EP1507801A1 - Procedes et compositions diriges contre la modulation de la chimiosensation - Google Patents

Procedes et compositions diriges contre la modulation de la chimiosensation

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Publication number
EP1507801A1
EP1507801A1 EP03723027A EP03723027A EP1507801A1 EP 1507801 A1 EP1507801 A1 EP 1507801A1 EP 03723027 A EP03723027 A EP 03723027A EP 03723027 A EP03723027 A EP 03723027A EP 1507801 A1 EP1507801 A1 EP 1507801A1
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European Patent Office
Prior art keywords
antibody
receptor
complex
taste
antibodies
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EP03723027A
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German (de)
English (en)
Inventor
Shmuel Ben-Sasson
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TVD Taste Virtual Dimensions Inc
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TVD Taste Virtual Dimensions Inc
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Publication of EP1507801A1 publication Critical patent/EP1507801A1/fr
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    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • 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

Definitions

  • the sensation of taste is now known to be the culmination of a process mediated by a diverse collection of signal transduction mechanisms, which originate at taste receptor cells. These cells are generally present in groups of 50-150 within individual taste buds.
  • odor signals are transmitted to both the brain's higher cortex, which handles conscious thought processes, and to the limbic system, which generates emotional feelings. It is now known that a single odor receptor can recognize multiple odorants. Further, a single odorant is typically recognized by multiple receptors. Additionally, different odorants are recognized by different combinations of receptors. In contrast to taste, the electrical signal from odor receptor neurons diminishes quickly over time, even when the odor is still present. Accordingly, the neurons usually produce a much smaller electrical signal if exposed to the same odor twice in a short period of time.
  • Taste transduction is, for example, effectuated through the depolarization or hyperpolarization of taste cells. There are five basic tastes each with its own mechanism of transduction.
  • Sweet taste (Striem et al., 1989, Biochem. J., 260: 121-126; Tonosaki et al., 1988, Nature, 331: 354-356), bitter taste (Akabas et al, 1988, Science, 242:1047-1050;. Hwang et al., 1990, Proc. Natl. Acad. Sci. USA, 87:7395-7399) and umami (Chaudhari et al., 2000, Nat. Neurosci., 3: 113-119) have been reported to involve G protein-coupled receptors (GPCRs).
  • GPCRs G protein-coupled receptors
  • Sour taste is mediated by a proton channel (Gilbertson et al., 1993, Neuron, 10: 931-942; Gilbertson et al., 1992, J. Gen. Physiol, 100:803-824) and blockade of voltage- gated potassium channels is also thought to occur ( innamon et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 7023-7027).
  • Salty taste can be mediated through sodium channels (Heck et al., 1984, Science, 223:403-405; Avenet et al., 1988, J. Membrane BioL, 105:245-255).
  • Odorant transduction is initiated when an odorant interacts with specific receptors on the cilia of the ORN. Receptors then couple to a G protein to stimulate adenyl cyclase.
  • cAMP is the key messenger in the initial phase of odorant detection and signal transduction.
  • Gustducin has been recently cloned and the protein has been used in methods for identifying small molecular weight agents that inhibit or activate gustducin (United States Pat. No. 6,008,000 and patents related by priority, hereby incorporated by reference). The patent further discloses the use of antibodies to gustducin in the inhibition of ligand binding. Additionally, an amiloride-sensitive sodium channel (Epithelial Na Channel or ENaC) and a method of identifying substances that activate or inactivate that channel has been disclosed (United States Pat. No. 5,693,756, hereby incorporated by reference).
  • An antibody is an immunoglobulin molecule that is capable of highly specific interaction with the antigen that induced its synthesis.
  • Antibodies are diverse, with more than 10 10 possible variations, yet each antibody is designed to recognize only a specific antigen.
  • Antibodies have been previously generated against cellular receptors. Antibodies against the 2 nd extracellular loop (2EL) of many type la GPCR have been generated and have an agonistic effect on the receptor. However, this is not a general rule. In some cases, the agonistic effect is achieved by antibodies against 1EL, or by simultaneous application of antibodies against 1EL and 3EL. In some cases, the antibodies against 2EL function as antagonists.
  • 2EL 2 nd extracellular loop
  • the problems and deficiencies described above are solved by the present invention which relates to the modulation of taste or smell, specifically through methods designed to produce antibodies to taste and odor receptors.
  • Such antibodies will generally have much larger molecular weight than natural ligands or tastants or odorants.
  • Such antibodies are raised against specific epitopes in taste or odor receptors and are designed to utilize these natural receptors to "manipulate" the mind into experiencing taste or smell.
  • Such antibodies may simulate or mimic natural ligands, down-regulate their action or may function unlike any known ligands.
  • Antibodies may be used in combination with ligands, tastants, odorants, or other antibodies. Peptide sequences used for antibody generation need not be involved in ligand binding at all.
  • Antibodies of the instant invention will modulate the experience of taste or smell by binding, respectively, to taste receptors or odor receptors and either stimulating or inhibiting signal transduction emanating from the receptor. «
  • FIG. 1 Domain architecture of metabotropic glutamate receptor mGluRl.
  • the architecture ofTlR is the same.
  • the instant invention relies on combining knowledge regarding antibody production with an understanding of taste and odorant receptor structure and function in such a way that chemosensation modulation can be achieved. Accordingly, the embodiments of the invention rely on a manipulation of the known structural details of taste and odor receptors,, where such manipulation is directed towards methods for generating novel antibodies with the ability to interact with and modulate these receptors in a highly specific manner. It is, therefore, in this novel analysis of taste and odor receptor structure, as viewed from the perspective of antibody generation, that the invention is best described.
  • modulates refers to an up or down regulation of receptor signaling.
  • taste sensation refers to either a single taste type or may comprise a combination of taste types.
  • taste type refers to a taste generated by a specific type of taste receptor, i.e. sweet or salty.
  • unpleasant or “pleasant” may be used as subjective terms as applied to both taste and smell or may be objectified using standard assays in test subjects.
  • a "portion" as used herein in reference to a receptor describes, at the minimum, the smallest antigenic amino acid sequence.
  • a "natural ligand” as used herein refers to a tastant or an odorant, particularly those associated with or isolated from a food or drink.
  • General Types of Taste Receptors a. Sweet and amino acid taste receptors
  • Sweet and amino acid taste receptors are represented by three genes, T1R1-3 (Lewcock, J.W., and Reed, R.R. (2001) Neuron 31, 515). They belong to the 3 r family of GPCR (Bockaert, J., and Pin, J.P. (1999) EMBOJ. 18, 1723). In families 1 and 2, extracellular loops and transmembrane domains are involved in ligand binding (see fig.l). In contrast, in family 3 GPCR, the binding site is located within the VFT. The members of this family have a long N-terminal extracellular domain, which belongs to the ANF receptor family of ligand-binding regions (FIGURE A).
  • T1R3 is allelic to the sweet responsiveness locus, Sac (Bachmanov, A.A. et al. (2001) Chem. Senses 26, 925) (Nelson, G., et al. (2001) Cell 106, 381) (Sainz, E. et al. (2001) J. Neurochem. 77, 896) (Max, M. et al. (2001) Nat. Genet. 28, 58). Recently, the sequences of human T1R1-3 were published (Li X. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 4692).
  • the trapping of a ligand within the VFT leads to its closure, which prepares an interface for binding the appropriate domain in the second receptor leading to a dimer formation which, in order, leads to the conformational changes within the whole molecule.
  • This mechanism seems to be a widespread way to transform low affinity binding to strong downstream signals (Kunishima, N. (2000) Nature 407, 971).
  • T1R3 is allelic to the sweet responsiveness locus, Sac (Max, M. et al. (2001) Nat. Genet. 28, 58).
  • the functional activity of T1R in sweet sensation has been achieved only when T1R2 and T1R3 were coexpressed (Nelson, G. et al., (2001) Cell 106, 381). So, it seems that the two receptors form heterodimers. Most probably, they are disulfide-linked, since their N-termini contain the conservative cysteins involved in dimerization in Ca 2+ -sensitive receptor (CaR) (Hu, J. et al. (2000) J. Biol. Chem. ITS, 1 382) and glutamate receptor mGluRl (Robbins, M.
  • T1R family should not contain additional members (Nelson, G., et al. (2001) Cell, 106, 381).
  • T1R1 and T1R2 are expressed in different cells, and both are co-localized with T1R3.
  • the coexpressed T1R2 and T1R3 are functionally active. It is probable that other functionally active pairs may be formed.
  • the physiological data confirms, at least in mice, the existence of at least two cell subsets, which differ by their sensitivity to the specific peptide inhibitor of a response to the sweetener gurmarin (Ninomiya, Y. et al. (1999) J. Neurophysiol. 81, 3087).
  • family 3 of GPCR have a long N-terminal extracellular domain, which belongs to the ANF receptor family of ligand-binding regions.
  • the ANF receptor family includes the extracellular ligand binding domains of a wide range of non-GPCR receptors, including ionotropic glutamate receptors, as well as bacterial periplasmic binding proteins, involved in the transport of various types of molecules such as amino acids, ions, sugars or peptides (Kuryatov, A., et al. (1994) Neuron 12, 1291) (O'Hara, PJ. et al. (1993) Neuron 11, 41).
  • the domain is constituted of two lobes separated by a hinge region, and several studies including X-ray crystallography indicated that these two lobes closed like a Venus flytrap (VFT) upon binding of the ligand.
  • VFT Venus flytrap
  • the receptors that contain ANF receptor domains belong to distinct families whose overall structure and transduction pattern are completely different.
  • the first group that contains ANF receptor domains belongs to G protein coupled receptor (GPCR) type 3 family.
  • GPCR G protein coupled receptor
  • This family includes closely related metabotropic glutamate receptors, extracellular Ca 2+ -sensitive receptor (CaR), taste and vomeronasal receptors T1R and V1R, and more distantly related GABA(B) receptors.
  • GABA(B) GABA(B) receptors
  • all of these receptors contain a cysteine-rich juxtamembrane region and form disulfide-bonded dimers. Binding of a ligand leads to the closure of VFT and formation of an interface for dimerization of ANF receptor domains, leading to conformational changes and activation of the receptor.
  • GPCR form homodimers, while others may form heterodimers.
  • GABA(B) receptor heterodimeric complex only GABA(A) Rl binds a specific ligand, thus leading to a conformational change in GABA(B) R2 (Galvez, T. et al., (2000) EMBO J. 20, 2152).
  • CaR and mGluRl may also form disulfide-linked heterodimers that are sensitive to glutamate-mediatedinternalization (Gama, L. etal. (2001) J. Biol. Chem. 276, 39053). It seems that mouse T1R3 is functional when it forms aheterodimer with T1R2.
  • Metabotropic glutamate receptors may form heterodimers with adenosine Al receptors, which belong to a family 1 A GPCR.
  • the interaction in this case, is mediated by the interaction of the C-termini of two types of receptors (Ciruela, F. etal. (2001) J. Biol. Chem. 276, 18345).
  • the next of the groups is the membrane-associated guanylyl kinase receptor family (Wedel, BJ., and Garbers, D.L. (1997) FEBSLett. 410, 29).
  • This family includes three receptors of atrial natriuretic peptide and the less studied receptor for heat-stable E. coli enterotoxin.
  • Additional orphan receptors of this family have been discovered in rods (Goraczniak, R.M. et al. (1998) Biochem. Biophys. Res. Commun. 245, 447) and in olfactory cells (Duda, T. et al. (2001) Biochemistry 40, 12067) (Duda, T.
  • NPR natriuretic peptide receptor
  • the current model suggests that the NPRs are dimerized even in the absence of the ligand. This dimerization is mediated by the juxtamembrane cysteine-rich region, but not through interchain disulfide bonds. Binding of a ligand leads to tighter association of the ANF receptor domains, leading to the conformational changes and activation of guanylyl kinase activity. Introduction of the additional cysteine into the juxtamembrane region leads to the formation of an interchain disulfide bond and to the appearance of constitutively active receptor (Labrecque, J. et al. (1999) J. Biol. Chem. 274, 9752) (Mammen, A.L. et al. (1997) J. Neurosci. 17, 7531).
  • the ANF receptor domain seems to play a different role in the third group, the ionotropic glutamate receptors. They do not participate in glutamate binding, and their function is not clear. These receptors function as tetramers, and it seems that the ANF receptor domain is involved in the primary dimerization of iGluR, which is followed by tetramerization mediated by other regions (Ayalon, G., and Stern-Bach, Y. (2001) Neuron 31, 103). Antibodies (but not Fab fragments) against peptides within the ANF receptor domain of GluRl induce clustering of the receptors (Mammen, A.L. etal. (1997) J. Neurosci. 17, 7531).
  • the agonistic auto- and heteroantibodies against GluR2 or GluR3 are directed against the sequence located in the hinge region between the ANF receptor domain and the downstream PBPe domain (McDonald, S. et al. (1999) J. Mol. Recognit. 12, 219) (Carlson, N.G. et al. (1991) J. Biol. Chem. 272, 11295) (Carlson, N.G., et al. (2001) J. Neurosci. Res. 63, 480). The latter, which are homologous to bacterial periplasmic binding proteins, also fit to the Venus flytrap model. It seems that the antibody function is mediated by promotion of tighter association between the subunits within the pre-formed oligomeric complex.
  • T2R bitter taste receptors
  • the sour taste is mediated by direct depolarization of the taste receptor cells. It seems that different mechanisms are involved in sourness of the strong and week acids.
  • the sourness of strong acids is mediated by pericellular H + penetration to the basolateral region and activation of some ion channels in this region.
  • the acid-sensitive cation channel ASIC2 (BNaCl, BCN1) is expressed in taste bud cells (Ugawa, S. et al. (1998) Nature 395, 556), but is not necessary for sour taste transduction (Kinna on, S.C., etal. (2000) Olf action and Taste XIII. New York: Springer-Verlag, p. 80).
  • HCN1 and HCN4 Two hype olarization-activated cation channels, HCN1 and HCN4, are located at the basolateral part of the taste bud epithelium and may be involved in this process (Stevens, D.R. et al. (2001) Nature 413, 631).
  • the sour transduction is mediated by apical K + channel, which is closed at low pH (Kinnamon, S.C. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7023).
  • acetic acid is a more potent sour stimulus at the same pH.
  • acetic acid seems to penetrate the apical membrane in the non-dissociated form and decrease the intracellular pH (Lyall, V. et al. (2001) Am. J. Physiol, 281, C1105).
  • MCT monocarboxylic acid transporters
  • the salt taste is mediated by direct depolarization of the taste receptor cells.
  • salt taste is inhibited by amiloride which means that Na + ions penetrate the cell through amiloride-sensitive Na + channel (ENaC).
  • ENaC channel consists of three homologous subunits, , ⁇ , and ⁇ .
  • the constitutively expressed subunit is non-functional, while ⁇ and ⁇ subunits are up-regulated by aldosterone, thus forming, together with the subunit, a functional channel. All three subunits are expressed in rat taste bud cells (Lin, W. etal. (1999) J Comp. Neurol. 405, 406).
  • the ENaC subunits have a common transmembrane topology, with intracellular N- and C-termini, two transmembrane domain, and a long highly glycosylated extracellular part in between.
  • a monoclonal antibody RA6.3 against the amiloride-binding domain of ENaC has been raised.
  • the antibody mimicked amiloride in that it inhibited transepithelial Na + transport (Kleyman, T. R., et al. (1991) J. Biol. Chem. 266, 3907) (Kieber-Emmons, T. et al. (1999) J. Biol. Chem. 274, 9648).
  • the epitope for this antibody has been recently mapped.
  • the antibody is completely inhibited by a peptide DAVRE WYRFH YINLL SRL, corresponding to residues 246-263 of human or rat ENaCcc (Kieber-Emmons, T. et al. (1999) J Biol. Chem. 274, 9648). While it is clear, that in rats the salt taste is mediated by Na + penetration through ENaC channels, it does not seem a general rule. In some mouse strains, the NaCl-induced response is suppressed by amiloride, whereas in others it is not (Miyamoto, T. et al. (1999) Neurosci. Lett. 277, .13).
  • mGluRs m metabotropic glutamate receptors
  • brain-mGluR4 the specific taste-mGluR4 (Chaudhari, N. et al. (2000) Nat. Neurosci. 3, 113). The latter is a splice form with a truncated N-terminus. Its affinity to glutamate is in the millimolar range, similar to that in the taste buds.
  • the taste-receptor GluR4 belongs to the 3 rd family of GPCR (see above). Since mGluR are able to form heterodimers with other members of the same family (Gama, L. et al. (2001) J. Biol. Chem. 276, 39053), it is very probable that they may form heterodimers with T1R. This is confirmed by the fact that the glutamate taste is affected by a specific peptide inhibitor of sweet taste, gurmarin.
  • ORs Odorant receptors
  • ORs also belong to the GPCR super-family and several hundred ORs are encoded by the human genome (for a review see Mobaerts P. (1999) Science, 286: 707-711).
  • ORs have relatively short extracellular N-terminus, probably due to the interaction of the volatile hydrophobic odorants with the trans-membrane alpha-helices portion of the receptor (see Afshar M. et al. (1998) Biochemie, 80: 129-135). Therefore, with respect to modulating odorant sensation, the prime targets for antibody production are the extracellular loops of the ORs.
  • ELI ANHLLGSKSISFGGC (SEQ ID NO: 1)
  • ELI LSRLLSRKRAVPC (SEQ ID NO: 10)
  • EL2 CGPNVTNHFY (SEQ ID NO: 11)
  • EL2 CDLPQLFQLS (SEQ ID NO: 12)
  • EL2 CSSTQLNEL (SEQ ID NO: 13)
  • EL3 (C)RLGSTKLSDKDKA (SEQ ID NO: 14) OR Type 5, 05Fl_Human:
  • (C) a Cysteine added to the native sequence to enable cross-linking to a carrier protein.
  • Antibodies to taste receptors are generated based on analysis of the structural features of taste receptors. For example, in one embodiment, epitopes in TIR are chosen based on analysis of polymorphisms in mTlR3, by analogy with mGluRl or by analogy with ionotropic GluR. Antibodies may be generated using transgenic animals, preferably birds and mammals. Alternatively, animals may be inoculated with antigen to provoke an immune response that would comprise antibodies to the antigen.
  • the region LGSTE EATLN QRTQP NSIPC (SEQ ID NO: 20), corresponding to residues 43-62 of mTlR3, would be utilized as a candidate for raising antibodies.
  • antibodies of the instant invention are raised in chickens.
  • X-ray crystallography of the taste receptor is used to deduce which parts of the fragments are involved, for example, in VFT closure or dimerization.
  • antigenic sites can be found within the fragments of mGluRl . Each antigenic site would be located on the 3D structures of mGluRl with and without glutamate, and the sites that may interfere with VFP closure or dimerization of ANF receptor domains would be chosen.
  • the sequences from hTlRl, hTlR2 and hTlR3, which correspond to those chosen as interfering with VFP closure or dimerization and are also good antigens by antigenicity plot, would be chosen as good candidates.
  • a method of selecting a candidate antigenic site comprises: a) using an antigenicity plot to identify antigenic sites within the extracellular domains of the selected receptor; b) using the three dimensional structure of mGluRl with and without glutamate to identify a selected antigenic site that may interfere with VFP closure or dimerization of ANF receptor domains; c) identifying the candidate antigenic site as that selected antigenic site which i. corresponds to a sequence from hTlRl, hTlR2 or hTlR3; and ii. is a good antigen based on the antigenicity plot.
  • analogy with ionotropic GluR is used, for example, homology between the mGluR and TIR is used to reveal appropriate epitopes for antibody generation.
  • the homologous sequence in mGluRl is EGVLN IDDYK IQMN (SEQ ID NO: 21).
  • the homologous sequences of hTlR3 are QGSVP RLHDV GRFN (SEQ ID NO: 22) and LRTER LKTRW HTSDN QKPVS RC (SEQ ID NO: 23), at two sides from the N-glycosylation site.
  • hTlRl/ Ac-LQVRH RPEVT LCX-NH 2 (SEQ ID NO: 24), hTlRl/ Ac-ETKIQ WHGKD NQVPK SVC-NH2 (SEQ ID NO: 25),hTlRl/ Ac-(C)ETLS VKRQY P-NH 2 (SEQ ID NO: 26),hTlRl/ Ac-(C)GSSD DYGQL G-NH 2 (SEQ ID NO: 27), hTlRl/ Ac-SAQVG DERMQ C-NH 2 (SEQ ID NO: 28), hTlR2/ Ac-LHANM KGIVH LNFLQ VPMC-NH 2 (SEQ ID NO: 29), hTlR2/ Ac-(C)DELRD KVRFP-NH 2 (SEQ ID NO: 30), hTlR2/ Ac-(C)VSSDT YGRQN G-NH
  • One aspect of the invention is an antibody or complex of two or more antibodies that modulates chemosensation.
  • the antibody or complex of two or more antibodies stimulates taste sensation.
  • the antibody or complex of two or more antibodies inhibits taste sensation.
  • the antibody or complex of two or more antibodies stimulates odor sensation.
  • the antibody or complex of two or more antibodies inhibits odor sensation.
  • the taste sensation that is modulated is selected from the group consisting of:
  • the odor sensation that is modulated is selected from the group consisting of:
  • the antibody or at least one antibody in the complex is raised against a receptor or a portion thereof.
  • the receptor against which an antibody is raised is selected from the group consisting of: RECT
  • the antibody or at least one antibody in the complex is raised against any one of the group consisting of:
  • the extracellularly exposed region against which an antibody is raised is a portion of a GPCR.
  • the portion of the GPCR against which an antibody is raised is is an extracellular loop of GPCR.
  • the portion of the GPCR is an N-terminal region of the GPCR.
  • an antibody is raised against an amino acid sequence selected from the group consisting of: a) (QETLSVKRQY P (SEQ ID NO: 40); b) (C)GSSDDYGQLG (SEQ ID NO: 41; c) SAQVGDERMQC (SEQ ID NO: 42); d) (C)ELLSARETFP (SEQ ID NO: 43); e) (QPRADDSRLGKVQ (SEQ ID NO: 44); f) (QGSDDEYGRQGL (SEQ ID NO: 45); g) LQVRH RPEVT LC (SEQ ID NO: 46); h) ETKIQ WHGKD NQVPK SVC (SEQ ID NO: 47); i) LRTER LKIRW HTSDN QKPVS RC (SEQ ID NO: 48); j) (C)QGSV PRLHD VGRFN (SEQ ID NO: 49); k) LGEAE EAGLR SRTRP SSPVC (SEQ ID NO: 50);
  • At least two of the complexed antibodies are raised against a receptor or a portion of a receptor.
  • the antibodies in the complex are raised against different receptors or portions of receptors.
  • the complex comprises an antibody raised against a T1R2 receptor or portion thereof and an antibody raised against a T1R3 receptor or portion thereof.
  • the complex comprises an antibody raised against a TlRl receptor or portion tiiereof and an antibody raised against a T1R3 receptor or portion thereof.
  • one antibody in the complex stimulates chemosensation while a second antibody in the complex inhibits chemosensation.
  • the complex of antibodies is formed by a method selected from the group consisting of:
  • the antibody or any one or more of the antibodies which comprise the complex is derived from a bird egg of a bird immunized against an antigen derived from a receptor or portion thereof.
  • the immunized bird is a chicken.
  • Another aspect of the invention is a composition comprising an antibody or complex of antibodies as described above.
  • the antibodies maybe modified, for example, to be antibody fragments.
  • a fragment could be, for example, a Fab fragment or a dsFV fragment or a scFv fragment.
  • Techniques for generating antibody fragments are well known to one of skill in the art (for example, Antibodies: A Laboratory Manual by E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988).
  • antibodies may be enhanced or otherwise synthesized (for example, as described in United States Patent No. 6,300,064, hereby incorporated by reference).
  • the composition would be provided in a chewing gum like form or any other long lasting form which would allow the antibodies to interact with taste receptors.
  • the composition would be provided in a form useful for nasal drops or a nasal spray.
  • One aspect of the invention is a method of making an antibody or complex of antibodies comprising:
  • the immunized bird is a chicken.
  • the receptor used to immunize the bird is a taste receptor or an odorant receptor.
  • Another aspect of the invention is a transgenic non-human animal, wherein said animal is genetically engineered to produce an antibody that modulates chemosensation.
  • Another aspect of the invention is a method of making an antibody or complex of antibodies comprising:
  • Another aspect of the invention is a method for selecting a candidate antigenic site, said method comprising:
  • (i) corresponds to a sequence from hTlRl, hTlR2, or hTlR3; and (ii) is a good antigen based on the antigenicity plot.
  • any of four different approaches are used for choosing the epitopes for functional anti-TlR antibodies:
  • an antibody to a receptor is administered in combination with a ligand for the receptor to which the antibody was raised.
  • the ligand is a natural ligand.
  • a combination of antibodies to different receptors or different portions of the same receptor are administered in combination with a ligand or more than one hgand for the receptor or receptors to which the antibodies were raised.
  • the differences between the taster and the non-taster lines are located in the TM domain and at the C-terminus, but most of them are located in the N-terminal part.
  • the high concentration of substitutions is located in the region closely upstream of the ANF receptor domain. It has been suggested that one of the substitutions (I60T) creates the novel N-glycosylation site (consensus sequence NX(S/T)), which interferes the dimerization of the ANF receptor domain (Max, M. et l. (2001) Nat. Genet. 28, 58).
  • this region (LGSTE EATLN QRTQP NSIPC (SEQ ID NO: 62)), corresponding to residues 43-62 of mTlR3) maybe a good candidate for, most probably, an inhibiting antibody.
  • the corresponding human sequence LGEAE EAGLR SRTRP SSPVC (SEQ ID NO: 63) is present without introns in human chromosome 1.
  • hTlRl LQVRH RPEVT LC (SEQ ID NO: 64).
  • hTlR2 LHANM KGIVH LNFLQ VPMC (SEQ ID NO: 65).
  • mGluRl is the most studied family 3 GPCR. It is homologous to T1R3, T1R2 and TlRl.
  • the X-ray crystallography of its ANF receptor domain with and without glutamate confirms the suggested model: closure of the Venus flytrap (VFT) upon binding of the ligand and formation of the interface for dimerization ( Figure A).
  • the autoantibodies against the N-terminus of mGluRl function as antagonists (Smitt, P.S. et al. (2000) N. Eng. J. Med. 342, 21).
  • the heteroantibodies against the parts of ANF receptor domain of mGluRl function as antagonists (Shigemoto, R. et al. (1994) Neuron 12, 1245).
  • These antibodies have been prepared against the fragments, corresponding to large parts of mGluRl ANF receptor domain: residues 104-154 and residues 177-341.
  • the availability of the X-ray crystallography data made it possible to deduce, what parts of the fragments are involved in VFT closure or dimerization.
  • each antigenic site was located on the 3D structures of mGluRl with and without glutamate, and the sites that may interfere with VFP closure or dimerization of ANF receptor domains were chosen;
  • hTlRl ((QETLS VKRQY P (SEQ ID NO: 66); (C)GSSD DYGQL G (SEQ ID NO: 67), and SAQVG DERMQ C (SEQ ID NO: 68)); for hTlR2 ((C)DELRD KVRFP (SEQ ID NO: 69), (C)VSSDT YGRQN G (SEQ ID NO: 70) and (QPNQNM TSEER QRL (SEQ ID NO: 71)); and for hTlR3 ((C)ELLS ARETF P (SEQ ID NO: 72), (C)PRAD DSRLG KVQ (SEQ ID NO: 73) and (C)GSDD EYGRQ GL (SEQ ID NO: 74)).
  • mGluRl the region homologous to one of the chosen peptides interacts upon closure of VFP with the sequence homologous with that chosen by the first method.
  • the long extracellular N-terminus of ionotropic glutamate receptors contains two separate domains, both homologous to bacterial periplasmic binding proteins (see Fig. 1).
  • the more proximal to the TM domains is called PBPe. It is responsible for binding glutamate, as well as specific agonists, like AMP A and kainate.
  • the X-ray structure of the crystallized PBPe domain of GluR2 with and without glutamate confirms the Venus flytrap (VFT) model ( Figure B).
  • ANF receptor domain also called X-domain. Its function in the ionotropic glutamate receptors is unknown. It seems that it is involved in the primary dimerization of iGluR, which is followed by tetramerization mediated by other regions.
  • the epitope is closer to the former and is separated from PBDe domain by N-glycosylation site.
  • the mGluRl, TlRl, T1R2, and T1R3 contain N-glycosylation site 12-15 residues downstream of the end of ANF receptor domain. Li GluR3, this site is glycosylated. In TlRl, T1R2, and T1R3 the sequence predicts an efficient glycosylation site.
  • the ANF receptor domains of GluR3 and mGluRl share some homology, therefore one can expect that antibodies against a similar effect.
  • the homologous sequence in mGluRl is EGVLN IDDYK IQMN (SEQ ID NO: 75).
  • the homologous sequences of hTlR3 are QGSVP RLHDV GRFN (SEQ ID .NO: 76) and LRTER LKIRW HTSDN QKPVS RC (SEQ ID NO: 77), at two sides from the N-glycosylation site, hi hTlRl, only the sequence at one of the two sides from the N-glycosylation site is immunogenic: ETKIQ WHGKD NQVPK SVC (SEQ ID NO: 78) while in hTlR2, the corresponding sequence is: LKNIQ DISWH TVNNT IPMSM C (SEQ ID NO: 79).
  • the following list of peptides against sweet taste and amino acid receptors are provided by the analysis. Since the peptides correspond to the internal parts of the sequences, their N- and C-termini are, respectively, acetylated and amidated.
  • the (C) means that cysteine residue was added to the sequence.
  • Immunization is done using either a whole taste-bud preparation (e.g. porcine's circumvallate papilla rich in taste-buds) or immunization with selected peptides derived from taste receptors or both.
  • a whole taste-bud preparation e.g. porcine's circumvallate papilla rich in taste-buds
  • immunization with selected peptides derived from taste receptors or both.
  • the immunogenic peptide is covalently attached to a larger protein carrier.
  • the peptide is ordered from a company that specializes in solid-phase synthesis of peptides. The minimal amount is, for example, 5 mg peptide, at >90% purity.
  • cysteine residue is added to the sequence at one of the termini for coupling purposes. Each peptide is dissolved in 5 mM acetic acid, the amount of the free sulfhydryl groups measured by reaction with Ellman's reagent.
  • the peptides are coupled through their sulfhydryl groups to maleimide-modified keyhole limpet hemocyanine (KLH). The efficiency of coupling is tested with Ellman's reagent.
  • the KLH-conjugated peptides are injected intramuscularly to laying hens, 5-6 months old. Each peptide is injected to two hens. Before immunizations, a few preimmune eggs are collected. In each immunization, 60 ⁇ g of a peptide is injected intramuscularly into 4 points. The first immunization is performed with complete Freund's adjuvant. The boosts are performed with incomplete Freund's adjuvant at weeks 2 and 4 after the primary immunization. A week after the 2 nd boost, eggs are collected daily and stored at 4 °C. The eggs from each hen are stored separately.
  • the yolks are separated from the egg whites and washed with deionized water.
  • the yolks are diluted with 4 volumes of sterile deionized water (1 :50), stored for 6 h at 4 °C, and centrifuged at 3500g.
  • the resulting supernatant contains 20-30 % IgY.
  • the supernatants are filtered through glass filters. Since the supernatants are to be used for tasting experiments, the use of standard anti-bacterial agents should be avoided.
  • the preparations are stored frozen or lyophilized.
  • the further purification is performed by ammonium sulfate or sodium sulfate precipitation, dialysis, and, if necessary, affinity purification on the appropriate peptide, immobilized through its SH group to Sulfolink beads.
  • the elution is performed with 3.5 M or 4.5 M MgCl 2 , which can be removed from the IgY preparation by dialysis.
  • the IgY concentration is measured spectrophotometrically.
  • the experiments are performed in double blind manner. Since individual variations in the sensitivity to individual tasters may be expected, the taste experiments are performed on groups of several volunteers.
  • MAPs may be used instead of conjugation of KLH with another protein.
  • MAPs may be used instead of conjugation of KLH with another protein.
  • hT2R4-EL2 The most soluble peptide conjugated with KLH, others -MAP. hT2R4-EL2 could also be made with MAP.
  • the yolks are separated from the egg whites and washed with deionized water. For further elimination of yolks, three basic procedures are accepted:
  • the third one is preferable.
  • the resulting supernatant contains 20-30 % IgY and is stored frozen or lyophilized.
  • the further purification is performed by ammonium sulfate or sodium sulfate precipitation, dialysis, and, possibly, affinity purification on the appropriate peptide.
  • Anti-human-chemoreceptors antibodies tasting. The experiment includes negative controls with preimmune eggs (possibly, from the same hens), positive control with denatonium (in the case of hT2R4), each antibody alone, as well as the mixtures. The relative dilution of each antibody is evaluated according to the results of affinity purification. Both agonistic and antagonistic effect may be expected.
  • Synthetic peptides 10-20 amino acid residues long, were synthesized at >80% purity. The purity and identity of peptides was confirmed by reverse phase HPLC and mass- spectroscopy. To the peptides that do not contain cysteine, additional cysteine was included at one of the termini, for coupling purpose.
  • the peptides were coupled to maleimide-modified keyhole limpet hemocyanin (KLH) through their cysteine moieties.
  • KLH keyhole limpet hemocyanin
  • the immunization schedule was the follows:
  • the eggs were collected and stored at 4 °C. After 12 eggs per hen were collected, the yolks from each hen were pooled and lyophilized.
  • the efficacy of the immunization was tested by Enzyme-Unked immunoassay (ELIS A) using immobilized free peptides.
  • the bound antibody was detected with horseradish peroxidase-conjugated antibody against IgY.
  • TVD peptides 3/21, 4/21, 5/21, 6/21 and 7/21 are derived from the hTlR2 receptor (for sweet taste) while TVD peptides 1/21, 9/21 and 11/21 are derived from the hTlR3 receptor (common to sweet and umami).

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Abstract

L'invention concerne des procédés et des anticorps dirigés contre la modulation de la chimiosensation. Plus précisément, l'invention concerne des procédés et des anticorps dirigés contre la modulation de la sensation de goût et des procédés et des anticorps dirigés contre la sensation d'odorat.
EP03723027A 2002-05-20 2003-05-20 Procedes et compositions diriges contre la modulation de la chimiosensation Withdrawn EP1507801A1 (fr)

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PCT/IL2003/000409 WO2003102030A1 (fr) 2002-05-20 2003-05-20 Procedes et compositions diriges contre la modulation de la chimiosensation

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US7364867B2 (en) 2000-04-17 2008-04-29 The Mount Sinai School Of Medicine Method of identifying bitter compounds by employing TRP8, a transient receptor potential channel expressed in taste receptor cells
US7803982B2 (en) 2001-04-20 2010-09-28 The Mount Sinai School Of Medicine Of New York University T1R3 transgenic animals, cells and related methods
US20140147556A1 (en) * 2012-11-27 2014-05-29 Elwha Llc Edible or inhalable compositions having antibodies and methods of use

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