Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
• • 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
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
  • G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
• • 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
  • C07K2317/565—Complementarity determining region [CDR]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4719—G-proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

• the present invention relates to novel antibodies or antibody fragments capable of binding to the G alpha protein.
• G proteins are heterotrimeric proteins (3 subunits: alpha, beta and gamma) which are activated by RCPGs.
• GPCRs Through GPCRs, the G proteins have a role of transducing a signal from outside the cell to the inside of the cell (i.e. cellular response to an external stimulus). Their commonly described mechanism of action is summarized below:
• the alpha subunit of the G protein is bound to the nucleotide GDP (full G protein bound to GDP);
• the latter binds to the alpha subunit of the G protein and initiates a process of activation of the G protein consisting of two steps: 1) the release of GDP from the G protein to give a G protein empty, and the formation of an inactive RCPG / protein-G-empty complex, and 2) the binding of GTP which leads to the formation of an active G protein, in the form of GTP (full G protein bound to GTP).
• the G protein bound to the receptor is in a form called “empty form”. This state is described in the literature as being transient since it is described that the GTP nucleotide binds rapidly to the alpha subunit of the G protein.
• the beta / gamma subunits of the activated G protein dissociate from the alpha subunit;
• the alpha subunit of the full G protein bound to GTP then binds to the effectors to activate them.
• the effectors in turn activate signaling pathways leading to a cellular response;
• Antibodies have also been synthesized to detect the activation of GPCRs at the cell surface level [1]. Reference may also be made to patent EP2723764 B1 which proposes nano-antibodies which bind to the interface between the alpha G protein and the beta / gamma G protein, making it possible to stabilize the RCPG / G protein complex.
• the inventors have developed antibodies or fragments of antibodies for detecting the activation of an RCPG by the implementation of a RET method which uses a GTP labeled by a member of a pair of partners by RET.
• the inventors have also shown that these antibodies or antibody fragments have these properties because they are capable of binding to the G alpha protein in a very particular area.
• a first object of the present invention relates to an antibody or an antibody fragment capable of binding to the G alpha protein, which comprises:
• variable domain of a heavy chain comprising a CDR1 of amino acid sequence SEQ ID NO: 1, a CDR2 of amino acid sequence SEQ ID NO: 2, and a CDR3 of amino acid sequence SEQ ID NO : 3, and
• variable domain of a light chain comprising a CDR1 of amino acid sequence SEQ ID NO: 4, a CDR2 of DTS amino acid sequence (ie the three amino acids Asp Thr Ser), and a CDR3 of sequence d amino acids SEQ ID NO: 5.
• a second object of the present invention relates to an antibody or an antibody fragment which competes for binding to the alpha G protein with the antibody or antibody fragment of the first object of the invention.
• a third subject of the present invention relates to a composition comprising the antibody or the antibody fragment according to the invention.
• a fourth object of the present invention relates to a kit-of-parts (kit of reagents in French) comprising (i) the antibody or the antibody fragment according to the invention or the composition according to the invention and ( ii) a source of GTP labeled with a member of a pair of RET partners.
• a fifth object of the present invention relates to a nucleic acid sequence encoding the antibody or the antibody fragment according to the invention.
• a sixth object of the present invention relates to a vector comprising the nucleic acid sequence according to the invention.
• a seventh object of the present invention relates to a cell comprising the vector according to the invention or the nucleic acid sequence according to the invention.
• anti-G alpha protein antibody "anti-G alpha antibody” or “antibody capable of binding to the G alpha protein” (or antibody fragment) are interchangeable and denote an antibody (or a antibody fragment) which binds to the G alpha protein with sufficient affinity to be used as a detection agent (e.g. for the implementation of a RET), diagnostic and / or therapeutic by targeting the G alpha protein .
• a detection agent e.g. for the implementation of a RET
• antibody also called “immunoglobulin” denotes a heterotetramer consisting of two heavy chains of about 50-70 kDa each (called the H chains for Heavy) and two light chains of about 25 kDa each ( say L chains for Light), linked together by intra- and inter-chain disulfide bridges.
• Each chain consists, in the N-terminal position, of a region or variable domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, consisting of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4, for the heavy chain.
• Each variable domain generally comprises 4 “hinge regions” (called FRI, FR2, FR3, FR4) and 3 regions directly responsible for binding with the antigen, called “CDR” (called CDR1, CDR2, CDR3).
• the antibody may be, for example, a mammalian antibody, such as a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.
• chimeric antibody is meant an antibody in which the sequences of the variable regions of the light chains and of the heavy chains belong to a different species from that of the sequences of constant regions of the light chains and of the heavy chains.
• the sequences of the variable regions of the heavy and light chains are preferably of murine origin while the sequences of the constant regions of the heavy and light chains belong to a non-murine species.
• all species of non-murine mammals are likely to be used, and in particular man, monkey, suidae, bovidae, equidae, felidae, canidae or even birds, this list not being exhaustive.
• the chimeric antibodies according to the invention contain sequences of constant regions of heavy and light chains of human origin and sequences of variable regions of heavy and light chains of murine origin.
• humanized antibody is meant an antibody of which all or part of the sequences of the regions involved in the recognition of the antigen (the hypervariable regions or CDR: Complementarity Determining Region) and sometimes certain amino acids of the FR regions (regions Framework) are of non-human origin while the sequences of constant regions and variable regions not involved in antigen recognition are of human origin.
• human antibody is meant an antibody containing only human sequences, both for the variable and constant regions of the light chains and for the variable and constant regions of the heavy chains.
• antibody fragment means any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction comprising at least one disulfide bridge and which is capable of binding to the antigen recognized by the whole antibody , for example Fab, Fab ', F (ab') 2 , Fab'-SH.
• the enzymatic digestion of immunoglobulins by papain generates two identical fragments, which are called Fab fragments (Fragment antigen binding), and an Fc fragment (Crystallizable fragment).
• Enzymatic digestion of immunoglobulins by pepsin generates an F (ab ') 2 fragment and an Fc fragment split into several peptides.
• F (ab ') 2 is formed from two Fab' fragments linked by inter-chain disulfide bridges.
• the Fab parts consist of the variable regions and the CH1 and CL domains.
• the Fab 'fragment consists of the Fab region and of a hinge region.
• Fab'-SH refers to an Fab 'fragment in which the cysteine residue of the hinge region carries a free thiol group.
• affinity refers to the strength of all the non-covalent interactions between a molecule, for example an antibody or an antibody fragment and the recognized antigen, for example an antigen such as the protein.
• G alpha. Affinity is generally represented by the dissociation constant (Kd).
• the dissociation constant (Kd) can be measured by well known methods, for example in FRET or in SPR.
• identity is calculated by comparing two sequences aligned in a comparison window.
• the alignment of the sequences makes it possible to determine the number of positions (nucleotides or amino acids) in common for the two sequences in the comparison window.
• the number of positions in common is therefore divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of identity.
• the determination of the percent sequence identity can be done manually or by well known computer programs.
• the identity or the homology corresponds to at least one substitution, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions , of an amino acid residue, preferably at least one substitution of an amino acid residue carried out conservatively.
• "Conservatively accomplished amino acid residue substitution” involves replacing one amino acid residue with another amino acid residue, having a side chain having similar properties.
• the families of amino acids possessing side chains with similar properties are well known, one can quote for example the basic side chains (eg, lysine, arginine, histidine), the acid side chains (eg, aspartic acid, glutamic acid) , polar and uncharged side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine , tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
• the basic side chains eg, lysine, arginine, histidine
• the acid side chains eg, aspartic
• the antibodies or fragments of homologous antibodies or "variants of antibodies or of fragments of antibodies” that is to say antibodies or fragments of antibodies having the same function
• a person skilled in the art uses his general knowledge to determine the number of substitutions which can be made and their location in order to be able to preserve the function of the antibody or of the antibody fragment.
• the antibodies or antibody fragments can therefore be tested by binding methods, such as, for example, the ELISA method, the affinity chromatography method, etc.
• the antibody variants or antibody fragments can be generated, for example, by the “phage display” method making it possible to generate a phage library.
• a large number of methods are known in order to generate a “phage display” library and target the antibody variants or antibody fragments having the desired functional characteristics.
• purified and isolated is meant, with reference to an antibody or an antibody fragment according to the invention, that the antibody is present in the substantial absence of other biological macromolecules of the same type.
• purified as used herein preferably means at least 75 wt%, more preferably at least 85 wt%, still more preferably at least 95 wt%, and most preferably at least 98 wt%. antibody, relative to all the macromolecules present.
• G protein denotes a hetero-trimeric protein composed of three subunits called G alpha protein, G beta protein and G gamma protein.
• G alpha protein designates the alpha subunit of the G protein.
• the G alpha protein have two domains, the GTPase domain, and the alpha helix domain.
• G alpha proteins There are at least 20 different G alpha proteins, which can be classified into the following major protein families: G alphas (known to activate adenylate cyclase to increase cAMP synthesis), G alphai (known to inhibit adenylate cyclase), G alphaolf (associated with olfactory receptors), G alphat (known to transduce visual signals in the retina in conjunction with rhodopsin), G alphaq (known to stimulate phospholipase C) or the G alpha family !
• the G alpha protein can be chosen from the G alphai, G alphao and / or G alphaz protein.
• the G alphai protein can be chosen from the G alphail, G alphai2 and G alphai3 protein.
• the alphai G protein can be of human or animal origin.
• the antibody or the antibody fragment according to the invention binds to the G alphai, G alphao and / or G alphaz protein, for example it binds to the G alphail protein, to the G alphai2 protein. and / or the G protein alphai3.
• the alphail G protein of human origin carries the identifier UniProt P63096-1 for isoform 1 and the UniProt identifier P63096-2 for isoform 2.
• the gene encoding the G alphail protein of human origin is known as the name "GNAI1" (Gene ID: 2770, NCBI).
• GTP denotes guanosine triphosphate
• non-hydrolyzable or slowly hydrolyzable GTP denotes an analogue of GTP which is not hydrolyzed or only slightly hydrolyzed to GDP. Mention may be made, for example, of GTPgammaS (CAS no. 37589-80-3), GppNHp (CAS no. 148892-91-5) or GppCp (CAS no. 10470-57-2).
• non-hydrolyzable or slowly hydrolyzable GTP labeled with a member of a RET partner pair or “labeled non-hydrolyzable or slowly hydrolyzable GTP” are interchangeable and denote either a non-hydrolyzable or slowly hydrolyzable GTP labeled with a member of a donor RET partner pair ("donor GTP”), either a non-hydrolyzable or slowly hydrolyzable GTP labeled by a member of an acceptor RET partner pair (“acceptor GTP”)
• RET Resonance Energy Transfer
• FRET Fluorescence Resonance Energy Transfer
• FRET is defined as a non-radiative energy transfer resulting from a dipole-dipole interaction between an energy donor and an acceptor. This physical phenomenon requires energy compatibility between these molecules. This means that the donor's emission spectrum must overlap, at least partially, the absorption spectrum of the acceptor.
• FRET is a process which depends on the distance separating the two molecules, donor and acceptor: when these molecules are at proximity to each other, a FRET signal will be emitted.
• the dissociation constant (Kd) between an antibody and its target can be measured by FRET, for example as described in the examples.
• BRET (from the English “Bioluminescence Resonance Energy Transfer”) designates the transfer of energy between a bioluminescent molecule and a fluorescent molecule.
• a first object of the invention relates to an antibody or an antibody fragment capable of binding to the G alpha protein, which comprises:
• variable domain of a heavy chain comprising a CDR1 of amino acid sequence SEQ ID NO: 1, a CDR2 of amino acid sequence SEQ ID NO: 2, and a CDR3 of amino acid sequence SEQ ID NO : 3, and
• variable domain of a light chain comprising a CDR1 of amino acid sequence SEQ ID NO: 4, a CDR2 of DTS amino acid sequence (ie the three amino acids "Asp Thr Ser", that is to say the three amino acids aspartic acid, threonine and serine), and a CDR3 of amino acid sequence SEQ ID NO: 5.
• variable domain of the heavy chain can include:
• an IRF exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 6,
• an FR2 exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 7,
• an FR3 exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 8, and / or
• variable domain of the light chain can include:
• an IRF exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 10,
• an FR2 exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 11,
• an FR3 exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 12, and / or
• an FR4 exhibiting at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or even 100% homology with the amino acid sequence SEQ ID NO: 13.
• variable domain of the heavy chain includes:
• variable domain of the light chain includes:
• variable domain of the heavy chain can have at least 80% homology, preferably at least 90% homology, for example at least 95 % homology, at least 96%, at least 97%, at least 98%, at least 99% or 100% homology with the amino acid sequence SEQ ID NO: 14, and the variable domain of the chain slight can have at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99 % or 100% homology with the amino acid sequence SEQ ID NO: 15.
• the invention relates to an antibody or antibody fragment in which:
• variable domain of the heavy chain has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98 %, at least 99% or 100% homology homology with the amino acid sequence SEQ ID NO: 14;
• variable domain of the light chain has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98 %, at least 99% or 100% homology homology with the amino acid sequence SEQ ID NO: 15; and
• the CDR1 of the heavy chain variable domain consists of the amino acid sequence SEQ ID NO: 1
• the CDR2 of the heavy chain variable domain consists of the amino acid sequence SEQ ID NO: 2
• the CDR3 of the heavy chain heavy chain variable domain consists of the amino acid sequence SEQ ID NO: 3
• the CDR1 of the light chain variable domain consists of the amino acid sequence SEQ ID NO: 4
• the CDR2 of the variable domain of the light chain consists of the amino acid sequence DTS
• CDR3 of the light chain variable domain consists of the amino acid sequence SEQ ID NO: 5.
• variable domain of the heavy chain consists of the amino acid sequence SEQ ID NO: 14 (ie the variable domain of the heavy chain has 100% homology with the amino acid sequence SEQ ID NO : 14) and the variable domain of the light chain consists of the amino acid sequence SEQ ID NO: 15.
• the antibody described in the examples under the reference DSV36S comprises a variable domain of the heavy chain which consists of the amino acid sequence SEQ ID NO: 14 and a variable domain of the light chain which consists of the sequence d amino acid SEQ ID NO: 15.
• antibody or antibody fragment of reference The antibody or antibody fragment according to the first object described above is called “antibody or antibody fragment of reference” below.
• a second object of the invention relates to an antibody or an antibody fragment which competes for binding to the G alpha protein with the antibody or antibody fragment of reference, hereinafter "antibody or fragment competitor antibody ”.
• a “competition method” consists in testing an antibody (or an antibody fragment) for its capacities to block the binding between an antibody or fragment of reference antibody and an antigen or to enter into competition with an antibody or fragment of an antibody. reference antibody for binding to the antigen.
• an antibody that competes with the reference antibody or antibody fragment binds to the same epitope as the reference antibody or antibody fragment or to an epitope that is sufficiently close to the antibody. epitope recognized by the reference antibody or antibody fragment to prevent binding of the reference antibody or antibody fragment for reasons of steric hindrance.
• competition methods can be used to determine whether an antibody or antibody fragment competes with a reference antibody or antibody fragment, for example: by a competitive ELISA test, by a immuno-fluorescent method eg by a FRET or HTRF test ("Homogeneous Time Resolved Fluorescence"), by an immunoluminescence method, by a direct or indirect sandwich method, by direct or indirect radio immunoassay (RIA) in solid phase, by direct or indirect solid phase enzyme immunoassay (EIA), by surface plasmon resonance technology (eg BIACORE), by flow cytometry, by fluorescence polarization (eg between a fluorescent peptide and l 'antibody to test), etc.
• a competitive ELISA test by a immuno-fluorescent method eg by a FRET or HTRF test ("Homogeneous Time Resolved Fluorescence")
• an immunoluminescence method eg by a direct or indirect sandwich method
• RIA radio immunoassay
• EIA direct or
• the competitive ELISA method involves the use of a purified antigen bound to a solid surface or to cells, the test antibody that binds to unlabeled antigen, and an antibody or antibody fragment from marked reference.
• the antibody or antibody fragment of reference is present in an unsaturated concentration (with respect to its dissociation constant Kd for the Galpha protein) and the signal is measured at increasing concentrations of the antibody or of the fragment of. antibody to be tested.
• an antibody can block or inhibit (e.g. reduce) the specific binding of a reference antibody or antibody fragment to an antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more.
• binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
• the antibodies or antibody fragments which compete for binding to the alpha G protein with the antibody or reference antibody fragment are identified by the implementation of an HTRF test or by carrying out a fluorescence polarization test, preferably an HTRF test.
• an antibody or antibody fragment competes for binding to the alpha G protein with the reference antibody or antibody fragment, it is able to inhibit an HTRF signal generated by the antibody or antibody. reference antibody fragment.
• an antibody or an antibody fragment which does not compete for binding to the alpha G protein with the antibody or the reference antibody fragment is not capable of inhibiting a signal.
• HTRF generated by the antibody or the reference antibody fragment is identified by the implementation of an HTRF test or by carrying out a fluorescence polarization test, preferably an HTRF test.
• the antibodies or fragments of competing antibodies according to the invention can, for example, be obtained with the antibody or fragment of the reference antibody by the implementation of the protocol described in Example 1.
• the antibody DSV38S is a competing antibody according to the invention.
• the DSV38S antibody and / or the DSV36S antibody is / are excluded from the antibodies or fragments of competing antibodies according to the invention.
• antibodies or fragments of reference antibodies and the antibodies or fragments of competing antibodies as defined above are jointly called “antibodies or fragments of antibodies according to the invention” below.
• the antibody or the antibody fragment according to the invention can bind to the G alpha protein in an isolated form and / or present in a membrane environment, for example it can bind to a G alpha protein present in a preparation of membranes carrying one or more RCPGs and one or more G alpha proteins. It is not necessary for the alpha G protein to be complexed with the RCPG for the antibody according to the invention to be able to bind to the alpha G protein.
• the antibody or the antibody fragment according to the invention can bind to the G alpha protein with a dissociation constant (Kd) measured in FRET less than or equal to 20 nM.
• Kd dissociation constant
• a dissociation constant of less than 20 nM is preferable for the proper implementation of a RET.
• the antibody or the antibody fragment according to the invention can bind to the G alpha protein with a dissociation constant (Kd) measured in FRET less than or equal to 20 nM, for example an affinity constant less than or equal to 10 nM or even less than or equal to 5 nM, for example ranging from 0 to 20 nM (0 being excluded), ranging from 0 to 10 nM (0 being excluded), ranging from 0 to 5 nM (0 being excluded).
• a method for measuring the Kd of an antibody or of an antibody fragment according to the invention in FRET is described in Example 2.
• the antibody or the antibody fragment according to the invention can be used in a RET process with a GTP labeled with a member of a pair of RET partners.
• the antibody or the antibody fragment according to the invention is particularly advantageous in the implementation of a RET, in particular of a FRET, in particular for detecting the activation of a G alpha protein.
• the antibody or the antibody fragment according to the invention can be coupled to a molecule allowing its detection.
• the antibody or the antibody fragment according to the invention can be labeled with a member of a pair of RET partners.
• antibodies or antibody fragments according to the invention can be obtained by implementing the protocol described in Example 1.
• the inventors have also shown that the antibodies or antibody fragments according to the invention bind to the SwitchII domain of the G alpha protein, and more particularly to the 215-294 peptide of the G alpha protein.
• the antibody or the antibody fragment according to the invention can be labeled directly or indirectly according to methods well known to those skilled in the art, for example as described below, but preferably, the The antibody is labeled directly, by covalent bonding with a member of a pair of RET partners.
• RET partners denotes a pair consisting of an energy donor compound (hereinafter “donor compound”) and an energy acceptor compound (hereinafter “acceptor compound”); when in proximity to each other and when excited at the excitation wavelength of the donor compound, these compounds emit a RET signal. It is known that for two compounds to be partners of RET, the emission spectrum of the donor compound must cover partially the excitation spectrum of the acceptor compound.
• donor compound an energy donor compound
• acceptor compound an energy acceptor compound
• the direct labeling of the antibody or of the antibody fragment by a member of a pair of RET partners can be carried out by the known conventional methods of those skilled in the art, relying on the presence of reactive groups on the antibody or the antibody fragment.
• the following reactive groups can be used: terminal amino group, carboxylate groups of aspartic and glutamic acids, amine groups of lysines, guanidine groups of arginines, thiol groups of cysteines, phenol groups of tyrosines, indole rings of tryptophanes, thioether groups of methionines, imidazole groups of histidines.
• the reactive groups can form a covalent bond with a reactive group carried by the antibody or the antibody fragment.
• the appropriate reactive groups, carried by the antibody or the antibody fragment are well known to those skilled in the art, for example a donor compound or an acceptor compound functionalized by a maleimide group will for example be able to bind in such a manner. covalent with the thiol groups carried by the cysteines carried by the antibody or the antibody fragment. Likewise, a donor / acceptor compound bearing an N-hydroxysuccinimide ester will be able to covalently bind to an amine present on the antibody or antibody fragment.
• the antibody or the antibody fragment according to the invention can also be labeled with a fluorescent or bioluminescent compound indirectly, for example by introducing into the measurement medium an antibody or an antibody fragment, itself. even covalently linked to an acceptor / donor compound, this second antibody or antibody fragment specifically recognizing the antibody or the antibody fragment according to the invention.
• biotinylated antibody or antibody fragment can be prepared by techniques well known to those skilled in the art; the Cisbio Bioassays, for example, markets streptavidin labeled with a fluorophore, the trade name of which is “d2” (ref. 610SADLA).
• the antibody or the antibody fragment is labeled with (i) a fluorescent donor or luminescent donor compound, or (ii) a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher ).
• the antibody or the antibody fragment is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).
• the antibody or the antibody fragment is labeled with a fluorescent acceptor compound, for example chosen from allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, nitrobenzoxadiazole and a quantum dot, GFP, GFP variants chosen from GFP10, GFP2 and eGFP, YFP, YFP variants chosen from eYFP, YFP topaz, YFP citrine, YFP venus and YPet, mOrange and DsRed.
• a fluorescent acceptor compound for example chosen from allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, nitrobenzoxadiazol
• the antibody or the antibody fragment is labeled with a fluorescent donor compound, for example chosen from a europium cryptate, a europium chelate, a chelate of terbium, a terbium cryptate, a ruthenium chelate, a quantum dot, allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives and nitrobenzoxadiazole, preferably chosen from: a europium cryptate; a europium chelate; a terbium chelate; a terbium cryptate; a ruthenium chelate; and a quantum dot; the chelates and cryptates of europium and terbium being particularly preferred.
• a fluorescent donor compound for example chosen from a europium cryptate, a europium
• the antibody or the antibody fragment is labeled with a luminescent donor compound, for example chosen from Luciferase (read), Renilla Luciferase (Rluc), variants of Renilla Luciferase (Rluc8) and Firefly Luciferase.
• a luminescent donor compound for example chosen from Luciferase (read), Renilla Luciferase (Rluc), variants of Renilla Luciferase (Rluc8) and Firefly Luciferase.
• a third subject of the invention relates to a composition comprising the antibody or the antibody fragment described above.
• the composition according to the invention may further comprise a source of GTP labeled with a member of a pair of RET partners.
• the GTP marked with a member of a pair of RET partners is described below in the “kit-of-parts” section.
• a fourth object of the invention relates to a kit of reagents (kit-of-parts) comprising (i) the antibody or the antibody fragment according to the invention (reference or competitor as defined above ) and (ii) a source of GTP labeled with a member of a RET partner pair (hereinafter “labeled GTP”).
• kit-of-parts comprising (i) the antibody or the antibody fragment according to the invention (reference or competitor as defined above ) and (ii) a source of GTP labeled with a member of a RET partner pair (hereinafter “labeled GTP”).
• GTP can be a non-hydrolyzable or slowly hydrolyzable GTP, such as GTPgammaS (GTPyS or GTPgS), GppNHp and GppCp.
• GTPgammaS GTPyS or GTPgS
• GppNHp GppNHp
• GppCp GppCp
• GTP can be labeled directly or indirectly.
• the GTP is directly labeled.
• the direct labeling of GTP by a member of a pair of RET partners, for example a fluorescent compound when a FRET is used, can be carried out by the methods based on the presence of reactive groups on the GTP.
• the reactive groups can form a covalent bond with a reactive group carried by a member of a pair of RET partners.
• the appropriate reactive groups, carried by the member of a pair of RET partners, are well known to those skilled in the art, for example a donor compound or an acceptor compound functionalized by a maleimide group will for example be able to bind to covalently with thiol groups.
• a donor / acceptor compound bearing an N-hydroxysuccinimide ester will be able to covalently attach to an amine.
• the GTP is labeled with (i) a fluorescent donor compound, or (ii) a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).
• a fluorescent donor compound e.g., a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).
• the GTP is labeled with a fluorescent donor compound.
• the labeled GTP is a non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent donor compound chosen from GTPgN-C2 (GTP-gamma-N-C2), GTPgN-C3 (GTP-gamma -N-C3), GTPgN-octyl-C2 (GTP- gamma-N-octyl-C2), GTPgN-octyl-Cll (GTP-gamma-N-octyl-Cll), GTPgN-octyl-C3 (GTP- gamma- N-octyl-C3), GTPgO-hexyl-C2 (GTP-gamma-0-hexyl-C2), GTPgO-hexyl-C3 (GTP-gamma-0-hexyl-C3) or GTP-gN-octyl-thiosuccin
• the labeled GTP is a non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent acceptor compound chosen from GTPgN-octyl-Cy5, GTPgN-octyl-AF488, GTPgN-L15-Fluorescein, GTPgO-Linker- Cy5 (P) or GTPgS-Linker-Cy5 (R).
• GTP can also be labeled with a non-fluorescent acceptor compound (quencher).
• GTP-gN-octyl-thiosuccinimidyl-C2 is shown below.
• the GTP is labeled with a fluorescent donor compound and the antibody or the antibody fragment is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).
• the GTP is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher) and the antibody or the antibody fragment is labeled with a fluorescent donor or luminescent donor compound.
• (i) is an antibody which comprises a variable domain of the heavy chain which consists of the amino acid sequence SEQ ID NO: 14 and a variable domain of the light chain which consists of the acid sequence amino SEQ ID NO: 15 and (ii) is GTP-gN-octyl-thiosuccinimidyl-C2.
• a fifth object of the present invention relates to a nucleic acid sequence encoding the antibody or the antibody fragment according to the invention (reference or competitor).
• a sixth object of the present invention relates to a vector comprising a nucleic acid sequence according to the invention.
• a vector comprising a nucleic acid sequence according to the invention.
• Any type of vector suitable for the production of antibodies can be used within the framework of the invention.
• the vector is a recombinant vector.
• the vector will comprise the nucleic sequences necessary to produce the antibody according to the invention, for example promoter sequences, regulatory sequences, etc.
• the preparation of a suitable vector is widely described in the literature.
• a seventh object of the invention relates to a cell comprising a vector according to the invention or a nucleic acid sequence according to the invention.
• the cell according to the invention can be obtained by methods widely described in the literature, for example by transfecting a cell clone with a vector according to the invention or a nucleic acid sequence.
• the invention is not limited to a particular cell type. Any cell capable of producing antibodies can be used within the framework of the invention. They may be eukaryotic cells, such as mammalian cells, for example human or mouse cells, or prokaryotic cells, for example bacteria or even yeasts.
• FIG. 1 represents the reaction scheme of a FRET making it possible to measure the Kd of an anti-G alphail protein antibody.
• FIG. 2 represents curves which illustrate the affinity of the antibodies SC13533, DSV36S and DSV38S labeled with d2 for the human Gail protein.
• a very significant HTRF signal is obtained with all the antibodies in the presence and in the absence of a nucleotide (GTPgS or GDP).
• the results indicate that the DSV36S, DSV38S and SC13533 antibodies are able to bind the Gail protein.
• the curves make it possible to calculate the affinity (Kd) of these different antibodies for the Gail protein (Table 1).
• Figure 3 is a curve which illustrates the ability of unlabeled DSV36S, DSV38S and SC 13533 antibodies to inhibit binding of SC13533-d2 antibody to human Gil protein.
• the unlabeled SC 13533 antibody completely inhibited the HTRF signal obtained with the SC 13533-d2 antibody.
• the DSV36S and DV38S antibodies were not able to inhibit the signal generated by the SC 13533-d2. This demonstrates that these 2 antibodies do not bind to the same region of the Gail protein as the SC 13533 antibody.
• the DSV36S and DSV38S antibodies recognize different epitopes from the SC 13533 antibody.
• FIG. 4 represents the capacity of the commercial antibodies sc-13533, sc-56536 and AM05302PU-N to inhibit the binding of the DSV SC13533-d2 antibody to the human Gil protein.
• the commercial antibodies sc-13533, sc-56536 and AM05302PU-N are all non-competing antibodies to the DSV 36S antibody. All these antibodies therefore recognize an epitope different from the DSV 36S antibody.
• FIG. 5 represents the capacity of the commercial antibodies sc-13533, sc-56536 and AM05302PU-N to inhibit the binding of the DSV 3S-d2 antibody (generated in the laboratory) on the human Gil protein.
• unlabeled DSV 3S antibody completely inhibited the HTRF signal obtained with DSV 3S-d2 antibody.
• the sc-13533, sc-56536 and AM05302PU-N antibodies completely inhibited the HTRF signal obtained with the DSV 3S-d2 antibody.
• these results demonstrate that the commercial antibodies sc-13533, sc-56536 and AM05302PU-N are all antibodies non-competitors of the DSV 36S antibody. It is therefore the same for the DSV 3S antibody made in the laboratory. All these antibodies therefore recognize an epitope different from the DSV 36S antibody.
• Figure 6 is a curve which illustrates the ability of unlabeled DSV36S and DSV38S antibodies to inhibit binding of DSV36S-d2 antibody to Gail protein.
• unlabeled DSV36S antibody completely inhibited the HTRF signal obtained with DSV36S-d2 antibody.
• the DSV38S antibody also completely inhibited the signal generated by DSV36S-d2. This demonstrates that these 2 antibodies bind to the same region of the garlic G protein.
• the DSV38S antibody competes for binding to the G protein alphai with the DSV36S antibody.
• FIG. 7 represents the capacity of the commercial antibodies DSV 36S, DSV 26S, DSV 3S and DSV 39S to inhibit the binding of the antibody DSV36S-d2 on the human Gil protein.
• DSV36S antibody completely inhibited the HTRF signal obtained with DSV36S-d2 antibody.
• DSV 26S, DSV 3S and DSV 39S antibodies were not able to inhibit the signal generated by the DSV 36S-d2. This demonstrates that these three antibodies do not bind to the same region of the human Gail protein as the DSV 36S antibody. In conclusion, these antibodies recognize different epitopes of the DSV 36S antibody.
• FIGS. 8A and 8B represent the reaction scheme of a FRET making it possible to measure the selectivity of the DSV36S, DSV38S and SC13533 antibodies for different types of Ga proteins.
• FIG. 9 is a diagram which represents the FRET signal for different types of Ga proteins obtained with a pair of Anti-Twin-Strep-tag-Lumi4 Tb / Anti-FLAG-d2 antibodies, which makes it possible to verify that all the Ga proteins encoded by the plasmids were well over-expressed in the HEK293s. Indeed, the HTRF signal obtained with each of them was much higher than the HTRF signal obtained with the negative condition (“MOCK”) which corresponds to HEK293 transfected with a control plasmid not encoding any protein tagged FLAG + Twin-Strep- tag.
• MOCK negative condition
• FIG. 10 is a diagram which illustrates the selectivity of the DSV36S, DSV38S and SC13533 antibodies for the different types of Ga proteins.
• the DSV36S and DSV38S antibodies gave a positive HTRF signal when the proteins Gail, Gai2, Gai3, Gao and Gas were overexpressed but did not give an HTRF signal when the Gas, Gaq, Gal2 and Gal3 proteins were overexpressed.
• the SC13533 antibody gave a signal HTRF positive when the Gail and Gai3 proteins were overexpressed but did not give an HTRF signal when the Gai2, Gao, Gaz, Gas, Gaq, Gal2 and Gal3 proteins were overexpressed.
• FIG 11 is a diagram which illustrates the ability of DSV36S, DSV38S and SC13533 antibodies to generate a TR-FRET signal when associated with a fluorescent analogue of GTP (GTPgN-octyl-C2-europium cryptate ) on membrane preparations overexpressing a GPCR.
• GTPgS represents the non-specific signal of the assay (NS) in which the excess of unlabeled GTPgS (100mM) inhibited the binding of europium GTPgN-octyl-C2-Cryptate to the Ga and a proteins. therefore prevented the appearance of a FRET signal.
• the “buffer” condition showed that it was possible to obtain a moderate but significant FRET signal between the europium GTPgN-octyl-C2-Cryptate and the DSV36S and DSV 38S antibodies labeled with d2.
• SNC162 which caused the activation of the over-expressed DOR receptors in the membranes
• an increase in this FRET signal was observed linked to an increase in the binding of the fluorescent GTP analog on all or part Gail, Gai2, Gai3, Gao and Gaz proteins recognized by the DSV36S and DSV38S antibodies.
• FIG. 12 represents the capacity of the DSV 36S, DSV 26S, DSV 3S and DSV 39S antibodies to generate a TR-FRET signal when they are associated with a fluorescent analogue of GTP (GTPgN-octyl-C2-Cryptate d 'europium) on unactivated membrane preparations overexpressing a GPCR.
• GTPgN-octyl-C2-Cryptate d 'europium a fluorescent analogue of GTP
• the "Not Specify Signal" condition showed that a weak basal FRET signal between the fluorescent analog of GTP and the d2-labeled antibodies was detected.
• the "Negative control" condition represents the nonspecific signal of the assay in which the excess of unlabeled GTPgS (IOOmM) inhibited the binding of the fluorescent analog of GTP to the Ga proteins and therefore prevented the appearance of GTPgS. 'a FRET signal.
• the inhibition measured was total since the HTRF signal level measured for the conditions "Not specified signal” and "Negative. control ”is almost identical.
• an increase in the FRET signal linked to an increase in the binding of the fluorescent analogs of GTP on all or part of the recognized Gail, Gai2, Gai3, Gao and Gaz proteins. by the DSV36S antibody was measured.
• FIG. 13 represents the capacity of the DSV 36S and sc-13533 antibody to generate a TR-FRET signal when it is associated with a fluorescent analogue of GTP (GTPgO-Linker-Cy5 (P)) on unactivated membrane preparations overexpressing a GPCR.
• GTPgO-Linker-Cy5 (P) fluorescent analogue of GTP
• the "Not Specify Signal” condition showed that a weak basal FRET signal between the fluorescent analogue of GTP and the antibodies labeled with Terbium cryptate was detected.
• the "Negative control” condition represents the nonspecific signal of the assay in which the excess of unlabeled GTPgS (IOOmM) inhibited the binding of the fluorescent analog of GTP to the Ga proteins and therefore prevented the appearance of GTPgS. 'a FRET signal.
• FIGS. 14A and 14B illustrate 2 FRET formats which can be implemented with the antibodies according to the invention (Formats 2A and 2B).
• FIGS. 15A and 15B illustrate an activation test according to the 2A format on RCPG Delta Opioid with the detection pair: GTPgN-octyl-C2 + DSV36S-d2.
• FIGS. 16A and 16B illustrate an activation test according to the 2A format on RCPG Delta Opioid with the detection pair: GTPgN-octyl-C2 + DSV36S-d2.
• FIGS. 17A and 17B illustrate an activation test according to the 2A format on RCPG Delta Opioid with the detection pair: GTPgN-octyl-C2 + DSV38S-d2.
• Figures 18A and 18B illustrate an activation test according to the 2A format on Delta Opioid RCPG with the detection pair: GTPgN-octyl-Cll + DSV36S-d2.
• Figures 19A and 19B illustrate an activation test according to the 2A format on RCPG
• FIGS. 20A and 20B illustrate an activation test according to the 2A format on Delta Opioid RCPG with the detection pair: GTPgN-C2 + DSV36S-d2.
• Figures 21A, 21B illustrate an activation test according to the 2A format on RCPG Dopamine D2S with the detection pair: GTPgN-octyl-C2 + DSV36S-d2.
• FIGS. 22A and 22B illustrate an activation test according to format 2A on RCPG Dopamine D2S with the detection pair: GTPgN-octyl-C2 + DSV36S-d2.
• FIGS. 23A and 23B illustrate an activation test according to format 2A on RCPG Dopamine D2S with the detection pair: GTPgN-octyl-C2 + DSV36S-d2.
• Figures 24A and 24B illustrate an activation test according to the 2B format on RCPG
• Figures 25A and 25B illustrate an activation test according to format 2B on Delta Opioid RCPG with the detection pair: GTPgN-octyl-AF488 + DSV36S-Lumi4Tb.
• FIGS. 26A and 26B illustrate an activation test according to format 2A on RCPG Delta Opioid with the detection pair: GTP-gN-octyl-thiosuccinimidyl-C2 + DSV36S-d2.
• the membrane preparations of cells expressing the Delta Opioid receptor (DOR) were purchased from Euroscreen via a service.
• the DSV36S, DSV38S, DSV 26S, DSV 3S and DSV 39S antibodies were generated by Cisbio Bioassays and are available from Cisbio Bioassays on request (under the respective references DSV36S, DSV38S, DSV 26S, DSV 3S and DSV 39S) .
• the DSV36S antibody comprises a heavy chain variable domain which consists of the amino acid sequence SEQ ID NO: 14 and a light chain variable domain which consists of the amino acid sequence SEQ ID NO: 15. antibodies were labeled with compatible fluorescent probes for TR-FRET detection (red acceptor - d2 or Lumi4Tb donor).
• the antibodies SC13533 and SC56536 were purchased from Santa Cruz Biotechnology (ref SC13533 and ref SC56536).
• the AM05302PU-N antibody was purchased from Acris Antibodies GmbH (ref AM05302PU-N).
• the anti Twin-Strep-tag antibody was purchased from IBA Lifesciences (ref 2-1517-001) and labeled with a fluorescent probe compatible with TR-FRET detection (donor Lumi4Tb).
• the anti-FLAG-d2 antibody is available from Cisbio Bioassays (Ref 61FG2DLF)
• the GDP and GTPyS nucleotides were purchased from Sigma Aldrich (respective catalog references G7127 and G8634).
• the Delta Opioid GPCR agonist (SNC162) was purchased from Tocris Biosciences (reference 1529).
• the 384-well, low-volume, white plates with a white background and the 96-well black plates (with a black background) suitable for cell culture were purchased from Greiner Bio One (Catalog references 784075 and 665086 respectively).
• the non-hydrolyzable / slowly hydrolyzable GTP analog (GTPgN-octyl-C2) labeled with a donor fluorophore (europium cryptate) was synthesized by Cisbio Bioassays.
• the non-hydrolyzable / slowly hydrolyzable GTP analog (GTPgO-Linker-Cy5 (P)) labeled with an acceptor fluorophore (Cy5) was purchased from Jena Bioscience (ref NU-834-Cy5).
• the plasmids encoding the various human G proteins, Gail, Gai2, Gai3, Gao, Gas, Gas, Gaq, Gal2 and Gal3 fused in their N-terminal part to the Twin-Strep-tag and FLAG tags were synthesized and amplified by the company Genecust (service provision).
• the HEK293 cells were purchased from the ATCC.
• Opti-MEM medium Lipofectamine 2000 and poly-ornithine were purchased from Thermo Fisher Scientific (ref. 51985-026 and 11668-019) and Sigma Aldrich (ref. P4957, respectively). ).
• the HTRF signal was measured on the PHERAstar reader (BMG Labtech) with the following configuration:
• the HTRF Ratio was calculated according to the following formula:
• HTRF Ratio Signal at 665nm / Signal at 620nm * 10,000.
• Example 1 Protocol for obtaining anti-G alphail protein antibodies according to the invention
• TST-G alphail protein G alphail protein of UniProt P63096-1 sequence tagged N-terminally with the TwinStreptag (TST) (IBA) tag via a TEV linker
• TST TwinStreptag
• TST TwinStreptag
• mice were immunized by injection of the TST-G alphail protein previously diluted in buffer containing GTPgS (20 mM HEPES pH8, 100 mM NaCl, 3 mM MgCl2, 11 mM CHAPS, 100 mM GTPgS). The first injection was followed by three boosters at monthly intervals.
• GTPgS 20 mM HEPES pH8, 100 mM NaCl, 3 mM MgCl2, 11 mM CHAPS, 100 mM GTPgS.
• TST-G alphail protein previously diluted to 20 pg / mL in buffer containing GTPgS (20mM Tris HCl pH8.5, 140mM NaCl, 2mM EDTA, 10mM MgCl2, 0.1% BSA, GTPgS ImM) was adsorbed via the TwinStreptag tag on 96-well plates containing Strep-Tactin®XT (IBA, Catalog: 2-4101-001). For this, 100 mI of protein were added to each well and then incubated for 2 h at 37 ° C. followed by three washes in PBS 1 ⁇ buffer, 0.05% Tween20.
• the same punctures were tested on the ELISA test after pre-incubation with an excess of another orthogonal protein tagged with the TwinStrepTag (SNAPTag-TwinStrepTag).
• the anti tag antibodies bind to the tagged orthogonal protein and therefore not to the G alphail protein attached to the bottom of the wells; in which case no HRP signal or a decrease in HRP signal is detected.
• mice exhibiting the best antibody titers and the least drop in signal in the anti tag control case were selected for the following stage of lymphocyte hybridization, also called fusion.
• the spleen of the mice was collected and mixed lymphocytes and plasmocysts derived from this spleen was fused in vitro with a myeloma cell line in the presence of a cell fusion catalyst of the polyethylene glycol type.
• a mutant myeloma cell line lacking the HGPRT (Hypoxanthine Guanosin Phosphoribosyl Transferase) enzyme was used to allow selection of hybrid cells, called hybridomas.
• hybridomas were then cultured in culture plates. The supernatants of these hybridomas were then tested to evaluate their capacity to produce anti G alphail protein antibodies. For this, an ELISA test as described above was carried out.
• the test was carried out in parallel on TST-protein conditions. G alphail pre-incubated in the buffer containing either ImM GDP, ImM GTPgS or nucleotide-free. The best hybridomas were then cloned with a step of limiting dilution in order to obtain hybridoma clones.
• the hybridoma clones of interest were then injected into mice (intraperitoneal injection) in order to allow the production of antibodies in large quantities in the ascitic fluid.
• the antibodies were then purified by affinity chromatography on columns with resins exhibiting protein A.
• All the reagents are diluted in 50 mM TrisHCl buffer, pH 7.4, 10 mM MgCl2, 0.1% BSA, 10 mM NaCl.
• the Gail protein is prepared 2X to obtain a final concentration in the wells of 2.5nM.
• the GTPgS nucleotide is prepared 2X to obtain a final concentration in the wells of 10 ⁇ M.
• These 2 reagents are prepared in the same solution and pre-incubated for 30 minutes at room temperature before being distributed into the wells.
• the above purified antibodies are prepared 4X to target final concentrations in the wells between 0.01 and ImM.
• the DSV36S-d2 antibody is prepared 4X to aim for a final concentration of 10nM.
• the anti Twin-Strep-tag-Lumi4 Tb antibody is prepared 4X to obtain a final concentration in the wells of 0.5nM.
• the reagents are distributed in the 384-well plates as follows:
• the plates are incubated for 1 hour at room temperature before reading the HTRF signal.
• the antibodies according to the invention are capable of inhibiting the HTRF signal obtained with the DSV36S-d2 antibody. Conversely, the antibodies which are not according to the invention are not capable of inhibiting the signal generated by DSV36S-d2.
• Example 2 Determination of the Affinity of the SC13533 Antibodies. DSV36S. DSV38S labeled with d2 for Gail protein
• the Gail protein was prepared 2X to obtain a final concentration in the wells of 2.5nM.
• the GTPgS nucleotide was prepared 2X to obtain a final concentration in the wells of 100mM.
• These 2 reagents were prepared in the same solution and preincubated for 30 minutes at room temperature before being distributed into the wells.
• the DSV36S-d2, DSV38S-d2 and SC13533-d2 antibodies were prepared 4X to target the final concentrations in the wells of between 0.01 and 10nM depending on the antibodies.
• the anti Twin-Strep-tag-Lumi4 Tb antibody was prepared 4X to obtain a final concentration in the wells of 0.25nM.
• the reagents were distributed in the 384-well plates as follows:
• FIG. 2 shows the results obtained with the DSV36S, DSV38S and SC13533 antibodies labeled with d2.
• a very significant HTRF signal is obtained with all these antibodies in the presence and in the absence of a nucleotide (GTPgS or GDP).
• the antibody titration curves produced make it possible to calculate the affinity (Kd) of these various antibodies for the Gail protein. This was done through GraphPad Prism software by applying a “one site specifies binding” model to the HTRF data. The Kd values obtained are presented in Table 1.
• the Kd values show that the 3 antibodies have an excellent affinity for the Gail protein, the Kd values being between 0.1 and lnM whatever the state of the protein.
• Example 3 Capacity of unlabelled DSV36S antibodies. DSV38S, SC
• the Gail protein was prepared 2X to obtain a final concentration in the wells of 2.5nM.
• the GTPgS nucleotide was prepared 2X to obtain a final concentration in the wells of 10pM.
• the cold antibodies DSV36S, DSV38S and SC13533 were prepared 4X to aim for the final concentrations in the wells of between 0.01 and 100nM.
• SC 13533-d2 antibody was prepared 4X to aim for a final concentration of 10nM.
• the anti Twin-Strep-tag-Lumi4 Tb antibody was prepared 4X to obtain a final concentration in the wells of 0.5nM.
• the reagents were distributed in the 384-well plates as follows:
• the plates were incubated for 1 hour at room temperature before reading the HTRF signal.
• FIG. 3 shows the results obtained with the DSV 36S, DSV 38S and SC 13533 antibodies.
• the unlabeled SC 13533 antibody completely inhibited the HTRF signal obtained with the SC 13533-d2 antibody.
• the DSV36S and DV38S antibodies were not able to inhibit the signal generated by the SC 13533-d2. This demonstrates that these 2 antibodies do not bind to the same region of the Gail protein as the SC 13533 antibody.
• the DSV36S and DSV38S antibodies recognize different epitopes from the SC 13533 antibody.
• Figure 4 shows the results obtained with the antibodies SC13533, SC56536 and AM05302PU-N.
• Example 4 Capacity of unlabelled DSV3S antibodies. SC56536. AM05302PU-N and SC 13533 to inhibit the binding of the DSV3S-d2 antibody to the Gil protein
• the Gail protein was prepared 2X to obtain a final concentration in the wells of 2.5nM.
• the GTPgS nucleotide was prepared 2X to obtain a final concentration in the wells of 10pM.
• These 2 reagents were prepared in the same solution and preincubated for 30 minutes at room temperature before being distributed into the wells.
• the cold antibodies DSV3S, SC56536, AM05302PU-N and SC 13533_ were prepared 4X to target the final concentrations in the wells between 0.03 and 300nM.
• DSV3S-d2 antibody was prepared 4X to aim for a final concentration of 10nM.
• the anti Twin-Strep-tag-Lumi4 Tb antibody was prepared 4X to obtain a final concentration in the wells of 0.25nM.
• the reagents were distributed in the 384-well plates as follows:
• the plates were incubated for 1 hour at room temperature before reading the HTRF signal.
• FIG. 5 shows the results obtained. Logically, unlabeled DSV 3S antibody completely inhibited the HTRF signal obtained with DSV 3S-d2 antibody. Also, the sc-13533, sc-56536 and AM05302PU-N antibodies completely inhibited the HTRF signal obtained with the DSV 3S-d2 antibody. In conclusion, these results, associated with those presented in Example 3, demonstrate that the commercial antibodies sc-13533, sc-56536 and AM05302PU-N are all antibodies which are non-competitive with the DSV 36S antibody. It is therefore the same for the DSV 3S antibody made in the laboratory. All these antibodies therefore recognize an epitope different from the DSV 36S antibody.
• Example 5 Capacity of unlabeled DSV36S antibodies. DSV38S, DSV3S, DSV26S and DSV39S to inhibit the binding of the DSV36S-d2 antibody to the Gail protein
• the Gail protein was prepared 2X to obtain a final concentration in the wells of 2.5nM.
• the GTPgS nucleotide was prepared 2X to obtain a final concentration in the wells of 100mM.
• These 2 reagents were prepared in the same solution and preincubated for 30 minutes at room temperature before being distributed into the wells.
• the cold DSV36S, DSV38S, DSV26S, DSV3S, and DSV39S antibodies were prepared 4X to target the final concentrations in the wells between 0.001 and ImM.
• the DSV36S-d2 antibody was prepared 4X to aim for a final concentration of 10nM.
• the anti Twin-Strep-tag-Lumi4 Tb antibody was prepared 4X to obtain a final concentration in the wells of 0.25nM.
• the reagents were distributed in the 384-well plates as follows:
• the plates were incubated for 1 hour at room temperature before reading the HTRF signal.
• FIG. 6 shows the results obtained with the DSV36S and DSV38S antibodies.
• Logically, unlabeled DSV36S antibody completely inhibited the HTRF signal obtained with DSV36S-d2 antibody.
• the DSV38S antibody also completely inhibited the signal generated by DSV36S-d2. This demonstrates that these 2 antibodies bind to the same region of the Gail protein.
• Figure 7 shows the results obtained with the DSV36S, DSV26S, DSV3S and DSV39S antibodies. Logically, unlabeled DSV36S antibody completely inhibited the HTRF signal obtained with DSV36S-d2 antibody.
• DSV 26S, DSV 3S and DSV 39S antibodies were not able to inhibit the signal generated by the DSV 36S-d2. This demonstrates that these 3 antibodies do not bind to the same region of the human Gail protein as the DSV 36S antibody.
• DSV38S antibody competes for binding to the alphai G protein with the DSV36S antibody and therefore shares the same epitope.
• Day 1 transfection of the HEK293 cells with plasmids encoding the various human Gail, Gai2, Gai3, Gao, Gas, Gas, Gaq, Gal2 and Gal3 proteins
• Plasmids / Lipofectamine mixtures containing 150 ng / well of plasmids and 0.375 ml of lipofectamine were prepared in OptiMEM medium 30 minutes before their addition to the 96-well plate with a dark background.
• microplates were incubated for 24 hours at 37 ° C. and 5% CO 2 (controlled oven).
• the reagents were diluted in 50 mM TrisHCl buffer pH 7.4, 10 mM MgCl2, 0.1% BSA, 0.02% Triton X100.
• the DSV36S-d2, DSV38S-d2 and SC13533-d2 antibodies were prepared 4X to aim for a final concentration in the lOnM wells.
• the anti Twin-Strep-tag-Lumi4 Tb antibody was prepared 4X to obtain a final concentration in the wells of 0.5nM.
• the GDP and GTPgS nucleotides were prepared 4X to obtain a final concentration in the 10mM wells.
• the reagents were distributed on the 384-well plates as follows:
• microplates were incubated for 20 hours at room temperature before reading the HTRF signal.
• the overexpression of all the Ga proteins being validated it was then possible to determine the selectivity profile of the DSV36S, DSV38S and SC 13533 antibodies by measuring a possible FRET between these antibodies labeled with d2 and the Anti-Twin-Strep- antibody. tag-Lumi4 Tb.
• the DSV36S and DSV38S antibodies gave a positive HTRF signal when the Gail, Gai2, Gai3, Gao and Gaz proteins were overexpressed but did not give an HTRF signal when the Gas, Gaq, Gal2 and Gal3 proteins were overexpressed.
• the SC13533 antibody gave a positive HTRF signal when the Gail and Gai3 proteins were overexpressed but did not give an HTRF signal when the Gai2, Gao, Gaz, Gas, Gaq, Gal2 and Gal3 proteins were overexpressed.
• Example 7 Capacity of DSV36S Antibodies. DSV38S. DSV 26S. DSV 39S. DSV 3S and SC13533 to generate a TR-FRET signal when combined with a fluorescent analogue of GTP on membrane preparations overexpressing a G PCR
• HEK293 membranes expressing the DOR receptor were prepared 4X to aim for a final amount in the 10 ⁇ g wells.
• DSV36S-d2, DSV38S-d2, SC13533-d2 and anti-FLAG-d2 antibodies were prepared 4X to aim for a final concentration in the lOnM wells.
• Europium cryptate labeled GTPgN-octyl-C2 was prepared 4X to obtain a final concentration in the wells of 6nM.
• the GTPgO-linker-Cy5 was prepared 4X to obtain a final concentration in the wells of 50nM.
• the nucleotides GTPgS and GDP were prepared 4X to obtain a final concentration in the wells of 100mM.
• SNC 162 was prepared 4X to obtain a final concentration in the 10 pM wells.
• the reagents were distributed in the 384-well plates as follows:
• the plates were incubated for 20 hours at room temperature before reading the HTRF signal.
• FIG. 11 shows the results obtained when the europium analog GTPgN-octyl-C2-Cryptate is combined with the antibodies DSV36S-d2, DSV38S-d2 and SC13533-d2.
• GTPgS represents the non-specific signal of the assay (NS) in which the excess of unlabeled GTPgS (100mM) inhibited the binding of europium GTPgN-octyl-C2-Cryptate to the Ga and a proteins. therefore prevented the appearance of a FRET signal.
• the “buffer” condition showed that it was possible to obtain a moderate but significant FRET signal between europium GTPgN-octyl-C2-Cryptate and the DSV36S and DSV 38S antibodies labeled with d2.
• SNC162 which caused the activation of the over-expressed DOR receptors in the membranes
• an increase in this FRET signal was observed linked to an increase in the binding of the fluorescent GTP analog on all or part Gail, Gai2, Gai3, Gao and Gaz proteins recognized by the DSV36S and DSV38S antibodies.
• FIG. 12 shows the results obtained when one associates the analog GTPgN-octyl-C2-Cryptate of europium with the antibodies DSV36S-d2, DSV26S-d2, DSV39S-d2 and DSV3S- d2 on membrane preparations not activated or expressing a GPCR.
• the "Not Specify Signal” condition showed that a weak basal FRET signal between the fluorescent analog of GTP and the d2-labeled antibodies was detected.
• the "Negative control” condition represents the nonspecific signal of the assay in which the excess of unlabeled GTPgS (100mM) inhibited the binding of the fluorescent analog of GTP to the Ga proteins and therefore prevented the appearance of 'a FRET signal.
• the inhibition measured was total since the level of HTRF signal measured for the conditions "Not specified signal” and "Negative control" is almost identical.
• an increase in the FRET signal linked to an increase in the binding of the fluorescent analogs of GTP on all or part of the recognized Gail, Gai2, Gai3, Gao and Gaz proteins. by the DSV36S antibody was measured.
• the DSV 26S, DSV 3S and DSV 39S antibodies did not give any FRET signal when they were combined with GTPgN-octyl -C2-Cryptate europium.
• FIG. 13 shows the results obtained when the GTPgO-Iinker-Cy5 analog is combined with the DSV36S-Tb and SC13533-Tb antibodies on unactivated membrane preparations over-expressing a GPCR.
• the "Not Specify Signal” condition showed that a weak basal FRET signal between the fluorescent analogs of GTP and the antibodies labeled with Terbium cryptate was detected.
• the "Negative control” condition represents the nonspecific signal of the assay in which the excess of unlabeled GTPgS (IOOmM) inhibited the binding of the fluorescent analog of GTP to the Ga proteins and therefore prevented the appearance of GTPgS. 'a FRET signal.
• the inhibition measured was total since the level of HTRF signal measured for the conditions "Not specified signal” and "Negative control" is almost identical.
• an increase in the FRET signal related to an increase in the binding of the fluorescent analog of GTP on all or part of the proteins Gail, Gai2, Gai3, Gao and Gases recognized by the DSV36S antibody was measured.
• the sc-13533 antibody gave no FRET signal when combined with the GTPgO-Linker-Cy5 (P).
• Example 8 measurement of the activation of a GPCR by implementing a FRET with a labeled antibody according to the invention.
• the DSV36S antibody was labeled with fluorescent probes compatible for TR-FRET detection (red acceptor - d2 or Lumi4Tb donor).
• nucleotides GTP, GDP and GTPyS were purchased from Sigma Aldrich (respective catalog references G8877, G7127 and G8634).
• Non-hydrolyzable / slowly hydrolyzable GTP analogues labeled with donor or acceptor fluorophores (GTPgN-C2; GTPgN-C3; GTPgN-octyl-C2; GTPgN-octyl-Cll; GTPgN-octyl-C3; GTPgO-hexyl- C2; GTPgO-hexyl-C3; GTP-gN-octyl-thiosuccinimidyl-C2; GTPgN-octyl-Cy5; GTPgN-octyl-AF488) were synthesized by Cisbio Bioassays.
• Non-hydrolyzable / slowly hydrolyzable GTP analogues labeled with acceptor fluorophores GTPgO-Linker-Cy5 (P) and GTPgS-Linker-Cy5 (R) were purchased from Jena Bioscience under the respective references NU-834-CY5 and NU-1610-CY5.
• the anti-G alphai antibodies used for the detection were prepared 4X to aim for the final concentrations in the following wells: DSV36S-d2 antibody (10nM); DSV36S-Lumi4Tb antibody (0.5 or lnM); DSV38S-d2 antibody (10nM).
• DSV36S-d2 antibody 10nM
• DSV36S-Lumi4Tb antibody 0.5 or lnM
• DSV38S-d2 antibody 10nM.
• Non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with fluorescent donor or acceptor probes were prepared 4X to aim for the final concentrations in the wells mentioned in the legends of each figure.
• Anti-G alphai-donor or acceptor antibody 5 pL
• Buffer or Compounds to be tested (agonists and / or antagonists): 2 ⁇ l.
• the non-specific signal (fluorescence background noise) was measured with wells containing an excess of GTPgS (100 mM).
• the plates were incubated at 21 ° C for 20 h (except if otherwise specified in the figures) then the HTRF signal was measured on the PHERAstar reader (BMG Labtech) with the following configuration:
• Figure 14A illustrates the test principle using a non-hydrolyzable / slowly hydrolyzable analogue of GTP labeled with a donor RET partner and an anti G alpha protein antibody labeled with an acceptor RET partner in which activation of RCPG with an agonist compound induces an increase in the binding of the GTP-donor analog to the G protein and therefore an increase in the RET signal (2k format).
• Figure 14B illustrates the test principle using a non-hydrolyzable / slowly hydrolyzable analogue of GTP labeled with an acceptor RET partner and an anti G alpha protein antibody labeled with a donor RET partner in which the activation of RCPG with an agonist compound induces an increase in the binding of the GTP-acceptor analog to the G protein and therefore an increase in the RET signal (2 B format).
• FIG. 15A GTPgN-octyl-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 500mM NaCl; 0.1% BSA.
• FIG. 16A GTPgN-octyl-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 300mM NaCl; GDP 0.5mM; 0.1% BSA.
• FIG. 17A GTPgN-octyl-C2 (6nM final in the well); DSV38S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4;
• FIG. 18A GTPgN-octyl-Cll (6nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 300mM NaCl; GDP 0.5mM; 0.1% BSA.
• - Figure 19A GTPgO-hexyl-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 300mM NaCl; GDP 0.5mM; 0.1% BSA.
• - Figure 20A GTPgN-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 300mM NaCl; GDP 0.5mM; 0.1% BSA.
• the membranes were incubated in the absence or in the presence of a strong excess of GTPgS (100 mM).
• the difference in TR-FRET signal (Ratio HTRF) observed between these two conditions shows that the analogs GTPgN-octyl-C2, GTPgN-octyl-Cll, GTPgO-hexyl-C2, GTPgN-C2 are able to bind to the G protein alphai and generate a TR-FRET signal with the anti G alphai-acceptor antibody ( Figures 16A to 20A).
• Figure 16B shows a second condition where activation by a fixed concentration of RCPG agonist SNC162 (200nM) was inhibited by an increasing concentration of RCPG antagonist (Naltrindole). This activation inhibition is observed by the decrease in the TR-FRET signal (HTRF Ratio).
• FIG. 21A GTPgN-octyl-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-D2S membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 10mM NaCl; GDP ImM; 0.1% BSA.
• - Figure 22A GTPgN-octyl-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10 pg CH0-D2S membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 100 mM NaCl; 0.1% BSA.
• FIG. 23A GTPgN-octyl-C2 (6nM final in the well); DSV36S-d2 (final 10nM in well); 10 pg CH0-D2S membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 100 mM NaCl; GDP ImM; 0.1% BSA.
• FIG. 24A GTPgN-octyl-Cy5 (50nM final in the well); DSV36S-Lumi4Tb (final lnM in the well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 10mM MgCl2; 300mM NaCl; GDP 0.5mM; 0.1% BSA. Reading after 3 hours of incubation at 21 ° C.
• FIG. 25A GTPgN-octyl-AF488 (50nM final in the well); DSV36S-Lumi4Tb (final lnM in the well); 10pg CHO-DOR membranes / well; Buffer: 50mM TrisHCI pH7.4 ; 10mM MgCl2; 300mM NaCl; GDP 0.5mM; 0.1% BSA ⁇ Reading after 3 hours of incubation at 21 ° C.
• Activation test according to the 2A format on RCPG Delta Opioid fDOR ' increase in the TR-FRET signal between GTP-donor and anti-G alpha-acceptor protein antibody under stimulation of an agonist.
• FIG. 26A GTP-gN-octyl-thiosuccinimidyl-C2 (7.5nM final in the well); DSV36S-d2 (final 10nM in well); 10pg CHO-DOR membranes / well; Buffer: 50 mM TrisHCl pH 7.4; 60mM MgCl2; 150mM NaCl; 0.1% BSA.
• the increase in the TR-FRET signal (Ratio HTRF) generated by the stimulation with the agonist means that the proportion of G alpha protein form bound to the GTP-donor increases (ie that the form of the empty G alpha protein decreases).
• the RCPG receptor activated by its agonist causes the binding of the GTP-donor to the G protein which then passes in the GTP-donor form and causes the increase in the TR-FRET signal.

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