AU2007237257A1 - Hematopoietin receptors HPR1 and HPR2 - Google Patents

Description

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AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Immunex Corporation Actual Inventor(s): Timothy A. Bird, David J. Cosman, Robert F. Dubose, Bruce A. Mosley, Steven R Wiley Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: HEMATOPOIETIN RECEPTORS HPR1 AND HPR2 Our Ref: 817491 POF Code: 461115/44735 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 0eooq

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N HEMATOPOIETIN RECEPTORS HPRI AND HPR2

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Z The present application is a divisional application from Australian patent application number 2002223182, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to new human and murine hematopoietin receptor polypeptides HPR1 and t HPR2, and to methods of making and using HPRI and HPR2 polypeptides.

BACKGROUND OF THE INVENTION C The hematopoietin receptor polypeptides are a related group of Type I membrane protein receptors, and in some cases soluble forms of those receptors; this family of polypeptides has Svariously been called the cytokine receptor family or the hematopoietin receptor family. There are ,l other families of receptors that bind cytokines or growth factors, such as the IL-I receptor family, the TNF receptor family, and the EGF receptor family, but the hematopoietin receptor family is considered to be a distinct group or family of receptors based on certain characteristic structural features or motifs that are shared by members of this family. Some of the members of the hematopoietin receptor family are gpl30, the granulocyte colony-stimulating factor receptor (GCSFR), leukemia inhibitory factor receptor (LIF-R), the alpha chains and the common beta chain of the IL-3 and IL-5 receptors, etc.; the hematopoietin receptor family contains more than 20 different polypeptides.

Common structural features of the hematopoietin receptor family of polypeptides include at least one extracellular cytokine receptor domain, which usually contains four cysteines and a WSXWS motif (where W is tryptophan, S is serine, and X indicates any amino acid), and, in most members of the family, a transmembrane and a cytoplasmic domain. The extracellular cytokine receptor domain is involved in ligand-binding activity, and the intracellular domain of a 'signaling' subfamily of hematopoietin receptors has a signal transduction function, transmitting the signal generated by ligand binding to a signal transduction pathway that results in the expression of genes involved in cell proliferation, differentiation, and/or activation. These activities of the hematopoietin receptor polypeptide family are mediated through interactions with cytokine ligands and other ligand-binding receptor molecules, with ligand binding to the cytokine receptor domain of hematopoietin receptor polypeptides and facilitating homo-or heterotypic interactions between receptor polypeptides, bringing the cytoplasmic domains of receptors into proximity with each other. Many of the cytokine ligands (such as IL-2, IL-6, or ciliary neurotrophic factor or CNTF, for example) interact with more than one type of heteromeric hematopoietin receptor complex, often with differing affinities, and "common" hematopoietin receptor polypeptides such as gpl30 are involved in several different heteromeric receptor complexes that bind a variety of ligands. Because of their ligand-binding and intracellular signaling activities, hematopoietin receptor polypeptides are associated with a wide variety of conditions involving cytokine-influenced cell proliferation, differentiation, or activation. For example, interaction of the gpl30 hematopoietin receptor polypeptide with its binding partners is involved in the W Vge650O.O 68999M88W9475M9475 De pages doc 0 normal upregulation of cardiac myocyte proliferation ("hypertrophy') in response to biomechanical stress on the heart, as lack of gpl30 leads to heart failure under those conditions (Hiroia et al., 1999, Cell 97(2): 189-198). Hematopoietin receptors are also involved in the activation or stimulation of cells in response to environmental factors, for example the activation of hepatocytes in the acute-phase inflammatory response to injury (Taga and Kishimoto, 1992, Crit Rev Immunol. 11(5): 265-280; Neben and Turner, 1993, Stem Cells 11 Suppl 2: 156-162). Hematopoietin receptor family polypeptides generally arc constitutively expressed in many different cell types throughout development, but the lexpression levels of hematopoictin receptor polypcptides may be up- or downregulated in response to I stimuli, and some members of the family exhibit more restricted patterns of expression in particular Stissues. Characteristics and activities of the hematopoielin receptor polypeptide family are described further in the following references, which are incorporated by reference herein: Drachman and SKaushansky, 1995, Curr Opin Hematol. 22-28; Ihle, 1995. Nature 377(6550): 591-594; Taga and C1 Kishimoto, 1995, Curr Opin Immunol. 17-23; hle et al., 1995, Annu Rev Immunol. 13: 369-398; Theze, 1994, Eur Cytokine Nelw. 353-368; Ilile et al., 1994, Trends Bioclihc Sci. 19(5): 222-227; Cosman, 1993, Cytokine 95-106; and Onishi et at., 1998, Int Rev Immunol. 16(5-6): 617-634.

In order to develop more effective treatments for disorders such as neurological, cardiac, hematopoietic, immunological, hepatic. and pulmonary conditions and diseases involving cell proliferation, differentiation. or activation, including neoplastic transformation or proliferation of virusinfected or cancerous cells, information is needed about previously unidentified members of the hematopoictin receptor polypeptide family.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

SUMMARY OF THE INVENTION The present invention is based upon the discovery of new human hematopoietin receptor family members, HPRI and HPR2.

The present invention provides an isolated nucleic acid encoding a polypeptide that signals through one or more STAT polypeptides, said polypeptide comprising: a) a cytokine-receptor domain; b) one or more fibronectin domains; c) a transmembrane domain; and d) an intracellular domain comprising the amino acid sequence set forth as 557-745 of SEQ ID NO:4, wherein said amino acid sequence comprises a tyrosine that can be phosphorylated by a kinase.

The invention provides an isolated polypeptide consisting of, consisting essentially of, or more preferably, comprising an amino acid ,cquencc sclectcd front the group consisting of: Z(a) the amino acid sequence of SEQ TID NO:4; amino acids 56 through 77 of SEQ ID NO: 1; an amino acid sequence selected from the group consisting of: amino acids I through of SEQ ID NO: I1; amino acids 5 through 40 of SEQ ID NO:2; amino acids I through 32 of SEQ ID NO:4; amino acids I through 241 of S1EQ ID NO:4; amino acids I through 525 of SEQ ID NO:4; amino acids 20 throuuh 32 of SEQ ID NO:4; amino acids 33 through 134 of SEQ ID NO:4; amino acids Xaal through Xaa2 of SEQ 11) NO:4. wherein Xua is selected fromt the group consisting of amino acids 33 through 43 of SEQ ID NO:4 and Xaa2 is selcted from the group consisting of amino icids 228 throuigh 241 of SFQ It- NC):4, aminn acids 33 through 239 of SEQ c~l ID NO:4; amino acids 33 through 241 of SEQ ID NO:4. aminu acids 33 through 525 of SEQ ID NO:4; amino acids 33 through 745 of SEQ ID NO:.4; amino acids 44 through 94 of.SEQ ID NQ:4; amino acids 139 through 241 of SEQ ID NO:4; amino acids 242 through 326 of SEQ ID NO:4; amino acids 242 through 514 of SEQ ID NO:4; amino acids 337 through 419 of SEQ ID NO:4; 2a. 5 amino acids 433 through 514 of SEQ ID NO:4; amino acids 526 through 556 of SEQ ID NO:4; 0 amino acids 533 through 552 of SEQ ID NO:4; amino acids 553 through 745 of SEQ ID NO:4; zamino acids 557 through 745 of SEQ ID NO:4; amino acids 563 through 573 of SEQ ID NO:4; amino acids 563 through 641 of SEQ ID NO:4; amino acids 567 through 581 of SEQ ID NO:4; amino acids 588 through 639 of SEQ ID NO:4; and amino acids 631 through 641 of SEQ ID 10 NO:4; fragments of the amino acid sequences of any of comprising at least contiguous amino acids; fragments of the amino acid sequences of any of comprising at least Scontiguous amino acids; 0 15 fragments of the amino acid sequences of any of having HPRI polypeptide activity; fragments of the amino acid sequences of any of comprising cytokine receptor domain amino acid sequences; an allelic variant of any of amino acid sequences comprising at least 20 amino acids and sharing amino acid identity with the amino acid sequences of any of wherein the percent amino acid identity is selected from the group consisting of: at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%; an amino acid sequence of any of wherein the polypeptide comprising said amino acid sequence also comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:I11, amino acids 652 though 745 of SEQ ID NO:4, a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 comprising at least 20 contiguous amino acids; a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 comprising at least 30 contiguous amino acids; a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 that is at least 25% of the length of the sequence of amino acids 652 though 745 of SEQ ID NO:4; a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 that is at least 50% of the length of the sequence of amino acids 652 though 745 of SEQ ID NO:4; and a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 comprising at least one tyrosine residue; an amino acid sequence of any of wherein the polypeptide comprising said amino acid sequence does not comprise an amino acid sequence selected from the group consisting of amino acids 239 through 252 of SEQ ID NO:13; amino acids 643 through 652 of SEQ ID NO:14; and amino acids 652 through 662 of SEQ ID an amino acid sequence of wherein a polypeptide comprising said amino acid sequence of binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any of and an amino acid sequence having HPR1 polypeptide activity.

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0 The present invention provides an isolated polypeptide having HPRI polypeptide activity comprising an amino acid sequence selected from the group consisting of: O the amino acid sequence of SEQ ID NO:4: Z an amino acid sequence selected from the group consisting of: amino acids 652 O 5 though 745 of SEQ ID NO:4, a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 comprising at least 20 contiguous amino acids; a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 comprising at least 30 contiguous amino acids; a fragment of the l sequence of amino acids 652 though 745 of SEQ ID NO:4 that is at least 25% of the length of the C, sequence of amino acids 652 though 745 of SEQ ID NO:4; a fragment of the sequence of amino acids 652 though 745 of SEQ ID NO:4 that is at least 50% of the length of the sequence of amino 10 acids 652 though 745 of SEQ ID NO:4; and a fragment of the sequence of amino acids 652 though 0 745 of SEQ ID NO:4 comprising at Jeast eight contiguous amino acids and comprising at least one N tyrosine residue; an amino acid sequence comprising at least 8 amino acids and sharing amino acid identity with the amino acid sequences of wherein the percent amino acid identity is selected from the group consisting of at least 70%, at least 75%, at least 80%, at least 85%. at least at least 95%, at least 97.5%, at least 99%, and at least 99.5%; an amino acid sequence comprising both an amino acid sequence of or and an amino acid sequence selected from the group consisting of: amino acids 1 through 55 of SEQ ID NO:1; amino acids 56 through 77 of SEQ ID NO:1; amino acids 5 through 40 of SEQ ID NO.2; amino acids 1 through 32 of SEQ ID NO:4; amino acids I through 241 of SEQ ID NO:4; amino acids 1 through 525 of SEQ ID NO:4; amino acids 20 through 32 of SEQ ID NO:4; amino acids 33 through 134 of SEQ ID NO:4; amino acids Xaal through Xaa2 of SEQ ID NO:4, wherein Xaal is selected from the group consisting of amino acids 33 through 43 of SEQ ID NO:4 and Xaa2 is selected from the group consisting of amino acids 228 through 241 of SEQ ID NO:4; amino acids 2533 through 238 of SEQ I N:4; amino acds 33 through 241 of SEQ ID N:4; amino acds 33 through 525 of SEQ ID NO:4; amino acids 33 through 745 of SEQ ID NO:4; amino acids 44 through 94 of SEQ ID NO:4; amino acids 139 through 241 of SEQ ID NO:4; amino acids 242 through 326 of SEQ ID NO:4; amino acids 242 through 514 of SEQ ID NO:4; amino acids 337 through 419 of SEQ ID NO:4; amino acids 433 through 514 of SEQ ID NO:4; amino acids 526 through 556 of SEQ ID NO:4; amino acids 533 through 552 of SEQ ID NO:4; amino acids 553 through 745 of SEQ ID NO:4; amino acids 557 through 745 of SEQ ID NO:4; amino acids 563 through 573 of SEQ ID NO:4; amino acids 563 through 641 of SEQ ID NO:4; amino acids 567 through 581 of SEQ ID NO:4; amino acids 588 through 639 of SEQ ID NO:4; amino acids 631 through 641 of SEQ ID NO:4; SEQ ID NO:10; and SEQ ID NO:11; an amino acid sequence comprising both an amino acid sequence of or and a fragment of SEQ ID NO:4 comprising cytokine receptor domain amino acid sequences; an allelic variant of any of and an amino acid sequence of wherein a polypeptide comprising said amino acid sequence of binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any of (bc).

sequence of any of The present invention provides an isolated polypeptide having HPRI polypeptide activity comprising an amino acid sequence selected from the group consisting of: the amio acid sequence of SEQ ID NO:12; 0 an amino acid sequence selected from the group consisting of: amino acids 633 Z 5 though 726 of SEQ ID NO: 12, a fragment of the sequence of amino acids 633 though 726 of SEQ ID NO:12 comprising at least 20 contiguous amino acids; a fragment of the sequence of amino acids 633 though 726 of SEQ ID NO:12 comprising at least 30 contiguous amino acids; a fragment of the sequence of amino acids 633 though 726 of SEQ ID NO:12 that is at least 25% of the length In of the sequence of amino acids 633 though 726 of SEQ ID NO:12; a fragment of the sequence of 10 amino acids 633 though 726 of SEQ ID NO:12 that is at least 50% of the length of the sequence of amino acids 633 though 726 of SEQ ID NO:12; and a fragment of the sequence of amino acids 633 though 726 of SEQ ID NO:12 comprising at least eight contiguous amino acids and comprising at least one tyrosine residue; (C1 an amino acid sequence comprising at least 8 amino acids and sharing amino acid identity with the amino acid sequences of(b), wherein the percent amino acid identity is selected from the group consisting of: at least 70%. at least 75%, at least 80%. at least 85%, at least at least 95%, at least 97.5%, at least 99%, and at least 99.5%; an amino acid sequence comprising both an amino acid sequence of(b) or and an amino acid sequence selected from the group consisting of: amino acids 1 through 28 of SEQ ID NO:12; amino acids 1 through 224 of SEQ ID NO:12; amino acids 1 through 509 of SEQ ID NO:12; amino acids 13 through 28 of SEQ ID NO:12; amino acids 29 through 124 of SEQ ID NO:12; amino acids Xaal through Xaa2 of SEQ ID NO:12, wherein Xaal is selected from the group consisting of amino acids 29 through 39 of SEQ ID NO:12 and Xaa2 is selected from the group consisting of amino acids 211 through 224 of SEQ ID NO:12; amino acids 29 through 128 of SEQ ID NO:12; amino acids 29 through 224 of SEQ ID NO:12; amino acids 29 through 509 of SEQ ID NO:12; amino acids 29 through 726 of SEQ ID NO:12; amino acids 129 through 224 of SEQ ID NO:12; amino acids 225 through 309 of SEQ ID NO:12; amino acids 225 through 499 of SEQ ID NO:12; amino acids 320 through 403 of SEQ ID NO:12; amino acids 417 through 508 of SEQ ID NO:12; amino acids 510 through 533 of SEQ ID NO:12; amino acids 517 through 532 of SEQ ID NO:12; amino acids 534 through 726 of SEQ ID NO:12; amino acids 547 through 557 of SEQ ID NO:12; amino acids 547 through 622 of SEQ ID NO:12; and amino acids 612 through 622 of SEQ ID NO:12; an amino acid sequence comprising both an amino acid sequence of or and a fragment of SEQ ID NO:12 comprising cytoline receptor domain amino acid sequences; an allelic variant of any of and an amino acid sequence of wherein a polypeptide comprising said amino acid sequence of binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any of Preferably, such polypeptides are isolated HPRI polypeptides or isolated polypeptides having HPR1 Z polypeptide activity.

Other aspects of the invention are isolated nucleic acids encoding polypeptides of the invention, with a preferred embodiment being an isolated nucleic acid consisting of, consisting essentially of, or more preferably, comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:3; c( SEQ ID C nucleotides 132 through 2366 of SEQ ID NO:3; and C' allclic variants of An additional preferred embodiment of the invention is an isolated nucleic acid consisting of, consisting essentially of, or more preferably, comprising a nucleotide sequence selected from the group consisting of nucleotides 1 through 137 of SEQ ID NO:3, nucleotides 138 through 228 of SEQ ID NO:3, nucleotides 229 through 346 of SEQ ID NO:3, nucleotides 347 through 528 of SEQ ID NO:3, nucleotides 529 through 680 of SEQ ID NO:3, nucleotides 681 through 846 of SEQ ID NO:3, nucleotides 847 through 926 of SEQ ID NO:3, nucleotides 927 through 1143 of SEQ ID NO:3, nucleotides 1144 through 1326 of SEQ ID NO:3, nucleotides 1327 through 1428 of SEQ ID NO:3, nucleotides 1429 through 1575 of SEQ ID NO:3, nucleotides 1576 through 1716 of SEQ ID NO:3, nucleotides 1717 through 1810 of SEQ ID NO:3, nucleotides 1811 through 1892 of SEQ ID NO:3, and nucleotides 1893 through 2480 of SEQ ID NO:3.

The invention provides an isolated polypeptide consisting of, consisting essentially of, or more preferably, comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:21; an amino acid sequence selected from the group consisting of: amino acids 1 through 177 of SEQ ID NO:16; amino acids 216 through 245 of SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; and amino acids 349 through 356 of SEQ ID NO:23; an amino acid sequence selected from the group consisting of: amino acids 1 through 23 of SEQ ID NO:21; amino acids 1 through 124 of SEQ ID NO:21; amino acids 1 through 318 of SEQ ID NO:21; amino acids 1 through 331 of SEQ ID NO:21; amino acids 1 through 355 of SEQ ID NO:21; amino acids Xaal through Xaa2 of SEQ ID NO:21, wherein Xaal is selected from the group consisting of amino acids 24 through 30 of SEQ ID NO:21 and Xaa2 is selected from the group consisting of amino acids 115 through 124 of SEQ ID NO:21; amino acids 24 through 124 of SEQ ID NO:21; amino acids 24 through 331 of SEQ ID NO:21; amino acids 24 through 355 of SEQ ID NO:21; amino acids Xaa3 through Xaa4 of SEQ ID NO:21, wherein Xaa3 is selected from the group consisting of amino acids 125 through 133 of SEQ ID NO:21 and Xaa4 is selected from the group consisting of amino acids 309 through 331 of SEQ ID NO:21; amino acids 125 through 219 of SEQ ID NO:21; amino acids 125 through 331 of SEQ ID NO:21; amino acids 133 through 309 of SEQ ID NO:21; amino acids 224 through 320 of SEQ ID NO:21; amino acids 224 through 331 of SEQ ID NO:21; amino acids 319 through 565 of SEQ ID NO:21; amino acids Xaa5 through Xaa6 of SEQ ID NO:21, wherein Xaa5 is selected from the group consisting of amino acids 376 0 through 393 of SEQ ED NO:21 and Xaa6 is selected from the group consisting of amino acids 618 through 629 of SEQ ID NO:21; amino acids 376 through 629 of SEQ ID NO:21; amino acids 393 through 440 of SEQ ID NO:21; amino acids 393 through 618 of SEQ ID NO:21; and amino acids 397 through 611 of SEQ ID NO:21; fragments of the amino acid sequences of any of comprising at least l contiguous amino acids; C<1 fragments of the amino acid sequences of any of comprising at least C' contiguous amino acids; S(f) fragments of the amino acid sequences of any of having HPR2 polypeptide activity; C fragments of the amino acid sequences of any of comprising cytokine receptor domain amino acid sequences; an allelic variant of any of amino acid sequences comprising at least 20 amino acids and sharing amino acid identity with the amino acid sequences of any of wherein the percent amino acid identity is selected from the group consisting of: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%. at least 99%, and at least 99.5%; an amino acid sequence of any of wherein the polypeptide comprising said amino acid sequence also comprises an amino acid sequence selected from the group consisting of: amino acids 1 through 177 of SEQ ID NO:16; amino acids 216 through 245 of SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; amino acids 349 through 356 of SEQ ID NO:23; amino acids 319 through 565 of SEQ ID NO:21; amino acids Xaa5 through Xaa6 of SEQ ID NO:21, wherein is selected from the group consisting of amino acids 376 through 393 of SEQ ID NO:21 and Xaa6 is selected from the group consisting of amino acids 618 through 629 of SEQ ID NO:21; amino acids 376 through 629 of SEQ ID NO:21; amino acids 393 through 440 of SEQ ID NO:21; amino acids 393 through 618 of SEQ ID NO:21; amino acids 397 through 611 of SEQ ID NO:21; amino acids 381 though 629 of SEQ ID NO:21; a fragment of the sequence of amino acids 381 though 629 of SEQ ID NO:21 comprising at least 20 contiguous amino acids; a fragment of the sequence of amino acids 381 though 629 of SEQ ID NO:21 comprising at least 30 contiguous amino acids; a fragment of the sequence of amino acids 381 though 629 of SEQ ID NO:21 that is at least 25% of the length of the sequence of amino acids 381 though 629 of SEQ ID NO:21; a fragment of the sequence of amino acids 381 though 629 of SEQ ID NO:21 that is at least 50% of the length of the sequence of amino acids 381 though 629 of SEQ ID NO:21; a fragment of the sequence of amino acids 381 though 629 of SEQ ID NO:21 comprising at least one of the following: an HPR2 Box 1 motif, an HPR2 Box 2 motif, and an HPR2 Box 3 motif; and a fragment of the sequence of amino acids 381 though 629 of SEQ ID NO:21 comprising at least one tyrosine residue; an amino acid sequence of any of wherein the polypeptide comprising said U amino acid sequence does not comprise amino acids 381 through 384 of SEQ ID NO:26; an amino acid sequence of wherein a polypeptide comprising said amino acid sequence of binds to an antibody that also binds to a polypeptide comprising an amino acid O 5 sequence of any of and an amino acid sequence of having HPR2 polypeptide activity.

c Preferably, such polypeptides are isolated HPR2 polypeptides or isolated polypeptides having HPR2 polypeptide activity.

SThe present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:23; SEQ ID an amino acid sequence selected from the group consisting of: an amino acid sequence comprising at least 20 contiguous amino acids of SEQ ID NO:23 and comprising the contiguous amino acids 318 and 319 of SEQ ID NO:23; and amino acids 349 through 356 of SEQ ID an amino acid sequence comprising at least 8 amino acids and sharing amino acid identity with the amino acid sequences of wherein the percent amino acid identity is selected from the group consisting of: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%; an amino acid sequence comprising both an amino acid sequence of or and an amino acid sequence selected from the group consisting of: amino acids 1 through 177 of SEQ ID NO: 16; amino acids 216 through 245 of SEQ ID NO:16; SEQ ID NO: 17; and SEQ ID NO: 18; an amino acid sequence comprising both an amino acid sequence of or and an amino acid sequences of any of comprising cytokine receptor domain amino acid sequences; an allelic variant of any of and an amino acid sequence of wherein a polypeptide comprising said amino acid sequence of binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any The present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:27; SEQ ID NO:27 from which amino acids 297 through 316 or amino acids 317 through 336 have been deleted; an amino acid sequence comprising 20 or more contiguous amino acids of or and an amino acid sequence comprising 30 or more contiguous amino and sharing at least amino acid identity with the amino acid sequences of Other aspects of the invention are isolated nucleic acids encoding polypqflidcs of the invention, with a preferred embodiment being an isolated nucleic acid consisting of, consisting essentially of, or more preferably, comprising a nucleotide sequence selected from the group consisting of- SEQ ID NO: 19; SEQ ID SEQ ID NQ:22; c-i(d) SEQ ID NO:24; and allelic variants of 17-. An additional preferred embodiment of the invention is an isolated nucleic acid consisting of, ci 10 consisting essentially of, or more preferably, comprising a nucleotide sequence selected from the group consisting of nucleotides 107 through 175 of SEQ ID NO:19, nucleotides 107 through 478 of SEQ MD c-i NQ:19, nucleotides 107 through 1060 of SEQ M1 NO:19, nucleotides 107 through 1099 of SEQ MD NO:19, nucleotides 107 through 1171 of SEQ ED NO:19, nucleotides 176 through 478 of SEQ MD NO:19, nucleotides 176 through 1099 of SEQ ED NO:19, nucteotides 176 through 1171 of SEQ ED 15N:9 uloie 79truh73o E N:9 uloie 7 truh19 fSQE NO:19, nucleotides 479 through 7033 of SEQ ED NO:19. nucleotides 4796 through 1069 of SEQ EID NO:19, nucleotides 503 through 1033 of SEQ MD NO:19, nucleotides 7761 through 106 of SEQ ED NO:19, nucleotides 7762 through 10993 of SEQ ED NO:19, nucleotides 1061 through 1401 of SEQ ID NO: 19, nucleotides 1283 through 1963 of SEQ ED NO: 19,an nucleotides 129 through 126 of SEQ) IOD NO: 19.

The invention also provides isolated genomic nucleic acids corresponding to the nucleic acids of the invention.

Another aspect of the invention provides isolated nucleic acids, prefrembly having a length of at least 15 nucleotides, that hybridize under conditions of moderate stringency to the nucleic acids encoding polypeptides of the invention. In preferred embodimnenrs of the invention, such nucleic acids encode a polypeptide having HPRI and/or BMR polypeptide activity, or comprise a nucleotide sequence thit shares nucleotide sequence identity with the nucleotide sequences of the nucleic acids of the invention, wherein the percent nucleotide sequence identity is selected from the group consisting of. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%.

Further provided by the invention are expression vectors and recombinant host cells 0 comprising at least one nucleic acid of the invention, and preferred recombinant host cells wherein said Z nucleic acid is integrated into the host cell genome.

C Also provided is a process for producing a polypeptide encoded by the nucleic acids of the invention, comprising culturing a recombinant host cell under conditions promoting expression of said polypeptide, wherein the recombinant host cell comprises at least one nucleic acid of the invention. A n preferred process provided by the invention further comprises purifying said polypeptide. In another aspect of the invention, the polypeptide produced by said process is provided.

SFurther aspects of the invention are isolated antibodies that bind to the polypeptides of the -invention, preferably monoclonal antibodies, also preferably humanized antibodies or humanized antibodies, and preferably wherein the antibody inhibits the activity of said polypeptides.

C, The invention additionally provides a method of designing an inhibitor of the polypeptides of the invention, the method comprising the steps of determining the three-dimensional structure of any such polypeptide, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predicted reactive site, and determining the polypeptideinhibiting activity of the molecule.

In a further aspect of the invention, a method is provided for identifying compounds that alter HPR1 and/or HPR2 polypeptide activity comprising mixing a test compound with a polypeptide of the invention; and determining whether the test compound alters the HPR1 and/or HPR2 polypeptide activity of said polypeptide.

In another aspect of the invention, a method is provided identifying compounds that inhibit the binding activity of HPRL and/or HPR2 polypeptides comprising mixing a test compound with a polypeptide of the invention and a binding partner of said polypeptide; and determining whether the test compound inhibits the binding activity of said polypeptide.

In preferred embodiments, the binding partner is a four alpha helix bundle cytokine; more preferably, the binding partner is selected from the group consisting of IL-6, OSM, LIF, CNTF, CLC, IL-12p35, and IL-23pl9, and most preferably the binding partners are a soluble hematopoietin receptor such as EBI-3, soluble IL-6R alpha, cytokine-like factor-1 (CLF), I-12p40, or a soluble form of HPR1 and/or HPR2 in conjunction with a four alpha helix bundle cytokine.

The invention also provides a method for increasing ligand-binding activity, comprising providing at least one compound selected from the group consisting of the polypeptides of the invention and agonists of said polypeptides; with a preferred embodiment of the method further comprising increasing said activity in a patient by administering at least one polypeptide of the invention.

Further provided by the invention is a method for decreasing ligand-binding activity, z comprising providing at least one antagonist of the polypeptides of the invention; with a preferred embodiment of the method further comprising decreasing said activity in a patient by administering at C least one antagonist of the polypeptides of the invention, and with a further preferred embodiment wherein the antagonist is an antibody that inhibits the activity of any of said polypeptides.

The invention additionally provides a method for treating a cell proliferation condition comprising administering at least one compound selected from the group consisting of the polypeptides of the invention and agonists of said polypeptides; with a preferred embodiment wherein the cell proliferation condition is selected from the group consisting of pancytopenia, leukopenia, anemia, Sthrombocytopenia, neurodegenerative disorders, and osteoporosis resulting from a lack of boneforming cells.

The invention additionally provides a method for treating a metabolic condition comprising administering at least one compound selected from the group consisting of the polypeptides of the invention and agonists of said polypeptides; with a preferred embodiment wherein the metabolic condition is obesity.

The invention additionally provides a method for treating a reproductive hormone condition comprising administering at least one compound selected from the group consisting of the polypeptides of the invention and agonists of said polypeptides; with a preferred embodiment wherein the condition is selected from the group consisting of deficient mammary development and infertility.

In other aspects of the invention, a method is provided for treating a cell proliferation condition comprising administering an antagonist of the polypeptide of the invention; with a preferred embodiment wherein the cell proliferation condition is selected from the group consisting of leukemia, tumour metastasis, and osteoporosis resulting from an excess of bone-resorbing cells.

In other aspects of the invention, a method is provided for treating a metabolic condition comprising administering an antagonist of the polypeptide of the invention; with a preferred embodiment wherein the metabolic condition is selected from the group consisting of cachexia, wasting, and AIDS-related weight loss.

In other aspects of the invention, a method is provided for treating cancer conditions stimulated by reproductive hormones comprising administering an antagonist of the polypeptide of the invention; with a preferred embodiment wherein the condition is selected from the group consisting of breast cancer and prolactinoma.

In another embodiment of the invention, methods are provided for using HPR1 and IHPR2 polypeptides and antagonists thereof as adjuvants.

SA further embodiment of the invention provides a use for the polypeptides of the invention in the preparation of a medicament for treating a cell proliferation condition; with a preferred embodiment wherein the cell proliferation condition is selected from the group consisting of pancytopenia, leukopenia, anemia, thrombocytopenia, neurodegenerative disorders, and osteoporosis.

-8- A further embodiment of the invention provides a use for the polypeptides of the invention in O the preparation of a medicament for treating a metabolic condition; with a preferred embodiment Z wherein the metabolic condition is obesity.

C A further embodiment of the invention provides a use for the polypeptides of the invention in the preparation of a medicament for treating a reproductive hormone condition; with a preferred embodiment wherein the condition is selected from the group consisting of deficient mammary Vn development and infertility.

DETAILED DESCRIPTION OF THE INVENTION Similarities of HPR1 and HPR2 Structure to Other Hematopoietin Receptor Family Members We have identified HPR1 and HPR2, new human hematopoietin receptor polypeptides having 15 structural features characteristic of this polypeptide family; the amino acid sequence of an HPR1 polypeptide is provided in SEQ ID NO:4 and the amino acid sequence of three alternatively spliced forms of HPR2 polypeptide are provided in SEQ ID NOs 21, 23, and 25. We have also identified the murine homologue of human HPR1; the amino acid sequence of Mus musculus HPR1 is presented in SEQ ID NO:12. (The use of "HPRI" without a species designation refers to HPR1 polypeptides generally, for example, human and/or murine, mammalian, or vertebrate HPR1. polypeptides.) Alignments showing the sequence similarities between HPRI, HPR2, and other hematopoietin receptors are presented in Tables 1, 2, and 3 in Example 1 below.

The typical structural elements common to members of the hematopoietin receptor polypeptide family include an extracellular region comprising at least one cytokine receptor domain, and in most members of the family, a cytoplasmic region that in at least a subset of the hematopoietin receptor polypeptides comprises domains involved in intracellular signaling functions. A signal sequence is found at the N-terminus of hematopoietin receptor family polypeptides, and is followed, in N-to-C order, by an immunoglobulin (Ig)-like domain (in some members of the family), a cytokine receptor domain, three copies of a fibronectin repeat (in some members of the family), a transmembrane domain or a glycosyl-phosphatidyl inositol (GPI) linkage to the membrane (except in soluble members of the family, which in most cases are soluble splice variant forms of transmembrane or membrane-linked hematopoietin receptor polypeptides), and a cytoplasmic domain (which is not present in soluble forms). The extracellular domain of hematopoietin receptor polypeptides extends from the N terminus to the transmembrane domain of the protein, and includes the cytokine receptor domain and any Ig-like domains (approximately 100 amino acids in length) or fibronectin repeats (such as fibronectin type Im repeats which are approximately 81-83 amino acids in length and are separated by spacer sequences of approximately 10 to 13 amino acids) that may be present in certain of the hematopoietin receptor polypeptides. There are key residues within the cytokine receptor domain, the two or four conserved cysteine residues and the WSXWS motif; substitutions of these residues are likely to be associated with an altered function or lack of that function for the polypeptide. The cytokine receptor domain, which is approximately 200 amino acids in length, can be subdivided into two roughly equal subdomains an N-terminal 'conserved cysteine' domain and a more C-terminal -9- WSXWS' domain separated by a proline-rich 'linker' stretch of four amino acids that allows the two z subdomains to form a ligand binding site between them (Bravo and Heath, 2000, EMBO J. 19(11): 2399-2411).

The intracellular domain (also called "cytoplasmic domain") of the hematopoietin receptor polypeptides (in those family members that contain such a domain), extends from the transmembrane domain of the protein to the C terminus, and in the signaling receptor subgroup, includes regions involved in intracellular signal transduction functions. Although the amino acid sequence of the Sintracellular domain varies considerably between hematopoietin receptor polypeptides, there are a few regions that show some similarity between the members of the family and which have been determined to be involved in binding to members of the signal transduction cascade. "Box 1" is a stretch of 9 to 12 amino acids that begins about 9 amino acids C-terminal to the transmembrane domain, and has within it a conserved Ar-P-X-AI-P-X-P motif, where Ar is an aromatic amino acid (Trp, Phe, or Tyr) and Al is an aliphatic amino acid (Ala, Gly, Val, Leu, or Ile). About 8 amino acids C-terminal to Box 1 there is a conserved aromatic amino acid (usually Trp but also Phe or Tyr), and approximately 15 to 60 amino acids further C-terminal there is a motif of about 11 to 13 amino acids, "Box While Box 1 is present in most of the hematopoietin receptor polypeptides, the Box 2 motif is present in a subset of the hematopoietin receptor family including gpl30, GCSFR, LIF-R, the erythropoietin receptor (EPO-R), and several others. Mutations to residues within Box 1 or Box 2, or to the conserved aromatic residue between the Box 1 and Box 2 motifs, have inactivated the ability of the mutated receptor to stimulate cell proliferation upon the addition of ligand. A further conserved domain has been identified in the cytoplasmic domains of signaling cytokine receptors such as gpl30, LIF-R, and G-CSFR: "Box 3".

The Box 3 motif is about 10 to 15 amino acids located between approximately 70 and 150 amino acids C-terminal of the transmembrane domain, and has a rough match to a (P/)VXGXGYXXQ consensus sequence. Cytoplasmic regions of these receptors containing Box 3 have been associated with a macrophage differentiation promoting activity (in the case of gpl30) and a granulocyte differentiation promoting activity (in the case of G-CSFR) (Soede-Bobok and Touw, 1997, J Mol Med 75: 470-477); however, members of the LIP/IL-6 gpl30-sharing family of hematopoietin receptors can also be involved in suppression of differentiation (see Ernst et 1999, J Biol Chem 274(14): 9729-9737).

Finally, the cytoplasmic domains of signaling hematopoietin receptor polypeptides contain several tyrosine residues that are potential sites for phosphorylation. Although hematopoietin receptors themselves do not generally have a protein kinase activity, they interact with and are phosphorylated by kinases within the JAK/STAT signal transduction pathways. Mutations in the Box 1 motif abolish the ability of certain of the signaling hematopoietin receptors to bind members of the Janus kinase (JAK) family, particularly JAK2 or JAK1 (Taner et aL, 1995, J Biol Chem 270(12): 6523-6530).

Hematopoietin receptor-ligand interactions also activate the ERK/MAPK pathway, most likely through the phosphorylation of tyrosine residues in the cytoplasmic domains as the tyrosines at cytoplasmic positions 118 of gpl30 (amino acid 759 of SEQ ID NO:8) and 115 of LIF-R (amino acid 974 of SEQ ID NO:6) are present within SHP2 binding sites (Schiemann et aL, 1997, J Biol Chem 272(26): 16631- 16636). The cytoplasmic tyrosine residues of signaling hematopoietin receptors and the amino acids 0 around them are also important motifs for the recruitment and phosphorylation of signal-transducing Z STAT polypeptides (Hirano et aL, 2000, Oncogene 19: 2548-2556).

Human HPRI polypeptide has a signal sequence extending from approximately amino acid through amino acid 32 of SEQ ID NO:4, with the mature polypeptide produced by cleavage of this signal sequence predicted to have an amino acid sequence beginning at amino acid 33 of SEQ ID SNO:4. Human HPR1 has a cytokine receptor domain extending approximately from amino acid 33 through amino acid 241 of SEQ ID NO:4; three fibronectin repeats from approximately amino acid 242 of SEQ ID NO:4 to about amino acid 515 of SEQ ID NO:4; a transmembrane domain that begins approximately between amino acids 526 and 533 of SEQ ID NO:4 and extends to approximately between amino acids 552 and 556 of SEQ ID NO:4 (defining a smaller 'core' transmembrane domain C from amino acid 533 to amino acid 552 of SEQ ID NO:4 and an extended transmembrane domain from amino acid 526 to amino acid 556 of SEQ ID NO:4); and a cytoplasmic domain extending from the end of the transmembrane domain beginning roughly between amino acids 553 and 557 of SEQ ID NO:4) and extending through the carboxyl terminus of the polypeptide (amino acid 745 of SEQ ID NO:4). Therefore, human HPR1 polypeptide has an overall structure consistent with other hematopoietin receptor family members. The four conserved cysteine residues within the human iHPR1 cytokine receptor domain are located at positions 43, 53, 81, and 94 of SEQ ID NO:4, and the human HPR1 WSXWS motif is located from amino acid 224 through amino acid 228 of SEQ ID NO:4. The human HPR1 N-terminal cytokine receptor subdomain containing four conserved cysteine residues extends approximately from amino acid 33 of SEQ-ID NO:4 to amino acid 134 of SEQ ID NO:4; the proline-rich linker is amino acids 135 through 138 of SEQ ID NO:4; and the WSXWS-containing

C-

terminal cytokine receptor subdomain extends from amino acid 139 to about amino acid 241 of SEQ ID NO:4. In human HPR1, as in several members of the hematopoietin receptor family, the cytokine receptor domain is followed by three fibronectin type E repeats; these repeats are located within the human HPRI amino acid sequence of SEQ ID NO:4 at the following approximate locations: amino acids 242 to 244 through 324 to 326, amino acids 336 to 337 through 419 to 422, and amino acids 430 to 433 through 514 to 515. Within its intracellular domain, human HPRI polypeptide contains a good match to the Box 1 conserved motif from amino acid 563 through amino acid 573 of SEQ ID NO:4, a conserved downstream Trp residue (amino acid 581 of SEQ ID NO:4), and a Box 2 motif from amino acid 631 to amino acid 641 of SEQ ID NO:4. The cytoplasmic domains of signaling hematopoietin receptor polypeptides contain several tyrosine residues that are potential sites for phosphorylation; in human HPR1, such tyrosines are located at positions 652, 683, and 721 of SEQ ID NO:4. Human HPRI contains several instances of an Asp-containing motif within its cytoplasmic region. In the area overlapping the Box 2 location, human HPRI has repeated amino acid sequences as shown in the following table; these sequences form a consensus sequence of DKL(NIV)(T/AI), where Al is an aliphatic residue as described above. Other signaling hematopoietin receptors such as murine HPRI (at -11amino acids 600 through 604 of SEQ ID NO:12) and gpl30 also contain at least one similar Asp- 0 containing sequence in the region around and following the Box 2 location.

SRepeat Sequence Location in SEQ ID NO:4 C" DKLNL amino acids 588 through 592 DSVNT amino acids 597 through 601 DRILK amino acids 603 through 607 SDKLVI_ amino acids 614 through 618 DKLWVV amino acids 619 through 623 DEART I amino acids 635 through 639 Variants, presumably splice variants, of human HPR1 are described in WO 00/75314: a 2 5 2-aminoacid form a 652-amino-acid form and a 6 62-amino-acid form ("NR10.3").

The 252-amino-acid form of HPRI (SEQ ID NO:13) is identical to SEQ ID NO:4 through amino acid 238. and then has a divergent amino acid sequence from amino acid 239 through 252 of SEQ ID NO:13. This 252-amino-acid form of human HPR1 therefore does not contain the fibronectin type l repeats found in the full-length 745-amino-acid HPR1 of SEQ ID NO:4, or the transmembrane domain or the intracellular region of the SEQ ID NO:4 polypcptide. The 6 52-amino-acid form of HPRI (SEQ ID NO:14) is identical to SEQ ID NO:4 through amino acid 642, and then has a divergent amino acid sequence from amino acid 643 through 652 of SEQ ID NO:14.; and the 6 62-amino-acid form of HPR1 (SEQ ID NO:15) is identical to SEQ ID NO:4 through amino acid 651, and then has a divergent amino acid sequence from amino acid 652 through 662 of SEQ ID NO:15. The 652- and 662-amino-acid forms of human HPR1 therefore do not contain the tyrosine residues at positions 652, 683, and 721 of the intracellular region of the SEQ ID NO:4 polypeptide which are potential substrates for phosphorylation by kinases, such as those of the ERK/MAPK signaling pathways.

The Mus musculus HPRI amino acid sequence of SEQ ID NO:12 has a signal sequence beginning approximately between amino acid 13 and amino acid 16 of SEQ ID NO:12 and extending approximately through amino acid 28 of SEQ ID NO:12, with the mature polypeptide produced by cleavage of this signal sequence predicted to have an amino acid sequence beginning at amino acid 29 of SEQ ID NO:12. Murine HPR1 has a cytokine receptor domain extending approximately from amino acid 29 through amino acid 224 of SEQ ID NO:12; three fibronectin repeats from approximately amino acid 225 of SEQ ID NO:12 to about amino acid 499 of SEQ ID NO:12; a transmembrane domain that begins approximately between amino acids 510 and 517 of SEQ ID NO:12 and extends to approximately between amino acids 532 and 533 of SEQ ID NO:12 (defining a smaller 'core' transmembrane domain from amino acid 517 to amino acid 532 of SEQ ID NO:12 and an extended transmembrane domain from amino acid 510 to amino acid 533 of SEQ ID NO:12); and a cytoplasmic domain extending from the end of the transmembrane domain beginning roughly between amino acids 533 and 534 of SEQ ID NO:12) and extending through the carboxyl terminus of the polypeptide (amino acid 726 of SEQ ID NO:12). Therefore, murine HPR1 polypeptide has an overall structure consistent with other hematopoietin receptor family members. There are two conserved cysteine residues within the murine HPRI cytokine receptor domain located at positions 39 and 49of SEQ ID NO:12, and there are two additional cysteines in this region (although at non-conserved positions) at -12amino acids 90 and 97 of SEQ ID NO:12. The murine HPR1 WSXWS motif is located from amino 0 acid 207 through amino acid 211 of SEQ ID NO:12. The murine HPR1 N-terminal cytokine receptor subdomain containing two conserved cysteine residues (and two additional cysteine residues) extends Sapproximately from amino acid 29 of SEQ ID NO:12 to amino acid 124 of SEQ ID NO:12; the prolinerich linker is amino acids 125 through 128 of SEQ ID NO:12; and the WSXWS-containing C-terminal cytokine receptor subdomain extends from amino acid 129 to about amino acid 224 of SEQ ID NO:12.

t In murine HPRI, as in several members of the hematopoietin receptor family, the cytokine receptor domain is followed by three fibronectin type Ml repeats; these repeats are located within the marine C HPR1 amino acid sequence of SEQ ID NO:12 at the following approximate locations: amino acids 225 to 227 through 307 to 309 amino acids 319 to 320 through 403 to 406, and amino acids 413 to 0 15 417 through 498 to 499. Within its intracellular domain, murine HPR1 polypeptide contains a good match to the Box 1 conserved motif from amino acid 547 through amino acid 557 of SEQ ID NO:12, a conserved downstream Trp residue (amino acid 565 of SEQ ID NO:12), and a Box 2 motif from amino acid 612 through amino acid 622 of SEQ ID NO:12. The cytoplasmic domains of signaling hematopoietin receptor polypeptides contain several tyrosine residues that are potential sites for phosphorylation; in murine HPR1, such tyrosines are located at positions 633, 674, and 701 of SEQ ID NO:12.

Human HPR2 polypeptide has a signal sequence extending from approximately amino acid 11 through amino acid 23 of SEQ ID NO:21, with the mature polypeptide produced by cleavage of this signal sequence predicted to have an amino acid sequence beginning at amino acid 24 of SEQ ID NO:21. The membrane-spanning (629 amino acids) form of HPR2 has an N-terminal Ig-like domain extending approximately from amino acid 24 through amino acid 124 of SEQ ID NO:21, a cytokine receptor domain extending approximately from amino acid 125 through an amino acid from 320 to 331 of SEQ ID NO:21; a transmembrane domain that begins approximately at amino acid 356 of SEQ ID NO:21 and extends to approximately amino acid 375 of SEQ ID NO:21; and a cytoplasmic domain extending from the end of the transmembrane domain beginning approximately at amino acid 376 of SEQ ID NO:21) and extending through the carboxyl terminus of the polypeptide (amino acid 629 of SEQ ID NO:21). Therefore, HPR2 polypeptide has an overall structure consistent with other hematopoietin receptor family members. The N-terminal Ig-like domain contains six cysteine residues at positions 30, 52,59, 101, 105, and 115 of SEQ ID NO:21, the most conserved of which appear to be the two cysteines at positions 52 and 101; the cysteines at positions 30, 115 (and to a lesser extent, at 105) also align with cysteines at similar positions in Ig or Ig-like domains. The HPR2 Ig-like domain appears to have the greatest degree of sequence similarity with members of the LIR (leukocyte Ig-like receptor) polypeptide family, particularly LIR-3 and LIR-4. The two conserved cysteine residues within the human HPR2 cytokine receptor domain are located at amino acid positions 133 and 144 of SEQ ID NO:21, and the HPR2 version of the WSXWS motif which has a glutamine residue at the second position of the motif rather than a serine residue, is located from amino acid 304 through amino acid 308 of SEQ ID NO:21. The HPR2 N-terminal cytokine receptor subdomain containing the two -13conserved cysteine residues extends approximately from amino acid 125 of SEQ IID NO-.21 to amino z ad 219 of SEQ IID NO:21; the proline-rich linker (in this case, proline- and alanine-ridi) isamn 223 of SEQ ID NO:21; and the 'WQXWS-contining C-terminal cytokine receptor c-i subdomain extends from amino acid 224 through an amino acid from 320 to 331 of SEQ MD NQtl.

EMPR does not contain the fibronectin type Ml repeats found in human and murine HPR1. Within its intracellular domain, the membrane-spanning (629 amino acids) form of HPIR2 contains a good match into the Box I conserved motif from amino acid 393 through amino acid 403 of SEQ II) N0-21, does not contain a Trp residue between Box 1 and Box2, andas aBox 2motif r mno aid 4 3 0 to ar acid 440 of SEQ ID NO:21. There are also two matches to the Box 3 motif in this membrane-sparming HPR2 polypeptide, at amino acids 478 through 491 and at amino acids 605 through 618 of SEQ ID) NO:21. 7he cytoplasmic domains of signaling hematopoietin receptor polypeptides contain several c-i tyrosine residues that am potential sites for pbosphorylauion; in human HPR2, such tyrosines are located at amino acid positions 397 (within the Box 1 motif), 429 (immediately N-terminal to the Box 2 motif), 450, 463, and 476 Oust N-termixal of the most N-terminal Box 3 motif), and amino acids 484 and 611 (eaof h) fsttwo amino c ds ihn a Box 3 mtf f SID NO In seveal respects, the membrane-spanning form of HPR2 shows similarity to the LIFz-R hematopoietin receptor.

both of these molecules have an Ig-like domain that is followed by a cytokine receptor domain having two (as compared. to four) conserved cysteines; and both have Box 1, Box 2, and Box 3 motifs in their intracellular domains, and do not have a tryptophan residue between Box 1 and Box I The FIPR2-ex9 polypeptide of 1SEQ ID NO0:25 (356 amino acids), created by alternative splicing which removes exon 9 of the HIPR2 coding sequence (see Example I below), is identical to the HPR2 629-amino-acid form from amino acid 1 through amino acid 348, but then diverges in sequence for the eight amino acids from amino acid 349 to the C terminus at amino acid 356. The HPR2-cx9 form does not contain a transmembrane region, and is expected to be a secreted form of HPR2 containing the HPR2 extracellular Ig-like. and cytokine receptor domains. The HPR2-ex8-ex& polypeptidecof SEQID NO:23{(565 amino acids), cted by alternative splicing which removes exons 8 and 9 of the HPR2 coding sequence (see Example I below), is identical to the H1PR2 629-amino-acid form from amino acid 1 through amino acid 318, is missing the next 64 amino acids which include the transmeaxhraDe domain, but then shows identity between amino acid 319 through amino acid 565 of SEQ ID NO:23 and the C-terminal region of the 629-amino-acid form of BPR2. The HPR2-ex8-exg form is also expected to be a secreted form of BPR2 containing not only the 11PR2 extracellular Ig-Like and cytokine receptor domains, but also the C-terminal portion Of the IHPR2 protein which includes the Box 1, Box 2, and Box 3 motifs. A variant, presumably a splice variant. of human FHPR2 is described in WO 00/73451: a 384-amino-acid form This 3 8 4 -amino-acid form of BPR2 (SEQ MD N026)--s.idntical to SEQ ID NOz2l through amino acid 380.,and then has a divergent amino acid sequence from amino acid 381 through 384 of SEQ ID NQ:26. This 3 84-amino-acid form of human JAPR2 therefore does not contain the intracellular region of the SEQ ID NO:21 HPR2 polypeptide, 14which contains the Boxl, 2, and 3 motifs and intracellular tyrosine residues that are involved in the O signaling (or signal transduction) function of the SEQ ID NO:21 HPR2 polypcptide.

Z The Mus musculus HPR2 amino acid sequence of SEQ ID NO:27 has a signal sequence beginning approximately between amino acid 8 and amino acid 11 and extending through amino acid 23 of SEQ ID NO:27, with the mature polypeptide produced by cleavage of this signal sequence predicted to have an amino acid sequence beginning at amino acid 24 of SEQ ID NO:27. Mus musculus HPR2, like the membrane-spanning form of human HPR2, has an N-terminal Ig-like domain extending approximately from amino acid 24 through amino acid 124 of SEQ ID NO:27, a cytokine C receptor domain extending approximately from amino acid 125 through an amino acid from 341 to 350 of SEQ ID NO:27; a transmembrane domain that begins approximately between amino acid 373 and amino acid 380 of SEQ ID NO:27 and extends through approximately between amino acid 394 and C( amino acid 395 of SEQ ID NO:27 (defining a smaller 'core' transmembrane domain from amino acid 380 to amino acid 394 of SEQ ID NO:27 and an extended transmembrane domain from amino acid 373 to amino acid 395 of SEQ ID NO:27); and a cytoplasmic domain extending from the end of the transmembrane domain (Le. beginning approximately at amino acid 395 or at amino acid 396 of SEQ ID NO:27) and extending through the carboxyl terminus of the polypeptide (amino acid 644 of SEQ ID NO:27). Therefore, murine HPR2 polypeptide has an overall structure consistent with other hematopoictin receptor family members. The N-terminal Ig-like domain contains six cysteine residues at positions 30, 52, 59, 101, 105, and 115 of SEQ ID NO:27, the most conserved of which appear to be the two cysteines at positions 52 and 101; the cysteines at positions 30, 115 (and to a lesser extent, at 105) also align with cysteincs at similar positions in Ig or Ig-like domains. As with human HPR2, the murine HPR2 Ig-like domain appears to have the greatest degree of sequence similarity with members of the LIR (lcukocyte Ig-like receptor) polypeptide family. The two conserved cysteine residues within the human HPR2 cytokine receptor domain are located at amino acid positions 133 and 144 of SEQ ID NO:27, and the murine HPR2 version of the "WSXWS" motif, which like human HPR2 has a glutamine residue at the second position of the motif rather than a serine residue, is located from amino acid 324 through amino acid 328 of SEQ ID NO:27. The murine HPR2 polypeptide contains an insert of 20 amino acids relative to the human HPR2 polypeptide; this insert region extends from amino acid 297 through amino acid 316 of SEQ ID NO:27, and is a perfect repeat of amino acids 317 through 336 of SEQ ID NO:27. Therefore, in the SEQ ID NO:27 form of murine HPR2, there is a second WQXWS motif at amino acids 304 through 308 of SEQ ID NO:27. The murine HPR2 N-terminal cytokine receptor subdomain containing the two conserved cysteine residues extends approximately from amino acid 125 of SEQ ID NO:27 to amino acid 219 of SEQ ID NO:27; the proline-rich linker (in this case, proline- and alanine-rich) is amino acids 220 through 223 of SEQ ID NO:27; and the C-terminal cytokine receptor subdomain containing the two repeats of the WQXWS motif extends from amino acid 224 through an amino acid from 340 to 350 of SEQ ID NO:27. Murine HPR2 does not contain the fibronectin type III repeats found in human and murine HPR1. Within its intracellular domain, this membrane-spanning form of murine HPR2 contains a good match to the Box 1 conserved motif from

(N

amino acid 412 through amino acid 422 of SEQ ID NO:27, does not contain a Trp residue between Box S1 and Box 2 and has a Box 2 motif from amino acid 449 to amino acid 459 of SEQ ID NO:27. There are also two matches to the Box 3 motif in this murine membrane-spanning HPR2 polypeptide, at (amino acids 498 through 511 and at amino acids 620 through 633 of SEQ ID NO:27. The cytoplasmic domains of signaling hematopoietin receptor polypeptides contain several tyrosine residues that are potential sites for phosphorylation; in murine HPR2, such tyrosines are located at amino acid positions 416 (within the Box 1 motif), 448 (immediately N-terminal to the Box 2 motif), 469, and 496 (just Nterminal of the most N-terminal Box 3 motif), and amino acids 504 and 626 (each of these last two amino acids is within a Box 3 motif) of SEQ ID NO:27. There is an additional intracellular tyrosine located at position 542 of SEQ ID N0:27. As with the membrane-spanning form of human HPR2, murine HPR2 shows similarity to the LI-R hematopoietin receptor.

Each of the HPR1 and the HPR2 groups of related polypeptides therefore contains a distinct subset of the several features characteristic of at least some members of the hematopoietin receptor family. The skilled artisan will recognize that the boundaries of the regions of the HPRI and HPR2 polypeptides described above are approximate and that the precise boundaries of such domains, as for example the boundaries of the transmembrane region (which can be predicted by using computer programs available for that purpose), can also differ from member to member within the hematopoietin receptor polypeptide family.

The hematopoietin receptor polypeptide family is highly to moderately conserved between species, with the family members within a particular species exhibiting some sequence conservation, particularly with respect to the conserved domains and residues described above. Subfamilies of the hematopoietin receptor polypeptide family can be defined on the basis of structure, for example the Iglike domain containing members, or the fibronectin repeat containing members. It is also possible to group hematopoietin receptor polypeptides according to the length of the cytoplasmic domain, with those receptors having a longer cytoplasmic domain being more likely to be signaling receptors.

Subgroups of the hematopoietin receptor family can also be defined on the basis of a shared common signaling receptor present in several different combinations of heteromeric receptors. For example, the signaling receptor is found in separate complexes with LIF-R. IL-6R alpha or a soluble form of IL-R alpha, and CNTFR alpha; monomeric forms or multimeric combinations of these receptor components bind to IL-6, OSM, LIF, and/or CNTF; thus a "gpl30-sharing group" subfamily would include these hematopoietin receptor polypeptides and be associated with this group of cytokines.

Another group of hematopoietin receptors are those which associate with a ligand comprising at least two soluble polypeptides. For example, the IL-12 receptor associates with the combination of the polypeptide, similar in structure to soluble forms of hematopoietin receptors such as soluble IL-6R alpha, and the four alpha helix bundle p35 polypeptide. The IL-12 p40 subunit can also associate with another four alpha helix bundle cytokine called p19; when p40 binds p19 the resulting combination has been named "IL-23" and has been shown to bind to the IL-12R beta 1 receptor subunit, but not the signaling IL-12R beta 2 receptor subunit (Oppmann et aL, 2000, Imnunity 13: 715-725). Thus the -16rp40-p19 complex is likely to bind a different IL-12RB2-like signaling receptor subunit, such as HPR2, 0 HPRI, GCSFR, or gpl30. As another example, CNTFR alpha, gpl30, and LIFR can each associate Z with a combination of the soluble receptor cytokine-like factor-1 (CLF-1) and cardiotrophin-like Scytokine (CLC), with CLF-I and CLC analogous to p40 and p35, respectively (Elson et at, 2000, Nat Neurosci 867-872). The cytokin receptor domains of HPRI and HPR2 are similar in sequence to those of gpl30, IL-6R beta, IL-12RB2, GCSFR, LIFR, leptin receptor, prolactin receptor, and other n members of the hematopoietin receptor family, with HPRI showing the greatest degree of similarity to and IL-6R beta, and HPR2 showing the greatest degree of similarity to gpl30 and IL-12RB2.

S* Because HPRI and HPR2 each have a substantial cytoplasmic domain and are most similar in sequence to gpl30, HPRI and HPR2 are likely to be new signaling members of the "gpl30-sharing" subfamily of hematopoietin receptors; however, HPR2 may also share attributes of the IL-12RB2 receptor subunit, such as involvement in modulation of the balance between Thl and Th2 immune responses.

Expression of HPRI and HPR2 has been detected by PCR amplification from tissue-specific cDNA libraries in several cell types including COS-1 cells, 293MSR cells, the B cell lines CB23 and MP-1, the B cell lymphoma lines Daudi, and Raji, the T cell leukemia line HSB2, and the promonocytic leukemia line U937. HPR2 mRNA expression appears to be more prevalent than HPRI expression in the B cell derived lines, while HPRI mRNA expression appears to be more prevalent than HPR2 expression in the T cell derived and monocyte lines. EBI-3 is a p40-like soluble hematopoietin receptor polypeptide; FACS analysis has shown that EBI-3-Fc fusion polypeptides bind to cells expressing HPR1 and HPR2 such as COS-1 cells, 293MSR cells, and CB23 and MP-1 cells, indicating that EBI-3 is a potential binding partner of HPR1 and HPR2, most likely in conjunction with a four alpha helix bundle cytokine such as IL-6, OSM, LIF, CNTF, CLC, IL-12p35, or IL,23p19.

Biological Activities and Functions of HPRI and HPR2 Polypeptides PCR amplification from tissue-specific cDNA libraries was performed to detect HPRI or HPR2 cDNA sequences. The results of these experiments show that HPRI transcripts are expressed in a wide variety of fetal and adult human cells, including testis, lung, placenta, pancreas, prostate, peripheral blood cells, thymus, stomach, and skin cells; as well as in various cell lines including U937 cells, the leukemia cell line HSB2, LX-1/OI-117 lung carcinoma cells, GI-112 colon adenocarcinoma cells, the B cell lines MP-1 and CB23, COS-1 cells, and 293MSR cells. HPR2 transcripts are present in a similarly diverse group of adult and fetal human cell types, including placenta, lung, kidney, pancreas, prostate, testis, colon, LX-1/GI-117 lung carcinoma cells, tonsil/CX-1 cells, lymph node, GI- 112 colon adenocarcinoma cells, heart, brain, spleen, thymus, ovary, small intestine, fetal brain, fetal lung/heart, fetal spleen, fetal thymus, esophagus, stomach, and skin; and in various cell lines such as the B cell lines MP-1 and CB23, Daudi cells, Raji cells, HSB2 cells, COS-1 cells, and 293MSR cells.

Typical biological activities or functions associated with HPRI and HPR2 polypeptides are ligand-binding activity, intraccllular signaling activity, cell proliferation stimulatory activity, cell proliferation inhibitory activity, cell differentiation stimulatory activity, and cell differentiation -17inhibitory activity. HPR and HPR2 polypeptides having ligand-binding activity bind to cytokine or z growth factor ligand molecules of the four alpha helix bundle family of cytokines, and in particular are likely to bind cytokines such as IL-6, OSM, LIF, CNTF, CLC, IL-12p35, and IL-23p19, and/or soluble C hematopoietin receptors such as EBI-3, soluble IL-6R alpha, cytokine-like factor-I (CLF), IL-12p40, or a soluble form of HPRI and/or HPR2. This ligand-binding activity is associated with the extracellular cytokine receptor domain of HPR1 polypeptides. Thus, for uses requiring ligand-binding activity, preferred HPRI and HPR2 polypeptides include those having at least one cytokine receptor domain and exhibiting ligand-binding activity. Preferred HPR1 and HPR2 polypeptides further include oligomers or fusion polypeptides comprising at least one cytokine receptor portion of one or more HPR1 and/or HPR2 polypeptides, and fragments of any of these polypeptides that have ligand-binding activity. The ligand-binding activity of HPR1 and HPR2 polypeptides may be determined, for example, by any standard assay to measure binding of labeled ligand or by a competitive binding assay, all of which are described more extensively below. HPRI and HPR2 polypeptides having intracellular signaling activity bind ligand molecules when in association with other receptor polypeptides to form a homo- or heteromeric complex, with ligand binding initiating a signaling cascade. The intracellular signaling activity is associated with the cytoplasmic domain of certain HPRI and HPR2 polypeptides. Thus, for uses requiring intracellular signaling activity, preferred HPR1 and HPR2 polypeptides include those having the cytoplasmic domain, and in particular having certain conserved domains (such as the Box 1 motif, the Trp residue at position 581 of SEQ ID NO:4, the Box 2 motif, the Asp-containing motifs between amino acids 588 and 639 of SEQ ID NO:4, or the Box 3 motif) and conserved cytoplasmic tyrosine residues, and exhibiting intracellular signaling biological activity. Preferred HPR1 and HPR2 polypeptides further include oligomers or fusion polypeptides comprising at least one cytoplasmic portion of one or more HPR1 and/or HPR2 polypeptides, and fragments of any of these polypeptides that have intracellular signaling activity. The intracellular signaling activity of HPRI and HPR2 polypeptides may be determined, for example, through assays to detect phosphorylation of the HPR1 polypeptide, the HPR2 polypeptide, or downstream polypeptides in signaling cascades such as the JAK/STAT or ERK/MAPK pathways, or in assays that measure biological activities related to the signal transmission, such as stimulation or suppression of cell proliferation, differentiation, or activation. One example of an assay to measure cytokine-binding and cell-proliferation activity involves expressing a polypeptide of the invention in Ba/F3 cells, exposing the polypeptide-expressing cells to radioactively labeled cytokine, and measuring specific cytokine binding to cells and uptake of 3H-thymidine by cells in response to cytokine, as described in Presky et aL, 1996, Proc NatlAcad Sci USA 93: 14002-14007. Further examples of such assays are described herein and in Ernst et 1999, J Biol Clem 274(14): 9729-9737. Soluble forms of hematopoietin receptors comprising one or more extracellular domains of the hematopoietin receptor, such as soluble forms of HPR1 and HPR2, may also be used in assays to measure their effect on cell growth, proliferation, differentiation, or activation; in such assays the cells are contacted with the soluble form of the receptor and their growth, -18proliferation, differentiation, or activation is measured, for example by measuring the incorporation of 0 radioactive thymidine or by microscopic examination of treated and untreated cells.

Z The terms "HPR1 polypeptide activity" and "HPR2 polypeptide activity," as used herein, Sinclude any one or more of the following: ligand-binding activity and intracellular signaling activity (which includes effects on cell growth, proliferation, differentiation, or activation), as well as the ex vivo and in vivo activities of HPRI and HPR2 polypeptides. The degree to which HPR1 and HPR2 t polypeptides and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay methods as disclosed herein; those of skill in the art will appreciate that i other, similar types of assays can be used to measure HPR1 and HPR2 biological activities.

Another aspect of the biological activity of HPR1 and HPR2 polypeptides is the ability of members of these polypeptide families to bind particular binding partners such as cytokines, other hematopoietin receptor polypeptides, and intracellular signaling polypeptides, with the cytokine receptor domain binding to cytokines and the intracellular signaling domain binding to intracellular signaling polypeptides such as members of the JAK and SHP polypeptide families. The term "binding partner," as used herein, includes ligands, receptors, substrates, antibodies, other hematopoietin receptor polypeptides, the same HPRI or HPR2 polypeptide (in the case of homotypic interactions), and any other molecule that interacts with an HPR1 or an HPR2 polypeptide through contact or proximity between particular portions of the binding partner and the HPR1 or HPR2 polypeptide.

Because the cytokine receptor domains of HPR1 and HPR2 polypeptides bind to cytokines, an HPR1 or HPR2 cytokine receptor domain when expressed as a separate fragment from the rest of an HPR1 or HPR2 polypeptide, or as a soluble polypeptide, fused for example to an immunoglobulin Fc domain, is expected to disrupt the binding of endogenous HPRI and/or HPR2 polypeptides to their binding partners. By binding to one or more binding partners, the separate cytokine receptor domain polypeptide likely prevents binding by the native HPR1 and/or HPR2 polypeptide(s), and so acts in a dominant negative fashion to inhibit the biological activities mediated via binding of HPR1 and/or HPR2 polypeptides to cytokines. Assays for evaluating the biological activities and partner-binding properties of HPRl and HPR2 polypeptides are described further herein.

HPR1 and HPR2 polypeptides are involved in cell proliferation, differentiation, or activation diseases or conditions, that share as a common feature ligand-binding activity in their etiology. More specifically, the following cell proliferation conditions are those that are known or are likely to involve the biological activities of HPR1 and/or HPR2 polypeptides: pancytopenia, leukopenia, anemia, thrombocytopenia, neurodegenerative disorders, osteoporosis resulting from a lack of bone-forming cells, leukemia, tumour metastasis, and osteoporosis resulting from an excess of bone-resorbing cells.

In addition, the following metabolic conditions involving hematopoietin receptor ligands such as leptin are those that are known or are likely to involve the biological activities of HPR1 and/or HPR2 polypeptides: obesity, cachexia, wasting, and AIDS-related weight loss. Also, the following prolactinrelated conditions are those- that are known or are likely to involve the biological activities of HPR1 and/or HPR2 polypeptides: deficient mammary development, infertility, breast cancer, and -19prolactinoma. Blocking or inhibiting the interactions between members of the HPR1 and HPR2 0 polypeptide families and their substrates, ligands. receptors, binding partners, and or other interacting polypeptides is an aspect of the invention and provides methods for treating or ameliorating these diseases and conditions through the use of inhibitors of EPRI and/or HPR2 polypeptide activity.

Examples of such inhibitors or antagonists are described in more detail below. For certain conditions involving too little HPRI or HPR2 polypeptide activity, methods of treating or ameliorating these conditions comprise increasing the amount or activity of HPR1 or HPR2 polypeptides by providing Sisolated HPR1 or HPR2 polypeptides or active fragments or fusion polypeptides thereof, or by providing compounds (agonists) that activate endogenous or exogenous HPR1 or HPR2 polypeptides.

0 HPR1 and HPR2 Polypeptides 15 An HPR1 polypeptide is a polypeptide that shares a sufficient degree of amino acid identity or similarity to the human HPR1 polypeptide of SEQ ID NO:4 or the murine HPR1 polypeptide of SEQ ID NO:12 to be identified by those of skill in the art as a polypeptide likely to share particular structural domains and/or have biological activities in common with the HPRI polypeptides of SEQ ID NO:4 and SEQ ID NO:12 and/or bind to antibodies that also specifically bind to other HPR1 polypeptides. An HPR2 polypeptide is a polypeptide that shares a sufficient degree of amino acid identity or similarity to the HPR2 polypeptides of SEQ ID NOs 21, 23, 25, and 27 to be identified by those of skill in the art as a polypeptide likely to share particular structural domains and/or have biological activities in common with the HPR2 polypeptides of SEQ ID NOs 21, 23, 25, and 27 and/or bind to antibodies that also specifically bind to other HPR2 polypeptides. HPRI and HPR2 polypeptides can be isolated from naturally occurring sources, or have the same structure as naturally occurring HPR1 or HPR2 polypeptides, or can be produced to have structures that differ from naturally occurring HPR1 or HPR2 polypeptides. Polypeptides derived from any HPR1 or HPR2 polypeptide by any type of alteration (for example, but not limited to, insertions, deletions, or substitutions of amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three-dimensional structure or self-association state; and changes to its association with other polypeptides or molecules) are also HPR1 or HPR2 polypeptides, respectively.

Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those of the HPRI and HPR2 polypeptides described herein, but into which modifications are naturally provided or deliberately engineered. A polypeptide that shares biological activities in common with members of the HPRI and/or HPR2 polypeptide family is a polypeptide having HPR1 and/or HPR2 polypeptide activity. Examples of biological activities exhibited by HPR1 and/or HPR2 polypeptides include, without limitation, ligand-binding activity and intracellular signaling.

The present invention provides both full-length and mature forms of HPR1 and HPR2 polypeptides. Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated. The amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame ("ORP) of a cDNA

O

molecule. Several full-length polypeptides can be encoded by a single genetic locus if multiple mRNA O forms are produced from that locus by alternative splicing or by the use of multiple translation Z initiation sites. The "mature form" of a polypeptide refers to a polypeptide that has undergone post- Stranslational processing steps such as cleavage of the signal sequence or proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example by cleavage of the signal sequence at multiple sites, or by differential regulation of proteases t that cleave the polypeptide. The mature form(s) of such polypeptide can be obtained by expression, in a suitable mammalian cell or other host cell, of a nucleic acid molecule that encodes the full-length Spolypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protcase cleavage sites. The HPR1 and HPR2 polypeptides of the invention also include those that result from •N post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield alternative splice forms of HPR1 or HPR2 such as a truncated but biologically active polypeptide or, for example, a naturally occurring soluble form of the polypeptide. Also encompassed within the invention are variations attributable to proteolysis such as differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptide (generally from 1-5 terminal amino acids).

The invention further includes HPRI and HPR2 polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems COS-1 or CHO cells) can be similar to or significantly different from a native polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coli, provides nonglycosylated molecules. Further, a given preparation can include multiple differentially glycosylated species of the polypeptide. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).

Species homologues of HPR1 and HPR2 polypeptides and of nucleic acids encoding them are also provided by the present invention. As used herein, a "species homologue" is a polypeptide or nucleic acid with a different species of origin from that of a given polypeptide or nucleic acid, but with significant sequence similarity to the given polypeptide or nucleic acid, as determined by those of skill in the art. Species homologues can be isolated and identified by making suitable probes or primers from polynucleotides encoding the amino acid sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of HPR1 and HPR2 polypeptides and nucleic acids encoding them; that is, naturally-occurring alternative forms of such polypeptides and nucleic acids in which differences in amino acid or nucleotide sequence are attributable to genetic polymorphism (allelic variation among individuals within a population).

Fragments of the HPR1 and HPR2 polypeptides of the present invention are encompassed by the present invention and can be in linear form or cyclized using known methods, for example, as -21- 5 described in Saragovi, et al, Bio/Technology 10, 773-778 (1992) and in McDowell, et al., J. Amer.

SChem. Soc. 114 9245-9253 (1992). Polypeptides and polypeptide fragments of the present invention, Sand nucleic acids encoding them, include polypeptides and nucleic acids with amino acid or nucleotide sequence lengths that are at least 25% (more preferably at least 50%, or at least 60%, or at least and most preferably at least 80%) of the length of an HPR1 polypeptide or of an HPR2 polypeptide, and have at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with that HPRI or HPR2 polypeptide or encoding nucleic acid, where sequence identity is Sdetermined by comparing the amino acid sequences of the polypeptides when aligned so as to O maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are polypeptides and polypeptide fragments, and nucleic acids encoding them, that contain or encode a segment preferably comprising at least 8, or at least 10, or preferably at least 15, or more preferably at least 20, or still more preferably at least 30, or most preferably at least 40 contiguous amino acids.

Such polypeptides and polypeptide fragments may also contain a segment that shares at least sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with any such segment of any of the HPR1 or HPR2 polypeptides, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The percent identity can be determined by visual inspection and mathematical calculation. Preferably, the comparison is done using a computer program. An exemplary, preferred computer program is the Genetics Computer Group (GCG-, Madison, WI) Wisconsin package version 10.0 program, 'GAP.' The preferred default parameters for the 'GAP' program includes: The GCO implementation of comparison matrices for nucleotides and amino acids; such as a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gibskov and Burgess, Nucl. Acids Res.

14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; no penalty for end gaps; and no maximum penalty for long gaps. Another program useful for determining percent identify is the BESTFIT program, also available from the University of Wisconsin as part of the GCG computer package. Default parameters for using the BESTFIT program are the same as those described above for using the GAP program. Other programs used by those skilled in the art of sequence comparison can also be used, such as, for example, the UW-BLAST 2.0 algorithm or the BLASTN program version 2.0.9, available for use via the National Library of Medicine website: ncbinlm.nih.gov/gorf/wblast2.cgi. Standard default parameter settings for UW-BLAST 2.0 are described at the following Internet site: blastwustl.edu/blast/README.ht5sRtferencD In addition, the BLAST algorithm uses the BLOSUM62 amino acid scoring matix, ad optional para=e-rs that can -22be used are as follows: inclusion of a filter to mask segments of the query sequence that have low 0 compositional complexity (as determined by the SEG program of Wootton and Fedcrhen (Computers Sand Chemistry, 1993); also see Wootton and Federhen, 1996, Analysis of compositionally biased regions in sequence databases, Methods EnzymwL 266: 554-71) or segments consisting of shortperiodicity internal repeats (as determined by the XNU program of Claverie and States (Computers and Chemistry, 1993)), and a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is Sgreater than this E-score threshold, the match will not be reported.); preferred E-score threshold values 'are 0.5, or in order of increasing preference, 0.25, 0.1, 0.01, 0.001, 0.0001, le-10. le-20, le-30, le-50, le-75, or le-100.

C "An isolated polypeptide consisting essentially of an amino acid sequence" means that the polypeptide may have, in addition to said amino acid sequence, additional material covalently linked to either or both ends of the polypeptide, said additional material preferably between 1 and 10,000 additional amino acids covalently linked to either end, each end, or both ends of polypeptide, and more preferably between 1 and 1,000 additional amino acids covalently linked to either end, each end, or both ends of the polypeptide, and most preferably between 1 and 100 additional amino acids covalently linked to either end, each end, or both ends of the polypeptide. In preferred embodiments, covalent linkage of additional amino acids to either end, each end, or both ends of the polypeptide results in a novel combined amino acid sequence that is neither naturally occurring nor disclosed in the art The present invention also provides for soluble forms of BPR1 and HPR2 polypcptides comprising or consisting essentially of certain fragments or domains of these polypeptides, and particularly those comprising the extracellular domain or one or more fragments of the extracellular domain. Soluble polypeptides are polypeptides that are capable of being secreted from the cells in which they are expressed. In such forms part or all of the intracellular and transmembrane domains of the polypeptide are deleted such that the polypeptide is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of polypeptides of the invention can be identified in accordance with known techniques for determination of such domains from sequence information. Soluble HPR1 and HPR2 polypeptides also include those polypeptides which include part of the transmembrane region, provided that the soluble HPR1 or HPR2 polypeptide is capable of being secreted from a cell, and preferably retains HPR1 and/or HPR2 polypeptide activity. Soluble HPRI and HPR2 polypeptides further include oligomers or fusion polypeptides comprising the extracellular portion of at least one HPR1 or HPR2 polypeptide, and fragments of any of these polypeptides that have HPR1 and/or HPR2 polypeptide activity. A secreted soluble polypeptide can be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a -23soluble form of the polypeptide. The use of soluble forms of HPR1 or HPR2 polypeptides is Sadvantageous for many applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Moreover, soluble polypeptides are generally more suitable than membrane-bound forms for parenteral administration and for many enzymatic procedures.

In another aspect of the invention, preferred polypeptides comprise various combinations of HPR1 and/or HPR2 polypeptide domains, such as the cytokine receptor domain and the intracellular signaling domain. Accordingly, polypeptides of the present invention and nucleic acids encoding them include those comprising or encoding two or more copies of a domain such as the cytokine receptor O domain, two or more copies of a domain such as the intracellular signaling domain, or at least one copy of each domain, and these domains can be presented in any order within such polypeptides.

Further modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the polypeptide sequences can include the alteration, substitution, replacement, insertion or deletion of a selected amino acid. For example, one or more of the cysteine residues can be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, U.S.

Pat. No. 4,518,584). As another example, N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain.

Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in polypeptides include those described in U.S. Patent 5.071,972 and EP 276.846. Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives can be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein. Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide or a substantial equivalent thereof. One example is a variant that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, as described in U.S. Patent No. 5,512,457 and as set forth herein.

-24- Other derivatives include covalent or aggregative conjugates of the polypeptides with other O polypeptidcs or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal Z fusions. Examples of fusion polypeptides are discussed below in connection with oligomers. Further, Sfusion polypeptides can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG® t peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant polypeptide. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAGe peptide in the presence of certain divalent metal cations, as described in U.S. Patent 5,011,912. The 4E1 1 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259.

C1 Monoclonal antibodies that bind the FLAG® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.

Encompassed by the invention are oligomers or fusion polypeptides that contain an HPR1 polypeptide and/or an HPR2 polypeptide, one or more fragments of HPRI and/or HPR2 polypeptides, or any of the derivative or variant forms of HPR1 and HPR2 polypeptides as disclosed herein. In particular embodiments, the oligomers comprise soluble HPR1 and/or HPR2 polypeptides. Oligomers can be in the form of covalently linked or non-covalently-linked multimers, including dimers, trimers, or higher oligomers. In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites. In an alternative embodiment the invention is directed to oligomers comprising multiple HPRI and/or HPR2 polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides, such peptides having the property of promoting oligomcrization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.

In embodiments where variants of the HPR1 and/or HPR2 polypeptides are constructed to include a membrane-spanning domain, they will form a Type I membrane polypeptide. Membranespanning HPR1 and/or HPR2 polypeptides can be fused with extracellular domains of receptor polypeptides for which the ligand is known. Such fusion polypeptides can then be manipulated to control the intracellular signaling pathways triggered by the membrane-spanning HPR1 or HPR2 polypeptide. HPR1 and HPR2 polypeptides that span the cell membrane can also be fused with agonists or antagonists of cell-surface receptors, or cellular adhesion molecules to further modulate HPR1 and/or HPR2 intracellular effects. In another aspect of the present invention, interleukins can be situated between the preferred HPR1 or HPR2 polypeptide fragment and other fusion polypeptide domains.

Immunoglobulin-based Oligomers. The polypeptides of the invention or fragments thereof can be fused to cmolecules such as immunoglobulins for many purposes, including increasing the valency of polypeptide binding sites. For example, fragments of an HPR1 polypeptide and/or of an 5 HPR2 polypeptide can be fused directly or through linker sequences to the PC portion of an z immunoglobulin. For a bivalent form of the polypeptide, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes can also be used to generate such fusions. For C example, a polypeptide-IgM fusion would generate a decavalent form of the polypeptide of the invention. The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides made up of the Pc region of an antibody comprising any or all of the CH domains of the Fc region.

Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also U included. Preferred Fc polypeptides comprise an Fc polypeptide derived from a human IgGl antibody.

As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins.

Preparation of fusion polypeptides comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al..(PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Polypeptides", in Current Protocols in lnnumology, Suppl. 4, pages 10.19.1 10.19.11, 1992). Methods for preparation and use of immunoglobulin-based oligomers are well known in the art One embodiment of the present invention is directed to a dimer comprising two fusion polypeptides created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fe fusion polypeptide is inserted into an appropriate expression vector. Polypeptide/Fc fusion polypeptides are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules. One suitable Fc polypeptide, described in PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgI1 antibody. Another useful Fc polypeptide is the Pc mutein described in U.S. Patent 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994). The amino acid sequence of this mutein is identical to that of the native Fe sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. The above-described fusion polypeptides comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Polypeptide A or Polypeptide G columns. In other embodiments, the polypeptides of the invention can be substituted for the variable portion of an antibody heavy or light chain. If fusion polypeptides are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four HPR1 and/or HPR2 extracellular regions.

Peptide-linker Based Oligomers. Alternatively, the oligomer is a fusion polypeptide comprising multiple HPR1 and/or HPR2 polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233. A DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique.

-26- For example, a chemically synthesized oligonucleotide encoding the linker can be ligated between the O sequences. In particular embodiments, a fusion polypeptide comprises from two to four soluble HPRI Sand/or HPR2 polypeptides, separated by peptide linkers. Suitable peptide linkers, their combination Swith other polypeptides, and their use are well known by those skilled in the art Leucine-Zippers. Another method for preparing the oligomers of the invention involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the lt polypeptides in which they are found. Leucine zippers were originally identified in several DNAbinding polypeptides (Landschulz et al., Science 240:1759, 1988), and have since been found in a Svariety of different polypeptides. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. The zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, often with four or C"1 five leucine residues interspersed with other amino acids. Use of leucine zippers and preparation of oligomers using leucine zippers are well known in the art.

Other fragments and derivatives of the sequences of polypeptides which would be expected to retain polypeptide activity in whole or in part and may thus be useful for screening or other immunological methodologies can also be made by those skilled in the art given the disclosures herein.

Such modifications are believed to be encompassed by the present invention.

Nucleic Acids Encoding HPR1 Polypeptides and Nucleic Acids Encoding HPR2 Polypeptides Encompassed within the invention are nucleic acids encoding HPR1 polypeptides and nucleic acids encoding HPR2 polypeptides. These nucleic acids can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source. Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for the isolation of nucleic acids or as query sequences for database searches, can be obtained by "back-translation" from the amino acid sequences, or by identification of regions of amino acid identity with polypeptides for which the coding DNA sequence has been identified. The well-known polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding an HPRI or HPR2 polypeptide or a desired combination of HPR1 and/or HPR2 polypeptide fragments. Oligonucleotides that define the desired termini of the combination of DNA fragments are employed as 5' and 3' primers.

-The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombnant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et. aL, eds., Academic Press, Inc. (1990).

Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the invention include full-length genes or cDNA -27molecules as well as a combination of fragments thereof. The nucleic acids of the invention are z preferentially derived from human sources, but the invention includes those derived from non-human species, as well.

SAn "isolated nucleic acid" is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally-occurring sources. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or Soligonucleotides for example, it is understood that the nucleic acids resulting from such processes are cisolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form O of a separate fragment or as a component of a larger nucleic acid construct In one preferred embodiment, the invention relates to certain isolated nucleic acids that are substantially free from contaminating endogenous material. The nucleic acid molecule has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.

"An isolated nucleic acid consisting essentially of a nucleotide sequence" means that the nucleic acid may have, in addition to said nucleotide sequence, additional material covalently linked to either or both ends of the nucleic acid molecule, said additional material preferably between 1 and 100,000 additional nucleotides covalently linked to either end, each end, or both ends of the nucleic acid molecule, and more preferably between 1 and 1,000 additional nucleotides covalently linked to either end, each end, or both ends of the nucleic acid molecule, and most preferably between 10 and 100 additional nucleotides covalently linked to either end, each end, or both ends of the nucleic acid molecule. In preferred embodiments, covalent linkage of additional nucleotides to either end, each end, or both ends of the nucleic acid molecule results in a novel combined nucleotide sequence that is neither naturally occurring nor disclosed in the art. An isolated nucleic acid consisting essentially of a nucleotide sequence may be an expression vector or other construct comprising said nucleotide sequence.

The present invention also includes nucleic acids that hybridize under moderately stringent conditions, and more preferably 'highly stringent conditions, to nucleic acids encoding HPR1 polypeptides and/or nucleic acids encoding HPR2 polypeptides described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook,, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, chapters 9 and 11; and Current Protocols 28in Molecular Biology, 1995, Ausubel et al., eds., John Wiley Sons, Inc., sections 2.10 and 6.3-6.4), 0 and can be readily determined by those having ordinary skill in the art based on, for example, the Z length and/or base composition of the DNA. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH hybridization buffer of about 50% formamide, 6 x SSC, and a hybridization temperature of about degrees C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 degrees and washing conditions of about 60 degrees C, in x SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as Sabove, but with washing at approximately 68 degrees C, 0.2 x SSC, 0.1% SDS. SSPE (lxSSPE is 0.15M NaC1, 10 mM NaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCI and 15 mM sodium citrate) in the hybridization and wash buffers; washes are C(7 performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, Sambrook et al., 1989). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10.degrees C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C) of A T bases) of #G C bases).

For hybrids above 18 base pairs in length, Tm (degrees C) 81.5 1 6 .6(logio [Nal) 0.41(% G C) (600/N), where N is the number of bases in the hybrid, and [Naf is the concentration of sodium ions in the hybridization buffer ([Nal for IxSSC 0.165M). Preferably, each such hybridizing nucleic acid has a length that is at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with the nucleic acid of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while minimizing sequence gaps as described in more detail above.

The present invention also provides genes corresponding to the nucleic acid sequences disclosed herein. "Corresponding genes" or "corresponding genomic nucleic acids" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA nucleic acid sequences are -29derived and can include contiguous regions of the genome necessary for the regulated expression of 0 such genes. Corresponding genes can therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer' or suppressor elements. Corresponding genomic nucleic acids can include 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence upstream of the first nucleotide of the genomic sequence C corresponding to the initiation codon of the HPR1 coding sequence or of the HPR2 coding sequence, C and 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence downstream of the last nucleotide of the genomic sequence corresponding to the termination codon of the HPR1 coding sequence or of the HPR2 coding sequence. The corresponding genes or genomic nucleic acids can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.

An "isolated gene" or an "isolated genomic nucleic acid" is a genomic nucleic acid that has been separated from the adjacent genomic sequences present in the genome of the organism from which the genomic nucleic acid was isolated.

Methods for Making and Purifying HPR1 and HPR2 Polypeptides Methods for making HPR1 and HPR2 polypeptides are described below. Expression, isolation, and purification of the polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods. The isolated nucleic acid of the invention can be operably linked to an expression control sequence such as the pDC409 vector (Giri et al., 1990, EMBO 13: 2821) or the derivative pDC412 vector (Wiley et al., 1995, Immunity 3: 673). The pDC400 series vectors are useful for transient mammalian expression systems, such as CV-1 or 293 cells. Alternatively, the isolated nucleic acid of the invention can be linked to expression vectors such as pDC312, pDC316, or pDC317 vectors, which are useful for stable mammalian expression systems, such as CHO cells or their derivatives. Other expression control sequences and cloning technologies can also be used to produce the polypeptide recombinantly, such as the pMT2 or pED expression vectors (Kaufman et al., 1991, Nucleic Acids Res. 19: 4485-4490; and Pouwels et al., 1985, Cloning Vectors: A Laboratory Manual, Elsevier, New York) and the GATEWAY Vectors (lifetech.com/Content/Tech-Online/molecularbiology/manuals_pps/11797016.pdf, Life Technologies; Rockville, MD). Many suitable expression control sequences are known in the art. General methods of expressing recombinant polypeptides are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As used herein "operably linked" means that the nucleic acid of the invention and an expression control sequence are situated within a construct, vector, or cell in such a way that the polypeptide encoded by the nucleic acid is expressed when appropriate molecules (such as polymerases) are present. As one embodiment of the invention, at least one expression control sequence is operably linked to the nucleic acid of the invention in a recombinant host cell or progeny 0 thereof, the nucleic acid and/or expression control sequence having been introduced into the host cell Z by transformation or transfectioni for example, or by any other suitable method. As another embodiment of the invention, at least one expression control sequence is integrated into the genome of a recombinant host cell such that it is operably linked to a nucleic acid sequence encoding a polypeptide of the invention. In a further embodiment of the invention, at least one expression control lr sequence is operably linked to a nucleic acid of the invention through the action of a trans-acting factor such as a transcription factor, either in vitro or in a recombinant host cell.

SIn addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. The choice of signal peptide or leader can depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. To illustrate, Cl examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-l receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846. A DNA sequence for a signal peptide (secretory leader) can be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion polypeptide comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell. The skilled artisan will also recognize that the position(s) at which the signal peptide is cleaved can differ from that predicted by computer program, and can vary according to such factors as the type of host cells employed in expressing a recombinant polypeptide. A polypeptide preparation can include a mixture of polypeptide molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.

Established methods for introducing DNA into mammalian cells have been described (Kaufman, RJ., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine- Plus lipid reagent, can be used to transfect cells (Feigner et al., Proc. Natl. Acad Sci USA 84:7413- 7417, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1- 3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185:487-511, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. NatL Acad. Sci. USA 77:4216-4220, 1980). Other examples of selectable markers that can be incorporated into an expression vector include cDNAs -31conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be 0 selected on the basis of resistance to these compounds.

SAlternatively, gene products can be obtained via homologous recombination, or "gene targeting," techniques. Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to 10 induce expression of the endogenous nucleic acid sequence of interest (see, for example, U.S. Patent No. 5,272,071). The location of integration into a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene. In a preferred embodiment, the present invention also contemplates the introduction of exogenous transcriptional O control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product, again, without the need for isolation of the gene sequence itself from the host cell.

A number of types of cells can act as suitable host cells for expression of the polypeptide.

Mammalian host cells include, for example, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al.

(EMBO J. 10: 2821, 1991), human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Alternatively, it is possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria.

Potentially suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombo, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides.

Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments can be accomplished using known chemical or enzymatic methods. The polypeptide can also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/nsect cell expression systems are commercially available in kit form from, Invitrogen. San Diego, Calif., U.S.A. (the MaxBac@ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Tecllology 6:47 (1988). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from nucleic acid constructs disclosed herein. A host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell".

-32- The polypeptide of the invention can be prepared by culturing transformed host cells under O culture conditions suitable to express the recombinant polypeptide. The resulting expressed Z polypeptide can then be purified from such culture from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the polypeptide can also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, heparinn toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction I> chromatography using such resins as phcnyl ether, butyl ether, or propyl ether, or immunoaffinity chromatography. Alternatively, the polypeptide of the invention can also be expressed in a form which will facilitate purification. For example, it can be expressed as a fusion polypeptide, such as those of maltose binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, NJ.) and InVitrogcn, respectively. The polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (FLAG@) is commercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide. The polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the present invention as an "isolated polypeptide"; such isolated polypeptides of the invention include isolated antibodies that bind to HPR1 and/or HPR2 polypeptides, fragments, variants, binding partners etc. The polypeptide of the invention can also be expressed as a product of transgenic animals, as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the polypeptide.

It is also possible to utilize an affinity column comprising a polypeptide-binding polypeptide of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention. In this aspect of the invention, polypeptido-binding polypeptides, such as the anti-polypeptide antibodies of the invention or other polypeptides that can interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of polypeptide-binding polypeptides of the invention to a solid phase contacting surface can be accomplished by any means, for example, -33magnetic microspheres can be coated with these polypeptide-binding polypeptides and held in the z incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding polypeptides thereon. Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding polypeptide and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cellsurface binding partner. Alternatively, mixtures of cells suspected of containing polypeptide- O expressing cells of the invention first can be incubated with a biotinylated polypeptide-binding polypeptide of the invention. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the polypeptide-binding cells to the beads. Use of avidin-coated beads is known in the art. See Berenson, et al. J. CelL Biochem., 10D:239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.

The polypeptide can also be produced by known conventional chemical synthesis. The synthetically-constructed polypeptide sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with HPR1 and/or HPR2 polypeptides can possess biological properties in common therewith, including HPR1 and/or HPR2 polypeptide activity. Thus, they can be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The desired degree of purity depends on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.

Antagonists and Agonists of HPR1 and/or HPR2 Polypeptides Any method which neutralizes HPR1 and/or HPR2 polypeptides or inhibits expression of the HPRI and/or HPR2 genes (either transcription or translation) can be used to reduce the biological activities of HPRI and/or ,HPR2 polypeptides. In particular embodiments, antagonists inhibit the binding of at least one HPRI polypeptide and/or at least one HPR2 polypeptide to cells, thereby inhibiting biological activities induced by the binding of those HPR1 or HPR2 polypeptides to the -34cells. In certain other embodiments of the invention, antagonists can be designed to reduce the level of O endogenous HPR1 and/or HPR2 gene expression, using well-known antisense or ribozyme Z approaches to inhibit or prevent translation of HPR1 and/or HPR2 mRNA transcripts; triple helix approaches to inhibit transcription of HPR1 and/or HPR2 genes; or targeted homologous recombination to inactivate or "knock out" the HPR1 gene(s), the HPR2 gene(s), or their endogenous promoters or enhancer elements. Such antisense, ribozyme, and triple helix antagonists can be n designed to reduce or inhibit either unimpaired, or if appropriate, mutant HPR1 and/or HPR2 gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation. Antisense approaches involve C the design of oligonucleotides (either DNA or RNA) that are complementary to an HPR1 and/or to an HPR2 mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of a nucleic acid, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the nucleic acid, forming a stable duplex (or triplex, as appropriate). In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA can thus be tested, or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Preferred oligonucleotides are complementary to the 5' end of the message, the 5' untranslated sequence up to and including the AUG initiation codon. However, oligonucleotides complementary to the 5- or 3'non- translated, non-coding regions of the HPR1 or HPR2 gene transcript(s) could be used in an antisense approach to inhibit translation of endogenous HPR1 and/or HPR2 mRNA. Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or doublestranded. Chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of nucleotides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open, end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound (see, U.S. Pat No. 5,985,664). Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers". The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.

The oligonucleotide can include other appended groups such as peptides for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, Letsinger et aL, 1989, Proc Natl Acad Sci USA 86:6553-6556; Lemaitre et 1987, Proc Natl Acad Sci 84:648-652; PCT Publication No. W088/09810), or hybridization-triggered cleavage agents or intercalating agents.

S(See, Zou, 1988, Pharm. Res. 5:539-549). The antisense molecules should be delivered to cells which express the HPRI and/or HPR2 transcript in vivo. A number of methods have been developed C- for delivering antisense DNA or RNA to cells; antisense molecules can be injected directly into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs.

Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol fl or pol II promoter. The use of such a O 15 construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous HPRI and/or HPR2 gene transcripts and thereby prevent translation of the HPRI and/or HPR2 mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.

Ribozyme molecules designed to catalytically cleave HPRI and/or HPR2 mRNA transcripts can also be used to prevent translation of HPR1 and/or HPR2 mRNA and expression of HPRI and/or HPR2 polypeptides. (See, PCT International Publication W090/11364 and US Patent No.

5,824,519). The ribozymes that can be used in the present invention include hammerhead ribozymes (Haseloff and Gerlach, 1988, Nature, 334:585-591), RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (International Patent Application No. WO 88/04300; Been and Cech, 1986, Cell, 47:207-216). As in the antisense approach, the ribozymes can be composed of modified oligonucleotides for improved stability, targeting, etc.) and should be delivered to cells which express HPRI and/or HPR2 polypeptides in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol II or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous HPRI and/or HPR2 messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Alternatively, endogenous HPRI and/or HPR2 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target HPRI and/or HPR2 gene. (See generally, Helene, 1991, Anticancer Drug Des., 569-584; Helene, et al., 1992. Ann. N.Y. Acad. Sci.. 660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815).

-36- Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be O prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include Z techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known it the art such as for example solid phase phosphoramidite chemical synthesis.

Oligonucleotides can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As l examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et 1988, Nucl. Acids Res. 16:3209. Methylphosphonate oligonucleotides can be prepared by use of controlled Spore glass polymer supports (Sarin et 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451).

Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety rC of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Endogenous target gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination see Smithies, et al., 1985, Nature 317, 230-234; Thomas and Capecchi. 1987, Cell 51, 503-512; Thompson, et al., 1989, Cell 313-321). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene see Thomas and Capecchi 1987 and Thompson, 1989, supra), or in model organisms such as Caenorhabditis eleganswhere the "RNA interference" ("RNAi") technique (Grishok, Tabara, and Mello, 2000, Genetic requirements for inheritance of RNAi in C. elegans, Science 287 (5462): 2494-2497), or the introduction of transgenes (Dernburg et at., 2000, Transgene-mediated cosuppression in the C. elegans germ line, Genes Dev. 14 1578-1583) are used to inhibit the expression of specific target genes.

However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate vectors such as viral vectors.

Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the nucleic acid sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense nucleic acids or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol.

58: 1-39). Transgenic animals that have multiple copies of the gene(s) corresponding to the nucleic -37acid sequences disclosed herein, preferably produced by transformation of cells with genetic constructs 0 that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or C- that change temporal or spatial patterns of gene expression, are also provided (see European Patent No.

0 649 464 BI). In addition, organisms are provided in which the gene(s) corresponding to the nucleic acid sequences disclosed herein have been partially or completely inactivated, through insertion of n extraneous sequences into the corresponding gene(s) or through deletion of all or part of the Scorresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, Spreferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc Natl Acad Sci USA 90(16): 7431-7435; Clark et al., 1994, Proc Natl Acad Sci USA 91(2): 719-722), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336: 348-352; U.S. Pat.

Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396; 5,616,491; and 5,679,523). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals.

Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the polypeptide product(s) of the corresponding gene(s).

Also encompassed within the invention are HPRI and HPR2 polypeptide variants with partner binding sites that have been altered in conformation so that the TPR1 or -PR2 variant will still bind to its partner(s), but a specified small molecule will fit into the altered binding site and block that interaction, or the HPR1 or HPR2 variant will no longer bind to its partner(s) unless a specified small molecule is present (see for example Bishop et aL, 2000, Nature 407: 395-401). Nucleic acids encoding such altered HPR1 or HPR2 polypeptides can be introduced into organisms according to methods described herein, and can replace the endogenous nucleic acid sequences encoding the corresponding HPRI or HPR2 polypeptide. Such methods allow for the interaction of a particular HPRI or HPR2 polypeptide with its binding partners to be regulated by administration of a small molecule compound to an organism, either systemically or in a localized manner.

The HPR1 and HPR2 polypeptides themselves can also be employed in inhibiting a biological activity of HPRI and /or of HPR2 in in vitro or in vivo procedures. Encompassed within the invention are cytokine receptor domains of HPR1 and HPR2 polypeptides that act as "dominant negative" inhibitors of native HPR1 and/or HPR2 polypeptide function when expressed as fragments or as components of fusion polypeptides. For example, a purified polypeptide domain of the present invention can be used to inhibit binding of HPRI or HPR2 polypeptides to endogenous binding partners. Such use effectively would block HPR1 and/or HPR2 polypeptide interactions and inhibit HPR1 and/or HPR2 polypeptide activities. In still another aspect of the invention, a soluble form of an HPR1 and/or HPR2 binding partner is used to bind to an endogenous HPR1 and/or HPR2 polypeptide, and competitively inhibit activation of that endogenous HPR1 and/or HPR2 polypeptide. Furthermore, antibodies which bind to HPRI and/or HPR2 polypeptides often inhibit HPR1 and/or HPR2 -38polypeptide activity and act as antagonists. For example, antibodies that specifically recognize one or O more epitopes of HPR1 and/or HPR2 polypeptides, or epitopes of conserved variants of HPR1 and/or Z HPR2 polypeptides, or peptide fragments of an HPR1 and/or HPR2 polypeptide can be used in the C invention to inhibit HPR1 and/or HPR2 polypeptide activity. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

Alternatively, purified and modified HPR1 and/or HPR2 polypeptides of the present invention can be administered to modulate interactions between HPR1 and/or HPR2 polypeptides and HPRI and/or HPR2 binding partners that are not membrane-bound. Such an approach will allow an alternative method for the modification of HPRI- and/or HPR2-influenced bioactivity.

In an alternative aspect, the invention further encompasses the use of agonists of HPRI and/or HPR2 polypeptide activity to treat or ameliorate the symptoms of a disease for which increased HPRI and/or HPR2 polypeptide activity is beneficial. Such diseases include but are not limited to pancytopenia, leukopenia, anemia, thrombocytopenia, neurodegenerative disorders, osteoporosis resulting from a lack of bone-forming cells, obesity, deficient mammary development, and infertility.

In a preferred aspect, the invention entails administering compositions comprising an HPR1 or HPR2 nucleic acid or an HPRI or HPR2 polypeptide to cells in vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism such as a vertebrate or mammal. Preferred therapeutic forms of HPRI and HPR2 are soluble forms, as described above. In still another aspect of the invention, the compositions comprise administering an HPR-encoding nucleic acid or an HPR2-encoding nucleic acid for expression of an HPR1 or HPR2 polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant decreased) endogenous activity of an HPR1 or HPR2 polypeptide. Furthermore, the invention encompasses the administration to cells and/or organisms of compounds found to increase the endogenous activity of HPRI andlor HPR2 polypeptides. One example of compounds that increase HPR1 and/or HPR2 polypeptide activity are agonistic antibodies, preferably monoclonal antibodies, that bind to HPR1 and/or HPR2 polypeptides or binding partners, which may increase HPRI and/or HPR2 polypeptide activity by causing constitutive intraccllular signaling (or "ligand mimicking"), or by preventing the binding of a native inhibitor of HPR1 and/or HPR2 polypeptide activity.

Antibodies to HPR1 and/or HPR2 Polypeptides Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). In the present invention, specifically binding antibodies are those that will specifically recognize and bind with HPR1 and/or HPR2 polypeptides, homologues, and variants, but not with other molecules. In one preferred embodiment, the antibodies arc specific for -39the polypeptides of the present invention and do not cross-react with other polypeptides. In this 0 manner, the HPR1 and HPR2 polypeptides, fragments, variants, fusion polypeptides, etc., as set forth above can be employed as "immunogens" in producing antibodies immunoreactive therewith.

More specifically, the polypeptides, fragment, variants, fusion polypeptides, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding (Janeway and Travers, Imnumw Biology 3-9 (Garland SPublishing Inc., 2nd ed. 1996)). Because folded polypeptides have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the polypeptide and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (Janeway and Travers, Imnumo Biology 2:14 (Garland Publishing Inc., 2nd ed.

1996)). Epitopes can be identified by any of the methods known in the art. Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below.

Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of the polypeptides of the invention, whether the epitopes have been isolated or remain part of the polypeptides, both polyclonal and monoclonal antibodies can be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988); Kohler and Milstein, Pat No.

4376,110); the human B-cell hybridoma technique (Kosbor et al., 1984, J Inununol 133: 3001-3005; Cole et al., 1983, Proc Natl Acad Sci USA 80:2026-2030); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques.

The hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. For the production of antibodies, various host animals can be immunized by injection with one or more of the following: an HPR1 or HPR2 polypeptide, a fragment of an HPRI or O HPR2 polypeptide, a functional equivalent of an HPR1 or HPR2 polypeptide, or a mutant form of an Z HPR1 or HPR2 polypeptide. Such host animals can include but are not limited to rabbits, mice, and C rats. Various adjuvants can be used to increase the immunologic response, depending on the. host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, It peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Coryncbacterium parvum. The monoclonal C antibodies can be recovered by conventional techniques. Such monoclonal antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA. IgD and any subclass thereof.

In addition, techniques developed for the production of "chimeric antibodies" (Takeda et al., (1 1985, Nature, 314: 452-454; Morrison et al., 1984, Proc Nati Acad Sci USA 81: 6851-6855; Boulianne et al., 1984, Nature 312: 643-646; Neuberger et al., 1985, Nature 314: 268-270) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a porcine mAb and a human immunoglobulin constant region. The monoclonal antibodies of the present invention also include humanized versions of murine monoclonal antibodies.

Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al.

(Bio/Techlology 7:934, 1989), and Winter and Harris (TIPS 14:139, Can, 1993). Useful techniques for humanizing antibodies are also discussed in U.S. Patent 6,054,297. Procedures to generate antibodies transgenically can be found in GB 2,272,440, US Patent Nos. 5,569,825 and 5,545,806, and related patents. Preferably, for use in humans, the antibodies are human or humanized; techniques for creating such human or humanized antibodies are also well known and are commercially available from, for example, Medarex Inc. (Princeton, NJ) and Abgenix Inc. (Fremont, CA). In another preferred embodiment, fully human antibodies for use in humans are produced by screening a phage display library of human antibody variable domains (Vaughan et al., 1998, Nat Biotechnol. 16(6): 535-539; and U.S. Patent No. 5,969,108).

Antigen-binding antibody fragments which recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can -41 be generated by reducing the disulfide bridges of the (ab)2 fragments. Alternatively, Fab expression z libraries can be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy Sidentification of monoclonal Fab fragments with the desired specificity. Techniques described for the production of single chain antibodies Pat. No. 4,946,778; Bird, 1988, Science 242:423426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward etal., 1989, Nature 334:544- 546) can also be adapted to produce single chain antibodies against HPRI and/or HPR2 gene products.

eg Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Such single chain antibodies can also be useful intracellularly as 'intrabodies), for example as described by Marasco et al. Immunol Metlods 231:223-238. 1999) for genetic therapy in HIV infection. In addition, antibodies to the HPRI and/or HPR2 polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the HPRI and/or HPR2 polypeptide and that may bind to the binding partner(s) of HPRI and/or HPR2 polypeptides, using techniques well known to those skilled in the art (See, eg., Greenspan Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8).2429-2438).

Antibodies that are immunoreactive with the polypeptides of the invention include bispecific antibodies antibodies that are immunoreactive with the polypeptides of the invention via a first antigen binding domain, and also immunoreactive with a different polypeptide via a second antigen binding domain). A variety of bispecific antibodies have been prepared, and found useful both in vitro and in vivo (see, for example, U.S. Patent 5,807,706; and Cao and Suresh, 1998, Bioconjugate Chem 9: 635-644). Numerous methods of preparing bispecific antibodies are known in the art, including the use of hybrid-hybridomas such as quadromas, which are formed by fusing two differed hybridomas, and triomas, which are formed by fusing a hybridoma with a lymphocyte (Milstein and Cuello, 1983, Nature 305: 537-540; U.S. Patent 4,474,893; and U.S. Patent 6,106,833). U.S. Patent 6,060,285 discloses a process for the production of bispecific antibodies in which at least the genes for the light chain and the variable portion of the heavy chain of an antibody having a first specificity are transfected into a hybridoma cell secreting an antibody having a second specificity. Chemical coupling of antibody fragments has also been used to prepare antigen-binding molecules having specificity for two different antigens (Brennan et al., 1985, Science 229: 81-83; Glennie et al., J. Immunol., 1987, 139:2367-2375; and U.S. Patent 6,010,902). Bispecific antibodies can also be produced via recombinant means, for example, by using. the leucine zipper moieties from the Fos and Jun proteins (which preferentially form heterodimers) as described by Kostelny et al. Innol. 148:1547-4553; 1992). U.S. Patent 5,582,996 discloses the use of complementary interactive domains (such as leucine zipper moieties or other lock and key interactive domain structures) to facilitate heterodimer formation in the production of bispecific antibodies. Tetravalent, bispecific molecules can be prepared by fusion of DNA encoding the heavy chain of an F(ab)2 fragment of an antibody with either DNA encoding the heavy chain of a second F(ab')2 molecule (in which the CH1 domain is replaced by a CH3 domain), or with DNA encoding a single chain FV fragment of an antibody, as described in U.S. Patent 5,959,083.

Expression of the resultant fusion genes in mammalian cells, together with the genes for the -42corresponding light chains, yields tetravalent bispecific molecules having specificity for selected O antigens. Bispecific antibodies can also be produced as described in U.S. Patent 5,807,706. Generally, Z the method involves introducing a protuberance (constructed by replacing small amino acid side chains Swith larger side chains) at the interface of a first polypeptide and a corresponding cavity (prepared by replacing large amino acid side chains with smaller ones) in the interface of a second polypeptide.

Moreover, single-chain variable fragments (sFvs) have been prepared by covalently joining two l. variable domains; the resulting antibody fragments can form dimers or trimers, depending on the length of a flexible linker between the two variable domains (Kortt et al., 1997, Protein Engineering S10:423-433).

Screening procedures by which such antibodies can be identified are well known, and can involve immunoaffinity chromatography, for example. Antibodies can be screened for agonistic Cl ligand-mimicking) properties. Such antibodies, upon binding to cell surface HPR1 and/or HPR2, induce biological effects transduction of biological signals) similar to the biological effects induced when the HPRI and/or HPR2 binding partner binds to cell surface HPRI and/or HPR2.

Agonistic antibodies can be used to induce HPRI- and/or HPR2-mediated intracellular signaling or cell proliferation. Bispecific antibodies can be identified by screening with two separate assays, or with an assay wherein the bispecific antibody serves as a bridge between the first antigen and the second antigen (the latter is coupled to a detectable moiety). Bispecific antibodies that bind HPR1 and/or HPR2 polypeptides of the invention via a first antigen binding domain will be useful in diagnostic applications and in treating cell proliferation, differentiation, or activation diseases or conditions.

Examples of polypeptides (or other antigens) that the inventive bispecific antibodies bind via a second antigen binding domain include: four alpha helix bundle cytokines such as IL-6, OSM, LIP, CNTF, CLC, IL-12p35, and IL-23pl9; soluble hematopoietin receptors such as EBI-3, soluble IL-6R alpha, cytokine-like factor-i (CLF), IL-12p40, or a soluble form of HPRI and/or HPR2; and soluble hematopoietin receptors such as EBI-3 etc. in conjunction with a four alpha helix bundle cytokine.

Those antibodies that can block binding of the HPR1 and/or HPR2 polypeptides of the invention to binding partners for HPR1 and/or HPR2 can be used to inhibit HPR1- and/or HPR2mediated intracellular signaling or cell proliferation that results from such binding. Such blocking antibodies can be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of HPR1 and/or HPR2 to certain cells expressing an HPR1 and/or HPR2 binding partner. Alternatively, blocking antibodies can be identified in assays for the ability to inhibit a biological effect that results from binding of soluble HPR1 and/or HPR2 to target cells. Antibodies can be assayed for the ability to inhibit HPR1 and/or HPR2 binding partner-mediated cell stimulatory pathways, for example. Such an antibody can be employed in an in vitro procedure, or administered in vivo to inhibit a biological activity mediated by the entity that generated the antibody. Disorders caused or exacerbated (directly or indirectly) by the interaction of HPR1 and/or HPR2 with cell surface binding partner receptor thus can be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting HPR1 and/or HPR2 binding -43partner-mediated biological activity. Monoclonal antibodies are generally preferred for use in such Stherapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed.

Compositions comprising an antibody that is directed against HPRI and/or HPR2, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described below for compositions containing HPRI and/or HPR2 polypeptides.

Also provided herein are conjugates comprising a detectable diagnostic) or therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures. The antibodies of the invention can also be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo. The antibodies also can be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.

Rational Design of Compounds that Interact with HPR1 and/or HPR2 Polypeptides The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, inhibitors, agonists, antagonists, etc. Any of these examples can be used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J (1991) Biotechnology 9:19-21). In one approach, the three-dimensional structure of a polypeptide of interest, or of a polypeptide-inhibitor complex, is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous polypeptides. In both cases, relevant structural information is used to design analogous HPR1- and/or HPR2-like molecules, to identify efficient inhibitors, or to identify small molecules that bind HPRI and/or HPR2 polypeptides. Useful examples of rational drug design include molecules which have improved activity or stability as shown by Braxton S and Wells JA (1992 Biochemistry 31:7796-7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda SB et al (1993 J Biochem 113:742-746). The use of HPR1 and/or HPR2 polypeptide structural information in molecular modeling software systems to assist in inhibitor design and in studying inhibitor-HPR1 polypeptide and/or inhibitor-HPR2 polypeptide interaction is also encompassed by the invention. A particular method of the invention comprises analyzing the threedimensional structure of HPR1 and/or HPR2 polypeptides for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein.

It is also possible to isolate a target-specific antibody, selected by functional assay, as described further herein, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass polypeptide -44crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, O pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids Z would be expected to be an analog of the original receptor. The anti-id could then be used to identify C^ and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.

Assays of HPR1 and HPR2 Polypeptide Activities The purified HPRI and HPR2 polypeptides of the invention (including polypeptides, C polypeptides, fragments, variants, oligomers, and other forms) are useful in a variety of assays. For example, the HPR1 and HPR2 molecules of the present invention can be used to identify binding Spartners of HPR1 and/or HPR2 polypeptides, which can also be used to modulate intracellular 15 signaling, cell proliferation, or immune cell activity. Alternatively, they can be used to identify nonbinding-partner molecules or substances that modulate intracellular signaling, cell proliferation, or immune cell activity.

Assays to Identify Binding Partners. HPR1 and HPR2 polypeptides and fragments thereof can be used to identify binding partners. For example, they can be tested for the ability to bind a candidate binding partner in any suitable assay, such as a conventional binding assay. To illustrate, the HPR1 or HPR2 polypeptide can be labeled with a detectable reagent a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like). The labeled polypeptide is contacted with cells expressing the candidate binding partner. The cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.

One example of a binding assay procedure is as follows. A recombinant expression vector containing the candidate binding partner cDNA is constructed. CV1-EBNA-1 cells in 10 cm 2 dishes are transfected with this recombinant expression vector. CV-1/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 driven from the CMV Immediate-early enhancer/promoter. CVI-EBNA-1 was derived from the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan etal., (EMBOJ. 10:2821. 1991). The transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4 x 104 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37 0 C with various concentrations of, for example, a soluble polypeptide/Fc fusion polypeptide made as set forth above. Cells then are washed and incubated with a constant saturating concentration of a 1I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37*C. After extensive washing, cells are released via trypsinization. The mouse anti-human IgO employed above is directe4 against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide/Fc polypeptide that has bound to the cells. In all assays, non-specific binding of 'i-antibody is assayed z in the absence of the Fc fusion polypcptide/Fc, as well as in the presence of the Pc fusion polypeptide and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody. Cell-bound '"l-antibody is C quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Am. N.Y. Acad Sci 51:660, 1949) are generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer.

Binding can also be detected using methods that are well suited for high-throughput screening C procedures, such as scintillation proximity assays (Udenfriend et aL, 1985, Proc Natl Acad Sci USA 82: 8672-8676), homogeneous time-resolved fluorescence methods (Park et al., 1999, Anal Biochem 269: 94-104), fluorescence resonance energy transfer (FRET) methods (Clegg RM, 1995, Curr Opin Biotechnol 6: 103-110), or methods that measure any changes in surface plasmon resonance when a bound polypeptide is exposed to a potential binding partner, using for example a biosensor such as that supplied by Biacore AB (Uppsala, Sweden). Compounds that can be assayed for binding to HPRI and/or HPR2 polypeptides include but are not limited to small organic molecules, such as those that are commercially available often as part of large combinatorial chemistry compound 'libraries' from companies such as Sigma-Aldrich (St. Louis, MO), Arqule (Woburn, MA), Enzymed (Iowa City, IA), Maybridge Chemical Co.(Trevillett, Cornwall, UK), MDS Panlabs (Bothell, WA), Pharmacopeia (Princeton, NJ), and Trega (San Diego, CA). Preferred small organic molecules for screening using these assays are usually less than 10K molecular weight and can possess a number of physicochemical and pharmacological properties which enhance cell penetration, resist degradation, and/or prolong their physiological half-lives (Gibbs, 1994. Pharmaceutical Research in Molecular Oncology, Cell 79(2): 193-198). Compounds including natural products, inorganic chemicals,-and-biologially-active materials such as proteins and toxins can also be assayed using these methods for the ability to bind to HPR1 and/or HPR2 polypeptides.

Yeast Two-Hybrid or Interaction Trap" Assays. Because HPR1 and HPR2 polypeptides bind or potentially bind to another polypeptide (such as, for example, in a receptor-ligand interaction), the nucleic acid encoding the HPRI or HPR2 polypeptide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify nucleic acids encoding the other polypeptide with which binding occurs, or to identify inhibitors of the binding interaction. Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Competitive Bindin Assays. Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant can be determined by assaying for the variant's ability to compete with the native polypeptide for binding to the candidate binding partner.

Competitive binding assays can be performed by conventional methodology. Reagents that can be employed in competitive binding assays include radiolabeled HPRI or HPR2 and intact cells expressing HPRI and/or HPR2 (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble HPRI or HPR2 fragment can be used to compete with a soluble HPR1 variant and/or a soluble HPR2 variant for binding to cell surface receptors. Instead of intact cells, one could -46substitute a soluble binding partner/Fc fusion polypeptide bound to a solid phase through the 0 interaction of Polypeptide A or Polypeptide G (on the solid phase) with the Fc moiety.

Z Chromatography columns that contain Polypeptide A and Polypeptide G include those available from Pharmacia Biotech, Inc., Piscataway, NJ.

Assays to Identify Modulators of Intracellular Signaling, Cell Proliferation, or Immune Cell Activity. The influence of HPR1 or HPR2 on intracellular signaling, cell proliferation, or immune cell lt activity can be manipulated to control these activities in target cells. For example, the disclosed HPRI and HPR2 polypeptides, nucleic acids encoding the disclosed HPR1 and HPR2 polypeptides, or Cagonists or antagonists of such polypeptides can be administered to a cell or group of cells to induce, enhance, suppress, or arrest intracellular signaling or cell proliferation by the target cells. Identification O 15 of HPR1 and HPR2 polypeptides, agonists or antagonists that can be used in this manner can be carried out via a variety of assays known to those skilled in the art. Included in such assays ar those that evaluate the ability of an HPR1 or HPR2 polypeptide to influence intracellular signaling, cell proliferation, or immune cell activity. Such an assay would involve, for example, the analysis of immune cell interaction in the presence of an HPR1 polypeptide and/or an HPR1 polypeptide. In such an assay, one would determine a rate of intracellular signaling or cell proliferation in the presence of the HPRI and/or HPR2 polypeptide and then determine if such intracellular signaling or cell proliferation is altered in the presence of a candidate agonist or antagonist or another HPR1 or HPR2 polypeptide. Exemplary assays for this aspect of the invention include cytokine secretion assays, cell proliferation assays, and mixed lymphocyte reactions involving antigen presenting cells and T cells.

These assays are well known to those skilled in the art.

In another aspect, the present invention provides a method of detecting the ability of a test compound to affect the intracellular signaling or cell proliferation activity of a cell. In this aspect, the method comprises: contacting a first group of target cells with a test compound including an HPR1 polypeptide and/or an HPR2 polypeptide, or a fragment or fragments thereof, under conditions appropriate to the particular assay being used; measuring the net rate of intracellular signaling or cell proliferation among the target cells; and observing the net rate of intracellular signaling or cell proliferation among control cells contacting the HPR1 andJor HPR2 polypeptides or fragments thereof, in the absence of a test compound, under otherwise identical conditions as the first group of cells. In this embodiment, the net rate of intracellular signaling or cell proliferation in the control cells is compared to that of the cells treated with both a test compound and the HPR1 and/or HPR2 polypeptide(s). The comparison will provide a difference in the net rate of intracellular signaling or cell proliferation such that an effector of intracellular signaling or cell proliferation can be identified.

The test compound can function as an effector by either activating or up-regulating, or by inhibiting or down-regulating, intracellular signaling or cell proliferation, and can be detected through this method.

Cell Proliferation. Cell Death Cell Differentiation, and Cell Adhesion Assays. A polypeptide of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting), or cell differentiation (either inducing or inhibiting) activity, or may induce production of other cytokines in -47certain cell populations. Many polypeptide factors discovered to date have exhibited such activity in 0 one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cell stimulatory activity. The activity of a polypeptide of the present invention is evidenced by any one of a number of routine factor-dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DAIG, TIO, 139, B9/11, BaF3, M09/G, M+ (preB 2E8, RB5, DAI, 123, T1 165, IM, CILL2, TF-1, Mole and CML The activity of an HPR1 or HPR2 In polypeptide of the invention may, among other means, be measured by the following methods: Assays for T-cell or thvmocMt roliferation include without limitation those described in: Current ProtocolsinL Immunology, Coligan el al. eds, Greene Publishing Associates and Wiley- Interscience (pp. 3.1-3.19: it vitro assays for mouse lymphocyte function; Chapter 7: Immunologic studies in humans); Takal et al, J. Immunol. 137: 3494-3500, 1986; Bertagnolli et al., J. Inimunol. 145: r~l 1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.

Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.

Assays for cmtline production and/orl poliferation of oee cls. ]Mphnoeclsr thvMocyles include, without limitation, those described in: Kruisbeek and Shevach, 1994, Polyla T cell stimulation, in Current Protocols in LImmunology, Coligan et al. eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto; and Schreiber, 1994, Measurement of mouse and human interferon gamma in Current Protocols in Inmmunology, Coligan et al, eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto.

Assays for rolifmrtion and differentiation of beMatoopei ad Inhpitcclsicue without limitation, those described in: Bottoinly et al., 1991, Measurement of human and murine interleukin 2 and interleukin 4, in Current Protocols in Immzunology, Coligan et al. eds. Vol 1 pp.

6.3.1-6.3.12, John Wiley and Sons, Toronto; deVries et al, J Exp Med 173: 1205-1211, 199 1; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc Nat! Acad Sci.USA 80: 2931-2938, 1983; Nordan, 1991, Measurement of mouse and human interleukin 6, in Current Protocols in Immunology Coligan et al eds. Vol I pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto; Smith et al., Proc Nail Aced Sci USA 83: 1857-1861, 1986; Bennett et al, 1991, Measurement of human interleukin 11, in Current Protocols in Immunology Coligan et al. eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto; Ciarletta et al., 1991, Measurement of mouse and human Interleukin 9, in Current Protocols in Immunology Coligan et al. eds. Vol 1 pp. 6.13. 1, John Wiley and Sons, Toronto.

AssaXs for T-cell clone responses to antigens (which will identify, among others, polypeptides that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in inununology, Coligan et al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter 3: In vitro assays for mouse lymphocyte function-, Chapter 6: Cytokines and their cellular receptors; Chapter 7: Immunologic studies in humans); Weinberger et al., Proc Nat! Mcad Sci USA 77: 6091-6095, 1980; Weinberger et al., Eur. J. Imrmun. 11:405-411, 1981; Takai et al., J. Immunoil. 137:3494-3500, 1986; Takai et al., L' Irnmunol. 140:508-512, 1988 -48- Assays for thymocyte or Eplenecyte cytotoxicitY include, without limitation, those described 0 in: Current Protocols in linunwzo~ogy, Coligan et al. eds, Greene Publishing Associates and Wiley- Z hnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmnann et Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-5 12, 1988; Herrmann tfl et al., Proc. Nat!. Acad. Sci. USA 78:2488-2492, 1981; Henrmnn et al., J. lImnunol. 128:1968-1974, 1982; Handa ot al., L. Immunol. 135:1564-1572, 1985; T1akai et al., 1. Iminunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et J. Imniunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et J. Imnaunol. 153:3079-3092, 1994.

Assays for T-cell-dependent immunolobulin respnses and isotvve switching (which will (71 identify, among others, polypeptides that modulate T-cell dependent antibody responses and that affect ThlIrh profiles) include, without limitation, those described in: Maliszewski, J Immunol 144: 3028- 3033, 1990; and Mond and Brunswick, 1994, Assays for B cell function: in vitro antibody production, in Current Protocols in Inumunology Coligan et al. eds. Vol 1 pp. 3.8.1-3.8.16. John Wiley and Sons, Toronto.

Mixed lvmvhocye reaction (MR) assays (which will identify, among others, polypaptides tha generate predominantly Thi and CTL responses) include, without limitation, those described in: Current Protocols in Inununology, Coligan et a. eds, Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et 1. Immunol. 137:3494-3500, 1986; Takai et J.

Imniunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dnedent assas (which will identify, among others, polypeptides expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J.

Immnunol 134:536-544, 1995; Inaba et J Exp Med 173:549-559, 199 1; Macatonia et al., J Immunol 154:5071-5079, 1995; Porgador et al., J Exp Med 182:255-260, 1995; Nair et al., J Virology 67:4062- 4069, 1993; Huang at al., Science 264:961-965, 1994; Macatonia et al., I Exp Med 169:1255-1264, 1989; Bhardwaj at aL., J Cliii Invest 94:797-807, 1994; and Inaba et al., I Exp Med 172:631-640,1990.

Assays for lMphocyta survivalapootosis (which will identify, among others, polypeptides that prevent apoptosis after superantigen induction and polypeptides that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz at Cytometry 13:.795- 808, 1992; Gorczyca at al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53: 1945- 1951, 1993; Itoh at al., Cell 66:233-243, 199 1; Zacharchuk, J Imjmunol 145:4037-4045, 1990; Zamai at Cytometry 14:891-897, 1993; Gorczyca at al., International Journal of Oncology 1:639-648, 1992.

Assays for polotides that influence early teo of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cell Inixunol 155:111-122, 1994; Gnly at al., Blood 85:2770-2778, 1995; Told et al., Proc Nat! Acad Sci.

USA 88:7548-7551, 1991 -49 for embryoic stem cell differentiatio (which will identif among others, 0 polypeptides that influence emnbryonic differentiation hematopajesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify, among others, polypeptides that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, 1994, In Culture of Hematopoiefic Cells, Freshney et al. eds. pp. 265-268, Wiley-Liss, Inc., New York, NY; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hernatopoietic colony forming cells with high proliferative potential, McNiece and Bridddll, 1994, In Culture of Hemattopoietic Cells, Freshney et al. eds. pp. 23-39, Wiley- Liss, Inc., New York, NY; Neben et al., Experimental Hematology 22:353-359, 1994; Ploemacher, Cl 1994, Cobblestone area forming cell assay, I Culture of Jiatopoietic Cells, Freshney et al. eds. pp.

1-21, Wiley-Liss, Inc., New York, NY; Spooncer et al., 1994, Long term bone marrow cultures in the presence of stromnal cells, In Culture of Heinatopoietic Cells, Freshney et a. eds. pp. 163-179, Wiley- Liss, Inc., New York, NY; Sutherland, 1994, Long term culture initiating cell assay, In Culture of Henmopoietic Cells, Freshney et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY.

Assay for hemostatic and thrombolytic activit include, without limitation, those described in: Linet et al., 1. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (199 Schaub, Prostaglandins 35:467-474, 1988.

Assays for etor-lcavi ativty include without limitation those described hr Current Protocols i immunology Coligan et a. eds, Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of cellular adhesion under static conditions 7.28.1-7.28.22), Talcaj et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169: 149-160 1989; Stoltenborg et al., J. Inimunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

Diagnostic and Other Uses of HPR1 and HPR2 Polypeptides and Nucleic Acids The nucleic acids encoding the HPR1 and HPR2 polypeptides provided by the present invention can be used for numerous diagnostic or other useful purposes. The nucleic acids of the invention can be used to express recombinant polypeptide for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (whcn labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel nucleic acids; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-polypeptide antibodies using DNA immunization 50 techniques; as an antigen to raise anti-DNA antibodies or elicit another immune response, and. for gene O therapy. Uses of HPR1 and HPR2 polypeptides and fragmented polypeptides include, but are not Z limited to, the following: purifying polypeptides and measuring the activity thereof; delivery agents; therapeutic and research reagents; molecular weight and isoelectric focusing markers; controls for peptide fragmentation; identification of unknown polypeptides; and preparation of antibodies. Any or all nucleic acids suitable for these uses are capable of being developed into reagent grade or kit format lt for commercialization as products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular SCloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, E. F.

Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987 Probes and Primers. Among the uses of the disclosed HPR1 and HPR2 nucleic acids, and combinations of fragments thereof, is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., 1989 and are described in detail above.

Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for non-human genetic libraries. Such libraries would include but are not limited to cDNA libraries, genomic libraries, and even electronic EST (express sequence tag) or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify non-human HPR1 and HPR2 homologues.

Chromosome Mapping. The nucleic acids encoding HPR1 and HPR2 polypeptides, and the disclosed fragments and combinations of these nucleic acids, can be used by those skilled in the art using well-known techniques to identify the human chromosome to which these nucleic acids map.

Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various well-known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (ow resolution).

Alternatively, the genomic sequences corresponding to nucleic acids encoding a cytokine polypeptide of the invention are mapped by comparison to sequences in public and proprietary databases, such as GenBank (ncbi.nlm.nih.gov/BLAST), Locuslink (ncbi.nlm.nih.gov:80/LocusLinl/). Unigene (ncbi.nlm.nih.gov/cgi-bin/UniGene), AceView (ncbi.nlm.nih.gov/AceView), Gene Map Viewer (ncbi.nlm.nih.gov/genemap), Online Mendelian Inheritance in Man (OMIM) (ncbi.nlm.nih.gov/Omim), and proprietary databases such as the Celera Discovery System (celera.com). These computer analyses of available genomic sequence information can provide the identification of the specific chromosomal -51location of human and/or murine genomic sequences corresponding to sequences encoding HPRI or 0 HPR2 polypeptides of the invention, and the unique genetic mapping relationships between HPR1 or Z HPR2 genomic sequences and the genetic map locations of known human genetic disorders C. Diagnostics and Gene Therapy. The nucleic acids encoding HPR1 and HPR2 polypeptides, and the disclosed fragments and combinations of these nucleic acids can be used by one skilled in the art using well-known techniques to analyze abnormalities associated with the genes corresponding to these polypeptides. This enables one to distinguish conditions in which this marker is rearranged or deleted. In addition, nucleic acids of the invention or a fragment thereof can be used as a positional marker to map other genes of unknown location. The DNA can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes corresponding to the nucleic acids of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes. Defective genes can be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene.

Methods of Screening for Binding Partnes. The HPRI and HPR2 polypeptides of the invention each can be used as reagents in methods to screen for or identify binding partners. For example, the HPRI and HPR2 polypeptides can be attached to a solid support material and may bind to their binding partners in a manner similar to affinity chromatography. In particular embodiments, a polypeptide is attached to a solid support by conventional procedures. As one example, chromatography columns containing functional groups that will react with functional groups on amino acid side chains of polypeptides are available (Pharmacia Biotech, Inc., Piscataway, NJ). In an alternative, a polypeptide/Fc polypeptide (as discussed above) is attached to Protein A- or Protein Gcontaining chromatography columns through interaction with the Fc moiety. The HPR1 and HPR2 polypeptides also find use in identifying cells that express a binding partner on the cell surface.

Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing potential bindingpartner-expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing the binding partner on the cell surface bind to the fixed polypeptides, and unbound cells are washed away. Alternatively, HPR1 and HPR2 polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression. After incubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined. In a further alternative, mixtures of cells suspected of expressing the binding partner are incubated with biotinylated polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads. Procedures for using avidincoated beads are known (see Berenson, et al. J. Cell Biochem., 10D:239, 1986). Washing to remove unbound material, and the release of the bound cells, -52are performed using conventional methods. In some instances, the above methods for screening for or O identifying binding partners may also be used or modified to isolate or purify such binding partner Z molecules or cells expressing them.

C Measuring Biological Activity. HPR1 and HPR2 polypeptides also find use in measuring the biological activity of HPRl-binding and/or HPR2-binding polypeptides in terms of their binding affinity. The polypeptides thus can be employed by those conducting "quality assurance" studies, e.g., lt to monitor shelf life and stability of polypeptide under different conditions. For example, the polypeptides can be employed in a binding affinity study to measure the biological activity of a binding partner polypeptide that has been stored at different temperatures, or produced in different cell types.

The polypeptides also can be used to determine whether biological activity is retained after modification of a binding partner polypeptide chemical modification, truncation, mutation, etc.).

C' The binding affinity of the modified polypeptide is compared to that of an unmodified binding polypeptide to detect any adverse impact of the modifications on biological activity of the binding polypeptide. The biological activity of a binding polypeptide thus can be ascertained before it is used in a research study, for example.

Carriers and Delivery Agents. The polypeptides also find use as carriers for delivering agents attached thereto to cells bearing identified binding partners. The polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types found to express binding partners on the cell surface) in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application.

Among the toxins are ricin, abrin, diphtheria toxin, Pseudoinonas aeruginosa exotoxin A, ribosomal inactivating polypeptides, mycotoxins such as trichothecenes, and derivatives and fragments single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, "I, 3'I, ""ITc, and 7 Examples of radionuclides suitable for therapeutic use are II, 21 'At, nBr, 86 Re, 2 12Pb, 21 2 Bi, 09 Pd, "Cu, and "Cu. Such agents can be attached to the polypeptide by any suitable conventional procedure. The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the polypeptide or agent can be derivatized to generate or attach a desired reactive functional group. The derivatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling polypeptides are known.

Radionuclide metals can be attached to polypeptides by using a suitable bifunctional chelating agent, for example. Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.

-53- Treating Diseases Using HPR1 and/or HPR2 Polypeptides and Antagonists Thereof 0 It is anticipated that the HPR1 and HPR2 polypeptides, fragments, variants, antagonists, Z agonists, antibodies, and binding partners of the invention will be useful for treating medical conditions and diseases including, but not limited to, cell proliferation, metabolic, and reproductive hormone related conditions as described further herein. The therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and Svariants, fragments, and binding partners of HPRI and/or HPR2 polypeptides may have effects similar rto or different from HPR1 or HPR2 polypeptides. For example, an antagonist of the ligand-binding activity of HPR1 and/or HPR2 polypeptides may be selected for treatment of conditions involving ligand-binding activity, but a particular fragment of a given HPRI or HPR2 polypeptide may also act as an effective dominant negative antagonist of that activity. Therefore, in the following paragraphs "HPR1 and HPR2 polypeptides or antagonists" refers to all HPR1 and HPR2 polypeptides, fragments, variants, antagonists, agonists, antibodies, and binding partners etc. of the invention, and it is understood that a specific molecule or molecules can be selected from those provided as embodiments of the invention by individuals of skill in the art, according to the biological and therapeutic considerations described herein.

Provided herein are methods for using HPR1 and HPR2 polypeptides or antagonists, compositions or combination therapies to treat various hematologic and oncologic disorders. For example, HPR1 and HPR2 polypeptides or antagonists are used to treat various forms of cancer, including acute myelogenous leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer-associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia and hypercalcemia. Additional diseases treatable with the subject HPR1 and HPR2 polypeptides or antagonists, compositions or combination therapies are solid tumors, including sarcoma, osteosarcoma, and carcinoma, such as adenocarcinoma (for example, breast cancer) and squamous cell carcinoma. In addition, the subject compounds, compositions or combination therapies are useful for treating leukemia, including acute myelogenous leukemia, chronic or acute lymphoblastic leukemia and hairy cell leukemia. Other malignancies with invasive metastatic potential can be treated with the subject compounds, compositions and combination therapies, including multiple myeloma. In addition, the disclosed HPR1 and HPR2 polypeptides or antagonists, compositions and combination therapies can be used to treat anemias and hematologic disorders, including anemia of chronic disease, aplastic anemia, including Fanconi's aplastic anemia; idiopathic thrombocytopenic purpura (ITP); myelodysplastic syndromes (including refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation); myelofibrosis/myeloid metaplasia; and sickle cell vasocclusive crisis.

Various lymphoproliferative disorders also are treatable with the disclosed HPR1 and HPR2 polypeptides or antagonists, compositions or combination therapies. These include, but are not limited to autoimmune lymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia, hairy cell -54leukemia, chronic lymphatic leukemia, peripheral T-cell lymphoma, small lymphocytic lymphoma, O mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive T cell Z lymphoma, histiocytic lymphoma, Hodgkin's disease, diffuse aggressive lymphoma, acute lymphatic S leukemias, T gamma lymphoproliferative disease, cutaneous B cell lymphoma, cutaneous T cell lymphoma mycosis fungoides) and Sdzary syndrome.

In addition, the subject invention provides HPRI and HPR2 polypeptides or antagonists, Scompositions and combination therapies for the treatment of non-arthritic medical conditions of the bones and joints. This encompasses osteoclast disorders that lead to bone loss, such as but not limited to osteoporosis, including post-menopausal osteoporosis, periodontitis resulting in tooth loosening or loss, and prosthesis loosening after joint replacement (generally associated with an inflammatory response to wear debris). This latter condition also is called "orthopedic implant osteolysis." Another Cl condition treatable by administering HPR1 and HPR2 polypeptides. or antagonists, is temporal mandibular joint dysfunction (TMJ).

The disclosed HPRI and HPR2 polypeptides or antagonists, compositions and combination therapies furthermore are useful for treating neurodegenerative conditions such as acute polyneuropathy; anorexia nervosa; Bell's palsy; chronic fatigue syndrome; transmissible dementia, including Creutzfeld-Jacob disease; demyelinating neuropathy; Guillain-Barre syndrome; vertebral disc disease; Gulf war syndrome; myasthenia gravis; chronic neuronal degeneration; and stroke, including cerebral ischemic diseases.

Administration of HPR1 and HPR2 Polypeptides and Antagonists Thereof This invention..provides -compounds,- compositions,--and methods for -treating- a patient, preferably a mammalian patient, and most preferably a human patient, who is suffering from a medical disorder, and in particular an HPR1- or HPR2-mediated disorder. Such HPR1- or HPR2-mediated disorders include conditions caused (directly or indirectly) or exacerbated by binding between HPRI and/or HPR2 and a binding partner. For purposes of this disclosure, the terms "illness," "disease," "medical condition," "abnormal condition" and the like are used interchangeably with the term "medical disorder." The terms "treat", "treating", and "treatment" used herein includes curative, preventative prophylactic) and palliative or ameliorative treatment. For such therapeutic uses, HPR1 and HPR2 polypeptides and fragments, HPR1 and HPR2 nucleic acids encoding the HPRI and HPR2 polypeptides, and/or agonists or antagonists of the HPR1 and/or HPR2 polypeptides such as antibodies can be administered to the patient in need through well-known means. Compositions of the present invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments. In particular embodiments, the composition comprises a soluble polypeptide or an oligomer comprising soluble HPR1 and/or HPR2 polypeptides.

Therapeutically Effective Amount In practicing the method of treatment or use of the present invention, a therapeutically effective amount of a therapeutic agent of the present invention is administered to a patient having a condition to be treated, preferably to* treat or ameliorate diseases o associated with the activity of an BPR1 and/or HPR2 polypeptide. 'Therapeutic agent" includes Z without limitation any of the HPR1 or HPR2 polypeptides, fragments, and variants; nucleic acids encoding the HPRI and HPR2 polypeptides, fragments, and variants; agonists or antagonists of the HPR1 and HPR2 polypeptides such as antibodies; HPR1 and/or HMR polypeptide binding partners; complexes formed from the 1HPRl and/or HPR2 polypeptides, fragments, variants, and binding partners, etc. As used herein, the term "therapeutically effective amount" means the total amount of I> each therapeutic agent or other active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, treatent, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual therapeutic agent or active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. As used herein, the phrase "administering a therapeutically effective amount" of a therapeutic agent means that the patient is treated with said therapeutic agent in an amount and for a time sufficient to induce an improvement and preferably a sustained improvemnent, in at least one indicator that reflects the severity of the disorder. Ani improvement is considered "sustained" if the patient exhibits the improvement on at least two occasions separated by one or more weeks. The degree of improvement. is determined based on signs or symptoms, and determinations can also employ questionnaires that are administered to the patient, such as quality-oflife questionnaires. Various indicators that reflect the extent of the patient's illness can be assessed for determining whether the amount and time of the treatment is sufficient. The baseline value for the chosen indicator or indicators is established by examination of the patient prior to administation of the first dose of the therapeutic agent. Preferably, the baseline examination is done within about 60 days of administering the first dose. If the therapeutic agent is being administered to treat acute symptoms, the first dose is administered as soon as practically possible after the injury has occurred. Improvement is induced by administering therapeutic agents such as HPRI and/or HR2 polypeptides or antagonists until the patient manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, for one, two, or three months or longer, or indefinitely. A period of one to six weeks, or even a single dose, often is sufficient for treating injurie or acute conditions. Although the extent of the patient's illness after treatment may appear improved according to one or more indicators treatment may be continued indefinitely at the same level or at a reduced dose or filequency. Once treatment has been reduced or discontinued, it later may be resumed at the original level if symptoms should reappear.

Dosing. One skilled in the pertinent art will recognize that suitable dosages will vary, depending upon such factors as the nature and severity of the disorder to be treated, the patient's body weight, age, general condition, and prior illnesses and/or treatments, and the route of administration.

56 Preliminary doses can be determined according to animal tests, and the scaling of dosages far human o administration is performed according to art-accepted practices such as standard dosing trials. For Z example, the therapeutically effective dose can be estimated initially from cell culture assays. The dosage will depend on the specific activity of the compound and can be readily determined by routine experimentation. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture, while minimizing toxicities.

(71 Such information can be used to more accurately determine useful doses in humans. Ultimately, the attending physician will decide the amount of polypeptide of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of polypeptide of the present invention and observe the patient's response. Larger doses of polypeptide of the present invention can be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ng to about 100 mng (preferably about 0. 1 ng to about 10 mg, more preferably about 0. 1 microgram to about 1 mg) of polypeptide of the present invention per kg body weight. In one embodiment of the invention, BPRI and/or HPR2 polypeptides or antagonists are administered one time per week to treat the various medical disorders disclosed herein, in another embodiment is administered at least two times per week.

and in another embodiment is administered at least three times per week. If injected, the effective amount of IIPRi or HPR2 polypeptides or antagonists per adult dose ranges from 1-20 mgin 2 and preferably is about 5-12 mg/rn. Alternatively, a flat dose can be administered, whose amount may range from 5-100 mg/dose. Exemplary dose ranges for a flat dose to be administered by subcutaneous injection are 5-25 mg/dose. 25-50 mg/dose and 50-100 mg/dose. In one embodiment of the invention, the various indications described below are treated by administering a preparation acceptable for injection containing HPRl and/or HPR2 polypeptides or antagonists at 25 mg/dose, or alternatively, containing 50 mng per dose. The 25 mg or 50 mg dose can be administered repeatedly, particularly for chronic conditions. If a route of administration other than injection is used, the dose is appropriately adjusted in accord with standard medical practices. In many instances, an improvement in a patient's condition will be obtained by injecting a dose of about 25 mng of HPRl or HPR2 polypeptides or antagonists one to du=e times per week over a period of at least three weeks, or a dose of 50 mng of FHR1 or H1PR2 polypeptides or antagonists one or two times per week for at least three weeks, though treatment for longer periods may be necessary to induce the desired degree of improvement For incurable chronic conditions, the regimen can be continued indefinitely, with adjustments being made to dose and frequency if such are deemed necessary by the patient's physician. The foregoing doses are examples for an adult patient who is a person who is 18 years of age or older. For pediatric patients (age 4-17), a suitable regimen involves the subcutaneous injection of 0.4 mg/kg, up to a maximum dose of 25 mg of HPRI or IIPR2 polypeptides or antagonists, administered by subcutaneous injection one or more times per week. If an antibody against an HPR1 and/or BPR2 polypeptide is used as the HPR1 57 and/or HPR2 polypeptide antagonist, a preferred dose range is 0.1 to 20 mg/kg, and more preferably is 0 1-10 mg/kg. Another preferred dose range for an anti-HPR1 polypeptide and/or anti-HPR2 Z polypeptide antibody is 0.75 to 7.5 mg/kg of body weight Humanized antibodies are preferred, that is, antibodies in which only the antigen-binding portion of the antibody molecule is derived from a nonhuman source. Such antibodies can be injected or administered intravenously.

Formulations. Compositions comprising an effective amount of an HPR1 and/or HPR2 polypeptide of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources), in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The term "phannaccutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents or thickening agents. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents, preservatives, emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Renington's Plurmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.

Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat No. 4,235.871; U.S. Pat No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat No. 4,737,323. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, so that the characteristics of the carrier will depend on the selected route of administration. In one preferred embodiment of the invention, sustained-release forms of HPR1 and/or HPR2 polypeptides are used. Sustained-release forms suitable for use in the disclosed methods include, but are not limited to, HPRI and/or HPR2 polypeptides that are encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles Sdescribed in U.S. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant.

Combinations of Theraeutic Comounds. An HPR1 or HPR2 polypeptide of the present invention may be active in multimers heterodimers or homodimers) or complexes with itself or other polypeptides. As a result, pharmaceutical compositions of the invention may comprise a -58polypeptide of the invention in such multimeric or complexed form. The pharmaceutical composition 0 of the invention may be in the form of a complex of the polypeptide(s) of present invention along with Z polypeptide or peptide antigens. The invention further includes the administration of HPR1 and/or HPR2 polypeptides or antagonists concurrently with one or more other drugs that are administered to the same patient in combination with the HPR1 and/or HPR2 polypeptides or antagonists, each drug being administered according to a regimen suitable for that medicament. "Concurrent administration" l encompasses simultaneous or sequential treatment with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and C the other(s) are administered intermittently. Components can be administered in the same or in separate compositions, and by the same or different routes of administration. Examples of components that can be included in the pharmaceutical composition of the invention are: cytokines, lymphokines, C\K or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL7, IL-8, IL-9, IL-10, IL-11, IL-12, 113, IL-14, IL-15, IL-17, IL-18, IL-23, IFN, TNFO, TNFI, TNF2, G- CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition can further contain other agents which either enhance the activity of the polypeptide or compliment its activity or use in treatment Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with polypeptide of the invention, or to minimize side effects. Additional examples of drugs to be administered concurrently include but are not limited to antivirals, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, non-steroidal anti-inflammatories, pentoxifylline, thalidomide, and disease-modifying antirheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline, penicillamine, sulfasalazine and gold compounds such as oral gold, gold sodium thiomalate, and aurothioglucose. Additionally, HPR1 and/or HPR2 polypeptides or antagonists can be combined with a second HPR1 and/or HPR2 polypeptide/antagonist, including an antibody against an HPRI and/or HPR2 polypeptide, or an HPR1 polypeptide-derived peptide or HPR2 polypeptide-derived peptide that acts as a competitive inhibitor of native HPR1 and/or HPR2 polypeptides.

Routes of Administration. Any efficacious route of administration may be used to therapeutically administer HPR1 and HPR2 polypeptides or antagonists thereof, including those compositions comprising nucleic acids. Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes by bolus injection or by continuous infusion., and also includes localized administration, at a site of diseasd or injury. Other suitable means of administration include sustained release from implants; aerosol inhalation and/or insufflation.; cyedrops; vaginal or rectal suppositories; buccal preparations; oral preparations, including pills, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays, ointments or other suitable techniques. Alternatively, polypeptideaceous

HPRI

and HPR2 polypeptides or antagonists may be administered by implanting cultured cells that express the polypeptide, for example, by implanting cells that express HPRI and/or HPR2 polypeptides or -59antagonists. Cells may also be cultured ex vivo in the presence of polypeptides of the present invention 0 in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then Z be introduced in vivo for therapeutic purposes. In another embodiment, the patient's own cells are induced to produce HPRI and/or HPR2 polypeptides or antagonists by transfection in vivo or ex vivo with a DNA that encodes HPR1 and/or HPR2 polypeptides or antagonists. This DNA can be introduced into the patient's cells, for example,, by injecting naked DNA or liposome-encapsulated DNA that encodes HPR1 and/or HPR2 polypeptides or antagonists, or by other means of transfection.

Nucleic acids of the invention can also be administered to patients by other known methods for Sintroduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). When HPR1 and/or HPR2 polypeptides or antagonists are administered in combination with one or more other biologically active compounds, these can be administered by the Csame or by different routes, and can be administered simultaneously, separately or sequentially.

Oral Administration. When a therapeutically effective amount of polypeptide of the present invention is administered orally, polypeptide of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention can additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% polypeptide of the present invention, and preferably from about 25 to 90% polypeptide of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils can be added. The liquid form of the pharmaceutical composition can further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of polypeptide of the present invention, and preferably from about 1 to 50% polypeptide of the present invention.

Intravenous Administration. When a therapeutically effective amount of polypeptide of the present invention is administered by intravenous, cutaneous or subcutaneous injection, polypeptide of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.

The preparation of such parenterally acceptable polypeptide solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to polypeptide of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention can also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the polypeptide of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of 0 intravenous therapy using the pharmaceutical composition of the present invention.

Z Bone and Tissue Administration. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament disorders, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically n acceptable form. Further, the composition can desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a polypeptide of the invention which can also optionally be included in the composition as described above, can alternatively or additionally, be administered simultaneously or sequentially with the composition in C"1 the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the polypeptide-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices can be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions can be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure polypeptides or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics Matrices can be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymcthyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).

The amount of sequestering agent useful herein is 0.5-20 wt preferably 1-10 wt based on total formulation weight, which represents the amount necessary to prevent desorbtion of the polypeptide from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, polypeptides of the invention can be combined with other agents beneficial to the treatment of the bone -61and/or cartilage defect, wound, or tissue in question. Progress can be monitored by periodic assessment 0 of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and Z tetracycline labeling.

SVeterinary Use. In addition to human patients. HPR1 and HPR2 polypeptides and antagonists are useful in the treatment of disease conditions in non-human animals, such as pets (dogs, cats, birds, primates, etc.), domestic farm animals (horses cattle, sheep, pigs, birds, etc.), or any animal Sthat suffers from a TNFao-mediated inflammatory or arthritic condition. In such instances, an appropriate dose can be determined according to the animal's body weight For example, a dose of S0.2-1 mg/kg may be used. Alternatively, the dose is determined according to the animal's surface area, an exemplary dose ranging from 0.1-20 mg/m 2 or more preferably, from 5-12 mg/m 2 For small animals, such as dogs or cats, a suitable dose is 0.4 mg/kg. In a preferred embodiment, HPR1 and/or SHPR2 polypeptides or antagonists (preferably constructed from genes derived from the same species as the patient), are administered by injection or other suitable route one or more times per week until the animal's condition is improved, or they can be administered indefinitely.

Manufacture of Medicaments. The present invention also relates to the use of HPR1 and HPR2 polypeptides, fragments, and variants; nucleic acids encoding the HPR1 or HPR2 polypcptides, fragments, and variants; agonists or antagonists of the HPR1 and/or HPR2 polypeptides such as antibodies; HPR1 and/or HPR2 polypeptide binding partners; complexes formed from the HPR1 and/or HPR2 polypeptides, fragments, variants, and binding partners, etc, in the manufacture of a medicament for the prevention or therapeutic treatment of each medical disorder disclosed herein.

EXAMPLES

The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.

EXAMPLE 1 A. Identification of HPR1, a New Member of the Human Hematopoletin Receptor Family A data set was received from Celera Genomics (Rockville, Maryland) containing a listing of amino acid sequences predicted to be encoded by the human genome. This data set was searched with a BLAST algorithm to identify hematopoietin receptor family polypeptides. Several amino acid sequences, including two overlapping amino acid sequences (SEQ ID NO: 1 and SEQ ID NO:2), were identified as comprising partial amino acid sequences of a new human hematopoietin receptor polypeptide, HPR1. These amino acids sequences were used to identify a DNA sequence (SEQ ID NO:3) encoding an HPR1 polypeptide having the amino acid sequence shown in SEQ ID NO:4; nucleotides 132 through 2366 of SEQ ID NO:3 encode SEQ ID NO:4, with nucleotides 2367 through 2369 corresponding to a stop codon. The HPR1 coding sequence (nucleotides 132 through 2369 of SEQ ID NO:3) is presented as SEQ ID NO:5. The HPR1 sequences of SEQ ID NOs 3 and 5 were confirmed by three independent PCR amplification experiments from a U937 cDNA library. These HPR1 coding sequences were compared with publicly available preliminary human genomic DNA sequences, and the following chromosome 5 contigs were identified as containing HPR1 coding sequences: AC022265.3, AC008914.3, AC008857.4, and AC016596.4. The human genomic region -62trcorresponding to these contigs also includes the gene for gpl30, which suggests that gpl30 and HPRI may derive from a common ancestral gene by gene duplication. The approximate positions of the exons containing HPR1 coding sequence in the AC022265.3 contig are shown in the table below, along

O

z with their locations relative to SEQ ID NOs 3 and 5; note that the 5' and 3' untranslated regions may extend further along the contig sequence beyond those portions that correspond to SEQ ID NOs 3 and c( 10 5, as indicated by the parentheses around the AC022265.3 endpoints in the table. Due to the preliminary sequence and assembly of the contig sequence, the exons within the contig are not always Sin the right order or orientation with respect to each other, and may contain sequence variations due to inaccurate sequence data or allelic polymorphism.

S

Corresponding positions of HPRI gene exons in human contig AC022265.3 and in cDNA sequences: Position in AC022265.3 Position in SEQ ID NO:3 _Position in SEQ ID Exon 1 (128423)-128559 1-137/1-6 Exon 2 134501-134591 138-228 7-97 Exon 3 143777-143894 229-346 98-215 Exon 4 147256-147437 347-528 216-397 Exon 5 51249-51098 529-680 398-549 Exon 6 44322-44157 681-846 550-715 Exon 7 16473-16394 847-926 716-795 Exon 8 30331-30115 927-1143 796-1012 Exon 9 178626-178808 1144-1326 1013-1195 Exon 10 179879-179980 1327-1428/ 1196-1297 Exon 11 180785-180931 1429-1575 1298-1444 Exon 12 183052-183192 1576-1716/ 1445-1585 Exon 13 185997-186090 1717-1810/ 1586-1679 Exon 14 187367-187448 1811-1892/1680-1761 Exon 15 189165-(189747) 1893-2480 1762-2238 A nucleic acid encoding a polypeptide with a high degree of amino acid similarity (approximately 61% amino acid identity) to human HPR1 was isolated from Mus musculus. The Mus HPR1 amino acid sequence is presented as SEQ ID NO:12, and due to its high level of similarity with human HPR1, is considered to be the murine homologue of human HPR1. PCR amplification of cDNA sequences corresponding to mRNAs encoding murine HPRI identified a cDNA molecule encoding SEQ ID NO:12; the nucleotide sequence of this murine HPR1 cDNA is presented as SEQ ID NO:28. Nucleotides 1 through 2178 of SEQ ID NO:28 encode SEQ ID NO:12, with nucleotides 2179- 2181 corresponding to a stop codon. Variants of the murine HPR1 amino acid sequence that are likely allelic variants have been identified in which the T residue at position 121 of SEQ ID NO:28 is changed to a residue, resulting in a change from the Phe residue at position 41 of SEQ ID NO:4 to a Leu residue, and in which the residue at position 1666 of SEQ ID NO:28 is changed to an 'A' residue, resulting in a change from the Asp residue at position 556 of SEQ ID NO:4 to an Asn residue.

Several splice variations of the HPR1 sequences have been identified in human genomic sequences and are included within the scope of the invention. For example, amino acids 1 through of SEQ ID NO:1 match the amino acid sequence of HPR1 presented in SEQ ID NO:4, while amino -63- 0 5 acids 56 through 77 of SEQ ID NO: 1 may be a portion of an alternatively spliced exon added following the exon/intron boundary identified between nucleotides 846 and 847 of SEQ ID NO:3 (nucleotides O 715 and 716 of SEQ ID NO:5). In an additional potential splice variant, an amino acid sequence Z ending in the amino acids of SEQ ID NO:10 could be substituted for the amino acids leading up to and Sincluding the lysine at position 190 of SEQ ID NO:4. However, such a splice variant would require an additional exon/intron boundary approximately between nucleotides 701 and 702 of SEQ ID NO:3 (nucleotides 570 and 571 of SEQ ID NO:5). In a further potential splice variant, the amino acid In sequence of SEQ ID NO: 11 could be substituted for amino acids 238 through 266 of SEQ ID NO:4 by replacing exon 7 with an alternative exon encoding the SEQ ID NO: 11 amino acids. In this potential Svariant, 29 amino acids C-terminal to the WSXWS motif and including the N-terminal portion of the most N-terminal fibronectin type III repeat (as shown in Table 1) would be replaced with 15 amino Sacids, resulting in deletion of a portion of the most N-terminal fibronectin type III repeat, including two highly conserved Trp residues.

Additional variations of HPR1 polypeptides are provided as naturally occurring genomic variants of the HPR1 sequences disclosed herein; such variations may be incorporated into an HPRI polypeptide or nucleic acid individually or in any combination, or in combination with alternative splice variation as described above. As one example, amino acids 5 through 40 of SEQ ID NO:2 match SEQ ID NO:4, with amino acid 4 of SEQ ID NO:2 likely representing an allelic variation, where the change from the Asn residue position 187 of SEQ ID NO:4 to a Thr residue in SEQ ID NO:2 could be caused by a single change from to at position 691 of SEQ ID NO:3 or 560 of SEQ ID This variation and others are listed in the table below: Amino Acid Change Position in Position in SEQ ID NO:3 SEQ ID NO:4 Nucleotide Change Position in SEQ ID Thr Ala 83 A G 378 247 Asp Asn 168 G A 633 502 Asn Thr 187 A C 691 560 Ser Pro 361 T C 1212/1081 Ala Gly 362 C 1216 /1085 Ser Asn 510 G A 1660 1529 Asn Asp 517 A G 1680 1549 Arg Gly 679 A G 2166 2035 B. Identification of HPR2, a New Member of the Human Hematopoietin Receptor Family A data set was received from Celera Genomics (Rockville, Maryland) containing a listing of amino acid sequences predicted to be encoded by the human genome. This data set was searched with a BLAST algorithm to identify hematopoietin receptor family polypeptides. Several amino acid sequences, including SEQ ID NO:16, were identified as comprising partial amino acid sequences of a new human hematopoietin receptor polypeptide, HPR2. These amino acids sequences were used to identify a DNA sequence (SEQ ID NO:19) encoding an HPR2 polypeptide having the amino acid sequence shown in SEQ ID NO:21; nucleotides 107 through 1993 of SEQ ID 19 encode SEQ ID -64- NO:21, with nucleotides 1994 through 1996 corresponding to a stop codon. The HPR2 coding O sequence (nucleotides 107 through 1996 of SEQ ID NO:19) is presented as SEQ ID NO:20. The HPR2 Z sequences of SEQ ID NOs 19 and 20 were confirmed by independent PCR amplification experiments from a human lymph node cDNA library and a CB23 B cell line cDNA library. These PCR amplification experiments also identified two additional splice variants of the HPR2 cDNA sequence referred to as HPR2-ex8-ex9 and HPR2-ex9; the coding sequences for HPR2-ex8-ex9 and HPR2-ex9 are presented as SEQ ID NOs 22 and 24, respectively, and the amino acid sequences they encode are presented as SEQ ID NOs 23 and 25, respectively. The HPR2 cDNA sequences of SEQ ID NOs 19, and the HPR2-ex8-ex9 cDNA of SEQ ID NO:22 were present in both the lymph node and CB23 cDNA libraries, while the HPR2-ex9 cDNA of SEQ ID NO'24 was only present in the lymph node library.

CThese HPR2 coding sequences were compared with publicly available preliminary human genomic DNA sequences, and the following chromosome 1 contigs were identified as containing HPR2 coding sequences: GenBank accession numbers AL109843 (lp31.2-32.1) and AL389925. The human genomic region corresponding to the AL389925 contig also includes the gene for IL-12RB2, which suggests that IL-12RB2 and HPR2 may derive from a common ancestral gene by gene duplication. The approximate positions of the exons containing HPR2 coding sequence in the AL109843 and AL389925 contigs are shown in the table below, along with their locations relative to SEQ ID NOs 19, 20, 22, and 24; note that the 5' and 3' untranslated regions may extend further along the contig sequence beyond those portions that correspond to SEQ ID NOs 19, 20, 22, and 24, as indicated by the parentheses around the AL109843 and AL389925 endpoints in the table. Due to the preliminary nature of the sequence data and assembly of the contig sequence, the exons within the genomic contigs may contain sequence variations due to inaccurate sequence data or allelic polymorphism.

Corresponding positions of HPR2 gene exons in human genomic contigs AL109843 and AL389925 and in HPR2 coding sequences: Position in AL109843 Position in SEQ ID NO:19 20 22/ 24 Exon 1 (34088)-34164 1-77 UTR, not in SEQ ID NOs 20, 22, and 24) Exon 2 35715-35813 78-176 1-70/ 1-70 1-70 Exon 3 36965-37261 177-473 71-367 /71-367 71-367 Exon 4 50459-50582 474-597 368491 368-491 368-491 Exon 5 68360-68520 598-758 492-652 492-652 492-652 Exon 6 74533-74678 759-904 653-798 653-798 I 653-798 Exon 7 87197-87353 905-1061 799-955 /799-955 1799-955 Exon 8 104336-104425 1062-1151 956-1045 (not present) 956-1045 Exon 9 107802-107904 1152-1254 1046-1148 (not present) (not present) Position in AL389925 Position in SEQ ID NO:19 20 22 24 Exon 10 8847-8937 1255-1345 1149-1239/'G'-957-1047 1046-1071 Exon 11 11488-(12972) 1346-2830/ 1240-1890 1048-1698 (not present) In the HPR3-ex9 splice variant, note that the absence of the exon 9 sequence (103 nucleotides) changes the reading frame towards the 3' end of the coding sequence for the HPR2-ex9 form (SEQ ID NO:24) 65 crelative to that of the HPR2 coding sequence of SEQ ID NO:20, leading to a different amino acid 0 sequence in the HPR2-ex9 C-terminal portion and a stop codon after amino acid 356 (compared to 629 Z amino acids in HPR2). For the HPR2-ex8-ex9 form, the splice is made at a slightly different exon splice acceptor site than for the HPR2 form, so that an extra r' residue is included at the start of exon in the HPR2-ex8-ex9 form, restoring the reading frame to be the same as in the 3' end of the HPR2 sequence. The C-terminal 248 amino acids of HPR2-ex8-ex9 form are therefore the same as the Cterminal 248 amino acids of HPR2 form, and although the coding sequence of the HPR2-ex8-ex9 form is missing both exons 8 and 9 (except for the last residue of exon the resulting HPR2-ex8-ex9 form polypeptide is longer (565 amino acids) than the HPR2-ex9 form polypeptide (356 amino acids).

Several splice variations of the HPR2 sequences have been identified in human genomic sequences and are included within the scope of the invention. For example, amino acids 118 through 215 of SEQ ID NO:16 match the amino acid sequence of HPR2 presented in SEQ ID NO:21, while amino acids 1 through 117 of SEQ ID NO:16 may correspond to an alternatively spliced exon added upstream of exon 3 at the exon/intron boundary identified between nucleotides 176 and 177 of SEQ ID NO:19). Amino acids 216 through 245 of SEQ ID NO:16 may correspond to an additional alternatively spliced exon added between exon 3 and exon 4 at the exon/intron boundary identified between nucleotides 473 and 474 of SEQ ID NO:19). Amino acids 340 through 344 of SEQ ID NO:16 may correspond to an alternatively spliced exon added downstream of exon 5 at the exon/intron boundary identified between nucleotides 758 and 759 of SEQ ID NO:19). In a further potential splice variant, an alternative.exon or exons encoding the amino acid sequence of SEQ ID NO:17 could be substituted for exon 6, resulting in the replacement of amino acids 217 through 267 of SEQ ID NO:21 with the SEQ ID NO:17 amino acids. In this potential variant, 51 amino acids N-terminal to the WSXWS motif, including the proline-rich region (as shown in Table 1) between the two cytokine receptor subdomains, would be replaced with 39 amino acids, resulting in deletion of a portion of the more C-terminal cytokine receptor subdomain which includes a highly conserved Trp residue. In an additional potential splice variant, an alternative exon could be added downstream of exon 4 at the exon/intron boundary identified between nucleotides 597 and 598 of SEQ ID NO:19) so that an amino acid sequence starting in the amino acids of SEQ ID NO:18 could be substituted for amino acids following and including the scrine at position 164 of SEQ ID NO:21. Multiple splice variations as described above can be included in a single splice variant, for example, replacing exon 6 with an alternative exon or exons encoding the amino acid sequence of SEQ ID NO:17, and also deleting exons 8 and/or 9 as described above.

Additional variations of HPR2 polypeptides are provided as naturally occurring genomic variants of the HPR2 sequences disclosed herein; such variations may be incorporated into an HPR2 polypeptide or nucleic acid individually or in any combination, or in combination with alternative splice variation as described above. As one example, a change from the Leu residue position 310 of SEQ ID NO:21 to a Pro residue could be caused by a single change from T to at position 1035 of SEQ ID NO:19. This variation and another are listed in the table below: -66- Amino Acid Change Position in SEQ ID NO:21 Nucleotide Position in SEQ ID NO:19 O Change LZeu->Pro 310 T->C 1035 0 applicable) (not alicable) A->G 2172 UTR) c-i 5 A nucleic acid encoding a polypeptide with a high degree of amino acid similarity (approximately 69% amino acid identity) to human HPR2 was isolated from Mus musculus. The Mus HPR2 amino acid sequence is presented as SEQ ID NO:27, and due to its high level of similarity with human HPR2, is considered to be the murine homologue of human HPR2. PCR amplification of C 10 cDNA sequences corresponding to mRNAs encoding murine HPR2 identified a cDNA molecule encoding SEQ ID NO:27; the nucleotide sequence of this murine HPR2 cDNA is presented as SEQ ID NO:29. Nucleotides 1 through 1932 of SEQ ID NO:29 encode SEQ ID NO:27, with nucleotides 1933- 1935 corresponding to a stop codon. The murine HPR2 amino acid sequence of SEQ ID NO:27 appears to have a 20-amino acid insertion at amino acids 297 through 316 of SEQ ID NO:27 relative to human HPR2 of SEQ ID NO:21, based on an alignment of the human and murine polypeptide sequences; this insertion is identical to amino acids 317 through 336. Given the number of alternatively spliced forms identified for human HPR2, it is possible that this insertion in murine HPR2 relative to the human HPR2 of SEQ ID NO:21 is the result of alternative splicing. One embodiment of the invention is a form of murine HPR2 in which one of these repeated WQPWS-containing motifs has been deleted; that is, polypeptides in which the amino acid sequence ending with amino acid 296 of SEQ ID NO:27 is contiguous with the amino acid sequence beginning with amino acid 317 of SEQ ID NO:27, or polypeptides in which the amino acid sequence ending with amino acid 316 of SEQ ID NO:27 is contiguous with the amino acid sequence beginning with amino acid 337 of SEQ ID NO27.

C. Comparison of HPR1 and HPR2 to Other Hematopoletin Receptor Polypeptides.

The amino acid sequences of human HPR1 (SEQ ID NO:4), murine HPR1 (SEQ ID NO:12), and human HPR2 (SEQ ID NO:21) were compared with the amino acid sequences of these other hematopoietin receptor family members LIF-R, the interleukin 12 beta 2 receptor chain (IL-12RB2), and GCSFR (SEQ ID NO:6 SEQ ID NO.9, respectively) using the GCG "pretty" multiple sequence alignment program, with amino acid similarity scoring matrix blosum62, gap creation penalty 8, and gap extension penalty 2. Alignments of these sequences are shown in Table 1, and include consensus residues which are identical among at least three of the amino acid sequences in the alignment The capitalized residues in the alignment are those which match the consensus residues.

The numbering of amino acid residues in Table 1 corresponds to the position of those residues in the HPR1 amino acid sequence (SEQ ID NO:4). Note that only a portion of the HPR2 amino acid sequence is shown in Table 1, as HPR2 does not contain fibronectin type I repeats in its extracellular domain. HPR1 and HPR2 sequences corresponding to the intracellular Box 1 and Box 2 motifs are shown in Table 2. Sequences of eleven amino acids similar to the Box 1 or 2 motif of other hematopoietin receptors were identified for HPR1 and HPR2, and placed into a column with these motif sequences (with no gaps introduced). Similarly, HPR2 sequences corresponding to the -67intracellular Box 3 motif are shown in Table 3. Sequences of fourteen amino acids similar to the Box 3 0 motif of other hematopoietin receptors were identified for HPR2, and placed into a column with these motif sequences (with no gaps introduced). The numbering of each sequence on Tables 2 and 3 corresponds to their position in the complete amino acid sequence for that HPR polypeptide. The consensus residues are those that are present in three or more (for Table 2) or two or more (for Table 3) sequences at that position in the motif.

Amino acid substitutions and other alterations (deletions, insertions, etc.) to HPRI and HPR2 amino acid sequences (for example, SEQ ID NOs 4, 12, and 21) are predicted to be more likely to alter Sor disrupt HPRI or HPR2 polypeptide activities if they result in changes to the capitalized residues of the amino acid sequences as shown in Tables 1, 2, and 3, and particularly if those changes do not substitute an amino acid of similar structure (such as substitution of any one of the aliphatic residues Ala, Gly, Leu, Ile, or Val for another aliphatic residue), or a residue present in other hematopoietin receptor polypeptides at that conserved position. Conversely, if a change is made to an HPR1 or HPR2 amino acid sequence resulting in substitution of the residue at that position in the alignment from one of the other Table 1, 2, or 3 hematopoietin receptor polypeptide sequences, it is less likely that such an alteration will affect the function of the altered HPR1 or HPR2 polypeptide. For example, the consensus residue at position 42 in Table 1 is serine, and one of the hematopoietin receptors (LPI-R) has an asparagine at that position. Substitution of asparagine or the chemically similar glutamine for serine at that position is considered to be less likely to alter the function of the polypeptide than substitution of tryptophan or tyrosine etc. Embodiments of the invention include HPR1 and HPR2 polypeptides and fragments of HPRI and HPR2 polypeptides, comprising altered amino acid sequences. Altered HPR1 or HPR2 polypeptide sequences share at least 30%, or more preferably at least 40%, or more preferably at least 50%, or more preferably at least 55%, or more preferably at least or more preferably at least 65%, or more preferably at least 70%, or more preferably at least or more preferably at least 80%, or more preferably at least 85%, or more preferably at least 90%. or more preferably at least 95%, or more preferably at least 97.5%, or more preferably at least 99%, or most preferably at least 99.5% amino acid identity with one or more of the hematopoietin receptor amino acid sequences shown in Tables 1, 2, and 3.

Table 1: Alignment of HPR1 and HPR2 extracellular domains with those of other hematopoietin receptors C conserved cysteine Bsm proline-rich linker' between cytokine receptor subdomains WSXWS motif a fibronectin type I repeats m* transmembrane domain sEQ

ID

NO: HS HPRI 4 PaKPeNISCV r.KNLTC TWsPGkETs yTqYTv KrtafGekh78 Mus HPR1 12 PtKPeNiSCV fYfd.rNLTC TWrPekETn. dTSYiv tltySyGK.

gp130 8 PeKPkNLSCi vne .KkmrC eWdGrETHL eTnfTL Ksewa fa GCSFR 9 PaiPhNLSC1 mnlttssLiC qWePGpET9H TSfTL KsfkSrGnCq Hs HPR2 21 PdiPdevtCV iYes TWnaGklTi dTk hv .K -68- IL-12RB2 7 PeclPgNLSCi cqkgeqgtvaC TerGrdTHL yTeYTL gi. S=Knl LIF-R- 6 PdtPq LnCe th.dlKeiiC sWnPGrvTaL vraTSYPL vesfS.CKv consensus ___P-KP-MLSCV -Y KNLTC T-PG-ETHL TSYTL K---S-GKc-- 126 Hs HPR1 4 IdnCttnssts enrasCsffL PRiti. .pdN YtieVeA~lg dGvikShmtv Mus HPR1 says dnateasysf PRscamppdi csVeVQAgNg dGkvkSditv gP13O 8 ldCkakr.... dtptsCtvdy stvvfv... .N ieVWVeAENA LGkvtSdhjn GCSFR 9 1tacdsildcv pkd ishCci PRkhllly N miWV AEN~A LGtSmSoaLc Hs HPR2 21 eteeeqqylt ssY i. stdslgggkk YlvwVQAaNA L~neeskcL(7 ]L-12RB2 7 twqgckdiv CdYldfainL tpespe. .sN ftakVtAvrNs LGsSsSlpst LIF-R 6 rlkraeaptn esYg11fcjnL Pngei YnftlnAhNp LGrSqgStiL.

consensus -Y L PR N Y-VWVQAENA LG-S-S--L- 127 172 Hs HPR1 4 wrLenlaKtE PPkIfRVKPv lgi. .1cm ipieWikPel apvssdLKvt Mus HPR1 12 whLislaKtE PPiIlsVnPI nxrm f iq .kPre ktr f DLvCm 1- fdpvykVKPn PPhnlsVins eel... ssi lkLtWtnPsj ksv.IiLKyn GCSFR_ 9 ldpmpvVKIE PPmlrtmdPs peaappgagc 1QLcW.ePwgt pglhInaKCE Hs HPR2 21 ihLdfllViPs aavlsRaetI natvpki. .iyd. qtekvsCE IL-12RB2 -7 ftfIDIVrPI PPwdiRiKfgi kasvsrct.. LyWrd... eglv. .Llnr LIF-R 6 vnitekVyPh tPtsfkVKdl nsta vkLsWhlPgr. nfakinf iCE consensus__ L-DIVKPE PP-I-RVKPI Q L-W--p I-LKCE S 173 221 Hs HPRI. 4 LRfRTvrNS. t sWmeVnFakN rkdiaiqtynL tGLQPFTEYV iaIRCavkes Mus HPR1 12 LRfRTvNS.s rWteVnP.eN ck.. .gvcnL tGLqaFTEYV laIRfrfnds g213O 8 icgYRTkda.s tWsgip.ped tastrssftv cadLkPFTEYV PrIRCmkEda GCSFR 9 LRhkpqrgea sWalVg. .p lplealayeL cGjlPaTaYt lQIRCirwpl Hs HPR2 21 mRYkattncit .WnvkeF. .d tnf tyvggcse fvLePnikYV FQVRCg.Etg IL-12PM2 7 LRYRpsNSr. .Wnm. .N vtkakgrhdL 1dLkPFTEYe FQIssklhly LIF-R 6 ieikksNSvgl egmVti.kg venssylvaL dkLnPyTlYt FrIRCstEtf consensus LRYRT-NS-- L -GL-PFTEYV PQIRC--E-- 222 261 Hs HPR1 4 K. fWSDWSgE kmTqgTeEEaP Le RvLkP aeadGrRpVr -Mus HPR1 12 r .YWSkWSkE etrvrmEEvP h vLD 1W. RiLeP adamGdRkVr gPl3O 8 KGYWSDWSeE asai yEdrP aps NW...ykiDP sht ~ytVc GCSFR 9 v~hWSDWqSps le1rtEraP tv..rLD 'IWWr ~LDP RtVg Hs HPR2 21 KrYWqpWSsl ffhkTpEtvP cvtskaf qh l IL-12RB2 7 KGsWSDWSes ra ~EEeP tml .D vWvmCRhiD.. ysrqgis LIF-R 6 .wkWSkWSnk kahlTt~asP TJrew ssd laili -consensus KGYWSDWS-E T-EE-P LD TW---R-LDP G-R-V- H-s HPRI 4 LIWKkarqap vleKtLGYni wyypesnTn. LTEti~tTngl lelhLgges -Mus HPR1 12 LIWKkargap vle~tfGYhi rf aensTn. LTEinNiTtq cellLns a gl13O 18 LvWKtLPpfE AnGKILdYeV tltrwksh:l. cnytvqaT.. kltvnLtndr GCSFR 9 LfW~pvPleE dsGrlcGYVV swrpsggaga ilplcNtTel sctfhLpsea IL-12RB2 7 LfWKnLsvsE ArGKILhYxV tleltggka n rnitghts wttviprtgrn LIF-R 6 iYPLPinE AnGKILsYnV scssdeeTcxs LsEipd.pqh kaeirLdcnd consensus--__ L-WK-LP--E A-GKILGY-V LTE- 312 359 Ms HPR1 4 fwvsmisyqS IGKSpvatLr IPaigEksfgf cievmaAcva ed.gLvVkwp -Mus HPR1 12 hsVSVtsfNS IGKSgeTiLr IPdvhEktfq yiksxugAyia eD.lLvVnWp cn,130 8 ylatltvrNI vGKSdaavLt IPacdfcxath pvrndlkAfpk dn.xnLwvewt GCSFR 9 cxeValvAyNS aGtSrPTpv. .vfsEsrgp altrlhAmar fthsLwVgwe- IL-12RB2 7 waVaVsAaN~S kGsSlP'rrin IzunicEagill aprcqvsAnse cmidnilVtwQ LIF-R 6 yiigVvAkNS vGsSpPskia smeipnddl. .kieqvvg mgkgilltWh consensu VSV-A-US -GKS-PT-L-

L-V-WO

-69 ___360 404 Hs HPR1 4 ssal... .dVn twmlEWf dv d.SePttlsw e-svSqaTnw TI DqkLypF Mus HPR1 12L ssip aVd twivEWIpea amSkfoalsw e. svs1: w1 T1!1 1 D1l ngkLK-PF 8 tPre... .sVk kYilEWcvls dka.1PcitdW edg tr iyr rNLaes GCSFR 9 pPnp w CYVIEWq 13sasnsnktW rme' at fllkeNjrPF I-2B 7 PrdsyeYVv3Wre1h dtM in wirs vys alise-NiKsv LIF-R 6~ d nAMtC dYVIKWc.ns srSePclldW r SzpnsTet vesDefrpq consensus -YVIEW---- P W T--T--DNL1XPF 405__444 HS HPR1 4 WCelVPks;7rkGv IC T Iq aKec v Mus HPR1 12 kelpksarnG ingrveyS gp13O 8 d CIPIdv Cv yirgpTspyr GCSFR 9 r fevtP Le asthiphiM as A~asq t vhltl l liI IL-12RB2 7 nshYe 1 inrvtY vlm IL TaA.Ggessh4 neref k anwmafva s LIF-R 6 kiri~fad kTr lvr d k eS 2L il consensus LSL- Tabl 2: Bo5 489 o oisI heItaellrdmisofHPi P2 n te hematopoietin receptors SEQ IM No Box 1 motif Hs JIPRi 4 563-thlcWPtVPNP..573 Mus HPR1 .12 517 -tplCCPDVPNP-527 -HPR2 21 393-pkwl eDiPNm-403 LIF-R 6 866-KetfvPDiPUP-876 9P13O B 648-KkhiWPnvpdP...658 GCSFR 9 S55-KlP1WPsVPdP-665 Consensus K- -WPDvpNp Box 2 M~otif 6 31-eifTdEArtgq-641 582 -VvTEEg c-c592 430-Vd miteiKei-440 9 10-VleTrsAf 1(1-920 693 -VlveiEanU D-703 6 V- -TEF.A-KK- Table 3: Box 3 motifs i the intracellular domains of HPR2 and other heniatopoletin receptors 70 0 EXAMPLE 2: Monoclonal Antibodies That Bind Polypeptides of the Invention This example illustrates a method for preparing monoclonal antibodies that bind HPRI or HPR2 polypeptides. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified HPR1 or HPR2 polypeptide or an immunogenic fragment thereof. Purified HPR1 or HPR2 polypeptide can be used to generate monoclonal antibodies t immunoreactive therewith, using conventional techniques such as those described in U.S. Patent 4,411,993. Briefly, mice are immunized with HPR1 or HPR2 polypeptide immunogen emulsified in S complete Freund's adjuvant, and injected in amounts ranging from about 10 to about 100 micrograms subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional HPR1 or HPR2 polypeptide emulsified in incomplete Freund's adjuvant Mice are c periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for anti-HPRI or anti-HPR2 antibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay), or inhibition of binding of HPRI or HPR2 polypeptide to an HPRI and/or HPR2 binding partner.

Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of HPR1 or HPR2 polypeptide in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened by ELISA for reactivity against purified HPR1 or HPR2 polypeptide by adaptations of the techniques disclosed in Engvall et al., (Immunochen 8:871, 1971) and in U.S. Patent 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., ImmunoL 144:4212, 1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti-HPR1 or anti-HPR2 monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography.

Alternatively, affinity chromatography based upon binding of antibody to Polypeptide A or Polypeptide G can also be used, as can affinity chromatography based upon binding to HPR1 or HPR2 polypeptide.

EXAMPLE 3 Antisense Inhibition of HPR1 and/or HPR2 Expression In accordance with the present invention, a series of oligonucleotides are designed to target different regions of HPRI and/or HPR2 human or murine mRNA molecules, using the nucleotide sequences of SEQ ID NOs 3, 5, 19, 20, 22, 24, 28, and 29 as the bases for the design of the oligonucleotides. The oligonucleotides are selected to be approximately 10, 12, 15, 18, or more -71- 4

(N

preferably 20 nucleotide residues in length, and to have a predicted hybridization temperature that is at 0 least 37 degrees C. Preferably, the oligonucleotides are selected so that some will hybridize toward the region of the mRNA molecule, others will hybridize to the coding region, and still others will hybridize to the 3' region of the mRNA molecule.

The oligonucleotides may be oligodeoxynucleotides, with phosphorothioate backbones (internucleoside linkages) throughout, or may have a variety of different types of intemucleoside linkages. Generally, methods for the preparation, purification, and use of a variety of chemically Smodified oligonucleotides are described in U.S. Patent No. 5,948,680. Modified oligonucleosides may Salso be used in oligonucleotide synthesis, as well as mixed backbone compounds having, for instance, alternating MMI and PO= or P=S linkages, which are prepared as described in US. Pat Nos.

5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289. Formacetal- and thioformacetal-linked (1 oligonucleosides may also be used and are prepared as described in U.S. Pat Nos. 5,264,562 and 5,264,564; and ethylene oxide linked oligonucleosides may also be used and are prepared as described in U.S. Pat No. 5,223,618. Peptide nucleic acids (PNAs) may be used as in the same manner as the oligonucleotides described above, and are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic Medicinal Chemistry, 1996, 4, 5-23; and U.S. Pat Nos. 5,539,082, 5,700,922, and 5,719,262. Chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides/oligonucleosides of the invention are synthesized according to U.S. Pat No. 5,623,065.

The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. Preferably, the effect of several different oligonucleotides should be tested simultaneously, where the oligonucleotides hybridize to different portions of the target nucleic acid molecules, in order to identify the oligonucleotides producing the greatest degree of inhibition of expression of the target nucleic acid.

Antisense modulation of HPR1 and/or HPR2 nucleic acid expression can be assayed in a variety of ways known in the art For example, HPR1 and HPR2 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR).

Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation and Northern blot analysis are taught in, for example, Ausubel. F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. Other methods of quantitative PCR analysis are also known in the art. HPRI and HPR2 protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA, or fluorescence-activated cell sorting (FACS). Antibodies directed to HPR1 and/or HPR2 polypeptides can be prepared via -72conventional antibody generation methods such as those described herein. Immunoprecipitation methods, Western blot (immunoblot) analysis, and enzyme-linked immunosorbent assays MEUSA) are standard in the art (see, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, 10.8.1-10.8.21, and 11.2.1-11.2.22, John Wiley Sons, Inc., 1991).

Al] publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it Will be readily apparent to those of ordinary sill in the aft in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Sequences Presented in the Sequence Listing SEQ ID NO Tpe; Description SEQ ID NO:l Amino acid Partial human HPRl amino acid sequence SEQ U) NO:2 Amino acid Partial human HPRI amino acid sequence SEQ D NO:3 Nucleotide Human HPR1 cDNA sequence SEQ MD NO:4 Amino acid Human HPR1 amino acid sequence (745 amino acids) SEQ ID NO:S Nucleotide Human HPRI coding sequence S ID) NO:6 Amino acid Human LIF-R amino acid sequence (ClenBank NP 002301) SEQ ID NO:7 Amino acid Human IIA12RB2 amino acid sequence (GenBank NP 001550) SEQ ID NO:8 Amino acid Human gp 130 amino acid sequence (GenBank NP 00O(2175) SEQ ID) NO:9 Amino acid Human GCSFR amino acid sequence (SWILSS-PROT'099062) SEQ ED NO: 10 Amino acid Portion of possible alternatively spliced form of human BPR1I SEQ ID NO: 11 Amino acid Portion of possible alternatively spliced form of human HPRl SEQ ED NO: 12 Amino acid Mus musculus HPR1 amino acid sequence SEQ D NO: 13 Amino acid Possible 252-an human BPRI. variant (WO 00/75314) SEQ ED NO: 14 Amino acid Possible 652-aa human HPRI variant (WO 00175314) MEQID NO:15 Amino acid Possible 662-aa human HPR1 variant (WO 00/75314) L IJJj LI A ATrblnfl api,! f W -1 I J n L= e~UL mwiuauvriy apiceu rorm orbuman kikR2 S1EQ ID NO: 17 SEO UD NO: 18 Amino acid in a i SEO ED NO: ig Nudeotidt~ S-EQ ID N0:20 Nucleotide SEQ ID NO:21 Amino acid SEQ ID N0:22 Nucleotide SEQ ID NO:23 SEQ ID N0.24 SEQ ID NO:25 Amino acid Nucleotide Portion of possible alternatively spliced form of human HPR2 Portion of possible alternatively spliced form of human HPR2 Human HRPR2. cDNA sequence exons I through I11 Human HPR2 coding sequence (encodes 629-an form) Human HPR2 amino acid sequence (629 amino acids) Human HPR2-.ex8-ex9 coding sequence (encodes 565-a form) Human HPR2-ex8-ex9 amino acid sequence (565 amino acids) Human HPR2-ex9 coding sequence (encodes 356-aa form) Human HPR2-ex9 amino acid sequence (356 amino acids) Possible 384-aa human HPR2 variant (WO 00/7345 1) Mus musculus HRPR2 amino acid sequence Mus musculus HPR1 coding sequence Mus musculus HPR2 coding sequence SEQ ED N0.26 SEQ DNO:27 SEQ 0D NO:28 SEQ ED NO029 aci Amino acid Amino acid Nucleotide INucleOtide 73

Claims (1)

1. 557-745 of SEQ ID NO:4, wherein said amino acid sequence comprises a tyrosine that can be phosphorylated by a kinase. 2. The isolated nucleic acid of claim 1, wherein the polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4. 3. The isolated nucleic acid of claim 2, wherein the polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4. 4. The isolated nucleic acid of claim 3, wherein the polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4. The isolated nucleic acid of claim 4, wherein the polypeptide comprises amino acids 33-745 of SEQ ID NO:4. 6. An expression vector comprising a nucleic acid of any of claims 1 to 7. A recombinant host cell comprising an expression vector of claim 6. 8. A process of producing a polypeptide, comprising culturing a host cell of claim 7 under conditions promoting expression of said polypeptide. W VNgePA50000 OO9\689475\51475 Dr. pages do 9. An isolated nucleic acid encoding a polypeptide comprising amino acids 557-745 Sof SEQ ID NO:4. An expression vector comprising a nucleic acid of claim 9. 11. A recombinant host cell comprising an expression vector of claim C C 12. A process of producing a polypeptide, comprising culturing a host cell of claim 11 Sunder conditions promoting expression of said polypeptide. 13. An isolated polypeptide comprising amino acids 557-745 of SEQ ID NO:4. 14. The isolated polypeptide of claim 13, wherein the polypeptide comprises amino acids 33-745 of SEQ ID NO:4. The isolated polypeptide of claim 14, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4. 16. A method of detecting an antibody that binds human hematopoietin receptor 1 (HPR1), the method comprising: a) contacting a host cell containing an expression vector comprising a nucleic acid encoding a polypeptide comprising amino acids 33-745 of SEQ ID NO:4 with a composition comprising an antibody; and b) detecting binding of an antibody to HPR1. W:Wiger50000 69999589475\889475 D, pagesdoc SEQUENCE LISTING <110> Immunex Corporation Cosman, David J. Mosley, Bruce A. Bird, Timothy A. DuBose, Robert F. Wiley, Steven R. <120> HEMATOPOIETIN RECEPTORS HPR1 AND HPR2 <130> 3160-WO <160> 29 <170> Patentln version 3.1 <210> <211> <212> <213> PRT Homno sapiens <400> 1 Met 1 Glu Val Asn Ala Lys Asn Arg Asp Lys Asn Gin Thr Tyr Asn Leu Thr Cys Ala Val Gly Leu Gin Pro Phe Thr 25 Glu Tyr Val Ile Ala Leu Arg Gln Glu Lys Lys Giu Ser Lys Trp Ser Asp Trp Met Gly Met Thr Giu Glu Gly Lys Leu Leu Ala Ile Pro Val Leu Ser Ala Thr Gly Val 70 Gly Leu Leu Trp Ala Arg Leu <210> 2 <211> 42 <212> PRT <213> Homo sapiens <400> 2 Met Giu Val Thr Phe Ala Lys Asn Arg Asp Lys Asn Gin Thr Tyr Asn Leu Thr Cys Ala Val Gly Leu Gin Pro Phe Thr Giu Tyr Vai Ile Ala Leu Arg Lys Glu Ser Lys Phe Leu Glu <210> 3 <211> 2480 <212> DNA <213> Homno sapiens <400> 3 cccacatctt agtgtggata gtgggaggtg gagttgcctt tcccctgata catgaagctc cctgggcact gtggatgctc agcctgagaa catttcctgt caggaaagga aaccagttat aacatgataa ttgtacaacc tccttccaag aataacgatc atggtgtaat taaatctcat cacctaagat tttccgtgtg ggataaagcc tgagttggcg cagtcaacag taccagctgg aaacgtacaa cctcacgggg cggtcaagga gtcaaagttc aagaagctcc atgtggcctg gaaggccagt gcggttgtta ttggctacaa catatggtac ctactaacca gcagcttgaa cttataattc tcttgggaag aatcatttca gtgcattgag agtggcaaag ccctgctcta actcagagcc caccaccctt agcaagataa attaaaacct acaaagttgg cgagccatat gtcctgagac caaggtggag ttcccaagag tgagagaaag gtggaaaagg attctccaag aattaaagtc tgatgcaaat tctccccagc ccttcactct gtctactact acccagtaca aatagttcta ccagataatt atgacatact aaaccagttt cctgtttcat atggaagtca ctgcagcctt tggagtgact gaac tgtgga tggaagaagg tatccagaaa ctgcatctgg tctccagtgg gtcatgcagg gacgtgaaca tcctgggaat ttctggtgct tccatccagg aacattggcg ggtatcatct acagtcaatt cagattgttc cctt tgagcc cttcatgtgt gcaaattcag ataggaaaaa cagttaagag caagtgaaaa ataccattga ggagattaga tgggcatcaa ctgatttaaa acttcgctaa ttacagaata ggagccaaga gagtcc tgaa caagaggagc gcaacac taa gaggcgagag ccaccctgag cctgcgttgc cttggatgat ctgtgtctca ataacatctc cttatgccaa tgaagacggt gcaac tacac ccagcatctt ttcctgtcct agcagaacat taacc tgggg cc tggcagc t tttaacctgc aacttacgct tcgtgcttcg ggtggaagc t gaacatagcg acgaatgatt atacacactt gaaccgtaag tgtcatagct aaaaatggga accagc tgag cccagtcc ta cc tcacagaa cttttgggtg gattccagct tgaggaccag tgaatggttt ggccacgaac tgtgtatcca agaaggcgtt cacgatcaca catcttttac gcag tacggc gacttgtgct ctgtggaaca atgatgtgga ctgccagcta act tggagtc tttggagaaa tgctcttttt gaaaatggag aaaactgaac caaattgaat cgattcagga gataaaaacc ctgcgatgtg atgactgagg gcggatggaa gagaaaacac acaatgaaca tc ta tga tt t attcaagaaa ctagtggtga ccggatgtgg tggacgatcc atgttgcatg ccatcagaag tggaaagaga caagctgaag ctggagtccc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 tgaaacgaaa acgggaccag cttctctgat aaaaacccaa gtatagccac actctgtgaa tgat tgacaa ccagaacggg tcaggcctga cgcccggaaa aagagcagc t ccccaaatcc t tccagagca cagtgtggat cctgccacat gacctcttac cataaatttc tggtggaggc caaattgact atggcatgga cacagaagac gttggtggtg tcaggaaaac ttgtcccctg atcccaatac tctcttttct atatttgaaa caccaaggga ggcccttgcc cctgcctagg attgttcagg aagacattgt cttcttattc catctgtgtt gatgatttca aggatcttaa aactttggga aat ttaggag gggaaaagtt ctacgttcga ggtcaaagtt aattcagtga gaagtc taaa tcatggccag cattcagtgt tcattatcct ggcccaccgt aggataagct aaccatgttc atgttctgca gggaaaagaa t tgaggagc t ggatgccaga tagtaccaga cagccaggga tgcgaccata caccagtgct ctttgagatt gacagtggca tcccaaccct aaacctgaag cacccccagt agaaattttc tgggtatgtg cccagtttca ggggacccgc tcatctgtgt atttcttgtg gcatgagacc gggggaaccg atcctcataa tatggtctca gctgaaagta gagtctgatg gacaagttgg acagatgaag acctgcccct cctgagattc ccagaagcca gaggaaggag tctgaaaaac ctcggggcct 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2480 agagaagatg tcaagactcg gcacgcagcg cttgcttggc <210> 4 <211> 745 <212> PRT <213> Homo sapiens <400> 4 Met Lys Leu Ser Pro 1 Gin Pro Ser Cys Val Asn Leu Gly Met Met Trp Thr Trp Ala Ala Leu Pro Trp Met Leu Pro Leu Cys Lys Phe Tyr Tyr Arg Ala Lys Pro Glu Ile Ser Cys Val Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys Thr Ser Tyr Thr Gin Tyr Thr Val Lys Thr Tyr Ala Phe Glu LYS His Asp Asn Cys Thr Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser Cys Ser Phe Phe Leu Pro Ala Giu Asn 115 Arg 100 Ile Thr Ile Pro Asp 105 Asn Tyr Thr Ile Giu Vai Giu 110 Tyr Trp Arg Gly Asp Gly Val Ile 120 Lys Ser His Met Thr 125 Leu Giu 130 Asn Ile Ala Lys Giu Pro Pro Lys Ile 140 Phe Arg Val Lys Val Leu Gly Ile Lys 150 Arg Met Ile Gin Giu Trp Ile Lys Glu Leu Ala Pro Ser Ser Asp Leu Lys 170 Tyr Thr Leu Arg Phe Arg 175 Thr Val Asn Lys Asp Lys 195 Thr Ser Trp Met Giu 185 Val Asn Phe Ala Lys Asn Arg 190 Pro Phe Thr Asn Gin Thr Tyr Asn 200 Leu Thr Gly Leu Gin 205 Glu Tyr 210 Val Ile Ala Leu Cys Ala Val Lys Giu 220 Ser Lys Phe Trp Asp Trp Ser Gin Giu 230 Lys Met Gly Met Giu Glu Glu Ala Cys Giy Leu Giu Trp Arg Val Leu Lys 250 Pro Ala Glu Ala Asp Gly 255 Arg Arg Pro Leu Giu Lys 275 Val1 260 Arg Leu Leu Trp Lys 265 Lys Ala Arg Gly 270 Glu Ser Asn Thr Leu Gly Tyr Asn 280 Ile Trp Tyr Tyr Pro 285 Thr Asn 290 Leu Thr Glu Thr Asn Thr Thr Asn Gin 300 Gin Leu Giu Leu Leu Gly Gly Giu Ser 310 Phe Trp Val Ser Met Ile Ser Tyr Asn 315 Leu Gly Lys Ser Pro 325 Val Ala Thr Leu Arg 330 Ile Pro Ala Ile Gln Giu 335 Lys Ser Phe Gin Leu Val 355 Cys Ile Giu Val Met 345 Gin Ala Cys Vai Ala Giu Asp 350 Asn Thr Trp Val Lys Trp Gin Pro Ala Leu Asp Met Ile 370 Giu Trp Phe Pro Asp 375 Val Asp Ser Giu Pro 380 Thr Thr Leu Ser Trp 385 Giu Ser Vai Ser Ala Thr Asn Trp Ile Gin Gin Asp Lys 400 Leu Lys Pro Phe Trp 405 Cys Tyr Asn Ile Vai Tyr Pro Met Leu His 415 Asp Lys Val Vai Pro Ser 435 Giy 420 Giu Pro Tyr Ser Ile 425 Gin Ala Tyr Aia 430 Gly Val Lys Giu Giy Pro Giu Thr Lys Vai Giu Asn Thr Vai 450 Thr Ile Thr Trp Lys 455 Giu Ile Pro Lys Giu Arg Lys Gly Ile 465 Ile Cys Asn Tyr Thr 470 Ile Phe Tyr Gin Ala 475 Giu Giy Giy Lys Phe Ser Lys Thr Asn Ser Ser Ile Leu 490 Gin Tyr Giy Leu Giu Ser 495 Leu Lys Arg Ala Giy Gly 515 Lys 500 Thr Ser Tyr Ile Gin Val Met Ala 510 Leu Ser Phe Thr Asn Gly Thr Ile Asn Phe Lys Thr 525 Ser Vai 530 Phe Gu Ile Ile Ile Thr Ser Leu Gly Gly Gly Leu Leu 545 Ile Leu Ile Ile Leu Thr Val Ala Tyr 550 Leu Lys Lys Pro Asn 560 Lys Leu Thr His Leu 565 Cys Trp Pro Thr Val1 570 Pro Asn Pro Ala Giu Ser 575 Ser Ile Ala Thr Trp His Gly Asp Asp Phe Lys Asp Lys Leu Asn Leu 580 Asp Asp Ser Val 585 Thr Glu Asp Arg Lys 0Th Ser 595 Asn 600 590 Ile Leu Lys Pro 605 Leu Val Val Asn Cys Ser 610 Thr Pro Ser Asp Lys 615 Leu Val Ilie Asp Gly Asn Val Leu Glu Ile Phe Thr Glu Ala Arg Thr Gin Giu Asn Asn Leu 645 Giy Gly Glu Lys Asn 650 Gly Tyr Val Thr Cys Pro 655 Phe Arg Pro Ser Pro Glu 675 Asp 660 Cys Pro Leu Gly Lys 665 Ser Phe Giu Glu 670 Ser Arg Met Ile Pro Pro Gly Lys 680 Ser Gin Tyr Leu Arg 685 Pro Glu 690 Gly Thr Arg Pro Ala Lys Glu Gin Leu Phe Ser Gly Gin 705 Ser Leu Val Pro Asp 710 His Leu Cys Glu Gly Ala Pro Asn Tyr Leu Lys Asn Ser 725 Val Thr Ala Arg Phe Leu Val Ser Glu LYS 735 Leu Pro Glu His Thr Lys Giy Glu Val <210> <211> 2238 <212> DNA <213> Homo sapiens <400> atgaagctct ctccccagcc tggatgctcc cttcactctg atttcctgtg tctactacta accagttata cccagtacac tgtacaacca atagttctac ataacgatcc cagataatta ttcatgtgtt caaattcagc taggaaaaat agt taagaga aagtgaaaat taccattgag aacc tgggga ctggcagctc ttaacctgca acttacgctt cgtgcttcgt gtggaagctg tgatgtggac tgccagctaa cttggagtcc ttggagaaaa gCtcttttt aaaatggaga c tgggcac tg gcc tgagaac aggaaaggaa acatgataat ccttccaaga tggtgtaatt aaatc tcata ttccgtgtga gagt tggcgc accagc tgga c tcacggqgc tcaaagttct tgtggcctgg atatggtact cagc ttgaac cttgggaagt tgcattgagg cctgctctag accacccttt ttaaaacctt gagccatatt aaggtggaga gagagaaagg ttctccaaga acctcttaca ataaatttca ggtggaggcc aaattgactc tggcatggag acagaagaca ttggtggtga caggaaaaca tgtcccctgg tcccaatacc ctcttttctg tgacatactg aaccagt t tt ctgtttcatc tggaagtcaa tgcagccttt ggagtgactg aactgtggag ggaagaaggc atccagaaag tgcatctggg ctccagtggc tcatgcaggc acgtgaacac cctgggaatc tctggtgcta ccatccaggc acattggcgt gtatcatc tg cagtcaattc ttgttcaggt agacattgtc ttcttattct atctgtgttg atgatttcaa ggatcttaaa actttgggaa atttaggagg ggaaaagttt tacgttcgag gtcaaagttt gagattagag gggcatcaaa tgatttaaaa cttcgctaag tacagaatat gagccaagaa agtcctgaaa aagaggagcc caacactaac aggcgagagc caccctgagg ctgcgttgct ttggatgatt tgtgtctcag taacatctct ttatgccaaa gaagacggtc caac tacacc cagcatcttg catggccagc attcagtgtc cattatcctg gcccaccgtt ggataagcta accatgttcc tgttctgcaa ggaaaagaat tgaggagc tc gatgccagag agtaccagat aacatagcga cgaatgattc tacacacttc aaccgtaagg gtcatagctc aaaatgggaa ccagctgagg ccagtcctag ctcacagaaa ttttgggtgt attccagcta gaggaccagc gaatggtttc gccacgaact gtgtatccaa gaaggcgt tc acgatcacat atcttttacc cagtacggcc accagtgctg tttgagatta acagtggcat cccaaccctg aacc tgaagg acccccagtg gaaattttca gggtatgtga ccagtttcac gggaccegcc catctgtgtg aaactgaacc aaattgaatg gattcaggac ataaaaacca tgcgatgtgc tgactgagga cggatggaag agaaaacact caatgaacac ctatgatttc ttcaagaaaa tagtggtgaa cggatgtgga ggacgatcca tgttgcatga catcagaagg ggaaagaga t aagctgaagg tggagtccct ggggaaccga tcctcataac atggtctcaa c tgaaagtag agtctgatga acaagt tggt cagatgaagc cstgcccctt ctgagattcc cagaagccaa aggaaggagc acctaagatt gataaagcct agtcaacagt aacgtacaac ggtcaaggag agaagctcca aaggccagtg tggctacaac tactaaccag ttataattct atcatttcag gtggcaaagc ctcagagccc gcaagataaa caaagttggc tcctgagacc tcccaagagt tggaaaagga gaaacgaaag cgggaccagc ttctctgatt aaaacccaac tatagccaca c tc tgtgaac gattgacaag cagaacgggt caggcctgat gcccggaaaa agagcagc tt cccaaatcca 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 tatttgaaaa attcagtgac agccagggaa tttcttgtgt ctgaaaaact tccagagcac 2220 2238 accaagggag aagtctaa <210> 6 <211> 1097 <212> PRT <213> Homo sapiens <400> 6 met 1 Met Asp Ile Tyr 5 Val Cys Leu Lys Arg Pro Ser Trp Met Val Asp Asn Lys Arg Phe Ile Leu Met Arg Thr Ala Ser Asn 25 Phe Gin Trp Leu Leu Ser Thr Lys Lys Gly Leu Tyr Leu Met Gin Val Asn Ser Gin Ala Pro His Asp Leu Lys Val Thr Asn Asn Gin Val Trp Asn Cys Ser Trp Lys Aia Ser Giy Thr Gly Gly Thr Asp Tyr Giu Val Cys Ile Giu Arg Ser Arg Ser Tyr Gin Leu Giu Lys Thr Ser Ile Lys Asn Ser Leu 115 Ile 100 Pro Ala Leu Ser Gly Asp Tyr Giu 110 Thr Leu Asn His Asp Phe Gly Ser Thr Ser Lys Glu Gin 136 Asn Val Ser Leu Ile 135 Pro Asp Thr Pro Giu Ile Leu Asn Leu 140 Lys Trp Asn Asp Arg Ser 145 Ala Asp Phe Ser Ser Thr Leu Tyr Gly Ser Val Phe Pro 165 His Arg Ser Asn Val1 170 Ile Trp Glu Ile Lys Val 175 Leu Arg Lys Ser Met Glu Leu Vai 185 Lys Leu Vai Thr His Asn Thr 190 Thr Leu Asn Gly Lys Asp Thr Leu His His Trp Ser Trp Ala Ser Asp 200 Met Pro 210 Leu Giu Cys Ala Ile 215 His Phe Val Giu Arg Cys Tyr Ile Asp 225 Asn Leu His Phe Ser 230 Giy Leu Giu Giu Ser Asp Trp Ser Val Lys Asn Ile Trp Ilie Pro Asp Ser 250 Gin Thr Lys Val -Phe Pro 255 Gin Asp Lys Ser Gin Giu 275 Ile Leu Val Gly Ser 265 Asp Ile Thr Phe 270 Asn Cys Pro Lys Val Leu Ser Leu Ile Gly His Thr 285 Leu Ilie 290 His Leu Asp Giy Giu 295 Asn Val Ala Ile Ile Arg Asn Ile 305 Asn Val Ser Aia Ser Ile Phe Giy Thr 325 Ser 310 Giy Thr Asn Vai Phe Thr Thr Giu Asp 320 Val Ile Phe Ala Tyr Pro Pro Asp Thr Pro 335 Gin Gin Leu Trp Asn Pro 355 Asn 340 Cys Giu Thr His Leu Lys Giu Ile Ile Cys Ser 350 Ala Thr Ser Gly Arg Val Thr Leu Vai Gly Pro Tyr Thr 370 Leu Val Giu Ser Phe 375 Ser Gly Lys Tyr Arg Leu Lys Arg Aia 385 Glu Ala Pro Thr Giu Ser Tyr Gin Leu Phe Gin Met Pro Asn Gin Giu Tyr Asn Phe Thr Leu Asn 410' Ala His Asn Pro Leu 415 Giy Arg Ser Pro His Thr 435 Ser Thr Ile Leu Val Asn 425 Ile Thr Giu Lys Val Tyr 430 Ser Thr Ala Pro Thr Ser Phe Val Lys Asp Ile Asn 445 0 z Val Lys 450 Leu Ser Trp His Leu 455 Pro Gly Asn Phe LyleAsPhAa 460 Lys Ile Asn Phe Leu 465 Cys Giu Ile Giu Ile 470 Lys Lys Ser Asn Ser 475 Val Gin Giu Gin Arg 480 Asn Val Thr Ile Lys 485 Giy Val Giu Asn Ser 490 Ser Tyr Leu Vai Ala Leu 495 Asp Lys Leu Thr Giu Thr 515 Asn 500 Pro Tyr Thr Leu Thr Phe Arg Ile Lys Gin His Phe Trp Lys Trp Lys Trp Ser Asn Leu Thr 530 Thr Giu Ala Ser Ser Lys Gly Pro Thr Trp Arg Giu Trp 545 Ser Ser Asp Gly Lys 550 Asn Leu Ile Ile Tyr 555 Trp Lys Pro Leu Ile Asn Glu Ala Asn 565 Gly Lys Ile Leu Ser 570 Tyr Asn Val Ser Cys Ser 575 Ser Asp Giu Lys Ala Giu 595 Thr Gin Ser Leu Ser 585 Giu Ile Pro Asp Pro Gin His 590 Ser Val Val Ile Arg Leu Asp Asn Asp Tyr Ile Ile 605 Ala Lys 610 Asn Ser Val Gly Ser Pro Pro Ser Ile Ala Ser Met Glu 625 Ile Pro Asn Asp Leu Lys Ile Giu Val Val Gly Met Gly 640 Lys Gly Ile Leu Leu 645 Thr Trp His Tyr Pro Asn Met Thr Cys Asp 655 Tyr Val Ilie Asp Trp Arg 675 Trp Cys Asn Ser Ser Arg Ser Giu Pro 665 670 Ile Glu Ser Lys Val Pro Ser Asn 680 Ser Thr Giu Thr Val 685 Asp Glu 690 Phe Arg Pro Gly Arg Tyr Asn Phe Phe 700 Leu Tyr Gly Cys Arg 705 Asn Gin Gly Tyr Leu Leu Arg Ser Met 715 Ile Gly Tyr Ile Giu Leu Ala Pro Ile 725 Val Ala Pro Asn Thr Vai Glu Asp Thr Ser 735 Ala Asp Ser Arg Gly Phe 755 Ile 740 Leu Val Lys Trp Giu 745 Asp Ile Pro Val Glu Giu Leu 750 Gly Giu Arg Leu Arg Gly Tyr Leu 760 Phe Tyr Phe Gly Asp Thr 770 Ser Lys Met Arg Leu Giu Ser Gly Arg Ser Asp Ile 780 Leu Arg Ile Ala Lys Val1 785 Lys Asn Ile Thr Ile Ser Gin Lys Thr 795 Asp 800 Leu Gin Gly Lys Ser Tyr His Leu Leu Arg Ala Tyr Thr Asp 815 Gly Gly Val Ser Val Gly 835 Giy 820 Pro Giu Lys Ser Tyr Val Val Thr 830 Val Ala Val Leu Ile Ile Ala Ile 840 Leu Ile Pro Val Ile Vai 850 Gly Val Val Thr Ser Ile Leu Cys Tyr 855 Lys Arg Giu Trp Lys Giu Thr Phe Tyr 870 Pro Asp Ile Pro Asn 875 Pro Giu Asn Cys Ala Leu Gin Phe Lys Ser Val Cys Giy Ser Ser Ala Leu Lys 895 Thr Leu Giu Thr Arg Ser 915 Asn Pro Cys Thr Asn Asn Val Giu 910 Ile Ser Pro Aia Phe Pro Lys Glu Aso Thr Glu Ile 925 Vai Ala Giu Arg Pro Giu 930 Asp 935 Arg Ser Asp Ala Giu Pro Glu Asn His 940 Val1 945 Val Val Ser Tyr Cys 950 Pro Pro Ile Ile Giu Giu Giu Ile Pro Asn 955 960 Pro Ala Ala Asp Ala Gly Gly Thr Ala Gin Val Ile Tyr Ile Asp 970 975 Val Gin Ser Asn Asp Pro 995 Met 980 Tyr Gin Pro Gin Ala Lys Pro Giu Giu Giu Gin Giu 985 990 Gly Tyr Lys Pro Gin Met His Leu Pro 1000 1005 Val Giy Gly Ala Ile Asn 1010 Lys Thr 1025 Leu Vai 1040 Ile Vai 1055 Leu Ile 1070 Gly Trp 1085 Ser Thr Val Giu Ala Gly Tyr Arg Ser Pro Asp Ser Ser Phe Gly Ser Pro Pro Lys Asp Ser Phe Thr Asn Asp 1015 Pro 1030 Pro 1045 Pro 1060 Giu 1075 Phe 1090 Ile Ala Ala Giu Gin Ala Asn Val Arg Ser Ile Asp Cys Ser Ile Asn Asp Ser Pro Lys Phe Gin Asn Lys Giu Asp Leu Asp 1020 Asn Thr Trp Asn 1035 Ser Asn Ser Glu 1050 Ser Arg Gin Phe 1065 Ser Asn Gly Gly 1080 Pro Asn Asp 1095 <210> 7 <211> 979 <212> PRT <213> Homo sapiens <400> 7 Met Ala Leu Phe Ala Val Phe Gin Thr Thr Phe Phe Leu Thr Leu Leu Ser Leu Arg Thr Pro Val Thr Tyr Gin Ser Giu Leu Aia Glu Arg Leu Pro Leu Ser Leu Lys Val Thr Asn Ser Thr Arg Gin Ser Leu His Leu Gin Trp Thr Val His 55 Asn Leu Pro Tyr His Gln Glu Leu Lys Met Val Phe Gin Ile Gin 70 Ile Ser Arg Ile Glu Thr Ser Asn Vai Trp Val Gly Asn Tyr Ser Thr Thr Val Trp Asn Gin Val Leu His Trp Ser Trp Arg Ile Lys 115 Glu 100 Ser Giu Leu Pro Glu Cys Ala Thr His Phe Val 110 Pro Asn Phe Ser Leu Val Asp Ala Lys Phe Pro Glu 125 Trp Ser 130 Asn Trp Ser Ser Glu Glu Val Ser Gin Asp Ser Thr Gly 145 Gin Asp Ile Leu Phe 150 Val Phe Pro Lys Lys Leu Val Glu Glu 160 Gly Thr Asn Val Thr 165 Ile Cys Tyr Val Arg Asn Ile Gin Asn Asn 175 Val Ser Cys Pro His Val 195 Leu Glu Gly Lys Ile His Gly Glu Gln Leu Asp 190 Ile Arg Asn Thr Ala Phe Asn Leu 200 Asn Ser Val Pro Lys Gly 210 Thr Asn Ile Tyr Cys 215 Glu Ala Ser Gin Gly 220 Asn Val Ser Glu Gly 225 Met Lys Gly Ile Val 230 Leu Phe Val Ser Lys 235 Val Leu Glu Glu Pro 240 Lys Asp Phe Ser Glu Thr Glu Asp Lys Thr Leu His Cys Thr 255 Trp Asp Pro Gin Ser Tyr 275 Thr Asp Thr Ala Leu Gly Trp Ser Lys Gin Pro Ser 265 270 Thr Leu Phe Glu Ser 280 Phe Ser Gly Glu Lys 285 Lys Leu Cys Thr His 290 Lys Asn Trp Cys Asn 295 Trp Gin Ile Thr Gin Asp Ser Gin Giu 300 Tyr Asn Phe Thr Ile Ala Giu Asn Tyr 315 Leu Arg Lys Arg Ser 320 Val Asn Ilie Leu Asn Leu Thr His Arg 330 Val Tyr Leu Met Asn Pro 335 Phe Ser Vai Trp Lys Val 355 Asn 340 Phe Giu Asn Val Ala Thr Asn Ala 350 Cys Gin Ile His Ser Ilie Arg Asn Phe Thr Tyr Giu Leu 370 His Gly Giu Gly Lys 375 Met Met Gin Tyr Val Ser Ile Lys Val1 385 Asn Gly Giu Tyr Phe 390 Leu Ser Giu Leu Pro Ala Thr Giu Met Ala Arg Val Arg 405 Cys Ala Asp Ala Ser 410 His Phe Trp Lys Trp Ser 415 Giu Trp Ser Ala Pro Asp 435 Gly 420 Gin Asn Phe Thr Thr 425 Leu Glu Ala Ala Pro Ser Giu 430 Vai Trp Arg Ilie Val 440 Ser Leu Giu Pro Gly Asn His Thr 445 Val Thr 450 Leu Phe Trp Lys Pro 455 Leu Ser Lys Leu His 460 Ala Asn Gly Lys Leu Phe Tyr Asn Val Val Giu Asn Leu 475 Asp Lys Pro Ser Ser Giu Leu His Ile Pro Ala Pro Ala 490 Asn Ser Thr Lys Leu Ile 495 Leu Asp Arg Gly Ala Ser 515 Ser Tyr Gin Ile Val Ile Ala Asn 510 Pro Giu Asn Pro Ala Ser Val Val Ile Ser Ala Lys Giu 530 Val Giu Glu Giu Arg Ile Ala Gly Thr 535 Giu 540 Giy Gly Phe Ser Leu 545 Ser Trp Lys Pro Gin 550 Pro Gly Asp Val Ilie 555 Gly Tyr Val Vai Asp 560 Trp Cys Asp His Thr 565 Gin Asp Vai Leu Asp Phe Gin Trp Lys Asn 575 Vai Giy Pro Pro Giy Val 595 Asn 580 Thr Thr Ser Thr Ile Ser Thr Asp 590 Thr Lys Arg Arg Tyr Asp Phe Arg 600 Ile Tyr Giy Leu Ilie Ala 610 Cys Leu Leu Giu Lys Thr Gly Tyr Gin Giu Leu Ala Pro 625 Ser Asp Asn Pro Vai Leu Val Asp Leu Thr Ser His Phe Thr Leu Ser Lys Asp Tyr Ser Thr 650 Giu Ser Gin Pro Giy Phe 655 Ile Gin Giy Pro Arg Phe 675 His Vai Tyr Leu Lys 665 Ser Lys Ala Arg 670 Cys Cys Lys Giu Lys Aia Vai Leu 680 Ser Asp Gly Ser Tyr Lys 690 Ile Asp Asn Pro Giu Lys Ala Leu Ile 700 Val Asp Asn Leu Lys 705 Pro Giu Ser Phe Giu Phe Phe IleThr 715 Pro Phe Thr Ser Gly Giu Gly Pro Ala Thr Phe Thr Lys 730 Val Thr Thr Pro Asp Giu 735 His Ser Ser Leu Leu Ilie 755 Leu Ile His Ilie Leu 745 Leu Pro Met Val 750 Ile Lys Giu Met Vai Met Cys Tyr 760 Leu Lys Ser Gin Thr Cys Tyr Pro Asp Ilie Pro Asp Pro Tyr Lys Ser Ser Ile Leu Ser Leu 785 Ile Lys Phe Lys Giu 790 Asn Pro His Leu Ile Ile Met Asn 795 Ser Lys Pro Giu Val1 Asp Cys Ilie Pro Ala Ile Giu Val Gly Thr 815 Lys Ilie Gin Thr Lys Pro 835 Phe 820 Leu Gly Thr Arg Ser Leu Thr Glu 830 Asn His Ser Asn Tyr Leu Tyr Leu Pro Thr Giu Gly Pro 850 Gly Pro Cys Ilie Phe Giu Asn Leu Thr 860 Tyr Asn Gin Ala Ala 865 Ser Asp Ser Gly Ser 870 Cys Gly His Val Pro 875 Val Ser Pro Lys Pro Ser Met Leu Gly 885 Leu Met Thr Ser Pro 890 Giu Asn Val Leu Lys Ala 895 Leu Giu Lys Thr Ser Leu 915 Tyr Met Asn Ser Leu 905 Gly Giu Ile Pro 910 Phe Giy Asp Asn Tyr Val Ser Leu Ala Ser Pro Lys Asp 930 Ser Leu Pro Thr Asn Pro Val Glu Ala Pro His Cys Ser Giu Tyr 945 Lys Met Gin Met Val Ser Leu Arg Ala Leu Pro Pro Pro 960 Thr Giu Asn Ser Leu Ser Ser Ile Leu Leu Asp Pro Giy Giu 975 His Tyr Cys <210> 8 <211> 918 <212> PRT <213> Homo sapiens <400> 8 Met 1 Leu Thr Leu Gin 5 Thr Trp Vai Val Gin 10 Ala Leu Phe Ile Phe Leu Thr Thr Giu Pro Giu Ser Thr Gly Giu Leu Leu Asp Pro Cys Gly Tyr Ile Ser Ala Vai Cys Pro Val Val Gin His Ser Asn Phe Vai Leu Lys Giu Lys Cys Asp Tyr Phe His Asn Aia Asn Tyr Val Trp Lys Thr His Phe Thr Ile Lys Glu Gin T'yr Ile Ile Asn Arg Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser Leu Asn Ile Gin 100 Leu Thr Cys Asn Ile Leu Thr Phe Gly Gin Leu Giu 105 110 Gin Asn Val 115 Tyr Gly Ilie Thr Ile 120 Ile Ser Gly Leu Pro 125 Pro Glu Lys Pro Lys 130 Asn Leu Ser Cys Val Asn Giu Giy Lys 140 Lys Met Arg Cys Glu 145 Trp Asp Giy Gly Glu Thr His Leu Thr Asn Phe Thr Lys Ser Giu Trp Thr His Lys Phe Asp Cys Lys Ala Lys Arg 175 Asp Thr Pro Asn Ile Giu 195 Ser Cys Thr Val Tyr Ser Thr Val Tyr Phe Val 190 Lys Val Thr Val. Trp Val Giu Ala 200 Giu Asn Ala Leu Gly 205 Ser Asp 210 His Ile Asn Phe Asp 215 Pro Val Tyr Lys Val 220 Lys Pro Asn Pro Pro 225 His Asn Leu Ser Val1 230 Ile Asn Ser Giu Leu Ser Ser Ile Lys Leu Thr Trp Thr Asn 245 Pro Ser lie Lys 250 Ser Val Ile Ile Leu Lys 255 Tyr Asn Ile Pro Pro Glu 275 Gin 260 Tyr Arg Thr Lys Asp 265 Ala Ser Thr Trp Ser Gln Ile 270 Val Gin Asp Asp Thr Ala Ser Arg Ser Ser Phe Thr 285 Leu Lys 290 Pro Phe Thr Glu Val Phe Arg Ile Arg 300 Cys Met Lys Glu Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser 305 310 Glu 315 Glu Ala Ser Gly Thr Tyr Glu Asp Arg 325 Pro Ser Lys Ala Ser Phe Trp Tyr Lys Ile 335 Asp Pro Ser Thr Leu Pro 355 Thr Gin Gly Tyr Thr Val Gin Leu Val Trp Lys 350 Tyr Glu Val Pro Phe Glu Ala Gly Lys Ile Leu Asp 365 Thr Leu 370 Thr Arg Trp Lys His Leu Gin Asn Tyr 380 Thr Val Asn Ala Thr 385 Lys Leu Thr Val Asn 390 Leu Thr Asn Asp Arg 395 Tyr Leu Ala Thr Leu 400 Thr Val Arg Asn Val Gly Lys Ser Asp Ala Ala Val Leu 410 Thr Ile 415 Pro Ala Cys Phe Pro Lys 435 Phe Gin Ala Thr His 425 Pro Val Met Asp Leu Lys Ala 430 Pro Arg Glu Asp Asn Met Leu Trp 440 Val Glu Trp Thr Thr 445 Ser Val 450 Lys Lys Tyr Ile Leu 455 Glu Trp Cys Val Ser Asp Lys Ala Pro Cys Ile Thr Asp 465 Trp 470 Gin Gin Glu Asp Thr Val His Arg Thr 480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485 49049 495 Thr Pro Val Tyr Leu Lys 515 Ala Asp Gly Pro Gly 505 Ser Pro Glu Ser Ile Lys Ala 510 Arg Thr Lys Gin Ala Pro Pro Lys Gly Pro Thr Lys Val 530 Gly Lys Asn Glu Vai Leu Glu Trp Gin Leu Pro Val Asp 545 Val Gin Asn Gly Phe 550 Ile Arg Asn Tyr Thr 555 Ile Phe Tyr Arg Ile Ile Gly Asn Giu 565 Thr Ala Val Asn Val1 570 Asp Ser Ser His Thr Giu 575 Tyr Thr Leu Ala Ala Tyr 595 Ser Leu Thr Ser Asp 585 Thr Leu Tyr Met 590 Phe Thr Phe Thr Asp Giu Gly Gly 600 Lys Asp Giy Pro Thr Thr 610 Pro Lys Phe Ala Gly Giu Ile Giu Ile Val Val Pro Val1 625 Cys Leu Ala Phe Leu 630 Leu Thr Thr Leu Leu 635 Giy Vai Leu Phe Phe Asn Lys Arg Asp 645 Leu Ile Lys Lys His 650 Ile Trp Pro Asn Val Pro 655 Asp Pro Ser Arg His Asn 675 Ser His Ile Ala Gin 665 Trp Ser Pro His 670 Gly Asn Phe Phe Asn Ser Lys Asp 680 Gin Met Tyr Ser Thr Asp 690 Val Ser Val Val Ile Giu Ala Asn Lys Lys Pro Phe Pro 705 Giu Asp Leu Lys Ser 710 Leu Asp Leu Phe Lys 715 Lys Giu Lys Ile Thr Giu Giy His Ser 725 Ser Gly Ile Gly Gly 730 Ser Ser Cys Met Ser Ser 735 Ser Arg Pro Thr Ser Ser 755 Ser Ile 740 Ser Ser Ser Asp 745 Giu Asn Glu Ser Ser Gin Asn 750 Giy Tyr Arg Thr Val Gin Tyr Ser 760 Thr Val Val His Ser 765 His Gin 770 Val Pro Ser Val Gin 775 Val Phe Ser Arg Giu Ser Thr Gin Leu Leu Asp Ser Giu 790 Giu Arg Pro Giu Leu Gin Leu Val Asp 800 His Vai Asp Giy Giy 805 Asp Giy Ile Leu Arg Gin Gin Tyr Phe Lys 815 Gin Asn Cys Arg Ser Lys 835 Ser 820 Gin His Giu Ser Pro Asp Ile Ser 830 Vai Arg Leu Gin Val Ser Ser Val 840 Asn Giu Giu Asp Lys Gin 850 Gin Ile Ser Asp His 855 Ile Ser Gin Ser Cys Giy Ser Gly 860 Ala Phe Gly Pro Gin Giy 880 Met 865 Lys Met Phe Gin Giu 870 Val Ser Ala Aia Thr Giu Giy Gin Vali 885 Giu Arg Phe Giu Vai Giy Met Giu Ala Aia 895 Thr Asp Giu Giy Giy Tyr 915 Gly 900 Met Pro Lys Ser Leu Pro Gin Thr Vai Arg Gin 910 Met Pro Gin <210> 9 <211> 836 <212> PRT <213> Homo sapiens <400> 9 Met Ala Arg Leu Giy Asn Cys Ser Leu Thr Trp Aia Ala Leu Ile Ile Leu Leu Leu Pro Gly Ser Leu Giu Giu 25 Cys Giy His Ile Ser Vai Ser Cys Ile Ile Ala Pro Ile Val His Leu Gly Asp 40 Pro Ile Thr Ala Ser Lys Gin Asn Cys Ser His ASP Pro Giu Pro Gin Ile Leu Trp Arg Giy Giy Ala Giu Leu Thr Gin Giu Ser Gin Pro Gly Giy Arg Gin Arg Leu Ser Ile Ile Thr Leu His Leu Asn His Thr Gin Ala Phe Leu Asp Gin Val 115 Cys Cys Leu Asn Gly Asn Ser Leu 110 Pro His Asn Giu Leu Arg Ala Gly 120 Tyr Pro Pro Ala Leu Ser 130 Cys Leu Met Asn Leu Thr Thr Ser Ser Leu Ile Cys Gin Trp 135 140 His Leu Pro Thr Ser Phe Thr Leu Lys Ser 155 160 Giu 145 Pro Gly Pro Giu Thr 150 Phe Lys Ser Arg Asn Cys Gin Thr Gin 170 Gly Asp Ser Ile Leu Asp 175 Cys Val Pro Leu Leu Leu 195 Asp Gly Gin Ser His 185 Cys Cys Ile Pro Arg Lys His 190 Giu Asn Ala Tyr Gin Asn Met Ile Trp Val Gin Leu Gly 210 Thr Ser Met Ser Gin Leu Cys Leu Pro Met Asp Vai Val 225 Lys Leu Giu Pro Pro 230 Met Leu Arg Thr ASP Pro Ser Pro Giu 240 Ala Ala Pro Pro Gin 245 Ala Giy Cys Leu Leu Cys Trp Giu Pro Trp 255 Gin Pro Giy Leu His Ile Asn Gin Lys Cys Glu Leu Arg His Lys Pro 270 Pro Leu Giu Gin Arg Gly 275 Glu Ala Ser Trp Leu Val Gly Pro Ala Leu 290 Gin Tyr Glu Leu Gly Leu Leu Pro Thr Ala Tyr Thr 305 Trp Gin Ile Arg Cys Ser Pro Ser Leu 325 Arg Trp Pro Leu Gly His Trp Ser Glu Leu Arg Thr Thr 330 Glu Arg Ala Pro Thr Val 335 Arg Leu Asp Gin Leu Phe 355 Thr 340 Trp Trp Arg Gin Arg 345 Gin Leu Asp Pro 350 Gly Arg Ile Trp Lys Pro Val Leu Glu Glu Asp Ser 365 Gin Gly 370 Tyr Val Val Ser Arg Pro Ser Gly Ala Gly Ala Ile Leu 385 Pro Leu Cys Asn Thr Glu Leu Ser Thr Phe His Leu Pro 400 Ser Glu Ala Gin Glu 405 Val Ala Leu Val Tyr Asn Ser Ala Gly Thr 415 Ser Arg Pro Thr Arg Leu 435 Thr 420 Pro Val Val Phe Ser 425 Giu Ser Arg Gly Pro Ala Leu 430 His Ala Met Ala Arg 440 Asp Pro His Ser Leu Trp Val Gly 445 Trp Glu 450 Pro Pro Asn Pro Pro Gin Gly Tyr Val 460 Ile Giu Trp Gly Leu 465 Gly Pro Pro Ser Ser Asn Ser Asn Thr Trp Arg Met Glu 480 Gin Asn Gly Arg Thr Gly Phe Leu Lys Glu Asn Ile Arg Pro 495 Phe Gin Leu Gu Ile Ile Val Thr 505 Pro Leu Tyr Gin Asp Thr Met 510 0 z Gly Pro Ser 515 Gin His Val Tyr Ala 520 Tyr Ser Gin Glu AlPrSe Met 525 Ala Pro Ser His Ala 530 Pro Glu Leu His Leu 535 Lys His Ile Gly Lys 540 Thr Trp Ala Gin Leu 545 Giu Trp Val Pro Pro Pro Giu Leu Gly 555 Lys Ser Pro Leu His Tyr Thr Ile Trp Thr Asn Ala Asn Gin Ser Phe Ser Ala 575 Ile Leu Asn Ala Ser Leu 595 Ala 580 Ser Ser Arg Gly Val Leu His Gly Leu Giu Pro 590 Ala Gly Ala Tyr His Ile His Met Ala Ala Ser Thr Asn 610 Ser Thr Val Leu Thr 615 Leu Met Thr Leu Pro Giu Gly Ser Giu 625 Leu His Ile Ile Leu 630 Gly Leu Phe Gly Leu Leu Leu Leu Leu 635 Cys Leu Cys Gly Ala Trp Leu Cys Cys 650 Ser Pro Asn Arg Lys Asn 655 Pro Leu Trp, Trp Val Pro 675 Ser Val Pro Asp Ala His Ser Ser Leu Gly Ser 670 Pro Gly Leu Thr Ile Met Giu Asp Ala Phe Gin Leu 685 Gly Thr 690 Pro Pro Ile Thr Leu Thr Val Leu Glu Giu Asp Glu Lys 700 Glu Thr Cys Gly Leu Lys 705 Pro Val Pro Trp Ser His Asn Ser Pro Thr Leu Val Gin 725 Thr Tyr Val Leu Gin 730 Gly Asp Pro Arg Ala Val 735 Ser Thr Gin Gin Ser Gin Ser Gly 745 Thr Ser Asp Gin Vai Leu TIyr 750 Gly Gin Leu 755 Leu Gly Ser Pro Thr 760 Ser Pro Gly Pro Gly 765 His Tyr Leu Arg Cys 770 Asp Ser Thr Gin Pro 775 Leu Leu Ala Gly Leu 780 Thr Pro Ser Pro Ser Tyr Glu Asn Leu 790 Trp Phe Gin Ala Ser 795 Pro Leu Gly Thr Leu 800 Pro Leu 815 Val Thr Pro Ala Ser Gin Giu Asp Cys Val Phe Gly Leu Asn Phe Pro 820 Leu Leu Gin Gly Arg Val His Gly Met Giu Ala 830 Leu Gly Ser Phe 835 <210> <211> 7 <212> PRT <213> Homo sapiens <400> Trp Lys Ser Thr Ser Val Lys <210> 11 <211> <212> PRT <213> Homo sapiens <400> 11 Glu Gly Lys Leu Leu Pro Ala Ile Pro Val Leu Ser Ala Leu Lys 1 5 10 <210> <211> <212> 12 726 PRT <213> Mus musculus <400> 12 Met Lys Pro Leu Gly 1 5 Tip Ala Phe Ser Phe Val Asn Ala Gly Met Trp Thr Leu Ala Leu Leu Cys Lys Phe 25 Ser Leu Ala Val Leu Pro Thr Lys Pro Giu Asn Ile Ser Cys Val Phe Tyr Phe Asp Arg Asn Leu Thr Trp Arg Pro Glu 40 Giu Thr Asn Asp Ser Tyr Ile Val Cys Thr Lys 55 Thr Leu Thr Tyr Ser Tyr Gly Lys Ser Asn Tyr Ser Asp Asn Ala Giu Ala Ser Tyr Phe Pro Arg Ser Cys Ala Met Pro Pro Asp Ile Cys Ser Val Asp Ile Thr 115 Val Gin Ala Gin Giy Asp Gly Lys 110 Giu Pro Pro Tyr Trp His Leu Ser Ile Ala Lys Ile Ilie 130 Leu Ser Val Asn Ile Cys Asn Arg Phe Gin Ile Gin Trp 145 Lys Pro Arg Giu Lys 150 Thr Arg Gly Phe Pro Leu Val Cys Met 155 Arg Phe Arg Thr Val1 165 Asn Ser Ser Arg Trp 170 Thr Glu Val Asn Phe Giu 175 Asn Cys Lys Tyr Val Leu 195 Val Cys Asn Leu Thr Gly Leu Gin Ala 185 Phe Thr Giu 190 Tyr Trp Ser Ala Leu Arg Phe Arg 200 Phe Asn Asp Ser Arg 205 Lys Trp 210 Ser Lys Giu Giu Thr 215 Arg Val Thr Met Giu 220 Giu Val Pro His Leu Asp Leu Trp Arg 230 Ile Leu Giu Pro Asp Met Asn Gly Arg Lys Val Arg Leu Trp Lys Lys Arg Gly Ala Pro Val Leu 255 Giu Lys Thr Gly Tyr His Ile Tyr Phe Ala Giu Asn Ser Thr 270 Asn Leu Thr 275 Giu Ile Asn Asn Ile Thr Thr Gin Gin 280 Tyr 285 Glu Leu Leu Leu Met 290 Ser Gin Aia His Ser 295 Vai Ser Val Thr Phe Asn Ser Leu Giy 305 Lys Ser Gin Giu Ile Leu Arg Ile ASP Val His Giu Thr Phe Gin Tyr Ile 325 Lys Ser Met Gin Ala Tyr Ile Aia Giu Pro Leu 335 Leu Val Vai Asn Trp Gin Ser Ser Ile Pro Aia Val Asp Thr Trp Ile 350 Val Giu Trp 355 Leu Pro Giu Aia Aia Met Ser Lys Phe Pro Aia Leu Ser 360 365 Trp, Giu 370 Ser Val Ser Gin Thr Asn Trp Thr Giu Gin Asp LYS Lys Pro Phe Thr Tyr Asn Ile Ser Tyr Pro Val Leu Giy 400 His Arg Vai Gly Giu 405 Pro Tyr Ser Ile Gin Ala Tyr Aia Lys 410 GJlu Giy 415 Thr Pro Leu Giy Pro Giu Thr Arg Vai Giu Asn Ile 425 430 Arg Asn Gly Thr Ala Thr 435 Ile Thr Trp Lys Glu 440 Ile Pro Lys Ser Ala 445 Phe Ile 450 Asn Asn Tyr Thr Phe Tyr Gin Ala Gly Giy Lys Glu Ser Lys Thr Val Asn 470 Ser His Ala Leu Gin Cys Asp Leu Giu 475 Ser 480 Leu Thr Arg Arg Thr 485 Ser Tyr Thr Val Val Met Ala Ser Thr Arg 495 Ala Gly Gly Asn Gly Val Arg Ile 505 Asn Phe Lys Thr Leu Ser Ile 510 Ser Val Phe Giu Val Val Leu Leu Thr Ser Leu Val Giy Gly Gly Leu 520 Leu Leu 530 Leu Ser Ilie Lys Thr 535 Val Thr Phe Gly Arg Lys Pro Asn Arg 545 Leu Thr Pro Leu Cys Pro Asp Val Asn Pro Ala Giu Ser Leu Ala Thr Trp Leu Gly Asp Giy 565 Lys Lys Ser Asn Met Lys 575 Glu Thr Gly Pro Val Pro 595 Asn 580 Ser Gly Asn Thr Giu 585* Asp Val Val Leu 590 Phe Glu Asn Ala Asp Leu Ile Lys Leu Val Val Asn 605 Phe Leu 610 Giu Val Val Leu Thr 615 Giu Glu Ala Gly Gly Gin Ala Ser Ile Leu Giy Gly Glu 625 Asp Giy Pro Pro Gly 645 Asn Giu Tyr Val Ser Pro Ser Arg Pro 640 Lys Ser Phe Lys Pro Ser Ile Leu Thr Giu 655 Val Ala Ser Giu 660 Asp Ser His Ser Cys Ser Arg Met Ala Asp Giu 670 Ala Tyr Ser 675 Giu Leu Ala Arg Pro Ser Ser Ser Cys Gin Ser Pro 685 Gly Leu 690 Ser Pro Pro Arg Giu 695 Asp Gin Ala Gin Asn 700 Pro Tyr Leu Lys Ser Val Thr Thr Arg 710 Giu Phe Leu Val His 715 Glu Asn Ile Pro Glu 720 His Ser Lys Gly Giu Val 725 <210> 13 <211> 252 <212> PRT <213> Homo sapiens <400> 13 Met 1 Lys Leu Ser Pro 5 Gin Pro Ser Cys Val1 10 Asn Leu Gly Met Met Trp Thr Trp Ala Ala Leu Pro Trp Met Leu Pro Ser Leu Cys Lys Phe Ser Leu Ala Tyr Tyr Arg Ala Lys Pro Glu Ile Ser Cys Val Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys Thr Ser Tyr Thr Gln Tyr Thr Val Lys Thr Tyr Ala Phe Glu Lys His Asp Cys Thr Thr Asn Ser Ser Thr Ser Glu Arg Ala Ser Cys Ser Phe Phe Leu Pro Ala Glu Asn 115 Arg 100 Ie Thr Ile Pro Asn Tyr Thr Ile 110 Tyr Trp Arg Gly Asp Gly Val Ile 120 Lys Ser His Met Thr 125 Leu Glu 130 Asn Ile Ala Lys Glu Pro Pro Lys Ile 140 Phe Arg Val Lys Val Leu Gly Ile Arg Met Ile Gin Glu Trp Ile'Lys Pro 160 Giu Leu Ala Pro Ser Ser Asp Leu Tyr Thr Leu Arg Phe Arg 175S Thr Val Asn Lys Asp Lys 195 Thr Ser Trp Met Val Asn Phe Ala 190 Pro Phe Thr Asn Gin Thr Tyr Asn 200 Leu Thr Gly Leu Giu Tyr 210 Val Ile Ala Leu Arg 215 Cys Ala Val Lys Glu Ser Lys Phe 220 Glu Giu Giu Gly Trp Lys 240 Ser 225 Asp Trp Ser Gin Lys Met Gly Met Thr 235 Leu Leu Pro Ala Ile Pro Val Leu Ser Thr Leu Val 245 250 <210> 14 <211> 652 <212> PRT <213> Homo sapiens <400> 14 Met Lys Leu Ser Pro Gin Pro Ser Cys Asn Leu Gly Met Met Trp Thr Trp Ala Ala Leu Pro Leu Trp Met Leu Pro Ser Leu Cys Lys Phe Ser Leu Ala Ala Lys Pro Giu Asn 40 Ile Ser Cys Val Tyr Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Ser Pro Gly Lys Giu Thr Ser Tyr Thr Gin Tyr Thr Val Lys Thr Tyr Ala Phe Gly Giu Lys His Asp Cys Thr Thr Asn Ser Thr Ser Giu Arg Ala Ser Cys Ser Phe Phe Leu Pro Ala Glu Asn 115 Ile Thr Ile Pro Asn Tyr Thr Ile 110 Tyr Trp Arg Gly Asp Gly Val Lys Ser His Met Leu Giu 130 Asn Ile Ala Lys Thr 135 Glu Pro Pro Lys Ile Phe Arg Val Lys Pro 145 Val Leu Gly Ile Arg Met Ile Gin Ile Glu Trp Ile Lys 155 Glu Leu Ala Pro Ser Ser Asp Leu Lys 170 Tyr Thr Leu Arg Phe Arg 175 Thr Val Asn Lys Asp Lys 195 Thr Ser Trp Met Giu 185 Val Asn Phe Ala 190 Pro Phe Thr Asn Gin Thr Tyr Leu Thr Gly Leu Glu Tyr 210 Val Ile Ala Leu Arg 215 Cys Ala Vai Lys Ser Lys Phe Trp Asp Trp Ser Gin Giu 230 Lys Met Gly Met Thr 235 Giu Giu Glu Ala Cys Giy Leu Giu Trp Arg Val Leu Lys 250 Pro Ala Glu Ala Asp Gly 255 Arg Arg Pro Leu Glu Lys 275 Val1 260 Arg Leu Leu Trp Lys Ala Arg Gly 270 Giu Ser Asn Thr Leu Gly Tyr Ile Trp Tyr Tyr Pro 285 Thr Asn 290 Leu Thr Glu Thr Met 295 Asn Thr Thr Asn Gin Leu Giu Leu Leu Gly Giy Giu Ser 310 Phe Trp Val Ser Met 315 Ile Ser Tyr Asn Leu Gly Lys Ser Val Ala Thr Leu Arg 330 Ile Pro Ala Ile Gin Giu 335 Lys Ser Phe Gin 340 Cys Ile Giu Val Gin Ala Cys Val Ala Giu Asp 350 Gin Leu Val 355 Val Lys Trp Gin Ser Ala Leu Asp Val1 365 Asn Thr Trp met Ile 370 Giu Trp Phe Pro Asp 375 Val Asp Ser Giu Thr Thr Leu Ser Giu Ser Val Ser Gin 390 Aia Thr Asn Trp Thr 395 Ile Gin Gin Asp Leu Lys Pro Phe Cys Tyr Asn Ile Ser 410 Val Tyr Pro met Leu His 415 Asp Lys Val Val Pro Ser 435 Gly 420 Giu Pro Tyr Ser Gin Ala Tyr Ala Lys Giu Gly 430 Gly Val Lys Giu Gly Pro Glu Lys Val Giu Asn Ile 445 Thr Val 450 Thr Ilie Thr Trp Lys 455 Glu Ile Pro Lys Ser 460 Giu Arg Lys Gly Ile 465 Ile Cys Asn Tyr Thr 470 Ile Phe Tyr Gin Ala Glu Gly Gly 475 Gin Tyr Gly Leu Lys Gly 480 Phe Ser Lys Thr Asn Ser Ser Ile Leu 490 Giu Ser 495 Leu Lys Arg Ala Gly Gly 515 Lys 500 Thr Ser Tyr Ilie Gin Val Met Ala 510 Leu Ser Phe Thr Asn Gly Thr Ile Asn Phe Lys Ser Vai 530 Phe Glu Ile Ilie Ile Thr Ser Leu Ile Gly Gly Gly Leu Leu 545 Ile Leu Ilie Ilie Leu Thr Val Ala Tyr Giy Leu Lys Lys Pro 550 555 Lys Leu Thr His Leu 565 Cys Trp Pro Thr Val1 570 Pro Asn Pro Ala Giu Ser 575 Ser Ilie Aia Lys Giu Ser 595 Trp His Gly Asp Asp 585 Phe Lys Asp Lys 590 Leu Lys Pro Asp Asp Ser Vai Thr Giu Asp Arg Cys Ser 610 Thr Pro Ser Asp Leu Vai Ile Asp Lys Leu Val Vai 620 Giu Aia Arg Thr Asn Gly Asn Val Leu Giu Ile Phe Thr Giy 640 Gin Giu Lys Gin Arg Arg Giy Lys Glu Trp Asp 650 <210> <211> 662 <212> PRT <213> Homo sapiens <400> Met Lys Leu Ser Pro Gin Pro Ser Cys Vai Asn Leu Giy Met Met Trp Thr Trp Ala Ala Leu Pro Leu Trp Met Leu Pro Ser 25 Leu Cys Lys Phe Ser Leu Ala Tyr Tyr Arg Ala Lys Pro Glu Ile Ser Cys Val Lys Asn Leu Thr Cys Thr Ser Pro Gly Lys Thr Ser Tyr Thr Gin Tyr Thr Val Lays Arg 70 Thr Tyr Ala Phe Gly Giu Lys His Asp Cys Thr Thr Asn Ser Ser Thr Ser Glu Arg Ala Ser Cys Ser Phe Phe Leu Pro Ala Glu Asn 115 Ile Thr Ile Pro Asn Tyr Thr Ile 110 Tyr Trp Arg Gly Asp Gly Val Ile 120 Lys Ser His Met Thr 125 Leu Giu 130 Asn Ile Ala Lys Glu Pro Pro Lys Phe Arg Val Lys Pro 145 Val Leu Gly Ile Arg Met Ile Gin Ile Glu Trp Ile Lys 155 Pro 160 Glu Leu Ala Pro Val 165 Ser Ser Asp Leu Tyr Thr Leu Arg Phe Arg 175 Thr Val Asn Thr Ser Trp Met Val Asn Phe Ala Lys Asn Arg 190 Pro Phe Thr Lys Asp Lys 195 Asn Gin Thr Tyr Asn Leu Thr Gly Leu 200 Gin 205 Glu Tyr 210 Val Ile Ala Leu Arg 215 Cys Ala Val Lys Giu 220 Ser Lys Phe Trp Ser 225 Asp Trp Ser Gin Lys Met Gly Met Giu Glu Glu Ala Pro 240 Cys Gly Leu Giu Leu 245 Trp Arg Val Leu Pro Ala Giu Ala Asp Gly 255 Arg Arg Pro Leu Glu Lys 275 Val1 260 Arg Leu Leu Trp Lys Ala Arg Gly Ala Pro Val 270 Giu Ser Asn Thr Leu Gly Tyr Ile Trp Tyr Tyr Thr Asn 290 Leu Thr Glu Thr Asn Thr Thr Asn Gin 300 Gin Leu Glu Leu His 305 Leu Gly Gly Glu Ser 310 Phe Trp Val Ser Met 315 Ile Ser Tyr Asn Lev Gly Lys Ser Val Ala Thr Leu Ile Pro Ala Ile Gin Glu 335 Lys Ser Phe Gin Leu Val 355 Gin 340 Cys Ile Glu Val Gin Ala Cys Val Ala Glu Asp 350 Asn Thr Trp Val Lys Trp Gin Ser Ala Leu Asp Met Ile 370 Giu Trp Phe Pro Val Asp Ser Giu Thr Thr Leu Ser Trp 385 Leu Giu Ser Val Ser Lys Pro Phe Trp 405 Gin 390 Aia Thr Asn Trp Thr 395 Ile Gin Gin Asp Cys Tyr Asn Ile Ser 410 Vai Tyr Pro Met Leu His 415 Asp Lys Val Val Pro Ser 435 Gly Giu 420 Pro Tyr Ser Ile 425 Gin Ala Tyr Aia Lys Glu Gly 430 Gly Val Lys Glu Gly Pro Giu Lys Val Glu Asn Thr Val 450 Thr Ile Thr Trp Giu Ile Pro Lys Glu Arg Lys Giy Ile 465 Ile Cys Asn Tyr Thr 470 Ile Phe Tyr Gin Ala Giu Giy Gly Lys 475 Phe Ser Lys Thr Val1 485 Asn Ser Ser Ile Leu 490 Gin Tyr Gly Leu Giu Ser 495 Leu Lys Arg Ala Gly Giy 515 Thr Ser Tyr Ile Val1 505 Gin Val Met Ala Ser Thr Ser 510 Leu Ser Phe Thr Asn Gly Thr Ile Asn Phe Lys Ser Val 530 Phe Giu Ile Ile Leu 535 Ile Thr Ser Leu Gly Giy Gly Leu Leu 545 Lys Ile Leu Ile Ile Leu Thr His Leu 565 Thr Vai Ala Tyr Leu Lys Lys Pro Cys Trp Pro Thr Pro Asn Pro Ala Giu Ser 575 Ser Ile Ala Lys Glu Ser 595 Trp His Gly Asp Asp 585 Phe Lys Asp Lys 590 Leu Lys Pro Asp Asp Ser Vai Thr Giu Asp Arg Cys Ser 610 Thr Pro Ser Asp Lys 615 Leu Val Ile Asp Leu Val Val Asn 625 Gin Giy Asn Val Leu Giu Asn Asn Leu 645 Gin 630 Giu Ile Phe Thr Glu Ala Arg Thr Giy Gly Glu Lys Asn 650 Gly Thr Arg Ile Leu Ser 655 Ser Cys Pro Thr Ser Ile 660 <210> 16 <21i> 344 <212> PRT <213> Homno sapiens <400> 16 Asn Pro Lys Asn Glu 1 5 Ser Ser Glu Asn Ile 10 Arg Giu Arg Leu Ser Leu Pro Ser Thr Leu Gin Gin Asn Phe Giy 25C Thr Leu Asn Phe Trp Phe Gin Arg Ser His Asn Phe His Asn Leu Thr Thr Glu Glu Gly Pro Ser Thr Pro Ile Gly Thr Leu Lys Pro Gly Leu Val Ile Ala Val Arg Lys Leu Leu Met Asn Asp Ser 70 Asp Gin Gly Gly Lys Leu Thr Thr Gly Phe Thr Pro Gin Gin Leu Ala Asn Thr Asn Gin Gly Leu Ser Arg Cys Leu Ser Ile Lys Leu 115 Phe Lys Lys Val Arg Ala Met Leu Met Met Lys 110 His Ile Trp Lys Arg Ilie Thr Asn 120 Ile Asn Cys Ser Gly 125 Val Glu 130 Pro Ala Thr Ile Phe 135 Lys Met Giy Met Ile Ser Ile Tyr Cys 145 Gin Ala Ala Ile Asn Cys Gin Pro Lys Leu His Phe Tyr 160 Lys Asn Gly Ile Lys 165 Giu Arg Phe Gin Thr Arg Ile Asn Lys Thr 175 Thr Ala Arg Tyr Cys Thr 195 Trp Tyr Lys Asn Leu Glu Pro His Ala Ser Met 190 Leu Ile Cys Ala Glu Cys Pro His Phe Gin Giu Gly Lys 210 Asp Ile Ser Ser Gly 215 Phe Cys Ile Thr Asp 220 Tyr Ser Gin Lys Pro 225 Ser Gln Val Leu Ala 230 Gly Gly Pro Leu Ser 235 Pro Asn Pro Thr Gly Asn Val Giu Pro Pro Asp Ile Pro Asp Giu Val Thr 250 Cys Val 255 Ilie Tyr Giu Leu Thr Tyr 275 Ser Gly Asn met Thr 265 Cys Thr Trp Asn 270 Ser Leu Giu Ile Asp Thr Lys Tyr 280 Vai Vai His Val Thr Glu Glu 290 Glu Gin Gin Leu Thr Ser Ser Tyr Ile Asn Ile Ser 300 Thr Asp Ser Leu Gin 305 Gly Gly Lys Lys 310 Met Giu Glu Ser Tyr Leu Val Trp Val Gin Ala 315 320 Lys Gin Leu Gin Ile His Leu 330 335 Ala Asn Ala Leu Gly 325 Asp Asp Ile <210> 17 <211> 39 <212> PRT <213> Homno sapi <400> 17 Ile Glu Asp Leu 1 Pro His Giu Arg ens Ser Ile Asn Val Met Ala Ala Asn Ile Leu Glu Thr 5 10 Thr Arg Asp Thr Asn Met Lys Gin Ser Ala Phe Glu Asn Asn Phe Ser Gin Ile Phe Gly Thr Val <210> 18 <211> 12 <212> PRT <213> Homo sapiens <400> 18 Ser Asn Trp Leu Ala Leu Lys Gly Asp Giu Glu Lys 1 5 <210> 19 <211> 2830 <212> DNA <213> Homno sapiens <400> 19 aaagaagaca tgacacagcc aacaagggtg gcagcctggc tctgaagtgg aattatgtgc ttcaaacagg ttgaaagagg gaaacagtct tttcctgctt ccagacatga atcaggtcac tattcaatgg gatgcagtaa tagcccttta catactcttc agctggtgtc atggaggaat tacaaatata aactgctctg gccacatctg ggtagaacca gccacaattt ttaagatggg 120 180 240 tatgaatatc tttttataaa tcggctttgg tcccaaacat agatattcct ctggaatgct agagacagaa attacaaggt agagtcaaaa ttccagggct tctatatatt aatggcatca tataaaaact tttcaagaga gatgaagtaa gggaagc tca gaagagcaac ggcaagaagt caac tgcaaa gagactataa aacaacaatt gaaaaggttt gaatgttaaa gccaaacatt gccttggagt agcattccaa ccttacttct tatgttgtca aagaagga tc cagcaatgtt gcaggtccta caagcctaca aaactcgcta accccaaatt ttccttaaca aaagcatcct tct tggagaa C tcagtagag agaacagacc gccatctatt actcttggaa gaatttgaca aagtacgtat tcactgtttt catgacacat gacaacagag attctttctt ttattgttaa gtgaaaatgc tatgttgatc gac tacaaga ttcgacaata tcaaattttc cttaaaccac aattttgctt ttaagcctca gaggaaacca C tgct tcctg aatacttatt aagtagagct gccaagcagc aagaaagat t ttctggaacc cactgatatg cctgtgtcat cctacataga agtatctcac acttggtttg ttcacctgga atgctacagt cctgtgaaat ccaattttac t tcaagtgag ttcataaaac ggaattctgg gagacattgg tgat tgggat taccaaagtg tacaggaaaa ccatgattac aggagaatac ctacagttgt tgcc tgaggg cagttgattc tttctgtttc tattaaatca ccatgctttt a tgaa t ttg t t tccacaaaa gtgtggtcaa aattaagaac tcaaatcaca acatgcttct tggaaaagac ttatgaatat cacaaaatac ctcaagctat ggtccaagca tgatatagtg gcccaagacc gagatacaag atatgtgcaa atgtcaagaa acc tgaaaca gctaacagtt act tt tat tg atttaacaga gctttatgaa tagtgaactt agagataaaa aggacccctg atatattcct aagccatctc cttagactca aagtgtgaat aggagaatgc ggaaaatgat ctcctgtttg tattttggaa aatcaatatg tgccaaccaa aggattaata atgtactgca atttcttctg tcaggcaaca gtggtacatg attaacatct gcaaacgcac ataccttctg ataatttatt gctacaacaa cagtcagaat acaggcaaaa gttccccagg gcttccatct ggaatgatcg tcattccgaa gatattccta atgaataata gaaatcttca gagacaagag gatctcaaca agcaataata ggaaataatc tcactaagca agttctcctg tcacccagtg gggatcgtga agccacttca agaaagc tgc ggaaacttca aaacaacagc ctgctgaatg gatatccgcc tgacttgcac tgaagagttt ccactgattc taggca tgga cagccgtcat gggatagtca accaaac ttg tctacttgga ggtactggca tcacatcaaa ctacagggca tctttgctgt ctgggattaa atatgaaaaa attccagtga tcccagaaca actacccgca ctggatataa atgaaattac ccaggttaca acacaatatt acatacaaaa aaactattcc atgaggagtt ataggatttc cttgcaatct 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1860 1920 1980 2040 0 z gaacttgggt tttccctgca atagaaattg aattctgcct ctttttgaaa aaaatgtatt cacatacaaa gggattgctg agtgacattt ttaattttag ggaacatgct tattttttat ctctctattg gaaaattttc cagcactttg ggccaatatg aggtgcttgt ggcagaggtt aaactctgtc tcttcacatg gaccatatga ctgtgctcct ccattcttct tcatggtcac agagtcaac t tgtacagaaa ctttaaaata gtaggc tgag ctgaaaccct aatcccagct gcac tgagc t gacacatgtt taagcatatg accatcacca gcctcatttc acatacaggc atttcctctt gggtaaataa gaatcattag gtaggtggat gtctctacta act tgggagg gagattgtgc ttcatttccc tttcagttct tgtaagaat t ttaaaattag acaaaaacag tattttccct tgcaaaatac gccaggcgtg cacctgaggt aaat tacaaa c tgaggcagg cactgcactc ttggataaat accaatcttg cccgggagct agaattaagg cattatgtgg cattgaaaga ctggtagtaa gtggctcatg caggagttcg aattagccgg agaatcactt cagcctgggc acctaggtag tttccagagt ccatgccttt tcccgaaggt acgcctcatg tgcaaaacag aataaatgct ct tgtaatcc agtccagcct ccatggtggc gaaccaggaa aacaagagca 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2830 <210> <211> 1890 <212> DNA <213> Homo sapiens <400> atgaatcagg tcactattca tgtcatggag gaattacaaa atttttaaga tgggtatgaa ccaaggaaac ttcattttta aataaaacaa cagctcggct tgcactgctg aatgtcccaa tctggatatc cgccagatat aacatgactt gcacctggaa catgtgaaga gtttagagac atctccactg attcattaca gcactaggca tggaagagtc tctgcagccg tcatttccag tattgggata gtcaaacaac atgggatgca tataaactgc tatctctata taaaaatggc ttggtataaa acattttcaa tcctgatgaa tgctgggaag agaagaagag aggtggcaag aaaacaactg ggctgagact aattgaaaag gtaatagccc tctggccaca tattgccaag atcaaagaaa aactttctgg gagacactga gtaacctgtg ctcacctaca caacagtatc aagtacttgg caaattcacc ataaatgcta gtttcctgtg tttacatact tctgggtaga cagcaattaa gatttcaaat aaccacatgc tatgtggaaa t cat tta tga tagacacaaa tcacctcaag tttgggtcca tggatgatat cagtgcccaa aaatgagata cttcagctgg accagccaca gaactgccaa cacaaggatt ttctatgtac agacatttct atattcaggc atacgtggta ctatattaac agcagcaaac agtgatacct gaccataatt caaggctaca acaaaccaaa gaattctact aaaaggtact caggtcacat atctctacag atcgtctttg cgaactggga cctaatatga aataattcca ttcatcccag agagactacc aacactggat aataatgaaa aatcccaggt agcaacacaa cctgacatac agtgaaacta gtgaatgagg ttcaatagga cttggaatgt tggagccaaa ggcagccttg caaaagcatt ggcaccttac ctgttatgtt t taaaagaag aaaacagcaa gtgagcaggt aacacaagcc cgcaaaactc ataaacccca t tact tcc tt tacaaaagca tatttcttgg aaaac tcagt ttccagaaca agttgccatc tttcactctt taaagaatt t cattaagtac gagttcactg ccaacatgac ttctgacaac gtcaattctt gatcttattg tgttgtgaaa cctatatgtt tacagac tac gctattcgac aatttcaaat aacacttaaa tcctaatttt agaattaagc agaggaggaa gaccctgctt tattaatact ggaaaagtag gacaccaatt gtatttcaag ttttttcata acatggaatt agaggagaca tctttgattg ttaataccaa atgctacagg gatcccatga aagaaggaga aatactacag tttctgcctg ccaccagttg gc tt t ttctg ctcatattaa accaccatgc cctgatgaat tattttccac t tacatatgt tgagatgtca aaacacc tga c tgggc taac ttggactttt ggatatttaa agtggcttta aaaatagtga ttacagagat atacaggacc ttgtatatat agggaagcca attccttaga tttcaagtgt a tcaaggaga t tttggaaaa ttgtCtcctg aaaatatttt gcaacagtca agaaacaggc aacagttccc agttgcttcc at tgggaatg cagatcattc tgaagatatt acttatgaat aaaagaaatc cc tggagaca tcctgatctc tctcagcaat ctcaggaaat gaattcacta atgcagttct tgattcaccc tttggggatc ggaaagccac 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1890 <210> 21 <211> 629 <212> PRT <213> Homo sapiens <400> 21 Met Asn Gin Val Thr 1 5 Ile Gin Trp Asp Val Ile Ala Leu Tyr Ile Leu Phe Ser His Ilie Trp Ser Ile Tyr Trp Cys His Gly Gly Ile 25 Thr Asn Ile Asn Met Asn Ile Val Glu Pro Ala Thr 40 Ile Phe Lys Met Gly Cys Gin Ala Ile Lys Asn Cys Gin Pro Arg Lys Leu His Phe Tyr Lys Asn Gly 70 Ilie Lys Giu Arg Phe Gin Ile Thr Asn Phe Leu Giu Arg Asn Lys Thr Thr Ary Leu Trp Tyr Lys Pro His Ala Ser Met Leu Ilie Cys 115 Cys Thr Ala Giu Cys 105 Pro Lys His Phe Asp Ile Pro Gly Lys Asp Ile Ser 120 Ser Gly Tyr Pro Pro 125 Asp Giu 130 Val Thr Cys Val Tyr Giu Tyr Ser Gly Asn Met Thr 140 Thr Lys Tyr Vai Cys Trp Asn Ala Gly Lys 150 Leu Thr Tyr Ile Val1 160 His Val Lys Ser Leu 165 Glu Thr Giu Giu Gin Gin Tyr Leu Thr Ser 175 Ser Tyr Ile Leu Vai Trp 195 Ile Ser Thr Asp Leu Gin Gly Gly 190 Glu Ser Lys Val Gin Aia Ala Ala Leu Gly Met Gin Leu 210 Gin Ilie His Leu Asp 215 Asp Ile Val Ile Ser Ala Ala Val Ser Arg Ala Giu Thr 230 Ile Asn Ala Thr Val1 235 Pro Lys Thr Ile Ile 240 Tyr Trp Asp Ser Thr Thr Ile Glu Val Ser Cys Giu Met Arg 255 Tyr Lys Ala Asn Phe Thr 275 Thr Asn Gin Thr Asn Val Lys Giu 270 Pro Asn Ile Tyr Val Gin Gin Ser 280 Giu Phe Tyr Leu Giu 285 Lys Tyr 290 VJal Phe Gin Val Arg 295 Cys Gin Giu Thr Gly 300 Lys Arg Tyr Trp Gin 305 Pro Trp Ser Ser Phe Phe His Lys rhr 315 Pro Giu Thr Val Gin Val Thr Ser Lys 325 Ala Phe Gin His Asp 330 Thr Trp Asn Ser Gly Leu 335 Thr Val Ala Asp Ile Gly 355 Ile Ser Thr Gly His 345 Leu Thr Ser Asp 350 Met Leu Ser Leu Leu Leu Gly Met 360 Ile Vai Phe Ala Val1 365 Ilie Leu 370 Ser Leu Ile Gly Phe Asn Arg Ser Arg Thr Gly Ile Lys 385 Arg Arg Ile Leu Leu Ile Pro Lys Leu Tyr Giu Asp Pro Asn Met Lys Asn 405 Ser Asn Vai Val Lys Met Leu Gin Glu 410 Asn Ser 415 Giu Leu Met Asn Asn Ser Ser Giu 425 Gin Val Leu Tyr Val Asp Pro 430 Lys Pro Thr Met Ile Thr 435 Asp Tyr Lys 450 Giu Ile Lys Glu Ile Phe Ilie Pro Glu 440 His 445 Lys Giu Asn Thr 455 Giy Pro Leu Giu Thr 460 Arg Asp Tyr Pro Gin 465 Asn Ser Leu Phe Asp 470 Asn Thr Thr Val Tyr Ile Pro Asp Leu 480 Asn Thr Giy Tyr Lys 485 Pro Gin Ile Ser Phe Leu Pro Giu Giy Ser 495 His Leu Ser Val Asp Ser 515 Asn 500 Asn Asn Giu Ile Ser Leu Thr Leu Lys Pro Pro 510 Lys His Pro Leu Asp Ser Giy Asn Pro Arg Leu Asn Phe 530 Aia Phe Ser Vai Ser Ser Vai Asn Ser 535 Leu 540 Ser Asn Thr Ile Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn Gin Giy Giu Cys Ser Ser 550 555 Pro Asp Ile Gin Asn 565 Ser Val Glu Giu Thr Thr Met Leu Leu Giu 575 Asn Asp Ser Glu Phe Val 595 Pro 580 Ser Glu Thr Ile Glu Gln Thr Leu Leu Pro Asp 590 Pro Ser Ile Ser Cys Leu Giy Ile 600 Val Asn Giu Giu Leu 605 Asn Thr 610 Tyr Phe Pro Gin Asn 615 Ile Leu Glu Ser Phe Asn Arg Ile Ser 625 Leu Leu Giu Lys <210> 22 <2ii> 1698 <212> DNA <213> Homo sapiens <400> 22 atgaatcagg tcactattca tgtcatggag gaattacaaa atttttaaga ccaaggaaac aa taaaacaa. tgcactgctg tctggatatc aacatgactt catgtgaaga. atctccactg gcac taggca tctgcagccg tattgggata acaaaccaaa. aaattctact aaaaggtact. tgggtatgaa ttcattttta cagc tcggc t aatgtcccaa cgccagatat gcacctggaa gtttagagac attcattaca tggaagagtc tcatttccag gtcaaacaac cttggaatgt tggagccaaa ggcagccttg atgggatgca tataaactgc tatctctata. taaaaatggc ttggtataaa. acattttcaa tcctgatgaa tgctgggaag agaagaagag aggtggcaag aaaacaactg ggctgagact aat tgaaaag taaagaattt cat taagtac gagttcactg gtaatagccc tctggccaca tattgccaag atcaaagaaa aactttctgg gagacactga. gtaacctgtg ctcacctaca. caacagtatc aagtacttgg caaat tcacc ataaatgcta gtttcctgtg gacaccaatt gtatttcaag ttttttcata tttacatact tctgggtaga. cagcaattaa gatttcaaat aaccacatgc tatgtggaaa tcatttat~ga. tagacacaaa. tcacctcaag tttgggtcca tggatgatat cagtgcccaa aaatgagata ttacatatgt tgagatgtca aaacacctga cttcagctgg accagccaca gaactgccaa cacaaggatt ttctatgtac agacatttct. atattcaggc atacgtggta ctatattaac agcagcaaac agtgatacct gaccataat e caaggc taca gcaacagtca agaaacaggc aacagggat t aaaagaagga aacagcaatg gagcaggtcc cacaagccta caaaactcgc aaaccccaaa acttccttaa caaaagcatc t ttc ttggag aactcagtag ccagaacaga ttgccatcta tcactcttgg tc ttat tgt t ttgtgaaaat tatatgttga cagactacaa tattcgacaa tttcaaattt cact taaacc ctaattttgc aattaagcct aggaggaaac ccctgcttcc ttaatactta aaaagtag aataccaaag gc tacaggaa tcccatgatt gaaggagaat tactacagtt tctgcctgag accagttgat tttttctgtt catattaaat caccatgctt tgatgaattt ttttccacaa tggctttatg aatagtgaac acagagataa acaggacccc gtatatattc ggaagccatc tccttagact tcaagtgtga caaggagaat ttggaaaatg gtCtcctgtt aatattttgg aagatattcc ttatgaataa aagaaatctt tggagacaag ctgatctcaa tcagcaataa caggaaataa attcactzaag gcagttctcc attcacccag tggggatcgt aaagccactt taatatgaaa taattccagt catcccagaa agac tacccg cactggatat taatgaaatt tcccaggtta caacacaata tgacatacaa tgaaactatt gaatgaggag caataggatt 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1698 <210> 23 <211> 565 <212> PRT <213> Homo sapiens <400> 23 Met Asn Gin Val Thr Ile Gin Trp Asp Ala Val Ile Ala Leu Tyr Ile Leu Phe Ser His Ile Trp Trp Cys His Gly gly Thr Asn Ile Asn Met Asn Ile Val Glu Pro Ala Ile Phe Lys Met Gly Ser Ile Tyr Cys Gin Ala Ala Ile Lys Asn Cys Pro Arg Lys Leu His Phe Tyr Lys Asn Gly 70 Ile Lys Glu Arg Gin Ilie Thr Arg Ile Asn Lys Thr Thr Arg Leu Trp Tyr Asn Phe Leu Giu Pro His Ala Ser Met Cys Thr Ala Glu Pro Lys His Phe Gin Glu Thr 110 Leu Ile Cys 115 Gly Lys Asp Ile Ser Gly Tyr Pro As iPr Asp Ile Pro Asp Giu 130 Val Thr Cys Val Ile 135 Tyr Giu Tyr Ser Gly Asn Met Thr Cys 140 Thr Lys Tyr Val Val Thr 145 Trp Asn Ala Gly Lys 150 Leu Thr Tyr Ile Asp 155 His Vai Lys Ser Giu Thr Giu Giu Giu 170 Gin Gin Tyr Leu Thr Ser 175 Ser Tyr Ile Leu Val Trp, 195 Ile Ser Thr Asp Ser 185 Leu Gin Gly Giy Lys Lys Tyr 190 Giu Ser Lys Val Gin Ala Ala Ala Leu Gly Met Gin Leu 210 Gin Ile His Leu Asp Ile Vai Ile Ser Ala Ala Val Ile 225 Ser Arg Ala Giu Thr 230 Ile Asn Ala Thr Pro Lys Thr Ile Ile 240 Tyr Trp Asp Ser Thr Thr Ile Giu Lys 250 Val Ser Cys Giu Met Arg 255 Tyr Lys Ala Asn Phe Thr 275 Thr Asn Gin Thr Trp 265 Asn Val Lys Giu Phe Asp Thr 270 Pro Asn Ile Tyr Val Gin Gin Ser 280 Giu Phe Tyr Leu Lys Tyr 290 Val Phe Gin Val Cys Gin Giu Thr Lys Arg Tyr Trp Gin 305 Pro Trp Ser Ser Leu 310 Phe Phe His Lys Pro Giu Thr Gly Lys Arg Arg Ile Pro Asn Met Lys 340 Leu Leu Ile Pro Trp Leu Tyr Giu Asp Ile 335 Asn Ser Asn Val Val Lys Met Leu Gin 345 Giu Asn Ser 350 Giu Leu Met 355 Asn Asn Asn Ser Ser 0Th Gin Val Leu Tyr 365 Val Asp Pro Met Ile 370 Thr Giu Ilie Lys Giu 375 Ile Phe Ile Pro Giu 380 His Lys Pro Thr Asp 385 Tyr Lys Lys Giu Thr Gly Pro Leu Giu 395 Thr Arg Asp Tyr Pro 400 Gin Asn Ser Leu Phe Asp Asn Thr Thr 405 Vai Tyr Ilie Pro Asp Leu 415 Asn Thr Giy His Leu Ser 435 Tyr 420 Lys Pro Gin Ile Asn Phe Leu Pro Giu Gly Ser 430 Asn Asn Asn Giu Thr Ser Leu Thr Leu Lys Pro Pro 445 Val Asp 450 Ser Leu Asp Ser Asn Asn Pro Arg Gin Lys His Pro Asn Phe Ala Phe Ser 465 Phe Leu Gly Giu Leu 485 Val 470 Ser Ser Vai Asn Ser .475 Leu Ser Asn Thr Ser Leu Ile Leu Gin Giy Giu Cys Ser Ser 495 Pro Asp Ile Asn Asp Ser 515 Asn Ser Val Giu Giu 505 Giu Thr Thr Met 510 Leu Pro Asp Pro Ser Giu Thr Ile 520 Pro Giu Gin Thr Leu 525 Giu Phe 530 Vai Ser Cys Leu Giy 535 Ile Vai Asn Giu Leu Pro Ser Ile Thr Tyr Phe Pro Asn Ilie Leu Glu His Phe Asn Arg Ile 560 Ser Leu Leu Giu Lys 565 24 <211> i071 <212> DNA <2i3> Homno sapiens <400> 24 atgaatcagg tgtcatggag atttttaaga ccaaggaaac aa taaaacaa tgcactgctg tctggatatc aacatgactt catgtgaaga atctccactg gcac taggca tctgcagccg tattgggata acaaaccaaa gaattctact aaaaggtact caggtcacat atctctacag tcac tattca gaat tacaaa tgggtatgaa ttcattttta cagctcggct aatgtcccaa cgccagatat gcacctggaa gtttagagac attcattaca tggaagagtc tcatttccag gtcaaacaac cttggaatgt tggagccaaa ggcagccttg caaaagcatt ggcaccttac atgggatgca tataaac tgc tatctctata taaaaatggc ttggtataaa acattttcaa tcctgatgaa tgc tgggaag agaagaagag aggtggcaag aaaacaactg ggctgagact aattgaaaag taaagaattt cattaagtac gagttcactg ccaacatgac ttctggatta gtaatagccc tctggccaca tat tgccaag atcaaagaaa aactttctgg gagacac tga gtaacctgtg c tcacc taca caacagtatc aagtacttgg caaat tcacc ataaatgcta gtttcctgtg gacaccaatt gtatttcaag ttttttcata acatggaatt aaagaaggat tttacatact tc tgggtaga cagcaattaa ga t ttcaaa t aaccacatgc ta tgtggaaa tcatttatga tagacacaaa tcacctcaag tttgggtcca tggatgatat cagtgcccaa aaatgagata ttacatatgt tgagatgtca aaacacctga ctgggc taac cttattgtta cttcagctgg accagccaca gaac tgccaa cacaaggatt ttctatgtac agacatttct atattcaggc atacgtggta ctatattaac agcagcaaac agtgatacct gaccataatt caaggctaca gcaacagtca agaaacaggc aacagttccc agttgcttcc a 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1071 <210> <211> 356 <212> PRT <213> Homo sapiens <400> Met Asn Gin Val Thr 1 5 Ile Gin Trp Asp Ala 10 Val Ile Ala Leu Tyr Ile Leu Phe Ser His Ile Trp Trp Cys His Gly Gly Thr Asn Ile Asn Cys Ser Gly Met Asn Ile Val Glu Pro Ala Ile Phe Lys Met Ser Ile Tyr Cys Gin Ala Ala 55 Ile Lys Asn Cys Gin Pro Arg Lys Leu His Phe Tyr Lys Asn Gly 70 Ile Lys Giu Arg Phe Gin Ile Thr Asn Phe Leu Giu Arg Asri Lys Thr Thr Ala Arg Leu Trp Tyr Lys 90 Pro His Ala Ser Met Tyr 100 Cys Thr Ala Giu Pro Lys His Phe Gin Giu Thr 110 Leu Ilie Cys Giy Lys Asp Ilie Ser Ser Gly Tyr Pro Pro Asp Ile Pro Asp Giu 130 Val Thr Cys Val Ile 135 Tyr Giu Tyr Ser Gly 140 Asn Met Thr Cys Thr 145 Trp Asn Ala Gly L~ys 150 Leu Thr Tyr Ile Asp 155 Thr Lys Tyr Vai His Val Lys Ser Giu Thr Giu Glu Gin Gin Tyr Leu Thr Ser 175 Ser Tyr Ile Leu Val Trp 195 Asn 180 Ile Ser Thr Asp Leu Gin Gly Gly Lys Lys Tyr 190 Giu Ser Lys Val Gin Ala Ala Asn 200 Ala Leu Gly Met Giu 205 Gin Leu 210 Gin Ile His Leu Asp 215 Asp Ile Val Ile Ser Ala Ala Val Ile 225 Tyr Ser Arg Ala Giu Trp Asp Ser Gin 245 Ile Asn Ala Thr Pro Lys Thr Ile Ile 240 Thr Thr Ile Giu Val Ser Cys Giu Met Arg 255 Tyr Lys Ala Asn Phe Thr 275 Thr 260 Thr Asn Gin Thr Asn Val Lys Giu Phe Asp Thr 270 Pro Asn Ile Tyr Val Gin Gin Giu Phe Tyr Leu Lys Tyr 290 Val Phe Gin Vai Cys Gin Giu Thr Giy 300 Lys Arg Tyr Trp Gin Pro Trp, Ser Ser Leu Phe Phe His Lys Thr Pro Giu Thr Vai Pro 315 320 Gin Val Thr Ser Lys 325 Ser Ile 340 Ala Phe Gin His Thr Trp Asn Ser Gly Leu 335 Thr Val Ala Ser Thr Gly His Leu Thr Ser Giy*Leu Lys Giu 345 350 Giy Ser Tyr Cys 355 <210> 26 <2i1> 384 <212> PRT <213> Homo sapiens <400> 26 Met Asn Gin Val 1 Thr 5 Ile Gin Trp Asp Val Ile Ala Leu Tyr Ile Leu Phe Ser His Ile Trp Cys His Gly Gly Thr Asn Ile Asn Met Asn Ile Val Giu Pro Ala Thr 40 Ile Phe Lys Met Giy Ser Ile Tyr Cys Gin Ala Ala 55 Ile Lys Asn Cys Gin Pro Arg Lys Leu His Phe Tyr Lys Asn Ile Lys Giu Arg Gin Ile Thr Arg Asn Lys Thr Thr Ala Arg Leu Trp Tyr Asn Phe Leu Giu Pro His Ala Ser Met Tyr 100 Cys Thr Ala Giu Pro Lys His Phe Gin Giu Thr 110 Leu Ile Cys 115 Gly Lys Asp Ile Ser Gly Tyr Pro Asp Ile Pro Asp Giu 130 Val Thr Cys Vai Ile 135 Tyr Giu Tyr Ser Giy Asn Met Thr Cys 140 Thr Lys Tyr Val Val Thr Trp Asn Ala Gly 145 Leu Thr Tyr Ile His Val Lys Ser Leu 165 Giu Thr Glu Giu Glu 170 Gin Gin Tyr Leu Thr Ser 175 Ser Tyr Ile Asn Ile Ser Thr Asp 180 Leu Gin Gly Gly Lys Lys Tyr 190 Giu Ser Lys Leu Val Trp 195 Val Gin Ala Ala Asn 200 Ala Leu Giy Met Gin Leu 210 Gin Ile His Leu Asp Ile Val Ile Ser Ala Ala Val Ser Arg Ala Giu Ile Asn Ala Thr Pro Lys Thr Ile Tyr Trp Asp Ser Gin 245 Thr Thr Ilie Giu Lys 250 Val Ser Cys Giu Met Arg 255 Tyr Lys Ala Asn Phe Thr 275 Thr Asn Gin Thr Asn Val Lys Giu Phe Asp Thr 270 Pro Asn Ile Tyr Val Gin Gin Giu Phe Tyr Leu Glu 285 Lys Tyr 290 Val Phe Gin Val Arg 295 Cys Gin Giu Thr Gly 300 Lys Arg Tyr Trp Gin 305 Pro Trp Ser Ser Pro 310 Phe Phe His Lys Thr 315 Pro Giu Thr Val Gin Val Thr Ser Ala Phe Gin His Asp 330 Thr Tr-p Asn Ser Gly Leu 335 Thr Val Ala Ile Ser Thr Gly Leu Thr Ser Asp Asn Arg Gly 350 Asp Ile Gly 355 Leu Leu Leu Gly Met Ilie Val Phe Ala Val Met Leu Ser 360 365 Ile Leu 370 Ser Leu Ile Gly Ile 375 Phe Asn Arg Ser Phe 380 Pro Asn Trp Asp <210> 27 <211> 644 <212> PRT <213> Mus musculus <400> 27 Met 1 Ser His Leu Thr Leu Gin Leu His 5 Val Ile Ala Leu Tyr Val Leu Phe Arg Trp Cys His Gly Gly Thr Ser Ile Asn Cys Ser Gly Ile Asn Val Asp Met Trp Val Glu Pro Gly Giu Ile Phe Gin Met Gly Ser Ile Tyr Cys Gin Giu Leu Lys His Cys Pro Arg Asn Leu Tyr Phe Tyr Lys Asn Phe Lys Giu Giu Asp Ile Thr Arg Asn Arg Thr Thr Ala Arg Ile Trp Tyr Gly Phe Ser Glu Pro His Ala Tyr Met Leu Ile Cys 115 His 100 Cys Thr Ala Glu Pro Giy His Phe 110 Asp Ala Pro Gly Lys Asp Ile Ser Gly His Pro Pro 125 Ser Asn 130 Leu Thr Cys Val Ile 135 Tyr Giu Tyr Ser Gly 140 Asn Met Thr Cys Thr 145 Trp Asn Thr Gly Lys 150 Pro Thr Tyr Ile Thr Lys Tyr Ile His Vai Lys Ser Giu Thr Giu Giu Glu 170 Gin Gin Tyr Leu Ala Ser 175 Ser Tyr Vai Leu Val Trp 195 Ile Ser Thr Asp Ser 185 Leu Gin Gly Ser Arg Lys Tyr 190 Asn Ser Gin Val Gin Ala Val Asn 200 Ser Leu Gly Met Gin Leu 210 His Val His Leu Asp Ile Val Ile Ser Ala Ser Ile Ile 225 Ser Arg Ala Glu Thr 230 Thr Asn Asp Thr Val Pro Lys Thr Ile 235 Tyr Trp Lys Ser Lys 245 Thr met Ile Giu Val Phe Cys Giu Met Arg 255 Tyr Lys Thr Asn Phe Thr 275 Thr 260 Thr Asn Gin Thr Trp 265 Ser Vai Lys Giu 270 Pro Asp Ser Tyr Vai Gin Gin Ser 280 Giu Phe Tyr Leu Giu 285 Lys Tyr 290 Gin Pro 305 Val Phe Gin Val Trp Ser Ser Pro 310 Arg 295 Cys Gin Glu Thr Lys Arg Asn Trp Phe Val His Gin Thr 315 Ser Gin Giu Thr Lys Arg Asn Trp Pro Trp Ser Ser Phe Val His Gin Thr Ser 335 Gin Thr Val Met Giu Met 355 Gin Val Thr Ala Ser Ser His Giu 350 Ala Ser Gly Leu Ser Ala Thr Ile 360 Phe Arg Gly His Pro 365 Asn His 370 Gin Asp Ilie Gly Leu 375 Leu Ser Gly Met Vai 380 Phe Leu Ala Ile Leu Pro Ile Phe Leu Ile Gly Ilie Phe 395 Asn Arg Ser Leu Ilie Gly Ile Lys Lys Vai Leu Leu Ile Pro Lys Trp,Leu Tyr 415 Giu Asp Ile Giu Lys Ser 435 Pro 420 Asn Met Glu Asn Ser Asn Val Ala Lys Leu Leu Gin 425 430 Asn Ala Ser Giu Gin Ala Leu Tyr 445 Val Phe Giu Asn Val Asp 450 Pro Val Leu Thr Ile Ser Giu Ile Pro Leu Glu His Pro Thr Asp Tyr Pro Tr As Tyr Gu Giu Arg Leu GyLeLeGu Gly Leu Leu Glu Arg Asp Cys Pro Leu 485 Leu Asn 500 Gly Met Leu Ser Thr Ser 490 Ser Ser Val Val TPyr 495 Ile Pro Asp Pro Gly Gly 515 Thr Gly Tyr Lys 505 Pro Gin Val Ser Asn Val Pro 510 Pro Thr Ser Asn Leu Phe Ilie Asn 520 Arg Asp Giu Arg Asp 525 Leu Giu 530 Thr Thr Asp Asp His 535 Phe Ala Arg Leu Lys Thr Tyr Pro Asn 540 Asn Lys Thr Leu Ile Gin Phe Ser Ala Ser Met Ala Leu Leu Asp Giu Leu Leu Val Leu Asn Gin 570 Giy Giu Phe Asn Ser Leu 575 Asp Ile Lys Asp Ser Pro 595 Asn 580 Ser Arg Gin Giu Giu 585 Thr Ser Ile Vai 590 Ser Asp Giu Ser Giu Thr Ilie Pro 600 Ala Gin Thr Leu Leu 605 Phe Val 610 Ser Cys Leu Ala Ile 615 Gly Asn Giu Asp Pro Ser Ile Asn Ser 625 Tyr Phe Pro Gin Val Leu Giu Ser Phe Ser Arg Ile Leu Phe Gin Lys <210> 28 <211> 2181 <212> DNA <213> Mus musculus <400> 28 atgaagcctc tgggtgtgaa cgctggaata atgtggacct tggcactgtg ggcattctct ttcctctgca aattcagcct ggcagtcctg ccgactaagc cagagaacat ttcctgcgtc ttttacttcg acagaaatct gacttgcact tggagaccag agaaggaaac caatgatacc agctacattg tgactttgac ttactcctat ggaaaaagca attatagtga caatgctaca gaggcttcat attcttttcc ccgttcctgt gcaatgcccc cagacatctg cagtgttgaa 0 z gtacaagctc tccatagcaa Ctccagatac cggttcagaa gtctgcaacc ttcaatgact gaagttccac aggaaggtgc ggctaccaca accacccagc tttaattctc accttccagt tggcaaagct atgtcgaagt gagcaagata caccgagttg ggtcctgaga attcctaaga ggtggaaaag c tgacacgaa aacggggtga acatctctag agaaagccaa agtttagcca tctgggaaca aagctggtag ggtcaggcga gacggtcccc gactccaca ccttcgtctt ccgtatttga aaaatggaga aaaccgaacc aatggaaacc ctgtcaacag tcacaggact caagatat tg atgtcctgga gattgctgtg tacagtactt agtatgaac t ttggcaagtc acattaagag ccattcctgc tccctgccct aac taaaacc gagagccgta ccagggtgga gtgctaggaa aactctccaa ggacctctta gaataaact t ttggaggagg accggt tgac catggctcgg cagaagacgt tgaactttga gcattttggg cagggaaaag gcacgtgttc cc tgtcagag aaaattcggt tggtaaagtt acctataatt gcgtgaaaag tagccgctgg tcaggctttc gagcaagtgg cctgtggaga gaagaaggca tgcagagaac gcttctgatg ccaagagacc catgcaggcc ggtggacact t tcc tgggaa tttcacatgc ttcaatccaa gaacatcggt tggatttatc gactgttaac tactg t ttgg caagacattg ccttcttcta tcccctgtgt agatggtttc ggtcctaaaa gaattttctg aggagaagcg ttttaaagag cagaatggcg tccagggcta gacaaccagg aaa tc tgaca ttaagtgtga ac tcgtgggt acggaagtca acagaatatg agcaaagaag attctggaac agaggagccc agcactaacc agccaggcac atcctgagga tacatagccg tggatagtgg tctgtgtctc tataatatat gcttatgcca c tgaggacag aacaattaca tctcatgccc gtca tggcca tcaatcagtg cttagcatca tgtcctgatg aagaagtcaa ccatgtcccg gaagtagttt aatgagtatg ccttccattt gacgaggcgt tcgcctcccc gaatttcttg tcacatattg atccaatttg ttcctttagt attttgaaaa tcctggctct aaaccagagt cagcagacat ccgtcttgga tcacagagat actctgtgtc tcccagatgt agcccctgtt agtggctccc aggtcacgaa cagtgtatcc aagaaggaac ccacgatcac tgcagtgtga gcaccagagc tgtttgaaat aaacagtgac ttcccgaccc atatgaagga tccccgcgga tgacagagga tgacc tcccc taactgaggt actcagaatt gtgaagacca tgcatgagaa gcatttaatc taatagaatg atgcatgctt ctgtaaacag acgattcagg gactatggag gaacggagac gaaaacattt aaacaacatc cgtgacttct ccatgagaag ggtggtgaac agaagctgcc ctggaccatc agtgttggga tccattaaaa a tggaaggag ccaagctgaa cctggagtct tggaggtacc tgtccttcta ttttggcctc tgctgaaagt gactggaaac tctcattgac agctgggaag gtctaggccc tgcttctgaa ggccaggcag agctcagaat tgtcccagag 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 cacagcaaag gagaagtctg a 28 2181 <210> 29 <211> 1935 <212> DNA <213> Mus musculus <400> 29 atgagtcacc tcacacttca tgtcacggag gaatcacaag atttttcaga tgggcataaa ccaaggaatc tttactttta aatagaacaa cagctcggat tgcactgctg aatgtcctgg tctggacatc caccggatgc aacatgacat gcacctggaa catgtgaaga gtttggagac atctccactg actcactgca tccctaggca tggagaactc tctgcgtcca tcatttccag tactggaaaa gcaaaactat acaaaccaaa cgtggagtgt gaattctacc tggagccaga aaaagaaact ggcagccttg aaaagaaact ggcagccttg caggttacag caaaatcatc ttcagaggac atcctgcttc ttcttggcca tcatgttgcc ataggaatta aaaggaaagt aatatggaaa atagcaatgt aatgccagtg agcaggccct cccctggaac acaaacccac agagactgtc ctctaggaat aacactggat acaaacccca gctgcatgtg tataaactgc tgtttctata taaaaatggc ttggtataaa tcattttcaa ccccagcaat cactgggaag agaagaagaa aggcagcagg acaacaacta ggctgagact gattgagaaa taaagaattt cagcaagtat gagttccccc gagttccccc ccacgaacct aggtaatcat gattttttct tttactgatg tgcaaaatta gtatgtggat agattacaaa gttgtctacc ggtttcaaat gtgatagccc tctggtgaca tattgccaag ttcaaagaag ggcttttcgg gagacactga ctgacatgtg cctacctaca caacaatatc aagtatttgg cacgtccatc acaaacgata gtattctgtg gacgccaatt gtatttcaag tttgtccacc ttcgtccacc cagaagatgg caagacattg ctgattggga atcccaaagt t tacaggaaa cctgtcctta gaagaaaggc agttcttctg gttcctcctg tgtgggttga aagcccttaa aatttgatat aacctcatgc tttgtgggaa tcatttatga tagataccaa ttgcctcaag tatgggtcca tggatgatat ctgtacccaa agatgagata tcacatatgt tgcgatgtca aaacttccca aaacttccca agatgctcag gac tt ttgtc tatttaacag ggctttatga aaagtgtatt cagaga taag tcacaggact ttgtgtatat gaggaaacc t cttcagatgg gcctggtgaa gcac tgccga cacaaggat t ctatatgcat agacatttcc atactcaggc gtatattgtg ctatgttaag agctgtcaat agtgatacct gaccatagtt caaaacaaca acagcagtca agaaac tgg t agaaactggt gacagtttcc tgctacaatc gggaatggtc atcacttcga agatattcct tgagaatgat tgaaatctct ccttgagaca tcctgacctc tttcattaac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 agagatgaaa acatatccca cttgatgaat tcaagacagg gcgcagac tc ccatctatta ctcttccaaa gagaccctac atcccttgag accacagatg accactttgc cagattgaaa acttccaatt ttctgcttca agtatggctt tactaaacaa aacactaatt tgtgcctcgt tttaaatcaa ggagaattca attctcttga cataaaaaac aggaaaccag catcgttttg caaagtgact cacccagtga aactatccca tgttgtctga tgaatttgtc tcctgtttgg caattgggaa tgaagacttg attcttactt tccacagaac gttttggaaa gccatttcag tagaatttca agtag 1620 1680 1740 1800 1860 1920 1935