. HDX of the ΰb-igi dimer without (c) and with (d) Nb5 is shown with all the identified peptides colored by their percentage of deuterium exchange (e) Differential HDX data mapped into the crystal structure of ΰb-igi (PDB ID: 5KDO) indicate peptides with increased (greencyan) and reduced (purple) deuterium incorporation upon Nb5 binding. Interestingly, ΰb-igi peptides that revealed changes in their solvent accessibility during HDX analyses displayed a partial interface with the Ga subunit (right, pink). The Ga subunit was omitted from the left panel for clarity.

Figure 3. Crystal structure of ΰbigi in complex with Nb5.

(a) The asymmetric unit contains two molecules of both ΰb-igi (blue and pink) and Nb5 (greencyan and red) shown in cartoon representation. Key interfaces between ΰb-igi and Nb5 are denoted by grey ellipsoids (b) Side and top views of the Gb₁g₁-Nb5 complex showing the CDR3 region (red) of Nb5 inserted into the ΰb-i-rGorbIIbG. The 2|Fo| - |Fc| density (blue) around the CDR3 region was calculated after the final refinement and is contoured at 1.5o. (c) ΰbg hot-spot is shared by several ΰbg regulatory proteins, including Gat (pink), phosducin (PDC, cyan) and G Protein-Coupled Receptor Kinase 2 (GRK2, dark grey) (d) Overall structure of the heterotrimeric G protein (PDB ID: 5KDO). (e) Overlay of the interface between the G iYi-Nb5 complex and heterotrimeric G protein. Schematic representation shows the extent of overlapping (in greencyan background, black residues) between the non-canonical qbigi- Nb5 complex interface (greencyan residues) and the interface between qbigi and Gat (pink residues).

Figure 4. GP selectivity of Nb5.

(a) Multiple sequence alignment of key amino acid residues of qb that interact with Nb5. *The amino acid numbering of qbd has an off-set of -50. (b) Nb5-mediated qbg extraction from mouse brain. Solubilized and partially purified extracts from mouse brain (left) were subjected to either Nb5- (right, lanes 1 and 2) or Nb17- (right, lanes 3 and 4) mediated immobilized-metal affinity purification of qbg. (c) In-gel protein digestion of qb subtypes (band marked in b with asterisks) purified from mouse brain. Peptides were separated, analyzed and searched against a full mouse proteome to identify unique peptides from qb-i, ΰb2, ΰb3, and ΰb₄. (d) Schematic diagram of the effect of Gbg-binding proteins on the BRET assay. Cotransfection of HEK293T/17 cells with nbhue-qbg and masGRK3ct-Nluc produced a high BRET signal through their direct interaction (left). Introduction of Gbg-binding proteins (e.g. the Ga subunit) competed with masGRK3ct-Nluc, lowering the BRET signal (right) (e) Effects of Nb5 on the interaction of qbg and the C-terminus of GRK3. The maximum BRET signal was determined by co-transfection of different nbhue-qb subtypes + GY_å pairs and masGRK3ct-Nluc-HA (grey). A minimum BRET signal also was determined after co-transfection of Venus- Qbig2 and masGRK3ct-Nluc with an excess amount of GaoA (pink). Effects of Nb5 and Nb17 were examined by co-transfection of nbhue-qbg and masGRK3ct-Nluc with either Nb5 (greencyan) or Nb17 (purple). Experiments were performed with qb subtypes 1-4+ GY_å pairs. Each bar represents the mean of 6 replicates. Similar results were obtained in three independent experiments. Results are expressed as the mean ± SEM. One-way ANOVA with Tukey’s post hoc multiple comparison test relative to the Gb1g2/GRK3ct control, ***P < 0.001 , n = 6 replicates (f) Western blot quantification of the expression levels of ΰb1-4, GRK3, Ga, Nb5, and Nb17 (Full blots are shown in Fig 9a).

Figure 5. Inhibition of GPy-mediated GIRK signaling by Nb5.

(a) Representative whole-cell recording of a GIRK2+ striatal medium spiny neuron (MSN) treated with 10 mM of intracellular Nb5. A single electrical stimulation evoked a D2R-IPSC that showed a significant amplitude reduction 15 min post-treatment with Nb5 (greencyan) as compared to the averaged trace observed at 5 min post-treatment with Nb5 (grey) (b) Representative trace of an M4-IPSC that displays an amplitude reduction 15 min post-treatment with Nb5 (greencyan) as compared to the averaged trace observed at 5 min post-treatment with Nb5 (grey). But no significant change was noted in D2R-IPSC exposed to 10 mM intracellular Nb17 (c) or without nanobody treatment (d). Time course measurements showing a decrease in D2R-IPSC (e) and M4R-IPSC (f) amplitudes upon their exposure to 10 pM Nb5. Time course measurements of D2R-IPSC amplitudes recorded with Nb17(g) or without nanobody treatment (h). Bar graph quantification of D2R-IPSC (n=9) (i) and M2R-IPSC (n=8) (j) amplitudes recorded with Nb5. Bar graph quantification of D2R-IPSC amplitudes (n=5) recorded with 10 pM Nb17 (k), or without nanobody treatment (I), revealing no significant change in amplitudes over 15 min. Results are expressed as the mean ± SEM. (m) Proposed model of GIRK inhibition by Nb5.

Figure 6. Nb5 as a control -switch for GPCR-mediated Qbg signaling.

(a) Schematic diagram of the GPCR-mediated qbg signaling network that controls various cellular functions (b) Parental CHO-APJ cells, CHO-APJ cells transfected with pcDNA3.1 (+) empty vector, transfected CHO-APJ-Nb5 and CHO-APJ-Nb17 cells were treated with 1 mM apelin over 0-5 min to investigate the effect of Nb5 on the phosphorylation of ERK1/2 and AKT. Nb5 significantly decreased the phosphorylation of both ERK1/2 and AKT as compared to the Nb17 control (compare lanes marked with asterisks). Full blots are reported in Supplementary Fig 4b. (c) No significant effect of Nb17 transfection (purple) was observed on the phosphorylation of either ERK1/2 or AKT as compared to parental CHO- APJ cells (black) and CHO-APJ cells transfected with pcDNA3.1 (+) empty vector (red). Results are expressed as the mean ± SEM, n = 3 replicates (d) Schematic diagram showing the effect of Ga, and qbg signaling on cellular cAMP levels (e) Dose-response curves of apelin-induced inhibition of cAMP accumulation in parental CHO-APJ cells (grey) and CHO-APJ cells expressing either Nb5 (greencyan) or Nb17 (purple). Apelin concentration is plotted on a semi-log scale. Results are expressed as the mean ± SEM, n = 4 replicates (f) Schematic diagram of GPCR-mediated second-messenger regulation by Ga_q and Gas. (g) The effect of nanobodies on Ga_q signaling events was determined in HEK293T/17 cells transfected with M3R and CalFluxVTN. Each trace represents the mean of the responses measured in six wells (h) Basal BRET ratio and ACh-induced maximum amplitude shown as a bar graph. Similar results were obtained in three independent experiments. Results are expressed as the mean ± SEM, n = 6 replicates. Two-way ANOVA with Tukey’s post hoc multiple comparison test (i) Effect of nanobodies on Gas signaling was evaluated in HEK293T/17 cells transfected with D1 R and Nluc-Epac-VV. Each trace represents the mean of the responses measured in six wells (j) Basal BRET ratio and dopamine-induced maximum amplitude shown as a bar graph. Each bar represents the mean of three independent experiments. Results are expressed as the mean ± SEM, n=6 replicates. Two-way ANOVA with Tukey’s post hoc multiple comparison test.

Figure 7. Interaction of Nb5 with the QTb1g1 dimer.

Gb1g1-Nb5 was crystallized in space group, P21 , where a majority of crystal contacts were mediated through Nb5 within the aqueous layers (a) Top view of the Gb1g1-Nb5 complex crystal lattice in a direction perpendicular to the long C-axis. Each asymmetric unit is composed of two Gb1g1-Nb5 molecules that form crystal contacts with neighboring molecules (b) Arg-101 in the complementarity-determining region 3 of Nb5 serves as a key that locks the qbI-rGorbIIbG and forms an intricate hydrogen-bonding network with water molecules in the qbI-rGorbIIbG cavity (c) Lys-663 in the C-terminal loop of G Protein-Coupled Receptor Kinase 2 (GRK2) forms a similar lock and key interaction in the Gb1g2-GRK2 complex (PDB accession: 10MW⁵³). Figure 8. : Effect of Nb5 on GPCR-mediated Qbg signaling.

(a) Wild-type CHO-APJ cells were treated with 1 m M apelin over 0-30 min at 37°C, and the optimum time point of 5 min was selected for further experiments based on the phosphorylation levels of both ERK1/2 and AKT. (b) Western blot analysis showed similar expression levels of the apelin receptor in parental CHO-APJ cells, CHO-APJ-vector cells, CHO-APJ-Nb5 and CHO-APJ-Nb17 cells.

Figure 9: Full Western blots.

(a) Western blots showing the quantification of the expression levels of ΰb1-4, GRK3, Ga, Nb5, and Nb17. (b) Western blots demonstrating reduced phosphorylation of both pERK1/2 and pAKT in CHO- APJ-Nb5 cells as compared to the CHO-APJ-NM 7 cells.

Figure 10. Nanobodies directed towards Qb1g1 dimer.

(a) Multiple sequence alignment of the three members of the Nb5 family, Nb5, Nb6 and Nb7 having an identical complementarity determining region 3 (CDR3). Complementarity determining regions 1 , 2 and 3 are marked with black, green, and red lines, respectively (b) Coomassie stained SDS- polyacrylamide gel showing the ability of Nb5, Nb6 and Nb7 to selectively trap the Qb1g1 dimer.

DETAILED DESCRIPTION TO THE INVENTION

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 only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing 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 other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 1 14), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

The terms“protein”,“polypeptide”,“peptide” are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa). By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide. By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polypeptide" refers to a polypeptide which has been purified from the molecules which flank it in a naturally-occurring state, e.g., a antigen-binding chimeric protein which has been removed from the molecules present in the production host that are adjacent to said polypeptide. An isolated protein can be generated by amino acid chemical synthesis or can be generated by recombinant production. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

As used herein, the term“protein complex” or“complex” refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. A protein complex, as used herein, typically refers to associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex are linked by non-covalent interactions. A protein complex can be a non-covalent interaction of only proteins, and is then referred to as a protein-protein complex; for instance, a non-covalent interaction of two proteins, of three proteins, of four proteins, etc. More specifically, a heterotrimeric G protein complex GDP-Gc^y dissociates into GDP-Ga and the qbg complex upon activation by a GPCR, to convert the GDP-Ga into GTP-Ga and qbg downstream signaling. As used herein, a protein complex can also be a non-covalent interaction of at least one protein and at least other macromolecule, such as a nucleic acid, and is then referred to as a protein-nucleic acid complex; for instance, a non-covalent interaction of one protein and one nucleic acid, two proteins and one nucleic acid, two proteins and two nucleic acids, etc. It will be understood that a protein complex can be multimeric. Protein complex assembly can result in the formation of homo-multimeric or hetero- multimeric complexes. Moreover, interactions can be stable or transient. The term “multimer(s)”, “multimeric complex”, or “multimeric protein(s)” comprises a plurality of identical or heterologous polypeptide monomers. Polypeptides can be capable of self-assembling into multimeric assemblies (i.e.: dimers, trimers, hexamers, pentamers, octamers, etc.) formed from self-assembly of a plurality of a single polypeptide monomers (i.e.,“homo-multimeric assemblies”).

The term“antibody” as used herein, refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen.‘Antibodies’ can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "active antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more CDRs accounting for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies, domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. An additional requirement for "activity" of said fragments in the light of the present invention is that said fragments are capable of binding qbg complex, and preferably inhibit qbg signaling, more preferably selectively inhibit qbg signaling.

The term“antibody”,“antibody fragment” and“active antibody fragment” as used herein refer to a protein comprising an immunoglobulin domain or an antigen binding domain capable of specifically binding the qbg dimer. The antibodies or active antibody fragments of the invention, can be labeled by an appropriate label, said label can for instance be of the enzymatic, colorimetric, chemiluminescent, fluorescent, or radioactive type, or can be coupled to a functional moiety, or cell penetrant carrier.

Antibodies are typically tetramers of immunoglobulin molecules. The term“immunoglobulin (Ig) domain”, or more specifically“immunoglobulin variable domain” (abbreviated as“IVD”) means an immunoglobulin domain essentially consisting of four“framework regions” which are referred to in the art and herein below as“framework region 1” or“FR1”; as“framework region 2” or“FR2”; as“framework region 3” or“FR3”; and as“framework region 4” or“FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or“CDRs”, which are referred to in the art and herein below as “complementarity determining region 1” or“CDR1”; as“complementarity determining region 2” or“CDR2”; and as“complementarity determining region 3” or“CDR3”, respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, with binding to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

A“patient” or“subject”, for the purpose of this invention, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, or an injury, also designated’’patient” herein. In another embodiment, a subject is a subject ready to receive a transplant or allograft, also designated as a“patient eligible for receiving an allograft”.

The term“treatment” or“treating” or“treat” can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, injury, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders.

With“G proteins” are meant the family of guanine nucleotide-binding proteins involved in transmitting chemical signals outside the cell, and causing changes inside the cell. G proteins are key molecular components in the intracellular signal transduction following ligand binding to the extracellular domain of a GPCR. They are also referred to as“heterotrimeric G proteins”, or“large G proteins”. G proteins consist of three subunits: alpha (a), beta (b), and gamma (y) and their classification is largely based on the identity of their distinct a subunits, and the nature of the subsequent transduction event. Further classification of G proteins has come from cDNA sequence homology analysis. G proteins bind either guanosine diphosphate (GDP) or guanosine triphosphate (GTP), and possess highly homologous guanine nucleotide binding domains and distinct domains for interactions with receptors and effectors. Different subclasses of Ga proteins, such as Gas, Gai, Gaq and Ga12, amongst others, signal through distinct pathways involving second messenger molecules such as cAMP, inositol triphosphate (IP3), diacylglycerol, intracellular Ca²⁺ and RhoA GTPases. To illustrate this further, the a subunit (39— 46 kDa) contains the guanine nucleotide binding site and possesses GTPase activity; the b (37 kDa) and y (8 kDa) subunits are tightly associated and function as a bg heterodimer. There are 23 types (including some splicing isoforms) of a subunits, 6 of b, and 1 1 of y currently described. Typically, in nature, G proteins are in a nucleotide-bound form. More specifically, G proteins (or at least the a subunit) are bound to either GTP or GDP depending on the activation status of a particular GPCR. Agonist binding to a GPCR promotes interactions with the GDP-bound qabg heterotrimer leading to the exchange of GDP for GTP on Ga, and the functional dissociation of the G protein into Ga-GTP and GPy subunits. The separate Ga-GTP and G y subunits can modulate, either independently or in parallel, downstream cellular effectors. The intrinsic GTPase activity of Gy leads to hydrolysis of GTP to GDP and the re-association of Ga-GDP and G y subunits, and the termination of signaling. Thus, G proteins serve as regulated molecular switches capable of eliciting bifurcating signals through a and bg subunit effects. The switch is turned on by the receptor and it turns itself off within a few seconds, a time sufficient for considerable amplification of signal transduction.

In a first aspect, the invention relates to an antibody or an active antibody fragment specifically binding to the qbg complex, at an interface or binding site overlapping the Ga binding site.

The invention is based on a study that identified specific Nbs that inhibit qbg complex signaling, and surprisingly did not affect Ga signaling by binding to the qbg complex site overlapping the Ga binding site. It is known in the art that qbg dimer signaling also regulates Ga, via binding of Ga at the‘hotspot’ region present on the qbg dimer. In fact, this identification of the hotspot involves specific amino acids in qbg involved in individual target recognition, but does not explain the molecular basis for Gbg-dependent recognition of diverse effector structures. Multiple, distinct peptides were identified that apparently bound to the same surface on qbg, which indicates that those large protein surfaces are subjected to selection in naive random peptide-binding screens, and only a small portion of the overall surface mediates binding of diverse sets of peptide sequences, forming the preferred protein binding surface, or in structural biology terms the concept of energetic“hot spots”. Hot spots are defined as to provide key energetic residues for binding at a protein-protein interface, but also have intrinsic physical-chemical characteristics that are optimal for mediating multiple protein-protein interactions. Some characteristics of these surfaces are flexibility and the opportunity for mediating multiple types of chemical interactions (ionic, hydrophobic) without strict geometric requirements for binding. In this way a single binding site can accommodate multiple structural and chemical motifs (for a review see Smrcka, 2008, Cell Mol Life Sci. 65(14): 2191— 2214). The qb subunit belongs to a large family ofWD40 repeat proteins with a circular b-bladed propeller structure. This structure allows Qbg to interact with a broad range of proteins to play diverse roles. So the Qbg multitarget recognition through its“hot spot” accommodates multiple modes of binding. Because each target has a unique recognition mode for Qbg subunits, it suggests that these interactions could be selectively manipulated, for instance by small molecules, peptides, or biologicals such as antibodies or active antibody fragments. Indeed, in the case of the present invention, the antibody or active antibody fragment of the invention specifically binds an epitope on Qb that is located in the hotspot region, overlapping the Ga binding site, but not affecting Gbg-mediated signaling of Ga, and in addition affecting other natural effector protein binding. Therefore, said antibody or active antibody fragment of the invention is superior in its properties as a non-natural binder of the qbg complex of selectivity, specificity and affinity. By the term“specifically binds,” as used herein with respect to an antigen-binding, antibody, antibody- derived active antibody fragment or immunoglobulin domain, is meant a binding that recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample, and also referred to as an“antigen-binding domain” or "antigen-binding protein”. For example, an antibody or an active antibody fragment that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms“specific binding” or“specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antigen-binding protein or antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antigen-binding protein or antibody is specific for epitope“A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled“A” and the antigen-binding protein, will reduce the amount of labeled A bound to the antigen-binding protein. The term "specificity", as used herein, refers to the ability of a binding protein, in particular an antigen-binding domain, immunoglobulin or an immunoglobulin fragment, such as an antibody, an immunoglobulin single variable domain (ISVD), a VHH or Nanobody®, to bind preferentially to one antigen, versus a different antigen, and does not necessarily imply high affinity. Other examples of antigen-binding proteins also include synthetic binding proteins, more specifically also monobodies (e.g. for a review see Sha et al., 2017, Protein Science. 26:910-924.).

In one embodiment said antibody or active antibody fragment specifically binding qbg via interaction with qb, is competitive for Ga binding to qbg since the binding site is at least partially overlapping. Surprisingly, the Nb family binding this overlapping binding site, which is probably located on the qbg hotspot, is capable of binding to qbg without affecting Ga signaling. Since antibody or active antibody fragments, as well as Nanobodies are known to confer high specificity, but also high affinity for their targets, an impact on the Ga regulation would be expected for the antibody or Nb binding to said overlapping binding site. However, a meticulous balance of providing an antibody with an affinity high enough to outcompete endogenous downstream regulators, but low enough to allow competition with Ga binding to qbg (KD of about 1 nM) may therefore provide a key solution obtained in this invention to specifically affect qbg signaling independent from Ga, or from affecting Ga signaling . The structural features of the CDR3 binding to said epitope are representative for such a balance for the antibody or active antibody fragments of the invention.

In one embodiment, said antibody or active antibody fragment binds a Qb subunit involving the epitope comprising the amino acids 80-99 and 1 1 1-1 18 of said Qb subtypes b1-4. The epitope as defined herein results in high affinity interaction of the Nanobodies of the invention with any of the 4 b subtypes, although few amino acid residues within said region of 80-99 and 1 1 1-1 18 are not identical but similar among the subtypes. In another embodiment, said epitope further allows interaction of the antibody or active antibody fragment with amino acid residues 57-59, 75-76, 100-101 , 1 19, 142-147, 186-188, 204, 228-230, 246, 270, 274, 290, and 314-316 of said Qb subtypes b1-4. The binding site is in this way defined in a less strict sense, and in fact includes the full range of weaker and stronger binding residues. The weaker binding may not be required for the effect on Qbg signaling. Therefore, the epitope is defined herein as the minimal number of amino acid residues that are essential or critical to obtain binding to the Qbg, with as a consequence inhibition of its signaling (without affecting Ga signaling). Specifically, said epitope is formed as a conformational binding site, and has not been described as such for any naturally occurring Qb oί ΰbg binder. The conformational epitope, however, is very conserved cross-species for Qb subtypes. An “epitope”, as used herein, refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, 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 multi-dimensional nuclear magnetic resonance. A“conformational epitope”, as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3-dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. Similarly, conformational epitopes may here 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.

In a specific embodiment, said antibody or active antibody fragment binds human ΰb1-4 (SEQ ID NO:6- 9), or bovine ΰb1-4 (b1 represented herein by SEQ ID NO: 15) , or mouse ΰb1-4 (SEQ ID NO: 10-13), or mammal ΰb1-4, or ΰb subtypes 1-4 in any subject. In one embodiment, said antibody or active antibody fragment does not bind ΰb5 (SEQ ID NO: 14). A preferred embodiment relates to the antibody or active antibody fragment wherein the epitope is present on the ΰbg complex, on the ΰb subtypes b1 -4 as presented in the amino acid sequences of human ΰb1-4, as provided by SEQ ID NO:6-9. >SEQ ID NO:6: guanine nucleotide binding protein (G protein), beta polypeptide 1 , isoform CRA_a [Homo sapiens] EAW56150.1

MSELDQLRQEAEQLKNQIRDARKACADATLSQITNNIDPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRLLVSASQD GKLI IWDSYTTNKVHAIPLRSSWVMTCAYAPSGNYVACGGLDNICSIYNLKTREGNVRVSRELAGHTGYLSCCRFL DDNQIVTSSGDTTCALWDIETGQQTTTFTGHTGDVMSLSLAPDTRLFVSGACDASAKLWDVREGMCRQTFTGHESD INAICFFPNGNAFATGSDDATCRLFDLRADQELMTYSHDNI ICGITSVSFSKSGRLLLAGYDDFNCNVWDALKADR AGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLKIWN

>SEQ ID NO:7: guanine nucleotide binding protein beta 2 [Homo sapiens] AAM15919.1

MSELEQLRQEAEQLRNQIRDARKACGDSTLTQITAGLDPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRLLVSASQD GKLI IWDSYTTNKVHAIPLRSSWVMTCAYAPSGNFVACGGLDNICSIYSLKTREGNVRVSRELPGHTGYLSCCRFL DDNQI ITSSGDTTCALWDIETGQQTVGFAGHSGDVMSLSLAPDGRTFVSGACDASIKLWDVRDSMCRQTFIGHESD INAVAFFPNGYAFTTGSDDATCRLFDLRADQELLMYSHDNI ICGITSVAFSRSGRLLLAGYDDFNCNIWDAMKGDR AGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLKIWN

>SEQ ID NO:8: guanine nucleotide binding protein beta-3 subunit [Homo sapiens] AAA52582.1

MGEMEQLRQEAEQLKKQIADARKACADVTLAELVSGLEWGRVQMRTRRTLRGHLAKIYAMHWATDSKLLVSASQD GKLIVWDSYTTNKVHAIPLRSSWVMTCAYAPSGNFVACGGLDNMCSIYNLKSREGNVKVSRELSAHTGYLSCCRFL DDNNIVTSSGDTTCALWDIETGQQKTVFVGHTGDCMSLAVSPDFNLFISGACDASAKLWDVREGTCRQTFTGHESD INAICFFPNGEAICTGSDDASCRLFDLRADQELICFSHESI ICGITSVAFSLSGRLLFAGYDDFNCNVWDSMKSER VGILSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN

>SEQ ID NO:9: guanine nucleotide binding protein beta subunit 4 [Homo sapiens] AAG18442.1

MSELEQLRQEAEQLRNQIQDARKACNDATLVQITSNMDSVGRIQMRTRRTLRGHLAKIYAMHWGYDSRLLVSASQD GKLI IWDSYTTNKMHAIPLRSSWVMTCAYAPSGNYVACGGLDNICSIYNLKTREGNVRVSRELPGHTGYLSCCRFL DDSQIVTSSGDTTCALWDIETAQQTTTFTGHSGDVMSLSLSPDMRTFVSGACDASSKLWDIRDGMCRQSFTGHVSD INAVSFFPNGYAFATGSDDATCRLFDLRADQELLLYSHDNI ICGITSVAFSKSGRLLLAGYDDFNCNVWDTLKGDR AGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLRIWN

>SEQ ID NO:10: guanine nucleotide binding protein beta subunit 1 [Mus musculus] sp|P62874|GBB1

MSELDQLRQEAEQLKNQIRDARKACADATLSQITNNIDPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRLLVSASQD GKLI IWDSYTTNKVHAIPLRSSWVMTCAYAPSGNYVACGGLDNICSIYNLKTREGNVRVSRELAGHTGYLSCCRFL DDNQIVTSSGDTTCALWDIETGQQTTTFTGHTGDVMSLSLAPDTRLFVSGACDASAKLWDVREGMCRQTFTGHESD INAICFFPNGNAFATGSDDATCRLFDLRADQELMTYSHDNI ICGITSVSFSKSGRLLLAGYDDFNCNVWDALKADR AGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLKIWN

>SEQ ID NO:11 : guanine nucleotide binding protein beta subunit 2 [Mus musculus] sp|P62880|GBB2

MSELEQLRQEAEQLRNQIRDARKACGDSTLTQITAGLDPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRLLVSASQD GKLI IWDSYTTNKVHAIPLRSSWVMTCAYAPSGNFVACGGLDNICSIYSLKTREGNVRVSRELPGHTGYLSCCRFL DDNQI ITSSGDTTCALWDIETGQQTVGFAGHSGDVMSLSLAPDGRTFVSGACDASIKLWDVRDSMCRQTFIGHESD INAVAFFPNGYAFTTGSDDATCRLFDLRADQELLMYSHDNI ICGITSVAFSRSGRLLLAGYDDFNCNIWDAMKGDR AGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLKIWN

>SEQ ID NO:12: guanine nucleotide binding protein beta subunit 3 [Mus musculus] sp|Q6101 1 |GBB3

MGEMEQLRQEAEQLKKQIADARKACADITLAELVSGLEWGRVQMRTRRTLRGHLAKIYAMHWATDSKLLVSASQD GKLIVWDTYTTNKVHAIPLRSSWVMTCAYAPSGNFVACGGLDNMCSIYNLKSREGNVKVSRELSAHTGYLSCCRFL DDNNIVTSSGDTTCALWDIETGQQKTVFVGHTGDCMSLAVSPDYKLFISGACDASAKLWDVREGTCRQTFTGHESD INAICFFPNGEAICTGSDDASCRLFDLRADQELTAYSQESI ICGITSVAFSLSGRLLFAGYDDFNCNVWDSLKCER VGILSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN

>SEQ ID NO:13: guanine nucleotide binding protein beta subunit 4 [Mus musculus] sp|P29387|GBB4

MSELEQLRQEAEQLRNQIQDARKACNDATLVQITSNMDSVGRIQMRTRRTLRGHLAKIYAMHWGYDSRLLVSASQD GKLI IWDSYTTNKMHAIPLRSSWVMTCAYAPSGNYVACGGLDNICSIYNLKTREGNVRVSRELPGHTGYLSCCRFL DDGQI ITSSGDTTCALWDIETGQQTTTFTGHSGDVMSLSLSPDLKTFVSGACDASSKLWDIRDGMCRQSFTGHISD INAVSFFPSGYAFATGSDDATCRLFDLRADQELLLYSHDNI ICGITSVAFSKSGRLLLAGYDDFNCSVWDALKGGR SGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLRIWN >SEQ ID NO:14: guanine nucleotide binding protein beta subunit 5 [Mus musculus] sp | P62 881 1 GBB5 MCDQTFLVNVFGSCDKCFKQRALRPVFKKSQQLNYCSTCAE IMATDGLHENETLASLKSEAESLKGKLEEERAKLH DVELHQVAERVEALGQFVMKTRRTLKGHGNKVLCMDWCKDKRRIVSSSQDGKVIVWDSFTTNKEHAVTMPCTWVMA CAYAPSGCAIACGGLDNKCSVYPLTFDKNENMAAKKKSVAMHTNYLSACSFTNSDMQI LTASGDGTCALWDVESGQ LLQSFHGHGADVLCLDLAPSETGNTFVSGGCDKKAMVWDMRSGQCVQAFETHESDVNSVRYYPSGDAFASGSDDAT CRLYDLRADREVAIYSKES I I FGASSVDFSLSGRLLFAGYNDYT INVWDVLKGSRVS I LFGHENRVSTLRVSPDGT AFCSGSWDHTLRVWA

>SEQ ID NO:15: bovine guanine nucleotide binding protein beta subunit 1 sp|P62871 |GBB1

MSELDQLRQEAEQLKNQIRDARKACADATLSQI TNNI DPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRLLVSASQD GKLI IWDSYTTNKVHAI PLRSSWVMTCAYAPSGNYVACGGLDNICS IYNLKTREGNVRVSRELAGHTGYLSCCRFL DDNQIVTSSGDTTCALWDIETGQQTTTFTGHTGDVMSLSLAPDTRLFVSGACDASAKLWDVREGMCRQTFTGHESD INAICFFPNGNAFATGSDDATCRLFDLRADQELMTYSHDNI ICGI TSVSFSKSGRLLLAGYDDFNCNVWDALKADR AGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLKIWN

In another embodiment, the antibody or active antibody fragment of the present invention promotes dissociation of Ga-GDP (or GDP-Ga, as used interchangeably herein) from the qbg dimer. Indeed, the binding of said antibody or active antibody fragment may have a similar or lower affinity for qbg as compared to the affinity of Ga-GDP for qbg, but dependent on its concentration, the conditions and the particular situation, the heterologous antibody or active antibody fragment is able to shift the association/dissociation equilibrium of the heterotrimer, which is normally regulated by activity of the GPCRs, to a status wherein the Ga-GDP (being converted into Ga-GTP thereby) and qbg dimer (bound to said antibody) is promoted or stimulated.

A specific embodiment relates to the antibody or active antibody fragment of the invention, with an affinity for qb that corresponds to a Kp that is at least 5nM and is maximally 50 nM. More specifically, said KD corresponds to an affinity similar or lower than the affinity of Ga to said binding site on qb, and corresponds to an affinity that is similar or higher than the affinity of regulatory effector proteins of qbg, as such that said antibody or active antibody fragment of the invention is capable of competing and outperforming these regulatory proteins, to affect the qbg signaling downstream of said effector proteins. In a specific embodiment, said KD is between about 5 nM and about 50 nM, or between about 10 nM and about 45 nM, or between about 15 nM and about 40 nM, or between about 20 nM and about 35 nM, or between about 25 nM and about 30 nM. In specific embodiments, said antibody or active antibody fragment has an affinity of binding the qbg dimer that is at least corresponding to a KD in the range of 10⁷ M, or at least 10⁸ M, or most preferable at least 10⁹ M.

The term "affinity", as used herein, generally refers to the degree to which an antibody or other binding protein (as defined further herein) binds to a target protein so as to shift the equilibrium of target protein and binding protein toward the presence of a complex formed by their binding. Thus, for example, where an antibody and an antigen are combined in relatively equal concentration, an antibody of high affinity will bind to the antigen so as to shift the equilibrium toward high concentration of the resulting complex. The dissociation constant KD is commonly used to describe the affinity between a ligand and a target protein, or an antibody and its antigen. Typically, the dissociation constant has a value that is lower than 10⁵ M. Preferably, the dissociation constant is lower than 10⁶ M, more preferably, lower than 10⁷ M. Most preferably, the dissociation constant is lower than 10⁸ M, or even lower than 10⁹ M. Other ways of describing the affinity between an antibody and its target are the association constant (Ka), the inhibition constant (Ki), or indirectly by evaluating the potency of ligands by measuring the half maximal inhibitory concentration (ICso) or half maximal effective concentration (ECso). It will be appreciated that within the scope of the present invention, the term“affinity” is used in the context of the antibody or active antibody fragment that binds a (conformational) epitope of the Qb and/or Qbg complex, more particularly the antibody or active antibody fragment is “functional” in binding its target via the CDR regions of its immunoglobulin (Ig) domain, even more preferably“functional” in inhibiting or blocking or reducing the activity of its target protein, the Qbg complex.

Accordingly, as used herein, the term“functional antibody” or“active antibody fragment” or“conformation- selective antibody” in the context of the present invention refers to an antibody or active antibody fragment that is functional in binding to its target protein, Qb, or the Qbg complex, optionally in a conformation- selective manner. The terms“specifically bind”,“selectively bind”,’’preferentially bind”, and grammatical equivalents thereof, are used interchangeably herein. The terms “conformational specific” or “conformational selective” are also used interchangeably herein.

In another embodiment, the antibody or active antibody fragment which specifically binds the Qbg complex is active in inhibiting Qbg signaling. More specifically, the antibody or active antibody fragment which specifically binds the Qbg complex is active in inhibiting Qbg signaling that does not affect Ga signaling. In one embodiment, the antibody or active antibody fragment specifically binding the Qbg complex and thereby actively inhibiting Qbg signaling, is without impact on the Gbg-mediated Ga signaling. The term “inhibiting” or“blocking” or“reducing”, as used herein interchangeably, refers to the fact that the antibody or active antibody fragment can inhibit the function and/or activity of its target protein, qbg complex. In case of qbg complex, this means that the downstream signaling activity is inhibited. Importantly, inhibition or decrease in qbg complex signaling may also be evaluated as an increase of another downstream parameter.“Signaling” as used here may mean to be involved in the transfer or the inhibition of the transfer of an activated receptor to a reporter gene or downstream effector gene or gene product. In that respect, a molecule that is not directly involved in signaling itself, but that, by binding on the receptor or by modulating downstream G protein activity can inhibit another molecule from binding and inducing the signaling pathway is also considered as a signaling molecule.“Inhibitory” can mean full inhibition (no downstream signaling activity is observable) or may mean partial inhibition. For instance, inhibition can mean 10% inhibition, 20% inhibition, 25% inhibition, 30% inhibition, 40% inhibition or more. Particularly, inhibition will be at least 50%, e.g. 50% inhibition, 60% inhibition, 70% inhibition, 75% inhibition, 80% inhibition, 90% inhibition, 95% inhibition or more. % inhibition typically will be evaluated against a suitable control (e.g. treatment with an irrelevant Nanobody, or a wild-type subject versus a diseased subject), as will be readily chosen by the skilled person.

More specifically, the qbg dimers are involved in multiple aspects of GPCR-mediated signaling and regulation. In addition to their role in downstream signaling, qbg subunits interact with GPCRs and Ga subunits and are critical for GPCR-dependent G protein activation. The diverse and expanding roles for Qbg in cell signaling are numerous and have been reviewed (Smrcka, 2008, Cell Mol Life Sci. 65(14): 2191-2214). Qbg was shown to activate a cardiac potassium channel normally activated by a muscarinic cholinergic receptor after stimulation by acetylcholine. Qbg is also the key activator of the pheromone response downstream from the G protein coupled pheromone receptor. Many Qbg effectors have been identified, including adenylyl cyclase (AC) isoforms, G protein-coupled receptor kinase 2 (GRK2), phospholipase C (PLC) _2 and _3 isoforms, inwardly rectifying potassium channels (GIRK), phosphoinositide 3-kinase _ (PI3K_), and A/-type calcium channels. The binding sites for said effector/binding proteins share a critical interaction interface on the top of the torus of qb created by the b propeller fold that binds to switch II helix of the Ga subunits, mentioned herein as the qbg hotspot. Effectors such as PLC_2, ACM, GRK2, and GIRK channels share a common binding surface on qbg but also reveals that Gbg-interacting proteins use unique combinations of residues within this common binding surface to mediate binding. So far, small molecules and peptides specifically interfering on a specific effector interaction or activity have been looked for³³. Many of the Qbg-ΐq^bΐ couplings are involved in diseases and disruption of these interactions has been shown to be of potential therapeutic benefit, in for preventing heart failure, arterial restenosis, hypertension, drug addiction, cancer metastasis, and prostate cancer. In many of these therapeutic proof of concept efforts, the GRK2ct (C terminus of GRK2) and the qbg binding peptide QEHA (sequence derived from ACM) were used, but also small molecules that bind to qbg (M1 19/gallein) were shown to be effective in animal models of inflammation, analgesia and heart failure. If multiple GPCRs are involved in the development of disease, targeting a single GPCR may not be effective; rather, inhibiting the therapeutically relevant signaling pathway(s) downstream of a group of receptors could achieve this goal. An example is chemokine receptors in rheumatoid arthritis, where common qbg signaling systems are downstream of multiple chemokine receptor subtypes. Inhibiting qbg signaling may be more efficacious than targeting a single GPCR. Although qbg binding compounds are somewhat selective for downstream signaling pathways, it is unlikely that compounds will be found that bind to qbg and only inhibit single effector because of the overlapping nature of the binding surface, which may limit to the specificity, but on the other hand, it could benefit to the efficacy.

In fact, the antibody or active antibody fragment of the present invention has been shown to bind different subtypes of qb, ΰb1-4, which are present in different cell types, and therefore, a number of target downstream of ΰb1-4 is within the scope of the antibody or active antibody fragment of the invention. Moreover, in a specific embodiment, said antibody or active antibody fragment of the invention is a nonnative binder that inhibits downstream qbg signaling in neuronal cells via blocking the activation of the effector G-protein-gated inward rectifier potassium (GIRK) channel. Another embodiment relates to the antibody or active antibody fragment of the invention, wherein the binding to qbg affects the cellular pathways that are often dysregulated in cancer. In one embodiment, the activation of phosphoinositide-3- kinase (PI3K)-protein kinase B / AKT signaling is blocked by binding of the antibody of the invention to the qbg complex in the present of activated apelin receptor. And another embodiment discloses an antibody or active antibody fragment of the invention, wherein said qbg binding upon apelin receptor activation blocks MAP/ERK pathways. Or in an alternative embodiment, both PI3K-AKT and MAP/ERK are inhibited when the antibody or active antibody fragment is present.

In fact, the invention discloses for the first time an antibody or active antibody fragment as a novel nonnatural binding tool to selectively target the hotspot of Qbg signaling. The advantages for using said antibodies or active antibody fragments of the present invention over peptides or small molecules are represented in the inherent properties of antibodies, binding with high affinity, thereby providing a tool that can be used to specifically target certain conformational epitopes in a selective manner. Moreover, the inherent properties of nanobodies, as small protein binders preferentially targeting conformational epitopes, is a further advantage to affect Qbg signaling in an elegant way as highly selective modulators or inhibitors The combination of targeting the‘hot spot’ area, which is in itself a‘multitarget’ or‘multibinding site’ via the use of a very specific and highly selective biological approach results in a tool that allows modulation of GPCR signaling, via Qbg signaling in a meticulous manner.

One embodiment relates to an antibody or active antibody fragment, which is an immunoglobulin single variable domain (ISVD), comprising the amino acid sequence that comprises 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4. An“immunoglobulin domain” of this invention also refers to“immunoglobulin single variable domains” (abbreviated as "ISVD"), equivalent to the term“single variable domains”, and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from“conventional” immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH- sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody^®, Nanobodies^® and Nanoclone^® are registered trademarks of Ablynx N.V. For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008/020079.“VHH domains”, also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al (1993) Nature 363: 446-448). The term“VHH domain” has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as“VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as“VL domains”). For a further description of VHHs and Nanobody , reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001 ), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591 , WO 99/37681 , WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301 , EP 1 134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551 , WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more“Hallmark residues” in one or more of the framework sequences. A further description of the Nanobody, including humanization and/or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or “Nanobody fusions”, multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody and their preparations can be found e.g. in WO 08/101985 and WO 08/142164. Nanobodies form the smallest antigen binding fragment that completely retains the binding affinity and specificity of a full-length antibody²¹. Nbs possess exceptionally long complementarity-determining region 3 (CDR3) loops and a convex paratope, which allow them to penetrate into hidden cavities of target antigens^{22 25}.

In a specific embodiment, said ISVD of the invention comprises a CDR3 with an amino acid sequence of SEQ ID NO:1 , or an amino acid sequence with at least 80 % identity thereof, or with maximally one amino acid different to SEQ ID NO:1. In one embodiment, said ISVD of the invention has CDR3 with an amino acid sequence of SEQ ID NO: 1.

>SEQ ID NO:1 : CDR3 amino acid sequence of ISVD family specific for Qbg complex binding

VGRSRGY Said CDR3 sequence represents an essential feature of a family of ISVDs, more particularly Nbs, specifically binding the Qbg dimer at the same binding site. A Nanobody family is defined herein as a group of Nanobody amino acid sequences with high similarity, or even identical, in the CDR3 sequence. By default, Nanobodies belong to the same family when binding to the same target epitope. Variations in a Nanobody family may be interesting if expression/stability/crystallization of a representative of that family is poor, small deviations like single amino acid mutations occurring within one family may improve these properties. One embodiment relates to the ISVDs of the invention, comprising SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a homologue with at least 80 % amino acid identity thereof, or a humanized variant thereof. Said amino acid sequences provide by SEQ ID NOs:2-4 represent 3 Nb members of one family as part of the invention. The Nb5 as disclosed in the invention comprises SEQ ID NO:2, and also called CA10957. In addition, Nb6 as disclosed in the invention comprises SEQ ID NO:3 and Nb7 comprises SEQ ID NO:4, also called CA10958, and CA10959, respectively, reveal an identical CDR3, as provided by SEQ ID NO:1 , and specifically bind Qbg dimer.

>SEQ ID NO:2: ISVD Nb5 amino acid sequence [CA10957] specific for Qbg complex binding

QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITRGGRTNYADSVKGRFTLSRDNAKN

TVYLQMNSLKPEDTAVYYCNVGRSRGYWGQGTQVTVSS

>SEQ ID NO:3: ISVD Nb6 amino acid sequence [CA10958] specific for Qbg complex binding

QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMNWYRQAPGKRRELVAAITNGGSTNYADSVKGRFTISRDNAKN

TVYLQMNSLKPEDTAVYYCNVGRSRGYWGQGTQVTVSS

>SEQ ID NO:4: ISVD Nb7 amino acid sequence [CA10959] specific for Qbg complex binding

QVQLVESGGGLVQAGESLRLSCAASGNIFSINATNWYRQAPGKQRELVAGITTRGSTNYADSVKGRFTISRDNAKN

TVYLQMNSLKPEDTAVYYCNVGRSRGYWGQGTQVTVSS

Furthermore, the role of framework regions in specific binding to the target is rather limited, and allows to include variation in the FR sequences to obtain a similar efficacy of said ISVDs (see for instance De Groeve et al., 2010, J. Nuclear Medicine. 51 (5):p.782; Saerens et al., 2005, J. Mol. Biol. 352, 597-607). In a specific embodiment, said ISVD comprises a homologue of at least 80 % identity to the SEQ ID NOs: 2, 3 or 4, or at least 85 % identity, at least 90 % identity, at least 95 % identity, or at least 99 % identity. More specifically, the homologues their differences in amino acid sequence as compared to the ISVD of SEQ ID NOs: 2-4 will be found in the Framework regions, for the reason provided above, and may be limited to conserved amino acid substitutions.

In a specific embodiment, said ISVD of the invention comprises Nb5, represented by SEQ ID NO:2. Alternatively, said ISVD of the invention comprises SEQ ID NO:5, or a homologue with at least 80 % identity thereof, or a humanized variant of SEQ ID NO:5, or of said homologue.

Immunoglobulin single variable domains such as Nanobody (including VHH domains) can be subjected to humanization, i.e. increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as Nanobody (including VHH domains) may be immunoglobulin single variable domains that are as generally defined for in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody (including VHH domains) may be partially humanized or fully humanized.

The antibody or active antibody fragment of the present invention may further comprise in some embodiments a detection agent, such as a tag or a label. For instance, the Nbs as exemplified were also tagged, by the 6-His-EPEA double tag (as presented in SEQ ID NO: 17; for EPEA tag: see also WO201 1/147890A1 ). Such a tag allows affinity purification and detection of the antibody or active antibody fragments of the invention.

Some embodiments comprise the antibody or active antibody fragment, further comprising a label or tag, or more specifically, the antibody or active antibody fragment is labelled with a detectable marker. The term detectable label or tag, as used herein, refers to detectable labels or tags allowing the detection and/or quantification of the antibody or active antibody fragments as described herein, and is meant to include any labels/tags known in the art for these purposes. Particularly preferred, but not limiting, are affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S- transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin and cyanine); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs); phosphorescent labels; a metal chelator; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease or glucose oxidase); radioisotopes. Also included are combinations of any of the foregoing labels or tags. Technologies for generating labelled polypeptides and proteins are well known in the art. An antibody or active antibody fragment of the invention, coupled to, or further comprising a label or tag allows for instance immune-based detection of said antibody or active antibody fragment. Immune-based detection is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as described above. See, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149 and 4,366,241. In the case where multiple antibodies are reacted with a single array, each antibody can be labelled with a distinct label or tag for simultaneous detection. Yet another embodiment may comprise the introduction of one or more detectable labels or other signalgenerating groups or moieties, or tags, depending on the intended use of the labelled or tagged antibody or active antibody fragment of the present invention. Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labelled antibodies or active antibody fragments of the invention may 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 in vivo imaging purposes, depending on the choice of the specific label.

In other embodiments, the antibody or active antibody fragment may further comprise a functional moiety, such as for instance a Blood-brain-barrier crossing moiety. In other embodiments the antibody or active antibody fragment may further comprise a cell penetrant carrier, such as examples. In other embodiments, said antibody or active antibody fragment may further comprise a combination of a tag or label, a functional moiety, and/or a cell penetrant carrier.

Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the antibody or active antibody fragment of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, an antibody or active antibody fragment of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated antibody may be used as a reporter, for example in a system where a detectable signal- producing agent is conjugated to avidin or streptavidin. In another embodiment, the antibody or active antibody fragment as used in the present invention is coupled to or fused to a functional moiety, in particular a therapeutically active agent, either directly or through a linker. As used herein, a “therapeutically active agent” means any molecule that has or may have a therapeutic effect (i.e. curative or stabilizing effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the art 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, 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 (for example covalently) to the antibody or active antibody fragment, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly( ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly( ethyleneglycol) or mPEG). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a immunoglobulin single variable domain of the invention, a immunoglobulin single variable domain of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of an antibody or active antibody fragment of the invention, all using techniques of protein engineering known per se to the skilled person. Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the antibody or active antibody fragment. Another technique for increasing the half-life of a binding domain may comprise the engineering into bifunctional or bispecific domains (for example, one antibody or active antibody fragment against the target qbg complex and one against a serum protein such as albumin) or into fusions of antibody fragments, in particular immunoglobulin single variable domains, with peptides (for example, a peptide against a serum protein such as albumin).

Various tactics to assist drugs to cross the blood brain barrier (BBB) including osmotic disruption of the BBB and chemical modification of prodrugs have been disclosed. Additionally, nanoparticles (NPs)- mediated drug delivery is emerging as an effective and non-invasive system to treat cerebral disease (Zhou et al., 2018, J. Controlled Release, 270: 290-303). Biologic drugs can be re-engineered as brain- penetrating neuropharmaceuticals using BBB molecular Trojan horse technology. Certain peptidomimetic monoclonal antibodies that target endogenous receptors on the BBB, such as the insulin or transferrin receptor, enable the re-engineering of biologic drugs that cross the BBB (for a review see Pardridge 2015, Clinic. Pharmac. & Therapuetics. 97 (4): 347).

Furthermore, the antibody or active antibody fragment of the invention may further comprise a cell penetrant carrier, which is capable of entering a cell through a sequence which mediates cell penetration (or cell translocation). So the antibody or active antibody fragment further comprising a cell penetrant carrier involves the recombinant or synthetic attachment of a cell penetration sequence or molecule. Thus, the molecule (or polypeptide) may be further fused or chemically coupled to a sequence facilitating transduction of the fusion or chemical coupled proteins into prokaryotic or eukaryotic cells. Sequences facilitating protein transduction are known to the person skilled in the art and include, but are not limited to Protein Transduction Domains. It has been shown that a series of small protein domains, termed protein transduction domains (PTDs), cross biological membranes efficiently and independently of transporters or specific receptors, and promote the delivery of peptides and proteins into cells. Preferably, said sequence is selected from the group comprising TAT protein from human immunodeficiency virus (HIV- 1 ), a polyarginine sequence, penetratin and a short amphipathic peptide carrier, Pep-1. Still other commonly used cell-permeable peptides (both natural and artificial peptides) are disclosed in Joliot A. and Prochiantz A. (2004) Nature Cell Biol. 6 (3) 189-193. Another aspect of the invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or active antibody fragment of the invention. One embodiment discloses an expression cassette comprising said nucleic acid molecule. More specific embodiments disclose the expression cassette wherein elements for cell- or tissue-specific expression are present. Further embodiments relate to a vector comprising said expression cassette or said nucleic acid molecule. More particular, said vector may be a viral vector, even more particular a lentiviral or AAV vector.

“Nucleotide sequence”,“DNA sequence” or“nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, the (reverse) complement DNA, and RNA. It also includes known types of modifications, for example, methylation,“caps” substitution of one or more of the naturally occurring nucleotides with an analog. By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. “Coding sequence” is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5’ to 3’ in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. Such cassettes can be constructed into a "vector”. The term“vector”, "vector construct," "expression vector," or "gene transfer vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, and includes any vector known to the skilled person, including any suitable type, but not limited to, for instance, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. The construction of expression vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clif ton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

Furthermore, an alternative embodiment relates to the use of said nucleic acid molecule, expression cassette, or vector of the invention encoding said antibody or active antibody fragment, for production of an intrabody. An intracellular antibody or“intrabody” is an antibody or a fragment of an antibody that is heterologously expressed within a designated intracellular compartment, a process which is made possible through the in frame incorporation of intracellular trafficking signals. Intrabodies exert their functions upon exquisitely specific interaction with target antigens. This results in interruption or modification of the biological functions of the target protein. An intrabody can be expressed in any shape or form such as an intact IgG molecule or a Fab fragment. More frequently, intrabodies are used in genetically engineered antibody fragment format and structures of scFv intrabodies, single domain intrabodies, or bispecific tetravalent intradiabodies. For a review see Zhu, and Marasco, 2008 (Therapeutic Antibodies. Handbook of Experimental Pharmacology 181. _c Springer-Verlag Berlin Heidelberg).

The antibody or active antibody fragment of the invention, possibly encoded by a nucleic acid molecule or expression cassette of the invention present on a vector of the invention, resulting in an intrabody upon expression within a suitable host system, could also serve as a tool to further investigate GPCR or G protein signaling, as well as a therapeutic, when an applicable form of gene delivery is identified. A skilled person is aware about the currently applied methodologies of administration and delivery (also see Zhu and Marasco 2008).

In another embodiment, the nucleic acid of the invention, or the expression cassette or vector may also be included in a kit, for instance to apply as a tool in G protein signaling studies, or for G protein biochemistry such as purification.

In one aspect of the invention, a host cell is disclosed, comprising the antibody or active antibody fragment of the invention. The host cell may therefore comprise the nucleic acid molecule of the invention, or the expression cassette, or the vector of the invention. Host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of DNA, such as nucleic acid molecules, expression cassettes or expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2* Ed. (R.l. Freshney. 1987. Liss, Inc. New York, N.Y.). The host cell may also be a recombinant host cell, which involves a cell which has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction. A DNA construct capable of enabling the expression of the antibody or active antibody fragment of the invention can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989), Maniatis et al. (1982), Wu (ed.) (1993) and Ausubel et al. (1992).

Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g. review Jarvis, Virology Volume 310, Issue 1 , 25 May 2003, Pages 1-7). Plant cells may for instance but non-limiting include tobacco cells, tomato cells, maize cells, algae cells, among others. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals.

In one embodiment said host cell is a transgenic mouse. One experiment in transgenic mice is done for instance to demonstrate the effect of the antibody or active antibody fragment of the invention, such as Nb5 exemplified below, on the GPCR signaling in Rod photoreceptor cells of the retina. Electroretinography and optical coherence tomography are performed before and after light-induced retinal damage in the transgenic mice to observe the rescuing effect of the Nb5. So the antibody or active antibody fragment of the invention, herein exemplified with Nb5, is therefore considered as an instrumental tool for studying the cellular localization and visualization of GPCR signaling in photoreceptor cells. Interestingly, the use of said antibody or active antibody fragment of the invention, such as Nb5, for visualizing GPCR signaling events is not limiting to the retina, but can be of use as tool in various cell types. Preferably, cells are eukaryotic cells, for example cultured cell lines, for example mammalian cell lines, preferably human cell lines, that endogenously or recombinantly express a GPCR and/or G protein of interest. The nature of the cells used will typically depend on the ease and cost of producing the native protein(s), the desired cellular properties, the origin of the target protein, the intended application, or any combination thereof. Animal or mammalian host cells suitable for harboring, expressing, and producing antibody or active antibody fragment of the invention include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61 ), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; and Kolkekar et al., 1997, Biochemistry, 36: 10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR- CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721 ,121 ); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651 ); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59, or GnTI KO HEK293S cells, Reeves et al. 2002, PNAS, 99: 13419); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1 , ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587; VERO, ATCC CCL-81 ); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251 ); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51 ); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4 cells. According to a particular embodiment, the cells are mammalian cells selected from Hek293 cells or COS cells.

Another aspect of the invention, relates to a method for identifying or producing a compound that modulates or alters G protein signaling, more specifically qbg signaling, comprising the steps of: a) providing a host cell of the present invention, and transfecting said cell with the GPCR of interest (if not yet present in said host cell); b) adding a test compound to said (transfected) host cell of the invention; and c) evaluate the effect of said test compound addition on the G protein signaling, more specifically on qbg and/ or Ga signaling in said cell, and compare to a host cell without the test compound.

Optionally, said evaluation may result in a selection for host cells of interest wherein qbg and/ or Ga signaling is altered as compared to the control. In a particular embodiment, the GPCR of interest may already be present in the host cell of step a), which allow to use said host cell without a need to additionally transfect with the GPCR of interest.

The presence of the ΰbg-erb ίίo antibody or active antibody fragment of the invention in the host cell of the method provides a tool useful in distinguishing the effect on qbg signaling and/or Ga signaling. In particular, several downstream signaling events can easily be evaluated by including a reporter or assay component in the method of interest. For instance, non-limiting examples of detection of effects of downstream Ga signaling, include the detection of increase of intracellular Ca²⁺ or G Y-mediated inositol monophosphate (IP1 ) for Gaq-linked receptors or the increase or decrease of cAMP for Gas- and Gai- coupled receptors, respectively, or are based on the detection of b-arrestin recruitment.

As used herein, the term“evaluating” includes“determining”,’’measuring”,’’assessing”,“monitoring” and “assaying” and are used interchangeably and include both quantitative and qualitative determinations. The term“compound” or“test compound” or“candidate compound” or“drug candidate compound” as used herein describes any molecule, either naturally occurring or synthetic that is tested in an assay, such as a screening assay or drug discovery assay, or specifically in the method for identifying a compound capable of modulating G protein signaling. As such, these compounds comprise organic and inorganic compounds. The compounds may be“small molecules”, which refers to a low molecular weight (e.g., < 900 Da or < 500 Da) organic compound. The compounds also include polynucleotides, lipids or hormone analogs that are characterized by low molecular weights. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) 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, test compound libraries may 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 be found further in the specification.

Screening assays for drug discovery can be solid phase or solution phase assays, e.g. a binding assay, such as radioligand binding assays. It will be appreciated that in some instances high throughput screening of test compounds is preferred and that the 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 inverse agonists and/or modulators form part of the invention. For high- throughput purposes, compound libraries may be used such as allosteric compound libraries, peptide libraries, antibody libraries, fragment-based libraries, synthetic compound libraries, natural compound libraries, phage-display libraries and the like. Methodologies for preparing and screening such libraries are known in the art. In one 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 display a desired characteristic activity. A“compound library” is a collection of stored chemicals usually used ultimately in high-throughput screening A “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical“building blocks” such as reagents. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics. Thus, in one further embodiment, the screening methods as described herein above further comprises modifying a test compound which has been shown to modulate G protein signaling, and determining whether the modified test compound affects Qbg or Ga signaling when residing in the host cell.

Another aspect of the invention relates to a solid substance, or resin or solid surface, or solid supports as used interchangeably herein, said solid substance comprising the antibody or active antibody fragment of the present invention.

In cases where an assessment of the binding to the qbg complex will be preferred, or applied, this will be facilitated by immobilization of the antibody or active antibody fragment of the present invention onto a suitable solid surface or support that can be arrayed, used in affinity chromatography, among other applications. Non-limiting examples of suitable solid supports include beads, columns, slides, chips or plates. More specifically, the solid supports may be particulate (e. g. beads or granules, generally used in affinity columns) or in sheet form (e. g. membranes or filters, glass or plastic slides, microtitre assay plates, dipstick, capillary fill devices or such like) which can be flat, pleated, or hollow fibres 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 (32-63 urn or 35-70 urn) supplied by Biotage (a division of Dyax Corp.), agarose or polyacrylamide supports, for example the Sepharose range of products supplied by Amersham Pharmacia Biotech, or the Affi-Gel supports supplied 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 utilised include; dextran, collagen, polystyrene, methacrylate, calcium alginate, controlled pore glass, aluminium, titanium and porous ceramics. Alternatively, the solid surface may comprise part of a mass dependent sensor, for example, a surface plasmon resonance detector. Further examples of commercially available supports are discussed in, for example, Protein Immobilisation, R.F. Taylor ed., Marcel Dekker, Inc., New York, (1991 ). Immobilization may be either non-covalent or covalent. In particular, non-covalent immobilization or adsorption on a solid surface of the antibody or active antibody fragment of the invention may occur via a surface coating with any of an antibody, or streptavidin or avidin, or a metal ion, recognizing a molecular tag attached to the antibody or active antibody fragment of the invention, according to standard techniques known by the skilled person (e.g. biotin tag, Histidine tag, etc.). Alternatively, the antibody or active antibody fragment of the invention may be attached to a solid surface by covalent cross-linking using conventional coupling chemistries. A solid surface may naturally comprise cross-linkable residues suitable for covalent attachment or it may be coated or derivatised to introduce suitable cross-linkable groups according to methods well known in the art. In one particular embodiment, sufficient functionality of the immobilised antibody or active antibody fragment is retained following direct covalent coupling to the desired matrix via a reactive moiety that does not contain a chemical spacer arm. Further examples and more detailed information on immobilization methods of antibody (fragments) on solid supports are discussed in Jung et al. (2008, Analyst. 133(6):697-701 ). Notably, the mutation of a particular amino acid (in a protein with known or inferred 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 reengineer a specific protein to alter the distribution of surface available amino acids involved in the chemical coupling, in effect controlling the orientation of the coupled protein. In case of an antibody or an antibody fragment, such as a nanobody, introduction of mutations in the framework region is preferred, minimising disruption to the antigen-binding activity of the antibody (fragment). Conveniently, the immobilised proteins may be used in im mu noadsorption processes such as immunoassays, for example ELISA, or immunoaffinity purification processes by contacting the immobilised antibody or active antibody fragments of the present invention with a test sample (i.e. comprising the test compound, amongst other) according to standard methods conventional in the art. Alternatively, and particularly for high-throughput purposes, the immobilized antibody or active antibody fragments of the present invention can be arrayed or otherwise multiplexed. Preferably, the immobilised antibody or active antibody fragments of the present invention are used for the screening and selection of compounds that mimic the Qbg complex.

The antibody or active antibody fragment of the present invention, as well as the solid substance of the present invention can be used as universal tools for the structural and functional characterization of G protein complexes, more particularly Qbg dimers, activated by G-protein coupled receptors when the latter are bound to various natural or synthetic ligands, for investigating the downstream dynamic features of G protein activation, as well as for screening and drug discovery efforts that make use of qbg complexes.

In a specific embodiment, said antibody or active antibody fragment of the invention, such as the exemplified Nb5, can be used for selective purification of qbg subtypes form virtually any tissue, such as bovine retinas and mouse brain, but basically any cell type or tissue or cell culture is usable. The antibody or active antibody fragment of the invention, such as the exemplified Nb5, may be cross-linked with 6 % cross-linked agarose for instance, to allow G protein purification. Said solid surface or cross-linked antibody or active antibody fragment of the invention may also be included in a kit for affinity purification purposes.

In various alternative embodiments, said antibody or active antibody fragment, the solid substance of the present invention are used for affinity chromatography, affinity purification, immunoprecipitation, in vivo- imaging, protein detection, immunochemistry, surface-display, FRET-type applications, or functional and/or structural analysis.

Moreover, in particular embodiments, said antibody or active antibody fragment of the present invention, as well as the nucleic acid molecule, expression cassette, vector, or solid substance of the present invention can be applied or used as a tool to differentiate Ga from qbg signaling. In fact, the use will allow to affect qbg signaling, whereas no effect on Ga is expected from the presence of the antibody or active antibody fragment of the invention. This tool provides a unique biological feature to measure the specificity of certain GPCR-mediated activation means.

In another aspect of the invention, said antibody or active antibody fragment of the present invention, as well as the nucleic acid molecule, expression cassette, or vector of the present invention is useful as a medicament. The term“medicament”, as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms“disease” or“disorder” refer to any pathological state, in particular to the diseases or disorders as defined herein. In a specific embodiment, the antibody or active antibody fragment of the present invention, as well as the nucleic acid molecule, expression cassette, or vector are used in treatment of a disease selected from the group consisting of cancer, metastasis, neurological and neuromuscular diseases. The advantage of the antibody or active antibody fragment of the invention on the one hand provides a very broad application in many cell types and tissues, since the antibody or active antibody fragments specifically bind the Qb subtypes b1 , 2, 3 and 4, allowing a broad coverage of multiple effects and in multiple disease areas. On the other hand, some of the major questions still concerns how signaling specificity is maintained with such a promiscuous signaling protein and what its molecular significance is in view of the very large isoform diversity of these Qbg combinations. Given the biological potential of these proteins as therapeutic targets, answering these questions could contribute significantly to development of novel pharmacologic approaches to therapeutics for a number of important diseases. In the present invention, an antibody or active antibody fragment specifically or selectively inhibiting a number of downstream signaling routes reveals a broad potential in therapeutic value, but also raises the above question on how to attribute specificity to avoid off-target effects.

Another embodiment relates to a pharmaceutical composition including the antibody or active antibody fragment of the invention, or nucleic acid molecule, expression cassette, or vector of the invention. The term“pharmaceutical compositions” relates to one or more compounds of the invention, in particular, the antibody or active antibody fragment inhibiting the Qbg complex, or the DNA encoding said antibody or antibody fragment, and a pharmaceutically acceptable carrier or diluent. Said pharmaceutical composition being used as a medicament or diagnostic. These pharmaceutical compositions can be utilized to achieve the desired pharmacological effect by administration to the patient. The present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound, or derivative thereof, of the present invention, including their use as a medicament or diagnostic. A pharmaceutically effective amount of compound is preferably that amount which produces a result or exerts an influence on the particular condition being treated. In general, "therapeutically effective amount", "therapeutically effective dose" and "effective amount" means the amount needed to achieve the desired result or results. One of ordinary skill in the art will recognize that the potency and, therefore, an "effective amount" can vary depending on the identity and structure of the compound of the invention. One skilled in the art can readily assess the potency of the compound. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Suitable carriers or adjuvantia 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. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al. ("Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-31 1 ), Strickley, R.G ("Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1 " PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324- 349), and Nema, S. et al. ("Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51 (4), 166-171 ). The term“excipient” is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent", in particular a "pharmaceutically acceptable vehicle", includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles.

In another embodiment of the invention, said antibody or active antibody fragment of the present invention, as well as the nucleic acid molecule, expression cassette, vector, or solid substance of the present invention is useful as a diagnostic.

In a particular embodiment, kits are provided which contain means to detect Qbg protein, including the antibody or active antibody fragment of the invention, allowing to detect or modulate Qbg signaling in a system, which may be an in vitro or in vivo system. It is envisaged that these kits are provided for a particular purpose, such as for modulating Qbg, or for in vivo imaging, or for diagnosis an altered GPCR response in a subject. In another embodiment, said kit is provided which contains means including a nucleic acid molecule, an expression cassette or a vector or solid substance of the invention.

The means further provided by the kit will depend on the methodology used in the application, and on the purpose of the kit. For instance, detection of a labelled antibody or active antibody fragment, or nucleic acid molecules, can be on nucleic acid or protein level. For protein-based detection, the kits typically will contain labelled or coupled antibodies. Likewise, for detection at the nucleic acid level, the kits may contain labels for nucleic acids such as primers or probes. Further control antibodies or nucleic acids may also be provided in the kit. A standard, for reference or comparison, a GPCR signaling component, a reporter gene or protein or other means for using the kit may also be included. Of course, the kit may further comprise pharmaceutically acceptable excipients, buffers, an instruction manual and so on.

Finally, the invention provides a major advantage over the known small molecule and peptide inhibitor compounds so far identified, due to their high affinity and selectivity. For instance, the M1 19 class of small- molecule inhibitors, which includes M1 19, gallein and M1 19K, are potent qbg antagonists. They remain the most extensively validated inhibitors of qbg signaling. Studies to date indicate that these molecules bind to a qbg hot spot on the top surface of the b subunit with apparent affinities in the high nM to low mM range¹⁰⁴. The M1 19 class of inhibitors displays a limited number of chemical moieties that interact with the Qbg dimer. Therefore, structure activity relationship studies to achieve Qb selectivity would have limited practicality. Conventional antibodies already provide an improvement in selectivity, and on top of that, antibody fragments, such as immunoglobulin single variable domain antibodies or Nanobodies are capable of reaching binding pockets not reachable by larger proteins. So, antibodies or active antibodies fragment of the invention, such as the exemplified Nb5, bind Qbg with a low nM affinity and show a larger area of interaction with the Qbg dimer (>1030 A²). These features indicate that antibodies or active antibody fragment of the invention, such as Nb5, might serve as a better template for structure activity relationship studies to achieve Qb selectivity. The C-terminal domain of GRK2 ^ARK-ct), PDC and several affinity matured peptides, including the SIRK peptide family, comprise an alternative class of potent Qbg inhibitors that are mechanistically similar to Nb5¹⁰⁵-¹⁰⁷. While bARK-ct and PDC have a large area of interaction with the Qbg dimer (>1000 A²), no atomic level information is available for SIRK or other peptides except that they bind to the same Qbg hot spot with high nM affinities³⁹⁴¹ and selectively inhibit the binding of Qbg effector molecules. Several cell-permeating versions of SIRK peptides have been shown to effectively modulate ERK1/2 and MAPK signaling in arterial smooth muscle cells³⁸. Treatment strategies involving antibodies or active antibody fragment of the invention, such as the exemplified Nb5, bARK-ct, PDC and members of the SIRK peptide family would all require gene transduction methods, however this may be worthy of significant effort given their potential to treat various Gbg-related disorders^107-108. Furthermore, while most of the small-molecule and peptide inhibitors typically have short half-life after administration, antibodies, as well as active antibody fragments, such as Nanobodies may be engineered to have a broad range of half-life varying from 30 min to 3 weeks¹⁰⁹’¹¹⁰, increasing their therapeutic applications from acute to chronic indications.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for engineered cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

EXAMPLES

Example 1. Generation of Nanobodies directed towards ΰb1g1.

A total of 17 different Nanobody clones were identified after ELISA selection wherein the wells were coated with purified bovine Qb1 y1 dimer. Nbs were divided into 14 families based on their amino acid sequence. All Nbs were produced as soluble His-tagged protein products in the E. coli periplasmic region. Initial binding analyses performed with an immobilized-Ni2+ affinity chromatography pull down assay identified 3 Nbs (SEQ ID NOs: 2-4) that bound the Qb1g1 dimer. Interestingly, all 3 qbΐgΐ-roeίΐίnb Nbs belonged to the same Nanobody family with an identical complementarity determining region 3 (CDR3) (SEQ ID NO: 1 ) and displayed similar biochemical properties (Figure 10). The Nanobody with highest expression levels, Nb5 (SEQ ID NO: 2) , was used for further characterization. Additionally, an irrelevant Nanobody, Nb17 (SEQ ID NO: 5) was chosen as a negative control due to its non-reactivity with bovine rod outer segments (ROS) proteins.

Example 2. Nb5 shifts the equilibrium of heterotrimeric G_t towards dissociated Ga_t and Nb5-bound Gpiy1 subunits.

The effect of Nb5 on the heterotrimeric state equilibrium of Gt was determined by assessing its ability to bind and pull out the b1g1 subunits of Gt (ΰb1g1 ) from no-salt extracts of ROS. No-salt extracts were chosen over purified Gt to ensure its heterotrimeric configuration as the purification procedure could cause partial dissociation of Gt subunits. As expected, complete Gt heterotrimer dissociation was observed in the presence of light-activated rhodopsin (Rh*) and GTP. As a result, Nb5 bound to the released ΰb1g1 dimer and the Gb1g1-Nb5 complex was obtained in the eluate upon immobilized-Ni²⁺ affinity chromatography purification (Fig. 1a Jane 1 and Fig. 1 b). In contrast, the displaced alpha subunit of Gt (Gat) was found in the flowthrough (Fig. 1a, lane 2). Interestingly, similar results were obtained in the absence of either Rh* or Rh* and GTP during the purification (Fig. 1a, lanes 3-6). Moreover, an irrelevant Nanobody, Nb17 failed to cause dissociation of the Gt heterotrimer (Fig. 1a, compare lanes marked with asterisks). Additionally, no non-specific interactions were seen between ΰb1 g1 and the Ni²⁺-NTA purification resin (Fig. 1a, lanes 9 and 10). These experiments indicate an Nb5-mediated shift in the equilibrium of heterotrimeric Gt in solution towards its dissociated subunits.

Next, the effect of Nb5 on the heterotrimeric state of membrane-bound Gt was investigated. Here, high- salt extracts from dark-adapted ROS were used that contain an excess of free ΰb1g1 (Fig. 1c, lane 5). As expected, light activation of ROS resulted in dissociation of heterotrimeric Gt and release of Gat into the high-salt extracts (Fig. 1c, lane 1 ). When ROS were washed with high-salt buffer containing a 2 molar excess of Nb5 with or without GTP, a similar release of Gat was observed. In contrast, high-salt extracts of ROS treated with Nb17 had no effect on either the heterotrimeric state of Gt or the release of Gat from ROS membranes (Fig. 1 c, compare lanes marked with asterisks). The effect of Nb5 on the Gt activation ability of Rh* was evaluated by monitoring the increase of intrinsic tryptophan fluorescence in Gat in the presence of Rh* at pH 7.0. Assay conditions were chosen such that the Gt activation rate was the same as that determined by GTPys-induced complex dissociation⁴⁶. A typical elevation of Gat intrinsic fluorescence was noted upon GTPys-induced complex dissociation with Rh* (Fig. 1d). Interestingly, a 5 min pretreatment of heterotrimeric Gt with a 2-fold molar excess of Nb5 resulted in a significant decrease in the Gt activation rate (Fig. 1d,e). In contrast, no significant effect was observed upon pre-treatment with Nb17 (Fig. 1d,e). This indicates that Nb5 alone can trap ΰb1g1 after its spontaneous dissociation from Gat, and thereby reduces the effective heterotrimeric population of Gt during Gt activation assays. Additionally, the observed changes in Gt activation kinetics with Nb5 are consistent with single turnover experiments of Rh* with different concentrations of heterotrimeric Gt, that demonstrate a decrease in Gt activation rates with decreasing Gt concentrations (Fig. 1f). Overall, these results indicate that Nb5 has a high affinity towards the Qb1g1 dinner which shifts the dynamic equilibrium of heterotrimeric Gt towards dissociated Gat and Nb5-bound ΰb1g1 complex in both solution and membranes.

Example 3. Nb5 competes with Qbg regulatory proteins for Qbg binding.

To examine the kinetics underlying the assembly of the Qb1 y1-Nb5 complex, surface plasmon resonance (SPR) was employed with varied concentrations of native bovine qbΐ gΐ . The binding kinetics analysis revealed a rapid on-rate constant (Kon = 4.4 x 105 M-1 s-1 ) and a slow off-rate constant (Koff = 1 .9 x 10- 2 s¹) with a KD of 43 nM (Fig. 2a). Analysis of the protein-protein interactions gave a similar KD of 53 nM (Fig. 2b). The low-nanomolar affinity of Nb5 for Qb1g1 means a possible competition between Nb5 and other Qb1g1 regulatory proteins that bind Qb1g1 with similar affinities⁴⁷·⁴⁸. Differential hydrogen/deuterium exchange (HDX) was used to investigate the binding dynamics between Qb1g1 and Nb5. Full HDX profiles of qbΐ gΐ alone were compared to those of the 6b1g1-I^5 complex (Fig. 2c, d). Binding of Nb5 to the qbΐgΐ dimer induced a statistically significant (P < 0.01 ) increase in protection from solvent exchange in the regions of residues 80-99 and 1 1 1-1 18 of the Qb1 subunit versus Nib qbΐgΐ dimer alone (Table 1 ). In addition, regions with a statistically significant decrease in solvent protection were also observed (Fig. 2e, left). The differential HDX data indicate that the binding interface between Nb5 and the Qb1g1 dimer or epitope involves at a minimum, residues 80-99 and 1 1 1-1 18 of Qb1 (Fig. 2e, left). Interestingly, the binding site of Nb5 on the Qb1g1 dimer implied by the HDX data closely resembles that of Gat with the qbΐgΐ dimer (Fig. 2e, right). This suggests a possible binding interface overlap between Nb5 and Gat for the qbΐgΐ dimer. To further investigate the binding between Qb1g1 and Nb5, the complex between Qb1g1 and Nb5 was crystallized. The crystal structure of the 6b1g1-I^5 complex refined at 2.34 A bore a strong resemblance to a heterotrimeric G protein structure. Two molecules of 6b1g1-I^5 complex formed an asymmetric unit (Fig. 3a) wherein most of the crystallographic contacts were mediated by Nb5 (Fig. 7a). This agrees with a role of Nanobodies as crystallization chaperones in the structural determination of challenging target proteins^49-52. An intriguing feature of the Gb1g1-Nb5 complex is insertion of the CDR3 loop of Nb5 into the qbI-rGorbϋbG, which occupies most of the binding interface (Fig. 3b). This finding is consistent with the nanomolar affinity observed in the SPR kinetics profiling of the Gb1g1-Nb5 complex (Fig.2a,b). Interestingly, Nb5 occupies the same hot-spot region on the Qb1g1 dimer that is shared by other qbg regulatory proteins, including phosducin (PDC) and G Protein-Coupled Receptor Kinase 2 (GRK2) (Fig. 3a,b,c). Also, insertion of the Nb5 CDR3 loop into the qbI-rGorbϋbG (interface area of 1030 A2) is reminiscent of the interaction between the C-terminal loop of GRK2 and Qb1g2 dimer (interface area of 1080 A2; PDB accession: 10MW 53). Notably, Arg-101 in the Nb5 CDR3 loop serves as a key that locks the qbI-rGorbIIbG. A similar interaction mechanism is mediated by Lys-663 of GRK2 in the Gb1g2-GRK2 complex (Fig. 7b, c). Complexation significance analysis 54 that indicates the significance of a protein assembly formation showed a score of 0.27 for the Gb1g1-Nb5 complex vs 0.10 for the 6b1g2- GRK2 complex. The shape complementarity (Sc) index 55 analysis of the binding interface formed by Nb5 and GRK2 with the qbg dimer displayed Sc scores of 0.80 and 0.57, respectively (1.0 is a perfect match). Although these results indicate that both Nb5 and GRK2 have a similar mode of binding with the ΰbg dimer, Nb5 is likely to have a structural advantage over GRK2. Similarly, Nb5 had a slight structural advantage over PDC for Qb1g1 binding (Sc index = 0.73). Interestingly, Arg-101 in the Nb5 CDR3 loop also forms an intricate hydrogen-bonding network with water molecules that extends throughout the Qb1- propeller cavity (Fig.7b). Consistent with the differential HDX analyses, the crystal structure of the Qbΐgΐ- Nb5 complex revealed a significant overlap between the binding interfaces formed by Nb5 and Gat with qbΐgΐ (Fig. 3b,c,d,e). Further, Sc analyses of the interface formed by Gat with Qb1g1 displayed a Sc index of 0.76 and an interface area of 1080 A2 that are comparable to the 6b1g1-I^5 complex. Overall, these results suggest a structural advantage of Nb5 over most qbg regulatory proteins except Ga, for qbg binding. Furthermore, a higher affinity of Ga (apparent KD = ~1 nM 56) for the qbg dimer as compared to Nb5 (KD = 43 nM) supports the inability of Nb5 to affect Ga signaling.

Table 1 : Sequences of Qb1g1 peptide fragments showing normalized deuterium uptake for the Qb1g1 complex alone and the Qb1g1 complex bound to Nb5.

LAGHTGYLSC [139:148) 10.33±0.56 NA

511.51 +2 9 7.2 11.28 N.S.

6.79+1.91 6.61+3.85 iWSSGDTTCAL [157:168] 59.40±7.6

1168.60 +1 11 8.8 13.85 53.15±2.78 6 N.S. WDiETGQQTTTF [169:1801 714.11 *2 11 8.8 11.40 5.5340.73 7.74+4.96 N.S.

714.67^¨2

11.74

WDIETGQQTTTFTGHTGDVMSL 12.43 23,6641.4

1213.40 +2 21 16.8 22.76+0.52 N.S.

[169:1901 -14.4 0

LSLAPDTRl [190:198] 11.82

29,5642.6

985.67 +1 7 5.6 29.17+1.30 N.S.

0

11.97

FVSGACDASAKL [199:2101 13.73

584.75 +2 „ 21.17+2.3

8.8 20.05+1.07 N.S.

1169.72 +1 ' 4

16,21

WDVREGM [211:217] 12.12

446.72 +2_f, 51.11+4.3

4.8 50.68+4.49 N.S.

892.60 *1 °

12.55 8

CRQTFTGHESDINA [218:231] 16.90+1,2

789.90 *2 13 10.4 11.14 19.17*1.68 N.S.

1

ICFFPNGNA [232:240] 9.87- 41.19+0.1

982.58 *1 7 5.6 40.88+2.74 N.S.

12.13 3

FATGSDDATCRL [241:252] 10.99

16.03+0.1

628.77 *2 11 8.8 14.31*2.81 N.S.

0

11.61

FDLRADQEL [253:261] 15.82+3.3

553.97 +2 8 6.4 12.14 16.28+3.27 N.S.

9

FDLRADQELMT [253:263] 11.75

670.27 +2 16.54+2.0

10 8 18.16*1.06 N.S.

12.74 2

YSHDNIIC [264:2711 964.54 +1₇

11.22 17.30+3.0

5.6 19.11+1.03 N.S.

482.95 *2 ' 3

GITSVSF [272:278J 11.77+0.9

710.41 *1 8 4.8 12.37 10.85+0.23 N.S.

9

SKSGRLLLAG [279:288J 13.11+0.3

501.63 +2 9 7.2 11.32 14.06+3.44 N.S.

5

LLAGYDDF [285:2921 10.53 41.40+2.4

913.50 *1 7 5.6 43.53+1.73 N.S.

12.64 6

NCNVWDALKADRAGVL [293:3Q8J 12.25

873.23 *2 ,, 17.43+1.8

12 19.05+2.76 N.S.

582.83 *3¹⁵ 12.85 8

AGHDNRVSCl [309:3181 9.93- 16.41+0.6

536.70 +2 9 7.2 10.87+1.41

10.70 3

GVTDDGMA [319:3261 10.22

13.60+0.4

765.37 +1 7 5.6 11,09*1.34

9

10.86

VATGSWOSF [327:3351 12.28

13 23+0 6

969.50 +1 8 6.4 13.63+0.59 N S

8

12.53

IK!WN |336;340J 10.96

673.53 +1 .

3.2 0.22+0.44 0.81+0.58 N.S.

337.22 *2⁴

12.08

PVIN1EDL [342:348J 12.96

47.69+2.6

912.68 +1 6 4.8 51.05+1.67 N.S.

1

13.11

LTEKOKUCMEVDGL [349:3621 845.62 *2 67.88+4.6

13 10.4 11.15 71.37+0.84 N.S.

564.23 +3 0

EVDQLKKEVTL [358:3681 11.53

651.60 +2 20.21 ±3.1

10 8 21.97+1.36 N.S.

434.87 *3 5

12.01

ERMLVSKC 1389:3761 10.44

965.72 +1 7 5.6 29.34+1.42^27'°³ N.S.

11.55

YVEERSGEDPLVKGIPEOKNPFKELK 11.54

30.18+0.8

G [383:4091 1025.61 +3 23 18.4 31.25+0.6? 4 N.S.

12.10

Column 1 shows the peptide sequences from the Qb1 g1 primary amino acid sequence. The numbers shown in brackets indicate the positions of peptides in the primary protein sequence. Column 2 reveals the mass over charge (m/z) ratio of the ion used to identify the peptide based on its MS/MS spectrum. Column 3 displays the charge of the ion from Column 2. Column 4 indicates the maximum number of theoretically H/D exchangeable sites in the peptide fragment (max = number of non-proline peptide bonds in the peptide fragment). Column 5 shows the deuterium uptake normalized to 80% of the theoretical maximum exchangeable sites. The 80% normalization reflects the dilution used during the sample preparation in D20 (see Methods). Column 6 reports the retention time (in min) of the listed peptides. Columns 7 and 8 indicate the percentage deuterium uptake of the exchangeable sites for the Qb1 g1 complex and Nb5-bound Qb1g1 complex, respectively. Column 9 shows the difference of deuterium uptake in Qb1 g1 with and without Nb5. Column 9 is colored according to the percentage change in the deuterium uptake observed between Qb1 g1 complex and Nb5-bound Qb1 g1 complex (<-5%, light purple; -5%<x<-10%, purple; +5%, light greencyarr, +5%<x<+10%, greencyan).

Example 4. Nb5 binds to various GP subtypes.

Assessment of the binding interface between the Gpi y1 dimer and Nb5 revealed critical amino acid residues that are highly conserved among GP subtypes 1 -4 (Fig. 4a). To further investigate the putative binding of Nb5 with different GP subtypes, Nb5-mediated GP purification was carried out with C57BL/6J mouse brain. As expected , Nb5 bound and pulled out the GP subtypes from mouse brain whereas Nb17 had no binding partner (Fig. 4b). In-gel protein digestion followed by mass-spectrophotometry (MS) analyses of the GP gel band (Fig. 4b, asterisks) identified unique peptides from GP1 , GP2, GP3 and GP4 subtypes (Fig. 4c, Table 2). However, unique peptides from the GP5 subtype were not observed due to its low sequence similarity with other qb subtypes. Notably, MS-identified peptides were searched against both the full mouse proteome and the primary sequences of qb subtypes to eliminate false-positives.

To further examine qb selectivity in a living cell, an optical assay was employed to monitor the interaction of Venus-tagged qbg and the nano Luciferase tagged C-terminal 6b1g2-ίh1bG3q1^ domain of G protein-coupled Receptor Kinase 3 (masGRK3ct-Nluc)⁵⁷. Direct interaction of these sensor pairs increases the bioluminescence resonance energy transfer (BRET) ratio in transfected cells. As a result, co-transfection with a qbg^^^ protein such as Ga, competes with the masGRK3ct-Nluc sensor and decreases the BRET ratio (Fig. 4d, pink). Indeed, exogenous Ga₀ markedly diminished the BRET ratio (Fig. 4e, pink). Similarly, but to a lesser extent, Nb5 suppressed the BRET ratio in cells transfected with all nbhue-qbg complexes tested (Fig. 4e, greencyan) suggesting an interaction between Nb5 and qb subtypes 1-4. In contrast, Nb17 had no effect on the BRET ratio suggesting no interaction with qb subtypes (Fig. 4e, purple). Additionally, western blot analyses of the transfected components were performed to verify similar expression levels of the exogenous proteins (Fig. 4f). Interestingly, the transfection of either Ga or Nb5, but not Nb17 slightly increased the expression levels of nbhu5-6b1-4 subunits, suggesting the formation of a proteolytically stable complex of Ga and Nb5 with nbhue-qbg dimer. Overall, these experiments suggest that Nb5 binds to all combinations of qb subtypes 1 -4 and G y and suppresses qbg signaling mediated through protein-protein interactions near the qbg hotspot. These results are suggestive of Nb5’s potential broad utility to influence qbg signaling in various cell types.

Table 2: List of proteins identified from the in-gel protein digestion and mass-spectrophotometry (MS) analyses of the qb gel band.

The MS-identified peptides were searched against the full mouse proteome to eliminate false- positives. Column 1 shows the Uniprot accession IDs. Column 2 describes the proteins that were identified based on their MS/MS spectrum. Column 3 displays the sequest score which determines the quality of hits based on the number of ions in the MS/MS spectrum that match with the experimental data. Column 4 shows the percentage of the protein sequence covered by identified peptides. Column 5 shows the number of peptide sequences that are unique to a protein group and do not occur in the proteins of any other group. Column 6 reports the total number of distinct peptide sequences identified in the protein group. Column 7 displays the number of peptide spectrum matches (PSMs) that reports the total number of identified peptide spectra matched for the protein. Columns 8 report the molecular weight of the identified proteins. The G j3 subtypes are highlighted in grey. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009503.

Example 5. Nb5 inhibits GPY-mediated GIRK signaling in striatum neurons.

GIRK channels, found primarily in central nervous system neurons and atrial myocytes, respond to GPCR- mediated signaling through qbg binding events. Activation of GIRKs affects the flow of K⁺ ions across cell membranes that attenuate cellular electrical excitability. The capability of Nb5 to modulate qbg signaling was examined in medium spiny neurons (MSNs) from mouse striatum. GIRK channels (GIRK2; kir3.2) were virally overexpressed in the MSNs and the resulting outward currents were used as indicators of GPCR-mediated GIRK2 activation. Indirect pathway MSNs (iMSNs) expressed D2 dopamine receptors (D2Rs)⁵⁵ whereas direct pathway MSNs (dMSNs) expressed M4 muscarinic acetylcholine receptors

(M4Rs)^{58 60}. Both D2Rs and M4Rs are Gi/o278 coupled receptors capable of gating GIRK channels through qbg signaling. A single electrical stimulation in the striatum evoked the release of dopamine from neuronal dopaminergic terminals, resulting in a D2R-mediated inhibitory post-synaptic current (D2R- IPSC) in postsynaptic GIRK2 positive iMSNs. Interestingly, inclusion of 10 mM Nb5 in the recording pipette attenuated D2R-IPSCs by about 60% (208.12 ± 47.08 pA in 1 min; 75.18 ± 9.40 pA after 15 min, n = 9, P < 0.05, Student’s f-test) (Fig. 5a,e,i). In contrast, including an irrelevant Nanobody, Nb17 at the same concentration had no significant effect on the D2R285 IPSCs amplitudes (210.05 ± 44.81 pA in 1 min; 186.66 ± 34.04 pA after 15 min, n = 5, P > 0.05, Student’s f-test) (Fig. 5c,g,k). Similarly, there was no effect on the D2R-IPSCs in postsynaptic GIRK2 positive iMSNs in the absence of nanobody treatment (271.50 ± 73.95 pA in 1 min; 257.20 ± 50.23 pA after 15 min, n = 7, P > 0.05, Student’s t-test) (Fig. 5d,h,l). To ensure that the effect of Nb5 was not D2R-specific, M4R287 mediated inhibitory post-synaptic current (M4R-IPSC) measurements were also made on postsynaptic GIRK2 positive dMSNs. As expected, Nb5 attenuated M4-IPSCs evoked by electrical stimulation by about 60% (430.84 ± 81.09 pA in 1 min; 179.68 ± 22.70 pA after 15 min, n = 8, P < 0.05, Student’s f-test) (Fig. 5b,f,j). The effect of Nb5 in these GIRK inhibition assays was more demonstrable compared to its effects in the BRET assays. This likely is due to differences in the mode of delivery and thereby the effective concentration of Nb5 attained in cells in the two assay systems. While, Nb5 was co-transfected in human embryonic kidney cells 293 in BRET assays, GIRK recordings were performed by introducing an internal solution containing 10 mM of Nb5 directly to the neuronal exons. These results, together with the crystal structure of the Qb1 y1-Nb5 complex demonstrate that Nb5 interferes with qbg dimer association to GIRK channels, thereby suppressing ΰbg- regulated GIRK signaling (Fig. 5m).

Example 6. Nb5 acts as a control -switch for GPCR-mediated Qbg signaling.

Next, the effect of Nb5 was investigated on cellular pathways that are often dysregulated in cancer. Here the phosphoinositide-3-kinase (PI3K)-protein kinase B/AKT (PI3K-PKB/AKT)^{29 36} and mitogen activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)^{42 43} pathways were examined that are co-regulated by GPCR-mediated qbg signaling (Fig. 6a). The PI3K-PKB/AKT and MAPK/ERK pathways respond to a variety of cellular stimuli, and hence play a key role in diverse cellular processes, including cell survival, growth, proliferation, angiogenesis, metabolism and migration. Because of the importance of these cellular pathways in cancer progression and metastasis, a Chinese hamster ovary cell line stably expressing human apelin receptor (APJ) was transfected with Nb5 cDNA and its effect upon the phosphorylation of AKT and ERK proteins downstream of APJ activation was assessed. Parental CHO- APJ cells were treated with 1 mM apelin over 0-30 min. Cell lysates then were collected and analyzed by immunoblotting to obtain optimum conditions to observe GPCR-mediated phosphorylation changes. Such changes were found most pronounced 5 min post-treatment with apelin (Fig. 8a). Interestingly, a significant decrease in the phosphorylation of both AKT and ERK1/2 proteins was observed in CHO-APJ- Nb5 cells 5 min post-treatment with apelin (Fig. 6b, c). In contrast, CHO-APJ-NM 7 cells showed no visible change in phosphorylation levels of either AKT or ERK proteins during the same time interval (Fig. 6b, compare lanes marked with asterisks). Additionally, no significant change was observed in the expression level of APJ with either Nb5 or Nb17 transfection (Fig. 8b). These results demonstrate a suppressive effect of Nb5 on APJ-mediated Qbg signaling, further supporting its capability to modulate G Y-mediated pathways governed by multiple GPCRs.

APJ signaling is regulated by the Gi/₀ class of G proteins wherein both G and qbg act either together or separately to affect AC-mediated intracellular cAMP production (Fig. 6d)⁶⁸·^70-71. To determine the effect of Nb5 on intracellular cAMP levels, the forskolin-stimulated accumulation of cAMP was measured in response to apelin treatment. Treatment of CHO-APJ cells with apelin inhibited the forskolin-stimulated accumulation of cAMP in a dose-dependent manner (Fig. 6e). CHO-APJ cells co-expressing Nb5 (greencyan) showed a reduced inhibition of cAMP accumulation at a higher dosage of apelin when compared to either parental CHO-APJ cells (grey) or CHO-APJ cells expressing the negative control Nb17 (purple). This result demonstrates that Nb5 modulates the apelin-induced inhibition of cAMP accumulation in CHO-APJ cells. In addition, Nb5 presumably had no effect on the G i/o-GTP-mediated cAMP signaling after the loss of the Gbg-mediated cAMP component in cells transfected with Nb5 (Fig. 6e, greencyan, Table 3). These results also are consistent with the higher affinity of Ga compared to Nb5 for the qbg dimer. Overall, the binding of Nb5 to ΰbg negatively regulates Gi/₀ signaling, at least partially by inhibiting Gbg-associated cAMP signaling.

To further confirm the specificity of Nb5 towards Gbg-induced signaling, we tested its effects on two well- established agonist-induced Ga signaling pathways (Fig. 6f)^{57, 121}. Here, Ga_q signaling was monitored in HEK293T/17 cells transfected with the M3 muscarinic acetylcholine receptor (M3R) and using a Ca²⁺ sensor (CalFluxVTN). Ga_s signaling was monitored in HEK293T/17 cells transfected with the dopamine D1 receptor (D1 R) and using a cAMP sensor (Nluc-Epac-VV). The transfected cells were then stimulated with acetylcholine or dopamine to activate M3R or D1 R, respectively. Real-time monitoring of the resultant responses clearly demonstrated that Nb5 had no effect on the elevation of intracellular Ca²⁺ induced by the activation of M3R-Ga_q (Fig. 6 g, h) or on D1 R-Gas-induced cAMP production (Fig. 6 i, j) in living cells.

Table 3: Measurements of the apelin-induced inhibition of cAMP accumulation in CHO-APJ-Nb5, CHO-APJ-

Nb5 and parental CHO-APJ cells.

Column 1 shows the concentration of apelin. Column 2 reports the mean percentage intracellular cAMP/total cAMP. Column 3 displays the standard deviation associated with the mean percentage intracellular cAMP/total cAMP reported in column 2. Discussion

The role of Qbg signaling in various cellular functions is relatively newly defined and diverse as compared to Ga signaling. The Qbg dimer functions as a negative regulator when bound to the Ga subunit. Additionally, the qbg dimer regulates many downstream events depending on its interaction with different effector molecules. Many of these downstream events are dysregulated in neurological disorders and cancer, making the qbg dimer a critical drug target. However, the ability of qbg subunits to play essential roles in various cellular functions, including the formation of heterotrimeric G proteins, implies the potential for numerous side effects when qbg is the pharmacological target. Here we found that Nb5, a Nanobody against Qb1 antigen, can cause selective inhibition of Gbg-mediated signaling while leaving basal Go mediated signaling intact. Gt activation analyses showed that Nb5 causes a GPCR-independent shift in the dynamic equilibrium of heterotrimeric Gt into its dissociated subunits in both membranes and in solution. Both in vitro SPR kinetics and Gt activation assays suggest a tight interaction between Nb5 and the qbg dimer qbg binding proteins, including Ga, PDC, GRKs, and PI3K are key regulators of GPCR signaling known to interact with qbg dimer with nanomolar affinities. A similar binding affinity of Nb5 with Qb1 g1 ensures competition between Nb5 and these qbg regulatory proteins in modulating Gbg-mediated signaling events. Indeed, combined differential HDX and X-ray crystallography studies demonstrate a possible competition between Nb5 and qbg regulatory proteins due to their overlapping binding interfaces. In fact, Sc index analysis that precisely measures correlations of directions suggests a structural advantage of Nb5 over most qbg regulatory proteins except Ga for qbg binding. Furthermore, the binding interface between Nb5 and Qb1g1 has the highest Sc index among several tested protein-Nb interactions^{61 63}. Nb5-mediated qb purification from mouse brain and cell-based BRET assays demonstrate the specificity of Nb5 towards qb subtypes 1-4. No evidence for binding between Nb5 and ΰb5 was observed, likely because the sequence of Qb5 differs from that of other qb subtypes with only 53% sequence identity to its closest qb subtype versus 80-90% sequence identity between other qb subtypes. Overall, these results identify Nb5 as a potential negative regulator of qbg signaling in various cell types.

Many neurological and neuromuscular disorders including Parkinson disease, Alzheimer disease, bipolar disorder, multiple sclerosis, and other age-related disorders are aggravated by dysregulated ion channel function. Ion channels like GIRK play a crucial role in maintaining ion homeostasis in neurons, determining their membrane potential and neurotransmitter secretion. The lack of endogenous GIRK channels in MSNs and their effective coupling to various GPCR signaling pathways makes them a useful tool for rapid sensing and monitoring of Gbg-regulated GIRK signaling⁶⁰·⁶⁴. Whole-cell patch clamping of GIRK2 overexpressed MSNs was performed to investigate the role of Nb5 as a negative regulator of ΰbg- mediated signaling. Treatment with Nb5 decreased both D2R- and M4R-IPSCs amplitudes, and thus inhibited Gbg-regulated GIRK channel opening. This serves as one of the first examples wherein a Nanobody modulates a GPCR-mediated Qbg-e^hqΐ^ event in any cell type. Additional evidence emanates from Gbg-mediated intracellular signaling wherein Nb5 reduces downstream phosphorylation events in both AKT and ERK kinase pathways. Notably, Nb5 does not completely ablate either GIRK activation or downstream AKT and ERK phosphorylation, suggesting its capability to suppress dysregulated pathways with critical roles in cancer progression and metastasis. The Qbg dimer classically was thought to inhibit adenylyl cyclase (AC) activity by binding and inhibiting stimulatory Ga subunits. However, recent studies have shown that Qbg can stimulate several AC isoforms through cross-talk between Ga- and Gbg-mediated cAMP signaling³⁸’^{65 69}. Co-expression of Nb5 in CHO-APJ cells showed a 58% reduction in apelin-mediated inhibition of cAMP accumulation when compared to either parental cells or CHO-APJ co-expressing Nb17. This result demonstrates that Nb5 modulates the forskolin- stimulated accumulation of cAMP in CHO-APJ cells by inhibiting the Gbg-mediated cAMP signaling component. Additionally, Nb5 showed no significant effect on either the M3R-Gaq-induced intracellular Ca²⁺ elevation or D1 R-Gas-induced cAMP production. These results show that Nb5 does not alter the GTP-bound Gaq and Gas-mediated signaling component in living cells. Overall, Nb5 serves as a dynamic scavenger of the ΰbg dimer and thereby causes partial inhibition of Gbg-mediated signaling. The remaining Nb5-free ΰbg supports the canonical Ga-GTP-mediated signaling that includes the GPCR- mediated GDP-GTP exchange from Ga, and signaling termination by GTP hydrolysis and re-association of Ga-GDP with qbg. However at higher concentrations, Nb5 might affect the Ga-GTP signaling component due to increased sequestration of qbg dimers (Fig. 1 d,e).

In summary, the ability of Nanobodies to act as crystallization chaperones by either stabilizing intrinsically flexible regions or shielding aggregating surfaces in a protein is well established⁷²·⁷³, but their ability to serve functional roles in cellular signaling has not been well studied. This work highlights the functional importance of these genetically-encodable antibody fragments in controlling various aspects of GPCR- signaling pathways. Interestingly, through its high affinity binding epitope on qb, Nb5 acts as a specific inhibitor of various Gbg-mediated signaling events that regulate several‘undruggable’ targets such as GIRK channels, ERK, and AKT kinases. The ability of Nb5 to suppress but not completely ablate qbg signaling makes it a beneficial tool for future nanobody-based therapeutics to modulate cellular signaling. A proof of principle is provided in these examples, and serves as the first ones wherein a nanobody modulates a GPCR-mediated ΰbg-e^hqI^ event in any cell type. The ease of production and genetic manipulation of nanobodies are advantageous characteristics for achieving the goal of engineering variants with higher specificity toward different qb subtypes and even qbg combinations. This opens new avenues for Nanobody-assisted modulation of cellular signaling to treat various excitatory neurological conditions and cancer progression. However, the development of Nanobody-based clinical therapies relies heavily on the advancement of specific and efficient gene transduction methods such as CRISPS/Cas9 and viral-gene transduction. Additional strategies for intracellular Nanobody delivery include coupling the Nbs to cell-penetrating peptides such as penetratin⁷⁴. Elucidating Nanobody translocation across the CNS could also provide a tremendous advantage in treating neurological disorders. Such strategies might include internalization via either clathrin-coated vesiclesl⁷⁵ or Trojan horse technology using transferrin and insulin receptors⁷⁶. Altogether, such nanobody-based therapeutics will find a niche in cases where‘undruggable’ protein targets are involved in multiple signaling pathways and where conventional therapeutic approaches induce unacceptable side effects. Methods

Reagents.

Antibodies recognizing phosphorylated-ERKs 1/2 (apT202/pY204-ERK1/2, catalog no. 9106), total ERKs 1/2 (aERK1/2, catalog no. 4695), phosphorylated-Akt (apS473-Akt, catalog no. 4058), and total Akt (aAkt, catalog no. 9272), were from Cell Signaling Technology (Danvers, MA). Anti-APJ antibody (3C3-7, catalog no. MABN1846) was from Millipore Sigma (St. Louis, MO). Polyclonal aFlag antibody (catalog no. A190- 101 A) was from Bethyl Laboratories, Inc. (Montgomery, TX). Anti-Flag antibody (catalog no. F7425), aHA antibody (catalog no. 1 1867423001 ), aGAPDH antibody (catalog no. MAB374) and aGFP antibody (catalog no. 1 1814460001 ) were from Millipore Sigma (St. Louis, MO) and used for Western blotting analysis in Fig. 4f. Anti-Gao antibody (catalog no. sc-387) was from Santa Cruz (Dallas, TX). IRDye- conjugated anti-mouse IgGs (catalogs P/N 925-32210 and P/N 925-68070), and anti-rabbit IgGs (P/N 925-3221 1 and P/N 925-68071 ) were from LI-COR (Lincoln, NE). HRP conjugated anti-goat IgG were from ThermoFisher Scientific, Inc (Waltham, MA). Apelin-13 (catalog no. 057-18) was from Phoenix Pharmaceuticals, Inc (Burlingame, CA). IBMX (catalog no. 2845) and Forskolin (catalog no. 1099) were from R&D systems, Inc (Minneapolis, MN). CHO-K cells and HEK293T/17 cells (catalog nos. CRL-9618 and CRL1 1268, respectively) were from the American Type Culture Collection (Manassas, VA) and have no mycoplasma contamination.

A pCMV5 plasmid encoding GaoA was a gift from Dr. Hiroshi Itoh (Nara Institute of Science and Technology, Japan). Plasmids encoding Venus 156-239-G 1 , and Venus 1-155-Gy2 were gifts from Dr. Nevin A. Lambert (Augusta University)⁷⁷. The masGRK3ct-Nluc constructs were reported previously⁵⁷. Amino acids 156-239 of Venus were fused to a GGSGGG linker at the N-terminus of Qb2 (GenBank: AF501883), Qb3 (GenBank: M31328), or Qb4 (GenBank: AF300648) to construct Venus 156-239-6b subunits. Nb5-Flag and Nb17-Flag constructs were based on the primary sequences of Nb5 (SEQ ID NO: 2) and Nb17 (SEQ ID NO: 5) wherein the 6x His-tag was replaced by the FLAG-tag (SEQ ID NO: 16). Human M3 muscarinic acetylcholine receptor and human dopamine D1 receptor in pcDNA3.1 (+) were purchased from cDNA Resource Center (Bloomsberg, PA). Nluc-Epac-VV and CalFluxVTN in pcDNA3.1 (+) were reported previously 57,121 Adeno-associated virus (AAV) AAV9.hSyn.tdTomato.T2A.mGIRK2-1-A22A.WPRE.bGH was obtained from the University of Pennsylvania Viral Core⁶⁰’⁶⁴.

>SEQ ID NO:5: ISVD Nb17 amino acid sequence [CA1 1 101]

QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFT

ISRDNAKNTVYLQMNSLKPEDTAVYYCNAENSIPIGDLARRVWDYWGQGTQVTVSS

>SEQ ID NO:16: FLAG tag amino acid sequence

DYKDDDDK

>SEQ ID NO:17: 6-HIS-EPEA double tag amino acid sequence

HHHHHHEPEA Generation and phage selection of Nanobodies.

Nanobodies against the Qb1g1 dimer were prepared by a previously published protocol²¹. Briefly, a llama (Lama glama) was immunized weekly for six weeks with 1 mg of purified bovine ΰb1 y1 dimer. Peripheral blood lymphocytes from anti-coagulated blood were then used to prepare cDNA clones that served as templates for amplification of open reading frames encoding the variable domains of the heavy-chain only antibodies. PCR fragments were then ligated into the pMESy4 phage display vector and transformed in E. coli TG1 cells to create a Nanobody library of 4 x 109 transformants. After super infection with M13 helper phage 21 , the display library was added to antigen-coated wells of MaxiSorp ELISA plates and selections were performed in buffer containing 10 mM HEPES, pH 7.5, 100 mM NaCI, 2 mM MgCL, 1 mM EDTA and 1 mM DTT. After washing, phage were eluted by incubating the antigen-coated wells with 100 pi of trypsin (250 pg/ml) for 30 min. Freshly grown TG1 cells were then infected with the eluted phage and grown overnight at 37 °C. A total of 184 colonies were randomly picked and analyzed with ELISA. Testing for specific binding to the ΰb1g1 dimer resulted in 14 families wherein all Nanobodies were produced as soluble His-tagged proteins in the E. coli periplasmic region²¹.

Nanobodv expression and purification.

Nanobodies (Nb5 (SEQ ID NO:2) and Nb17 (SEQ ID NO:5)) were expressed and purified as described before²¹. Briefly, Nanobodies bearing a C-terminal His-tag were transformed in E. coli WK6 (Su-) cells. Small scale cultures of 5 ml were grown in LB media containing 2% glucose, 1 mM MgCL and 50 pg/ml ampicillin. Large scale cultures of 2 L were grown to OD6oo = 0.9-1.0 at 37 °C in TB media containing 0.1 % glucose, 1 mM MgCL, and 50 pg/ml ampicillin. Cultures were induced with 1 mM IPTG and grown overnight at 28 °C. Cells were harvested by centrifugation at 1 1 ,000 g for 20 min at 4 °C. The periplasmic fraction was extracted by re-suspending cells in ice-cold TES buffer containing 0.2 M Tris, pH 8.0, 0.5 mM EDTA, and 0.5 M sucrose. Following a 1 h incubation at 4 °C, cells were supplemented with 4X diluted TES buffer to achieve a final buffer composition of 0.1 M Tris, pH 8.0, 0.25 mM EDTA, and 0.25 M sucrose. After 1 h incubation at 4 °C, cells were removed by centrifugation at 1 1 ,000 g for 30 min at 4 °C. The periplasmic extract was filtered through a 0.22 pm vacuum driven filter and subjected to immobilized-Ni²⁺ affinity chromatography followed by size exclusion chromatography (SEC) on a Superdex 200 10/300 GL column equilibrated with 20 mM bis-Tris propane, pH 7.0 and 100 mM NaCI. Peak protein fractions were pooled and concentrated to 10 mg/ml.

Rh purification.

All experimental procedures were carried out in a darkroom under dim red light (>670 nm). Bovine rod outer segments (ROS) were prepared as described elsewhere⁷⁸·⁷⁹. ROS were washed with isotonic and hypotonic buffer to remove both soluble and membrane associated ROS proteins⁸⁰·⁸¹. Rh was purified by a protocol described previously⁸². Briefly, native Rh membranes were solubilized by a zinc/alkyl-glucoside extraction method and centrifuged at 100,000 for 40 min to extract Rh⁸³. Clear supernatants were loaded on a 1 D4-coupled CNBr-activated Sepharose 4B column and washed with buffer containing 10 mM MES, pH 6.4, 100 mM NaCI, and 0.02 % n-dodecyl b-D-maltoside (DDM) to either dispose of excess retinal or achieve further purification. Finally, purified Rh was eluted with 0.5 mg/ml of TETSQVAPA (SEQ ID NO: 18) nanopeptide (from the Rh C-terminal sequence).

Purification of heterotrim eric Gt.

Bovine rod outer segments (ROS) were prepared as described elsewhere⁷⁸·⁷⁹ in a darkroom under dim red light (>670 nm). ROS were resuspended in isotonic buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCI, 1 mM DTT and 5 mM MgC . Resuspended ROS were then centrifuged at 31 ,000 g at 4 °C for 25 min to remove soluble and some membrane-associated proteins. The pellet was then gently homogenized twice in hypotonic buffer containing 5 mM HEPES, pH 7.5, 1 mM EDTA, and 1 mM DTT by manually passing the solution through a glass-to-glass homogenizer. The homogenized suspension was centrifuged at 40,000 g for 30 min at 4 °C. Supernatants from the two hypotonic washes were pooled and centrifuged multiple times at 40,000 g for 30 min at 4 °C to completely remove any residual ROS pellet. The clear supernatant was dialyzed against the equilibrating buffer containing 10 mM HEPES, pH 7.5, 2 mM MgCl2, and 1 mM DTT for 3 h at 4 °C. For purification of heterotrimeric Gt, the hypotonic solution was loaded onto a 010/10 column (GE Healthcare) with 6 ml_ of pre-equilibrated propyl-agarose resin. Next, the column was washed with 30 resin volumes of the equilibration buffer followed by 2 resin volumes of the same buffer containing 50 mM NaCI. Bound proteins were eluted with 50 ml_ of equilibration buffer containing 0.5 M NaCI. The eluate containing heterotrimeric Gt was concentrated and loaded onto a Superdex 200 10/300 GL column equilibrated with equilibration buffer containing 0.1 M NaCI. Fractions containing heterotrimeric Gt were combined, concentrated to about 2 mg/mL and used for further analyses.

Nanobodv-mediated shift in heterotrimeric Gt equilibrium.

ROS were resuspended in isotonic buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCI, 1 mM DTT and 5 mM MgC . For Gt dissociation experiments in ROS membranes, the resuspended ROS (2 mg/ml Rh) were either illuminated with a 150-W fiber light in the presence of 250 mM GTP, co-treated with 250 mM GTP and 3 pM Nb5, or treated with either 3 pM Nb5 or 3 pM Nb17 for 30 min at 4 °C. The resuspensions were then centrifuged at 100,000 g at 4 °C for 20 min and the supernatants were analyzed by SDS-PAGE. In solution Gt dissociation experiments were conducted on supernatants obtained after ROS hypotonic washes. Hypotonic extracts were combined and centrifuged multiple times at 40,000g for 30 min at 4 °C to ensure complete removal of the residual ROS pellet. Next, the hypotonic extracts (1 pM Gt) were either supplemented with 1 pM Rh and illuminated with a 150-W fiber light in the presence of 250 pM GTP and 2 pM Nb5, co-treated with 250 pM GTP and 2 pM Nb5, or treated with either 2 pM Nb5 or 2 pM Nb17 for 30 min at 4 °C. Then these treated hypotonic extracts were subjected to immobilized- Ni²⁺ affinity chromatography. A small volume of Ni²⁺-NTA resin (250 pi) pre-equilibrated with 10 mM HEPES, pH 7.5, 0.1 M NaCI, 2 mM MgC and 1 mM DTT was added to the treated hypotonic extracts. After 1 h of incubation, the resin was washed with 50 resin volumes of equilibration buffer and bound proteins were eluted in equilibration buffer containing 300 mM imidazole, pH 7.5. Eluted fractions were then analyzed by SDS-PAGE. Gt activation assay.

Gt was extracted from frozen bovine ROS membranes as described elsewhere⁸⁴·⁸⁵. The intrinsic fluorescence increase from Gat was measured with a L55 luminescence spectrophotometer (PerkinElmer Life Sciences) operating at excitation and emission wavelengths of 300 and 335 nm, respectively 46,86- 88. The ratio of Gt to Rh was 20:1 , with Gt at a concentration of 1000 nM and Rh at 50 nM. Gt was preincubated with a two molar excess of either Nb5 or Nb17 to determine their effects on Rh*-mediated Gt activation rates. This was followed by the addition of 300 mM GTPyS (Sigma-Aldrich) to determine the GTPys induced complex dissociation and fluorescence changes⁸². Samples were bleached for 1 min with a Fiber-Light delivered through a 480 to 520 nm long pass wavelength filter prior to the fluorescence measurements. Gt activation rates were determined for the first 100 s in a Gt activation assay⁸².

GB1v1 alone and GB1v1-Nb5 complex purification.

Bovine ROS were washed with isotonic buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCI, 1 mM DTT and 5 mM MgCL. For GB1 y1 purification, ROS washed with isotonic buffer were illuminated with a 150-W fiber light (NCL-150, Volpi, USA) for 15 min and then washed twice with hypotonic buffer containing 5 mM HEPES, pH 7.5, 1 mM EDTA, and 1 mM DTT supplemented with 250 pM GTP. The hypotonic wash extracts were pooled and applied to a Blue Sepharose CL-GB column pre-equilibrated with 10 mM HEPES, pH 7.7, 6 mM MgCL, 1 mM EDTA and 1 mM DTT. GB1y1 obtained from the unbound flowthrough was further purified by SEC on a Superdex 200 10/300 GL column (GE Life sciences) equilibrated with 50 mM Tris-HCI, pH 7.5, 100 mM NaCI, 2 mM MgCL and 3 mM DTT. For purification of the GB1 y1- Nb5 complex, ROS washed with isotonic buffer were washed twice with hypotonic buffer and the washes were combined for further purification. Hypotonic wash extracts were then incubated with a 1 :2 molar ratio of Nb5 for 1 h at 4 °C. This was followed by the addition of Ni²⁺-NTA resin pre-equilibrated with 50 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5 mM imidazole, 2 mM MgCL and 1 mM DTT. Following 1 h of incubation, the resin was washed with 50 resin volumes of equilibration buffer and the GB1y1-Nb5 complex was eluted with equilibration buffer containing 300 mM imidazole. The Ni²⁺-NTA purified GB1y1-Nb5 complex was then loaded on a Superdex 200 10/300 GL column equilibrated with 50 mM Tris-HCI, pH 7.5, 100 mM NaCI, 2 mM MgCb, and 3 mM DTT.

GB1v1-Nb5 complex crystallization.

SEC fractions were concentrated to 4.9 mg/ml and then used for crystallization. The concentrated protein was supplemented with 2 mM DTT. Crystallization screens by the sparse matrix crystallization method⁸⁹ were carried out by both the hanging-drop and sitting drop vapor diffusion methods. Each hanging drop was prepared on a siliconized coverslip by mixing equal volumes of GB1y1-Nb5 complex and reservoir solution. The reservoir solution contained 25% (w/v) PEG 3350 in 0.1 M Bis-Tris-HCI, pH 5.5-5.7, and 0.2- 0.3 M MgCL. Crystals appeared in 2 days at 4 °C and reached 50-80 pm in their longest dimension within 5 days. Crystals were harvested directly from the mother liquor into dual thickness microloops (MiTeGen, LLC) and plunge-frozen in liquid nitrogen. Diffraction data collection and structural refinement.

X-ray data collection of the G 1y1-Nb5 complex was performed at -173 °C. Diffraction data were collected at the NE-CAT-24-ID-E beamline. Data were integrated with XDS and scaled using XSCALE 90. Initial phases for the G 1y1-Nb5 complex were obtained by molecular replacement using the G 1y1-phosducin complex and TssK Nanobody nb18 structures as search models (PDB accession: 1A0R 44, 5M2W 91 ) with the CCP4 program PHASER 92-94. Initial models were improved by multiple rounds of REFMAC ver. 5.8⁹² refinement against the G 1y1-Nb5 complex dataset and manual model adjustments with Coot 0.8.8⁹⁵. The final models had agreement factors Rfree and R cryst of 24.7% and 20%, respectively. The stereochemical quality of the G 1y1-Nb5 complex model was assessed with the Molprobity⁹⁶·⁹⁷ and wwPDB validation servers⁹⁸. Details of the diffraction data collection and structural refinement statistics are provided in Table 4. Coordinates and structure factor amplitudes were deposited in the PDB (PDB accession: 6B20). The dimer interface shape complementarity was calculated by Sc 55 and PISA 54.

Table 4: Diffraction data collection and structural refinement statistics for the Gpiy1-Nb5 complex.

†Values in parentheses are for the highest-resolution shell of data. ID, insertion device.

#Resolution bin at <l/o> of 2.1 is 2.70-2.62 A for comparison with the historical standards of x-ray data truncation. The resolution bin with <l/o> of 1.05 is used for the resolution cut-off to include the intensities that are significantly above the noise level. Extending the data beyond <l/o> values of >2 have been shown to improve structure determination in many cases with no negative impact on model building

‘Glycine Ramachandran outliers that were consistent with other Qb1 g1 crystal structures. Differential hvdroqen/deuterium exchange (HDX) of the GB1 v1 and GB1v1-Nb5 complex.

Amide H/D exchange was performed as described previously^{99 101}. Briefly, 10 pg of purified Qb1g1 or Qb1 y1-Nb5 complex were diluted in 70 pl_ of ice cold D2O to obtain a final concentration of 80% D2O. The solution was incubated on ice for 10 min to achieve steady state deuterium exchange conditions. This was followed by quenching the reaction with ice cold buffer containing D20 in formic acid (Sigma-Aldrich) to obtain a final pH of 2.5. Non-deuterated samples were quenched with buffer containing H2O in formic acid, pH 2.5. Samples were digested with freshly prepared 5-20 pg pepsin (Worthington, Lakewood, NJ) solution prepared in H2O. All samples were digested over a time range of 1-5 min on ice. Next, samples (100 pL) were loaded onto a C8 trap (2.1 mm, Thermo Scientific) and a C18 (20 mm^c 2.0 mm, Phenomenex) column using a temperature-controlled Accela 600 autosampler and pump (Thermo Scientific) with a temperature set to 4 °C. Separation was achieved with an Agilent 1 100 HPLC system (Agilent Technologies, Santa Clara, CA) at a flow rate of 0.1 ml/min. Peptides were eluted with a gradient from 98% of buffer A containing 0.1 % (v/v) formic acid in H2O and 2% of buffer B containing 0.1 % (v/v) formic acid in acetonitrile to 2% of buffer A and 98% of buffer B. The eluent was injected into a Thermo Finnigan LXQ (Thermo Scientific, Waltham, MA) MS equipped with an electrospray ionization source operated in the positive ion mode with other parameters adjusted as follows: activation type was set to collision induced dissociation, normalized collision energy to 35 kV, capillary temperature to 370 °C, source voltage to 5 kV, capillary voltage to 43 V, tube lens to 105 V, and then MS spectra were collected over a 200-2,000 mass range. To avoid contamination from previous runs, each production run was followed by a 10 pi mock injection of buffer A, followed by residual peptide elution with the gradient profile described above. Each run was also followed by a 20 min equilibration run with 98% buffer A and 2% buffer B.

Differential HDX data analysis.

mzXML files were generated from raw data with the MassMatrix file conversion tool. Peptides were identified by searching against the primary sequence of bovine G protein beta 1 (GBB1 ; Uniprot ID: P62871 ) and G protein gamma 1 (GNGT1 ; Uniprot ID: P02698) using MassMatrix v3.10. Search parameters for peptide identification were as follows: precursor ion tolerance, 3.0 Da; variable modifications, farnesylation and methylation of the N-terminus; minimal peptide length, 6 amino acids; minimal pp score, 5; pptag score, 1 .3; maximal number of combinations of different modification sites for a peptide match with modifications, 1 ; and maximal number of candidate peptide matches for each spectrum output in the result, 1. Raw data in the form of the relative signal intensity (percent) as a function of m/z were extracted with Xcalibur version 2.1.0. Qual Browser was used for recently described semi- automated peak detection, and a deconvolution procedure was performed with HXExpress¹⁰².

Surface plasmon resonance (SPR) analyses.

SPR data acquisition was carried out using the Biacore T100 SPR instrument (Biacore, GE Healthcare) at 25 °C. Purified Nb5 (50 ng ml¹) was injected at a flow rate of 10 pi min¹ in 10 mM sodium acetate, pH 5.5 to achieve capture level between 400-500 resonance units (RU) on a CM5 sensor chip according to directions in the manufacturer's amine coupling kit. After Nb5 immobilization, the surface was blocked with 1 M ethanolamine at pH 8.5, followed by regeneration using 50 mM NaOH. The interaction experiments were performed using running buffer containing 10 mM HEPES pH 7.5, 150 mM NaCI and 1 mM Tris(2- carboxyethyl)phosphine and 0.1 % Tween-20. Binding experiments were carried out using a GB-iyi concentration range of 0.6-312 nM in running buffer at a flow rate of 30 pl_ min¹. The association and dissociation kinetics for GB-igi were monitored for 140 s and 300 s, respectively. SPR data processing and analysis were performed using Biacore T100 Evaluation Software (GE, version 2.0.3). For kinetic analyses, data were locally fit to a 1 :1 Langmuir model to obtain on- and off-rate constants.

G protein extraction from mouse brain.

G proteins were extracted from mouse brain using a protocol described elsewhere with a few modifications¹⁰³. Brains from 32 C57BL/6J mice (Jackson laboratory) were thawed in 50 ml buffer containing 20 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT, 3 mM MgCL and halt protease inhibitor cocktail (ThermoFisher). Thawed brains were homogenized and centrifuged at 39,000 g for 20 min at 4 °C. Membrane pellets were homogenized in 50 ml buffer A containing 20 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and halt protease inhibitor cocktail and washed twice after adding buffer A by centrifugation at 39,000 g for 20 min. Pellets then were resuspended and washed by centrifugation in buffer A supplemented with 0.1 M NaCI and 0.1 % (w/v) sodium cholate. Next, membranes were homogenized and solubilized in buffer A containing 2% (w/v) sodium cholate for 2 h at 4 °C. The clear supernatant obtained after centrifugation at 186,000 g was loaded onto a DEAE Sepharose column equilibrated with buffer A containing 20 mM NaCI and 0.2% sodium cholate. Bound proteins were eluted with a linear gradient of NaCI (0 to 500 mM) in buffer A with 0.2% sodium cholate. The eluate was desalted, supplemented and incubated with a two molar excess of either Nb5 or Nb17 for 1 h and then subjected to immobilized-Ni²⁺ affinity chromatography. The Ni2+-NTA resin was equilibrated with buffer A containing 0.1 M NaCI, 0.2% sodium cholate and 5 mM imidazole and this was added to the eluate obtained during DEAE Sepharose purification. Following 1 h of incubation, the resin was washed with 50 resin volumes of buffer A containing 0.1 M NaCI, 0.2% sodium cholate and 5 mM imidazole. The Gbg-Nb5 complex then was eluted with buffer A containing 0.1 M NaCI, 0.1 % sodium cholate and 300 mM imidazole. The Ni²⁺- NTA purified Gbg-Nb5 complex was desalted and loaded onto 500 pi of Talon resin packed into a Pierce™ disposable column (ThermoFisher) equilibrated with buffer A containing 0.1 M NaCI, 0.1 % sodium cholate and 5 mM imidazole. The Talon resin was washed with 50 resin volumes of equilibration buffer and the purified Gbg-Nb5 complex was eluted with buffer A containing 0.1 M NaCI, 0.1 % sodium cholate and 300 mM imidazole.

In-gel protein digestion of GB subtypes obtained from mouse brain.

The excised SDS-PAGE band was destained in a solution containing 50% (v/v) ethanol with 5% (v/v) acetic acid in water. Next, the gel band was treated with acetonitrile and 5 mM DTT followed by its alkylation with iodoacetamide. Complete in-gel protein digestion was achieved by treatment with 50 ng of trypsin and chymotrypsin proteases prepared in 50 mM ammonium bicarbonate for 16 h at room temperature. Peptides were extracted by washing the gel twice with 30 pl_ of 50% (v/v) acetonitrile with 5% (v/v) formic acid in water. Extracts were combined and evaporated to <10 mI_ in a Speedvac and then resuspended in 1 % (v/v) acetic acid to achieve a final volume of 30 mI_. Next, the sample was loaded on to a Dionex 15 cm x 75 pm id Acclaim Pepmap C18, 2pm, 100 A reversed phase capillary chromatography column attached to a Finnigan LTQ-Obitrap Elite hybrid mass spectrometer system. Peptides were eluted with a gradient of 0.1 % (v/v) formic acid in acetonitrile at a flow rate of 0.3 pL/min and these were injected into the electrospray ionization source of the mass spectrometer on-line. The nano-electrospray ion source was operated at 1.9 kV. The digest was analyzed using the data-dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to obtain the amino acid sequence in successive instrument scans. Data were analyzed by using all CID spectra collected in the experiment to search whole mouse UniProtKB databases with the search program Sequest. Search parameters for peptide identification were as follows: minimum precursor mass, 350 Da; maximum precursor mass, 5000 Da; maximum missed cleavage sites, 2 for trypsin and 6 for chymotrypsin digestion; minimum peptide length, 6; maximum peptide length, 144; precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.8 Da; static modification, carbamidomethylation; dynamic modification, oxidation for trypsin and oxidation and phosphorylation for chymotrypsin digestion; maximum dynamic modifications per peptide, 4; and maximum Delta Cn (degree of match between the scores of the possible peptide spectral matches), 0.05. Sequest searches were also performed against five GB subtypes using the same parameters to confirm the identities of the peptides and the sequence coverage. GB subunit selectivity of Nb5.

HEK293T/17 cells were chosen for this analysis because of their high transfectability (PMID: 7690960). Cells were grown in culture medium (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum, MEM non-essential amino acids (Life Technologies), 1 mM sodium pyruvate, and antibiotics (100 units/ml penicillin and 100 pg/ml streptomycin) at 37°C in a humidified incubator containing 5% C02. For transfection, 6-cm culture dishes were coated during incubation for 10 min at 37°C with 2.5 ml of Matrigel solution (approximately 10 pg/ml growth factor-reduced Matrigel (BD Biosciences) in culture medium). Cells were seeded into the 6-cm dishes containing Matrigel solution at a density of 3.5 x 106 cells/dish. After 4 h, expression constructs (total 10 pg/dish) were transfected into the cells using PLUS (10 mI/dish) and Lipofectamine LTX (12 pl/dish) reagents. GaoA, Venus 1-155 GB, Venus-156-239-Gy2, masGRK3ct-Nluc, and Nanobody constructs were used at a 2:1 :1 : 1 : 12 ratio (ratio 1 = 0.42 pg of plasmid DNA). Empty vector pcDNA3.1 (+) was employed to normalize the amounts of transfected DNA. Cellular measurements of bioluminescence resonance energy transfer (BRET) between Venus-GBy and masGRK3ct-Nluc were performed to examine the GB selectivity of Nb5 in living cells. Sixteen to twenty- four hours post-transfection, HEK293T/17 cells were washed once with BRET buffer (PBS containing 0.5 mM MgCL and 0.1 % glucose) and detached by gentle pipetting with BRET buffer. Cells were harvested by centrifugation at 500 g for 5 min and resuspended in BRET buffer. Approximately 50,000 to 100,000 cells per well were distributed in 96-well flat bottomed white microplates (Greiner Bio-One). The Niue substrate, furimazine, purchased from Promega, was used according to the manufacturer's instructions. BRET measurements were made with a micro plate reader (POLARstar Omega; BMG Labtech). All measurements were performed at room temperature. The BRET ratio was determined by calculating the ratio of the light emitted by the Venus-G y (535 nm with a 30 nm band path width) over the light emitted by the masGRK3ct-Nluc (475 nm with a 30 nm band path width). BRET assays for real-time monitoring of cAMP and Ca²⁺ were performed in the same manner as described above. At approximately 16-24 h posttransfection, cells were stimulated with either 100 mM acetylcholine (ACh) or dopamine.

Stereotaxic injection.

All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee at Case Western Reserve University. To allow the expression of G protein-coupled inwardly rectifying potassium channels (GIRKs, Kir3.2) in striatal medium spiny neurons (MSNs), wild type (WT) C57BL6 mice (Jackson Laboratory) were injected with adeno-associated virus encoding mGIRK2 (AAV.GIRK2, TdTomato) into the dorsal striatum at postnatal day 21. Stereotaxic injections were performed while mice were under anesthesia. AAV.GIRK2, 300 nL, was injected into one hemisphere of the dorsal striatum. The injection coordinates were: AP +1.15 mm, ML +1.825 mm, DV -3.325 mm (relative to the bregma). Animals were allowed to recover for ~3 weeks after surgery.

Brain slice preparation.

Three weeks after surgery, mice were euthanized and coronal brain slices containing the striatum were made in ice cold cutting solution containing 75 mM NaCI, 2.5 mM KCI, 6 mM MgCL, 0.1 mM CaCL, 1.2 mM NaH2P04, 25 mM NaHC03, 2.5 mM D-glucose, and 50 mM sucrose bubbled with 95% O2 and 5% CO2. Slices were incubated for 1 h at 32 °C in artificial cerebrospinal fluid (ACSF) containing: 126 mM NaCI, 2.5 mM KCI, 1.2 mM MgCI₂, 2.4 mM CaCI₂, 1.2 mM NaH₂P0₄, 21.4 mM NaHCOs, 1 1.1 mM D- glucose and 10 pM MK-801 bubbled with 95% O2 and 5% CO2. Slices then were transferred to a recording chamber and perfused with ACSF at 32 °C containing picrotoxin (100 pM) and DNQX (10 pM).

Electrophvsioloqical recordings.

Striatal neurons were visualized with an Olympus BX51 fluorescence microscope, and GIRK2 expressing MSNs were identified by the presence of tdTomato. Electrical stimulation was applied with a monopolar extracellular stimulating electrode filled with ACSF. A single stimulation (1 ms, 20 - 35 pA) was used to evoke the release of neurotransmitters in the striatum. Whole-cell voltage clamp recordings were performed with an Axopatch 200B amplifier. Patch pipettes were filled with an internal solution containing 135 mM D-gluconate(K), 20 mM NaCI, 1.5 mM MgCI₂, 10 mM HEPES(K), pH 7.4, 10 mM BAPTA tetrapotassium, 1 mg/mL ATP, 0.1 mg/mL GTP, 1.5 mg/mL phosphocreatine, and 10 pM of either Nb5 or Nb17 (275 mOsm). Recordings were acquired with Axograph X (Axograph Scientific) at 10 kHz and filtered to 2 kHz for analysis. Neurons were held at -60 mV. No series resistance compensation was used, and neurons were discarded if the series resistance exceeded 15 MW.

Cell Culture and Transfection.

A Chinese hamster ovary cell-apelin receptor (CHO-APJ) stable cell line was established in the lab. Briefly, human apelin receptor (APJ) cDNA was synthesized (GenScript, Piscataway, NJ) and inserted into pLNCX2 (catalog no. 631503, Clontech, Inc.). Stable cell lines were generated by viral infection and G418 selection at a concentration of 800 pg/nnl. For expression of Nb5 and Nb17 in mammalian cells, cDNAs of the Nanobodies were sub-cloned into pcDNA3.1 (+) (Catalog no: V79020, Thermo Fisher Scientific, Inc) with a Flag tag at the C-terminus. CHO-APJ cells were transiently transfected with lipofectamine 2000 (Invitrogen). All cells were incubated overnight at 37 °C in serum-free medium containing 1 % BSA before treatment with 1 pm apelin or a vehicle control.

cAMP assay.

CHO-APJ cells expressing Nb5 or Nb17 were plated onto a 96-well plate at 80,000 cells/well and incubated at 37°C overnight in 5% CO2. On the day of the cAMP assay, cells were treated with IBMX at a final concentration of 750 mM for 20 min at room temperature. Cells then were treated with forskolin (20 mM), together with apelin-13 at various concentrations for 10 min. After these reactions, cells were lysed and their intracellular cAMP levels were measured according to the procedures of the CatchPoint Cyclic- AMP Fluorescent Assay Kit (Molecular Devices, Sunnyvale, CA USA). Fluorescence with excitation/emission at 530/590 nm was read with a Flexstation3 plate reader (Molecular Devices).

Statistics.

Differential HDX of Qb1g1 alone and the G 1y1-Nb5 complex was performed on three independent repeats and statistical significance was determined with two-tailed student’s f-test where P values of <0.01 were considered significant. The BRET assays were performed on six replicates where one-way ANOVA with Tukey’s post hoc multiple comparison test was applied relative to the Qb1 y2/GRK3ct control. The P values of <0.001 were considered significant and the results were expressed as mean ± SEM. Nb5 electrophysiological recordings were obtained from nine striatum neurons for the measurement of D2R- IPSCs and eight neurons for the measurements of M4R-IPSCs. Nb17 electrophysiological recordings were obtained from five striatum neurons for the measurement of D2R-IPSCs. The amplitudes obtained from individual recordings were then averaged and the significance ( P <0.05) was determined with two- tailed student’s f-test.

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Claims

1. An antibody or an active antibody fragment specifically binding the Qbg complex at an interface overlapping the Ga binding site.

2. The antibody or active antibody fragment of claim 1 , which binds the Qbg complex epitope comprising the amino acid residues 80-99 and 1 1 1-1 18 of the ΰbi-₄ subtypes as defined in SEQ ID NOs: 6-9.

3. The antibody or active antibody fragment of any one of claims 1 or 2, which promotes dissociation of Ga-GDP from the qbg complex.

4. The antibody or active antibody fragment of any one of claims 1 to 3, wherein said antibody has an affinity for the qbg complex corresponding to a KD between 5 nM and 50 nM.

5. The antibody or active antibody fragment of any one of claims 1 to 4, which is able to inhibit qbg signaling.

6. The antibody or active antibody fragment of any one claims 1 to 5, wherein said antibody is an immunoglobulin single variable domain (ISVD) comprising the amino acid sequence that comprises 4 framework regions (FR) and 3 complementary determining regions (CDR) according to the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

7. The ISVD of claim 6, comprising a CDR3 with SEQ ID NO: 1 , or an amino acid sequence with at least 80 % identity thereof.

8. The ISVD of claim 7, comprising SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:4, or a homologue with at least 80 % amino acid identity thereof, or a humanized variant of any one thereof.

9. The antibody or active antibody fragment of any one of claims 1 to 8, wherein said antibody or active antibody fragment bound to the qbg complex inhibits GIRK channel activation.

10. The antibody or active antibody fragment of any one of claims 1 to 8, wherein said antibody or active antibody fragment blocks activation of PI3K-AKT and/or activation of MAP ERK.

1 1. The antibody or active antibody fragment of any one of claims 1 to 10, further comprising a detection agent, such as a tag or a label, and/or a functional moiety, such as a BBB crossing moiety, and/or a cell penetrant carrier.

12. A nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or active antibody fragment of any one of claims 1 to 1 1.

13. An expression cassette comprising the nucleic acid molecule of claim 12.

14. The vector comprising the expression cassette of claim 13 or nucleic acid molecule of claim 12.

15. A solid substance comprising the antibody or active antibody fragment of any one of claims 1 to 1 1.

16. Use of an antibody or active antibody fragment of any one of claims 1 to 1 1 , or the solid substance according to claim 15, for affinity chromatography, affinity purification, immunoprecipitation, in- vivo imaging, protein detection, immunochemistry, surface-display, FRET-type applications or for structural analysis.

17. Use of an antibody or active antibody fragment of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, expression cassette of claim 13, the vector of claim 14, or the solid substance of claim 15, as a tool to differentiate Ga from Qbg signaling.

18. The antibody or active antibody fragment of any one of claims 1 to 1 1 , the nucleic acid molecule of claim 12, expression cassette of claim 13, the vector of claim 14, or the solid substance of claim 15, for use as a medicament or as a diagnostic.

19. A host cell comprising the antibody or active antibody fragment of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, expression cassette of claim 13, the vector of claim 14.

20. A method for identifying a compound that modulates G protein signaling, comprising the steps of: a. Providing the host cell of claim 19, transfecting said cell with the GPCR of interest b. Adding a test compound

c. Evaluating the effect of said test compound on G protein signaling in said cell as compared to a cell without the test compound.