CA2486423A1 - Antibodies and their use for the modulation of taste sensation - Google Patents
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- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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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.
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 50-1 SO within individual taste buds.
The sensation of smell is now known to be the culmination of a process mediated by olfactory receptor neurons (ORNs) 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.
RECTIFIED SHEET (RULE 91) 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 CC 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 (Kinnamon et al., 1988, Proc. Natl. Acad.
Sci. USA, 85: 7023.-7027). Salty taste can be mediated through sodium channels (Heck et al., 2984, 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 transductian.
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 gustduein 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 arriino 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 tune.
hnportantly, 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 imrnunoglobulin molecule that is capable of highly specific interaction with the antigen that induced its synthesis. Antibodies are diverse, with more than lOlo possible variations, yet each antibody is designed to recognize only a specifxe antigen.
Antibodies have been previously generated against cellular receptors.
Antibodies against the 2"a extracellular loop (2EL) of many type Ia 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 lEL, or by simultaneous application of antibodies against 1EL and 3EL. In some cases, the antibodies against 2EL
function as antagonists. In the case of j32 adrenergic receptor, it is clear that the functional effect of the RECTIFIED SHEET (RULE 91) 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.
In fact, for this receptor, dimerization has been shown to have a role in the normal function of this receptor.
Tablel. Antibodies against GPCR with functional effect.
Receptor AutoantibodiesAntibodiesFunctionReferences a ainst: a ainst: _ ~.-opiaidlEL + 3EL agonist Mace, G. et al., (1999) J.Biol.Chem., 274,20079 (3IAR 2EL 1EL; 2EL agonist Iwata, M. et al., (2001) Circ. Res.
88, 578; Magnusson, Y. et al., (1989) Clin. Exp. Immutzol.
78, 42;
Mabini, R. et aL, (1999) J.
Autoimmun. 13, 179.
j32~ 2EL agonist,Mijares, A. et al., Fab (2000) Mol.
antagonistPharmacol. 58, 373;
Lebesgue, D. et al., (1998) Eur. J.
Pharmacol. 348, 123; Mijares, A. et al., (1996) FEBS
Lett. 399, 188 mI 2EL agonist Borda, E. and Sterin-Borda, L.
2001 J. Autoimmxnun.
16, 143 nn2 2EL agonist Fu, M.L. et al., Receptors Channels _2, 1_21 aIAAR 2EL agonist Fu. M.L. et al., (1994) Clin. Exp.
Immunol. 97, 146 5-HT1A 2EL agonist Verdot, L. et al., (1995) J.
Neurochem 65, 319 5-HT4 2EL antagonistSalle, L. et al., 92001) J. Mol. Cell Cardiol. 33, 405 ATl 2EL 2EL agonist Fu, M.L. et aL, (1998) Receptors Channels 6, 99; Wallukat, G. et al., (1999 J. Clip. Invest.
103, 945.
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-xegulate their action or may function unlike any known ligands. Antibodies rnay 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 RECTIFIED SHEET (RULE 91) 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 of T1R 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 subj ective 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.
RECTIFIED SHEET (RULE 91) Gehe~al Types of Taste Reeepto~s a. Sweet and amino acid taste receptors Sweet and amino acid taste receptors are represented by three ~enes, T1R1-3 (Lewcock, J.W., and Reed, R.R. (2001) Neuron 31, 515). They belong to the 3r family of GPCR
(Bockaert, J., and Pin, J.P. (1999) EMB~J J. 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 Iigand-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) (Saint, E. et al. (2001) J. Neurochenx. 77, 896) (May, M. et al. (2001) Nat.
Genet. 28, 58). Recently, the sequences of human TlRl-3 were published (Li X.
et a1. (2002) P~oc. Natl. Acaa'. 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 (Mar, 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 Cant-sensitive receptor (CaR) (Hu, J. et al. (2000) J. Biol. Chem. 275, 16382) and glutamate receptor mGIuRl (R.obbins, M. J., et al. (1999) J. Neurochem. 72, 2539) (Ray, K. et al.
(2000) J. Biol.
Chem. 275, 34245). Likewise, umarni taste stimuli generated by amino acids, require both TIRl and T1R3, probably through a heterodimer formation (Li X. et al., (2002) Pnoc. Natl.
Aced. Sci. USA 99, 4692).
According to the human genorne 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 heterodirnerization of more distantly related GPCR
have been described (Jordan, B.A. et al. (2001) P~oe. Natl. Aced. Sci. USA 98, 343) (Rocheville, M. et al. (2000) Science 288, 154) (Game, 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).
RECTIFIED SHEET (RULE 91) 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 W 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, P.J. et al.
(1993) Neu~~o~t 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 Caa+-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 dirners. 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, anly GABA(A) R1 binds a specific ligand, thus leading to a conforrriational change in GABA(B) R2 (Galvez, T. et al., (2000) EMBQ ,T, 20, 21.52). CaR and mGluR1 may also form disulfide-linked heterodimers that are sensitive to glutamate-mediated internalization (Gama, L. et al. (2001) J. Biol. Chem..
276, 39053). It seems that mouse T1R3 is functional when it forms a heterodimer 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 lA GPCR. The interaction, in this case, is mediated by the interaction of the C-termini of two types of receptors (Ciruela,. F. et al. (2001) J. Biol.
Chem. 276, 18345).
The next of the groups is the membrane-associated guanylyl kinase receptor family (Wedel, B.J., and Garbers, D.L. (1997) FEBS Lett. 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 (Dada, T. et al. (2001) Biochemistry 40, 22067) (Dada, T. et al.
(2001)Biochemist~y 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 dirnerization.
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 juxtarnembrane cysteine-rich region, but not through interchain disulfide bonds. Binding of a ligand leads to tighter association of the ANF
RECTIFIED SHEET (RULE 91) 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. Neu~oSCi. 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 tetrarners, 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 GluR1 induce clustering of the receptors (Mammen, A.L. et al. (1997) J.
Neurosci. I7, 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) .l. Mol. Recognit. 12, 219) (Carlson, N.G. et al.
(1997) J. Biol. Chem, 272, I 1295) (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 oligomerie complex.
b. Bitter taste receptors A large family ofbitter taste receptors, T2R, has been recently discovered (Matsunami; H. et aZ (2000) Natu~~e 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 extxacellular loops are involved in ligand binding.
While the overall architecture of this family is similar to that of type I a 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 (BNaCI, BCN1) is expressed in taste bud cells (Ugawa, S. et al. (1998) Nature 395, 556), but is not necessary for sour taste transduction (Kinnamon, S.C., et al. (2000) Olfaction and Taste XIII.
New York: Springer-Verlag, p. 80). Two hyperpolarization-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) Natut~e 413, 631}. In the amphibian Nectu~us maculosus, the sour transduction is mediated by apical K+ channel, which is closed at low pH
(Kinnamon, S.C. et al. (1988) Proc. Natl. Aead. Sci. USA 85, 7023).
RECTIFIED SHEET (RULE 91) 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., 28I, CI I05).
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, oc, (3, and y. In the interstitial epithelium, the constitutively expressed a subunit is non-functional, while (3 and y subunits are up-regulated by aldosterone, thus forming, together with the a subunit, a functional channel. All three subunits are expressed in rat taste bud cells {Lin, W. et al. (1999) J. Comp. Neurol. 405, 406).
The ENaC subunits have a common transmembrane topology, with intracellular N-and C-termini, two transmernbrane domain, and a long highly glycosylated extracellular part in between. Using the anti-idiotype approach, a monoclonal antibody RA6.3 against the arniloride-binding domain of ENaC has been raised. The antibody mimicked amiloride in that it inhibited transepithelial Na+ transport (I~leyman, 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 YIN1L SRL, corresponding to residues 246-263 of human or rat ENaCa {Kieber-Emmons, T. et al. (1999) .I: 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 NaCI-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, Halpern, 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 >II 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.
RECTIFIED SHEET (RULE 91) (2000) Nat. Neurosci. 3, 113). The latter is a splice form with a truncated N-terminus. Its affipity to glutamate is in the millimolar range, similar to that in the taste buds. The taste-receptor GluR4 belongs to the 3rd family of GPCR (see above). Since mGluR are able to forxxi 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 traps-membrane alpha-helices portion of the receptor (see Afshar M. et aI. (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 traps-membrane alpha helices. For a comprehensive list of human ORs sequences see: Zozulya S.
et al. ,(2001) Genorne Biol. 2: 1-26 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 I, OlAI Human:
EL1: ANHLLGSKSISFGGC (SEQ ID NO: 1) EL2: CGNQEVANFY (SEQ 1D NO: 2) EL2: CDITPLLKLS (SEQ ID NO: 3) EL2: CSDII-IFHV (SEQ ID NO: 4) EL3: (C)YFRPLTNYSLKDAV (SEQ. ID NO: 5) OR Type 2, 02A4 Human:
EL1: (C)NLLHPAKPISFAGRMMQT (SEQ ID NO: 6) EL2: RPQKIYHF'FC (SEQ ID NO: 7}
EL2: CADTHINENM (SEQ ID NO: 8) EL3: (C)GPRYGNPKEQKKY (SEQ ID NO: 9) OR Type 3, 03A1 Human:
EL1: LSRLLSRKRANPC (SEQ m NO: 10}
EL2: CGPNVINIiFY (SEQ 1D NO: 11) EL2: CDLPQLFQLS (SEQ m NO: 12) EL2: CSSTQLNEL (SEQ ID NO: 13) EL3: (C)RLGSTKLSDKDKA (SEQ ID NO: 14) RECTIFIED SHEET (RULE 91) OR Type 5, OSF1 Human:
EL1: ADLLSEKKTISFAGC (SEQ m NO: 15) EL2: CDSNVII-IHFF (SEQ ID NO: 16) EL2: CDSPPLFKLS (SEQ m NO: 17) EL2: CSDTILKESI (SEQ ID NO: 28) EL3: CTYLRPSSSYSLNQDKV (SEQ ID NO: 19) (C) = a Cysteine added to the native sequence to enable cross-linking to a carrier protein.
Antibody Ge~ce~atiora Antibodies to taste receptors are generated based on analysis of the structural features of taste receptors. For example, in one embodiment, epitopes in T1R are chosen based on analysis of polymorphisms in mTlR3, by analogy with mGluR1 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 woWd comprise antibodies to the antigen.
In a preferred embodiment, the region LGSTE EATLN QRTQP NSIfC (SEQ 117 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 ofthe 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 mGluR1. Each antigenic site would be located on the 3D structures of mGluR1 with and without glutamate, and the sites that may interfere with VFP closure or dimerization of ANF
receptor domains wauld 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 mGIuRI 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, hTIR2 or hTIR3; and ii, is a good antigen based on the antigenicity plot.
In another embodiment, analogy with ionotropic GIuR 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 mGluR1 is EGVLN 1DDYK
IQMN
(SEQ ID NO: 21). The homologous sequences of hTlR3 are QGSVP RLHDV GRFN (SEQ
ID NO: 22) and LRTER LKIRW HTSDN QKPVS RC (SEQ ID NO: 23), at two sides from the N-glycosylation site.
RECTIFIED SHEET (RULE 91) In a preferred embodiment the following following peptides are used against sweet and umami receptors: hTlRI/ Ac-LQVRH RPEVT LCX-NHz (SEA m NO: 24), hTlR1!
Ac-ETKIQ WI~GKD NQVPK SVC-NH2 (SEA ID NO: 25), hTlR1! Ac-(C)ETLS VKRQY
P-NHa (SEA II7 NO: 26), hTlR1/ Ac-(C)GSSD DYGQL G-NH2 (SEA m NO: 27), hTIRI/
Ac-SAQVG DERMA C-NH2 (SEA ID NO: 28), hTIR2/ Ac-LHA.NM KGIVH LNFLQ .
VPMC-NH2 (SEA ID NO: 29), hTlR2/ Ac-(C)DELRD KVRFP-NHz (SEA TD NO: 30), hTlR2/ Ac-(C)VSSDT YGRQN G-NH2 (SEA m NO: 31), hTlR2/ Ac-(C)PNQNM TSEER
QRIrNH2 (SEA TD NO: 32), hTlR2/ Ac-LKNIQ DISWH TVNNT IPMSM C-NHZ (SEA lD
NO: 33), hTIR3/Ac-LGEAE EAGLR SRTRP SSPVC-NHa (SEA ID NO: 34), hTlR3/
Ac-(C)QGSV PRLHD VGRFN-NH2 (SEA B7 NO: 35), hTIR3/ Ac-LRTER LKIRW
HTSDN QKPVS RC-NHS (SEA ID NO: 36), hTlR3/ Ac-(C)ELLS ARETF P-NH2 (SEA ID
NO: 37), hTlR3/ Ac-(C)PRAD DSRLG KVQ-NHa (SEA ID NO: 38), hTlR3/ Ac-(C)GSDD
EYGRQ GL-NEI2 (SEA m 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.
In another 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 ador sensation;
(b) a pleasant odor sensation;
(c) a chemical odor sensation;
(d) an organic odor sensation;
(e) an inorganic odor sensation;
(fj 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 RECTIFIED SHEET (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 o~
(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.
Tn 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 GFCR.
In another preferred embodiment, an antibody is raised against an amino acid sequence selected from the group consisting of a) (C)ETLSVKRQY P (SEQ ID NO: 40);
b) (C)GSSDDYGQLG (SEQ ID NO: 41;
c) SAQVGDERMQC (SEQ m NO: 42);
d) (C)ELLSARETFP (SEQ ID NO: 43);
e) (C)PRADDSRLGKVQ (SEQ ID NO: 44);
~ (C)GSDDEYGRQGL (SEQ ID NO: 45);
g) LQVRH RPEVT LC (SEQ D7 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 117 NO: 51);
m) (C)DELRD KVRFP (SEQ m NO: 52);
n) (C)VSSDT YGRQN G (SEQ D7 NO: 53);
o) (C)PNQNM TSEER QRL (SEQ ID NO: 54);
p) LKNIQ DISWH TVNNT IPMSM C (SEQ ID NO: 55);
c~ (C)NTIY FVSSN TER (SEQ Da NO: 56);
r) (C)FPEL VTTRN NTSFN ISEG (SEQ m NO: 57);
s) Y AGMDM GTKSI (SEQ ID NO: 58);
t) (C)DL1KH RKMAP LD (SEQ ID. NO: 59);
u) (C)RKFF SQNAT IQKED TLAIQ (SEQ ID NO: 60); and v) (C)KVFL SSLKF HIRRF IF (SEQ ID NO: 61).
In one preferred embodiment, at Ieast 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.
RECTIFIED SHEET (RULE 91) In another preferred embodiment, the complex comprises an antibody raised against a T1R1 receptor or portion tl2ereof 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 chernosensation.
In 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 crosslinking; 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 may be 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, 1.988). 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).
Tn 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.
In a preferred embodiment, the receptor used to immunize the bird is a taste receptor or an odorant receptor.
RECTIFIED SHEET (RULE 91) Another aspect ofthe 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 methodfor 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 mGluR1 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-T1R antibodies:
1. by analysis of polymorphisms in mTlR3;
2. by analogy with mGIuRI;
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 50-1 SO within individual taste buds.
The sensation of smell is now known to be the culmination of a process mediated by olfactory receptor neurons (ORNs) 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.
RECTIFIED SHEET (RULE 91) 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 CC 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 (Kinnamon et al., 1988, Proc. Natl. Acad.
Sci. USA, 85: 7023.-7027). Salty taste can be mediated through sodium channels (Heck et al., 2984, 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 transductian.
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 gustduein 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 arriino 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 tune.
hnportantly, 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 imrnunoglobulin molecule that is capable of highly specific interaction with the antigen that induced its synthesis. Antibodies are diverse, with more than lOlo possible variations, yet each antibody is designed to recognize only a specifxe antigen.
Antibodies have been previously generated against cellular receptors.
Antibodies against the 2"a extracellular loop (2EL) of many type Ia 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 lEL, or by simultaneous application of antibodies against 1EL and 3EL. In some cases, the antibodies against 2EL
function as antagonists. In the case of j32 adrenergic receptor, it is clear that the functional effect of the RECTIFIED SHEET (RULE 91) 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.
In fact, for this receptor, dimerization has been shown to have a role in the normal function of this receptor.
Tablel. Antibodies against GPCR with functional effect.
Receptor AutoantibodiesAntibodiesFunctionReferences a ainst: a ainst: _ ~.-opiaidlEL + 3EL agonist Mace, G. et al., (1999) J.Biol.Chem., 274,20079 (3IAR 2EL 1EL; 2EL agonist Iwata, M. et al., (2001) Circ. Res.
88, 578; Magnusson, Y. et al., (1989) Clin. Exp. Immutzol.
78, 42;
Mabini, R. et aL, (1999) J.
Autoimmun. 13, 179.
j32~ 2EL agonist,Mijares, A. et al., Fab (2000) Mol.
antagonistPharmacol. 58, 373;
Lebesgue, D. et al., (1998) Eur. J.
Pharmacol. 348, 123; Mijares, A. et al., (1996) FEBS
Lett. 399, 188 mI 2EL agonist Borda, E. and Sterin-Borda, L.
2001 J. Autoimmxnun.
16, 143 nn2 2EL agonist Fu, M.L. et al., Receptors Channels _2, 1_21 aIAAR 2EL agonist Fu. M.L. et al., (1994) Clin. Exp.
Immunol. 97, 146 5-HT1A 2EL agonist Verdot, L. et al., (1995) J.
Neurochem 65, 319 5-HT4 2EL antagonistSalle, L. et al., 92001) J. Mol. Cell Cardiol. 33, 405 ATl 2EL 2EL agonist Fu, M.L. et aL, (1998) Receptors Channels 6, 99; Wallukat, G. et al., (1999 J. Clip. Invest.
103, 945.
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-xegulate their action or may function unlike any known ligands. Antibodies rnay 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 RECTIFIED SHEET (RULE 91) 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 of T1R 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 subj ective 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.
RECTIFIED SHEET (RULE 91) Gehe~al Types of Taste Reeepto~s a. Sweet and amino acid taste receptors Sweet and amino acid taste receptors are represented by three ~enes, T1R1-3 (Lewcock, J.W., and Reed, R.R. (2001) Neuron 31, 515). They belong to the 3r family of GPCR
(Bockaert, J., and Pin, J.P. (1999) EMB~J J. 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 Iigand-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) (Saint, E. et al. (2001) J. Neurochenx. 77, 896) (May, M. et al. (2001) Nat.
Genet. 28, 58). Recently, the sequences of human TlRl-3 were published (Li X.
et a1. (2002) P~oc. Natl. Acaa'. 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 (Mar, 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 Cant-sensitive receptor (CaR) (Hu, J. et al. (2000) J. Biol. Chem. 275, 16382) and glutamate receptor mGIuRl (R.obbins, M. J., et al. (1999) J. Neurochem. 72, 2539) (Ray, K. et al.
(2000) J. Biol.
Chem. 275, 34245). Likewise, umarni taste stimuli generated by amino acids, require both TIRl and T1R3, probably through a heterodimer formation (Li X. et al., (2002) Pnoc. Natl.
Aced. Sci. USA 99, 4692).
According to the human genorne 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 heterodirnerization of more distantly related GPCR
have been described (Jordan, B.A. et al. (2001) P~oe. Natl. Aced. Sci. USA 98, 343) (Rocheville, M. et al. (2000) Science 288, 154) (Game, 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).
RECTIFIED SHEET (RULE 91) 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 W 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, P.J. et al.
(1993) Neu~~o~t 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 Caa+-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 dirners. 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, anly GABA(A) R1 binds a specific ligand, thus leading to a conforrriational change in GABA(B) R2 (Galvez, T. et al., (2000) EMBQ ,T, 20, 21.52). CaR and mGluR1 may also form disulfide-linked heterodimers that are sensitive to glutamate-mediated internalization (Gama, L. et al. (2001) J. Biol. Chem..
276, 39053). It seems that mouse T1R3 is functional when it forms a heterodimer 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 lA GPCR. The interaction, in this case, is mediated by the interaction of the C-termini of two types of receptors (Ciruela,. F. et al. (2001) J. Biol.
Chem. 276, 18345).
The next of the groups is the membrane-associated guanylyl kinase receptor family (Wedel, B.J., and Garbers, D.L. (1997) FEBS Lett. 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 (Dada, T. et al. (2001) Biochemistry 40, 22067) (Dada, T. et al.
(2001)Biochemist~y 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 dirnerization.
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 juxtarnembrane cysteine-rich region, but not through interchain disulfide bonds. Binding of a ligand leads to tighter association of the ANF
RECTIFIED SHEET (RULE 91) 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. Neu~oSCi. 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 tetrarners, 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 GluR1 induce clustering of the receptors (Mammen, A.L. et al. (1997) J.
Neurosci. I7, 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) .l. Mol. Recognit. 12, 219) (Carlson, N.G. et al.
(1997) J. Biol. Chem, 272, I 1295) (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 oligomerie complex.
b. Bitter taste receptors A large family ofbitter taste receptors, T2R, has been recently discovered (Matsunami; H. et aZ (2000) Natu~~e 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 extxacellular loops are involved in ligand binding.
While the overall architecture of this family is similar to that of type I a 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 (BNaCI, BCN1) is expressed in taste bud cells (Ugawa, S. et al. (1998) Nature 395, 556), but is not necessary for sour taste transduction (Kinnamon, S.C., et al. (2000) Olfaction and Taste XIII.
New York: Springer-Verlag, p. 80). Two hyperpolarization-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) Natut~e 413, 631}. In the amphibian Nectu~us maculosus, the sour transduction is mediated by apical K+ channel, which is closed at low pH
(Kinnamon, S.C. et al. (1988) Proc. Natl. Aead. Sci. USA 85, 7023).
RECTIFIED SHEET (RULE 91) 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., 28I, CI I05).
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, oc, (3, and y. In the interstitial epithelium, the constitutively expressed a subunit is non-functional, while (3 and y subunits are up-regulated by aldosterone, thus forming, together with the a subunit, a functional channel. All three subunits are expressed in rat taste bud cells {Lin, W. et al. (1999) J. Comp. Neurol. 405, 406).
The ENaC subunits have a common transmembrane topology, with intracellular N-and C-termini, two transmernbrane domain, and a long highly glycosylated extracellular part in between. Using the anti-idiotype approach, a monoclonal antibody RA6.3 against the arniloride-binding domain of ENaC has been raised. The antibody mimicked amiloride in that it inhibited transepithelial Na+ transport (I~leyman, 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 YIN1L SRL, corresponding to residues 246-263 of human or rat ENaCa {Kieber-Emmons, T. et al. (1999) .I: 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 NaCI-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, Halpern, 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 >II 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.
RECTIFIED SHEET (RULE 91) (2000) Nat. Neurosci. 3, 113). The latter is a splice form with a truncated N-terminus. Its affipity to glutamate is in the millimolar range, similar to that in the taste buds. The taste-receptor GluR4 belongs to the 3rd family of GPCR (see above). Since mGluR are able to forxxi 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 traps-membrane alpha-helices portion of the receptor (see Afshar M. et aI. (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 traps-membrane alpha helices. For a comprehensive list of human ORs sequences see: Zozulya S.
et al. ,(2001) Genorne Biol. 2: 1-26 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 I, OlAI Human:
EL1: ANHLLGSKSISFGGC (SEQ ID NO: 1) EL2: CGNQEVANFY (SEQ 1D NO: 2) EL2: CDITPLLKLS (SEQ ID NO: 3) EL2: CSDII-IFHV (SEQ ID NO: 4) EL3: (C)YFRPLTNYSLKDAV (SEQ. ID NO: 5) OR Type 2, 02A4 Human:
EL1: (C)NLLHPAKPISFAGRMMQT (SEQ ID NO: 6) EL2: RPQKIYHF'FC (SEQ ID NO: 7}
EL2: CADTHINENM (SEQ ID NO: 8) EL3: (C)GPRYGNPKEQKKY (SEQ ID NO: 9) OR Type 3, 03A1 Human:
EL1: LSRLLSRKRANPC (SEQ m NO: 10}
EL2: CGPNVINIiFY (SEQ 1D NO: 11) EL2: CDLPQLFQLS (SEQ m NO: 12) EL2: CSSTQLNEL (SEQ ID NO: 13) EL3: (C)RLGSTKLSDKDKA (SEQ ID NO: 14) RECTIFIED SHEET (RULE 91) OR Type 5, OSF1 Human:
EL1: ADLLSEKKTISFAGC (SEQ m NO: 15) EL2: CDSNVII-IHFF (SEQ ID NO: 16) EL2: CDSPPLFKLS (SEQ m NO: 17) EL2: CSDTILKESI (SEQ ID NO: 28) EL3: CTYLRPSSSYSLNQDKV (SEQ ID NO: 19) (C) = a Cysteine added to the native sequence to enable cross-linking to a carrier protein.
Antibody Ge~ce~atiora Antibodies to taste receptors are generated based on analysis of the structural features of taste receptors. For example, in one embodiment, epitopes in T1R are chosen based on analysis of polymorphisms in mTlR3, by analogy with mGluR1 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 woWd comprise antibodies to the antigen.
In a preferred embodiment, the region LGSTE EATLN QRTQP NSIfC (SEQ 117 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 ofthe 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 mGluR1. Each antigenic site would be located on the 3D structures of mGluR1 with and without glutamate, and the sites that may interfere with VFP closure or dimerization of ANF
receptor domains wauld 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 mGIuRI 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, hTIR2 or hTIR3; and ii, is a good antigen based on the antigenicity plot.
In another embodiment, analogy with ionotropic GIuR 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 mGluR1 is EGVLN 1DDYK
IQMN
(SEQ ID NO: 21). The homologous sequences of hTlR3 are QGSVP RLHDV GRFN (SEQ
ID NO: 22) and LRTER LKIRW HTSDN QKPVS RC (SEQ ID NO: 23), at two sides from the N-glycosylation site.
RECTIFIED SHEET (RULE 91) In a preferred embodiment the following following peptides are used against sweet and umami receptors: hTlRI/ Ac-LQVRH RPEVT LCX-NHz (SEA m NO: 24), hTlR1!
Ac-ETKIQ WI~GKD NQVPK SVC-NH2 (SEA ID NO: 25), hTlR1! Ac-(C)ETLS VKRQY
P-NHa (SEA II7 NO: 26), hTlR1/ Ac-(C)GSSD DYGQL G-NH2 (SEA m NO: 27), hTIRI/
Ac-SAQVG DERMA C-NH2 (SEA ID NO: 28), hTIR2/ Ac-LHA.NM KGIVH LNFLQ .
VPMC-NH2 (SEA ID NO: 29), hTlR2/ Ac-(C)DELRD KVRFP-NHz (SEA TD NO: 30), hTlR2/ Ac-(C)VSSDT YGRQN G-NH2 (SEA m NO: 31), hTlR2/ Ac-(C)PNQNM TSEER
QRIrNH2 (SEA TD NO: 32), hTlR2/ Ac-LKNIQ DISWH TVNNT IPMSM C-NHZ (SEA lD
NO: 33), hTIR3/Ac-LGEAE EAGLR SRTRP SSPVC-NHa (SEA ID NO: 34), hTlR3/
Ac-(C)QGSV PRLHD VGRFN-NH2 (SEA B7 NO: 35), hTIR3/ Ac-LRTER LKIRW
HTSDN QKPVS RC-NHS (SEA ID NO: 36), hTlR3/ Ac-(C)ELLS ARETF P-NH2 (SEA ID
NO: 37), hTlR3/ Ac-(C)PRAD DSRLG KVQ-NHa (SEA ID NO: 38), hTlR3/ Ac-(C)GSDD
EYGRQ GL-NEI2 (SEA m 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.
In another 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 ador sensation;
(b) a pleasant odor sensation;
(c) a chemical odor sensation;
(d) an organic odor sensation;
(e) an inorganic odor sensation;
(fj 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 RECTIFIED SHEET (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 o~
(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.
Tn 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 GFCR.
In another preferred embodiment, an antibody is raised against an amino acid sequence selected from the group consisting of a) (C)ETLSVKRQY P (SEQ ID NO: 40);
b) (C)GSSDDYGQLG (SEQ ID NO: 41;
c) SAQVGDERMQC (SEQ m NO: 42);
d) (C)ELLSARETFP (SEQ ID NO: 43);
e) (C)PRADDSRLGKVQ (SEQ ID NO: 44);
~ (C)GSDDEYGRQGL (SEQ ID NO: 45);
g) LQVRH RPEVT LC (SEQ D7 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 117 NO: 51);
m) (C)DELRD KVRFP (SEQ m NO: 52);
n) (C)VSSDT YGRQN G (SEQ D7 NO: 53);
o) (C)PNQNM TSEER QRL (SEQ ID NO: 54);
p) LKNIQ DISWH TVNNT IPMSM C (SEQ ID NO: 55);
c~ (C)NTIY FVSSN TER (SEQ Da NO: 56);
r) (C)FPEL VTTRN NTSFN ISEG (SEQ m NO: 57);
s) Y AGMDM GTKSI (SEQ ID NO: 58);
t) (C)DL1KH RKMAP LD (SEQ ID. NO: 59);
u) (C)RKFF SQNAT IQKED TLAIQ (SEQ ID NO: 60); and v) (C)KVFL SSLKF HIRRF IF (SEQ ID NO: 61).
In one preferred embodiment, at Ieast 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.
RECTIFIED SHEET (RULE 91) In another preferred embodiment, the complex comprises an antibody raised against a T1R1 receptor or portion tl2ereof 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 chernosensation.
In 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 crosslinking; 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 may be 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, 1.988). 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).
Tn 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.
In a preferred embodiment, the receptor used to immunize the bird is a taste receptor or an odorant receptor.
RECTIFIED SHEET (RULE 91) Another aspect ofthe 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 methodfor 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 mGluR1 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-T1R antibodies:
1. by analysis of polymorphisms in mTlR3;
2. by analogy with mGIuRI;
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 ligand 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 ligand which binds a receptor involved in that same sensation. In a more preferred embodiment, the ligand is a natural ligand.
In 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 RECTIFIED SHEET (RULE 91) a sour taste receptor. ' In another embodiment, an antibody raised against an umarni 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 may be administered in combination with a ligand capable of stimulating another receptor.
In one embodiment, "in combination" can mean that either the antibody or the ligand 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 al. (2001) Nat. Genet. 28, S8). According to this model, this region (LGSTE EATLN QRTQP NSIPC (SEQ ID NO: 62)), corresponding to residues 43-62 of mTlR3) may be a good candidate for, most probably, an inhibiting antibody. The corresponding human sequence LGEAE EAGLR SRTRP SSPVG (SEQ ID NO: 63) is present without introns in human chromosome 1.
The appropriate sequence of hTlR1 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.
na.GluR1 is the most studied family 3 GPCR. It is homologous to T1R3, T1R2 and T1R1. The X-ray crystallography of its ANF receptor domain with and without glutamate confnms 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 mGluRlfunction as antagonists (Srnitt, P.S. et al. (2000} N. Eng. J. Med. 342, 21). The RECTIFIED SHEET (RULE 91) heteroantibodies against the parts of ANF receptor domain of mGluR1 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 mGIuRl 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 hTlRI hTlR2, and hTIR3, 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 hTlRI ((C)ETLS VKRQY P
(SEQ
ID NO: 66); (C)GSSD DYGQL G (SEQ ID NO: 67), and SAQVG DERMQ C (SEQ ID'NO:
6~)); for hTlR2 ({C)DELRD KVRFP (SEQ ID NO: 69), (C)VSSDT YGRQN G (SEQ ID
NO: 70) and (C)PNQNM 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 AMPA 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, TIRI, T1R2, and T1R3 contain N-glycosylation site 12-I S residues downstream of the end of ANF receptor domain. In GluR3, this site is glycosylated. In T1RI, T1R2, and T1R3 the sequence predicts an efficient glycosylation site.
The ANF receptor domains of GIuR3 and mGluR1 share some homology, therefore one can expect that antibodies against t~~~~r~~i~~~~f ~1R and T1R will also have a similar effect. The homologous sequence in mGluRl is EGVLN IDDYK IQMN (SEA 1D
NO: 75). The homologous sequences of hTIR3 are QGSVP RLHDV GRFN (SEA ID,NO:
76) and LRTER LKIRW HTSDN QKPVS RC (SEA ID NO: 77), at two sides from the N-glycosylation site. In hTlRl, only the sequence at one of the two sides from the N glycosylation site is ixnmunogenic: ETKIQ WHGKD NQVPK SVC (SEA ID NO: 78) while in hTIR2, the corresponding sequence is: LKNIQ DISWH TVNNT IPMSM C (SEA
DJ 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.
# Rece for quence Se I hTIRl -Ac-LQVRH RPEVT LC-NHa SE ID NO: 80) 2 hTlRl Ac-ETKI WHGKD NQVPK SVC-NHZ SEA ID NO: 81 3 hTlRl Ac-(C)ETLS VKR Y P-NH2 SEA ID NO: 82 4 hTlR1 Ac- C GSSD DYG L G-NH2 SEA ID NO: 83) hTlRI Ac-SAQVG DERMA C-NH2 (SEA ID NO: 84) 6 hTlR2 Ac-L~:fANM KG1VH LNFLQ VPMC-NHS (SEA ID NO:
8S) 7 hTlR2 Ac-(C)DELRD KVRFP NH2 (SEA ID NO: 8b7 8 hTIR2 Ac-(C)VSSDT YGRQN G-NH2 (SEA ID NO: 87) 9 hTIR2 Ac-(C)PNQNM TSEER QRL-NH2 (SEA II? NO: 88) hTlR2 Ac-LI~HQ DISWH TVNNT IPMSM C-NHZ (SEA ID NO:
89) I1 hTIR3 Ac-LGEAE EAGLR SRTRP SSPVC-NHx (SEA lD NO:
90) 12 hTlR3 Ac-(C)QGSV PRLHD VGRFN-NHa (SEA ID NO: 9I) 13 hTlR3 Ac-LRTER LKIRW HTSDN QKPVS RC-NHZ SE ID NO:
14 hTIR3 Ac-(C ELLS ARETF P-NH2 SEA ID NO: 93) hTlR3 Ac- C PRAD DSRLG KV -NHZ (SE ID NO: 94 16 hTlR3 Ac- C GSDD EYGRQ GL-NH2 SE ID NO: 95 RECTIFIED SHEET (RULE 91) Example S
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 applied to extra-cellular loops of all GPCR chemosensation receptors and to ARDs and the flanking sequences of all ANF-containing taste receptors:
a~ Chosen epitopes ## ReceptorLocation ResiduesPeptide Controlled taste _ 1 hT2R4 1EL 65-76 Ac-(C)NTIY FVSSN TER-NHz bitter SEA ID NO: 96 2 hT2R4 2EL I56-I73Ac-(C)FPEL VTTRN NTSFN bitter ISEG-NHz SEA I17 NO: 97 3 hT2R1 1EL 34-45 Ac-(C}DLIKH RKMAP LD-NH2 bitter (SE ID NO: 98 4 hT2R1 2EL 157-175AC-(C}RKFF SQNAT IQKED bitter TLAIQ-NH2 SE LD NO: 99) hT2R1 3EL 244-259Ac-(C)KVFL SSLKF HIRRF bitter IF-NHz SE ID NO: 100 6 hTlRI Upstream 5I-62 Ac-LQVRH RPEVT LC-NHa sweet the ARD (SEA TD NO: 101) 7 hTIRI Downstream481-498Ac-ETKIQ WHGKD NQVPK sweat the ARD SVC-NHa SE ID NO: 102 8 hTlR1 ARD 174-183Ac-(C)ETLS VKRQY P-NHa sweet SE TD NO: I03 9 hTlR1 ARD 215-224-Ac-(C)GSSD DYGQL G--NHa sweet (SE ID NO: 104) hTlR1 ARD 248-258Ac-SAQVG DERMA C-NHZ sweet (SE ID NO: I OS
11 hTlR3 Upstream 43-62 Ac-LGEAE EAGLR SRTRP sweet the ARD SSPVG-NFi2 (SE ID NO:
106) I2 hTlR3 Downstream462-474Ac-(C)QGSV PRLHD sweet the ARD VGRFN-NH2 (SEA ID NO:
107) 13 hTlR3 Downstream478-499Ac-LRTER LKIRW HTSDN sweet the ARD QKPVS RC-NHz (SEA ID NO:
108) 14 hTlR3 ARD I72-181Ac-(C)ELLS ARETF P-NH2 sweet (SEA
ID NO: 109) IS hTlR3 ARD 246-257Ac-(C)PRAD DSRLG KVQ-NH2 sweet (SE ID NO: 1 I O) I6 hTIR3 ARD 2I3-223Ac-(C)GSDD EYGRQ GL-NHz sweet (SE m NO: l I l) EL = extracellular loop; ARD=ANF receptor domain.
RECTIFIED SHEET (RULE 91) (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 (KLIT). 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 pg of a peptide is injected intramuscularly into 4 points.
The first immunization is perforn2ed 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 2nd 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 purif cation 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 spectrophotornetrically.
Example 6 Tasting experiments The modulating antibodies against taste receptors may have agonistic or antagonistic effect, both ofwhich 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 RECTIFIED SHEET (RULE 91) 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 T1R1 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.
1 Pre-immune I Y
2 Benzoate _ Denatonium 3 _ Anti- a tide 1, hen #1 4 Anti- a tide 1, hen #2 5 .Anti- a tide 2, hen #3 ~
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 ligand 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 ligand which binds a receptor involved in that same sensation. In a more preferred embodiment, the ligand is a natural ligand.
In 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 RECTIFIED SHEET (RULE 91) a sour taste receptor. ' In another embodiment, an antibody raised against an umarni 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 may be administered in combination with a ligand capable of stimulating another receptor.
In one embodiment, "in combination" can mean that either the antibody or the ligand 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 al. (2001) Nat. Genet. 28, S8). According to this model, this region (LGSTE EATLN QRTQP NSIPC (SEQ ID NO: 62)), corresponding to residues 43-62 of mTlR3) may be a good candidate for, most probably, an inhibiting antibody. The corresponding human sequence LGEAE EAGLR SRTRP SSPVG (SEQ ID NO: 63) is present without introns in human chromosome 1.
The appropriate sequence of hTlR1 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.
na.GluR1 is the most studied family 3 GPCR. It is homologous to T1R3, T1R2 and T1R1. The X-ray crystallography of its ANF receptor domain with and without glutamate confnms 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 mGluRlfunction as antagonists (Srnitt, P.S. et al. (2000} N. Eng. J. Med. 342, 21). The RECTIFIED SHEET (RULE 91) heteroantibodies against the parts of ANF receptor domain of mGluR1 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 mGIuRl 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 hTlRI hTlR2, and hTIR3, 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 hTlRI ((C)ETLS VKRQY P
(SEQ
ID NO: 66); (C)GSSD DYGQL G (SEQ ID NO: 67), and SAQVG DERMQ C (SEQ ID'NO:
6~)); for hTlR2 ({C)DELRD KVRFP (SEQ ID NO: 69), (C)VSSDT YGRQN G (SEQ ID
NO: 70) and (C)PNQNM 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 AMPA 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, TIRI, T1R2, and T1R3 contain N-glycosylation site 12-I S residues downstream of the end of ANF receptor domain. In GluR3, this site is glycosylated. In T1RI, T1R2, and T1R3 the sequence predicts an efficient glycosylation site.
The ANF receptor domains of GIuR3 and mGluR1 share some homology, therefore one can expect that antibodies against t~~~~r~~i~~~~f ~1R and T1R will also have a similar effect. The homologous sequence in mGluRl is EGVLN IDDYK IQMN (SEA 1D
NO: 75). The homologous sequences of hTIR3 are QGSVP RLHDV GRFN (SEA ID,NO:
76) and LRTER LKIRW HTSDN QKPVS RC (SEA ID NO: 77), at two sides from the N-glycosylation site. In hTlRl, only the sequence at one of the two sides from the N glycosylation site is ixnmunogenic: ETKIQ WHGKD NQVPK SVC (SEA ID NO: 78) while in hTIR2, the corresponding sequence is: LKNIQ DISWH TVNNT IPMSM C (SEA
DJ 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.
# Rece for quence Se I hTIRl -Ac-LQVRH RPEVT LC-NHa SE ID NO: 80) 2 hTlRl Ac-ETKI WHGKD NQVPK SVC-NHZ SEA ID NO: 81 3 hTlRl Ac-(C)ETLS VKR Y P-NH2 SEA ID NO: 82 4 hTlR1 Ac- C GSSD DYG L G-NH2 SEA ID NO: 83) hTlRI Ac-SAQVG DERMA C-NH2 (SEA ID NO: 84) 6 hTlR2 Ac-L~:fANM KG1VH LNFLQ VPMC-NHS (SEA ID NO:
8S) 7 hTlR2 Ac-(C)DELRD KVRFP NH2 (SEA ID NO: 8b7 8 hTIR2 Ac-(C)VSSDT YGRQN G-NH2 (SEA ID NO: 87) 9 hTIR2 Ac-(C)PNQNM TSEER QRL-NH2 (SEA II? NO: 88) hTlR2 Ac-LI~HQ DISWH TVNNT IPMSM C-NHZ (SEA ID NO:
89) I1 hTIR3 Ac-LGEAE EAGLR SRTRP SSPVC-NHx (SEA lD NO:
90) 12 hTlR3 Ac-(C)QGSV PRLHD VGRFN-NHa (SEA ID NO: 9I) 13 hTlR3 Ac-LRTER LKIRW HTSDN QKPVS RC-NHZ SE ID NO:
14 hTIR3 Ac-(C ELLS ARETF P-NH2 SEA ID NO: 93) hTlR3 Ac- C PRAD DSRLG KV -NHZ (SE ID NO: 94 16 hTlR3 Ac- C GSDD EYGRQ GL-NH2 SE ID NO: 95 RECTIFIED SHEET (RULE 91) Example S
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 applied to extra-cellular loops of all GPCR chemosensation receptors and to ARDs and the flanking sequences of all ANF-containing taste receptors:
a~ Chosen epitopes ## ReceptorLocation ResiduesPeptide Controlled taste _ 1 hT2R4 1EL 65-76 Ac-(C)NTIY FVSSN TER-NHz bitter SEA ID NO: 96 2 hT2R4 2EL I56-I73Ac-(C)FPEL VTTRN NTSFN bitter ISEG-NHz SEA I17 NO: 97 3 hT2R1 1EL 34-45 Ac-(C}DLIKH RKMAP LD-NH2 bitter (SE ID NO: 98 4 hT2R1 2EL 157-175AC-(C}RKFF SQNAT IQKED bitter TLAIQ-NH2 SE LD NO: 99) hT2R1 3EL 244-259Ac-(C)KVFL SSLKF HIRRF bitter IF-NHz SE ID NO: 100 6 hTlRI Upstream 5I-62 Ac-LQVRH RPEVT LC-NHa sweet the ARD (SEA TD NO: 101) 7 hTIRI Downstream481-498Ac-ETKIQ WHGKD NQVPK sweat the ARD SVC-NHa SE ID NO: 102 8 hTlR1 ARD 174-183Ac-(C)ETLS VKRQY P-NHa sweet SE TD NO: I03 9 hTlR1 ARD 215-224-Ac-(C)GSSD DYGQL G--NHa sweet (SE ID NO: 104) hTlR1 ARD 248-258Ac-SAQVG DERMA C-NHZ sweet (SE ID NO: I OS
11 hTlR3 Upstream 43-62 Ac-LGEAE EAGLR SRTRP sweet the ARD SSPVG-NFi2 (SE ID NO:
106) I2 hTlR3 Downstream462-474Ac-(C)QGSV PRLHD sweet the ARD VGRFN-NH2 (SEA ID NO:
107) 13 hTlR3 Downstream478-499Ac-LRTER LKIRW HTSDN sweet the ARD QKPVS RC-NHz (SEA ID NO:
108) 14 hTlR3 ARD I72-181Ac-(C)ELLS ARETF P-NH2 sweet (SEA
ID NO: 109) IS hTlR3 ARD 246-257Ac-(C)PRAD DSRLG KVQ-NH2 sweet (SE ID NO: 1 I O) I6 hTIR3 ARD 2I3-223Ac-(C)GSDD EYGRQ GL-NHz sweet (SE m NO: l I l) EL = extracellular loop; ARD=ANF receptor domain.
RECTIFIED SHEET (RULE 91) (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 (KLIT). 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 pg of a peptide is injected intramuscularly into 4 points.
The first immunization is perforn2ed 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 2nd 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 purif cation 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 spectrophotornetrically.
Example 6 Tasting experiments The modulating antibodies against taste receptors may have agonistic or antagonistic effect, both ofwhich 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 RECTIFIED SHEET (RULE 91) 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 T1R1 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.
1 Pre-immune I Y
2 Benzoate _ Denatonium 3 _ Anti- a tide 1, hen #1 4 Anti- a tide 1, hen #2 5 .Anti- a tide 2, hen #3 ~
6 Anti- ~ a tide 2, hen #4 7 Anti-peptide 1, hen #1 + anti-peptide 2, hen #3 8 Anti-peptide 1, hen #2 + anti-peptide 2, hen #3 9 Anti-peptide 1, hen #1 + anti-peptide 2, hen #4 10 Anti-peptide 1, hen #2 + anti-peptide 2, hen #4 11 Anti-peptide l, hen #1, then Denatonium Benzoate 12 Anti-peptide l, hen #2, then Denatonium Benzoate I3 Anti-peptide 2, hen #3, then Denatonium Benzoate 14 Anti-peptide 2, hen #4, then Denatonium Benzoate RECTIFIED SHEET (RULE 91) Example 7 (a) Chosen peptides.
Table 2. Chosen peptides fox T2R
Rece LocationResiduesPe tide for hT2R4 IEL 65-76 Ac-(C)NTTY FVSSN TER-NH2 .
SE ID NO: 112 hT2R4 2EL 1S6-173 Ac-F PELVT TRNNT SFNIS EG MAP
(SEQ ID NO: 113} KLH
or Ac-(C)FPEL VTTRN NTSFN
ISEG-NHZ (SE II7 NO: 114 hT2R4 3EL 254-264 Ac-Y AGMDM GTKSI (SEQ ID MAP
NO: 115) hT2Rl lEL 34-4S Ac-(C)DLIKH RKMAP LD-NH2 KLH
SEQ DJ NO: 116 hT2R1 2EL 157-175 AC-(C)RKFF SQNAT IQKED KLH
TLAIQ-NH2 SEQ ID NO: 117) hT2R1 3EL 244-259 Ac-(C)KVFL SSLKF HIRRF IF-NH2KLH
SE ff~ NO: 118 If some of these peptides are not hydrophilic enough, MAPS may be used instead of conjugation of KLH with another protein. Fox a review of the MAPS technology see Tam JP.
(1988) Proc. Natl. Acad. Sci. 85:5409-5413.
(b) hnmunizations Table 3. Several references of preparing IgY.
Age of Lineage Immunizations,Dose, Antigen Reference hens weeks 0,1,2,4,6,10,14,100 Whole Fortgens, P.H. et al. (1997) ... protein Immuhopha~macology 36, 160 days 0,2,4,6 500 Whole Tu, Y.Y. et al. (2001) Food rotein Res. Int. 34, 783 monthsPadua 0, 22d; 20d 40; GST-FP Di Lonardo, A. et 20 ~ al. (2001) Brown ~Irch. Yirol. 146, 180 days 0,2,4;8,12,... Whole Sun, S. {2001) Rapid protein Commuu. Mass.
S ectrom. 2 5, 708 20 weeksLohrxran0,10 d, 20d 60 Whole De Ceuninck, F. et al.
Brown protein (2001) J. Immunol.
Meth.
252, 153 0,2,4,6,8,10 Peptide-Williams, L.M. et al. (2001) KLH J. Neurae~tdoeYihol., 13, 94 25 weeksWhite 0,30 d 100 Virus Bizhanov, G., and Vendete Vyshniausl~is, G.
(2000) Tlet. Res.Commuh., 24, 203 RECTIFIED SHEET (RULE 91) 7 monthsLeghorn 0,2,3 ~ 100 Whole Vieira-da-Motta, A.
et al.
white rotein (2001 Pe tides 22, 6 monthsLeghorn 0,4,8,. .. 25 Haptene-Shelver, W.L. et al.
(1998) white KLH J. Chromato . 705, Leghorn 0,2,4 60 GST-FP Garnenisch, G. et al. (1999) white FASEB J. 13, 81 Leghorn 0,2,4,8,12,...100 Peptide-Knecht, W. et al.
(1996) white MAP Eur J. Biochem. 236, Peptide-Lee, S.C. et al. (1997) Peep.
KLH Biochetn. Biotech.
27, 227 Peptide-Campbell, W.G. et al.
KLH (I999) Am. J. Physiol.
276, Leghorn 0, 10 d, ... 300 Mixture Iwamoto, T. et al.
(1996) J.
white of Biol. Chem. 271, 13609 peptide-MAP
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-ELZ
could also be made with MAP.
2. Immunizations - week 0 - 60 ~.g in complete Freund's Adjuvant, further -weeks 2, 4 -60 ~.g in incomplete Freund's adjuvant in 4 points intramuscularly.
3. Before immunizations, a few preirnmune eggs are collected.
4. A week after the 2"d 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 axe accepted:
1. Chloroform separation.
2. Polyethylene glycol precipitation of IgY
3. The dilution with 4 volumes of deionized 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.
RECTIFIED SHEET (RULE 91) Example ~
Tests and assays.
I . ELISA 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 rnaleimide-modified keyhole limpet hemocyanin (KLH) through their cysteine moieties.
For immunization, two White Leghorn and Brown Leghorn hens S-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 Pre-immune eggs (2-3) + Tmmunisation 1 I (200 u,~) Week Timnunization II (200 ~.~g) Week Tmmunization III (100 p.g) Weak Egg collection III+2 week 6 eggs +ELISA
Week Egg collection III+2 week 6 eggs (stored 8 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-linked immunoassay (ELISA) 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 l~Iz (SEQ ID NO: I 19) TVD-3/21 Ac-EWSYQ SETSC FKR NH2 (SEQ ID NO: 120) RECTIFIED SHEET (RULE 91) TVD-4/21 Ac-(C)QWDR SQNPF Q-NHZ (SEQ 1D NO: 121) TVD-5/21 Ac-(C)QRQL I~NIQD-NHZ (SEQ m NO: 122) TVD-6/21 Ac-(C)DTYG RDNGQ-NHZ (SEQ m NO: 123) TVD-7/21 Ac-ETLPT LQPNQ C-NH2 (SEQ Il7 NO: 124) TVD-9121 Ac-HTSDN QKPVS RC (SEQ ID NO: 125) TVD-11/21 Ac-(C)ELLS ARETF P-NHZ (SEQ ID NO: 126) Ac- at the N-terminus means acetylated N terminus. NHZ 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 1/2I, 9/21 and 11/21 are derived from the hTlR3 receptor (conunon to sweet and umami).
Results:
The Ab containing powders (=lyophilized yolks) were dissolved in distilled water to a final concentration of 2-10% (dry yolk wtlvolume) 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°O) 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 ofhTlR2 were mixed together (e.g. Ab against 3/21 + 4/21 + 5121 + 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 dixected 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.
RECTIFIED SHEET (RULE 91) SEQUENCE LISTING
<110> TVD Taste Virtual Aimensions, Ina.
Ben-Sasson, Shmuel <120> Methods and Compositions Directed Towards Modulation of Chemosensation <130> 24836-501-061 <140> PCT/IL03/00409 <141> 2003-05-20 <150> 60/451,639 <151> 2003-03-04 <150> 60/382,.059 <151> 2002-05-20 <160> 126 <170> Patentln Ver. 2.1 <210> 1 <211> 15 <212> PRT
<213> Homo Sapiens <400> 1 Ala Asn Iiis Leu Leu Gly Ser Lys Ser 21e Ser Phe Gly G1y Cys <210> 2 <211> 10 <212> PRT
<213> Homo Sapiens <400> 2 Cys Gly Asn Gln Glu Val Ala Asn Phe Tyr <210> 3 <211> 10 <212> PRT
<213> Homo sapiens <400> 3 Cys Asp Tle Thr Pro Leu Leu Lys Leu Ser <210> 4 <211> 8 <212> PRT
<213> Homo Sapiens <400> 4 Cys Ser Asp Ile His Phe His Val RECTIFIED SHEET (RULE 91) <210> 5 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 5 Xaa Tyr Phe Arg Pra Leu Thr Asn Tyr Ser Leu Lys Asp Ala Val <220> 6 <2I1> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 6 Xaa Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe Ala Gly Arg Met Met Gln Thr c210> '7 <211> 10 c212> PRT
c213> Homo Sapiens c400> 7 Arg Pro G1n Lys Ile Tyr His Phe Phe Cys <210> 8 <211> 10 <212> PRT
<213> Homo Sapiens <400> 8 ' Cys Ala Asp Thr His Ile Asn Glu Asn Met c210> 9 <211> 14 <212> PRT
<213> Homo Sapiens <220>
RECTIFIED SHEET (RULE 91) <221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 9 X~a Gly Pro Arg Tyr Gly Asn Pro Lys Glu Gln Lys I~ys Tyr <210> 10 <211> 13 <212> PRT
<213> Homo Sapiens <400> ZO
Leu Ser Arg Leu Leu Ser Arg Lys Arg Ala Val Pro Cys <210> 11 <211> 10 <212> PRT
<213> Homo Sapiens <400> 11 Cys Gly Pro Asn Val Ile Asn His Plie Tyr <210> 12 <211> 10 <212> PRT
<213> Homo sapiens <400> 12 Cys Asp Leu Pro Gln Leu Phe Gln Leu Ser 1 5 3.0 <210> 13 <211> 9 <212> PRT
<213> Homo Sapiens <400> 13 Cys Ser Ser Thr Gln Leu Asn Glu Leu <210> 14 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 14 Xaa Arg Leu Gly Ser Thr Lys Leu Ser Asp Lys Asp Lys Ale RECTIFIED SHEET (RULE 91) <210> 15 <211> 15 <212> PRT' <213> Homo Sapiens <400> 15 Ala Asp Leu Leu Ser Glu Lys Lys Thr Ile Ser Phe Ala Gly Cys <210> 16 <211> 10 <212> PRT
<213> Homo Sapiens <400> 16 Cys Asp Ser Asn Val Ile His His Phe Phe 1 5 7.0 <210> 17 <211> 10 <212> PRT
<213> Homo Sapiens <400> .17 Cys Asp Ser Pro Pro Leu Phe Lys Leu Ser <210> 7.8 <211> 10 <212> PRT
<213> Homo Sapiens <400> 18 Cys Ser Asp Thr Ile Leu Lys Glu Sex Ile 1 5 la <210> 19 <211> 17 <212> PRT
<213> Homo Sapiens <400> 19 Cys Thr Tyr Leu Arg Pro Ser Ser Ser Tyr Ser Leu Asn Gln Asp Lys Val <210> 20 <212> 20 <212> PRT
<213> mouse RECTIFIED SHEET (RULE 91) <400> 20 Leu Gly Ser Thr Glu Glu Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn 1 5 l0 15 Ser Ile Pro Cys <210> 22 <211> 14 <212> PRT
<213> mouse <400> 21 Glu Gly Va1 Leu Asn Ile Asp Asp Tyr Lys Ile Gln Met Asn <210> 22 <211> 14 <212> PRT
<213> Homo sapiens <400> 22 Gln Gly Ser Val Pro Arg Leu His Asp Val Gly Arg Phe Asn <210> 23 <211> 22 <212> PRT
<213> Homo sapiens <400> 23 Leu Arg Thr G1u Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln 1 . 5 10 15 Lys Pro Val Ser Arg Cys <210>24 <211>15 <212>PRT
<213>Homo Sapiens <220>
<221>VARIANT
<222>(1) <223>Wherein Xaa.isan Acetyl Group <220>
<221>VARTANT
<222>(14) <223>Wherein Xaa any Amino is Acid <220>
<221>VARIANT
<222>(15) <223>Wherein Xaa an Amino Group is RECTIFIED SHEET (RULE 91) <400> 24 Xaa Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Xaa Xaa <210> 25 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (20) <223> Wherein Xaa is an Amino Group <400> 25 Xaa Glu Thr Lys Tle Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Xaa <210> 26 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <400> 26 Xaa Xaa Glu Thr Leu Ser Val Lys Arg Gln Tyr Pro Xaa <a1o> 27 <211> 13 <212> PRT
<213> Homo sapiens <220>
<221> VARTANT
<222> (1) RECTIFIED SHEET (RULE 91 ) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 27 Xaa Xaa Gly Ser Ser Asp Asp Tyr Gly G1n Leu Gly Xaa <210> 28 <211> 13 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
e222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 28 Xaa Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys Xaa <210> 29 <211> 21 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 29 Xaa Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn Phe Leu G1n Val Pro Met Cys.Xaa <210> 30 RECTIFIED SHEET (RULE 91 ) <211> 13 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
<222> (1) .
<223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 30 Xaa Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro Xaa 1 g 10 <210> 31 <211> 14 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 31 Xaa Xaa Val Ser Ser Asp Thr Tyr Gly Arg Gln Asn Gly Xaa <210> 32 <211> 16 <212> PRT
<213> Homo sagiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (2) <223> tr~herein Xaa is Cys or nothing <220>
<221> VARTANT
<222> (16) <223> P~herein Xaa is an Amino Group <400> 32 Xaa Xaa Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu Xaa 1 ' S 10 15 <210> 33 <211> 23 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (23) <223> Wherein Xaa is an Amino Group <400> 33 Xaa Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr 1 5 lp 15 Ile Pro Met Sex Met Cys Xaa <210> 34 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223,> P7herein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group <400> 34 Xaa Leu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Xaa <210> 35 <211> 17 RECTIFIED SHEET (RULE 91) <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa an Acetyl Group is <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa Cys or nothing is <220>
<221> VARIANT
<222> (17) <223> Wherein Xaa an Amino Group is <400> 35 Xaa Xaa G1n Gly Ser Val Pro Azg Leu His Asp Val Gly Arg Phe Asn 1 ~ 10 15 Xaa <210> 36 <211> 24 <212> PRT
<2.13> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (24) <223> Wherein Xaa is an Amino Group <400> 36 Xaa Leu Arg Thr GIu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn 1 5 10 ~ 15 Gln Lys Fro Val Ser Arg Cys Xaa <210> 37 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino. Group <400> 37 Xaa Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro Xaa <210> 38 <211> 15 <212> PRT
<213> Homo sapisns~
<220>
<221> VARTANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARTANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 38 Xaa Xaa Pro Arg Ala Asp Asp Ser'Arg Leu Gly hys Val Gln Xaa 1 5 10 . 15 <210> 39 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT .
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 39 Xaa Xaa G1y Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu Xaa RECTIFIED SHEET (RULE 91) <210> 40 <211> 1.1 <212 > PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 40 Xaa Glu Thr Leu Ser Val Lys Arg Gln Tyr Pro <210> 41 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 41 Xaa Gly Ser Ser Asp Asp Tyr Gly Gln Leu Gly <210> 42 <211> 11 <212> PRT
<213> Homo Sapiens <400> 42 Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys <210> 43 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 43 Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro <210> 44 <211> 13 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91) <220>
<221> VARIANT
<222> (1) <223> Tn~herein Xaa is Cys or nothing <400> 44 Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val Gln 1 5 ~0 <210> 45 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 45 Xaa Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu <210> 46 <211> 12 <212> PRT
<213> Homo Sapiens <400> 46 Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys <210> 47 <211> 18 <212> PRT
<213> Homo sapience <400> 47 Glu Thr Lys Ile Gln Trp 33is Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys <210> 48 <211> 22 <212> PRT
<213> Homo Sapiens <400> 48 Leu Arg Thr Giu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys RECTIFIED SHEET (RULE 91) <210> 49 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARTANT
<222> fl) <223> Wherein Xaa is Cys or nothing <400> 49 Xaa Gln Gly.Ser Val Pro Arg Leu His Asp Val Gly Arg Phe Asn <210> 50 <211> 20 <212> PRT
<213> Homo Sapiens <400> 50 Leu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys <210> 51 <211> 19 <212> PRT
<213> Homo Sapiens <400> 51 Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn Phe Leu G1n Val Pro M2t Cys <210> 52 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARTANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 52 Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro <210> 53 <211> 12 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91) ls/39 <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 53 Xaa Val Ser Ser Asp Thr Tyr Gly Arg Gln Asn Gly <210> 54 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 54 Xaa Pro Asn Gln Asn Met Thr.Ser Glu:Glu Arg:Gln Arg Leu <210> 55 <211> 21 <212> PRT
<213> Homo Sapiens <400> 55 Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr Ile Pro Met Ser Met Cys <210> 56 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 56 Xaa Asn Thr Ile Tyr Phe Val. Ser Ser Asn Thr Glu Arg <210> 57 <211> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (1) <223> Wherein Xaa is Cys or nothing <400> 57 Xaa Phe Pro Glu Leu Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Ile Ser Glu Gly <210> 58 <211> 11 <212> PRT
<213> Homo Sapiens <~00> 58 Tyr Ala Gly Met Asp Met Gly Thr Lys Ser Ile <210> 59 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 59 Xaa Asp Leu Ile Lys His Arg Lys Met Ala Pro Leu Asp <210> 60 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 60 Xaa Arg Lys Phe Phe Ser Gln Asn Ala Thr Ile Gln Lys Glu Asp Thr Leu Ala Tle Gln <210> 61 <211> 17 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (1) <223> wherein Xaa is Cys or nothing <400> 61 Xaa Lys Val Phe Leu Ser Ser Leu Lys Phe His.Ile Arg Arg Phe Ile Phe <210> 62 <211> 20 <212> PRT
<213> mouse <400> 62 Leu Gly Ser Thr Glu Glu Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn Ser I1e Pro Cys <210> 63 <211> 20 <212> PRT .
<213> Homo Sapiens <400> 63 Leu Gly G1u A1a Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Sex Ser Pro Val Cys <210> 64 <211> 12 <212> PRT
<213> Homo Sapiens <400> 64 Leu Gln Val Arg His Arg Pro GIu Val Thr Leu Cys <210> 65 <211> 19 <212> PRT
<213> Homo Sapiens <400> 65 Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn Phe Leu Gln Val Pro Met Cys <210> 66 RECTIFIED SHEET (RULE 91 ) <211> 11 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 66 Xaa Glu Thr Leu Sex Val Lys Arg Gln Tyr Pro <210> 67 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 67 Xaa Gly Sex Ser Asp Asp Tyr Gly Gln Leu Gly 1 ' ~ 10 <210> 68 <211> 11 <212> PRT
<213> Homo Sapiens <400> 68 Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys <210> 69 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 69 Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro <210> 70 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221>.VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (1) .
<223> Wherein Xaa is Cys or nothing <400> 70 Xaa Val Ser Ser Asp Thr Tyr Gly Arg Gln Asn GIy <210> 71 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 71 Xaa Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu <210> 72 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 72 Xaa GIu Leu Leu Ser Ala Arg Glu Thr Phe Pro <210> 73 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 73 Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val Gln <210> 74 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) RECTIFIED SHEET (RULE 91) <223> Wherein Xaa is Cys or nothing <400> 74 Xaa Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu <210> 75 <211> 14 <212> PRT
<213> mouse <400> 75 Glu Gly Val Leu Asn Ile Asp Asp Tyr Lys Il.e Gln Met Asn <210> 76 <211> Z4 <212> PRT
<213> Homo Sapiens <400> 76 Gln Gly Ser VaI Pro Arg Leu His Asp Val Gly Arg Phe Asn <210> 77 <211> 22 <212> PRT
<213> Homo Sapiens <400> 77 Leu Arg Thr GIu Arg Leu Lys zle Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys <210> 78 <211> 18 <212> PRT
<213> Homo Sapiens <400> 78 Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys <210> 79 <211> 21 <212> PRT
<213> Homo Sapiens <400> 79 Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr Tle RECTIFIED SHEET (RULE 91) Pro Met Ser Met Cys <210> 80 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 80 Xaa Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Xaa 1 .5 10 <210> 81 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (20) <223> Wherein Xaa is an Amino Group <400> 81 Xaa Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Xaa <210> 82 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) RECTIFIED SHEET (RULE 91 ) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 82 Xaa Xaa Glu Thr Leu Ser Val Lys Arg Gln Tyr Pro Xaa <210> 83 <211> 13 <2I2> PRT
<223> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARTANT
<222> (13) <223> Wherein xaa is an Amino Group <400> 83 Xaa Xaa Gly Ser Ser~Asp Asp~Tyr GlyGln Leu Gly Xaa <210> 84 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 84 Xaa Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys Xaa <210> 85 <211> 21 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91) <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 85 Xaa Leu His ATa Asn Met Lys Gly Ile Val His Leu Asn Phe Leu Gln Va1 Pro Met Cys Xaa <220> 86 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 86 Xaa Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro Xaa <210> 87 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>.
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (14) <223> Wherein Xaa is an Amirio Group <400> 87 Xaa Xaa Val Ser Ser Asp Thr Tyyr Gly Arg Gln Asn Gly Xaa <210> 88 <211> 16 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221.> VARIANT
<222> (16.) <223> Wherein Xaa is an Amino Group <400> 88 Xaa Xaa Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu Xaa <220> 89 <211> 23 <212> PRT
<213> Homo Sapiens ' <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (23) <223> Wherein Xaa is an Amino Group <400> 89 Xaa Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr Ile Pro Mat Ser Met Cys Xaa 2a <210> 90 <211> 22 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91 ) <220>
<221> VARIANT
<222> {1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is ,an Amino Group <400> 90 Xaa L~eu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Xaa <210> 91 <211> 17 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (17) <223> Wherein Xaa is an Amino Group <400> 91 Xaa Xaa Gln Gly Ser Val Pro Arg heu His Asp Val Gly Arg Phe Asn z ~ 10 15 Xaa <210> 92 <211> 24 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> {1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> {24) <223> Wherein Xaa is an Amino Group RECTIFIED SHEET (RULE 91) <400> 92 Xaa Leu Arg Thr Glu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn GTn Lys Pro Val Ser Arg Cys Xaa <210> 93 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys~or nothing <220> ' <221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 93 Xaa Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro Xaa <210> 94 <211> 15 <212> PRT
<213> Homo Sapiens <220> .
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220> .
<221> VARTANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 94 Xaa Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val Gln Xaa 1 ~ 5 10 15 <210> 95 <211> 14 <212> PRT
RECTIFIED SHEET (RULE 91) <213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 95 Xaa Xaa Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu Xaa <210> 96 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARTANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 96 , Xaa Xaa Asn Thr Ile Tyr Phe Val Ser Ser Asn Thr Glu Arg Xaa <210> 97 <211> 21 <212> PRT
<223> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222 > (2 ) <223> Wherein Xaa is Cys or nothing RECTIFIED SHEET (RULE 91 ) <2zo>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 97 Xaa Xaa Phe Pro Glu Leu.Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Ile Ser Glu Gly Xaa <210> 98 <211> 15 <212> PRT
<213> Flomo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 98 Xaa Xaa Asp Leu I1e Lys His Arg Lys Met A.la Pro Leu Asp Xaa <210> 99 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group <400> 99 Xaa Xaa Arg Lys Phe Phe Ser Gln Asn Ala Thr Ile Gln Lys Glu Asp RECTIFIED SHEET (RULE 91 ) Thr Leu Ala Ile Gln Xaa <220> 100 <211> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220> .
<221> VARIANT
<222> (29) <223> Wherein Xaa is ari Amino Group <400> 100 Xaa Xaa Lys Val Phe Leu Ser Ser Leu Lys Phe His Ile Arg Arg Phe zle Phe Xaa <220> 102 <222> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> {14) <223> Wherein Xaa is an Amino Group <400> 101 Xaa Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Xaa <210> 202 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91) <222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (20) <223> Wherein Xaa is an Amino Group <400> 102 Xaa Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Xaa <210>103 <211>13 <212>PRT
<213>Homo Sapiens <220>
<221>VA12TANT
<222>(1) <223>Wherein Xaa an Acetyl Group is <220>
<221>VARIANT
<222>(2) <223>Wherein Xaa Cys or nothing is <220>
<221>VARIANT
<222>(13) <223>Wherein Xaa an Amino Group is <400>103 Xaa Ser Val Lys Arg Gln Tyr Xaa Pro Xaa GIu Thr Leu 1 5 1.0 <210> 104 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group RECTIFIED SHEET (RULE 91) <400> 204 Xaa Xaa Gly Ser Ser Asp Asp Tyr Gly Gln Leu Gly Xaa <210> 105 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 105 Xaa Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys Xaa <210> 106 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group <400> 106 Xaa Leu Gly G1u Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Xaa <210> 107 <221> 17 <212> PRT
<213> Homo Sapiens <220>
<221> VA12IANT
<222> (1}
<223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222>.. (2) RECTIFIED SHEET (RULE 91) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (17) <223> Wherein Xaa is an Amino Group <400> 107 Xaa Xaa Gln Gly Ser Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Xaa <210> 108 <211> 24 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<227.> VARIANT
<222> (24) <223> Wherein Xaa is an Amino Group <400> 108 Xaa Leu Arg Thr Glu Arg Leu Lys Tle Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys Xaa <210> 109 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (23) <223> Wherein Xaa is an Amino Group <400> 109 Xaa Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro Xaa RECTIFIED SHEET (RULE 91 ) <210> 110 <211> 15 <212> PRT
<213> Homo sapiens <220>
<2:21> VARIANT
<222> {1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 110 Xaa Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly;Lys Val Gln Xaa 1 5 10 ~ 15 <210> 111 <211> 14 <212> PRT
<223> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein an Acetyl Group Xaa is <220>
<221> VARIANT
.
<222> (2) .
<223> Wherein Cys or nothing Xaa is <220>
<221> VARIANT
<222> (14) <223> Wherein an Amino Group Xaa is <400> 111 Xaa Xaa Gly Ser Asp Glu Tyr Gly Arg Gln Gly Asp Leu Xaa <210> 112 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> {1) <223> Wherein Xaa is an. Acetyl Group RECTIFIED SHEET (RULE 91) <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) .
<223> Wherein Xaa is an Amino Group <400> 112 Xaa Xaa Asn Thr Ile Tyr Phe Val Ser Ser Asn Thr Glu Arg Xaa <210> 113 <211> 19 <212> PRT
<213> fiomo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <400>.113 Xaa Phe Pro GIu Leu Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Tle Ser Glu Gly <210> 114 <211> 21 <212> PRT
<213> Tiomo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARTANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 114 Xaa Xaa Phe Pro Glu Leu Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Ile Ser Glu Gly Xaa RECTIFIED~SHEET (RULE 91) <210> 115 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <400> 115 Xaa Tyr Ala Gly Met Asp Met Gly Thr Lys S~er Ile <210> 116 <211> 15 <212 > PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group' <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 116 Xaa Xaa Asp Leu Ile Lys His Arg Lys Met Ala Pro Leu Asp Xaa <210> 117 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<222> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group RECTIFIED SHEET (RULE 91) <400> 117 Xaa Xaa.Arg Lys Phe Phe Ser Gln Asn Ala Thr Ile Gln Lys Glu Asp Thr Leu Ala Tle Gln Xaa <210> 118 <211> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (19) <223> Wherein Xaa is an Amino Group <400> 118 Xaa Xaa Lys Val Phe Leu Ser Ser Leu Lys Phe, His Tle Arg Arg Phe Ile Phe Xaa <210> 119 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 119 Xaa Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Xaa <210> 120 <211> 15 <212> PRT
<213> Homo sapiens RECTIFIED SHEET (RULE 91 ) <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 120 Xaa Glu Trp Ser Tyr Gln Ser Glu Thr Ser Cys Phe Lys Arg Xaa <210> 121 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 121 Xaa Xaa Gln Trp Asp Arg Ser G1n Asn Pro Phe Gln Xaa <210> 122 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (12) <223> Wherein Xaa is an Amino Group <400> 122 RECTIFIED SHEET (RULE 91) Xaa Xaa G1n Arg Gln Leu Lys Asn I1e Gln Asp Xaa <210> 123 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Whexein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (12) <223> Wherein Xaa is an Amino Group <400> 123 Xaa Xaa Asp Thr Tyr Gly Arg Asp Asn Gly Gln Xaa <210> 124 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 124 Xaa Glu Thr Leu Pro Thr Leu G1n Pro Asn Gln Cys Xaa <210> 125 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <400> 125 Xaa His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys RECTIFIED SHEET (RULE 91) <210> 126 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 126 Xaa Xaa Glu Leu Leu Sex Ala Arg Glu Thr Phe Pro Xaa RECTIFIED SHEET (RULE 91)
Table 2. Chosen peptides fox T2R
Rece LocationResiduesPe tide for hT2R4 IEL 65-76 Ac-(C)NTTY FVSSN TER-NH2 .
SE ID NO: 112 hT2R4 2EL 1S6-173 Ac-F PELVT TRNNT SFNIS EG MAP
(SEQ ID NO: 113} KLH
or Ac-(C)FPEL VTTRN NTSFN
ISEG-NHZ (SE II7 NO: 114 hT2R4 3EL 254-264 Ac-Y AGMDM GTKSI (SEQ ID MAP
NO: 115) hT2Rl lEL 34-4S Ac-(C)DLIKH RKMAP LD-NH2 KLH
SEQ DJ NO: 116 hT2R1 2EL 157-175 AC-(C)RKFF SQNAT IQKED KLH
TLAIQ-NH2 SEQ ID NO: 117) hT2R1 3EL 244-259 Ac-(C)KVFL SSLKF HIRRF IF-NH2KLH
SE ff~ NO: 118 If some of these peptides are not hydrophilic enough, MAPS may be used instead of conjugation of KLH with another protein. Fox a review of the MAPS technology see Tam JP.
(1988) Proc. Natl. Acad. Sci. 85:5409-5413.
(b) hnmunizations Table 3. Several references of preparing IgY.
Age of Lineage Immunizations,Dose, Antigen Reference hens weeks 0,1,2,4,6,10,14,100 Whole Fortgens, P.H. et al. (1997) ... protein Immuhopha~macology 36, 160 days 0,2,4,6 500 Whole Tu, Y.Y. et al. (2001) Food rotein Res. Int. 34, 783 monthsPadua 0, 22d; 20d 40; GST-FP Di Lonardo, A. et 20 ~ al. (2001) Brown ~Irch. Yirol. 146, 180 days 0,2,4;8,12,... Whole Sun, S. {2001) Rapid protein Commuu. Mass.
S ectrom. 2 5, 708 20 weeksLohrxran0,10 d, 20d 60 Whole De Ceuninck, F. et al.
Brown protein (2001) J. Immunol.
Meth.
252, 153 0,2,4,6,8,10 Peptide-Williams, L.M. et al. (2001) KLH J. Neurae~tdoeYihol., 13, 94 25 weeksWhite 0,30 d 100 Virus Bizhanov, G., and Vendete Vyshniausl~is, G.
(2000) Tlet. Res.Commuh., 24, 203 RECTIFIED SHEET (RULE 91) 7 monthsLeghorn 0,2,3 ~ 100 Whole Vieira-da-Motta, A.
et al.
white rotein (2001 Pe tides 22, 6 monthsLeghorn 0,4,8,. .. 25 Haptene-Shelver, W.L. et al.
(1998) white KLH J. Chromato . 705, Leghorn 0,2,4 60 GST-FP Garnenisch, G. et al. (1999) white FASEB J. 13, 81 Leghorn 0,2,4,8,12,...100 Peptide-Knecht, W. et al.
(1996) white MAP Eur J. Biochem. 236, Peptide-Lee, S.C. et al. (1997) Peep.
KLH Biochetn. Biotech.
27, 227 Peptide-Campbell, W.G. et al.
KLH (I999) Am. J. Physiol.
276, Leghorn 0, 10 d, ... 300 Mixture Iwamoto, T. et al.
(1996) J.
white of Biol. Chem. 271, 13609 peptide-MAP
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-ELZ
could also be made with MAP.
2. Immunizations - week 0 - 60 ~.g in complete Freund's Adjuvant, further -weeks 2, 4 -60 ~.g in incomplete Freund's adjuvant in 4 points intramuscularly.
3. Before immunizations, a few preirnmune eggs are collected.
4. A week after the 2"d 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 axe accepted:
1. Chloroform separation.
2. Polyethylene glycol precipitation of IgY
3. The dilution with 4 volumes of deionized 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.
RECTIFIED SHEET (RULE 91) Example ~
Tests and assays.
I . ELISA 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 rnaleimide-modified keyhole limpet hemocyanin (KLH) through their cysteine moieties.
For immunization, two White Leghorn and Brown Leghorn hens S-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 Pre-immune eggs (2-3) + Tmmunisation 1 I (200 u,~) Week Timnunization II (200 ~.~g) Week Tmmunization III (100 p.g) Weak Egg collection III+2 week 6 eggs +ELISA
Week Egg collection III+2 week 6 eggs (stored 8 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-linked immunoassay (ELISA) 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 l~Iz (SEQ ID NO: I 19) TVD-3/21 Ac-EWSYQ SETSC FKR NH2 (SEQ ID NO: 120) RECTIFIED SHEET (RULE 91) TVD-4/21 Ac-(C)QWDR SQNPF Q-NHZ (SEQ 1D NO: 121) TVD-5/21 Ac-(C)QRQL I~NIQD-NHZ (SEQ m NO: 122) TVD-6/21 Ac-(C)DTYG RDNGQ-NHZ (SEQ m NO: 123) TVD-7/21 Ac-ETLPT LQPNQ C-NH2 (SEQ Il7 NO: 124) TVD-9121 Ac-HTSDN QKPVS RC (SEQ ID NO: 125) TVD-11/21 Ac-(C)ELLS ARETF P-NHZ (SEQ ID NO: 126) Ac- at the N-terminus means acetylated N terminus. NHZ 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 1/2I, 9/21 and 11/21 are derived from the hTlR3 receptor (conunon to sweet and umami).
Results:
The Ab containing powders (=lyophilized yolks) were dissolved in distilled water to a final concentration of 2-10% (dry yolk wtlvolume) 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°O) 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 ofhTlR2 were mixed together (e.g. Ab against 3/21 + 4/21 + 5121 + 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 dixected 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.
RECTIFIED SHEET (RULE 91) SEQUENCE LISTING
<110> TVD Taste Virtual Aimensions, Ina.
Ben-Sasson, Shmuel <120> Methods and Compositions Directed Towards Modulation of Chemosensation <130> 24836-501-061 <140> PCT/IL03/00409 <141> 2003-05-20 <150> 60/451,639 <151> 2003-03-04 <150> 60/382,.059 <151> 2002-05-20 <160> 126 <170> Patentln Ver. 2.1 <210> 1 <211> 15 <212> PRT
<213> Homo Sapiens <400> 1 Ala Asn Iiis Leu Leu Gly Ser Lys Ser 21e Ser Phe Gly G1y Cys <210> 2 <211> 10 <212> PRT
<213> Homo Sapiens <400> 2 Cys Gly Asn Gln Glu Val Ala Asn Phe Tyr <210> 3 <211> 10 <212> PRT
<213> Homo sapiens <400> 3 Cys Asp Tle Thr Pro Leu Leu Lys Leu Ser <210> 4 <211> 8 <212> PRT
<213> Homo Sapiens <400> 4 Cys Ser Asp Ile His Phe His Val RECTIFIED SHEET (RULE 91) <210> 5 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 5 Xaa Tyr Phe Arg Pra Leu Thr Asn Tyr Ser Leu Lys Asp Ala Val <220> 6 <2I1> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 6 Xaa Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe Ala Gly Arg Met Met Gln Thr c210> '7 <211> 10 c212> PRT
c213> Homo Sapiens c400> 7 Arg Pro G1n Lys Ile Tyr His Phe Phe Cys <210> 8 <211> 10 <212> PRT
<213> Homo Sapiens <400> 8 ' Cys Ala Asp Thr His Ile Asn Glu Asn Met c210> 9 <211> 14 <212> PRT
<213> Homo Sapiens <220>
RECTIFIED SHEET (RULE 91) <221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 9 X~a Gly Pro Arg Tyr Gly Asn Pro Lys Glu Gln Lys I~ys Tyr <210> 10 <211> 13 <212> PRT
<213> Homo Sapiens <400> ZO
Leu Ser Arg Leu Leu Ser Arg Lys Arg Ala Val Pro Cys <210> 11 <211> 10 <212> PRT
<213> Homo Sapiens <400> 11 Cys Gly Pro Asn Val Ile Asn His Plie Tyr <210> 12 <211> 10 <212> PRT
<213> Homo sapiens <400> 12 Cys Asp Leu Pro Gln Leu Phe Gln Leu Ser 1 5 3.0 <210> 13 <211> 9 <212> PRT
<213> Homo Sapiens <400> 13 Cys Ser Ser Thr Gln Leu Asn Glu Leu <210> 14 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 14 Xaa Arg Leu Gly Ser Thr Lys Leu Ser Asp Lys Asp Lys Ale RECTIFIED SHEET (RULE 91) <210> 15 <211> 15 <212> PRT' <213> Homo Sapiens <400> 15 Ala Asp Leu Leu Ser Glu Lys Lys Thr Ile Ser Phe Ala Gly Cys <210> 16 <211> 10 <212> PRT
<213> Homo Sapiens <400> 16 Cys Asp Ser Asn Val Ile His His Phe Phe 1 5 7.0 <210> 17 <211> 10 <212> PRT
<213> Homo Sapiens <400> .17 Cys Asp Ser Pro Pro Leu Phe Lys Leu Ser <210> 7.8 <211> 10 <212> PRT
<213> Homo Sapiens <400> 18 Cys Ser Asp Thr Ile Leu Lys Glu Sex Ile 1 5 la <210> 19 <211> 17 <212> PRT
<213> Homo Sapiens <400> 19 Cys Thr Tyr Leu Arg Pro Ser Ser Ser Tyr Ser Leu Asn Gln Asp Lys Val <210> 20 <212> 20 <212> PRT
<213> mouse RECTIFIED SHEET (RULE 91) <400> 20 Leu Gly Ser Thr Glu Glu Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn 1 5 l0 15 Ser Ile Pro Cys <210> 22 <211> 14 <212> PRT
<213> mouse <400> 21 Glu Gly Va1 Leu Asn Ile Asp Asp Tyr Lys Ile Gln Met Asn <210> 22 <211> 14 <212> PRT
<213> Homo sapiens <400> 22 Gln Gly Ser Val Pro Arg Leu His Asp Val Gly Arg Phe Asn <210> 23 <211> 22 <212> PRT
<213> Homo sapiens <400> 23 Leu Arg Thr G1u Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln 1 . 5 10 15 Lys Pro Val Ser Arg Cys <210>24 <211>15 <212>PRT
<213>Homo Sapiens <220>
<221>VARIANT
<222>(1) <223>Wherein Xaa.isan Acetyl Group <220>
<221>VARTANT
<222>(14) <223>Wherein Xaa any Amino is Acid <220>
<221>VARIANT
<222>(15) <223>Wherein Xaa an Amino Group is RECTIFIED SHEET (RULE 91) <400> 24 Xaa Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Xaa Xaa <210> 25 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (20) <223> Wherein Xaa is an Amino Group <400> 25 Xaa Glu Thr Lys Tle Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Xaa <210> 26 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <400> 26 Xaa Xaa Glu Thr Leu Ser Val Lys Arg Gln Tyr Pro Xaa <a1o> 27 <211> 13 <212> PRT
<213> Homo sapiens <220>
<221> VARTANT
<222> (1) RECTIFIED SHEET (RULE 91 ) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 27 Xaa Xaa Gly Ser Ser Asp Asp Tyr Gly G1n Leu Gly Xaa <210> 28 <211> 13 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
e222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 28 Xaa Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys Xaa <210> 29 <211> 21 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 29 Xaa Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn Phe Leu G1n Val Pro Met Cys.Xaa <210> 30 RECTIFIED SHEET (RULE 91 ) <211> 13 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
<222> (1) .
<223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 30 Xaa Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro Xaa 1 g 10 <210> 31 <211> 14 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 31 Xaa Xaa Val Ser Ser Asp Thr Tyr Gly Arg Gln Asn Gly Xaa <210> 32 <211> 16 <212> PRT
<213> Homo sagiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (2) <223> tr~herein Xaa is Cys or nothing <220>
<221> VARTANT
<222> (16) <223> P~herein Xaa is an Amino Group <400> 32 Xaa Xaa Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu Xaa 1 ' S 10 15 <210> 33 <211> 23 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (23) <223> Wherein Xaa is an Amino Group <400> 33 Xaa Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr 1 5 lp 15 Ile Pro Met Sex Met Cys Xaa <210> 34 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223,> P7herein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group <400> 34 Xaa Leu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Xaa <210> 35 <211> 17 RECTIFIED SHEET (RULE 91) <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa an Acetyl Group is <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa Cys or nothing is <220>
<221> VARIANT
<222> (17) <223> Wherein Xaa an Amino Group is <400> 35 Xaa Xaa G1n Gly Ser Val Pro Azg Leu His Asp Val Gly Arg Phe Asn 1 ~ 10 15 Xaa <210> 36 <211> 24 <212> PRT
<2.13> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (24) <223> Wherein Xaa is an Amino Group <400> 36 Xaa Leu Arg Thr GIu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn 1 5 10 ~ 15 Gln Lys Fro Val Ser Arg Cys Xaa <210> 37 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino. Group <400> 37 Xaa Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro Xaa <210> 38 <211> 15 <212> PRT
<213> Homo sapisns~
<220>
<221> VARTANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARTANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 38 Xaa Xaa Pro Arg Ala Asp Asp Ser'Arg Leu Gly hys Val Gln Xaa 1 5 10 . 15 <210> 39 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT .
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 39 Xaa Xaa G1y Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu Xaa RECTIFIED SHEET (RULE 91) <210> 40 <211> 1.1 <212 > PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 40 Xaa Glu Thr Leu Ser Val Lys Arg Gln Tyr Pro <210> 41 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 41 Xaa Gly Ser Ser Asp Asp Tyr Gly Gln Leu Gly <210> 42 <211> 11 <212> PRT
<213> Homo Sapiens <400> 42 Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys <210> 43 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 43 Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro <210> 44 <211> 13 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91) <220>
<221> VARIANT
<222> (1) <223> Tn~herein Xaa is Cys or nothing <400> 44 Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val Gln 1 5 ~0 <210> 45 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 45 Xaa Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu <210> 46 <211> 12 <212> PRT
<213> Homo Sapiens <400> 46 Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys <210> 47 <211> 18 <212> PRT
<213> Homo sapience <400> 47 Glu Thr Lys Ile Gln Trp 33is Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys <210> 48 <211> 22 <212> PRT
<213> Homo Sapiens <400> 48 Leu Arg Thr Giu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys RECTIFIED SHEET (RULE 91) <210> 49 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARTANT
<222> fl) <223> Wherein Xaa is Cys or nothing <400> 49 Xaa Gln Gly.Ser Val Pro Arg Leu His Asp Val Gly Arg Phe Asn <210> 50 <211> 20 <212> PRT
<213> Homo Sapiens <400> 50 Leu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys <210> 51 <211> 19 <212> PRT
<213> Homo Sapiens <400> 51 Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn Phe Leu G1n Val Pro M2t Cys <210> 52 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARTANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 52 Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro <210> 53 <211> 12 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91) ls/39 <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 53 Xaa Val Ser Ser Asp Thr Tyr Gly Arg Gln Asn Gly <210> 54 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 54 Xaa Pro Asn Gln Asn Met Thr.Ser Glu:Glu Arg:Gln Arg Leu <210> 55 <211> 21 <212> PRT
<213> Homo Sapiens <400> 55 Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr Ile Pro Met Ser Met Cys <210> 56 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 56 Xaa Asn Thr Ile Tyr Phe Val. Ser Ser Asn Thr Glu Arg <210> 57 <211> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (1) <223> Wherein Xaa is Cys or nothing <400> 57 Xaa Phe Pro Glu Leu Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Ile Ser Glu Gly <210> 58 <211> 11 <212> PRT
<213> Homo Sapiens <~00> 58 Tyr Ala Gly Met Asp Met Gly Thr Lys Ser Ile <210> 59 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 59 Xaa Asp Leu Ile Lys His Arg Lys Met Ala Pro Leu Asp <210> 60 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 60 Xaa Arg Lys Phe Phe Ser Gln Asn Ala Thr Ile Gln Lys Glu Asp Thr Leu Ala Tle Gln <210> 61 <211> 17 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (1) <223> wherein Xaa is Cys or nothing <400> 61 Xaa Lys Val Phe Leu Ser Ser Leu Lys Phe His.Ile Arg Arg Phe Ile Phe <210> 62 <211> 20 <212> PRT
<213> mouse <400> 62 Leu Gly Ser Thr Glu Glu Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn Ser I1e Pro Cys <210> 63 <211> 20 <212> PRT .
<213> Homo Sapiens <400> 63 Leu Gly G1u A1a Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Sex Ser Pro Val Cys <210> 64 <211> 12 <212> PRT
<213> Homo Sapiens <400> 64 Leu Gln Val Arg His Arg Pro GIu Val Thr Leu Cys <210> 65 <211> 19 <212> PRT
<213> Homo Sapiens <400> 65 Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn Phe Leu Gln Val Pro Met Cys <210> 66 RECTIFIED SHEET (RULE 91 ) <211> 11 <212> PRT
<213> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 66 Xaa Glu Thr Leu Sex Val Lys Arg Gln Tyr Pro <210> 67 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 67 Xaa Gly Sex Ser Asp Asp Tyr Gly Gln Leu Gly 1 ' ~ 10 <210> 68 <211> 11 <212> PRT
<213> Homo Sapiens <400> 68 Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys <210> 69 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 69 Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro <210> 70 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221>.VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (1) .
<223> Wherein Xaa is Cys or nothing <400> 70 Xaa Val Ser Ser Asp Thr Tyr Gly Arg Gln Asn GIy <210> 71 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 71 Xaa Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu <210> 72 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 72 Xaa GIu Leu Leu Ser Ala Arg Glu Thr Phe Pro <210> 73 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is Cys or nothing <400> 73 Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val Gln <210> 74 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) RECTIFIED SHEET (RULE 91) <223> Wherein Xaa is Cys or nothing <400> 74 Xaa Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu <210> 75 <211> 14 <212> PRT
<213> mouse <400> 75 Glu Gly Val Leu Asn Ile Asp Asp Tyr Lys Il.e Gln Met Asn <210> 76 <211> Z4 <212> PRT
<213> Homo Sapiens <400> 76 Gln Gly Ser VaI Pro Arg Leu His Asp Val Gly Arg Phe Asn <210> 77 <211> 22 <212> PRT
<213> Homo Sapiens <400> 77 Leu Arg Thr GIu Arg Leu Lys zle Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys <210> 78 <211> 18 <212> PRT
<213> Homo Sapiens <400> 78 Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys <210> 79 <211> 21 <212> PRT
<213> Homo Sapiens <400> 79 Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr Tle RECTIFIED SHEET (RULE 91) Pro Met Ser Met Cys <210> 80 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 80 Xaa Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Xaa 1 .5 10 <210> 81 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (20) <223> Wherein Xaa is an Amino Group <400> 81 Xaa Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Xaa <210> 82 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) RECTIFIED SHEET (RULE 91 ) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 82 Xaa Xaa Glu Thr Leu Ser Val Lys Arg Gln Tyr Pro Xaa <210> 83 <211> 13 <2I2> PRT
<223> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARTANT
<222> (13) <223> Wherein xaa is an Amino Group <400> 83 Xaa Xaa Gly Ser Ser~Asp Asp~Tyr GlyGln Leu Gly Xaa <210> 84 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 84 Xaa Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys Xaa <210> 85 <211> 21 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91) <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 85 Xaa Leu His ATa Asn Met Lys Gly Ile Val His Leu Asn Phe Leu Gln Va1 Pro Met Cys Xaa <220> 86 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 86 Xaa Xaa Asp Glu Leu Arg Asp Lys Val Arg Phe Pro Xaa <210> 87 <211> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>.
<221> VARIANT
RECTIFIED SHEET (RULE 91 ) <222> (14) <223> Wherein Xaa is an Amirio Group <400> 87 Xaa Xaa Val Ser Ser Asp Thr Tyyr Gly Arg Gln Asn Gly Xaa <210> 88 <211> 16 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221.> VARIANT
<222> (16.) <223> Wherein Xaa is an Amino Group <400> 88 Xaa Xaa Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu Xaa <220> 89 <211> 23 <212> PRT
<213> Homo Sapiens ' <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (23) <223> Wherein Xaa is an Amino Group <400> 89 Xaa Leu Lys Asn Ile Gln Asp Ile Ser Trp His Thr Val Asn Asn Thr Ile Pro Mat Ser Met Cys Xaa 2a <210> 90 <211> 22 <212> PRT
<213> Homo Sapiens RECTIFIED SHEET (RULE 91 ) <220>
<221> VARIANT
<222> {1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is ,an Amino Group <400> 90 Xaa L~eu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Xaa <210> 91 <211> 17 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (17) <223> Wherein Xaa is an Amino Group <400> 91 Xaa Xaa Gln Gly Ser Val Pro Arg heu His Asp Val Gly Arg Phe Asn z ~ 10 15 Xaa <210> 92 <211> 24 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> {1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> {24) <223> Wherein Xaa is an Amino Group RECTIFIED SHEET (RULE 91) <400> 92 Xaa Leu Arg Thr Glu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn GTn Lys Pro Val Ser Arg Cys Xaa <210> 93 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys~or nothing <220> ' <221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 93 Xaa Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro Xaa <210> 94 <211> 15 <212> PRT
<213> Homo Sapiens <220> .
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220> .
<221> VARTANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 94 Xaa Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val Gln Xaa 1 ~ 5 10 15 <210> 95 <211> 14 <212> PRT
RECTIFIED SHEET (RULE 91) <213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (14) <223> Wherein Xaa is an Amino Group <400> 95 Xaa Xaa Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu Xaa <210> 96 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARTANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 96 , Xaa Xaa Asn Thr Ile Tyr Phe Val Ser Ser Asn Thr Glu Arg Xaa <210> 97 <211> 21 <212> PRT
<223> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222 > (2 ) <223> Wherein Xaa is Cys or nothing RECTIFIED SHEET (RULE 91 ) <2zo>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 97 Xaa Xaa Phe Pro Glu Leu.Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Ile Ser Glu Gly Xaa <210> 98 <211> 15 <212> PRT
<213> Flomo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 98 Xaa Xaa Asp Leu I1e Lys His Arg Lys Met A.la Pro Leu Asp Xaa <210> 99 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group <400> 99 Xaa Xaa Arg Lys Phe Phe Ser Gln Asn Ala Thr Ile Gln Lys Glu Asp RECTIFIED SHEET (RULE 91 ) Thr Leu Ala Ile Gln Xaa <220> 100 <211> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220> .
<221> VARIANT
<222> (29) <223> Wherein Xaa is ari Amino Group <400> 100 Xaa Xaa Lys Val Phe Leu Ser Ser Leu Lys Phe His Ile Arg Arg Phe zle Phe Xaa <220> 102 <222> 14 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> {14) <223> Wherein Xaa is an Amino Group <400> 101 Xaa Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Xaa <210> 202 <211> 20 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
RECTIFIED SHEET (RULE 91) <222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (20) <223> Wherein Xaa is an Amino Group <400> 102 Xaa Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Xaa <210>103 <211>13 <212>PRT
<213>Homo Sapiens <220>
<221>VA12TANT
<222>(1) <223>Wherein Xaa an Acetyl Group is <220>
<221>VARIANT
<222>(2) <223>Wherein Xaa Cys or nothing is <220>
<221>VARIANT
<222>(13) <223>Wherein Xaa an Amino Group is <400>103 Xaa Ser Val Lys Arg Gln Tyr Xaa Pro Xaa GIu Thr Leu 1 5 1.0 <210> 104 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group RECTIFIED SHEET (RULE 91) <400> 204 Xaa Xaa Gly Ser Ser Asp Asp Tyr Gly Gln Leu Gly Xaa <210> 105 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 105 Xaa Ser Ala Gln Val Gly Asp Glu Arg Met Gln Cys Xaa <210> 106 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group <400> 106 Xaa Leu Gly G1u Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Xaa <210> 107 <221> 17 <212> PRT
<213> Homo Sapiens <220>
<221> VA12IANT
<222> (1}
<223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222>.. (2) RECTIFIED SHEET (RULE 91) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (17) <223> Wherein Xaa is an Amino Group <400> 107 Xaa Xaa Gln Gly Ser Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Xaa <210> 108 <211> 24 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<227.> VARIANT
<222> (24) <223> Wherein Xaa is an Amino Group <400> 108 Xaa Leu Arg Thr Glu Arg Leu Lys Tle Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys Xaa <210> 109 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (23) <223> Wherein Xaa is an Amino Group <400> 109 Xaa Xaa Glu Leu Leu Ser Ala Arg Glu Thr Phe Pro Xaa RECTIFIED SHEET (RULE 91 ) <210> 110 <211> 15 <212> PRT
<213> Homo sapiens <220>
<2:21> VARIANT
<222> {1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 110 Xaa Xaa Pro Arg Ala Asp Asp Ser Arg Leu Gly;Lys Val Gln Xaa 1 5 10 ~ 15 <210> 111 <211> 14 <212> PRT
<223> Homo sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein an Acetyl Group Xaa is <220>
<221> VARIANT
.
<222> (2) .
<223> Wherein Cys or nothing Xaa is <220>
<221> VARIANT
<222> (14) <223> Wherein an Amino Group Xaa is <400> 111 Xaa Xaa Gly Ser Asp Glu Tyr Gly Arg Gln Gly Asp Leu Xaa <210> 112 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> {1) <223> Wherein Xaa is an. Acetyl Group RECTIFIED SHEET (RULE 91) <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) .
<223> Wherein Xaa is an Amino Group <400> 112 Xaa Xaa Asn Thr Ile Tyr Phe Val Ser Ser Asn Thr Glu Arg Xaa <210> 113 <211> 19 <212> PRT
<213> fiomo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <400>.113 Xaa Phe Pro GIu Leu Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Tle Ser Glu Gly <210> 114 <211> 21 <212> PRT
<213> Tiomo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARTANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (21) <223> Wherein Xaa is an Amino Group <400> 114 Xaa Xaa Phe Pro Glu Leu Val Thr Thr Arg Asn Asn Thr Ser Phe Asn Ile Ser Glu Gly Xaa RECTIFIED~SHEET (RULE 91) <210> 115 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <400> 115 Xaa Tyr Ala Gly Met Asp Met Gly Thr Lys S~er Ile <210> 116 <211> 15 <212 > PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group' <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 116 Xaa Xaa Asp Leu Ile Lys His Arg Lys Met Ala Pro Leu Asp Xaa <210> 117 <211> 22 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<222> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (22) <223> Wherein Xaa is an Amino Group RECTIFIED SHEET (RULE 91) <400> 117 Xaa Xaa.Arg Lys Phe Phe Ser Gln Asn Ala Thr Ile Gln Lys Glu Asp Thr Leu Ala Tle Gln Xaa <210> 118 <211> 19 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (19) <223> Wherein Xaa is an Amino Group <400> 118 Xaa Xaa Lys Val Phe Leu Ser Ser Leu Lys Phe, His Tle Arg Arg Phe Ile Phe Xaa <210> 119 <211> 15 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 119 Xaa Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Xaa <210> 120 <211> 15 <212> PRT
<213> Homo sapiens RECTIFIED SHEET (RULE 91 ) <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (15) <223> Wherein Xaa is an Amino Group <400> 120 Xaa Glu Trp Ser Tyr Gln Ser Glu Thr Ser Cys Phe Lys Arg Xaa <210> 121 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 121 Xaa Xaa Gln Trp Asp Arg Ser G1n Asn Pro Phe Gln Xaa <210> 122 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (12) <223> Wherein Xaa is an Amino Group <400> 122 RECTIFIED SHEET (RULE 91) Xaa Xaa G1n Arg Gln Leu Lys Asn I1e Gln Asp Xaa <210> 123 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Whexein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (12) <223> Wherein Xaa is an Amino Group <400> 123 Xaa Xaa Asp Thr Tyr Gly Arg Asp Asn Gly Gln Xaa <210> 124 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 124 Xaa Glu Thr Leu Pro Thr Leu G1n Pro Asn Gln Cys Xaa <210> 125 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <400> 125 Xaa His Thr Ser Asp Asn Gln Lys Pro Val Ser Arg Cys RECTIFIED SHEET (RULE 91) <210> 126 <211> 13 <212> PRT
<213> Homo Sapiens <220>
<221> VARIANT
<222> (1) <223> Wherein Xaa is an Acetyl Group <220>
<221> VARIANT
<222> (2) <223> Wherein Xaa is Cys or nothing <220>
<221> VARIANT
<222> (13) <223> Wherein Xaa is an Amino Group <400> 126 Xaa Xaa Glu Leu Leu Sex Ala Arg Glu Thr Phe Pro Xaa RECTIFIED SHEET (RULE 91)
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;
ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat 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;
ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat t) (C)DLIKH RKMAP LD;
u) (C)RKFF 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 T1R1 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 the group consisting of:
(a) covalent crosslinking;
(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.
t) (C)DLIKH RKMAP LD;
u) (C)RKFF 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 T1R1 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 the group consisting of:
(a) covalent crosslinking;
(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.
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 ligand 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 ligand 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 combination with a ligand capable of stimulating another receptor.
50. The method of according to any one of claims 44-49, wherein the ligand 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.
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;
ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat 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;
ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat t) (C)DLIKH RKMAP LD;
u) (C)RKFF 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 T1R1 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 the group consisting of:
(a) covalent crosslinking;
(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.
t) (C)DLIKH RKMAP LD;
u) (C)RKFF 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 T1R1 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 the group consisting of:
(a) covalent crosslinking;
(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.
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 ligand 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 ligand 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 combination with a ligand capable of stimulating another receptor.
50. The method of according to any one of claims 44-49, wherein the ligand 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.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US38205902P | 2002-05-20 | 2002-05-20 | |
| US60/382,059 | 2002-05-20 | ||
| US45163903P | 2003-03-04 | 2003-03-04 | |
| US60/451,639 | 2003-03-04 | ||
| PCT/IL2003/000409 WO2003102030A1 (en) | 2002-05-20 | 2003-05-20 | Antibodies and their use for the modulation of taste sensation___ |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2486423A1 true CA2486423A1 (en) | 2003-12-11 |
Family
ID=29715290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002486423A Abandoned CA2486423A1 (en) | 2002-05-20 | 2003-05-20 | Antibodies and their use for the modulation of taste sensation |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1507801A1 (en) |
| AU (1) | AU2003230176A1 (en) |
| CA (1) | CA2486423A1 (en) |
| WO (1) | WO2003102030A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201006846A (en) * | 2000-03-07 | 2010-02-16 | Senomyx Inc | T1R taste receptor and genes encidung same |
| WO2003004992A2 (en) * | 2001-07-03 | 2003-01-16 | The Regents Of The University Of California | Mammalian sweet and amino acid heterodimeric taste receptors |
-
2003
- 2003-05-20 CA CA002486423A patent/CA2486423A1/en not_active Abandoned
- 2003-05-20 WO PCT/IL2003/000409 patent/WO2003102030A1/en not_active Application Discontinuation
- 2003-05-20 AU AU2003230176A patent/AU2003230176A1/en not_active Abandoned
- 2003-05-20 EP EP03723027A patent/EP1507801A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP1507801A1 (en) | 2005-02-23 |
| WO2003102030A1 (en) | 2003-12-11 |
| AU2003230176A1 (en) | 2003-12-19 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |