WO2003102030A1 - Antibodies and their use for the modulation of taste sensation___ - Google Patents

Abstract

the present invention relates to methods and antibodies directed towards the modulation of chemosensation. Specifically, the present invention relates to methods and antibodies directed towards the modulation of taste sensation and methods and antibodies directed towards the modulation of smell sensation.

Description

ANTIBODIES AND THEIR USE FOR THE MODULATION OF TASTE SENSATION

Background of the Invention

1. Field of the Invention

The present invention relates to methods and compositions directed towards the modulation of chemosensation. Specifically, the present invention relates to methods and antibodies directed towards the modulation of taste sensation and methods and antibodies directed towards the modulation of smell sensation.

2. Background of the Related Art

Historically, the only way to "modulate" the taste of a food or drink was with the use of flavored additives that act in the same way as the natural tastants. For example, the bitter taste of some medicines can be partially hidden or "masked" by the addition of something sweet. Alternatively, substitutes for substances such, as salt and sugar have been designed to simulate the respective flavor without the dietary ramifications (for example, United States Pat. Nos. 6,159,529 and 5,756,543, hereby incorporated by reference). Similarly, the only way to "modulate" an odor was by the addition of odorants. However, the elucidation of molecular constituents involved in chemosensation provides, for the first time, the opportunity to develop novel strategies and agents for the modulation of taste and smell.

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.

The sensation of smell is now known to be the culmination of a process mediated by olfactory receptor neurons (OR s) located in the olfactory epithelium. ORNs are activated by G protein-coupled receptors and binding by an odorant leads to a G protein-coupled adenylyl cyclase cascade which, in turn, transmits the sensation of smell to the brain. An odorant receptor protein has several defining features and belongs to the general class of seven-transmembrane proteins. When an odor excites a neuron, the signal travels along the nerve cell's axon and is transferred to the neurons in the olfactory bulb. From the olfactory bulb, 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). 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).

However, the natural tastants or ligands of the prior art bind to amino acid residues scattered along a large stretch of the receptor sequence. Further, none of the aforementioned patents or publications suggest large molecular weight molecules, such as antibodies, or their use in the modulation of chemosensation in ways other than as inhibiting the binding of the natural ligands. Moreover, in order to create a continuous chemosensation, a tastant should be consumed continuously because of its low affinity to the corresponding receptor. This, in essence, is the mechanistic reason behind food craving. On the other hand, specific antibodies should have a high affinity towards such receptors and therefore the effect of arelatively small amount of them should last for a longer period of time.

Importantly, although there is a considerable amount of data available to describe the constituents involved in both taste and odor reception and transmission, as will be described further below, there continues to exist a need in the art for new products and methods that are involved in or affect chemosensation. In that respect, methods and compositions for capable of modulating chemosensation would be highly prized.

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 10¹⁰ 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. In the case of β2 adrenergic receptor, it is clear that the functional effect of the antibody is associated with dimerization, since the Fab fragment of the mAb behaved as antagonist, while the application of anti-mouse IgG partially restored the agonistic function, h fact, for this receptor, dimerization has been shown to have a role in the normal function of this receptor.

Table 1. Antibodies against GPCR with functional effect.

As can be seen, antibodies are able to interact with and effect GPCR function. However, the prior art has not applied these techniques to provide methods of modulating chemosensation. In fact, none of the aforementioned patents or publications suggest the use of large molecular weight molecules, such as antibodies, in the modulation of chemosensation in ways other than as inhibiting taste modulation by the natural ligands.

Brief Summary of the Invention

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, whether used alone or in combination with tastants or odorants, 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. «

Brief Description of the Drawings

Figure 1. Ligand-binding sites of different families of GPCR (from Bockaert, J., and Pin, J.P., 1999). Orange - ligand-binding sites; green conservative cysteins.

Figure 2. Domain architecture of metabotropic glutamate receptor mGluRl. The architecture ofTlR is the same.

Figure 3. Domain architecture of ionotropic glutamate receptor GluR3.

Detailed Description of the Invention

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.

The term "modulates" as used herein refers to an up or down regulation of receptor signaling.

The term "taste sensation" as used herein refers to either a single taste type or may comprise a combination of taste types.

The term "taste type" as used herein refers to a taste generated by a specific type of taste receptor, i.e. sweet or salty.

Terms for the sensations generated by specific chemosensation receptors may be referred to herein by using the receptor name or sequence followed, for example, by "type".

The terms "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 "domain" as used herein refers to a region on the three dimensional protein which is directly involved in ligand binding.

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).

In mouse or rat, three receptors have been cloned. 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).

According to the current view, the trapping of a ligand within the VFT (not necessarily with high affinity) 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 Ca²⁺-sensitive receptor (CaR) (Hu, J. et al. (2000) J. Biol. Chem. ITS, 1 382) and glutamate receptor mGluRl (Robbins, M. L, et al. (1999) J Neurochem. 72, 2539) (Ray, K. etal. (2000) J. Biol. Chem. 275, 34245). Likewise, uma i taste stimuli generated by amino acids, require both T1R1 and T1R3, probably through a heterodimer formation (Li X. et al., (2002) Proc. Natl. Acad. Sci. USA 99, 4692).

According to the human genome analysis, the T1R family should not contain additional members (Nelson, G., et al. (2001) Cell, 106, 381). In taste buds, 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.

Recently, many cases of heterodimerization of more distantly related GPCR have been described (Jordan, B.A. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 343) (Rocheville, M. et al. (2000) Science 288, 154) (Gama, L. et al. (2001) J. Biol. Chem. 276, 39053). Therefore, one cannot exclude that the members of T1R family might form heterodimers with other GPCR expressed in the same cells, and, possibly, with a taste bud form of GluR4, which is expressed in the same cells and also belongs to the family 3 of GPCR. 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). As described above, 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.

The receptors that contain ANF receptor domains belong to distinct families whose overall structure and transduction pattern are completely different.

As described above, the first group that contains ANF receptor domains belongs to G protein coupled receptor (GPCR) type 3 family. This family includes closely related metabotropic glutamate receptors, extracellular Ca²⁺-sensitive receptor (CaR), taste and vomeronasal receptors T1R and V1R, and more distantly related GABA(B) receptors. With the exception of GABA(B), 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.

Some type 3 GPCR form homodimers, while others may form heterodimers. In the 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.

In fact, there is new data that does not correspond with the simple concept of VFT dimerization. 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. Recently, 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. et al (2001) Biochemistry 40, 4654). The most studied is the natriuretic peptide receptor (NPR) group. It was initially hypothesized that the guanylyl cyclase activity of NPRs must require receptor dimerization. However, the situation of taste receptor differs from that in several aspects: the stoichiometry of binding is one ligand to two receptor molecules, and the ligand is not dimeric (Rondeau, J.-J., et al. (1995) Biochemistry 34, 2130).

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.

b. Bitter taste receptors

A large family of bitter taste receptors, T2R, has been recently discovered (Matsunami, H. et al. (2000) Nature 404, 601) (Adler, E. et al. (2000) Cell 100, 693). It belongs to type 4 family of GPCR, together with V1R vomeronasal receptors. This group is characterized by a very short N-terminal part, and highly diverse extracellular loops. This, together, makes it probable that the extracellular loops are involved in ligand binding.

While the overall architecture of this family is similar to that of type la family GPCR, which includes most of the GPCR to small ligands, there is no homology between the two families.

c. Sour 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). 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). In the amphibian Necturus maculosus, 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). In contrast to the strong acids, acetic acid is a more potent sour stimulus at the same pH. Moreover, the sourness of acetic acid is essentially the same as that of a buffer consisting of the acid plus its conjugate base, even though the latter has a higher 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).

It is not known whether acetic acid penetrates actively or passively. In general, acetic acid can be transported by monocarboxylic acid transporters (MCT) (Poole, R.C., and Halestrap, A. P. (1993) Am. J. Physiol. 264, C761). To date, seven MCT have been cloned (Price, N.T. et al. (1998) Biochem. J. 329, 321). However, there is no information whether any of them is located at the apical surface of taste receptor cells.

d. Salt taste receptors

Like the sour taste, the salt taste is mediated by direct depolarization of the taste receptor cells. In rats, salt taste is inhibited by amiloride which means that Na⁺ ions penetrate the cell through amiloride-sensitive Na⁺ channel (ENaC). The ENaC channel consists of three homologous subunits, , β, and γ. In the interstitial epithelium, the constitutively expressed subunit is non-functional, while β and γ 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. Using the anti-idiotype approach, 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). In humans, the effect of amiloride on salty taste is minor, if at all. It seems that there are individual differences in amiloride sensitivity. (Halpern, B.P., and Darlington, R.B. (1998) Chem. Senses 23, 501, Halpe n, B.P. (1998) Neurosci. Biobehav. Rev.23, 5).

e. Umami taste receptors

The concept of the basic taste "umami" refers to the taste of amino acids. The word was adopted from Japanese, the only language to have a term for it.

The umami taste is induced by monosodium glutamate, and it seems that Na⁺ may play a role in the animals whose salt taste is mediated by ENaC. Both NMDA receptor channels and group m metabotropic glutamate receptors (mGluRs) are present in fungiform taste cells and may be involved in transducing glutamate taste. There are two types of metabotropic mGluR: the brain type brain-mGluR4 and 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.

Odor Receptors

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). Unlike taste receptors, 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. The predicted amino-acid sequence of these loops in human ORs is known to a person of skill in the art based on the identification of the trans-membrane alpha-helices. For a comprehensive list of human ORs sequences see: Zozulya S. et al..(2001) Genome Biol. 2: 1-16 and Glusman G. et al., (2001) Genome Res. 108: 685-702.

As an illustration, the following are examples of amino acid sequences derived from the extra-cellular loops of several human ORs. The second extracellular loop (EL2) is usually the largest and, therefore, several peptide sequences are derived from it (additional OR sequences may be found at www.cmbi.kun.nl/7tn/seq/snakes.html).

OR Type 1, OlAl_Human:

ELI: ANHLLGSKSISFGGC (SEQ ID NO: 1)

EL2: CGNQEVANFY (SEQ ID NO: 2)

EL2: CDITPLLKLS (SEQ ID NO: 3)

EL2: CSDIHFHV (SEQ ID NO: 4)

EL3: (QYFRPLTNYSLKDAV (SEQ ID NO: 5)

OR Type 2, O2A4JHuman:

ELI: (QNLLHPAKPISFAGRMMQT (SEQ ID NO: 6)

EL2: RPQKLYHFFC (SEQ ID NO: 7)

EL2: CADTHINENM (SEQ ID NO: 8)

EL3: (OGPRYGNPKEQKKY (SEQ ID NO: 9)

OR Type 3, O3 AlJHuman:

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:

ELI: ADLLSEKKTISFAGC (SEQ ID NO: 15)

EL2: CDSNVIHHFF (SEQ ID NO: 16)

EL2: CDSPPLFKLS (SEQ ID NO: 17)

EL2: CSDTILKESI (SEQ ID NO: 18)

EL3: CTYLRPSSSYSLNQDKV (SEQ ID NO: 19)

(C) = a Cysteine added to the native sequence to enable cross-linking to a carrier protein.

Antibody Generation

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.

In a preferred embodiment, 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.

In a preferred embodiment, antibodies of the instant invention are raised in chickens.

In another embodiment, 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. For example, using an antigenicity plot, 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.

In a preferred embodiment, 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.

In another embodiment, analogy with ionotropic GluR is used, for example, homology between the mGluR and TIR is used to reveal appropriate epitopes for antibody generation. In a preferred embodiment, 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. hi a preferred embodiment the following following peptides are used against sweet and umami receptors: hTlRl/ Ac-LQVRH RPEVT LCX-NH₂ (SEQ ID NO: 24), hTlRl/ Ac-ETKIQ WHGKD NQVPK SVC-NH2 (SEQ ID NO: 25),hTlRl/ Ac-(C)ETLS VKRQY P-NH₂ (SEQ ID NO: 26),hTlRl/ Ac-(C)GSSD DYGQL G-NH₂ (SEQ ID NO: 27), hTlRl/ Ac-SAQVG DERMQ C-NH₂ (SEQ ID NO: 28), hTlR2/ Ac-LHANM KGIVH LNFLQ VPMC-NH₂ (SEQ ID NO: 29), hTlR2/ Ac-(C)DELRD KVRFP-NH₂ (SEQ ID NO: 30), hTlR2/ Ac-(C)VSSDT YGRQN G-NH₂ (SEQ ID NO: 31), hTlR2/ Ac-(C)PNQNM TSEER QRL-NH₂ (SEQ ID NO: 32), hTlR2/ Ac-LKNIQ DISWH TVNNT IPMSM C-NH₂ (SEQ ID NO: 33), hTlR3/Ac-LGEAE EAGLR SRTRP SSPVC-NH₂ (SEQ ID NO: 34), hTlR3/ Ac-(C)QGSV PRLHD VGRFN-NH₂ (SEQ ID NO: 35), hTlR3/ Ac-LRTER LKIRW HTSDN QKPVS RC-NH₂ (SEQ ID NO: 36), hTlR3/ Ac-(C)ELLS ARETF P-NH₂ (SEQ ID NO: 37), hTlR3/ Ac-(C)PRAD DSRLG KVQ-NH₂ (SEQ ID NO: 38), hTlR3/ Ac-(C)GSDD EYGRQ GL-NH₂ (SEQ ID NO: 39).

One aspect of the invention is an antibody or complex of two or more antibodies that modulates chemosensation.

In a preferred embodiment, the antibody or complex of two or more antibodies stimulates taste sensation. hi anotiier preferred embodiment, the antibody or complex of two or more antibodies inhibits taste sensation.

In another preferred embodiment, the antibody or complex of two or more antibodies stimulates odor sensation.

In another preferred embodiment, the antibody or complex of two or more antibodies inhibits odor sensation.

In another preferred embodiment, the taste sensation that is modulated is selected from the group consisting of:

(a) sweet taste sensation;

(b) sour taste sensation;

(c) salty taste sensation;

(d) bitter taste sensation; and

(e) umami or amino acid taste sensation.

In another preferred embodiment, the odor sensation that is modulated is selected from the group consisting of:

(a) an unpleasant odor sensation;

(b) a pleasant odor sensation;

(c) a chemical odor sensation;

(d) an organic odor sensation;

(e) an inorganic odor sensation;

(f) a natural odor sensation;

(g) a synthetic odor sensation.

In another preferred embodiment, the antibody or at least one antibody in the complex is raised against a receptor or a portion thereof.

In a preferred embodiment, the receptor against which an antibody is raised is selected from the group consisting of: _{RECT|F|ED SHE}ET (RULE 91) (b) odor receptors.

In a further preferred embodiment, the antibody or at least one antibody in the complex is raised against any one of the group consisting of:

(a) a taste receptor domain involved in ligand or tastant binding;

(b) a taste receptor domain not involved in ligand or tastant binding;

(c) an odor receptor domain involved in ligand or odorant binding;

(d) an odor receptor domain not involved in ligand or odorant binding; and

(e) an extracellularly exposed region.

In a preferred embodiment of the invention, the extracellularly exposed region against which an antibody is raised is a portion of a GPCR.

In a preferred embodiment of the invention, the portion of the GPCR against which an antibody is raised is is an extracellular loop of GPCR. In another preferred embodiment, the portion of the GPCR is an N-terminal region of the GPCR.

In another preferred embodiment, 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);

1) LHANM KGIVH LNFLQ VPMC (SEQ ID NO: 51); m) (C)DELRD KVRFP (SEQ ID NO: 52); n) (C)VSSDT YGRQN G (SEQ ID NO: 53); o) (QPNQNM TSEER QRL (SEQ ID NO: 54); p) LKNIQ DISWH TVNNT IPMSM C (SEQ ID NO: 55); q) (C)NTIY FVSSN TER (SEQ ID NO: 56); r) (C)FPEL VTTRN NTSFN ISEG (SEQ ID NO: 57); s) Y AGMDM GTKSI (SEQ ID NO: 58); t) (C)DLIKH RKMAP LD (SEQ ID NO: 59); u) (C)RKFF SQNAT IQKED TLAIQ (SEQ ID NO: 60); and v) (QKVFL SSLKF HIRRF IF (SEQ ID NO: 61).

In one preferred embodiment, at least two of the complexed antibodies are raised against a receptor or a portion of a receptor.

In another preferred embodiment, the antibodies in the complex are raised against different receptors or portions of receptors.

In another preferred embodiment, the complex comprises an antibody raised against a T1R2 receptor or portion thereof and an antibody raised against a T1R3 receptor or portion thereof. hi another preferred embodiment, the complex comprises an antibody raised against a TlRl receptor or portion tiiereof and an antibody raised against a T1R3 receptor or portion thereof.

In another preferred embodiment, one antibody in the complex stimulates chemosensation while a second antibody in the complex inhibits chemosensation. h one aspect of the invention, the complex of antibodies is formed by a method selected from the group consisting of:

(a) covalent crosslinking;

(b) non-covalent crosshnking; and

(c) adsorption of the two or more antibodies onto the surface of inert particles.

In another aspect of the invention, 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.

In a preferred embodiment, the immunized bird is a chicken.

Another aspect of the invention is a composition comprising an antibody or complex of antibodies as described above.

In another embodiment of the invention, 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). In a further embodiment, antibodies may be enhanced or otherwise synthesized (for example, as described in United States Patent No. 6,300,064, hereby incorporated by reference).

In one preferred embodiment, 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.

In one preferred embodiment, 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:

(a) immunizing a bird against a taste bud, a receptor or a portion thereof;

(b) harvesting an egg laid by the bird; and

(c) recovering the antibody from the egg.

In a preferred embodiment, the immunized bird is a chicken. h a preferred embodiment, 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:

(a) immunizing a mammal against a taste bud, a receptor or a portion thereof; and

(b) harvesting milk produced by the mammal.

In a preferred embodiment, the antibody or complex of antibodies is further purified from the milk.

In a further preferred embodiment of the described method, the mammal is a cow or goat.

Another aspect of the invention is a method for selecting a candidate antigenic site, said method comprising:

(a) using an antigenicity plot to identify antigenic sites within an extracellular portion of a receptor;

(b) using the three dimensional structure of mGluRl with and without glutamate to identify a selected antigenic site that may interfere with VFT closure or dimerization of ANF receptor domains; and

(c) identifying the candidate 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.

In a preferred embodiment, any of four different approaches are used for choosing the epitopes for functional anti-TlR antibodies:

1. by analysis of polymorphisms in mTlR3;

2. by analogy with mGluRl;

3. by analogy with ionotropic GluR; or

4. by targeting the extracellular loops of the "seven trans-membrane" (7TM) region.

In a preferred embodiment, an antibody to a receptor is administered in combination with a ligand for the receptor to which the antibody was raised. In a more preferred embodiment, the ligand is a natural ligand.

In another preferred embodiment, 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.

In another embodiment, an antibody to a receptor activated in the experience of a particular taste sensation or smell sensation is administered in combination with a hgand which binds a receptor involved in that same sensation. In a more preferred embodiment, the ligand is a natural ligand. hi a preferred embodiment, an antibody raised against a sweet taste receptor is administered in combination with a ligand for a sweet taste receptor. In another preferred embodiment, an antibody raised against a sour taste receptor is administered in combination with a ligand for a sour taste receptor, hi another embodiment, an antibody raised against an umami taste receptor is administered in combination with a ligand for an umami taste receptor. In another preferred embodiment, an antibody against a salty taste receptor is administered in combination with a ligand for a salty taste receptor. In another embodiment, an antibody raised against a bitter taste receptor is administered in combination with a ligand for a bitter taste receptor.

In another embodiment, an inhibitor of a receptor maybe administered in combination with a ligand capable of stimulating another receptor.

In one embodiment, "in combination" can mean that either the antibody or the hgand is administered first and the other is administered second. For example, an antibody to a sweet taste receptor may be administered either after of before administration of a ligand for a sweet receptor. Alternatively, "in combination" can mean that the antibody and the ligand are administered concomitantly. As would be apparent to one of skill in the art, such dosing schedules can be understood for each of the receptor and antibody types.

The invention may be illustrated by the following examples which are not intended to limit the scope of the invention in any way.

Example 1

Analysis of polymorphisms in mTlR3.

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). According to this model, 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.

The appropriate sequence of hTlRl is LQVRH RPEVT LC (SEQ ID NO: 64).

The appropriate sequence of hTlR2 is LHANM KGIVH LNFLQ VPMC (SEQ ID NO: 65).

Example 2

Analogy with mGluRl. 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.

Based on this data, the following strategy has been used:

1. using antigenicity plot, good antigenic sites were found within the fragments of mGluRl;

2. 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; and

3. the sequences from hTlRl hTlR2, and hTlR3, which correspond to those chosen in #2 and are also good antigens by antigenicity plot, have been chosen as possible candidates.

By this approach, three sequences have been chosen for 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)). Interestingly, in 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.

Example 3

Analogy with ionotropic glutamate receptor.

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).

The closer to the N-terminus is the 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 auto- and heteroantibodies antibodies against a region immediately downstream the ANF receptor domain of GluR3 and GluR2 function as agonists. It is not clear, however, whether their effect is mediated by changes in ANF receptor or PBPe domains. The epitope is closer to the former and is separated from PBDe domain by N-glycosylation site.

Interestingly, similarly to ionotropic GluR3, 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).

Example 4

Designing peptides based on the methods of the invention.

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.

Example 5

Immunization procedures.

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.

These procedures are detailed below; similar techniques can be apphed to extra-cellular loops of all GPCR chemosensation receptors and to ARDs and the flanking sequences of ah ANF-containing taste receptors: a) Chosen epitopes

EL = extracellu ar loop; ARD=ANF receptor domain. (b) Immunization

In general, the immunogenic peptide is covalently attached to a larger protein carrier. For example, 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. To the peptides that do not contain the internal cysteine, 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 μg 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.

(c) Purification of crude IgY

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. For short-term storage, 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. For long-term storage, 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 MgCl₂, which can be removed from the IgY preparation by dialysis. The IgY concentration is measured spectrophotometrically.

Example 6

Tasting experiments

The modulating antibodies against taste receptors may have agonistic or antagonistic effect, both of which can be revealed in tasting experiments. The tasting experiments must include negative controls with preimmune IgY and positive controls with the natural ligands of the appropriate receptors. The following substances are the natural ligands for the appropriate bitter receptors: hT2Rl 6-n-propyl-2-thiouracil hT2R4 Denatonium Benzoate For sweet receptors hTlR3 and hTlRl, different sweet substances are tested, beginning with sucrose, glucose, aspartame and saccharin. Several dilutions of the appropriate IgY preparations are tested. The experiments also include mixtures of several antibodies against the same receptors. It is likely that TlRl and T1R3 form heterodimers, therefore, in the case of these two receptors, the combinations of antibodies against both receptors are used.

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.

The typical tasting experiment is presented below on the example of anti-T2R4 receptor.

Example 7

(a) Chosen peptides.

If some of these peptides are not hydrophilic enough, MAPs may be used instead of conjugation of KLH with another protein. For a review of the MAPs technology see Tarn JP. (1988) Proc. Natl. Acad. Sci. 85:5409-5413.

(b) Immunizations

Table 3. Several references of preparing IgY.

The following method is used:

1. 5-6 months old laying hens, every lineage, at least two for each peptide.

The most soluble peptide (hT2R4-EL2) conjugated with KLH, others -MAP. hT2R4-EL2 could also be made with MAP.

2. Immunizations - week 0 - 60 μg in complete Freund's Adjuvant, further - weeks 2, 4 - 60 μg in incomplete Freund's adjuvant in 4 points intramuscularly.

3. Before immunizations, a few preimmune eggs are collected.

4. A week after the 2^nd ICA immunization, eggs collected daily and stored at 4 °C.

(c) Purification and storage.

The yolks are separated from the egg whites and washed with deionized water. For further elimination of yolks, three basic procedures are accepted:

1. Chloroform separation.

2. Polyethylene glycol precipitation of IgY

3. The dilution with 4 volumes of deiomzed water (1 :50), staying for 6 h at 4 °C, and centrifugation at 3500 X g.⁴

In this example, 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. Example 8

Tests and assays.

1. ELIS A with the appropriate peptide for the monitoring of the response.

2. Small-scale affinity purification - in order to evaluate the relative amount of specific antibodies against each peptide.

3. 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.

Example 9

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.

For immunization, two White Leghorn and Brown Leghorn hens 5-6 months old were used for each peptide. Prior to immunization, 2-3 pre-immune eggs were collected from each hen. First immunization was done with Complete Freund's Adjuvant following with Incomplete Freund's Adjuvant. Injections were done intramuscularly into the pectorial muscle and subcuntaneously at one point each.

The immunization schedule was the follows:

Week 1 Pre-immune eggs (2-3) + Immunisation I (200 μg)

Week 3 Immunization II (200 μg)

Week 6 Immunization III (100 μg)

Week 7 Egg collection III+l week 6 eggs +ELISA

Week 8 Egg collection iπ+2 week 6 eggs (stored up to 2 months)

Following the immunization, 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.

The following are the antigenic peptides tested:

TVD-1/21 Ac-EWSPE RSTRC FRR-NH₂ (SEQ ID NO: 119)

TVD-3/21 Ac-EWSYQ SETSC FKR-NH₂ (SEQ ID NO: 120) TVD-4/21 Ac-(C)QWDR SQNPF Q-NH₂ (SEQ ID NO: 121)

TVD-5/21 Ac-(C)QRQL KNIQD-NH₂ (SEQ ID NO: 122)

TVD-6/21 Ac-(C)DTYG RDNGQ-NH₂ (SEQ ID NO: 123)

TVD-7/21 Ac-ETLPT LQPNQ C-NH₂ (SEQ ID NO: 124)

TVD-9/21 Ac-HTSDN QKPVS RC (SEQ ID NO: 125)

TVD-11/21 Ac-(C)ELLS ARETF P-NH₂ (SEQ ID NO: 126)

Ac- at the N-terminus means acetylated N-terminus. NH₂ at the C-terminus means amidated

C-terminus.

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).

Results:

The Ab containing powders (=lyoρhilized yolks) were dissolved in distilled water to a final concentiation of 2-10% (dry yolk wt volume) and were then mixed in different combinations as described below. The various combinations were then mixed 1:1 with either diluted sucrose solution (2%-10%) or diluted mono-sodium glutamate solution (0.04%-0.2%) as compared with samples mixed with non-immune yolks or water. Healthy volunteers were asked to rate the intensity of the corresponding taste sensation without prior knowledge of the content of the tubes.

The results show that when Ab directed against various epitopes of hTlR2 were mixed together (e.g. Ab against 3/21 + 4/21 + 5/21 + 6/21 + 7/21 or Ab against 4/21 + 6/21 + 7/21) and administered in combination with sucrose, it caused a significant enhancement and extension of the sweet taste sensation. In a similar set of experiments it was found that that when Ab directed against various epitopes of hTlR3 were mixed together (e.g. Ab against 1/21 + 9/21 + 11/21) and administered in combination with mono-sodium glutamate, it caused a significant enhancement and extension of the umami taste sensation.

Claims

We claim:

1. An antibody or complex of two or more antibodies that modulates chemosensation.

2. The antibody or complex of two or more antibodies according to claim 1, wherein the antibody or complex of two or more antibodies stimulates taste sensation.

3. The antibody or complex of two or more antibodies according to claim 1, wherein the antibody or complex of two or more antibodies inhibits taste sensation.

4. The antibody or complex of two or more antibodies according to claim 1, wherein the antibody or complex of two or more antibodies stimulates odor sensation.

5. The antibody or complex of two or more antibodies according to claim 1, wherein the antibody or complex of two or more antibodies inhibits odor sensation.

6. The antibody or complex of two or more antibodies according to claim 2, wherein the taste sensation is selected from the group consisting of:

(a) sweet taste sensation;

(b) sour taste sensation;

(c) salty taste sensation;

(d) bitter taste sensation; and

(e) umami or amino acid taste sensation.

7. The antibody or complex of two or more antibodies according to claim 3, wherein the taste sensation is selected from the group consisting of:

(a) sweet taste sensation;

(b) sour taste sensation;

(c) salty taste sensation;

(d) bitter taste sensation; and

(e) umami or amino acid taste sensation.

8. The antibody or complex of two or more antibodies according to claim 4, wherein the odor sensation is selected from the group consisting of:

(a) an unpleasant odor sensation;

(b) a pleasant odor sensation;

(c) a chemical odor sensation;

(d) an organic odor sensation;

(e) an inorganic odor sensation;

(f) a natural odor sensation;

(g) a synthetic odor sensation.

9. The antibody or complex of two or more antibodies according to claim 5, wherein the odor sensation is selected from the group consisting of:

. (a) an unpleasant odor sensation;

(b) a pleasant odor sensation;

(c) a chemical odor sensation;

(d) an organic odor sensation;

(e) an inorganic odor sensation;

(f) a natural odor sensation; (g) a synthetic odor sensation.

10. The antibody or complex of two or more antibodies according to any one of claims 1-9, wherein the antibody or at least one antibody in the complex is raised against a receptor or a portion thereof.

11. The antibody or complex of two or more antibodies according to claim 10j wherein the receptor is selected from the group consisting of:

(a) taste receptors; and

(b) odor receptors.

12. The antibody or complex of two or more antibodies according to claim 11, wherein the antibody or at least one antibody in the complex is raised against any one of the group consisting of:

(a) a taste receptor domain involved in ligand or tastant binding;

(b) a taste receptor domain not involved in ligand or tastant binding;

(c) an odor receptor domain involved in ligand or odorant binding;

(d) an odor receptor domain not involved in ligand or odorant binding; and

(e) an extracellularly exposed region.

13. The antibody or complex of two or more antibodies according to claim 12, wherein the extracellularly exposed region is a portion of a GPCR.

14. The antibody or complex of two or more antibodies according to claim 13, wherein the portion of the GPCR is an extracellular loop of GPCR.

15. The antibody or complex of two or more antibodies according to claim 13, wherein the portion of the GPCR is an N-terminal region of the GPCR.

16. The antibody or complex of two or more antibodies according to claim 10, wherein the antibody is raised against an amino acid sequence selected from the group consisting of: a) (QETLSVKRQYP; b) (C)GSSDDYGQLG; c) SAQVGDERMQC; d) (C)ELLSARETFP; e) (C)PRADDSRLGKVQ; f) (QGSDDEYGRQGL; g) LQVRH PEVT LC; h) ETKIQ WHGKD NQVPK SVC; i) LRTER LKIRW HTSDN QKPVS RC; j) (C)QGSV PRLHD VGRFN; k) LGEAE EAGLR SRTRP SSPVC;

1) LHANM KGIVH LNFLQ VPMC; ) (QDELRD KVRFP; n) (C)VSSDT YGRQN G; o) (C)PNQNM TSEER QRL; p) LKNIQ DISWH TVNNT IPMSM C; q) (QNTIY FVSSN TER; r) (C)FPEL VTTRNNTSFN ISEG; s) Y AGMDM GTKSI; t) (C)DLKH RKMAP LD; u) (QRKFF SQNAT IQKED TLAIQ; and v) (C)KVFL SSLKF HIRRF IF.

17. The complex of antibodies according to claim 10, wherein at least two of the complexed antibodies are raised against a receptor or a portion of a receptor.

18. The complex of antibodies according to claim 10, wherein the antibodies in the complex are raised against different receptors or portions of receptors.

19. The complex of antibodies according to claim 18, wherein the complex comprises an antibody raised against a T1R2 receptor or portion thereof and an antibody raised against a T1R3 receptor or portion thereof.

20. The complex of antibodies according to claim 18, wherein the complex comprises an antibody raised against a TlRl receptor or portion thereof and an antibody raised against a T1R3 receptor or portion thereof.

21. The complex of antibodies according to claim 11, wherein one antibody in the complex stimulates chemosensation while a second antibody in the complex inhibits chemosensation.

22. The complex of antibodies according to any one of claims 1-21, wherein the complex is formed by a method selected from ihβ group consisting of:

(a) covalent crosslin dng;

(b) non-covalent crosslinking; and

(c) adsorption of the two or more antibodies onto the surface of inert particles.

23. The antibody or complex of antibodies according to any one of claims 1-22, wherein 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.

24. The antibody or complex of antibodies according to claim 23, wherein the bird is a chicken.

25. A composition comprising an antibody or complex of antibodies according to any of claims 1-22.

26. A composition comprising the antibody or complex according to claim 25, wherein the composition further comprises a ligand.

27. The composition according to claim 26, wherein the ligand binds to the same receptor as the antibody or an antibody comprised in the complex of antibodies.

28. The composition according to claim 26, wherein the ligand is selected from the group consisting of a tastant and an odorant.

29. The composition according to claim 28, wherein the tastant bind to the same receptor as the antibody or an antibody comprised in the complex of antibodies.

30. The composition according to claim 25, comprised:

(a) in a chewing gum like form;

(b) in a liquid beverage-like form;

(c) in nasal drops or a nasal spray form.

31. A method of making an antibody or complex of antibodies according to claims 1-22, the method comprising:

(a) immunizing a bird against a taste bud, a receptor or a portion thereof;

(b) harvesting an egg laid by the bird; and

(c) recovering the antibody from the egg.

32. The method according to claim 27, wherein the bird is a chicken.

33. The method according to claim 27, wherein the receptor is a taste receptor or an odorant receptor.

34. A transgenic non-human animal, wherein said animal is genetically engineered to produce an antibody that modulates chemosensation.

35. A method of making an antibody or complex of antibodies according to claims 1-22, the method comprising:

(a) immunizing a mammal against a taste bud, a receptor or a portion thereof; and

(b) harvesting milk produced by the mammal.

36. The method according to claim 35, wherein the antibody or complex of antibodies is further purified from the milk.

37. The method according to claim 35, wherein the mammal is a cow or goat.

38. A method for selecting a candidate antigenic site, said method comprising:

(a) using an antigenicity plot to identify antigenic sites within an extracellular portion of a receptor;

(b) using the three dimensional structure of mGluRl with and without glutamate to identify a selected antigenic site that may interfere with VFT closure or dimerization of ANF receptor domains; and

(c) identifying the candidate 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.

39. A method for selecting a candidate antigenic site, said method comprising:

(a) by targeting the extracellular loops of the "seven trans-membrane" (7TM) region.

(b) using an antigenicity plot to identify antigenic sites within the extracellular loops.

40. A method for enhancing chemosensation, comprising administering an antibody in combination with tastant or odorant.

41. A method for enhancing chemosensation, comprising administering an antibody raised against a receptor in combination with a ligand for the receptor.

42. A method for enhancing chemosensation, comprising administering a combination of antibodies to different receptors or different portions of the same receptor in combination with a ligand or more than one ligand for the receptor or receptors to which the antibodies were raised.

43. A method for enhancing chemosensation, comprising administering an antibody to a receptor activated in the experience of a particular taste sensation or smell sensation in combination with a ligand which binds a receptor involved in that same sensation.

44. A method for enhancing chemosensation, comprising administering an antibody raised against a sweet taste receptor in combination with a ligand for a sweet taste receptor.

45. A method for enhancing chemosensation, comprising administering an antibody raised against a sour taste receptor in combination with a hgand for a sour taste receptor.

46. A method for enhancing chemosensation, comprising administering an antibody raised against an umami taste receptor in combination with a hgand for an umami taste receptor.

47. A method for enhancing chemosensation, comprising administering an antibody against a salty taste receptor in combination with a ligand for a salty taste receptor.

48. A method for enhancing chemosensation, comprising administering an antibody raised against a bitter taste receptor in combination with a ligand for a bitter taste receptor.

49. A method for enhancing chemosensation, comprising administering an inhibitor of a receptor in combmation with a ligand capable of stimulating another receptor.

50. The method of according to any one of claims 44-49, wherein the hgand is a tastant.

51. The method according to claim 50, wherein the tastant is a natural tastant.

52. The method according to claim 50, wherein the tastant is a flavored additive.