US007795392B2

(12) United States Patent (10) Patent No.: US 7,795,392 B2 Kirchgessner et al. (45) Date of Patent: Sep. 14, 2010

(54) ORGANIC ANION TRANSPORT NCBI . Accession No. T73863 (gi:690538), Wilson, R.K., Mar. 2, 1995. (75) Inventors: Todd G. Kirchgessner, Flemington, NJ NCBI Entrez. Accession No. AIO27850 (gi:3246549), Strausberg, R., (US); Bonnie Hsiang, Pennington, NJ Oct. 30, 1998. (US); Yingjie Zhu, Killingworth, CT NCBI Entrez. Accession No. L19031 (gi:410310), Jacquemin, et al., Mar. 4, 1994. (US); Yuli Wu, Newtown, PA (US); NCBI Entrez. Accession No. P46720 (gi: 1171883), Jacquemin, et al., Zhaoqing Wang, Piscataway, NJ (US); May 1, 2005. Jean S. Lynch, Ringoes, NJ (US); Xin NCBI Entrez. Accession No. P46721 (gii: 1171882), Kullak-Ublick, et Huang, Princeton, NJ (US); Wen-Pin al., May 1, 2005. Yang, Princeton, NJ (US) NCBI Entrez. Accession No.T55488 (gi:657349), Hillier, et. al., Feb. 6, 1995. (73) Assignee: Bristol-Myers Squibb Company, NCBI Entrez. Accession No. U21943 (gi:885.977), Kullak-Ublick, et Princeton, NJ (US) al., Feb. 16, 1996. Hagenbuch, B., et al., Proc. Natl. Acad. Sci., vol. 88, Dec. 1991 pp. 10629-10633. (*) Notice: Subject to any disclaimer, the term of this Hagenbuch.B., et al., The American Society for Clinical Investiga patent is extended or adjusted under 35 tion, Inc. vol. 93, Mar. 1994, pp. 1326-1331. U.S.C. 154(b) by 155 days. Meier, P.J., et al., Hepatology vol. 26, No. 6, 1997, pp. 1667-1677. Jacquemin, E., et al., Proc. Natl. Acad. Sci., vol. 91, Jan. 1994, pp. (21) Appl. No.: 11/804,103 133-137. Noe, B.A., et al., Proc. Nat;. Acad. Sci., vol. 94, Sep. 1997, pp. (22) Filed: May 17, 2007 10346-103.50. Abe, T., et al., J. Biol. Chem. vol. 273, No. 18, (1998), pp. 11395 (65) Prior Publication Data 11401. Bossuyt. X., et al., J. Pharmacol. Exp. Ther. vol. 276, (1996), pp. US 2007/O24358.6 A1 Oct. 18, 2007 891-896. Bossuyt, X., et al., J. Hepatol. vol. 25. (1996) pp. 733-738. Related U.S. Application Data Kanai, N., et al., Am. J. Physiol. vol. 270, (1996), pp. F319-F325. (62) Division of application No. 10/736,936, filed on Dec. Kanai, N., et al., Am. J. Physiol. vol. 270, (1996), pp. F326-F331. Kontaxi, M., et al., J. Pharmacol. Exp. Ther. vol. 279, (1996), pp. 16, 2003, now Pat. No. 7,235,375, which is a division 1507-1513. of application No. 09/575,081, filed on May 19, 2000, Li, L., et al., J. Biol. Chem. vol. 273, No. 26, (1998), pp. 16184 now Pat. No. 6,692,934. 16191. (60) Provisional application No. 60/135,081, filed on May Kullak-Ublick, G.A., et al., Gastroenterology vol. 109, No. 4. (1995), pp. 1274-1282. 20, 1999. Kullak-Ublick, G.A., et al., Hepatology vol. 20, No. 2, (1994), pp. 411-416. (51) Int. Cl. Kullak-Ublick, G.A., et al., FEBS Lett., vol. 424, (1998), pp. 173 C07K L/00 (2006.01) 176. C07K I4/00 (2006.01) Wolkoff, A.W., Semin. Liver Dis... vol. 16, No. 2, (1996), pp. 121-127. CI2P 2/06 (2006.01) Abe, T., et al., J. Biol. Chem., vol. 273, No. 35 (1998), pp. 22395 CI2P 2L/04 (2006.01) 22401. (52) U.S. Cl...... 530/350; 435/69. 1; 435/69.7 Primary Examiner Olga NChemyshev (58) Field of Classification Search ...... None See application file for complete search history. (74) Attorney, Agent, or Firm Todd Spalding (56) References Cited (57) ABSTRACT FOREIGN PATENT DOCUMENTS The current invention discloses nucleic acid and amino acid WO WO9517.905 7, 1995 sequences for novel organic anion transfer proteins WO WO973.1111 8, 1997 (“OATPs). The invention encompasses the OATPs described WO WO99/O7891 2, 1999 herein, together with vectors containing the cDNA WO WO99, 12952 3, 1999 sequences, host cells containing the vectors and polypeptides having all or part of an OATP. Also encompasses are uses for OTHER PUBLICATIONS OATPs for targeting drugs to specific organs and for modu Chen, et al. "Identification of two hERR2-related novel nuclear lating the concentration of endogenous Substrates. receptors utilizing bioinformatics and inverse PCR”. , vol. 228, pp. 101-109 (1999). 6 Claims, 8 Drawing Sheets U.S. Patent Sep. 14, 2010 Sheet 1 of 8 US 7,795,392 B2

OATP2 OATP-RP Pn K Sm Lv L. P B H B C SiO T PrTy S Pn STYLEB, HESS,

9.5 7.5 4.4

2.4 1.4

FIG. A FIG 1B

OATP-RP2 OATP-RP4

Pn K Sin W L P B H B C SiO PryS Pn K Sin Lv L. P B H B C SiO T PrTyS

9.5 9.5 7.5 7.5 4.4 44 2.4 2.4 14 1.4

F.G. C FC D OATP-RP5 Pn K Smily L. P B H B C Si O T PrTy S

9.5 7.5 4.4

2.4 1.4

FIG. IE Tissue Key H: heart S: spleen B: brain Ty: thymus P: placenta Pr: prostate l: lung : testis LV: liver O: Ovary Sm: Skeleta Si: Small intestine muscle C: Colon K: kidney Bl: peripheral blood leukocytes Pn: pancreas

FIG. 1 (A-E) U.S. Patent Sep. 14, 2010 Sheet 2 of 8 US 7,795,392 B2

Mock O CN OATP2 O

cy cy O C c d (6u/uu/eoud) exedn vigZ

MOCK ? CN OATP2 CD & S & E S S (6u/uufeloud) eyeydn eleIOuOOlney-(H)

DHEAS N () O s pravastatin CC CN

- DHEAS S C O pravastatin

SN 9 O s N d (6u/uufeloud) exednjeoen-LH)

U.S. Patent Sep. 14, 2010 Sheet 5 of 8 US 7,795,392 B2

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U.S. Patent Sep. 14, 2010 Sheet 7 of 8 US 7,795,392 B2

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snstrestrojo sºnstrestrolo US 7,795,392 B2 1. 2 ORGANIC ANON TRANSPORT PROTEINS first cloning and identification of a member of the OATP transporterfamily, namely the rat oatp1. The first cloning and This application is a divisional application of U.S. Ser. No. identification of a OATP was reported in Kullak 10/736,936, filed Dec. 16, 2003, now U.S. Pat. No. 7,235,375, Ublick, G. A., et al., (1995) Gastroenterology, 109:1274 which is a divisional application of U.S. Ser. No. 09/575,081, 1282. Its expression was found in liver, kidney brain and other filed May 19, 2000, now U.S. Pat. No. 6,692,934, which organs. The authors concluded, based on Substrate specifici claims priority from provisional U.S. Application Ser. No. ties, that it was not the human orthologue of rat oatp1. 60/135,081, filed May 20, 1999, all of which are incorporated Substrate specificities of rat oatp1 are discussed in Kullak herein by reference in their entirety. Ublick, G. A. et al., (1994) Hepatology, 20:411-416, while 10 substrate specificities of human OATP are discussed in FIELD OF THE INVENTION Bossuyt, X., et al., (1996).J. Hepatol., 25:733–738. The invention claims isolated nucleic acid encoding all or Data was later discovered showing that rat oatp1 is a portion of novel members of the organic anion transport involved in the transport of steroids (Bossuyt, X., et al., (“OATP) designated OATP2, OATP-RP1, OATP (1996) J. Pharmacol. Exp. Ther, 276:891-896), and that 15 human OATP acts as a transporter for the psychoactive hor RP2, OATP-RP3, OATP-RP4 and OATP-RP5. Also claimed mone DHEAS (Kullak-Ublick, G. A., et al., (1998) FEBS are vectors containing the nucleic acid sequences, host cells Lett., 424:173-176). For a review of the OATP family and containing the vectors and polypeptides having all or part of organic anoin transport in the liver, see Wolkoff, A.W., (1996) the amino acid sequence of OATP2, OATP-RP 1, OATP-RP2, Semin. Liver Dis., 16:121-127. OATP-RP3, OATP-RP4 and OATP-RP5. Tissue expression A third rat OATP isoform that was shown to transport of the transporter is described as well as some of its substrates. thyroid hormones T3 and T4 was cloned and reported in Abe, Also claimed are uses for these novel OATPs, including for T. et al., (1998).J. Biol. Chem., 273:22395-22401. targeting drugs to specific tissues, for modulating the concen All references cited herein, whether supra or infra, are tration of endogenous Substrates, and for identifying a Sub hereby incorporated by reference in their entirety. strate capable of being transported by a novel OATP of the 25 invention. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION The present invention encompasses novel organic anion The liver functions in the clearance of a large variety of transport proteins (“OATP) and polynucleotides encoding metabolic products, drugs and other Xenobiotics by transport 30 said OATPs. The OATPs disclosed herein are designated ing them across the sinusoidal membrane into the hepatocyte. OATP2, OATP-RP2, OATP-RP3, OATP-RP4, OATP-RP5 Several classes of transport systems have been described that and OATP-RP1. A polynucleotide sequence of each OATP is mediate these processes including the Na+/taurocholate disclosed herein, along with the deduced amino acid cotransporter polypeptide, NTCP, in rat and human liver sequence. The cDNAs encoding the OATPs of the present (Hagenbuch, B., et al. (1991) Proc. Natl. Acad. Sci. USA 35 invention have been deposited with the American Type Cul 88: 10629-33; Hagenbuch, B. et al., (1994) J. Clin. Invest. ture Collection and given Accession Numbers ATCC 207213 93: 1326-31) and a family of organic anion transporting (OATP2), ATCC 207212 (OATP-RP2), ATCC 207209 polypeptides (OATPs) that are principally expressed in liver, (OATP-RP3), ATCC 207210 (OATP-RP4), ATCC 207211 kidney and brain, and transportabroad spectrum of substrates (OATP-RP5), and ATCC 207214 (OATP-RP1). in a sodium-independent manner (Meier, P. J., et al., (1997) 40 The present inventors sequenced the cDNAs encoding the Hepatology 26:1667-77; Wolkoff, A.W., (1996) Semin. Liver novel OATPs and determined the primary sequence of the Dis. 16:121-127). The distribution of this latter family of deduced proteins. Disclosed herein are the nucleic acid transporters in liver, kidney and choroid plexus in the brain is sequence (SEQID NO: 1) and amino acid sequence (SEQID thought to reflect common physiological requirements of NO:2) of OATP2: the nucleic acid sequence (SEQID NO:3) these organs for the clearance of a multitide of organic anions. 45 and amino acid sequence (SEQID NO:4) of OATP-RP2; the There are three OATP isoforms in the rat: roatp1 (Jacquemin, nucleic acid sequence (SEQ ID NO:5) and amino acid E., et al., (1994) Proc. Natl. AcadSci, USA 91:133-37); roatp2 sequence (SEQ ID NO:6) of OATP-RP3; the nucleic acid (Noe, B. A., et al., (1997) Proc. Natl. Acad. Sci. USA sequence (SEQID NO:7) and amino acid sequence (SEQID 94:10346–50; and roatp3 (Abe, T., et al., (1998).J. Biol. Chem. NO:8) of OATP-RP4; the nucleic acid sequence (SEQ ID 273:11395-401). In addition to bile acids, OATPs are known 50 NO:9) and amino acid sequence (SEQID NO:10) of OATP to transport a variety of other compounds. These include, RP5; and the nucleic acid sequence (SEQ ID NO:11) and depending on the transporter, unconjugated and conjugated amino acid sequence (SEQID NO:12) of OATP-RP 1. steroids Such as estrone Sulfate, estradiol-17B-glucuronide, The OATPs of the present invention can be produced by: aldosterone, and cardiac glycosides (Boussuyt, X., et al., (1) inserting the cDNA of a disclosed OATP into an appro (1996) J. Pharmacol. Exp. Ther: 276:891-6: Boussuyt, X. 55 priate expression vector; (2) transfecting the expression vec (1996).J. Hepatol. 25.733-8: Kanai, N., et al., (1996) Am. J. tor into an appropriate transfection host(s); (3) growing the Physiol. 270:F319-F325; Kanai, N., et al., (1996) Am. J. transfected host(s) in appropriate culture media; and (4) Physiol. 270:F326-F331; Noe, B.A., et al., (1997) Proc. Natl. assaying the transport activity in the transfected cells. Acad. Sci. USA 94:10346–50). Bromosulfophthalien (Jac The present invention therefore provides a purified and quemin, E., et al., (1994) Proc. Natl. Acad. Sci. USA 91:133 60 isolated nucleic acid molecule, preferably a DNA molecule, 7); mycotoxin (Kontaxi, M., et al., (1996).J. Pharmacol. Exp. having a sequence which codes for an OATP, or an oligo Ther. 279:1507-13); leukotriene C (Li, L., et al., (1998).J. nucleotide fragment of the nucleic acid molecule which is Biol. Chem. 273:16184-91); and thyroid hormone (Abe, T., et unique to an OATP of the invention. In a preferred embodi al., (1998) J. Biol. Chem. 273:11395) are additional sub ment of the invention, the purified and isolated nucleic acid Strates. 65 molecule has the sequence as shown in SEQ ID NO:1 Several proteins have been identified. Jacquemin.E., et al., (OATP2). In another preferred embodiment, the purified and (1994) Proc. Natl. Acad. Sci. U.S.A., 91:133-137 reported the isolated nucleic acid molecule has the sequence as shown in US 7,795,392 B2 3 4 SEQ ID NO:3 (OATP-RP2). In still another preferred under stringent conditions with the polynucleotides of the embodiment the purified and isolated nucleic acid molecule present invention may be, for example, allelic variants of the has the sequence as shown in SEQID NO:5 (OATP-RP3). In disclosed DNA sequences, or may be derived from other still another preferred embodiment of the present invention Sources. General techniques of nucleic acid hybridization are the purified and isolated nucleic acid molecule has the nucle 5 disclosed by Sambrook et al., “Molecular Cloning: A Labo otide sequence as shown in SEQ ID NO:7 (OATP-RP4). In ratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, still another preferred embodiment the purified and isolated Cold Spring Harbor, N.Y. (1984); and by Haymes et al., nucleic acid molecule has the sequence as shown in SEQID “Nucleic Acid Hybridization: A Practical Approach, IRL NO:9 (OATP-RP5). In still another preferred embodiment of Press, Washington, D.C. (1985), which references are incor the present invention the purified and isolated nucleic acid 10 porated herein by reference. molecule has the nucleotide sequence as shown in SEQ ID The present invention provides in another embodiment: (a) NO:11 (OATP-RP1). an isolated and purified nucleic acid molecule comprising a The invention also contemplates a double stranded nucleic sequence encoding all or a portion of a protein having the acid molecule comprising a nucleic acid molecule of the amino acid sequence as shown in SEQ ID NO:4 (OATP invention or an oligonucleotide fragment thereof hydrogen 15 RP2); (b) nucleic acid sequences complementary to (a); (c) bonded to a complementary nucleotide base sequence. nucleic acid sequences which are at least 80%, more prefer The terms "isolated and purified nucleic acid, “isolated ably at least 90%, more preferably at least 95%, and most and purified polynucleotide”, “substantially pure nucleic preferably at least 98% sequence identity to (a); or (d) a acid', and “substantially pure polynucleotide', e.g., Substan fragment of (a) or (b) that is at least 18 bases and which will tially pure DNA, refer to a nucleic acid molecule which is one hybridize to (a) or (b) under Stringent conditions. or both of the following: (1) not immediately contiguous with The present invention provides in another embodiment: (a) either one or both of the sequences, e.g., coding sequences, an isolated and purified nucleic acid molecule comprising a with which it is immediately contiguous (i.e., one at the 5' end sequence encoding all or a portion of a protein having the and one at the 3'end) in the naturally occurring genome of the amino acid sequence as shown in SEQ ID NO:6 (OATP organism from which the nucleic acid is derived; or (2) which 25 RP3); (b) nucleic acid sequences complementary to (a); (c) is substantially free of a nucleic acid sequence with which it nucleic acid sequences which are at least 80%, more prefer occurs in the organism from which the nucleic acid is derived. ably at least 90%, more preferably at least 95%, and most The term includes, for example, a recombinant DNA which is preferably at least 98% sequence identity to (a); or (d) a incorporated into a vector, e.g., into an autonomously repli fragment of (a) or (b) that is at least 18 bases and which will cating plasmid or virus, or into the genomic DNA of a 30 hybridize to (a) or (b) under Stringent conditions. prokaryote or eukaryote, or which exists as a separate mol The present invention provides in another embodiment: (a) ecule (e.g., a cDNA or a genomic DNA fragment produced by an isolated and purified nucleic acid molecule comprising a PCR or restriction endonuclease treatment) independent of sequence encoding all or a portion of a protein having the other DNA sequences. Substantially pure or isolated and amino acid sequence as shown in SEQ ID NO:8 (OATP purified DNA also includes a recombinant DNA which is part 35 RP4); (b) nucleic acid sequences complementary to (a); (c) of a hybrid gene encoding additional OATP sequence. nucleic acid sequences which are at least 80%, more prefer The present invention provides in one embodiment: (a) an ably at least 90%, more preferably at least 95%, and most isolated and purified nucleic acid molecule comprising a preferably at least 98% sequence identity to (a); or (d) a sequence encoding all or a portion of a protein having the fragment of (a) or (b) that is at least 18 bases and which will amino acid sequence as shown in SEQID NO:2 (OATP2); (b) 40 nucleic acid sequences complementary to (a); (c) nucleic acid hybridize to (a) or (b) under Stringent conditions. sequences which exhibitat least 80%, more preferably at least The present invention provides in another embodiment: (a) 90%, more preferably at least 95%, and most preferably at an isolated and purified nucleic acid molecule comprising a least 98% sequence identity to (a); or (d) a fragment of (a) or sequence encoding all or a portion of a protein having the (b) that is at least 18 bases and which will hybridize to (a) or 45 amino acid sequence as shown in SEQ ID NO:10 (OATP (b) under stringent conditions. RP5); (b) nucleic acid sequences complementary to (a); (c) The degree of homology (percent sequence identity) nucleic acid sequences which are at least 80%, more prefer between two sequences may be determined, for example, by ably at least 90%, more preferably at least 95%, and most comparing the two sequences using computer programs com preferably at least 98% sequence identity to (a); or (d) a monly employed for this purpose. One Suitable program is the 50 fragment of (a) or (b) that is at least 18 bases and which will GAP computer program described by Devereux et al., (1984) hybridize to (a) or (b) under Stringent conditions. Nucl. Acids Res. 12:387. The GAP program utilizes the align The present invention provides in another embodiment: (a) ment method of Needleman and Wunsch (1970).J. Mol. Biol. an isolated and purified nucleic acid molecule comprising a 48:433, as revised by Smith and Waterman (1981) Adv. Appl. sequence encoding all or a portion of a protein having the Math. 2:482. Briefly, the GAP program defines percent iden 55 amino acid sequence as shown in SEQ ID NO:12 (OATP tity as the number of aligned symbols (i.e., nucleotides or RP1); (b) nucleic acid sequences complementary to (a); (c) amino acids) which are identical, divided by the total number nucleic acid sequences which are at least 80%, more prefer of symbols in the shorter of the two sequences. ably at least 90%, more preferably at least 95%, and most As used herein the term "stringent conditions' encom preferably at least 98% sequence identity to (a); or (d) a passes conditions known in the art under which a nucleotide 60 fragment of (a) or (b) that is at least 18 bases and which will sequence will hybridize to: (a) an isolated and purified nucleic hybridize to (a) or (b) under Stringent conditions. acid molecule comprising a sequence encoding a protein The present invention also provides: (a) a purified and having the amino acid sequence as shown herein, or to (b) a isolated nucleic acid molecule comprising a sequence as nucleic acid sequence complementary to (a). Screening poly shown in SEQID NO: 1 (OATP2); (b) nucleic acid sequences nucleotides under stringent conditions may be carried out 65 complementary to (a); (c) nucleic acid sequences having at according to the method described in Nature, 313:402-404 least 80%, more preferably at least 90%, more preferably at (1985). Polynucleotide sequences capable of hybridizing least 95%, and most preferably at least 98% sequence identity US 7,795,392 B2 5 6 to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and non-human primate cells, or pig cells. In preferred embodi which will hybridize to (a) or (b) under stringent conditions. ments, the cell or cells include an OATP transgene, e.g., a The present invention further provides: (a) a purified and heterologous form of an OATP gene, e.g., a gene derived from isolated nucleic acid molecule comprising a sequence as (in the case of a non-human cell). The OATP trans shown in SEQ ID NO:3 (OATP-RP2); (b) nucleic acid gene can be misexpressed, e.g., overexpressed or underex sequences complementary to (a); (c) nucleic acid sequences pressed. In other preferred embodiments, the cell or cells having at least 80%, more preferably at least 90%, more include a gene which misexpresses an endogenous OATP preferably at least 95%, and most preferably at least 98% gene, e.g., a gene that expression of which is disrupted, e.g.,. sequence identity to (a); or (d) a fragment of (a) or (b) that is a knockout. Such cells can serve as a model for studying at least 18 bases and which will hybridize to (a) or (b) under 10 disorders which are related to mutated or misexpressed OATP stringent conditions. alleles for use in drug screening. The present invention further provides: (a) a purified and Still further, the invention provides plasmids which com isolated nucleic acid molecule comprising a sequence as prise the nucleic acid molecules of the invention. Also encom shown in SEQ ID NO:5 (OATP-RP3); (b) nucleic acid passed within the invention are vectors comprising the sequences complementary to (a); (c) nucleic acid sequences 15 nucleic acid sequences disclosed herein, as well as host cells having at least 80%, more preferably at least 90%, more comprising said vectors. preferably at least 95%, and most preferably at least 98% The present invention also includes a novel OATP of the sequence identity to (a); or (d) a fragment of (a) or (b) that is present invention, or an active part thereof. A biologically at least 18 bases and which will hybridize to (a) or (b) under competent or active form of the protein or part thereof is also stringent conditions. referred to herein as an “active OATP or part thereof. The present invention further provides: (a) a purified and The invention further contemplates antibodies having isolated nucleic acid molecule comprising a sequence as specificity against an epitope of an OATP of the present shown in SEQ ID NO:7 (OATP-RP4); (b) nucleic acid invention or part of the protein. These antibodies may be sequences complementary to (a); (c) nucleic acid sequences polyclonal or monoclonal. The antibodies may be labeled having at least 80%, more preferably at least 90%, more 25 with a detectable substance and they may be used, for preferably at least 95%, and most preferably at least 98% example, to detect a novel OATP of the invention in tissue and sequence identity to (a); or (d) a fragment of (a) or (b) that is cells. Additionally, the antibodies of the present invention, or at least 18 bases and which will hybridize to (a) or (b) under portions thereof, may be used to make targeted antibodies that stringent conditions. destroy OATP expressing cells (e.g., antibody-toxin fusion The present invention further provides: (a) a purified and 30 isolated nucleic acid molecule comprising a sequence as proteins, or radiolabelled antibodies). shown in SEQ ID NO:9 (OATP-RP5); (b) nucleic acid The invention also permits the construction of nucleotide sequences complementary to (a); (c) nucleic acid sequences probes which encode part or all of a novel OATP protein of the having at least 80%, more preferably at least 90%, more invention or a part of the protein. Thus, the invention also preferably at least 95%, and most preferably at least 98% 35 relates to a probe comprising a nucleotide sequence coding sequence identity to (a); or (d) a fragment of (a) or (b) that is for a protein, which displays the properties of a novel OATP at least 18 bases and which will hybridize to (a) or (b) under of the invention or a peptide unique to the protein. The probe stringent conditions. may be labeled, for example, with a detectable (e.g., radioac The present invention further provides: (a) a purified and tive) substance and it may be used to select from a mixture of isolated nucleic acid molecule comprising a sequence as 40 nucleotide sequences a nucleotide sequence coding for a pro shown in SEQ ID NO:11 (OATP-RP1); (b) nucleic acid tein which displays the properties of a novel OATP of the sequences complementary to (a); (c) nucleic acid sequences invention. having at least 80%, more preferably at least 90%, more The present invention also provides a transgenic non-hu preferably at least 95%, and most preferably at least 98% man animal (e.g., a rodent, e.g., a mouse or a rat, a rabbit or a sequence identity to (a); or (d) a fragment of (a) or (b) that is 45 pig) or embryo all of whose germ cells and Somatic cells at least 18 bases and which will hybridize to (a) or (b) under contain a recombinant molecule of the invention, preferably a stringent conditions. recombinant molecule comprising a nucleic acid molecule of The present invention additionally covers polynucleotides the present invention encoding an OATP of the invention or and amino acid sequences of the present invention having one part thereof. The recombinant molecule may comprise a or more structural mutations including replacement, deletion 50 nucleic acid sequence encoding an OATP of the present or insertion mutations. For example, a signal peptide may be invention with a structural mutation, or may comprise a deleted, or conservative amino acid Substitutions may be nucleic acid sequence encoding an OATP of the invention or made to generate a protein that is still biologically competent part thereofandone or more regulatory elements which differ or active. from the regulatory elements that drive expression of the The invention further contemplates a recombinant mol 55 native protein. In another preferred embodiment, the animal ecule comprising a nucleic acid molecule of the present has an OATP gene which is misexpressed or not expressed, invention or an oligonucleotide fragment thereof and an e.g., a knockout. Such transgenic animals can serve as a expression control sequence operatively linked to the nucleic model for studying disorders that are related to mutated or acid molecule or oligonucleotide fragment. A transformant misexpressed OATPs of the present invention. host cell including a recombinant molecule of the invention is 60 The invention still further provides a method for identify also provided. ing a substance which is capable of binding a novel OATP of In another aspect, the invention features a cell or purified the invention, comprising reacting a novel OATP of the inven preparation of cells which include a novel gene encoding an tion or part of the protein under conditions which permit the OATP of the present invention, or which otherwise misex formation of a complex between the Substance and a novel presses a gene encoding an OATP of the present invention. 65 OATP protein or part of the protein, and assaying for sub The cell preparation can consist of human or non-human stance-OATP complexes, for free substance, for non-com cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, plexed OATP, or for activation of an OATP. US 7,795,392 B2 7 8 An embodiment of the invention provides a method for OATP protein of the present invention, and agonists and/or identifying Substrates which are capable of binding to a novel antagonists of the novel OATPs as described above. OATP protein of the invention, isoforms thereof, or part of the protein, said method comprising reacting a novel OATP pro BRIEF DESCRIPTION OF THE FIGURES tein of the invention, isoforms thereof, or part of the protein, 5 with at least one substrate which potentially is capable of FIG. 1 is a Northern blot showing the mRNA tissue distri binding to the protein, isoform, or part of the protein, under bution of OATP2, OATP-RP1, OATP-RP2, OATP-RP4, and conditions which permit the formation of substrate-trans OATP-RP5. The tissues corresponding to the abbreviations porter protein complexes, and assaying for Substrate-trans above the lanes are indicated below. porter protein complexes, for free Substrate, for non-com 10 FIG.2 shows that OATP2 transports pravastatin, dehydroe plexed OATP protein, or for activation of an OATP. In a piandosterone sulfate (DHEAS), taurocholate and thyroid preferred embodiment of the method, substrates are identified hormone (T). FIG. 2A shows specific uptake of H-pravas which are capable of binding to and being transported by a tatin and H-DHEAS. FIG. 2B shows specific uptake of novel OATP protein of the invention, isoforms thereof, or part H-taurocholate. FIG. 2C shows specific uptake of 125II of the protein. 15 thyroid hormone (T4). The uptake of radiolabeled substrate The invention also provides methods for screening poten for 5 minutes into cells transfected with pCEPOATP-RP1 or tially useful pharmacological agonists or antagonists of the empty vector (MOCK) was determined in the absence (solid OATPs of the present invention. The method comprises test bars) and presence (open bars) of excess unlabeled Substrate. ing potential agents by adding the agent to be tested to a cell FIG.3 shows a sequence alignment of OATP family mem expressing a novel OATP of the present invention in the bers. The protein sequences of human OATP2 (SEQ ID presence of a compound known to be transported by an OATP NO:2), OATP-RP1 (SEQ ID NO:12), OATP-RP2 (SEQ ID of the invention, and measuring the augmentation or inhibi NO:4), OATP-RP3 (SEQ ID NO:6), OATP-RP4 (SEQ ID tion of transport of the known compound. NO:8), and OATP-RP5 (SEQID NO:10) are aligned with the An OATP of the present invention is also useful to identify following other known OATP family members: roatp2 (SEQ compounds that may be transported into an organ, e.g., the 25 ID NO:23), roatp3 (SEQ ID NO:24), rOAT-K1 (SEQ ID liver. Compounds that are found to be actively transported NO:25), roatp1 (SEQID NO:26), hCATP (SEQID NO:27); into the liver are useful as carriers for other therapeutics and hPGT (SEQ ID NO:28). Also shown is a consensus targeting the liver. sequence (SEQID NO:29) in bold. A consensus is indicated Also included within the scope of the present invention is a if at least 6 out of the 12 sequences are identical at a given composition which includes an OATP of the present inven 30 position. A residue is capitalized if it agrees with the consen tion, a fragment thereof (or a nucleic acid encoding said SS. OATP or fragment thereof) and one or more additional com ponents, e.g., a carrier, diluent or solvent. The additional DETAILED DESCRIPTION OF THE INVENTION component can be one that renders the composition useful for in vitro, in Vivo, pharmaceutical or veterinary use. 35 The following definitions apply to the terms used through Encompassed within the present invention are agonists and out this specification, unless otherwise defined in specific antagonists of an OATP of the present invention. Pharmaco instances: logical agonists or antagonists are useful to increase or "cloning isolation of a particular gene from genetic decrease the flow of compounds transported by an OATP of 40 material, for example a genome, genomic library, or cDNA the present invention. Said agonists and/or antagonists of the library into a plasmid or other vector; present invention are preferably administered with an accept "coding region' the region of a nucleic acid sequence able carrier, diluent or solvent. that codes for an active protein; In another aspect, the present invention relates to a method “OATP organic anion transport protein; of treating a mammal, e.g., a human, at risk for a disorder, e.g., 45 'stringent conditions” (as used concerning nucleic acid a disorder characterized by aberrant or unwanted level or hybridization)—Southern blotting washed in 0.1xSSC and biological activity of an OATP of the present invention. Addi 0.1% SDS at a temperature of at least about 65° C. See tionally, encompassed within the invention is a method of Maniatis et al., Molecular Cloning: A Laboratory Manual, treating a mammal, e.g., a human, at risk for disorders of the Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. liver. Since OATP2 is expressed exclusively in the liver, com 50 pounds that are optimized for OATP2 are useful to target (1982); one skilled in the relevant art would recognize that hepatic delivery. These compounds in themselves may be less stringent conditions (e.g., 1x or 2xSSC, 0.1% SDS) may useful therapeutics, or may be useful to chaperone other be employed in using the novel sequences disclosed herein to therapeutic compounds to the liver. In addition, blocking identify nucleic acid sequences encoding novel OATPs. “Northern blotting a method of identifying particular OATP2-compound interactions could provide benefit by 55 decreasing its first-pass extraction by the liver and, thus, RNA fragments by hybridization with a complementary increasing plasma concentrations and prolonging the sys nucleic acid, typically a cDNA or an oligonucleotide; temic half-life of a drug. “open reading frame' or “ORF' a DNA sequence con Also within the scope of the present invention are fusion taining a series of nucleotide triplets coding for amino acids proteins comprising all or a portion of an OATP of the present 60 and lacking any termination codes; invention. "plasmid' -cytoplasmic, autonomously replicating DNA The primary object of the present invention is the identifi elements found in microorganisms; cation of new human OATPs, as identified by the nucleic acid “promoter a region on DNA at which RNA polymerase and amino acid sequences disclosed herein. Additional binds and initiates transcription; and objects of the invention are the methods of using the cDNA, 65 “Southern blotting a method of identifying particular the OATP proteins, monoclonal antibodies specific for the DNA fragments by hybridization with a complementary novel OATPs, fusion proteins comprising a portion of the nucleic acid, typically a cDNA or an oligonucleotide; US 7,795,392 B2 10 “transport'—the movement of a Substance across a bio compound into an organ, e.g., the liver, or an agent or com logical membrane as determined by measuring the redistri pound that decreases the rate or amount of transport of a bution of Such a Substance across the membrane upon expo compound into an organ. The term “negative modulator Sure to a transporter. refers to a compound that is joined to a second compound to For definitions of other terms in this specification, see F. prevent the second compounds transport into or out of cells. Sherman et al., Laboratory Course Manual for Methods in The term “carrier as used herein refers to an agent or com Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring pound that is transported by an OATP of the present invention Harbor, N.Y. (1987) and Lewin, B., IV, Oxford Uni and that is capable of being joined to or associated with versity Press, Oxford (1990). For the definitions of abbrevia another compound to chaperone that other compound into an tions, see Aldrichimica Acta, Vol. 17, No. 1 (1984). 10 organ, e.g., the liver. A carrier includes an agent that is used to Use and Utility transport a compound into an organ that is otherwise not The amino acid sequences of the novel organic anion trans transported into said organ, and includes an agent that port proteins of the present invention are aligned with known increases the transport of a compound into an organ that is transporters of this family in FIG. 3. The degree of sequence 15 capable of being transported by an OATP. homology between the sequences of the present invention and One can administer OATP modulators and carriers to vari known organic anion transporters indicates that the proteins ous mammalian species, such as monkeys, dogs, cats, mice, of the present invention are organic anion transporters. rats, humans, etc. By known methods, persons skilled in the It is believed by those skilled in the art that OATP proteins pharmaceutical art can incorporate OATP modulators and may be involved in the transport of compounds into the liver. carriers in a conventional systemic dosage form, such as a Persons of ordinary skill in the art can use the OATP proteins tablet, capsule, elixir or injectable formulation. The above of the present invention to assay for agents that may increase dosage forms will also include any necessary physiologically or decrease the rate of transport of compounds into the liver, acceptable carrier material, excipient, lubricant, buffer, anti or for compounds that are transported by the OATPs of the bacterial, bulking agent (Such as mannitol), anti-oxidants present invention that are useful as carriers for other com 25 (ascorbic acid or sodium bisulfite) or the like. pounds that are desired to be carried to a specific organ (e.g., Process of Preparation the liver). In General Therefore, agents that increase or decrease the rate of Sub strate transport by the OATPs of the present invention, or This specification describes the cloning and functional expression of full-length human cDNA clones of OATPs. agents identified as carriers, are useful in the treatment of liver 30 disease. preferably the nucleic acid sequence of OATP2 (SEQ ID Because some of the OATPs of the present invention are NO:1), the amino acid sequence of OATP2 (SEQ ID NO:2), organ specific/selective (e.g., OATP2 liver; OATP-RP4— the nucleic acid sequence of OATP-RP2 (SEQID NO:3), the amino acid sequence of OATP-RP2 (SEQ ID NO:4), the heart and skeletal muscle, and OATP-RP5 brain and testis), nucleic acid sequence of OATP-RP3 (SEQ ID NO:5), the compound specificity is built into any specific Substrate of 35 these OATPs and into molecular carriers transported by these amino acid sequence of OATP-RP3 (SEQ ID NO:6), the OATPs. An agent transported by the above OATPs of the nucleic acid sequence of OATP-RP4 (SEQ ID NO:7), the present invention would thus be delivered to the tissues in amino acid sequence of OATP-RP4 (SEQ ID NO:8), the which they are expressed and not to tissues lacking the above nucleic acid sequence of OATP-RP5 (SEQ ID NO:9), the amino acid sequence of OATP-RP5 (SEQ ID NO:10), the OATPs, thereby achieving tissue specific targeting. 40 The OATP nucleic acids of the present invention, or anti nucleic acid sequence of OATP-RP1 (SEQ ID NO:11), and sense nucleic acids, may be useful therapeutic or diagnostic the amino acid sequence of OATP-RP1 (SEQ ID NO:12). agents. For Such genetherapy, the nucleic acids may be incor DNA clones comprising nucleotide sequences encoding porated into vectors and/or formulated as described below the OATPs described above were deposited with the Ameri and in further detail in the art. 45 can Type Culture Collection (ATCC) (10801 University The present invention also provides a basis for diagnostic Blvd., Manassas, Va. 20110-2209) on Apr. 20, 1999, and genetic screens for predicting response to drugs. At least one given the following ATCC Accession Numbers: 207209 of the transporters disclosed and claimed herein is a trans (OATP-RP3), 207210 (OATP-RP4), 207211 (OATP-RP5), porter of a known drug (i.e., OATP2 transports pravastatin 207212 (OATP-RP2), 207213 (OATP2), and 207214 (OATP into hepatocytes). Other transporters disclosed herein may 50 RP1). The deposit(s) referred to herein will be maintained similarly transport additional drugs into tissues. Persons under the terms of the Budapest Treaty on the International skilled in the art can: (1) Screen the transporter genes for Recognition of the Deposit of Microorganisms for purposes allelic variants (genotypes) in the general population by vari of Patent Procedure. These deposits are provided merely as ous sequencing methods; and (2) determine the association of convenience to those of skill in the art and are not an admis these transporter genotypes in patients with response to the 55 sion that a deposit is required under 35 U.S.C. S 112. The transported drug in clinical trials. Particular allelic variants sequence of the polynucleotides contained in the deposited may be more or less effective in transporting a drug, which materials, as well as the amino acid sequence of the of the would be related to drug efficacy. Thus, genotyping of the polypeptides encoded thereby, are incorporated herein by claimed transporters could form the basis of a clinical diag reference and are controlling in the event of any conflict with nostic test to predict a patient's response to drug therapy. 60 any description of sequences herein. A license may be Persons skilled in the art can use the polypeptides and required to make, use or sell the deposited materials, and no nucleic acids of this invention to prepare vectors, cells or cell Such license is hereby granted. lines, and antibodies. All of these are useful in assays for Nucleic Acids identification of OATP positive and negative modulators (i.e., With the disclosed OATP gene sequences in hand, one agonists and/or antagonists) and OATP carriers. The term 65 skilled in the art can obtain OATP nucleic acids of this inven “positive modulator as used herein refers to an agent or tion by known methods. Such methods include: (1) Southern compound that increases the rate or amount of transport of a and Northern blotting; (2) Western immunoblotting; (3)

US 7,795,392 B2 40

- Continued ATC GAC AAG GCC TGT CTG CTG TGG CAG GAC CAG TGT GGC CAG 2098 I D K. A C L L W Q D Q C G Q

CAG GGC. TCC TGC TTG. GTG TAC CAG AAT TCG GCC ATG AGC CGC 214 O Q G. S C L V Y O N S A M S R

TAC ATA CTC ATC ATG GGG CTC CTG TAC AAG GTG. CTG GGC GTC 2182 Y I L I M G L L Y K W L G W

CTC TTC TTT GCC ATA GCC TGC TTC TTA. TAC AAG CCC CTG TCG 2224 L. F. F. A. I. A C F L Y K P L S

GAG TCT, TCA GAT GGC CTG GAA ACT TOT CTG CCC AGC CAG TCC 2266 E S S D G L E T C L P S Q S

TCA GCC CCT GAC AGT, GCC ACA GAT AGC CAG CTC CAG AGC AGC 23O8 S A P D S A T D S Q L Q S S

GTC TGA, CCACCGCCCG CGCCCACCCG GCCACGGCGG, GCACTCAGCA 2354 W k

TTTCCTGATG ACAGAACAGT, GCCGTTGGGT GATGCAATCA CACGGGAACT 24O4.

TCTATTTGAC CTGCAACCTT CTACTTAACC TGTGGTTTAA AGTCGGCTGT 2.454

GACCTCCTGT CCCCAGAGCT GTACGGCCCT, GCAGTGGGTG. GGAGGAACTT 2504

GCATAAATAT, ATATTTATGG ACACACAGTT T.GCATCAGAA CGTGTTTATA 2554

GAATGTGTTT TATACCCGAT CGTGTGTGGT GTGCGTGAGG ACAAACTCCG 2604

CAGGGGCTGT GAATCCCACT GGGAGGGCGG CGGGCCTGCA GCCCGAGGAA 2654

GGCTTGTGTG TCCTCAGTTA AAACTGTGCA. TATCGAAATA TATTTTGTTA 2704

TTTAAGCCTG CGAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2754

AAAAAAAAA 2763

Persons skilled in the art can also modify the nucleic acids 35 Expression vectors are usually plasmids, but the invention coding for the OATPs of the present invention to prepare includes other vector forms that serve equivalent functions useful mutations. For example, one may modify the sequence and become known in the art Subsequently hereto. A person to provide additional restriction endonuclease recognition skilled in the art might also stably integrate a sequence encod sites in the nucleic acid. Such mutations may be silent or may ing an OATP into the of an appropriate host cell. change the amino acid encoded by the mutated codon. One 40 Expression vectors typically contain regulatory elements can prepare these modified nucleic acids, for example, by capable of affecting expression of an OATP. These regulatory mutating the nucleic acid coding for an OATP of the present elements can be heterologous or native OATP elements. Typi invention to result in deletion, Substitution, insertion, inver cally, a vector contains an origin of replication, a promoter, sion or addition of one or more amino acids in the encoded and a transcription termination sequence. The vector may polypeptide. For methods of site-directed mutagenesis, see 45 also include other regulatory sequences, including mRNA Taylor, J. W. et al. (1985), Nucl. Acids Res. 13, 8749-64 and stability sequences, which provide for stability of the expres Kunkel, J. A. (1985), Proc. Natl. Acad. Sci. USA 82: 482-92. sion product; secretory leader sequences, which provide for In addition, kits for site-directed mutagenesis are available secretion of the expression product; environmental feedback from commercial vendors (e.g., BioRad Laboratories, Rich sequences, which allow expression of the structural gene to be mond, Calif.; Amersham Corp., Arlington Heights, Ill.). For 50 modulated (e.g., by the presence or absence of nutrients or disruption, deletion and truncation methods, see Sayers, J. R. other inducers in the growth medium); marking sequences, et al. (1988), Nucl. Acids Res. 16: 791-800. which are capable of providing phenotypic selection in trans This invention also comprises modified nucleic acids, formed host cells; restriction sites, which provide sites for including (1) alternative splice exon variants; (2) allelic vari cleavage by restriction endonucleases; and sequences which ants; and (3) chimeric proteins in which the fusion construct 55 allow expression in various types of hosts, including prokary comprises an OATP or fragment thereof. Such modified otes, yeasts, fungi, plants and higher eukaryotes. nucleic acids can be obtained by persons of ordinary skill in An expression vector of this invention is at least capable of the art when armed with the present disclosure. directing the replication, and preferably the expression, of the Expression Vectors nucleic acids and protein of this invention. Suitable origins of This invention further concerns expression vectors com 60 replication include, for example, the Col E1, the SV40 viral, prising a nucleotide sequence encoding an OATP of the Epstein Barr viral, and the M13 origins of replication. Suit present invention. Preferably, the expression vectors com able promoters include, for example, the cytomegalovirus prise all or a portion of the nucleic acid sequence as shown in promoter, the lacZ promoter, the gal10 promoter and the SEQID NO:1, SEQID NO:3, SEQID NO:5, SEQID NO:7, Autographa Californica multiple nuclear polyhedrosis virus SEQ ID NO:9, or SEQID NO:11; preferred is a nucleotide 65 (AcMNPV) polyhedral promoter. Suitable termination sequence encoding an OATP as shown above (i.e., the coding sequences include, for example, the bovine growth hormone, region). SV40, lacz and AcMNPV polyhedral polyadenylation sig US 7,795,392 B2 41 42 nals. Examples of selectable markers include neomycin, this invention. Neither will all host cells function equally well ampicillin, and hygromycin resistance and the like. with the same expression system. However, one of ordinary Persons skilled in the art may insert DNA encoding An skill in the art may make a selection among expression vec OATP of the present invention into several commercially tors, DNA regulatory sequences, and host cells using the available vectors. Examples include vectors compatible with guidance provided herein without undue experimentation and mammalian cells, such as pcDNA3 or pCEP4; baculovirus without departing from the scope of the invention. vectors such as pBlueBac; prokaryotic vectors such as Polypeptides pcDNA2; and yeast vectors such as pYes2. For vector modi This invention further concerns polypeptides comprising fication techniques, see Sambrook et al. (1989), Molecular all or a portion of the amino acid sequences of OATPs of the Cloning: A Laboratory Manual, Second Edition, Cold Spring 10 present invention. The inventors prefer polypeptides com Harbor Laboratory, Cold Spring Harbor, N.Y. prising all or a portion of the amino acid sequences shown as Host Cells in SEQID NO:2 (OATP2), SEQID NO:4 (OATP-RP2), SEQ This invention additionally concerns host cells containing ID NO:6 (OATP-RP3), SEQID NO:8 (OATP-RP4), SEQID an expression vector that comprises a sequence encoding an NO:10 (OATP-RP5) or SEQID NO:12 (OATP-RP1). Where OATP preferably the OATP2, OATP-RP2, OATP-RP3, 15 a portion of an OATP of the present invention is used, pref OATP-RP4, OATP-RP5 or OATP-RP1 of the present inven erably the portion exhibits the same biological activity of the tion. The host cells preferably contain an expression vector OATP from which the portion is derived. For example, and which comprises all or part of the DNA sequence having the within the scope of the invention, are polypeptides that com nucleotide sequence substantially as shown in SEQID NO:1, prise all or a portion of OATP2, OATP-RP2, OATP-RP3, SEQID NO:3, SEQID NO:5, SEQID NO:7, SEQID NO:9, OATP-RP4, OATP-RP5 or OATP-RP1 that exhibit transport or SEQ ID NO:11, particularly the coding regions thereof. activity. The portions may contain one or more mutations so Suitable host cells include both prokaryotic cells (e.g., E. coli that the protein(s) fail(s) to exhibit transport activity, but that strains HB10, DH5a, XL1 Blue, Y1090 and JM101) and can be used to Screen for compounds that will modulate or eukaryotic cells (e.g., Spodoptera frugiperda insect cells, bind to the protein or portion thereof. CHO cells, COS-7 cells, HEK 293 cells, human skin fibro 25 Persons having ordinary skill in the art may prepare these blasts, and S. cerevisiae cells). polypeptides by methods known in the art. For example, one Persons skilled in the art may introduce expression vectors may use chemical synthesis, Such as the Solid phase proce into host cells by various methods known in the art. Exem dure described by Houghton et al. (1985), Proc. Natl. Acad. plary methods are transfection by calcium phosphate precipi Sci. 82: 513.1-5. Another method is in vitro translation of tation, electroporation, liposomal fusion, nuclear injection, 30 mRNA. One may also produce the polypeptides in the above and viral or phage infection. One may then culture the host described host cells, which is the preferred method. For cell under conditions permitting expression of large amounts example, one may synthesize DNA comprising all or a por of OATP. tion of SEQID NO:1, SEQID NO:3, SEQID NO:5, SEQID One may identify such modified host cells by any of five NO:7, SEQID NO:9, or SEQID NO:11 by PCR as described general approaches: 35 above, insert the synthesized DNA into an expression vector, (a) DNA-DNA hybridization with probes complementary transform a host cell with the expression vector, and culture to the sequence encoding an OATP (Southern blotting). the host cell to produce the desired polypeptides. (b) detection of marker gene functions, such as thymidine Persons skilled in the art can isolate and purify such kinase activity, resistance to antibiotics, and the like. A polypeptides by any one of several known techniques; for marker gene can be placed in the same plasmid as an OATP 40 example, ion exchange chromatography, gel filtration chro sequence under the regulation of the same or a different matography and affinity chromatography. Such techniques promoter. may require modification of the protein. For example, one (c) detection of mRNA transcripts by hybridization assays may add a histidine tag to the protein to enable purification on (e.g., Northern blotting or a nuclease protection assay using a a nickel column. probe complementary to the RNA sequence). 45 Persons skilled in the art can use the polypeptides of the (d) immunodetection of gene expression (e.g., by Western inventionina wide variety of ways. For example, one may use blotting with antibody to OATP). them to generate polyclonal or monoclonal antibodies. One (e) PCR with primers homologous to expression vector may then use such antibodies for immunodetection (e.g., sequences or sequences encoding OATP. The PCR produces a radioimmunoassay, enzyme immunoassay, or immunocy DNA fragment of predicted length, indicating incorporation 50 tochemistry), immunopurification (e.g., affinity chromatog of the expression system in the host cell. raphy) of polypeptides from various sources, or immuno Persons skilled in the art may determine DNA sequences therapy. by various known methods. See, for example, the dideoxy Persons skilled in the art may make modified OATP chain termination method in Sanger et al. (1977), Proc. Natl. polypeptides by known techniques. Such modifications may Acad. Sci. USA 74:5463-7 and the Maxam-Gilbert method in 55 cause higher or lower activity, permit higher levels of protein Maxam-Gilbert (1977), Proc. Natl. Acad. Sci. USA 74:560-4. production, or simplify purification of the protein. Such One may use the host cells of this invention in a variety of modifications may help identify specific OATP amino acids ways that are now apparent. One may use the cells to Screen involved in binding, which in turn may help rational drug for compounds that bind to or otherwise modulate or regulate design of OATP modulators. One can make amino acid sub the function of an OATP of the present invention, which 60 stitutions based on similarity in polarity, charge, Solubility, would be useful for modulation, for example activation or hydrophobicity, hydrophilicity and/or the amphipathic nature inactivation, of OATP2, OATP-RP2, OATP-RP3, OATP of the residues involved. For example, negatively charged RP4, OATP-RP5 or OATP-RP1 activity; to study signal trans amino acids include aspartic acid and glutamic acid; posi duction mechanisms and protein-protein interactions; and to tively charged amino acids include lysine and arginine; amino prepare OATP for the uses described below. 65 acids with uncharged polar head groups or nonpolar head Not all expression vectors and DNA regulatory sequences groups having similar hydrophilicity values include the fol will function equally well to express the DNA sequences of lowing: leucine, isoleucine, Valine, glycine, alanine; aspar US 7,795,392 B2 43 44 agine, glutamine; serine, threonine; phenylalanine, tyrosine. All such modified polypeptides are included within the scope TABLE 1 - continued of the invention. Preferred analogs include proteins that differ from the Conservative amino acid replacements novel OATPs of the present invention (or biologically active For Amino fragments thereof) by one or more conservative amino acid Acid Code Replace with any of : Substitutions or by one or more non-conservative amino acid Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, substitutions, deletions or insertions which do notabolish the D-His biological activity of the analog. Conservative Substitutions 10 Waline W D-Wall, Lieu, D-Leu, Ile, D-Ile, typically include the substitution of one amino acid for Met, D-Met another with similar characteristics, e.g., Substitutions within the following groups: valine, glycine; glycine, alanine; Other analogs within the invention are those with modifi Valine, isoleucine, leucine; aspartic acid, glutamic acid; cations which increase protein or peptide stability. Such ana asparagine, glutamine; serine, threonine; lysine, arginine: 15 logs may contain, for example, one or more non-peptide and phenylalanine, tyrosine. Other conservative amino acid bonds (which replace the peptide bonds) in the protein or substitutions can be taken from the table below. peptide sequence. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., TABLE 1. D-amino acids or non-naturally occurring or synthetic amino Conservative amino acid replacements acids, e.g., for Yamino acids. The inventors contemplate a number of other variations of For Amino the above-described polypeptides. Such variations include Acid Code Replace with any of: salts and esters of the polypeptides, as well as precursors of Alanine A D-Ala., Gly, beta-Ala., L - Cys, the aforementioned polypeptides (e.g., having N-terminal D-Cys 25 Substituents such as methionine, N-formylmethionine and Arginine R D-Arg, Lys, D-Lys, homo-Arg, leader sequences). The invention includes all such variations. D-homo-Arg, Met, Ile, D-Met, Method for Detecting Nucleic Acids D-Ile, Orn, D- Orn The present invention further concerns a method for detect Asparagine N D-Asn., Asp, D-Asp, Glu, D-Glu, 30 ing nucleic acids encoding OATP proteins. In this method, a Glin, D-Gln person of ordinary skill in the art (a) contacts nucleic acids of unknown sequence with a nucleic acid having a sequence Aspartic Acid D D-Asp, D-Asn., Asn., Glu, D-Glu, complementary to a known coding sequence (e.g., a sequence Glin, D-Gln of at least about 10 nucleotides from, e.g., SEQ ID NO:1, Cysteine C D-Cys, S-Me-Cys, Met, D-Met, 35 SEQID NO:3, SEQID NO:5, SEQID NO:7, SEQID NO:9, Thir D-Thr or SEQ ID NO:11, particularly the coding regions thereof), wherein the latter nucleic acid has a detectable marker; and Glutamine Q D-Gln, ASn D-ASn, Glu, D-Glu, (b) determines the presence of marker bound to any of the Asp, D-Asp nucleic acids of unknown sequence. The presence of bound Glutamic Acid E D-Glu, D-Asp, Asp, Asn., D-ASn, 40 marker indicates the presence of the desired nucleic acids. Glin, D-Gln One can apply this method to detect OATP nucleic acids from Glycine G Ala, D-Ala, Pro, D-Pro, B-Ala., other tissues (which may have different regulatory elements) Acp and nucleic acids from other species (e.g., monkey). Persons of ordinary skill in the art generally know how to Isoleucine I D-Ile, Wall, D-Wall, Lieu, D-Leu, 45 obtain nucleic acids to be analyzed in this method. For Met, D-Met genomic DNA, one can rapidly freeze tissue, crush the tissue Leucine L D-Leu, Wall, D-Wal Met, D-Met into readily digestible pieces, and incubate the crushed tissue in proteinase K and SDS to degrade most cellular proteins. Lysine K D-Lys, Arg, D-Arg, homo-Arg, One can then deproteinize the genomic DNA by successive D-homo-Arg, Met, D-Met, Ile, phenol/chloroform/isoamyl alcohol extractions, recover D-Ile, Orn, D- Orn 50 DNA by ethanol precipitation, dry it and resuspend it in Methionine M D-Met, S-Me-Cys, Ile, D-Ile, buffer. For RNA, one can lyse cultured cells in 4M guani Lieu, D-Leu, Wall, D-Wall dinium solution, draw the lysate through a 20-gauge needle, Phenylalanine F D-Phe, Tyr, D- Thr, L-Dopa, His, pellet the RNA through a cesium chloride step gradient, and D-His, Trp, D-Trp, Trans-3, 4, 55 remove the supernatant. The pellet should contain purified or 5-phenylproline, cis-3, 4, RNA. or 5-phenylproline The detectable marker may be a radioactive ion linked to Proline P D-Pro L-1-thioazolidine-4- one of the nucleotides of the complementary nucleic acid. carboxylic acid, D- or L-1- Common radioactive labels are P and S, although one oxazolidine-4-carboxylic acid 60 may also use other labels such as biotin. Persons skilled in the Serine S D-Ser Thr, D-Thr allo - Thir, art are aware of various methods to attach the labels to the Met, D-Met, Met (O), D-Met (O), complementary nucleic acid (e.g., the random primer method L-Cys, D- Cys for attachment of 'P or S). Threonine T D-Thr Ser D-Ser allo - Thir, Persons of ordinary skill in the art generally know how to Met, D-Met, Met (O), D-Met (O), 65 carry out Such a method of detecting nucleic acids. For Wall, D-Wall example, one may perform a Southern or northern blot using a radiolabeled OATP complementary oligonucleotide probe. US 7,795,392 B2 45 46 One can then detect hybridization by autoradiography. For example, the novel organic anion transporter disclosed Depending on the marker, one may also use other detection herein, OATP2, represents a potential therapeutic target due methods (e.g., spectrophotometry). to its ability to modulate the cellular uptake and potential Methods for Detecting OATP Modulators and Compounds secretion of a several biologically important organic anions, Transported By the OATPs of the Present Invention including bile acids and the androgen hormone dehydroepi This invention further concerns methods for detecting androsterone sulfate (“DHEAS”). Furthermore, since OATP2 modulators of the OATPs of the present invention, as well as transports at least one drug (i.e. pravastatin), and other mem methods for detecting compounds that are transported by the bers of this family are known to transport a variety of other OATPs of the present invention (e.g., compounds that are 10 xenobiotics, this transporter could be exploited to optimize transported into the liver that may be used as carriers for other the delivery of drugs into liver and away from other tissues. compounds). A screen for OATP modulators entails detecting binding of molecules (e.g., polypeptides, natural products, OATP2 is unique among the OATP family, in that it is the synthetic compounds) in cells expressing OATP protein. only known organic anion transporter that is expressed exclu sively in the liver. Thus, drugs optimized for this transporter Alternatively, a screen for OATP positive modulators and/or 15 negative modulators entails detecting the augmentation and/ could be targeted for hepatic delivery with greater selectivity or inhibition of transport of a known compound. A screen for than with any other known transporter. To generalize this OATP-transported compounds entails detecting the transport approach, it may be possible to identify a small molecule of molecules (e.g., polypeptides, natural products, synthetic “adaptor' that is efficiently recognized and transported by compounds) by an OATP. OATP2 (an OATP2-transported compound) that could be Cloning and sequencing of the OATPs of the present inven appended to other drugs for hepatic targeting even if the tion enables construction of cells useful in screening for natu parent compound is not transported by OATP2. ral products and synthetic compounds that bind to, modulate, Alternatively, if a therapeutic compound is taken up into and/or are transported by OATP activity. A process for detect the liver entirely or substantially by OATP2, one could inhibit ing OATP modulators requires transforming a Suitable vector 25 hepatic clearance and thereby elevate circulating concentra into compatible host cells as described previously herein. One tions, or increase the compounds half-life in the periphery, by treats such transformed cells with test Substances (e.g., Syn adding a functionality to said compound that disallows trans thetic compounds or natural products), and then measures port by OATP2. Likewise, if an endogenous substance uti activity in the presence and absence of the test Substance. 30 lizes OATP2 for liver uptake and clearance from the circula OATP Assay tion, a competitive or non-competitive OATP2 inhibitor could An assay for the measurement of OATP activity is per elevate plasma levels of said substance. As an example, formed as follows: HEK293 cells are plated in Dulbeccos DHEAS is an adrenal androgen that declines with age and on Modified Eagles Medium (DMEM) plus 10% fetal bovine the basis of Some animal data, it has been Suggested that serum plus penecillin and streptomycin, in poly-d-lysine 35 replacement of DHEAS deficiency may stimulate age-related coated dishes and co-transfected with OATP transporter immune deficiencies, increase cognitive function and insulin expression plasmids using Lipofectamine Plus (Life Tech sensitivity, and maintain bone mass. Inhibiting the hepatic nologies, Inc.). The cells and media are assayed for Substrate transport 24 hours later. Alternatively, cell lines engineered to clearance of endogenous DHEAS through blocking its inter stably express OATPs could be plated and assayed directly 40 actions with OATP2 could result in elevated hormone levels without transfection. To measure transport, media is removed in the absence of hormone Supplementation. and monolayers are assayed in triplicate by washing once in With the information provided herein, one skilled in the art serum-free DMEM and adding the same medium containing is able to identify molecules, both naturally occurring and H-substrate alone or in the presence of various concentra synthetic (including therapeutic drugs), that are transported tions of unlabeled test compounds. For OATP2, the H 45 by the OATPs, e.g., OATP2, disclosed herein. OATPs as a substrate could be H-pravastatin, H-taurocholate, or class generally exhibit broad substrate specificity (“polyspe H]-dehydroepiandrosterone sulfate, or 'I'-thyroid hor cific' transporters). Thus, it is anticipated that many addi mone (T4). Monolayers are incubated at room temperature tional substrates of these transporters will be identified. for 5 to 10 minutes depending on the transporter. Then the 50 Gene Therapy cells are rapidly washed once With ice cold DMEM contain Persons skilled in the art can also use sense and antisense ing 5% BSA, twice with DMEM plus 0.1% BSA and once nucleic acid molecules as therapeutic agents for OATP-re with DMEM alone. Cells are lysed in 0.1 N. NaOH and a lated indications. One may construct vectors that direct the fraction of the lysate is used to determine radiolabel incorpo synthesis of the desired DNA or RNA or formulate the nucleic ration by liquid Scintillation counting, and another is used to 55 acid as described in the art. determine protein concentration in the lysate using the Brad Several references describe the usefulness of antisense ford assay with BSA as a standard. The transport activity is molecule. See Toulme and Helene (1988), Gene 72: 51-8; expressed as moles of Substrate transported into cells/mg of Inouye (1988), Gene, 72: 25-34; Uhlmann and Peyman cell protein/minute. 60 (1990), Chemical Reviews 90: 543-584: Biotechnology Drug Targeting Newswatch (Jan. 15, 1996), p. 4: Robertson, Nature Biotech Also included within the present invention is tissue expres nology 15: 209 (1997); Gibbons and Dzau (1996), Science sion of an OATP of the present invention. The OATPs of the 272: 689-93. One can design them based on genomic DNA present invention are also useful for targeting drugs to certain and/or cDNA, 5' and 3’ flanking control regions, other flank organs that express an OATP described herein (e.g., the liver), 65 ing sequences, intron sequences, and nonclassic Watson and and for modulating the concentration of endogenous Sub Crick base pairing sequences used in formation of triplex Strates. DNA. Such antisense molecules include antisense oligode US 7,795,392 B2 47 48 oxyribonucleotides, oligoribonucleotides, oligonucleotide EST sequence, Genbank accession number T73863, encoded analogues, and the like, and may comprise at least about 15 to a partial cDNA with significant sequence identity with OATP. 25 bases. EST sequences encoding partial cDNAs for OATP-RP 1. Antisense molecules may bind noncovalently or covalently OATP-RP2, OATP-RP3, OATP-RP4, and OATP-RP5 were to the OATP DNA or RNA. Such binding could, for example, identified by searching the public EST databases and the cleave or facilitate cleavage of OATP DNA or RNA, increase Incyte, Inc. EST database for sequences homologous to degradation of nuclear or cytoplasmic mRNA, or inhibit tran human OATP. The EST clone IDs corresponding to OATP Scription, translation, binding of transactivating factors, or pre-mRNA splicing or processing. Antisense molecules may RP1 are 8201 17, 2668489, 1610706, 2972518, and 588148. 10 These clones represent a contig encoding only part of the full also contain additional functionalities that increase stability, transport into and out of cells, binding affinity, cleavage of the length cDNA. The Incyte EST clone IDs corresponding to target molecule, and the like. All of these effects would OATP-RP2 are 1664737 and 2641944. These clones repre decrease expression of OATP protein and thus make the anti sent a contig encoding only part of the full length cDNA. The sense molecules useful as OATP modulators. Incyte EST clone IDs corresponding to OATP-RP3 are 15 2493241, 2497845, and 2664024. These clones represent a EXAMPLES contig encoding only part of the full length cDNA. The Incyte EST clone IDs corresponding to OATP-RP4 are 1494683 and The following examples are included for understanding the 1685219. These clones represent a contig encoding only part present invention and are not intended to limit the scope of 20 of the full length cDNA. The Incyte EST clone ID corre Applicants invention, which is defined solely by the claims. sponding to OATP-RP5 is 925716. This clone encodes only part of the full length cDNA. Full length clones for each of the Example 1 above genes were obtained using the Gene Trapper cDNA Positive Selection System (LifeTechnologies, Inc.). In this Isolation of OATP2, OATP-RP1, OATP-RP2, 25 procedure, a single or multiple oligonucleotides complemen OATP-RP3, OATP-RP4 and OATP-RP5 Full Length tary to each of the EST contigs or individual EST sequences, cDNAs and Cloning Into Mammalian Expression were biotinylated at the 3'-end and used to hybridize to a Vectors single-stranded human cINA library constructed in pCM 30 VSport2 (LifeTechnologies, Inc.). The sequence of oligo Human OATP2 was identified by searching the public EST nucleotides used for each gene as well as the tissue source of databases for sequences homologous to human OATP. One the libraries screened are shown in Table 2.

TABL E 2

Oligonucleotides used to screen for OATP Full length cDNAs using Gene-Trapper Selection

Human cDNA Biotinylated capture Seq ID number of library Gene oligonucleotide (s) used oligonucleotide screened

OATP2 s' - ACCCTGTCTAGCAGGTTGCA-3' 3 liver

OATP-RP1 s' - CTGTCGGAGTCTTCAGATG-3' 4. brain

OATP-RP2 s" - TCCATCACAGCCTCCTACGC-3' 5 liver

OATP-RP3 5'-TGCCTCTACTCTGACCCTAG-3' 6 heart

OATP-RP4 5 - GGAGCAGTCATTGACACCAC-3 7 heart

5-TGCTGGGAGTACAACGTGACG-3 8

5'-ACAAGGAGGATGGACTGCAG-3 9

OATP-RP5 5 - CAGGAATCCCAGCTCCAGTG-3 2O brain

5 - GCTACAACCCAACTACTGGC-3 21

5 - GGGACTAACTGTGATACTGG-3 22 US 7,795,392 B2 49 50 Hybrids between the biotinylated oligonucleotides and tissues (FIG. 1). Transporters of this family previously single-stranded cDNA were captured on Streptavidin-coated described in the literature, namely human OATP rat oatp1, rat paramagnetic beads. After washing, the captured single oatp2 and rat oatp3, are all expressed in liver, kidney and stranded cDNA targets was released from the biotinylated brain. All of the above transport bile acids as well as a variety oligonucleotides and converted to dsDNA by DNA poly 5 of other substrates that are specific for subsets of these trans merase using the corresponding unbiotinylated oligonucle porters. In contrast, the expression of OATP2, which also otide. Following transformation and plating, several positive transports bile acids, is very hepato-specific; a major 3.2 kb clones for each gene were identified by PCR analysis. Full and several minor hybridizing bands were observed only in length cDNA clones were identified by sequencing. In the RNA from liver and no other tissue. The specific cell types case of OATP-RP1, a partial cDNA was obtained by the above 10 that express this transporter were examined by in situ hybrid technique (pSP-RP1A). Another cDNA clone that was part of ization of OATP2 riboprobe to human liver samples. Strong the OATP-RP1 contig was identified by searching the public hybridization signal was seen localized to hepatocytes EST databases (Genbank accession number A1027850). An throughout the liver lobule with no significant difference in EcoRI-NotI fragment of this clone containing the first 477 signal intensity among centrilobular, midZonal or periportal nucleotides of OATP-RP1 (SEQ ID NO:11) (obtained from 15 regions. No signal was observed in bile ducts, Kupffer cells, Research Genetics, Inc.) was ligated to EcoRI-Not I digested or blood vessels, nor in any cell types from human lung (data pSP-RP1A to generate the full length sequence. not shown). Two polymorphic positions were identified when sequenc OATP-RP 1 is expressed in nearly all tissues tested with ing multiple OATP-RP4 cDNA clones. Thus, nucleotide highest abundance in skeletal muscle, lung, placenta, and number 713 of SEQID NO:7 can be either a C, encoding Leu heart. OATP-RP2 is ubiquitously expressed in all tissues in SEQ ID NO:8, or a T, encoding a Phe in SEQ ID NO:8. tested. OATP-RP4 has a much more restricted pattern of Similarly, nucleotide number 2397 of SEQ ID NO:7 can be expression with abundant transcipts in skeletal muscle and either a G, encoding a Gly in SEQID NO:8, or a T, encoding heart and much less in prostate and thymus. The expression of a Val in SEQID NO:8. OATP-RP5 is likewise tissue specific, with brain and testes For expression studies, OATP2 cl DNA was cloned into the 25 being the only sites where transcripts were detected. expression vector pCEP4BR, a modified form of pCEP4 (In vitrogen, Inc.) in which the CMV promoter-driven expression Example 3 cassette has been inverted, and used in transient transfections. To accomplish this, OATP2 cDNA in pCMVSport2, corre Expression of OATP2 in Transfected Cells ponding to nucleotides 59 through 2361 of SEQ ID NO:1, 30 was excised by digestion with KpnI and Not. This fragment 293EBNA cells (Invitrogen, Inc.), an HEK293 cell deriva was cloned into KpnI-NotI digested pCEP4BR. This clone, tive, were transiently transfected with the OATP2 expression pCEP-OATP2 was used for transient transfection expression vector pCEP-OATP2, or the pCEP4 vector alone (MOCK) studies. and the transport of H-labeled substrates was determined 35 24 hours later. FIG. 2A shows specific uptake of H-prav Example 2 astatin and H-DHEAS. FIGS. 2B and 2C show the specific uptake of H-taurocholate and 125II-thyroid hormone Tissue and Cellular Distribution of OATP2, (T4), respectively. The uptake of radiolabeled substrate for 5 OATP-RP 1, OATP-RP2, OATP-RP4, and minutes into cells transfected with pCEP-OATP2 or empty OATP-RP5 40 vector (MOCK) was determined in the absence (solid bars) and presence (open bars) of excess unlabeled Substrate. Thus, The tissue distribution of OATP2, OATP-RP1, OATP-RP2, OATP2 is a liver specific human transporter of at least some OATP-RP4, and OATP-RP5 expression was determined by HMG CoA reductase inhibitors, bile acids, adrenal steroids, Northern blotting of poly A+ RNA from a variety of human and thyroid hormone.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 29

<21 Os SEQ ID NO 1 &211s LENGTH: 2830 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (135) . . (22O7

<4 OOs SEQUENCE: 1

cggacgcgt.g. gig.cggacgcg tdggtCCCC acgcgt.ccga Cttgttgcag ttgctgtagg 60

attictaaatc caggtgattg tttcaaactg agcatcaiaca acaaaaacat ttgitatgata 12O

totatatttic aatc atg gac caa aat caa cat ttg aat aaa aca gca gag 17O Met Asp Glin Asn Gln His Lieu. Asn Llys Thr Ala Glu 1. 5 1 O US 7,795,392 B2 51 52

- Continued gca Cala cott to a gag aat aag a.a.a. aca aga tac tgc aat gga ttg aag 218 Ala Glin Pro Ser Glu Asn Lys Lys Thir Arg Cys Asn Gly Luell Lys 15 2O 25 atg ttic ttg gca gct Ctg toa citc. agc titt att gct aag aca Cta ggt 266 Met Phe Luell Ala Ala Lell Ser Luell Ser Phe Ile Ala Lys Thir Luell Gly 3O 35 4 O gca att att atg a.a.a. agt t cc at C att Cat ata gaa cgg aga titt gag 314 Ala Ile Ile Met Lys Ser Ser Ile Ile His Ile Glu Arg Arg Phe Glu 45 SO 55 60 ata to c tot tot citt gtt ggit titt att gac gga agc titt gala att gga 362 Ile Ser Ser Ser Lell Wall Gly Phe Ile Asp Gly Ser Phe Glu Ile Gly 65 70 7s aat ttg citt gtg att gta titt gtg agt tac titt gga t cc a.a.a. Cta Cat 41 O Asn Luell Luell Wall Ile Wall Phe Wall Ser Phe Gly Ser Lys Luell His 8O 85 90 aga CC a aag tta att gga atc. ggt tgt ttic att atg gga att gga ggt 458 Arg Pro Lys Luell Ile Gly Ile Gly Cys Phe Ile Met Gly Ile Gly Gly 95 105 gtt ttg act gct ttg C Ca Cat ttic ttic atg gga tat tac agg tat tot SO 6 Wall Luell Thir Ala Lell Pro His Phe Phe Met Gly Tyr Arg Ser 11 O 115 12O a.a.a. gala act aat atc. gat toa to a gala aat toa a Ca tcg acc tta to c 554 Lys Glu Thir Asn Ile Asp Ser Ser Glu Asn Ser Thir Ser Thir Luell Ser 125 13 O 135 14 O act tgt tta att aat Cala att tta to a citc. aat aga gca to a cott gag Thir Cys Luell Ile Asn Glin Ile Luell Ser Luell ASn Arg Ala Ser Pro Glu 145 150 155 ata gtg gga a.a.a. ggit tgt tta aag gala tot 999 toa tac atg tgg at a 650 Ile Wall Gly Lys Gly Cys Lell Lys Glu Ser Gly Ser Met Trp Ile 160 1.65 17 O tat gtg ttic atg ggit aat atg citt cgt gga ata 999 gag act cc c at a 698 Wall Phe Met Gly Asn Met Luell Arg Gly Ile Gly Glu Thir Pro Ile 17s 18O 185 gta CC a 999 citt tot tac att gat gat titc gct a.a.a. gala gga Cat 746 Wall Pro Luell Gly Lell Ser Tyr Ile Asp Asp Phe Ala Glu Gly His 19 O 195 2 OO tot tot ttg tat tta ggit ata ttg aat gca ata gca atg att ggt CC a 794 Ser Ser Luell Lell Gly Ile Luell Asn Ala Ile Ala Met Ile Gly Pro 2O5 210 215 22O atc. att ggc titt a CC Ctg gga tot Ctg titt tot a.a.a. atg tac gtg gat 842 Ile Ile Gly Phe Thir Lell Gly Ser Luell Phe Ser Met Wall Asp 225 23 O 235 att gga tat gta gat Cta agc act at C agg ata act cott act gat tot 890 Ile Gly Wall Asp Lell Ser Thir Ile Arg Ile Thir Pro Thir Asp Ser 24 O 245 25 O cga tgg gtt gga gct tgg tgg citt aat ttic citt gtg tot gga Cta ttic 938 Arg Trp Wall Gly Ala Trp Trp Luell Asn Phe Luell Wall Ser Gly Luell Phe 255 26 O 265 t cc att att tot t cc ata C Ca ttic titt ttic ttg c cc Cala act CC a aat 986 Ser Ile Ile Ser Ser Ile Pro Phe Phe Phe Luell Pro Glin Thir Pro Asn 27 O 27s 28O a.a.a. CC a Cala a.a.a. gaa aga a.a.a. gct to a Ctg tot ttg Cat gtg Ctg gala 1034 Lys Pro Glin Glu Arg Ala Ser Luell Ser Lell His Wall Luell Glu 285 290 295 3OO a Ca aat gat gala aag gat Cala aca gct aat ttg a CC aat Cala gga a.a.a. 1082 Thir Asn Asp Glu Lys Asp Glin Thir Ala Asn Luell Thir Asn Glin Gly 3. OS 310 315 aat att acc a.a.a. aat gtg act ggt titt ttic cag tot titt a.a.a. agc at C 113 O Asn Ile Thir Lys Asn Wall Thir Gly Phe Phe Glin Ser Phe Lys Ser Ile 32O 3.25 33 O US 7,795,392 B2 53 54

- Continued citt act aat cc c Ctg tat gtt atg titt gtg citt ttg acg ttg tta Cala 178 Lell Thir Asn Pro Lell Wall Met Phe Wall Luell Lell Thir Luell Luell Glin 335 34 O 345 gta agc agc tat att ggit gct titt act tat gtc tto a.a.a. tac gta gag 226 Wall Ser Ser Ile Gly Ala Phe Thir Wall Phe Wall Glu 350 355 360

Cala cag tat ggt Cag cott toa tot aag gct aac atc. tta ttg gga gt C 274 Glin Glin Gly Glin Pro Ser Ser Lys Ala ASn Ile Lell Luell Gly Wall 365 37O 375 38O ata acc at a cott att titt gca agt gga atg titt tta gga gga tat at C 322 Ile Thir Ile Pro Ile Phe Ala Ser Gly Met Phe Lell Gly Gly Tyr Ile 385 390 395 att a.a.a. a.a.a. ttic a.a.a. Ctg aac acc gtt gga att gcc a.a.a. ttic to a tgt 37 O Ile Phe Lys Lell Asn Thir Wall Gly Ile Ala Phe Ser Cys 4 OO 405 41 O titt act gct gtg atg toa ttg to c titt tac Cta tta tat titt ttic at a 418 Phe Thir Ala Wall Met Ser Lell Ser Phe Luell Lell Tyr Phe Phe Ile 415 42O 425 citc. tgt gala aac a.a.a. toa gtt gcc gga Cta acc atg a CC gat gga 466 Lell Cys Glu Asn Lys Ser Wall Ala Gly Luell Thir Met Thir Asp Gly 43 O 435 4 4 O aat aat CC a gtg a Ca tot Cat aga gat gta CCa citt tot tgc aac 514 Asn Asn Pro Wall Thir Ser His Arg Asp Wall Pro Lell Ser Cys Asn 445 450 45.5 460 toa gac tgc aat tgt gat gaa agt Cala tgg gaa C Ca gtc tgt gga aac 562 Ser Asp Cys Asn Cys Asp Glu Ser Glin Trp Glu Pro Wall Cys Gly Asn 465 470 47s aat gga at a act tac atc. toa cc c tgt Cta gca ggit a.a.a. tot to a 610 Asn Gly Ile Thir Tyr Ile Ser Pro Cys Luell Ala Gly Lys Ser Ser 48O 485 49 O agt ggc aat a.a.a. aag cott ata gtg titt tac aac tgc agt ttg gala 658 Ser Gly Asn Lys Pro Ile Wall Phe ASn Cys Ser Cys Luell Glu 495 SOO 5 OS gta act ggt citc. Cag aac aga aat to a gcc Cat ttg ggt gala tgc 706 Wall Thir Gly Luell Glin Asn Arg Asn Ser Ala His Lell Gly Glu Cys 510 515 52O

C Ca aga gat gat gct tgt a Ca agg titt tac titt titt gtt gca at a 754 Pro Arg Asp Asp Ala Cys Thir Arg Phe Tyr Phe Phe Wall Ala Ile 525 53 O 535 54 O

Cala gt C ttg aat tta titt tto tot gca citt gga ggc a CC to a Cat gt C Glin Wall Luell Asn Lell Phe Phe Ser Ala Luell Gly Gly Thir Ser His Wall 5.45 550 555 atg Ctg att gtt a.a.a. att gtt Cala cott gala ttg a.a.a. toa citt gca Ctg 850 Met Luell Ile Wall Lys Ile Wall Glin Pro Glu Luell Ser Luell Ala Luell 560 565 st O ggit ttic CaC to a atg gtt ata cga gca Cta gga gga att Cta gct CC a 898 Gly Phe His Ser Met Wall Ile Arg Ala Luell Gly Gly Ile Luell Ala Pro sts 58 O 585 ata tat titt 999 gct Ctg att gat aca acg tgt ata aag tgg to c acc 946 Ile Tyr Phe Gly Ala Lell Ile Asp Thir Thir Cys Ile Lys Trp Ser Thir 590 595 6 OO aac aac tgt ggc a Ca cgt 999 to a tgt agg aca tat aat to c aca to a 994 Asn Asn Cys Gly Thir Arg Gly Ser Cys Arg Thir Asn Ser Thir Ser 605 610 615 62O titt to a agg gt C tac ttg ggc ttg tot to a atg tta aga gtC to a to a Phe Ser Arg Wall Tyr Lell Gly Luell Ser Ser Met Lell Arg Wall Ser Ser 625 630 635 citt gtt tta tat att ata tta att tat gcc atg aag a.a.a. a.a.a. tat Cala 209 O Lell Wall Luell Ile Ile Lell Ile Ala Met Lys Glin US 7,795,392 B2 55 56

- Continued

64 O 645 65 O gag a.a.a. gat at C aat gca t ca gala aat gga agt gtc atg gat gala gca 2138 Glu Lys Asp Ile Asn Ala Ser Glu Asn Gly Ser Wall Met Asp Glu Ala 655 660 665 aac tta gala to c tta aat aaa aat aaa Cat titt gtc cott tot gct gig 2186 Asn Luell Glu Ser Lieu. Asn Lys Asn Llys His Phe Wall Pro Ser Ala Gly 670 675 68O gca gat agt gala aca Cat tt talaggggaga aaaaaa.gc.ca cittctgct tc 2237 Ala Asp Ser Glu Thr His Cys 685 690 tgttgtttcca aacago attg cattgattica gtaagatgtt atttittgagg agttcCtggit 2297 cott to act a agaattitcca catcttittat gigtggaagta taaataagcc tatgaactta 2357 taataaaa.ca aactgtaggit agaaaaaatg agagtactica ttgttacatt atagotacat 24.17 atttgttggitt aaggittagac tatatgat co atacaaatta aagtgagaga catggittact 2477 gtgtaataaa agaaaaaata cittgttcagg taattictaat tottaataaa. acaaatgagt 2537 atcatacagg tagaggittaa aaaggaggag ctagatt cat atcctaagta aagagaaatg 2597 cctagtgtct attitt attaa acaaacaaac acagagtttg alactataata Ctalaggcctg 2657 aagttctagot tggatatatgctacaataat atctgttact CaCataaaat tatatattt C 2717 acagactitta t caatgtata attaacaatt atc.ttgttta agtaaattta gaatacattt 277 7 aagtattgttg gaagaaataa agacattcca at atttgcaa. aaaaaaaaaa. a.a.a. 283 O

<210 SEQ ID NO 2 &211s LENGTH: 691 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2

Met Asp Glin Asn Glin His Lieu. Asn Llys Thir Ala Glu Ala Glin Pro Ser 1. 15

Glu Asn Lys Lys Thir Arg Tyr Cys Asn Gly Lieu. Met Phe Lieu Ala

Ala Luell Ser Luell Ser Phe Ile Ala Lys Thr Lieu. Gly Ala Ile Ile Met 35 4 O 45

Ser Ser Ile Ile His Ile Glu Arg Arg Phe Glu Ile Ser Ser Ser SO 55 6 O

Lell Wall Gly Phe Ile Asp Gly Ser Phe Glu Ile Gly Asn Luell Lieu Wall 65 70 7s 8O

Ile Wall Phe Wall Ser Tyr Phe Gly Ser Lys Lieu. His Arg Pro Llys Lieu. 85 90 95

Ile Gly Ile Gly Cys Phe Ile Met Gly Ile Gly Gly Wall Luell Thir Ala 105 11 O

Lell Pro His Phe Phe Met Gly Tyr Tyr Arg Tyr Ser Lys Glu Thir Asn 115 12 O 125

Ile Asp Ser Ser Glu Asn. Ser Thir Ser Thir Lieu. Ser Thir Lieu. Ile 13 O 135 14 O

Asn Glin Ile Luell Ser Lieu. Asn Arg Ala Ser Pro Glu Ile Wall Gly Lys 145 150 155 160

Gly Luell Glu Ser Gly Ser Tyr Met Trp Ile Wall Phe Met 1.65 17O 17s

Gly Asn Met Luell Arg Gly Ile Gly Glu Thr Pro Ile Wall Pro Lieu. Gly 18O 185 19 O

Lell Ser Ile Asp Asp Phe Ala Lys Glu Gly His Ser Ser Leu Tyr US 7,795,392 B2 57 58

- Continued

195 2O5

Lell Gly Ile Luell Asn Ala Ile Ala Met Ile Gly Pro Ile Ile Gly Phe 21 O 215 22O

Thir Luell Gly Ser Lell Phe Ser Met Tyr Wall Asp Ile Gly Wall 225 23 O 235 24 O

Asp Luell Ser Thir Ile Arg Ile Thir Pro Thir Asp Ser Arg Trp Wall Gly 245 250 255

Ala Trp Trp Luell Asn Phe Lell Wall Ser Gly Luell Phe Ser Ile Ile Ser 26 O 265 27 O

Ser Ile Pro Phe Phe Phe Lell Pro Glin Thir Pro Asn Lys Pro Glin 27s 28O 285

Glu Arg Ala Ser Lell Ser Luell His Wall Luell Glu Thir Asn Asp Glu 29 O 295 3 OO

Lys Asp Glin Thir Ala Asn Lell Thir Asn Glin Gly Asn Ile Thir Lys 3. OS 310 315 32O

Asn Wall Thir Gly Phe Phe Glin Ser Phe Lys Ser Ile Lell Thir Asn Pro 3.25 330 335

Lell Wall Met Phe Wall Lell Luell Thir Luell Luell Glin Wall Ser Ser 34 O 345 35. O

Ile Gly Ala Phe Thir Wall Phe Wall Glu Glin Glin Gly 355 360 365

Glin Pro Ser Ser Lys Ala Asn Ile Luell Luell Gly Wall Ile Thir Ile Pro 37 O 375

Ile Phe Ala Ser Gly Met Phe Luell Gly Gly Tyr Ile Ile Phe 385 390 395 4 OO

Luell Asn Thir Wall Gly Ile Ala Phe Ser Phe Thir Ala Wall 4 OS 415

Met Ser Luell Ser Phe Lell Luell Tyr Phe Phe Ile Lell Cys Glu Asn 425 43 O

Ser Wall Ala Gly Lell Thir Met Thir Asp Gly Asn Asn Pro Wall 435 44 O 445

Thir Ser His Arg Asp Wall Pro Luell Ser Asn Ser Asp Asn 450 45.5 460

Cys Asp Glu Ser Glin Trp Glu Pro Wall Gly Asn Asn Gly Ile Thir 465 470

Ile Ser Pro Cys Lell Ala Gly Lys Ser Ser Ser Gly Asn 485 490 495

Pro Ile Wall Phe Asn Ser Luell Glu Wall Thir Gly Luell SOO 505

Glin Asn Arg Asn Tyr Ser Ala His Luell Gly Glu Pro Arg Asp Asp 515 52O 525

Ala Cys Thir Arg Lys Phe Tyr Phe Phe Wall Ala Ile Glin Wall Luell Asn 53 O 535 54 O

Lell Phe Phe Ser Ala Lell Gly Gly Thir Ser His Wall Met Luell Ile Wall 5.45 550 555 560

Ile Wall Glin Pro Glu Lell Ser Luell Ala Lell Gly Phe His Ser 565 st O sts

Met Wall Ile Arg Ala Lell Gly Gly Ile Luell Ala Pro Ile Tyr Phe Gly 585 59 O

Ala Luell Ile Asp Thir Thir Ile Trp Ser Thir Asn Asn Gly 595 6OO 605

Thir Arg Gly Ser Cys Arg Thir Asn Ser Thir Ser Phe Ser Arg Wall 610 615 62O

US 7,795,392 B2 65 66

- Continued

85 90 95

Gly Gly Ala Ile Lell Wall Ala Luell Ala Gly Lell Lell Met Thir Luell 105 11 O

Pro His Phe Ile Ser Glu Pro Tyr Arg Asp Asn Thir Ser Pro Glu 115 12 O 125

Asp Met Pro Glin Asp Phe Lys Ala Ser Luell Lell Pro Thir Thir Ser 13 O 135 14 O

Ala Pro Ala Ser Ala Pro Ser Asn Gly Asn Cys Ser Ser Thir Glu 145 150 155 160

Thir Glin His Luell Ser Wall Wall Gly Ile Met Phe Wall Ala Glin Thir Luell 1.65 17s

Lell Gly Wall Gly Gly Wall Pro Ile Glin Pro Phe Gly Ile Ser Ile 18O 185 19 O

Wall Asp Phe Ala His Asn Ser Asn Ser Pro Luell Lell Gly Ile Luell 195

Phe Ala Wall Thir Met Met Gly Pro Gly Luell Ala Phe Gly Luell Gly Ser 21 O 215 22O

Lell Met Lel Arg Lell Tyr Wall Asp Ile Asn Glin Met Pro Glu Gly Gly 225 23 O 235 24 O

Ile Ser Lel Thir Ile Asp Pro Arg Trp Wall Gly Ala Trp Trp Luell 245 250 255

Gly Phe Lel Ile Ala Ala Gly Ala Wall Ala Luell Ala Ala Ile Pro 26 O 265 27 O

Phe Phe Phe Pro Lys Glu Met Pro Glu Arg Glu Luell Glin Phe 27s 28O 285

Arg Arg Wall Lell Ala Wall Thir Asp Ser Pro Ala Arg Gly 29 O 295 3 OO

Asp Ser Ser Lys Glin Ser Pro Gly Glu Ser Thir Glin Asp 3. OS 310 315

Gly Luell Wall Glin Ile Ala Pro Asn Luell Thir Wall Ile Glin Phe Ile 3.25 330 335

Wall Phe Pro Arg Wall Lell Lell Glin Thir Luell Arg His Pro Ile Phe Luell 34 O 345 35. O

Lell Wall Wall Luell Ser Glin Wall Cys Luell Ser Ser Met Ala Ala Gly Met 355 360 365

Ala Thir Phe Luell Pro Phe Luell Glu Arg Glin Phe Ser Ile Thir Ala 37 O 375

Ser Ala Asn Lell Lell Ile Gly Luell Ser Phe Pro Ser Wall Ile 385 390 395 4 OO

Wall Gly Ile Wall Wall Gly Gly Wall Luell Wall Arg Lell His Luell Gly 4 OS 415

Pro Wall Gly Cys Gly Ala Lell Luell Luell Met Lell Luell Luell 425 43 O

Phe Phe Ser Luell Pro Lell Phe Phe Ile Gly Ser Ser His Glin Ile 435 44 O 445

Ala Gly Ile Thir His Glin Thir Ser Ala His Pro Gly Lell Glu Luell Ser 450 45.5 460

Pro Ser Met Glu Ala Ser Pro Luell Asp Gly Phe Asn Pro 465 470 47s

Wall Asp Pro Ser Thir Arg Wall Glu Tyr Ile Thir Pro His Ala 485 490 495

Gly Ser Ser Trp Wall Wall Glin Asp Ala Luell Asp Asn Ser Glin Wall SOO 505 51O

US 7,795,392 B2 73

- Continued ggcagggit Co taggcc act cqcgcggct gggcc acaga gtctactittg aaggcacct C 2589 atggittitt ca ggatgctgac agctgcaa.gc aac aggcact gccaaattica gggalacagtg 2649 gtggc.cagct taggatgg acatttctgg atacacatac acatacaaaa Cagaaaacat 2709 tttittaaaag aagttt cota aaataaaaaa aataaaaaaa aaaaaaaa 2757

<210s, SEQ ID NO 6 &211s LENGTH: 710 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Met Glin Gly Lys Llys Pro Gly Gly Ser Ser Gly Gly Gly Arg Ser Gly 1. 5 1O 15 Glu Lieu. Glin Gly Asp Glu Ala Glin Arg Asn Llys Llys Llys Llys Llys Llys 2O 25 3O Val Ser Cys Phe Ser Asn Ile Lys Ile Phe Leu Val Ser Glu. Cys Ala 35 4 O 45 Lieu Met Lieu Ala Glin Gly Thr Val Gly Ala Tyr Lieu Val Ser Val Lieu. SO 55 6 O Thir Thr Lieu. Glu Arg Arg Phe Asn Lieu. Glin Ser Ala Asp Val Gly Val 65 70 7s 8O Ile Ala Ser Ser Phe Glu Ile Gly Asn Lieu Ala Lieu. Ile Lieu. Phe Val 85 90 95 Ser Tyr Phe Gly Ala Arg Gly His Arg Pro Arg Lieu. Ile Gly Cys Gly 1OO 105 11 O Gly Ile Val Met Ala Lieu. Gly Ala Lieu. Lieu. Ser Ala Lieu Pro Glu Phe 115 12 O 125 Lieu. Thir His Glin Tyr Llys Tyr Glu Ala Gly Glu Ile Arg Trp Gly Ala 13 O 135 14 O Glu Gly Arg Asp Val Cys Ala Ala Asn Gly Ser Gly Gly Asp Glu Gly 145 150 155 160 Pro Asp Pro Asp Lieu. Ile Cys Arg Asn Arg Thr Ala Thr Asn Met Met 1.65 17O 17s Tyr Lieu. Lieu. Lieu. Ile Gly Ala Glin Val Lieu. Lieu. Gly Ile Gly Ala Thr 18O 185 19 O Pro Val Glin Pro Lieu. Gly Val Ser Tyr Ile Asp Asp His Val Arg Arg 195 2OO 2O5 Lys Asp Ser Ser Lieu. Tyr Ile Gly Ile Leu Phe Thr Met Leu Val Phe 21 O 215 22O Gly Pro Ala Cys Gly Phe Ile Leu Gly Ser Phe Cys Thr Lys Ile Tyr 225 23 O 235 24 O Val Asp Ala Val Phe Ile Asp Thir Ser Asn Lieu. Asp Ile Thr Pro Asp 245 250 255 Asp Pro Arg Trp Ile Gly Ala Trp Trp Gly Gly Phe Lieu. Lieu. Cys Gly 26 O 265 27 O Ala Lieu. Leu Phe Phe Ser Ser Lieu. Leu Met Phe Gly Phe Pro Glin Ser 27s 28O 285 Lieu Pro Pro His Ser Asp Pro Ala Met Glu Ser Glu Glin Ala Met Leu 29 O 295 3 OO Ser Glu Arg Glu Tyr Glu Arg Pro Llys Pro Ser Asn Gly Val Lieu. Arg 3. OS 310 315 32O His Pro Lieu. Glu Pro Asp Ser Ser Ala Ser Cys Phe Glin Glin Lieu. Arg 3.25 330 335 US 7,795,392 B2 75

- Continued Val Ile Pro Llys Val Thr Lys His Leu Lleu Ser Asn Pro Val Phe Thr 34 O 345 35. O Cys Ile Ile Lieu Ala Ala Cys Met Glu Ile Ala Val Val Ala Gly Phe 355 360 365 Ala Ala Phe Lieu. Gly Llys Tyr Lieu. Glu Glin Glin Phe Asn Lieu. Thir Thr 37 O 375 38O Ser Ser Ala Asn. Glin Lieu. Lieu. Gly Met Thir Ala Ile Pro Cys Ala Cys 385 390 395 4 OO Lieu. Gly Ile Phe Lieu. Gly Gly Lieu. Lieu Val Lys Llys Lieu. Ser Lieu. Ser 4 OS 41O 415 Ala Lieu. Gly Ala Ile Arg Met Ala Met Lieu Val Asn Lieu Val Ser Thr 42O 425 43 O Ala Cys Tyr Val Ser Phe Leu Phe Leu Gly Cys Asp Thr Gly Pro Val 435 44 O 445 Ala Gly Val Thr Val Pro Tyr Gly Asn Ser Thr Ala Pro Gly Ser Ala 450 45.5 460 Lieu. Asp Pro Tyr Ser Pro Cys Asn. Asn. Asn. Cys Glu. Cys Glin Thr Asp 465 470 47s 48O Ser Phe Thr Pro Val Cys Gly Ala Asp Gly Ile Thr Tyr Lieu. Ser Ala 485 490 495 Cys Phe Ala Gly Cys Asn. Ser Thr Asn Lieu. Thr Gly Cys Ala Cys Lieu. SOO 505 51O Thir Thr Val Pro Ala Glu Asn Ala Thr Val Val Pro Gly Lys Cys Pro 515 52O 525 Ser Pro Gly Cys Glin Glu Ala Phe Lieu. Thr Phe Lieu. Cys Val Met Cys 53 O 535 54 O Ile Cys Ser Lieu. Ile Gly Ala Met Ala Glin Thr Pro Ser Val Ile Ile 5.45 550 555 560 Lieu. Ile Arg Thr Val Ser Pro Glu Lieu Lys Ser Tyr Ala Lieu. Gly Val 565 st O sts Lieu. Phe Lieu. Lieu. Lieu. Arg Lieu. Lieu. Gly Phe Ile Pro Pro Pro Lieu. Ile 58O 585 59 O Phe Gly Ala Gly Ile Asp Ser Thr Cys Lieu Phe Trp Ser Thr Phe Cys 595 6OO 605 Gly Glu Glin Gly Ala Cys Val Lieu. Tyr Asp Asn Val Val Tyr Arg Tyr 610 615 62O Lieu. Tyr Val Ser Ile Ala Ile Ala Lieu Lys Ser Phe Ala Phe Ile Lieu. 625 630 635 64 O Tyr Thir Thr Thr Trp Gln Cys Lieu. Arg Lys Asn Tyr Lys Arg Tyr Ile 645 650 655 Lys Asn His Glu Gly Gly Lieu Ser Thr Ser Glu Phe Phe Ala Ser Thr 660 665 67 O Lieu. Thir Lieu. Asp Asn Lieu. Gly Arg Asp Pro Val Pro Ala Asn Glin Thr 675 68O 685 His Arg Thr Llys Phe Ile Tyr Asn Lieu. Glu Asp His Glu Trp Cys Glu 69 O. 695 7 OO

Asn Met Glu Ser Wall Lieu. 7 Os 71O

<210s, SEQ ID NO 7 &211s LENGTH: 3692 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAMEAKEY: misc feature

US 7,795,392 B2 79 80

- Continued

Gly Gly Arg Arg Arg Pro Luell Trp Luell Ala Wall Gly Gly Lieu. Luell 19 O 195 2 OO atc. gcc ttic gca gcc citc. ttic gcc tta cott CaC tto at C tog cc c 276 Ile Ala Phe Ala Ala Lell Phe Ala Luell Pro His Phe Ile Ser Pro 2O5 210 215 22O c cc tac cag Cala gag ttg aac gcc tog gcc c cc aac gac ggc Ctg 324 Pro Glin Glin Glu Lell Asn Ala Ser Ala Asn Asp Gly Luell 225 23 O 235 tgt cag ggt aac t cc a CC gcc act ttg gag cc.g gcc tgt cc.g 372 Cys Glin Gly Asn Ser Thir Ala Thir Luell Glu Pro Ala Cys Pro 245 25 O aag gac tog gga aat aat CaC tgg gt C tac gct tta ttic att Lys Asp Ser Gly Asn Asn His Trp Wall Tyr Ala Luell Phe Ile 255 26 O 265 tgc gcg cag citc. att gga atg ggc to c aca att tat acc Ctg 468 Cys Ala Glin Lell Ile Gly Met Gly Ser Thir Ile Thir Luell 27 O 27s gga CC a acc tta gat gac aat gt C aag a.a.a. gaa aac to c to c ttg 516 Gly Pro Thir Lell Asp Asp Asn Wall Lys Lys Glu Asn Ser Ser Luell 285 290 295 3OO

Cta gcc at C atg tat gtc atg gga gca citt ggc cott gca gtg gga 564 Luell Ala Ile Met Wall Met Gly Ala Luell Gly Pro Ala Wall Gly 3. OS 310 315

tta tta ggt gga citt citt att ggt titt tat gtt gat cc c aga aat 61.2 Luell Luell Gly Gly Lell Lell Ile Gly Phe Wall Asp Pro Arg Asn 32O 3.25 33 O cott gtt CaC citt gac cag aat gac cott cgt. titc att gga aac tgg tgg 660 Pro Wall His Luell Asp Glin Asn Asp Pro Arg Phe Ile Gly Asn Trp Trp 335 34 O 345 agt gga ttic citc. citt tgt gcc att gca atg titt citt gtg at a ttic CC a 7 OS Ser Gly Phe Luell Lell Cys Ala Ile Ala Met Phe Lell Wall Ile Phe Pro 350 355 360 atg titt act ttic C Ca a.a.a. aag citt CC a cott cga CaC aag a.a.a. aag a.a.a. 756 Met Phe Thir Phe Pro Lys Lys Luell Pro Pro Arg His Lys Lys Lys 365 37O 375 38O aag a.a.a. titt tot gtt gat gct gtt agt gat gac gat gtt Ctg aag 804 Lys Phe Ser Wall Asp Ala Wall Ser Asp Asp Asp Wall Luell Lys 385 390 395 gag to a aac aac agt gaa Cala gcg gac a.a.a. a.a.a. gtt tot tog atg 852 Glu Ser Asn Asn Ser Glu Glin Ala Asp Wall Ser Ser Met 4 OO 405 41 O gga titt gga aag gat gtc aga gac Cta CC a aga gca gct gtC agg at C 9 OO Gly Phe Gly Lys Asp Wall Arg Asp Luell Pro Arg Ala Ala Wall Arg Ile 415 42O 425 tta agc aac atg a Ca tto citt titt gtg agt ttg toa tac aca gct gag 948 Lell Ser Asn Met Thir Phe Lell Phe Wall Ser Luell Ser Thir Ala Glu 43 O 435 4 4 O agt gcc att gta act gct tto att acc ttic att c cc aag ttic at C gag 996 Ser Ala Ile Wall Thir Ala Phe Ile Thir Phe Ile Pro Lys Phe Ile Glu 445 450 45.5 460 toa cag titt ggt atc. C Ca gcc to c aat gcc agc atc. tac act 999 gtt Ser Glin Phe Gly Ile Pro Ala Ser Asn Ala Ser Ile Thir Gly Wall 465 470 47s att at C gt C cc c agt gct ggit gtt ggt att gtc citc. gga ggc tac att 2092 Ile Ile Wall Pro Ser Ala Gly Wall Gly Ile Wall Lell Gly Gly Ile 48O 485 49 O ata a.a.a. a.a.a. ttg a.a.a. citt ggit gcc aga gala tot gca a.a.a. Cta gca atg 214 O Ile Lys Luell Lys Lell Gly Ala Arg Glu Ser Ala Lys Luell Ala Met 495 SOO 5 OS

US 7,795,392 B2 83 84

- Continued ata agt to C tot gcg gaC ccg ggg ctg. gala gag agc ccc gct gcc ttg 3148 Ile Ser Ser Ser Ala Asp Pro Gly Lieu. Glu Glu Ser Pro Ala Ala Lieu. 83 O 835 84 O gag cc.g. ccc to C taagcttga aaatggaaga atttagttitt gttggttgaa 32OO Glu Pro Pro Ser 845 ttgaaaatgg cacttgaga aacaactgtg cottctitt to titt citt to t t t t t t t talacc 326 O tctacagaca caatcc ticaa accala Caaaa. cticagtatac acagcc.gcta t t cattgagg 332O gctggatacct caacaagac tgaga.gc.ctt toccc.gcttc t ct coaagaa ggagacgttc 3380 agctagattt gttcc cattt aattcaaag.c t catgct coc ctacgg taca 344 O ggctgaggta Cacggittagc aaaac catgg galaggggaat ggcggtgcat at Catta act 3500 aacact coaa acaaaggtga gcttgcc.cag gacttgg cat titccaaatca aagtttittag 3560 atatgaacac c tactgtgag ttctgctaca aag cacaaat gaatttgttct caactatgca 362O atttgattgg aaaaatgitat gtgcagdatg ttacatttac titt cacggaa taaag cagat 3 680 atgtttctga aa 3692

<210s, SEQ ID NO 8 &211s LENGTH: 848 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (33) . . (33) 223 OTHER INFORMATION: The Xa a" at location 33 stands for Lieu. o Phe. 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (.594). . (594) 223 OTHER INFORMATION: The Xa a' at location 594 stands for Gly, o Wall.

<4 OOs, SEQUENCE: 8 Met Asp Glu Gly Thr Gly Lieu. Glin Pro Gly Ala Gly Glu Gln Lieu. Glu 1. 5 1O 15

Ala Pro Ala Thir Ala Glu Ala Wall Glin Glu Arg Glu Pro Glu. Thir 25 3O

Xaa Arg Ser Lys Ser Lieu Pro Val Luell Ser Ser Ala Ser Cys Arg Pro 35 4 O 45

Ser Leu Ser Pro Thr Ser Gly Asp Ala ASn Pro Ala Phe Gly Cys Val SO 55 6 O

Asp Ser Ser Gly His Glin Glu Lieu. Lys Glin Gly Pro Asn Pro Lieu. Ala 65 70 7s

Pro Ser Pro Ser Ala Pro Ser Thr Ser Ala Gly Lell Gly Asp Cys Asn 85 90 95

His Arg Val Asp Lieu. Ser Llys Thr Phe Ser Wall Ser Ser Ala Lieu Ala 105 11 O

Met Lieu. Glin Glu Arg Arg Cys Lieu. Tyr Val Val Lell Thir Asp Ser Arg 115 12 O 125

Cys Phe Leu Val Cys Met Cys Phe Lieu. Thir Phe Ile Glin Ala Lieu Met 13 O 135 14 O

Val Ser Gly Tyr Lieu Ser Ser Val Ile Thir Thir Ile Glu Arg Arg Tyr 145 150 155 160

Ser Lieu Lys Ser Ser Glu Ser Gly Lieu. Lieu Wall Ser Phe Asp Ile 1.65 17O 17s

Gly Asn Lieu Val Val Val Val Phe Val Ser Tyr Phe Gly Gly Arg Gly US 7,795,392 B2 85 86

- Continued

18O 185 19 O

Arg Arg Pro Luell Trp Lell Ala Wall Gly Gly Luell Lell Ile Ala Phe Gly 195 2O5

Ala Ala Luell Phe Ala Lell Pro His Phe Ile Ser Pro Pro Tyr Glin Ile 21 O 215 22O

Glin Glu Luell Asn Ala Ser Ala Pro Asn Asp Gly Lell Glin Gly Gly 225 23 O 235 24 O

Asn Ser Thir Ala Thir Lell Glu Pro Pro Ala Pro Asp Ser Gly 245 250 255

Gly Asn Asn His Trp Wall Luell Ala Luell Phe Ile Ala Glin Ile 26 O 265 27 O

Lell Ile Gly Met Gly Ser Thir Pro Ile Thir Lell Gly Pro Thir Tyr 28O 285

Lell Asp Asp Asn Wall Lys Glu Asn Ser Ser Lell Tyr Luell Ala Ile 29 O 295 3 OO

Met Wall Met Gly Ala Lell Gly Pro Ala Wall Gly Tyr Luell Luell Gly 3. OS 310 315

Gly Luell Luell Ile Gly Phe Wall Asp Pro Arg Asn Pro Wall His Luell 3.25 330 335

Asp Glin Asn Asp Pro Arg Phe Ile Gly Asn Trp Trp Ser Gly Phe Luell 34 O 345 35. O

Lell Ala Ile Ala Met Phe Luell Wall Ile Phe Pro Met Phe Thir Phe 355 360 365

Pro Lys Luell Pro Pro Arg His Lys Phe 37 O 375

Ser Wall Asp Ala Wall Ser Asp Asp Asp Wall Luell Glu Ser Asn 385 390 395 4 OO

Asn Ser Glu Glin Ala Asp Wall Ser Ser Met Gly Phe Gly Lys 4 OS 415

Asp Wall Arg Asp Lell Pro Arg Ala Ala Wall Arg Ile Lell Ser Asn Met 425 43 O

Thir Phe Luell Phe Wall Ser Lell Ser Thir Ala Glu Ser Ala Ile Wall 435 44 O 445

Thir Ala Phe Ile Thir Phe Ile Pro Phe Ile Glu Ser Glin Phe Gly 450 45.5 460

Ile Pro Ala Ser Asn Ala Ser Ile Thir Gly Wall Ile Ile Wall Pro 465 470

Ser Ala Gly Wall Gly Ile Wall Luell Gly Gly Tyr Ile Ile Lys Luell 485 490 495

Luell Gly Ala Arg Glu Ser Ala Lys Luell Ala Met Ile Cys Ser Gly SOO 505

Wall Ser Luell Luell Phe Ser Thir Luell Phe Ile Wall Gly Cys Glu Ser 515 525

Ile Asn Luell Gly Gly Ile Asn Ile Pro Thir Thir Gly Pro Ser Luell 53 O 535 54 O

Thir Met Pro His Arg Asn Lell Thir Gly Ser Cys Asn Wall Asn Gly 5.45 550 555 560

Ile His Glu Glu Pro Wall Cys Gly Ser Asp Gly Ile Thir 565 st O sts

Phe Asn Pro Lell Ala Gly Cys Wall ASn Ser Gly Asn Luell Ser 585 59 O

Thir Xaa Ile Arg Asn Thir Glu Thir Wall Glin Ser Arg Glin 595 6OO 605 US 7,795,392 B2 87 88

- Continued

Wall Ile Thr Pro Pro Thr Val Gly Glin Arg Ser Glin Lieu. Arg Val Val 610 615 62O

Ile Val Lys Thr Tyr Le u. Asn. Glu Asin Gly Tyr Ala Val Ser Gly Lys 625 63 O 635 64 O

Cys Lys Arg Thr Cys As in Thir Lieu. Ile Pro Phe Leul Wall Phe Lieu. Phe 645 650 655

Ile Val Thr Phe Ile Thr Ala Cys Ala Glin Pro Ser Ala Ile Ile Wall 660 665 67 O Thr Lieu. Arg Ser Val Gl u Asp Glu Glu Arg Pro Phe Ala Leu Gly Met 675 68O 685

Glin Phe Wall Lieu. Lieu Airg Thr Lieu Ala Tyr Ile Pro Thr Pro Ile Tyr 69 O. 695 7 OO

Phe Gly Ala Val Ile As p Thr Thr Cys Met Leu Trp Gln Glin Glu. Cys 7 Os 71. O 72O Gly Val Glin Gly Ser Cys Trp Glu Tyr Asn Val Thir Ser Phe Arg Phe 72 73 O 73 Val Tyr Phe Gly Lieu. Al a Ala Gly Lieu Lys Phe Val Gly Phe Ile Phe 740 74. 7 O Ile Phe Leu Ala Trp Tyr Ser Ile Glu Asp Gly Lieu. Glin 760 765

Arg Arg Arg Glin Arg Gl ul Phe Pro Lieu. Ser Thr Val Ser Glu Arg Val 770 775 78O

Gly His Pro Asp Asn Al a Arg Thr Arg Ser Cys Pro Ala Phe Ser Thr 78s 79 O 79. 8OO

Gln Gly Glu Phe His Gl u Glu. Thir Gly Lieu. Glin Lys Gly Ile Glin Cys 805 810 815

Ala Ala Glin Thr Tyr Pr o Gly Pro Phe Pro Glu Ala Ile Ser Ser Ser 82O 825 83 O

Ala Asp Pro Gly Lieu Gl u Glu Ser Pro Ala Ala Lieu. Glu Pro Pro Ser 835 84 O 845

<210s, SEQ ID NO 9 &211s LENGTH: 3381 &212s. TYPE: DNA &213s ORGANISM: Homo is apiens 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (370). . (25 O5)

<4 OOs, SEQUENCE: 9 cgcaaagaaa tigctcaaaa gct tcagotc tittctgtgcc Ctgggagctg agatgcacgt. 6 O

Cagtggcctt gcc agcgtgg cca attct ct gctgactgcc agaaaaaaga ggc.caggaag 12 O aaagagga aa gagaagagat cgct Cagggg tgagaccatg c cott catct t t t c titt to c 18O ctaatc to ct ctdcttgttgt CCaCC Cacao t ct coccacc tggcaaaatt gttcaaaatt 24 O gctgtggagt ttacct cagt ttoctott to agt ctgtggit gtgtggit coa tocticttgct 3OO gagdacattgaaaggaactg gctat ctittg atc. tott cott ccagat caga gtcaaggaat 360 gtgtttata atg gac act t catcc aaa gala aat atc cag titg titc tdc aaa 411 Met Asp Thr Ser Ser Lys Glu Asn. Ile Glin Lieu. Phe Cys Llys 1. 5 10 act tca gtg caa cct git t gga agg cct tot titt aaa aca gaa tat coc 459 Thir Ser Wall Glin Pro Wa l Gly Arg Pro Ser Phe Lys Thr Glu Tyr Pro 15 2O 25 3O tcc to a gala gala aag Ca a cca tdc tgt ggit gaa Cta aag gtg tt C ttg Ser Ser Glu Glu Lys Gl in Pro Cys Cys Gly Glu Lieu Lys Val Phe Lieu. US 7,795,392 B2 89 90

- Continued

35 4 O 45

gcc ttg tot titt gtt tac titt gcc a.a.a. gca ttg gca gala ggc tat 555 Ala Luell Ser Phe Wall Phe Ala Lys Ala Lell Ala Glu Gly Tyr SO 55 60

Ctg aag agc acc atc. act Cag at a gag aga agg titt gat at C cott tot 603 Lell Lys Ser Thir Ile Thir Glin Ile Glu Arg Arg Phe Asp Ile Pro Ser 65 70 7s toa Ctg gtg gga gtt att gat ggt agt titt gaa att 999 aat citc. tta 651 Ser Luell Wall Gly Wall Ile Asp Gly Ser Phe Glu Ile Gly Asn Luell Luell 8O 85 9 O gtt at a aca titt gtt agc tac titt gga gcc a.a.a. citt CaC agg CC a a.a.a. 699 Wall Ile Thir Phe Wall Ser Phe Gly Ala Lys Lell His Arg Pro Lys 95 1 OO 105 110 ata att gga gca 999 tgt gta at C atg gga gtt gga a Ca Ctg citc. att 747 Ile Ile Gly Ala Gly Cys Wall Ile Met Gly Wall Gly Thir Luell Luell Ile 115 12O 125 gca atg cott cag tto tto atg gag cag tac a.a.a. tat gag aga tat tot 79. Ala Met Pro Glin Phe Phe Met Glu Glin Glu Arg Ser 13 O 135 14 O cott to c to c aat t cc act citc. agc at C tot cc.g tgt citc. Cta gag to a 843 Pro Ser Ser Asn Ser Thir Lell Ser Ile Ser Pro Cys Lell Luell Ser 145 15 O 155 agc agt tta C Ca gtt toa gtt atg gala a.a.a. toa a.a.a. to c at a 891. Ser Ser Luell Pro Wall Ser Wall Met Glu Ser Ser Ile 16 O 1.65 17O agt aac tgt gaa gtg gac act agc tot to c atg tgg att gtt 939 Ser Asn Cys Glu Wall Asp Thir Ser Ser Ser Met Trp Ile Wall 17s 18O 185 190 tto Ctg aat citt citt cgt gga at a gga gaa act c cc att cott 987 Phe Luell Asn Lell Lell Arg Gly Ile Gly Glu Thir Pro Ile Pro 195 2OO ttg ggc gcc tac Ctg gat gat titt gcc agt gaa gac aat gct Lell Gly Ala Tyr Lell Asp Asp Phe Ala Ser Glu Asp Asn Ala 21O 215 22 O tto tat att 999 tgt gtg Cag acg gtt gca att ata gga CC a titt Phe Gly Cys Wall Glin Thir Wall Ala Ile Ile Gly Pro Phe 225 23 O 235 ggit ttic Ctg tta ggc toa tta gcc a.a.a. Cta tat gtt gac ggc 131 Gly Phe Luell Luell Gly Ser Lell Cys Ala Luell Tyr Wall Asp Gly 24 O 245 250 titt gta aac Cta gat CaC ata acc att acc CCa a.a.a. gat cc c tgg 179 Phe Wall Asn Luell Asp His Ile Thir Ile Thir Pro Asp Pro Trp 255 26 O 265 27 O gta gga gcc tgg tgg citt ggc Cta at a gca gga atc. at a agt citt 227 Wall Gly Ala Trp Trp Lell Gly Luell Ile Ala Gly Ile Ile Ser Luell 27s 28O 285 citt gca gct gtg cott tto tgg tta CC a aag agt tta CC a aga to c 27s Lell Ala Ala Wall Pro Phe Trp Luell Pro Lys Ser Lell Pro Arg Ser 290 295 3OO

Cala agt aga gag gat tot aat tot to c tot gag a.a.a. t cc aag titt att 323 Glin Ser Arg Glu Asp Ser Asn Ser Ser Ser Glu Ser Lys Phe Ile 305 31 O 315 ata gat gat CaC a Ca gac tac Cala aca cc c cag gga gaa aat gca a.a.a. 3.71 Ile Asp Asp His Thir Asp Tyr Glin Thir Pro Glin Gly Glu Asn Ala Lys 32O 3.25 330 ata atg gala atg gca aga gat titt citt CC a toa Ctg aag aat citt titt 419 Ile Met Glu Met Ala Arg Asp Phe Luell Pro Ser Lell Lys Asn Luell Phe 335 34 O 345 350 gga aac CC a gta tac tto Cta tat tta aca agc act gtt cag ttic 467

US 7,795, 392 B2 93 94

- Contin lued a.a.a. CaC aga agt titt ata acc aag aga gala aga aca atg gtg tot aca 2427 His Arg Ser Phe Ile Thr Lys Arg Glu Arg Thir Met Wall Ser Thr 675 68O 685 aga ttic Cala aag gaa aat tac act aca agt gat cat citg cta Cala CCC 2475 Arg Phe Glin Lys Glu Asn Tyr Thr Thir Ser Asp His Lieu. Lieu. Gln Pro 690 695 7 OO aac tac tgg cca ggc aag gala act caa citt tagaaacatg atgactggaa 2525 Asn Tyr Trp Pro Gly Lys Glu Thr Gln Lieu. 7Os gtcatgtc.tt ctaattggitt gacattttgc aaacaaataa. attgtaatca aaagagctict 2585 aaatttgtaa titt citt to to Ctttcaaaaa. atgtc tactt tgttittggit c c taggcatta 2645 ggtaatataa citgataat at actgaaat at ataatggaag atgcagatga taaaactaat 2705 tittgaactitt ttaatttata taaattattt tatat cattt act tatttca citt tattittg 2765

Ctttgttgctic attgatatat attagctgta ctic ctagaag aacaattgtc tctattgtca 2825 cacatggitta tatttaaagt aatttctgaa Ctgtgtaatg tgtctagagt aagcaaatac 2.885 tgctaacaat talacticatac Cttgggitt CC ttcaagtatt act cotatag tattt to tcc. 29.45 catagotgtc ttcatctgtg tattittaata atgat cittag gatggagcag alacatggaga 3 OOS ggaagatttic attittaagct cct cottt to cittgaaatac aataattitat atagaaatgt gtag cagcaa. attatattgg ggattagaat tittgaattaa tagct ct cot act attaatt 31.25 tacatgtgct ttttgttgttgg cgctataagt gactatggitt gtaaagtaat aaaattgatg 3.185 ttaa catgcc caattattgt t ctitt tatga attcaatgaa tittaaaacta ttgttaaata 3.245 taatactgcc CCaCtttaat atatgtaagc aactt CCtac ttatacacga cgtgttccta 3305 aaacatgttt gaaaggtgaa tittctgaaag tot CC catala atgtaggtgt tacaiacagga 33.65 aaaaaaaaaa. aaaaaa. 3381

<210s, SEQ ID NO 10 &211s LENGTH: 712 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 10 Met Asp Thir Ser Ser Lys Glu Asn Ile Glin Lieu. Phe Cys Lys Thir Ser 1. 5 1O 15

Wall Glin Pro Val Gly Arg Pro Ser Phe Llys Thr Glu Tyr Pro Ser Ser 25 3O

Glu Glu Lys Gln Pro Cys Cys Gly Glu Lieu Lys Wall Phe Lieu. Cys Ala 35 4 O 45

Lell Ser Phe Val Tyr Phe Ala Lys Ala Lieu Ala Glu Gly Tyr Lieu Lys SO 55 6 O

Ser Thir Ile Thr Glin Ile Glu Arg Arg Phe Asp Ile Pro Ser Ser Luell 65 70 7s

Wall Gly Wall Ile Asp Gly Ser Phe Glu Ile Gly Asn Lieu. Luell Wall Ile 85 90 95

Thir Phe Wall Ser Tyr Phe Gly Ala Llys Lieu. His Arg Pro Llys Ile Ile 105 11 O

Gly Ala Gly Cys Val Ile Met Gly Val Gly Thr Lieu. Lieu. Ile Ala Met 115 12 O 125

Pro Glin Phe Phe Met Glu Gln Tyr Llys Tyr Glu Arg Tyr Ser Pro Ser 13 O 135 14 O

Ser Asn Ser Thir Lieu. Ser Ile Ser Pro Cys Lieu. Lieu. Glu Ser Ser Ser 145 150 155 160 US 7,795,392 B2 95 96

- Continued

Glin Luell Pro Wall Ser Wall Met Glu Ser Lys Ser Ile Ser Asn 1.65 17O 17s

Glu Glu Wall Asp Thir Ser Ser Ser Met Trp Ile Wall Phe Luell 18O 185 19 O

Gly Asn Luell Luell Arg Gly Ile Gly Glu Thir Pro Ile Glin Pro Luell Gly 195

Ile Ala Luell Asp Asp Phe Ala Ser Glu Asp Asn Ala Ala Phe 21 O 215 22O

Ile Gly Wall Glin Thir Wall Ala Ile Ile Gly Pro Ile Phe Gly Phe 225 23 O 235 24 O

Lell Luell Gly Ser Lell Ala Luell Tyr Wall Asp Ile Gly Phe Wall 245 250 255

Asn Luell Asp His Ile Thir Ile Thir Pro Asp Pro Glin Trp Wall Gly 26 O 265 27 O

Ala Trp Trp Luell Gly Lell Ile Ala Gly Ile Ile Ser Luell Luell Ala 285

Ala Wall Pro Phe Trp Lell Pro Ser Luell Pro Arg Ser Glin Ser 29 O 295 3 OO

Arg Glu Asp Ser Asn Ser Ser Ser Glu Ser Phe Ile Ile Asp 3. OS 310 315

Asp His Thir Asp Tyr Glin Thir Pro Glin Gly Glu Asn Ala Ile Met 3.25 330 335

Glu Met Ala Arg Asp Phe Lell Pro Ser Luell Asn Lell Phe Gly Asn 34 O 345 35. O

Pro Wall Tyr Phe Lell Lell Cys Thir Ser Thir Wall Glin Phe Asn Ser 355 360 365

Lell Phe Gly Met Wall Thir Tyr Pro Ile Glu Glin Glin 37 O 375

Gly Glin Ser Ser Ser Arg Ala Asn Phe Wall Ile Gly Lell Ile Asn Ile 385 390 395 4 OO

Pro Ala Wall Ala Lell Gly Ile Phe Ser Gly Gly Ile Wall Met Lys 4 OS 415

Phe Arg Ile Ser Wall Gly Ala Ala Luell Lell Gly Ser Ser 425 43 O

Wall Phe Gly Lell Lell Phe Luell Ser Luell Phe Ala Lell Gly Glu 435 44 O 445

Asn Ser Asp Wall Ala Gly Lell Thir Wall Ser Glin Gly Thir Pro 450 45.5 460

Wall Ser His Glu Arg Ala Luell Phe Ser Asp Asn Ser Arg Cys 465 470 47s

Ser Glu Thir Trp Glu Pro Met Gly Glu Asn Gly Ile 485 490 495

Thir Wall Ser Ala Lell Ala Gly Glin Thir Ser Asn Arg Ser SOO 505

Gly Asn Ile Ile Phe Asn Thir Wall Gly Ile Ala Ala 515 525

Ser Lys Ser Gly Asn Ser Ser Gly Ile Wall Gly Arg Glin Asp 53 O 535 54 O

Asn Gly Pro Glin Met Phe Luell Phe Luell Wall Ile Ser Wall Ile 5.45 550 555 560

Thir Ser Thir Lell Ser Lell Gly Gly Ile Pro Gly Ile Luell Luell 565 st O sts

US 7,795,392 B2 103 104

- Continued atatattttgttatttaa.gc ctd.cgaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2751 aaaaaaaaaa aa 2763

<210s, SEQ ID NO 12 &211s LENGTH: 722 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 12

Met Pro Lieu. His Glin Lell Gly Asp Pro Luell Thir Phe Pro Ser Pro 1. 5 15

Asn Ser Ala Met Glu Asn Gly Luell Asp His Thir Pro Pro Ser Arg Arg 25

Ala Ser Pro Gly Thir Pro Lell Ser Pro Gly Ser Lell Arg Ser Ala Ala 35 4 O 45

His Ser Pro Luell Asp Thir Ser Glin Pro Luell Cys Glin Luell Trp Ala SO 55 6 O

Glu His Gly Ala Arg Gly Thir His Glu Wall Arg Wall Ser Ala 65 70

Gly Glin Ser Wall Ala Gly Trp Trp Ala Phe Ala Pro Pro Cys Luell 85 90 95

Glin Wall Luell Asn Thir Pro Gly Ile Luell Phe Phe Lell Cys Ala Ala 105 11 O

Ala Phe Luell Glin Gly Met Thir Wall Asn Gly Phe Ile Asn Thir Wall Ile 115 12 O 125

Thir Ser Luell Glu Arg Arg Tyr Asp Luell His Ser Tyr Glin Ser Gly Luell 13 O 135 14 O

Ile Ala Ser Ser Tyr Asp Ile Ala Ala Luell Lell Thir Phe Wall 145 150 155 160

Ser Phe Gly Gly Ser Gly His Pro Arg Trp Lell Gly Trp Gly 1.65 17O 17s

Wall Luell Luell Met Gly Thir Gly Ser Luell Wall Phe Ala Lell Pro His Phe 18O 185 19 O

Thir Ala Gly Arg Tyr Glu Wall Glu Luell Asp Ala Gly Wall Arg Thir 195

Pro Ala Asn Pro Gly Ala Wall Ala Asp Ser Thir Ser Gly Luell Ser 21 O 215 22O

Arg Glin Luell Wall Phe Met Luell Gly Glin Phe Lell His Gly Wall Gly 225 23 O 235 24 O

Ala Thir Pro Luell Tyr Thir Lell Gly Wall Thir Lell Asp Glu Asn Wall 245 250 255

Ser Ser Cys Ser Pro Wall Ile Ala Ile Phe Thir Ala Ala 26 O 265 27 O

Ile Luell Gly Pro Ala Ala Gly Tyr Luell Ile Gly Gly Ala Luell Luell Asn 28O 285

Ile Tyr Thir Glu Met Gly Arg Arg Thir Glu Luell Thir Thir Glu Ser Pro 29 O 295 3 OO

Lell Trp Wall Gly Ala Trp Trp Wall Gly Phe Luell Gly Ser Gly Ala Ala 3. OS 310 315

Ala Phe Phe Thir Ala Wall Pro Ile Luell Gly Tyr Pro Arg Glin Luell Pro 3.25 330 335

Gly Ser Glin Arg Tyr Ala Wall Met Arg Ala Ala Glu Met His Glin Luell 34 O 345 35. O US 7,795,392 B2 105 106

- Continued

Asp Ser Ser Arg Gly Glu Ala Ser Asn Pro Asp Phe Gly Lys Thir 355 360 365

Ile Arg Asp Luell Pro Lell Ser Ile Trp Luell Luell Lell Lys Asn Pro Thir 37 O 375

Phe Ile Luell Luell Lell Ala Gly Ala Thir Glu Ala Thir Luell Ile Thir 385 390 395 4 OO

Gly Met Ser Thir Phe Ser Pro Phe Luell Glu Ser Glin Phe Ser Luell 4 OS 415

Ser Ala Ser Glu Ala Ala Thir Luell Phe Gly Tyr Lell Wall Wall Pro Ala 425 43 O

Gly Gly Gly Gly Thir Phe Lell Gly Gly Phe Phe Wall Asn Luell Arg 435 44 O 445

Lell Arg Gly Ser Ala Wall Ile Phe Luell Phe Thir Wall Wall 450 45.5 460

Ser Luell Luell Gly Ile Lell Wall Phe Ser Luell His Pro Ser Wall Pro 465 470

Met Ala Gly Wall Thir Ala Ser Gly Gly Ser Lell Lell Pro Glu Gly 485 490 495

His Luell Asn Luell Thir Ala Pro Asn Ala Ala Ser Cys Glin Pro SOO 505

Glu His Tyr Ser Pro Wall Gly Ser Asp Gly Lell Met Phe Ser 515 525

Lell Cys His Ala Gly Pro Ala Ala Thir Glu Thir Asn Wall Asp Gly 53 O 535 54 O

Glin Wall Asp Ser Ile Pro Glin Asn Luell Ser Ser 5.45 550 555 560

Gly Phe Gly His Ala Thir Ala Gly Cys Thir Ser Thir Glin Arg 565 st O sts

Pro Luell Luell Lell Wall Phe Ile Phe Wall Wall Ile Phe Phe Thir Phe 585 59 O

Lell Ser Ser Ile Pro Lell Thir Ala Thir Luell Arg Cys Wall Arg Asp 595 605

Pro Glin Arg Ser Phe Lell Gly Ile Glin Trp Ile Wall Wall Arg Ile 610 615

Lell Gly Gly Ile Pro Pro Ile Ala Phe Gly Trp Wall Ile Asp Lys 625 635 64 O

Ala Luell Luell Trp Asp Glin Gly Glin Glin Gly Ser Cys Luell 645 650 655

Wall Glin Asn Ser Met Ser Arg Ile Lell Ile Met Gly Luell 660 665 67 O

Lell Tyr Lys Wall Lell Wall Luell Phe Phe Ala Ile Ala Phe Luell 675 685

Lys Pro Luell Ser Ser Ser Asp Gly Luell Glu Thir Luell Pro 69 O. 695 7 OO

Ser Glin Ser Ser Ala Asp Ser Ala Thir Asp Ser Glin Luell Glin Ser 7 Os 71s 72O

Ser Wall

SEQ ID NO 13 LENGTH: TYPE: DNA ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 13

US 7,795,392 B2 109 110

- Continued gctaca accc aac tactggc 2O

<210s, SEQ ID NO 22 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 22 gggactaact gtgatactgg

<210s, SEQ ID NO 23 &211s LENGTH: 661 212. TYPE: PRT &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 23

Met Gly Lys Ser Glu Lys Arg Wall Ala Thir His Gly Wall Arg Cys Phe 1. 5 15

Ala Lys Ile Llys Met Phe Lieu Luell Ala Luell Thir Ala Tyr Wall Ser 2O 25 3O

Lys Ser Leu Ser Gly Thr Tyr Met Asn Ser Met Lell Thir Glin Ile Glu 35 4 O 45

Arg Glin Phe Gly Ile Pro Thr Ser Ile Wall Gly Lell Ile Asn Gly Ser SO 55 6 O

Phe Glu Ile Gly Asn Lieu. Lieu. Luell Ile Ile Phe Wall Ser Phe Gly 65 70

Thir Lys Lieu. His Arg Pro Ile Met Ile Gly Wall Gly Ala Wall Met 85 90 95

Gly Lieu. Gly Cys Phe Lieu. Ile Ser Luell Pro His Phe Lell Met Gly Glin 1OO 105 11 O

Tyr Glu Tyr Glu Thir Ile Leu Pro Thir Ser ASn Wall Ser Ser Asn Ser 115 12 O 125

Phe Phe Cys Val Glu Asn Arg Ser Glin Thir Luell Asn Pro Thir Glin Asp 13 O 135 14 O

Pro Ser Glu. Cys Val Lys Glu Met Ser Luell Met Trp Ile Wall 145 150 155 160

Lieu Val Gly Asn. Ile Ile Arg Gly Ile Gly Glu Thir Pro Ile Met Pro 1.65 17O

Lieu. Gly Ile Ser Tyr Ile Glu Asp Phe Ala Ser Glu Asn Ser Pro 18O 185 19 O

Lieu. Tyr Ile Gly Ile Lieu. Glu Thir Gly Met Thir Ile Gly Pro Luell Ile 195

Gly Lieu. Lieu. Lieu Ala Ser Ser Ala Asn Ile Tyr Wall Asp Ile Glu 21 O 215 22O

Ser Val Asn. Thir Asp Asp Lieu. Thir Ile Thir Pro Thir Asp Thir Arg Trp 225 23 O 235 24 O

Val Gly Ala Trp Trp Ile Gly Phe Luell Wall Ala Gly Wall Asn Ile 245 250 255

Lieu. Thir Ser Phe Pro Phe Phe Phe Phe Pro Thir Lell Pro Glu 26 O 265 27 O

Gly Lieu. Glin Glu Asn. Wall Asp Gly Thir Glu ASn Ala Lys Glu 27s 285

His Arg Llys Lys Ala Lys Glu Glu Gly Ile Thir Asp Phe 29 O 295 3 OO

Phe Val Phe Met Lys Ser Lieu. Ser Asn Pro Ile Met Luell Phe US 7,795,392 B2 111 112

- Continued

3. OS 310 315

Ile Luell Ile Ser Wall Lell Glin Phe Asn Ala Phe Ile Asn Ser Phe Thir 3.25 330 335

Phe Met Pro Lys Tyr Lell Glu Glin Glin Tyr Gly Ser Thir Ala Glu 34 O 345 35. O

Wall Wall Phe Luell Met Gly Lell Tyr Met Luell Pro Pro Ile Luell Gly 355 360 365

Luell Ile Gly Gly Lell Ile Met Phe Lys Wall Thir Wall 37 O 375

Lys Ala Ala His Lell Ala Phe Trp Luell Luell Ser Glu Tyr Luell Luell 385 390 395 4 OO

Ser Phe Luell Ser Tyr Wall Met Thir Asp ASn Phe Pro Wall Ala Gly 4 OS 41O 415

Lell Thir Thir Ser Tyr Glu Gly Wall Glin His Glin Lell Wall Glu Asn 425 43 O

Wall Luell Ala Asp Asn Thir Arg ASn Ser Thir Asn Thir 435 44 O 445

Trp Asp Pro Wall Cys Gly Asp Asn Gly Luell Ala Tyr Met Ser Ala 450 45.5 460

Lell Ala Gly Glu Lys Ser Wall Gly Thir Gly Thir Asn Met Wall Phe 465 470 48O

Glin Asn Ser Cys Ile Glin Ser Ser ASn Ser Ser Ala Wall Luell 485 495

Gly Luell Asn Gly Pro Asp Cys ASn Lell Glin Phe SOO 505

Lell Ile Ile Ala Ile Phe Gly Cys Phe Ser Lell Ala Ile 515 525

Pro Gly Tyr Met Wall Lell Lell Arg Ser Glu Glu Ser 53 O 535 54 O

Lell Gly Wall Gly Lell His Ala Phe Arg Ile Lell Ala Gly Ile 5.45 550 555 560

Pro Ala Pro Ile Tyr Phe Gly Ala Luell Asp Arg Thir Luell His 565 sts

Trp Gly Thir Luell Gly Glu Pro Ala Arg Met Asp 585 59 O

Ile Asn Ser Phe Arg Lell Tyr Luell Luell Pro Ala Ala Luell Arg 595 605

Gly Ala Ser Phe Wall Pro Ala Phe Phe Luell Arg Lell Thir Arg Thir 610 615 62O

Phe Glin Phe Pro Gly Asp Ile Glu Ser Ser Lys Thir Asp His Ala Glu 625 630 635 64 O

Met Luell Thir Lell Glu Ser Glu Cys Thir Glu Wall Luell Arg Ser 645 650 655

Wall Thir Glu Asp 660

<210s, SEQ ID NO 24 &211s LENGTH: 670 212. TYPE : PRT &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 24 Met Gly Glu Thr Glu Lys Arg Val Ala Thr His Glu Val Arg Cys Phe 1. 5 15 US 7,795,392 B2 113 114

- Continued

Ser Ile Lys Met Phe Lell Luell Ala Luell Thir Trp Ala Tyr Val Ser 25

Glin Ser Luell Ser Gly Ile Met Asn Thir Met Lell Thir Glin Ile Glu 35 4 O 45

Arg Glin Phe Asp Ile Pro Ile Ser Ile Wall Gly Phe Ile Asn Gly Ser SO 55 6 O

Phe Glu Ile Gly Asn Phe Lell Luell Ile Ile Phe Wall Ser Phe Gly 65 70

Thir Luell His Arg Pro Ile Met Ile Gly Wall Gly Wall Ile Met 85 90 95

Gly Luell Gly Cys Phe Lell Met Ser Luell Pro His Phe Lell Met Gly Arg 105 11 O

Glu Tyr Glu Thir Thir Ile Ser Pro Thir Ser Asn Lell Ser Ser Asn 115 12 O 125

Ser Phe Luell Met Glu Asn Arg Ser Glin Thir Lell Pro Thir Glin 13 O 135 14 O

Asp Pro Ala Glu Ile Glu Met Ser Lell Met Trp Ile Tyr 145 150 155 160

Wall Luell Wall Gly Asn Ile Ile Arg Gly Ile Gly Glu Thir Pro Ile Met 1.65 17O 17s

Pro Luell Gly Ile Ser Ile Glu Asp Phe Ala Ser Glu Asn Ser 18O 185 19 O

Pro Luell Tyr Ile Gly Ile Lell Glu Thir Gly Lys Wall Phe Gly Pro Ile 195

Wall Gly Luell Luell Lell Gly Ser Phe Ala Ser Ile Wall Asp Thir 21 O 215 22O

Gly Ser Wall Asn Thir Asp Asp Luell Thir Ile Thir Pro Thir Asp Thir Arg 225 23 O 235 24 O

Trp Wall Gly Ala Trp Trp Ile Gly Phe Luell Ile Ala Gly Wall Asn 245 250 255

Ile Luell Ser Ser Ile Pro Phe Phe Phe Phe Pro Thir Luell Pro 26 O 265 27 O

Glu Gly Luell Glin Asp Asp Wall Asp Gly Thir ASn Asn Asp Glu Glu 285

His Arg Glu Ala Lys Glu Glu Asn Arg Gly Ile Thir Asp 29 O 295 3 OO

Phe Luell Pro Phe Met Lys Ser Luell Ser ASn Pro Ile Met Luell 3. OS 310 315

Lell Ile Luell Thir Ser Wall Lell Glin Ile Asn Ala Phe Ile Asn Met Phe 3.25 330 335

Thir Phe Luell Pro Lell Glu Glin Glin Tyr Gly Ser Thir Ala 34 O 345 35. O

Glu Wall Wall Luell Lell Ile Gly Wall Asn Luell Pro Ile Ile 355 360

Gly Tyr Luell Luell Ile Gly Phe Ile Met Phe Ile Thir Wall 37 O 375

Lys Ala Ala Tyr Met Ala Phe Luell Ser Lell Phe Glu Tyr Luell 385 390 395 4 OO

Lell Phe Luell His Phe Met Ile Thir Cys Asp Asn Phe Pro Wall Ala 4 OS 41O 415

Gly Luell Thir Ala Lell Tyr Glu Gly Wall His His Pro Lel Tyr Wall Glu 425 43 O

Asn Wall Luell Ala Asp Asn Arg Gly Cys Ser Ser Thir Asn US 7,795,392 B2 115 116

- Continued

435 44 O 445

Ser Trp Asp Pro Wall Gly Asp Asn Gly Luell Ala Tyr Met Ser Ala 450 45.5 460

Cys Luell Ala Gly Cys Lys Ser Wall Gly Thir Gly Thir Asn Met Wall 465 470

Phe Glin Asn Ser Ile Arg Ser Ser Gly Asn Ser Ser Ala Wall 485 490 495

Lell Gly Luell Cys Lys Gly Pro Glu Ala Asn Luell Glin Tyr SOO 505

Phe Luell Ile Met Ser Wall Ile Gly Ser Phe Ile Ser Ile Thir Ala 515 525

Ile Pro Gly Met Wall Lell Luell Arg Ile Lys Pro Glu Lys 53 O 535 54 O

Ser Luell Gly Ile Gly Lell His Phe Thir Arg Wall Phe Ala Gly 5.45 550 555 560

Ile Pro Ala Pro Ile Phe Ala Luell Ile Asp Arg Thir Cys Luell 565 st O sts

His Trp Gly Thir Lell Glu Pro Gly Ala Arg Met Tyr 585 59 O

Asn Ile Asn Asn Phe Arg Arg Luell Wall Lell Pro Ala Ala Luell 595 605

Arg Gly Ser Ser Tyr Lell Pro Luell Phe Ile Lell Ile Luell Met Arg 610 615

Lys Phe Glin Phe Pro Gly Glu Asp Ser Ser Glu Thir Glu Luell Ala 625 630 635 64 O

Glu Met Ile Thir Wall Ser Glu Thir Asp Wall His Gly 645 650 655

Ser Pro Glin Wall Glu Asn Asp Gly Glu Luell Thir Arg Luell 660 665 67 O

<210s, SEQ ID NO 25 &211s LENGTH: 669 212. TYPE : PRT &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 25 Met Gly Asp Lieu Glu Gly Ala Ala Thir His Gly Ala Gly Cys Phe 1. 5 15

Ala Lys Ile Lys Wall Phe Lell Met Ala Luell Thir Ala Tyr Wall Ser 2O 25 3O

Ser Luell Ser Gly Thir Phe Met Ser Ser Met Lell Thir Glin Ile Glu 35 4 O 45

Arg Glin Phe Gly Ile Pro Thir Ala Ile Wall Gly Phe Ile Asn Gly Ser SO 55 6 O

Phe Glu Ile Gly Asn Lell Lell Luell Ile Ile Phe Wall Ser Phe Gly 65 70

Met Luell His Arg Pro Ile Wall Ile Gly Wall Gly Ala Wall Met 85 90 95

Gly Luell Gly Cys Phe Ile Ile Ser Luell Pro His Phe Lell Met Gly Arg 105 11 O

Glu Tyr Glu Thir Thir Ile Luell Pro Thir Ser Asn Lell Ser Ser Asn 115 12 O 125

Ser Phe Luell Met Glu Asn Glin Thir Glin Thir Lell Asn Pro Ala Glin 13 O 135 14 O US 7,795,392 B2 117 118

- Continued Asp Pro Ala Glu Wall Glu Wall Lys Ser Lieu Met Trp Ile Tyr 145 150 155 160

Wall Luell Wall Gly Asn Ile Ile Arg Gly Ile Gly Glu Thir Pro Ile Met 1.65 17O 17s

Pro Luell Gly Wall Ser Ile Glu Asn Phe Ala Ser Glu Asn Ser 18O 185 19 O

Pro Luell Tyr Ile Gly Ile Lell Glu Thir Gly Lys Met Ile Gly Pro Ile 195

Phe Gly Luell Luell Lell Gly Ser Phe Ala Ser Ile Wall Asp Thir 21 O 215 22O

Gly Ser Wall Asn Thir Asp Asp Luell Thir Ile Thir Pro Thir Asp Ile Arg 225 23 O 235 24 O

Trp Wall Gly Ala Trp Trp Ile Gly Phe Luell Wall Ala Gly Wall Asn 245 250 255

Ile Luell Ile Ser Ile Pro Phe Phe Phe Phe Pro Thir Luell Pro 26 O 265 27 O

Glu Gly Luell Glin Glu Asn Wall Asp Gly Thir Glu Asn Ala Glu Glu 285

Ser Thir Glu Pro Arg Asn Arg Gly Ile Thir Asp 29 O 295 3 OO

Phe Phe Pro Phe Lell Lys Ser Pro Wall Luell Glin Pro Asp Luell His Ala 3. OS 310 315

Wall His Pro Lys Wall Lell Glin Wall Asn Ala Phe Asn Ile Tyr Phe 3.25 330 335

Ser Phe Luell Pro Lell Glu Asn Glin Tyr Gly Ser Thir Ala 34 O 345 35. O

Glu Wall Ile Phe Lell Met Gly Wall Asn Luell Pro Ala Ile Ile 355 360 365

Gly Tyr Luell Ile Ala Gly Phe Met Met Phe Ile Thir Wall 37 O 375

Lys Thir Ala Ala Phe Lell Arg Phe Luell Ser Lell Ser Glu Tyr Ser 385 390 395 4 OO

Phe Gly Phe Asn Phe Lell Ile Thir Cys Asp Asn Wall Pro Wall Ala 4 OS 415

Gly Luell Thir Asn Ser Glu Arg Asp Glin Lys Pro Lell Tyr Luell Glu 42O 425 43 O

Asn Asn Wall Luell Ala Asn Thir Arg Cys Ser Cys Luell Thir 435 44 O 445

Thir Trp Asp Pro Wall Gly Asp Asn Gly Luell Ala Met Ser Ala 450 45.5 460

Cys Luell Ala Gly Glu Ser Wall Gly Thir Gly Thir Asn Met Wall 465 470 48O

Phe His Asn Ser Ile Glin Ser Pro Gly Asn Ser Ser Ala Wall 485 490 495

Lell Gly Luell Cys Asn Gly Pro Glu Thir Asn Luell Glin Tyr SOO 505

Lell Luell Ile Luell Ser Gly Phe Luell Ser Ile Luell Tyr Ser Phe Ala Ala 515 525

Ile Pro Gly Tyr Met Wall Phe Luell Arg Ile Lys Ser Glu Glu 53 O 535 54 O

Ser Luell Gly Ile Gly Ile His Ala Phe Ile Arg Wall Phe Ala Gly 5.45 550 555 560

Ile Pro Ala Pro Ile Phe Gly Ala Luell Ile Asp Arg Thir Luell US 7,795,392 B2 119 120

- Continued

565 st O sts

His Trp Gly Thir Glin Gly Ala Pro Gly Arg Arg Met Tyr Asp 58O 585 59 O

Ile Asn Ser Phe Arg Ile Tyr Luell Gly Met Ser Ala Ala Luell Arg 595 605

Gly Ser Ser Lell Pro Ala Phe Wall Ile Wall Ile Lell Thir Arg 610 615

Phe Ser Luell Pro Gly Lys Ile Asn Ser Ser Glu Met Glu Ile Ala Glu 625 630 635 64 O

Met Luell Thir Glu Glu Ser Glin Cys Thir Asp Wall His Arg Asn 645 650 655

Pro Phe Lys Asn Asp Gly Glu Luell Thir Lell 660 665

<210s, SEQ ID NO 26 &211s LENGTH: 670 212. TYPE : PRT &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 26

Met Glu Glu. Thir Glu Ile Ala Thir Glin Glu Gly Arg Luell Phe 1. 5 15

Ser Lys Met Lys Wall Phe Lell Luell Ser Luell Thir Ala Cys Luell Thir 25 3O

Ser Luell Ser Gly Wall Met Asn Ser Met Lell Thir Glin Ile Glu 35 4 O 45

Arg Glin Phe Asp Ile Ser Thir Ser Wall Ala Gly Lell Ile Asn Gly Ser SO 55 6 O

Phe Glu Ile Gly Asn Lell Phe Phe Ile Wall Phe Wall Ser Phe Gly 65 70

Thir Luell His Arg Pro Wall Wall Ile Gly Ile Gly Wall Ile Met 85 90 95

Gly Luell Gly Cys Lell Lell Met Ser Luell Pro His Phe Phe Met Gly Arg 105 11 O

Glu Tyr Glu Thir Thir Ile Ser Pro Thir Gly Asn Lell Ser Ser Asn 115 12 O 125

Ser Phe Luell Met Glu Asn Arg Thir Glin Thir Lell Pro Thir Glin 13 O 135 14 O

Asp Pro Ala Glu Wall Glu Met Ser Lell Met Trp Ile Cys 145 150 155 160

Wall Met Wall Gly Asn Ile Ile Arg Gly Ile Gly Glu Thir Pro Ile Wall 1.65 17O 17s

Pro Luell Gly Ile Ser Ile Glu Asp Phe Ala Ser Glu Asn Ser 18O 185 19 O

Pro Luell Tyr Ile Gly Ile Lell Glu Met Gly Lys Wall Ala Gly Pro Ile 195

Phe Gly Luell Luell Lell Gly Ser Ala Glin Ile Wall Asp Ile 21 O 215 22O

Gly Ser Wall Asn Thir Asp Asp Luell Thir Ile Thir Pro Ser Asp Thir Arg 225 23 O 235 24 O

Trp Wall Gly Ala Trp Trp Ile Gly Phe Luell Wall Ala Gly Wall Asn 245 250 255

Ile Luell Thir Ser Ile Pro Phe Phe Phe Luell Pro Ala Luell Pro 26 O 265 27 O US 7,795,392 B2 121 122

- Continued

Gly Glin Glin Glu Asn Wall Ala Wall Thir Asp Gly Lys Wall Glu 27s 285

Tyr Gly Gly Glin Ala Arg Glu Glu Asn Luell Gly Ile Thir Lys Asp 29 O 295 3 OO

Phe Luell Thir Phe Met Lys Arg Luell Phe Cys ASn Pro Ile Met Luell 3. OS 310 315

Phe Ile Luell Thir Ser Wall Lell Glin Wall Asn Gly Phe Ile Asn Lys Phe 3.25 330 335

Thir Phe Luell Pro Lys Lell Glu Glin Glin Gly Ser Thir Ala 34 O 345 35. O

Glu Ala Ile Phe Lell Ile Gly Wall Ser Luell Pro Pro Ile Luell 355 360 365

Gly Tyr Luell Ile Gly Gly Phe Ile Met Lys Phe Ile Thir Wall 37 O 375

Lys Ala Ala Tyr Lell Ala Phe Luell Ser Wall Phe Glu Luell 385 390 395 4 OO

Lell Phe Luell His Phe Met Luell Thir Cys Asp Asn Ala Ala Wall Ala 4 OS 415

Gly Luell Thir Thir Ser Gly Wall Glin His Glin Lell His Wall Glu 42O 425 43 O

Ser Wall Luell Ala Asn Thir Arg Ser Cys Ser Thir Asn 435 44 O 445

Thir Trp Asp Pro Wall Gly Asp Asn Gly Wall Ala Met Ser Ala 450 45.5 460

Cys Luell Ala Gly Cys Lys Phe Wall Gly Thir Gly Thir Asn Met Wall 465 470

Phe Glin Asp Ser Ile Glin Ser Luell Gly Asn Ser Ser Ala Wall 485 490 495

Lell Gly Luell Cys Lys Gly Pro Glu Ala Asn Arg Luell Glin Tyr SOO 505

Phe Luell Ile Luell Thir Ile Ile Ile Ser Phe Ile Ser Luell Thir Ala 515 525

Ile Pro Gly Met Wall Phe Luell Arg Wall Lys Ser Glu Glu Lys 53 O 535 54 O

Ser Luell Gly Wall Gly Lell His Thir Phe Ile Arg Wall Phe Ala Gly 5.45 550 555 560

Ile Pro Ala Pro Wall Phe Ala Luell Ile Asp Arg Thir Cys Luell 565 st O sts

His Trp Gly Thir Lell Glin Arg Gly Ala Arg Met Tyr 585 59 O

Asp Ile Asn Ser Phe Arg His Luell Gly Lell Pro Ile Ala Luell 595 605

Arg Gly Ser Ser Tyr Lell Pro Phe Phe Ile Lell Ile Luell Met Arg 610 615

Lys Phe Glin Phe Pro Gly Asp Asp Ser Ser Ala Thir Asp His Thir 625 630 635 64 O

Glu Met Met Luell Gly Glu Ser Glu His Thir Asp Wall His Gly 645 650 655

Ser Pro Glin Wall Glu Asn Asp Glu Luell Lys Thir Luell 660 665 67 O

<210s, SEQ ID NO 27 &211s LENGTH: 670 212. TYPE : PRT US 7,795,392 B2 123 124

- Continued

&213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 27

Met Gly Glu Thr Glu Lys Arg Ile Glu Thir His Arg Ile Arg Cys Luell 1. 5 1O 15

Ser Lys Luell Lys Met Phe Lieu. Leu Ala Ile Thr Cys Ala Phe Val Ser 25 3O

Thir Luell Ser Gly Ser Tyr Met Asn Ser Met Lieu. Thr Glin Ile Glu 35 4 O 45

Arg Glin Phe Asn Ile Pro Thr Ser Leu Val Gly Phe Ile Asin Gly Ser SO 55 6 O

Phe Glu Ile Gly Asn Lieu Lleu Lieu. Ile Ile Phe Val Ser Tyr Phe Gly 65 70 7s

Thir Luell His Arg Pro Ile Met Ile Gly Ile Gly Cys Val Val Met 85 90 95

Gly Luell Gly Cys Phe Leu Lys Ser Leu Pro His Phe Leu Met Asn Glin 105 11 O

Glu Tyr Glu Ser Thr Val Ser Val Ser Gly Asn Lieu Ser Ser Asn 115 12 O 125

Ser Phe Luell Met Glu Asn Gly Thr Glin Ile Lieu. Arg Pro Thr Glin 13 O 135 14 O

Asp Pro Ser Glu Cys Thr Lys Glu Val Lys Ser Leu Met Trp Val Tyr 145 150 155 160

Wall Luell Wall Gly Asn Ile Val Arg Gly Met Gly Glu Thr Pro Ile Luell 1.65 17O 17s

Pro Luell Gly Ile Ser Tyr Ile Glu Asp Phe Ala Lys Phe Glu Asn Ser 18O 185 19 O

Pro Luell Tyr Ile Gly Lieu Val Glu Thr Gly Ala Ile Ile Gly Pro Luell 195 2OO 2O5

Ile Gly Luell Luell Lieu Ala Ser Phe Cys Ala Asn Val Tyr Val Asp Thir 21 O 215 22O

Gly Phe Wall Asn Thr Asp Asp Lieu. Ile Ile Thr Pro Thr Asp Thr Arg 225 23 O 235 24 O

Trp Wall Gly Ala Trp Trp Phe Gly Phe Lieu. Ile Cys Ala Gly Val Asn 245 250 255

Wall Luell Thir Ala Ile Pro Phe Phe Phe Lieu. Pro Asn. Thir Lieu Pro 26 O 265 27 O

Glu Gly Luell Glu Thir Asn Ala Asp Ile Ile Lys Asn. Glu Asn. Glu Asp 28O 285

Glin Glu Glu Val Llys Lys Glu Lys Tyr Gly Ile Thir Lys Asp 29 O 295 3 OO

Phe Luell Pro Phe Met Lys Ser Leu Ser Cys Asn Pro Ile Tyr Met Luell 3. OS 310 315

Phe Ile Luell Wall Ser Wall Ile Glin Phe Asn Ala Phe Wall Asn. Met Ile 3.25 330 335

Ser Phe Met Pro Lys Tyr Lieu. Glu Gln Glin Tyr Gly Ile Ser Ser Ser 34 O 345 35. O

Asp Ala Ile Phe Lieu Met Gly Ile Tyr Asn Lieu Pro Pro Ile Cys Ile 355 360 365

Gly Tyr Ile Ile Gly Gly Lieu. Ile Met Lys Llys Phe Lys Ile Thr Wall 37 O 375 38O

Lys Glin Ala Ala His Ile Gly Cys Trp Lieu. Ser Lieu. Lieu. Glu Tyr Luell 385 390 395 4 OO US 7,795,392 B2 125 126

- Continued Lell Tyr Phe Leu Ser Phe Leu Met Thir Cys Glu Asn. Ser Ser Wall Wall 4 OS 415

Gly Ile Asn Thr Ser Tyr Glu Gly Ile Pro Glin Asp Lell Tyr Wall Glu 42O 425 43 O

Asn Asp Ile Phe Ala Asp Cys Asn Wall Asp Asn Cys Pro Ser 435 44 O 445

Ile Trp Asp Pro Val Cys Gly Asn Asn Gly Luell Ser Luell Ser Ala 450 45.5 460

Cys Lieu Ala Gly Cys Glu Thir Ser Ile Gly Thir Gly Ile Asn Met Wall 465 470

Phe Glin Asn Cys Ser Cys Ile Glin Thir Ser Gly Asn Ser Ser Ala Wall 485 490 495

Lell Gly Lieu. Cys Asp Llys Gly Pro Asp Ser Lell Met Luell Glin Tyr SOO 505

Phe Lieu. Ile Lieu. Ser Ala Met Ser Ser Phe Ile Ser Luell Ala Ala 515 52O 525

Ile Pro Gly Tyr Met Val Lieu. Leu Arg Met Lys Ser Glu Glu 53 O 535 54 O

Ser Leu Gly Val Gly Lieu. His Thr Phe Thir Arg Wall Phe Ala Gly 5.45 550 555 560

Ile Pro Ala Pro Ile Tyr Phe Gly Ala Luell Met Asp Ser Thir Cys Luell 565 st O sts

His Trp Gly. Thir Lieu Lys Cys Gly Glu Ser Gly Ala Arg Ile Tyr 58O 585 59 O

Asp Ser Thir Thr Phe Arg Tyr Ile Luell Gly Lell Pro Ala Ala Luell 595 6OO 605

Arg Gly Ser Ser Phe Val Pro Ala Luell Ile Ile Lell Ile Luell Luell Arg 610 615

Lys Cys His Lieu Pro Gly Glu Asn Ala Ser Ser Gly Thir Glu Luell Ile 625 630 635 64 O

Glu Thir Lys Wall Lys Gly Lys Glu Asn Glu Cys Asp Ile Tyr Glin 645 650 655

Ser Thr Val Lieu Lys Asp Asp Glu Luell Lys Thir Luell 660 665 67 O

<210s, SEQ ID NO 28 &211s LENGTH: 643 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 28

Met Gly Lieu. Lieu Pro Llys Lieu. Gly Wall Ser Glin Gly Ser Asp Thir Ser 1. 5 15

Thir Ser Arg Ala Gly Arg Cys Ala Arg Ser Wall Phe Gly Asn Ile 2O 25

Wall Phe Val Lieu. Cys Glin Gly Lieu. Luell Glin Luell Glin Luell Luell Tyr 35 4 O 45

Ser Ala Tyr Phe Lys Ser Ser Leu Thir Thir Ile Glu Arg Phe Gly SO 55 6 O

Lell Ser Ser Ser Ser Ser Gly Lieu. Ile Ser Ser Lell Asn Glu Ile Ser 65 70

Asn Ala Ile Lieu. Ile Ile Phe Wall Ser Tyr Phe Gly Ser Arg Wall His 85 90 95

Arg Pro Arg Lieu. Ile Gly Ile Gly Gly Luell Phe Lell Ala Ala Gly Ala 1OO 105 11 O US 7,795,392 B2 127 128

- Continued

Phe Ile Luell Thir Lell Pro His Phe Luell Ser Glu Pro Tyr Glin Tyr Thr 115 12 O 125

Lell Ala Ser Thir Gly Asn Asn Ser Arg Luell Glin Ala Glu Luell Cys Glin 13 O 135 14 O

Lys His Trp Glin Asp Lell Pro Pro Ser Cys His Ser Thir Thr Gin 145 150 155 160

Asn Pro Glin Glu Thir Ser Ser Met Trp Gly Lell Met Wall Wall Ala 1.65 17O 17s

Glin Luell Luell Ala Gly Ile Gly Thir Wall Pro Ile Glin Pro Phe Gly Ile 18O 185 19 O

Ser Wall Asp Asp Phe Ser Glu Pro Ser ASn Ser Pro Luell Tyr Ile 195

Ser Ile Luell Phe Ala Ile Ser Wall Phe Gly Pro Ala Phe Gly Tyr Lieu. 21 O 215

Lell Gly Ser Ile Met Lell Glin Ile Phe Wall Asp Gly Arg Wall Asn 225 23 O 235 24 O

Thir Ala Ala Wall Asn Lell Wall Pro Gly Asp Pro Arg Trp Ile Gly Ala 245 250 255

Trp Trp Luell Gly Lell Lell Ile Ser Ser Ala Luell Lell Wall Luell Thir Ser 26 O 265 27 O

Phe Pro Phe Phe Phe Phe Pro Arg Ala Met Pro Ile Gly Ala 27s 285

Ala Pro Ala Thir Ala Asp Glu Ala Arg Luell Glu Glu Ala Llys Ser 29 O 295 3 OO

Arg Gly Ser Luell Wall Asp Phe Ile Phe Pro Ile Phe Lieu. 3. OS 310 315 32O

Arg Luell Luell Met Asn Ser Lell Phe Wall Luell Wall Wall Lell Ala Gln Cys 3.25 330 335

Thir Phe Ser Ser Wall Ile Ala Gly Luell Ser Thir Phe Lell Asn Llys Phe 34 O 345 35. O

Lell Glu Lys Glin Tyr Gly Thir Ser Ala Ala Ala Asn Phe Lieu. Ile 355 360 365

Gly Ala Wall Asn Lell Pro Ala Ala Ala Luell Gly Met Lell Phe Gly Gly 37 O 375

Ile Luell Met Phe Wall Phe Ser Luell Glin Thir Ile Pro Arg Ile 385 390 395 4 OO

Ala Thir Thir Ile Ile Thir Ile Ser Met Ile Luell Wall Pro Lieu. Phe 4 OS 415

Phe Met Gly Cys Ser Thir Thir Wall Ala Glu Wall Pro Pro Ser 425 43 O

Thir Ser Ser Ser Ile His Glin Ser Pro Ala Arg Arg Asp Cys 435 44 O 445

Ser Cys Pro Asp Ser Ile Phe His Pro Wall Gly Asp Asn Gly Ile 450 45.5 460

Glu Tyr Luell Ser Pro Cys His Ala Gly Ser Asn Ile Asn Met Ser 465 470 47s

Ser Ala Thir Ser Lys Glin Lel Ile Luell ASn Ser Wall. Thir 485 490 495

Gly Gly Ser Ala Ser Ala Thir Gly Ser Cys Pro Wall Pro Cys Ala SOO 505 51O

His Phe Luell Luell Pro Ala Ile Phe Luell Ile Ser Phe Wall Ser Lieu. Ile 515 52O 525 US 7,795,392 B2 129 130

- Continued Ala Cys Ile Ser His Asn Pro Luell Tyr Met Met Wall Lieu. Arg Val Wall 53 O 535 54 O

Asn Glin Glu Glu Lys Ser Phe Ala Ile Gly Wall Glin Phe Luell Luell Met 5.45 550 555 560

Arg Luell Luell Ala Trp Lell Pro Ser Pro Ala Luell Tyr Gly Luell Thir Ile 565 st O sts

Asp His Ser Cys Ile Arg Trp Asn Ser Luell Cys Lell Gly Arg Arg Gly 585 59 O

Ala Ala Tyr Asp Asn Asp Ala Luell Arg Asp Arg Luell Gly 595 605

Lell Glin Met Gly Tyr Ala Luell Gly Met Luell Lell Lell Phe Ile 610 615

Ser Trp Arg Wall Lys Lys Asn Glu Tyr ASn Wall Glin Ala Ala 625 630 635 64 O

Gly Luell Ile

SEQ ID NO 29 LENGTH: 384 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Construct

<4 OOs, SEQUENCE: 29

Gly Glu Ala Cys Phe Ile Phe Luell Luell Ala Lell Ala Luell Gly 1. 5 15

Met Ser Luell Thir Glin Ile Glu Arg Arg Phe Ile Ser Ser Wall Gly Luell 25

Ile Gly Ser Phe Glu Ile Gly Asn Luell Luell Luell Ile Phe Wall Ser 35 4 O 45

Phe Gly Luell His Arg Pro Ile Gly Gly Cys Met Gly Luell Gly Luell SO 55 6 O

Lell Pro His Phe Lell Met Gly Tyr Glu Tyr Glu Asn Ser Luell Asn 65 70

Ser Luell Pro Asp Glu Glu Ser Luell Met Trp Ile Wall Wall 85 90 95

Gly Asn Ile Luell Arg Gly Ile Gly Glu Thir Pro Ile Pro Luell Gly Ile 105 11 O

Ser Ile Asp Asp Phe Ala Lys Glu Asn Ser Pro Lell Tyr Ile Gly 115 12 O 125

Ile Luell Thir Gly Pro Gly Lell Luell Gly Ser Ala Ile Wall Asp 13 O 135 14 O

Gly Wall Asn Thir Asp Lell Ile Thir Pro Asp Pro Arg Trp Wall Gly Ala 145 150 155 160

Trp Trp Gly Phe Lell Ala Gly Luell Ser Ile Pro Phe Phe Phe Phe 1.65 17O 17s

Pro Luell Pro Gly Wall Asp Phe Lell Lell Asn Pro Tyr 18O 185 19 O

Lell Luell Wall Glin Asn Gly Thir Phe Luell Pro Lys Lell Glu Glin Glin 195 2OO

Gly Ser Ser Ala Phe Lell Gly Luell Pro Cys Gly Gly Gly Ile Met 21 O 215 22O

Lys Phe Wall Ala Ala Luell Ala Ser Luell Tyr Lell Luell Phe Cys 225 23 O 235 24 O

Asn Wall Ala Gly Lell Thir Ser Gly Glu Ala Asp Asn Ser US 7,795,392 B2 131 132

- Continued

245 250 255

Trp Pro Wall Cys Gly Asn Gly Tyr Ser Ala Lell Ala Gly 26 O 265 27 O

Ser Gly Thir Gly Asn Wall Phe Asn Ser Ile Gly Asn Ser Ser 27s 285

Ala Wall Lieu. Gly Cys Pro Luell Phe Leu Ser Phe Ile Ser 29 O 295 3 OO

Lell Ile Pro Gly Tyr Met Wall Luell Arg Wall Glu Glu Ser 3. OS 310 315 32O

Lell Ala Gly His Arg Lieu. Ala Gly Ile Pro Ala Pro Ile Phe Gly 3.25 330 335

Ala Lieu. Ile Asp Thr Lieu. His Trp Gly Thir Gly Gly Ala 34 O 345 35. O

Arg Asp Phe Lell Gly Luell Ala Luell Arg Ser Luell Ile Lieu. 355 360 365

Lieu. Arg Lys Pro Ile Ser Ser Glu Glu Ser Thir His Asp Glu Thir 37 O 375

25 We claim: c) growing said transfected host cells in an appropriate 1. A purified or isolated protein comprising the amino acid culture media; and d) purifying the protein from said culture media. sequence of SEQID NO:2 (Organic Anion Transport Protein 3. A fusion protein comprising the protein of claim 1, 2, or OATP2) or an amino acid sequence having at least 95% 30 attached to a second polypeptide. identity to the amino acid sequence of SEQ ID NO:2 4. The protein of claim 1, comprising an amino acid (OATP2), wherein said protein has organic anion transport sequence having at least 98% identity to the amino acid activity. sequence of SEQID NO:2 (OATP2). 2. The protein of claim 1, produced by: 5. The protein of claim 1, comprising the amino acid a) inserting a nucleic acid sequence encoding said protein 35 sequence of SEQID NO:2 (OATP2). into an appropriate expression vector; 6. A protein encoded by the nucleic acid molecule in ATCC b) transfecting said expression vector into an appropriate Accession Number 207213. transfection host cell; k k k k k