Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• 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 ⁇ bg- ⁇ 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.
• G protein-coupled receptors 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 protein 6 ⁇ 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 ⁇ bg-e ⁇ hqI ⁇ is a central element of various neurological and cancer-related anomalies.
• 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.
• heterotrimeric G proteins maintain an equilibrium between their heterotrimeric and dissociated states by undergoing spontaneous GPCR-independent association/dissociation 11 13 .
• 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 modulators 14 . 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, respectively 15 .
• 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, respectively 15 .
• drug development for GPCR signaling pathways has been hampered by difficulties in identifying molecules with suitable selectivity 18 .
• Related GPCRs share a promiscuous ligand binding site for lipophilic ligands 19 , which allows several off-target effects when pursued therapeutically.
• mAbs monoclonal antibodies
• CNS central nervous system
• Antibody alternatives are now known as versatile tools for exploring the determinants of GPCR recognition.
• Nanobodies ® derived from the variable region of camelid heavy chain are endowed with favorable characteristics in terms of size, solubility, affinity and ease of production 21 .
• GPCR GPCR
• therapeutic Nbs are being discovered that target GPCR signaling 26 .
• b-arrestin specific antibody fragments all small-molecule-, antibody- and Nanobody-based approaches target GPCR-mediated signaling at the GPCR level 26 ⁇ 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.
• PI3K phosphoinositide- 3-kinase
• MAPK mitogen-activated protein kinase
• 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.
• Gt heterotrimeric visual G protein
• Differential hydrogen/ deuterium exchange and crystallography studies suggest a competition between the Nb and other ⁇ bg- regulatory proteins for a common binding site on the qbg dimer.
• binding was shown for qb subtypes 1-4, showing its broad applicability to various cell types.
• Nanobodies respond to all combinations of b- and g-subtypes and compete with other ⁇ bg- ⁇ uIqIqGg proteins for a common binding site on the qbg dimer.
• the Nb-binding suppresses the activation of both the ⁇ bg- regulated G-protein-gated inward rectifier potassium channels and the Gbg-mediated phosphoinositide- 3-kinase and mitogen-activated protein kinase pathways.
• the Nb-binding to qbg has no effect on Ga-GTP-mediated signaling events, i.e.
• 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.
• the invention relates to an antibody or an active antibody fragment specifically binding the ⁇ bg complex at an interface overlapping the Ga binding site.
• said antibody or active antibody fragment binds the ⁇ bg complex epitope conserved among the ⁇ bi- 4 subtypes, comprising the amino acid acids 80-99 and 1 1 1-1 18 of said ⁇ b subtypes b1-4.
• said antibodies or active antibody fragments promote dissociation of Ga-GDP from the ⁇ bg complex, upon GPCR activation.
• the antibody or active antibody fragment of the invention has an affinity for the ⁇ bg 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 ⁇ bg such as PDC and GRK2.
• said antibody or active antibody fragment is able to inhibit ⁇ bg signaling.
• said antibody or active antibody fragment is able to selectively inhibit ⁇ bg signaling, without affecting Ga signaling.
• 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 ⁇ bg complex activity.
• ISVD immunoglobulin single variable domain
• Another embodiment relates to said antibody or active antibody fragment of the invention bound to the ⁇ bg 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.
• 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.
• a detection agent such as a tag or a label
• a functional moiety such as a blood-brain-barrier (BBB) crossing moiety, or comprises a cell penetrant carrier, or any combination thereof.
• BBB blood-brain-barrier
• 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.
• 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.
• 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.
• 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.
• 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.
• a host cell comprising the antibody or active antibody fragment, the nucleic acid molecule, the expression cassette or the vector of the invention.
• 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.
• Nb5 alone can trap ⁇ bigi after its spontaneous dissociation from Gat, and thereby shifts the dynamic equilibrium of heterotrimeric Gt towards its dissociated subunits.
• Nb17 did not affect the heterotrimeric configuration of Gt (lanes 7 and 8, compare lanes marked with asterisks). Additionally, non-specific interactions between ⁇ b-igi and Ni 2+ -NTA resin were not seen (lanes 9 and 10).
• the eluate and flowthrough obtained from affinity chromatography purification are denoted as ⁇ ” and“FT”, respectively.
• 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).
• Binding of ⁇ b-igi to immobilized Nb5 in SPR equilibrium binding experiments 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 ⁇ b-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 ⁇ b-igi (PDB ID: 5KDO) indicate peptides with increased (greencyan) and reduced (purple) deuterium incorporation upon Nb5 binding.
• ⁇ b-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.
• the asymmetric unit contains two molecules of both ⁇ b-igi (blue and pink) and Nb5 (greencyan and red) shown in cartoon representation. Key interfaces between ⁇ b-igi and Nb5 are denoted by grey ellipsoids (b) Side and top views of the Gb 1 g 1 -Nb5 complex showing the CDR3 region (red) of Nb5 inserted into the ⁇ b-i-rGorbIIbG. The 2
• ⁇ bg hot-spot is shared by several ⁇ bg regulatory proteins, including Gat (pink), phosducin (PDC, cyan) and G Protein-Coupled Receptor Kinase 2 (GRK2, dark grey)
• PDC phosducin
• GRK2 G Protein-Coupled Receptor Kinase 2
• the maximum BRET signal was determined by co-transfection of different nbhue-qb subtypes + GY ⁇ 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 ⁇ 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
• Nb5 as a control -switch for GPCR-mediated Qbg signaling.
• (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.
• Gb1g1-Nb5 was crystallized in space group, P21 , where a majority of crystal contacts were mediated through Nb5 within the aqueous layers
• Each asymmetric unit is composed of two Gb1g1-Nb5 molecules that form crystal contacts with neighboring molecules
• 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
• 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: 10MW 53 ).
• Figure 8. Effect of Nb5 on GPCR-mediated Qbg signaling.
• Figure 9 Full Western blots.
• “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.
• polypeptide refers to a polymer of amino acid residues and to variants and synthetic analogues of the same.
• 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).
• recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
• isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
• 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.
• amino acid identity refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison.
• 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.
• 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
• protein complex refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein.
• a protein complex 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.
• 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.
• 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.
• 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”).
• antibody 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.
• 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.
• antibody refers 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.
• 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.
• IVDs immunoglobulin variable domain(s)
• a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site.
• VH heavy chain variable domain
• VL light chain variable domain
• 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.
• the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
• a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
• a Fab fragment such as a F(ab')2 fragment
• an Fv fragment such as a disulphide linked Fv or a scFv fragment
• a diabody all known in the art
• 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).
• the subject is a human, a rat or a non-human primate.
• the subject is a human.
• a subject is a subject with or suspected of having a disease or disorder, or an injury, also designated’’patient” herein.
• a subject is a subject ready to receive a transplant or allograft, also designated as a“patient eligible for receiving an allograft”.
• 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.
• 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.
• GDP guanosine diphosphate
• GTP guanosine triphosphate
• 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 Ca 2+ and RhoA GTPases.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• a particular structure e.g., an antigenic determinant or epitope
• an antigen-binding protein or antibody recognizes and binds to a specific protein structure rather than to proteins generally.
• 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.).
• 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.
• 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.
• 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.
• 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).
• said epitope is formed as a conformational binding site, and has not been described as such for any naturally occurring Qb o ⁇ ⁇ bg binder.
• the conformational epitope 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• said antibody or active antibody fragment binds human ⁇ b1-4 (SEQ ID NO:6- 9), or bovine ⁇ b1-4 (b1 represented herein by SEQ ID NO: 15) , or mouse ⁇ b1-4 (SEQ ID NO: 10-13), or mammal ⁇ b1-4, or ⁇ b subtypes 1-4 in any subject.
• said antibody or active antibody fragment does not bind ⁇ b5 (SEQ ID NO: 14).
• a preferred embodiment relates to the antibody or active antibody fragment wherein the epitope is present on the ⁇ bg complex, on the ⁇ b subtypes b1 -4 as presented in the amino acid sequences of human ⁇ b1-4, as provided by SEQ ID NO:6-9.
• 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.
• 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.
• 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.
• 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 10 7 M, or at least 10 8 M, or most preferable at least 10 9 M.
• affinity 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.
• 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.
• the dissociation constant has a value that is lower than 10 5 M.
• the dissociation constant is lower than 10 6 M, more preferably, lower than 10 7 M.
• the dissociation constant is lower than 10 8 M, or even lower than 10 9 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• qbg dimers are involved in multiple aspects of GPCR-mediated signaling and regulation.
• 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.
• Qbg effectors 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.
• AC adenylyl cyclase
• GRK2 G protein-coupled receptor kinase 2
• PLC phospholipase C
• PI3K_ phospholipase C
• A/-type calcium channels 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 for 33 . Many of the Qbg- ⁇ q ⁇ b ⁇ 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.
• GRK2ct C terminus of GRK2
• QEHA sequence derived from ACM
• small molecules that bind to qbg M1 19/gallein
• 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.
• 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.
• the antibody or active antibody fragment of the present invention has been shown to bind different subtypes of qb, ⁇ b1-4, which are present in different cell types, and therefore, a number of target downstream of ⁇ b1-4 is within the scope of the antibody or active antibody fragment of the invention.
• 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.
• GIRK G-protein-gated inward rectifier potassium
• 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.
• 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.
• PI3K phosphoinositide-3- kinase
• another embodiment discloses an antibody or active antibody fragment of the invention, wherein said qbg binding upon apelin receptor activation blocks MAP/ERK pathways.
• both PI3K-AKT and MAP/ERK are inhibited when the antibody or active antibody fragment is present.
• 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.
• nanobodies as small protein binders preferentially targeting conformational epitopes
• 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.
• 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.
• 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.
• the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.
• 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).
• a light chain variable domain sequence e.g., a VL-sequence
• a heavy chain variable domain sequence e.g., a VH-sequence or VHH sequence
• 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.
• 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.
• 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.
• the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof.
• Nanobody ® , Nanobodies ® and Nanoclone ® are registered trademarks of Ablynx N.V.
• VHH domains also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies
• Ig antigen binding immunoglobulin
• 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”).
• VHHs and Nanobody 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.
• Nanobody in particular VHH sequences and partially humanized Nanobody
• 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 antibody 21 .
• 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 .
• CDR3 complementarity-determining region 3
• 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.
• said ISVD of the invention has CDR3 with an amino acid sequence of SEQ ID NO: 1.
• 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.
• 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.
• 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.
• said ISVD of the invention comprises Nb5, represented by SEQ ID NO:2.
• 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.
• 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.
• a detection agent such as a tag or a label.
• 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.
• detectable label or tag 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.
• 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, biolumin
• any of the foregoing labels or tags 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.
• 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.
• the antibody or active antibody fragment may further comprise a functional moiety, such as for instance a Blood-brain-barrier crossing moiety.
• the antibody or active antibody fragment may further comprise a cell penetrant carrier, such as examples.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• a therapeutically active agent has a curative effect on the disease.
• Such functional groups 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).
• a suitable pharmacologically acceptable polymer such as poly( ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly( ethyleneglycol) or mPEG).
• 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).
• 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
• fusions of antibody fragments in particular immunoglobulin single variable domains
• peptides for example, a peptide against a serum protein such as albumin
• NPs nanoparticles
• BBB blood brain barrier
• NPs nanoparticles
• 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).
• 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.
• the molecule or polypeptide
• the molecule 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.
• protein transduction domains PTDs
• 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.
• TAT protein from human immunodeficiency virus (HIV- 1 )
• HAV- 1 human immunodeficiency virus
• polyarginine sequence a polyarginine sequence
• penetratin a polyarginine sequence
• a short amphipathic peptide carrier Pep-1.
• Still other commonly used cell-permeable peptides are disclosed in Joliot A. and Prochiantz A. (2004) Nature Cell Biol. 6 (3) 189-193.
• 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 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.
• 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.
• Codon 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.
• 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”.
• vector refers 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).
• plasmid vectors such as 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.).
• 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 extraordinarly 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.
• intrabodies are used in genetically engineered antibody fragment format and structures of scFv intrabodies, single domain intrabodies, or bispecific tetravalent intradiabodies.
• scFv intrabodies single domain intrabodies
• 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).
• 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.
• a host cell 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 ⁇ bg-erb ⁇ o 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.
• several downstream signaling events can easily be evaluated by including a reporter or assay component in the method of interest.
• detection of effects of downstream Ga signaling include the detection of increase of intracellular Ca 2+ 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• silica porous amorphous silica
• 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.
• macroporous polymers such as the pressure-stable Affi-Prep supports as supplied by Bio-Rad.
• supports that could be utilised include; dextran, collagen, polystyrene, methacrylate, calcium alginate, controlled pore glass, aluminium, titanium and porous ceramics.
• the solid surface may comprise part of a mass dependent sensor, for example, a surface plasmon resonance detector.
• a mass dependent sensor for example, a surface plasmon resonance detector.
• Immobilization may be either non-covalent or covalent.
• 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.).
• 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.
• 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.
• immobilization methods of antibody (fragments) on solid supports are discussed in Jung et al. (2008, Analyst. 133(6):697-701 ).
• 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.
• 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.
• a test sample i.e. comprising the test compound, amongst other
• the immobilized antibody or active antibody fragments of the present invention can be arrayed or otherwise multiplexed.
• 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 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.
• said antibody or active antibody fragment of the invention 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.
• 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.
• 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.
• 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.
• 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.
• the terms“disease” or“disorder” refer to any pathological state, in particular to the diseases or disorders as defined herein.
• 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 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.
• 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.
• 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.
• compositions including the antibody or active antibody fragment of the invention, or nucleic acid molecule, expression cassette, or vector of the invention.
• 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.
• “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” means the amount needed to achieve the desired result or results.
• an “effective amount” can vary depending on the identity and structure of the compound of the invention.
• 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.
• 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.
• 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.
• kits 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.
• said kit is provided which contains means including a nucleic acid molecule, an expression cassette or a vector or solid substance of the invention.
• kits 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.
• 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.
• 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 range 104 .
• 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.
• antibodies or active antibodies fragment of the invention such as the exemplified Nb5
• 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 Nb5 105 - 107 . While bARK-ct and PDC have a large area of interaction with the Qbg dimer (>1000 A 2 ), no atomic level information is available for SIRK or other peptides except that they bind to the same Qbg hot spot with high nM affinities 3941 and selectively inhibit the binding of Qbg effector molecules.
• 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 weeks 109 ’ 110 , increasing their therapeutic applications from acute to chronic indications.
• Example 1 Generation of Nanobodies directed towards ⁇ b1g1.
• 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.
• all 3 qb ⁇ g ⁇ -roe ⁇ nb 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).
• Nanobody with highest expression levels 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.
• ROS bovine rod outer segments
• Example 2 Nb5 shifts the equilibrium of heterotrimeric G t towards dissociated Ga t and Nb5-bound Gpiy1 subunits.
• the low-nanomolar affinity of Nb5 for Qb1g1 means a possible competition between Nb5 and other Qb1g1 regulatory proteins that bind Qb1g1 with similar affinities 47 ⁇ 48 .
• Differential hydrogen/deuterium exchange (HDX) was used to investigate the binding dynamics between Qb1g1 and Nb5.
• Full HDX profiles of qb ⁇ g ⁇ alone were compared to those of the 6b1g1-I ⁇ 5 complex (Fig. 2c, d).
• 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).
• PDC phosducin
• GRK2 G Protein-Coupled Receptor Kinase 2
• insertion of the Nb5 CDR3 loop into the qbI-rGorb ⁇ bG is reminiscent of the interaction between the C-terminal loop of GRK2 and Qb1g2 dimer (interface area of 1080 A2; PDB accession: 10MW 53).
• 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).
• Table 1 Sequences of Qb1g1 peptide fragments showing normalized deuterium uptake for the Qb1g1 complex alone and the Qb1g1 complex bound to Nb5.
• 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 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.
• 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.
• Nb17 had no effect on the BRET ratio suggesting no interaction with qb subtypes (Fig. 4e, purple).
• western blot analyses of the transfected components were performed to verify similar expression levels of the exogenous proteins (Fig. 4f).
• 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.
• PSMs peptide spectrum matches
• Example 5 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
• D2Rs D2Rs
• M4Rs 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.
• D2R- IPSC D2R-mediated inhibitory post-synaptic current
• 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.
• 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.
• Nb5 acts as a control -switch for GPCR-mediated Qbg signaling.
• PI3K-PKB/AKT phosphoinositide-3-kinase
• MAPK mitogen activated protein kinase
• ERK extracellular signal-regulated kinase
• a Chinese hamster ovary cell line stably expressing human apelin receptor 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).
• 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 .
• 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 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).
• Nb5 modulates the apelin-induced inhibition of cAMP accumulation in CHO-APJ cells.
• 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).
• Ga q signaling was monitored in HEK293T/17 cells transfected with the M3 muscarinic acetylcholine receptor (M3R) and using a Ca 2+ 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.
• Nb5 had no effect on the elevation of intracellular Ca 2+ 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.
• 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.
• 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.
• 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.
• Nb5 a Nanobody against Qb1 antigen, can cause selective inhibition of Gbg-mediated signaling while leaving basal Go mediated signaling intact.
• 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.
• Nb5-mediated qb purification from mouse brain and cell-based BRET assays demonstrate the specificity of Nb5 towards qb subtypes 1-4.
• Nb5 As a potential negative regulator of qbg signaling in various cell types.
• Nb5 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 ⁇ hq ⁇ 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.
• AC adenylyl cyclase
• recent studies have shown that Qbg can stimulate several AC isoforms through cross-talk between Ga- and Gbg-mediated cAMP signaling 38 ’ 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.
• Nb5 showed no significant effect on either the M3R-Gaq-induced intracellular Ca 2+ 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.
• Nb5 serves as a dynamic scavenger of the ⁇ bg dimer and thereby causes partial inhibition of Gbg-mediated signaling.
• the remaining Nb5-free ⁇ bg 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.
• Nb5 might affect the Ga-GTP signaling component due to increased sequestration of qbg dimers (Fig. 1 d,e).
• Nanobodies act as crystallization chaperones by either stabilizing intrinsically flexible regions or shielding aggregating surfaces in a protein is well established 72 ⁇ 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.
• 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.
• 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 ⁇ bg-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.
• 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 penetratin 74 .
• 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 vesiclesl 75 or Trojan horse technology using transferrin and insulin receptors 76 .
• 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.
• 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.
• 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
• 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 previously 57 . 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 Core 60 ’ 64 .
• Nanobodies against the Qb1g1 dimer were prepared by a previously published protocol 21 . Briefly, a llama (Lama glama) was immunized weekly for six weeks with 1 mg of purified bovine ⁇ b1 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.
• 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 ⁇ b1g1 dimer resulted in 14 families wherein all Nanobodies were produced as soluble His-tagged proteins in the E. coli periplasmic region 21 .
• 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.
• 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.
• 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-Ni 2+ 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 was purified by a protocol described previously 82 . Briefly, native Rh membranes were solubilized by a zinc/alkyl-glucoside extraction method and centrifuged at 100,000 for 40 min to extract Rh 83 .
• Bovine rod outer segments were prepared as described elsewhere 78 ⁇ 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.
• 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
• 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.
• the hypotonic solution was loaded onto a 010/10 column (GE Healthcare) with 6 ml_ of pre-equilibrated propyl-agarose resin.
• ROS were resuspended in isotonic buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCI, 1 mM DTT and 5 mM MgC .
• 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.
• hypotonic extracts 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- Ni 2+ affinity chromatography.
• Ni 2+ -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 elsewhere 84 ⁇ 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.
• Bovine ROS were washed with isotonic buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCI, 1 mM DTT and 5 mM MgCL.
• isotonic buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCI, 1 mM DTT and 5 mM MgCL.
• 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.
• 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.
• 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 Ni 2+ -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.
• 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 method 89 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. 5.8 92 refinement against the G 1y1-Nb5 complex dataset and manual model adjustments with Coot 0.8.8 95 .
• 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 Molprobity 96 ⁇ 97 and wwPDB validation servers 98 . 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
• 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.
• 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.
• Thermo Finnigan LXQ Thermo Scientific, Waltham, MA
• 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.
• 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.
• 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.
• 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 HXExpress 102 .
• 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_ min 1 . 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.
• GE Biacore T100 Evaluation Software
• G proteins were extracted from mouse brain using a protocol described elsewhere with a few modifications 103 .
• 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.
• 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 Ni 2+ - NTA purified Gbg-Nb5 complex was desalted and loaded onto 500 pi of Talon resin packed into a PierceTM 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.
• the excised SDS-PAGE band was destained in a solution containing 50% (v/v) ethanol with 5% (v/v) acetic acid in water.
• 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_.
• 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.
• 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).
• BRET bioluminescence resonance energy transfer
• ACh acetylcholine
• G protein-coupled inwardly rectifying potassium channels GIRKs, Kir3.2
• GIRKs G protein-coupled inwardly rectifying potassium channels
• MSNs striatal medium spiny neurons
• wild type (WT) C57BL6 mice Jackson Laboratory
• AAV.GIRK2, TdTomato adeno-associated virus encoding mGIRK2
• SA adeno-associated virus encoding mGIRK2
• AAV.GIRK2 300 nL
• 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.
• 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 MgCI 2 , 2.4 mM CaCI 2 , 1.2 mM NaH 2 P0 4 , 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).
• ACSF artificial cerebrospinal fluid
• 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 MgCI 2 , 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.
• CHO-APJ Chinese hamster ovary cell-apelin receptor
• APJ human apelin receptor
• 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.
• pcDNA3.1 (+) Catalog no: V79020, Thermo Fisher Scientific, Inc
• 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.
• 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.
• 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).
• 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.
• GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325, 321-326 (1987).
• RhoA-dependent fashion Dev Cell 19, 845-857 (2010).
• NRAMP transition-metal ion transporter
• rrens D.L., Altenbach, C., Yang, K., Hubbell, W.L. & Khorana, H.G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274, 768-770 (1996).
• TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex. Nat Microbiol 2, 17103 (2017).
• Dynamic peptides of human TPP1 fulfill diverse functions in telomere maintenance.
• VNAR Variable New Antigen Receptor
• Gbeta-subunits eliminates signaling mediated by all four families of G proteins. Proceedings of the National Academy of Sciences of the United States of America 102, 9493-9498 (2005).

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