WO1999019491A2

WO1999019491A2 - Human dnax interleukin-40 (dil-40)

Info

Publication number: WO1999019491A2
Application number: PCT/US1998/020932
Authority: WO
Inventors: J. Fernando Bazan; Robert A. Kastelein
Original assignee: Schering Corp
Current assignee: Merck Sharp and Dohme LLC
Priority date: 1997-10-14
Filing date: 1998-10-13
Publication date: 1999-04-22
Anticipated expiration: 2000-04-14
Also published as: AR016145A1; AU9786398A; ZA989343B; WO1999019491A3

Abstract

Purified genes encoding cytokines from a mammal, reagents related thereto including purified polypeptides, specific antibodies, and nucleic acids encoding this molecule are provided. Methods of using said reagents and diagnostic kits are also provided.

Description

HUMAN DNAX INTERLEUKIN-40 (DIL-40)

FIELD OF THE INVENTION

The present invention pertains to compositions related to proteins which function in controlling biology and physiology of mammalian cells, e.g., cells of a mammalian system. In particular, it provides purified genes, proteins, antibodies, and related reagents useful, e.g., to regulate activation, development, differentiation, and function of various cell types, including hematopoietic or neural cells.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host .

For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the "immune network". Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes , and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines , cytokines , or monokines , play a critical role in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which will lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, e.g., immune system disorders . Some of these factors are hematopoietic growth factors, e.g., granulocyte colony stimulating factor (G-CSF) . See, e.g., Thomson (ed. 1994) The Cytokine Handbook (2d ed. ) Academic Press, San Diego; Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating Factors Cambridge University Press; and Aggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response . The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents .

Cell lineages especially important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal) , and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T- cells) making up the immune network. These lymphocytes interact with many other cell types . In addition, the neurotrophins and their receptors exhibit similarities structurally and otherwise to the cytokines and their receptors. See, e.g., Ip and Yancopoulos (1996) Annual Rev. Neurosci. 19:491-515; Ip and Yancopoulos (1995) Progress in Brain Research

105:189-195; Patterson (1994) Proc . Nat ' 1 Acad. Sci. USA 91:7833-7835; Patterson and Nawa (1993) Cell 72:123-137; and Davis and Yancopoulos (1993) Current Opinion in Neurology 3:20-24. The cilliary neurotrophic factor (CNTF) exhibits a wide range of biological activities, but mice lacking the gene exhibit' relatively few problems. In addition, a substantial proportion of the Japanese population is homozygous for a null mutation of CNTF, but these individuals appear normal even in old age. These observations suggest that CNTF is not crucial for development, and may have a related counterpart.

Moreover, ablation of the identified receptor for CNTF resulted in a dramatic phenotype . See, e.g., Ip and Yancopoulos (1996) Annual Rev. Neurosci. 19:491-515, at 496. These suggest that an as yet undiscovered CNTF- related factor exists which shares the same receptor. This second factor, possibly that described herein, may be critical for normal development of the nervous system and for motor neurons, in particular. Research to better understand and treat various immune disorders has been hampered by the general inability to maintain cells of the immune or neural system in vitro. Immunologists have discovered that culturing these cells can be accomplished through the use of T-cell and other cell supernatants, which contain various growth factors, including many of the lymphokines .

From the foregoing, it is evident that the discovery and development of new lymphokines or neurotrophins could contribute to new therapies for a wide range of degenerative or abnormal conditions which directly or indirectly involve the immune or nervous system and/or hematopoietic or neuronal cells. In particular, the discovery and development of lymphokines which enhance or potentiate the beneficial activities of known lymphokines would be highly advantageous . The present invention provides new interleukin compositions and related compounds , and methods for their use .

SUMMARY OF THE INVENTION The present invention is directed to mammalian, e.g., rodent, canine, feline, primate, DNAX interleukin- 40 (DIL-40) and its biological activities. It includes nucleic acids coding for polypeptides themselves and methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences described herein, and/or by functional assays for growth factor- or cytokine-like activities, e.g., see Thomson The Cytokine Handbook 2d ed. , Academic Press, San Diego, applied to the polypeptides, which are typically encoded by these nucleic acids. Methods for modulating or intervening in the control of a growth factor dependent physiology or an immune response are provided.

The present invention is based, in part, upon the discovery of a new cytokine sequence exhibiting significant sequence and structural similarity to the cytokines. In particular, it provides a primate gene encoding a protein whose predicted mature size is about 193 amino acids. Functional equivalents exhibiting significant sequence homology will be available from other mammalian, e.g., pig, mouse, and rat, and non- mammalian species .

In various polypeptide embodiments, the invention provides: a substantially pure or recombinant DIL-40 polypeptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 2 or 4; a natural sequence DIL-40 of SEQ ID NO: 2 or 4; and a fusion polypeptide comprising DIL-40 sequence. In certain embodiments, the length is at least about 17 amino acids, 21 amino acids; or 25 amino acids. In other embodiments, the DIL-40: comprises a mature sequence of Table 1; or exhibits a post-translational modification pattern distinct from natural DIL-40; or the polypeptide: is from a warm blooded animal selected from a mammal, including a primate; comprises at least one polypeptide segment of SEQ ID NO: 2 or 4; exhibits a plurality of portions exhibiting the identity; is a natural allelic variant of DIL-40; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a mammalian DIL-40; exhibits a sequence over a length of at least about 20 amino acids to mammalian DIL-40; is glycosylated; has a molecular weight of at least 10 kD with natural glycosyla ion; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; or is a deletion or insertion variant from a natural sequence. Preferred embodiments include a composition comprising: a sterile DIL-40 polypeptide; or the DIL-40 polypeptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration. In fusion protein embodiments, the protein can have: mature protein sequence of Table 1; a detection or purification tag, including a FLAG, His6, or Ig sequence; and/or sequence of another cytokine or chemokine.

Kit embodiments include those with a DIL-40 polypeptide, and: a compartment comprising the polypeptide; and/or instructions for use or disposal of reagents in the kit. In binding compound embodiments, the compound may have an antigen binding site from an antibody, which specifically binds to a natural DIL-40 protein, wherein: the DIL-40 is a mammalian protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide of Table 1; is raised against a mature DIL-40; is raised to a purified rodent DIL-40; is immunoselected; is a polyclonal antibody; binds to a denatured DIL-40; exhibits a Kd of at least 30 μM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. Kits containing binding compounds include those with: a compartment comprising the binding compound; and/or instructions for use or disposal of reagents in the kit. Often the kit is capable of making a qualitative or quantitative analysis. Preferred compositions will comprise: a sterile binding compound; or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.

Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding a DIL-40 polypeptide or fusion protein, wherein: the DIL-40 is from a mammal; and/or the nucleic acid: encodes an antigenic peptide sequence of Table 1; encodes a plurality of antigenic peptide sequences of Table 1; exhibits identity over a length of at least 23 nucleotides to a natural cDNA encoding the segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a primate; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding the DIL-40; or is a PCR primer, PCR product, or mutagenesis primer. The invention also provides a cell, tissue, or organ comprising such a recombinant nucleic acid, and preferably the cell will be: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.

Kit embodiments include those with such nucleic acids, and: a compartment comprising the nucleic acid; a compartment further comprising the DIL-40 polypolypeptide; and/or instructions for use or disposal of reagents in the kit. Typically, the kit is capable of making a qualitative or quantitative analysis.

In certain embodiments, the nucleic acid: hybridizes under wash conditions of 30° C and less than 2M salt, or of 45° C and/or 500 mM salt, or 55° C and/or 150 mM salt, to SEQ ID NO: 1; or exhibits identity over at least: 30 nucleotides, 55 nucleotides, or 75 nucleotides, to a primate DIL-40.

The invention embraces a method of modulating physiology or development of a cell or tissue culture cells comprising contacting the cell with an agonist or antagonist of a mammalian DIL-40. The method may include contacting with an antagonist, including a binding composition comprising an antibody binding site which specifically binds a DIL-40.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

I . General The present invention provides amino acid sequences and DNA sequences encoding various mammalian proteins which are cytokines, e.g., which are secreted molecules which can mediate a signal between immune or other cells. See, e.g., Paul (1994) Fundamental Immunology (3d ed. ) Raven Press, N.Y. The full length cytokines, and fragments, or antagonists will be useful in physiological modulation of cells expressing a receptor. It is likely that DIL-40 has either stimulatory or inhibitory effects on hematopoietic or neuronal cells, including, e.g., lymphoid cells, such as T-cells, B-cells, natural killer (NK) cells, macrophages, dendritic cells, hematopoietic progenitors, glail cells, brain cells, etc.^" The proteins will also be useful as antigens, 'e.g., immunogens, for raising antibodies to various epitopes on the protein, both linear and conformational epitopes, in a natural or denatured conformation. DNA sequences were analyzed with detection of various sequences which exhibit characteristics of cytokines . Based upon sequence assembly of such sequences, a cDNA encoding this cytokine was identified from a human cell line. The molecule was designated human DNAX Interleukin-40 (DIL-40) .

The human gene encodes a small soluble cytokine-like protein, of about 193 amino acids. The signal sequence probably is about 15 residues, and would run from the Met to about Thr. See Nielsen, et al. (1997) Prot. Eng. 10:1-9; Table 1; and SEQ. ID. NO: 1 and 2. DIL-40 exhibits structural motifs characteristic of a member of the long chain cytokines, with helix A from about gly29 to about ala44; helix B from about leu93 to about asnlOδ; helix C from about leul29 to about leul41; and helix D from about serl57 to about alal94. Table 1: Nucleic acid (SEQ ID NO: 1) encoding DIL-40 from a human; translated amino acid sequence is SEQ ID NO: 2. Predicted most likely signal cleavage site is indicated, between VRT and SQQ. Sequence indicates that introns are likely, with possible frameshifts, between nucleotides 183/184 and 309/310. Nucleotides at positions 168 and 330 may result from errors.

ATG ACA CAC CTG AGC CTC CTA GGG CCC TTG CCC TGT GTC AGG ACA TCC 48 Met Thr His Leu Ser Leu Leu Gly Pro Leu Pro Cys Val Arg Thr Ser -15 -10 -5 1

CAG CAG CTC CCT GAG ACG CAG CAA GTG ACG ACT CCT GGG AAG AAG CCA 96 Gin Gin Leu Pro Glu Thr Gin Gin Val Thr Thr Pro Gly Lys Lys Pro

5 10 15

GTC TCA GTG GGC AGG CGT GAA GTC AGA GTC CCA GGC ACC GCT CTG GTC 144 Val Ser Val Gly Arg Arg Glu Val Arg Val Pro Gly Thr Ala Leu Val 20 25 30

CCC TCA CTC CTG TCT GTC TCT GTC TCT CCT GCA GCT CCA CAG TAC CAG 192 Pro Ser Leu Leu Ser Val Ser Val Ser Pro Ala Ala Pro Gin Tyr Gin 35 40 45

GGC AGC CCC TTT AGT GAC CCT GGC TTC TCA GCC CCT GAG CTC CAG CTC 240 Gly Ser Pro Phe Ser Asp Pro Gly Phe Ser Ala Pro Glu Leu Gin Leu

50 55 60 65

AGC AGC CTG CCT CCT GCC ACC GCC TTC TTT AAG ACC TGG CAC GCC CTG 288 Ser Ser Leu Pro Pro Ala Thr Ala Phe Phe Lys Thr Trp His Ala Leu

70 75 80

GAT GAC GGG GAA CGG CTG AGC CCC AGA GGG CCA TTG ACC CAG CAC CTC 336 Asp Asp Gly Glu Arg Leu Ser Pro Arg Gly Pro Leu Thr Gin His Leu

85 90 95

CAG CTC GTG GAG GAC GAC CAG AGC GAC CTG AAC CCT GGC AGT CCC ATC 384 Gin Leu Val Glu Asp Asp Gin Ser Asp Leu Asn Pro Gly Ser Pro lie 100 105 110

CTG CCG GCT CAG CTC GGG GCT GCG AGA CTC AGG GCC CAA GGC CCG CTG 432 Leu Pro Ala Gin Leu Gly Ala Ala Arg Leu Arg Ala Gin Gly Pro Leu 115 120 125

GGC AAT ATG GCC GCC ATC ATG ACG GCC CTG GGG CTG CCA ATC CCC CCA 480 Gly Asn Met Ala Ala lie Met Thr Ala Leu Gly Leu Pro He Pro Pro

130 135 140 145

GAA GAG GAT ACT CCA GGG CTT GCT GCC TTT GGG GCC TCG GCC TTT GAG 528 Glu Glu Asp Thr Pro Gly Leu Ala Ala Phe Gly Ala Ser Ala Phe Glu 150 155 160

AGG AAA TGT CGA GGC TAC GTG GTG ACC CGG GAA TAT GGC CAC TGG ACA 576

Arg Lys Cys Arg Gly Tyr Val Val Thr Arg Glu Tyr Gly His Trp^' Thr 165 170 175

GAC CGA GCT GTG AGA GAC TTG GCT CTG CTC AAG GCC AAG TAC TCA GCA 624

Asp Arg Ala Val Arg Asp Leu Ala Leu Leu Lys Ala Lys Tyr Ser Ala 180 185 190

TAG 627

alternative sequences (SEQ ID NO: 3 and 4) resulting from deletion of nucleotides 168 and 330 of SEQ ID NO: 1:

ATG ACA CAC CTG AGC CTC CTA GGG CCC TTG CCC TGT GTC AGG ACA TCC 48 Met Thr His Leu Ser Leu Leu Gly Pro Leu Pro Cys Val Arg Thr Ser -15 -10 -5 1 CAG CAG CTC CCT GAG ACG CAG CAA GTG ACG ACT CCT GGG AAG AAG CCA 96 Gin Gin Leu Pro Glu Thr Gin Gin Val Thr Thr Pro Gly Lys Lys Pro 5 10 15

CCC TCA CTC CTG TCT GTC TCT GTT CTC CTG CAG CTC CAG TAC CAG GGC 192 Pro Ser Leu Leu Ser Val Ser Val Leu Leu Gin Leu Gin Tyr Gin Gly 35 40 45

AGC CCC TTT AGT GAC CCT GGC TTC TCA GCC CCT GAG CTC CAG CTC AGC 240

Ser Pro Phe Ser Asp Pro Gly Phe Ser Ala Pro Glu Leu Gin Leu Ser

50 55 60 65

AGC CTG CCT CCT GCC ACC GCC TTC TTT AAG ACC TGG CAC GCC CTG GAT 288

Ser Leu Pro Pro Ala Thr Ala Phe Phe Lys Thr Trp His Ala Leu Asp

70 75 80 GAC GGG GAA CGG CTG AGC CTT GCC CAG AGG GCC ATT GAC CCA CAC CTC 336 Asp Gly Glu Arg Leu Ser Leu Ala Gin Arg Ala He Asp Pro His Leu 85 90 95

CAG CTC GTG GAG GAC GAC CAG AGC GAC CTG AAC CCT GGC AGT CCC ATC 384 Gin Leu Val Glu Asp Asp Gin Ser Asp Leu Asn Pro Gly Ser Pro He 100 105 110

GGC AAT ATG GCC GCC ATC ATG ACG GCC CTG GGG CTG CCA ATC CCC CCA 480 Gly Asn Met Ala Ala He Met Thr Ala Leu Gly Leu Pro He Pro Pro 130 135 140 145 GAA GAG GAT ACT CCA GGG CTT GCT GCC TTT GGG GCC TCG GCC TTT GAG 528 Glu Glu Asp Thr Pro Gly Leu Ala Ala Phe Gly Ala Ser Ala Phe Glu 150 155 160

AGG AAA TGT CGA GGC TAC GTG GTG ACC CGG GAA TAT GGC CAC TGG ACA 576 Arg Lys Cys Arg Gly Tyr Val Val Thr Arg Glu Tyr Gly His Trp Thr 165 170 175

GAC CGA GCT GTG AGA GAC TTG GCT CTG CTC AAG GCC AAG TAC TCA GCA 624 Asp Arg Ala Val Arg Asp Leu Ala Leu Leu Lys Ala Lys Tyr Ser Ala 180 185 190

TAG 627

Table 2: Comparison of related sequences. Human CNTF is SEQ ID NO: 5; rabbit CNTF is SEQ ID NO: 6; pig CNTF is SEQ ID NO: 7; mouse CNTF is SEQ ID NO: 8; rat CNTF is SEQ ID NO: 9; and chicken CNTF is SEQ ID NO: 10.

CNTF_HUMAN MAFTEHSPLTPHRRDLCSRSI LAR-KIRSDLTALTES CNTF_RABBI MAFMEHSALTPHRRELCSRTIWLAR-KIRSDLTALTES

CNTF_PIG MAFAEHSPLTPHRRDLCSRSIWLAR-KIRSDLTALMEA

CNTF_MOUSE MAFAEQSPLTLHRRDLCSRSIWLAR-KIRSDLTALMES

CNTF_RAT MAFAEQTPLTLHRRDLCSRSIWLAR-KIRSDLTALMES

CNTF_CHICK MAAADTPSATLRHHDLCSRGIRLAR-KMRSDVTDLLDI DIL-40.2 MTHLSLLGPLPCVRTSQQLPETQQVTTPGKKPVSVGRREVRVPGTALVPS

DIL-40 MTHLSLLGPLPCVRTSQQLPETQQVTTPGKKPVSVGRREVRVPGTALVPS

CNTF_HUMAN YVKHQGLNKNINLDSADGMPVASTDQ SELTEAERLQENLQAYRTFHVLL

CNTF_RABBI YVKHQGLNKNINLDSVDGVPMASTDQWSELTEAERLQENLQAYRTFHIML

CNTF_PIG YVKHQGLNENINLDSVDGVPMASTDR SELTEAERLQENLRAYRTFHVML

CNTF_MOUSE YVKHQGLNKNISLDSVDGVPVASTDR SEMTEAERLQENLQAYRTFQGML

CNTF_RAT YVKHQGLNKNINLDSVDGVPVASTDRWSEMTEAERLQENLQAYRTFQGML CNTF_CHICK YVERQGLDASISVAAVDGVPTAAVERWAEQTGTQRLLDNLAAYRAFRTLL

DIL-40.2 LLS VSVLLQL-QYQGSPFSDPGFSAPELQLSSLPPATAFFKT HALD

DIL-40 LLS VSVSPAAPQYQGSPFSDPGFSAPELQLSSLPPATAFFKTWHALD

CNTF_HUMAN A RLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQ

CNTF_RABBI A RLLEDQQVHFTPAEGDFHQAIHTLLLQVAAFAYQ

CNTF_PIG A RLLEDQREHFTPAEDDFHQAIHTIVLQVAAFAYQ

CNTF_MOUSE T KLLEDQRVHFTPTEGDFHQAIHTLTLQVSAFAYQ

CNTF_RAT T KLLEDQRVHFTPTEGDFHQAIHTLMLQVSAFAYQ

CNTF_CHICK A QMLEEQRELLGDTDAELGPALAAMLLQVSAFVYH

DIL-40.2 DGERLSLAQRAIDPHLQLVEDDQSDLNPGSPILPAQLGAARLRAQGPLGN

DIL-40 DGERLSPRG-PLTQHLQLVEDDQSDLNPGSPILPAQLGAARLRAQGPLGN

...*... . . . . * . 19491

12

CNTF_HUMAN IEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQ TVRS

CNTF_RABBI IEELMVLLECNIPPKDADGTPVIGGDG-LFEKKL GLKVLQELSH TVRS

CNTF_PIG LEELMVLLEHKVPPSEADGTPLSVGGGGLFEKKL GLKVLQELSQ TVRS

CNTF_MOUSE LEELMALLΞQKVPEKEADGMPVTIGDGGLFEKKLWGLKVLQELSQ TVRS

CNTF_RAT LEELMVLLEQKIPENEADGMPATVGDGGLFEKKLWGLKVLQELSQ TVRS

CNTF_CHICK LEELLELESRGAPAEEGSEPPAPPRLS-LFEQKLRGLRVLRELAQWAVRS

DIL-40.2 MAAIMTALGLPIPPEE-DTPGLAAFGASAFERKCRGYWTREYGH TDRA

DIL-40 MAAIMTALGLPIPPEE-DTPGLAAFGASAFERKCRGYWTREYGH TDRA

. * . * .

CNTF_HUMAN IHDLRFISSHQTGIPARGSHYIANNKKM

CNTF_RABBI IHDLRVISCHQTGIPAHGSHYIANDKEM

CNTF_PIG IRDLRVISSHQAGVPAHGSHHVAKDKKM

CNTF_MOUSE IHDLRVISSHHMGISAHESHYGAKQM--

CNTF_RAT IHDLRVISSHQMGISALESHYGAKDKQM

CNTF_CHICK VRDLRQLSKHGPGSGAALGLPESQ-^J--

DIL-40.2 VRDLALLKAK YSA

DIL-40 VRDLALLKAK YSA

The structural homology of DIL-40 to related neurotrophins and cytokine proteins suggests related function of this molecule. See, e.g., structure predictions made using, e.g., PHD program: accessed by http://www.embl-heidelberg.de/predictprotein/; or DSC program: accessed by http://bonsai.lif.icnet.uk/bmm/dsc/.

It has been suggested that another CNTFRα ligand exists, which has not yet been identified. See, e.g., Ip and Yancopoulos (1996) Annual Rev. Neurosci. 19:491-515; Ip and Yancopoulos (1995) Progress in Brain Research 105:189-195; and Davis and Yancopoulos (1993) Current Opinion in Neurology 3:20-24. This ligand, e.g., DIL-40, is likely to share many of the biological activities of the CNTFRα signaling, including, e.g., survival of chick ciliary ganglion neurons in vitro (see Adler, et al. (1979) Science 204:1434-1436; Lin, et al . (1989) Science 246:1023-1025; and Stδckli, et al. (1989) J. Cell Biol . 115:447-459); growth factor activity on a wide variety of peripheral and central neurons (see Ip, et al . (1991) J. Phvsiol. 85:123-130; and Manthorpe, et al . , pp. 443-473, in Fallon and Loughlin (eds. 1992) Neuro rophic Factors Academic Press, NY) ; proliferation of sympathetic neurons (see Ernsberger, et al. (1989) Neuron 2:1275-1284); differentiation of developing sympathetic neurons and induction of neuropeptide genes in neuronal cell lines (see Saadat, et al . (1989) J. Cell Biol . 108:1807-1816; Rao, et al. (1992) Dev. Biol. 150:281-293; Symes, et al . (1993) Proc. Nat ' 1 Acad. Sci. USA 90:572-576; and Farm and Patterson (1993) J. Neurochem. 61:1349-1355); survival effects on cultured embryonic motor neurons (see Arakawa, et al . (1990) J. Neurosci. 10:3507-3515; Oppenheim, et al . (1991) Science 251-1616-1618; and Martinou, et al . (1992) Neuron 8:737-744), ^'preganglionic sympathetic neurons (see Blottner', et al . (1989) Neurosci. Lett. 105:316-320), or sensory neurons (see Barbin, et al . (1984) J. Neurochem. 43:1468-1478; and Thaler, et al . (1994) Dev. Biol. 161:338-344); increase survival of cultured hippocampal neurons (see Ip, et al .

(1991) J. Neurosci. 11:3124-3134); protection from degeneration of particular neuronal populations in the CNS (see Hagg, et al . (1992) Neuron 8:145-158; and Hagg and Varon (1993) Proc. Nat ' 1 Acad. Sci. USA 90:6315- 6319) ; and regulation of the voltage gated ion channels in neuroblastoma cells and potentiate release of transmitters (see Lesser and Lo (1995) J. Neurosci. 15:253-261; and Stoop and Poo (1995) Science 263:695- 699) . Non-neuronal cell effects of CNTF include, e.g., differentiation of glial progenitor cells into type-2 astrocytes (see Hughes, et al. (1988) Nature 335:70-73; and Lillien, et al. (1988) Neuron 1:485-494); survival and maturation of oligodendrocytes (see Louis, et al. (1993) Science 259:689-692; and Barres, et al . (1993) Development 118:283-295); induction of acute-phase protein expression in hepatocyes (see Schooltinck, et al .

(1992) FEBS Lett. 314:280-284); and inhibiting differentiation of pluripotent embryonic stem cells (see Conover, et al. (1994) Development 119:559-565).

However, CNTF effects on motor neurons are most studied (see Arakawa, et al . (1990) J. Neurosci. 10:3507-3515; and Wong, et al . (1993) Eur. J. Neurosci. 5:466-474), including inhibition of degeneration of axotomized facial motor neurons and amelioration of motor deficit in mice with neuro uscular dysfunction (see Sendtner, et al. (1990) Nature 345:4401-4403; Sendtner, et al . (1992) Nature 358:502-504). Retrograde axonal transport to motor neurons of CNTF was observed by Curtis, et al. (1993) Nature 365:253-255.

Much evidence suggests that CNTF, and thus DIL-40, is a major factor in the injury response iri the nervous system. Dramatic changes in the level of expression of CNTF occur in many models of neural trauma in the CNS and PNS . Mechanical lesions in the brain resulted in a dramatic increase in CNTF mRNA and protein bordering the wound site (see Ip, et al . (1993) Neuron 10:89-102; and Asada, et al. (1995) J. Neurosci. 40:22-30) which could be localized to reactive astrocytes in the resulting glial scar (see Ip, et al . (1993) Eur. J. Neurosci. 5:25- 33; and Rudge (1992) Eur. J. Neurosci. 4:459-471). While CNTF may act as a trophic factor for damaged neurons at the sites of CNS injury, astrocytes potentially represent an important target site for CNTF after injury. In such a situation, it was shown that the expression of CNTFRα switched from a purely neuronal localization to include cells in the glial scar at the edge of the wound (Rudge, et al, . (1994) Eur. J. Neurosci. 6:693-705) suggesting that astrocytes, as well as fibroblasts, which are prevalent at the wound site, possess functional CNTF receptor complexes, and thus would have the ability to respond to CNTF localized to that area after trauma. In the PNS, high levels of CNTF mRNA and protein were localized within the Schwann cells of the sciatic nerve (Stδckli, et al. (1991) J. Cell Biol. 115:447-459; Rende, et al. (1992) Glia 5:25-32; and Friedman, et al . (1992) Neuron 9:295-305. The CNTF expressed by the Schwann cells is sequestered within the cytoplasm, presumably because CNTF lacks a signal sequence for secretion. Schwann cells may release CNTF by direct cell injury, e.g., disintegration.

Changes in levels of CNTF are a marker of neuronal injury in the light damage model with the retina. LaVail, et al . (1992) Proc. Nat ' 1 Acad. Sci. USA

89:11249-11253. A dramatic increase in the level of CNTF expression was coincidental with degeneration of photoreceptor cells .

Because it is likely that the DIL-40 is the second ligand for the CNTFRα, it will exhibit a positive signal in at least some of the above assays. See Ip and Yancopoulos (1996) Annual Rev. Neurosci. 19:491-515; Nawa, et al . (1990) Cold Spring Harbor Symposia on Quantitative Biology vol. 55, Cold Spring Harbor Press, CSH, NY; and Patterson and Nawa (1993) Cell 72:123-137. Structural relationship to the growth promoting activity (GPA) suggests that a family of related proteins exists. See Shelton (1996) Perspect. Dev. Neurobiol . 4:101-107. DIL-40 agonists, or antagonists, may also act as functional or receptor antagonists, e.g., which block

CNTF binding to their respective receptors, or mediating the opposite actions. Thus, DIL-40, or its antagonists, may be useful in the treatment of abnormal medical conditions, including immune or neurological disorders, e.g., T cell immune deficiencies, chronic inflammation, or tissue rejection, or in cardiovascular or neurophysiological conditions .

The natural antigens are capable of mediating various biochemical responses which lead to biological or physiological responses in target cells. The preferred embodiment characterized herein is from human, but other primate, or other species counterparts exist in nature. Additional sequences for proteins in other mammalian species, e.g., primates, canines, felines, and rodents, should also be available. See below. The descriptions below are directed, for exemplary purposes, to a human DIL-40, but are likewise applicable to related embodiments from other species .

II. Purified DIL-40 Human DIL-40 amino acid sequence, is shown as one embodiment within SEQ ID NO: 2 or 4. Other naturally occurring nucleic acids which encode the protein can be isolated by standard procedures using the provided sequence, e.g., PCR techniques, or by hybridization. These amino acid sequences, provided amino ^'to carboxy, are important in providing sequence information for the cytokine allowing for distinguishing the protein antigen from other proteins and exemplifying numerous variants. Moreover, the peptide sequences allow preparation of peptides to generate antibodies to recognize such segments, and nucleotide sequences allow preparation of oligonucleotide probes, both of which are strategies for detection or isolation, e.g., cloning, of genes encoding such sequences . As used herein, the term "human soluble DIL-40" shall encompass, when used in a protein context, a protein having amino acid sequence corresponding to a soluble polypeptide shown in SEQ ID NO: 2 or 4, or significant fragments thereof. Binding components, e.g., antibodies, typically bind to a DIL-40 with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM. Counterpart proteins will be found in mammalian species other than human, e.g., other primates, ungulates, or rodents. Non- mammalian species should also possess structurally or functionally related genes and proteins, e.g. , birds or amphibians .

The term "polypeptide" as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least about 12 amino acids, typically at least about 16 amino acids, preferably at least about 20 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids, e.g., 35, 40, 45, 50, etc. Such fragments may have ends which begin and/or end at virtually all positions, e.g., beginning at residues 1, 2, 3, etc., and ending at, e.g., 193, 192, 191, etc., in all practical combinations . Particularly interesting peptides have ends corresponding to structural domain boundaries, e.g., helices A, B, C, and/or D, or exon boundaries. See Table 1.

The term "binding composition" refers to molecules that bind with specificity to DIL-40, e.g., in an antibody-antigen interaction. The specificity may be more or less inclusive, e.g., specific to a particular embodiment, or to groups of related embodiments, e.g., primate, rodent, etc. It also includes compounds, e.g., proteins, which specifically associate with DIL-40, including in a natural physiologically relevant protein- protein interaction, either covalent or non-covalent . The molecule may be a polymer, or chemical reagent. A functional analog may be a protein with structural modifications, or it may be a molecule which has a molecular shape which interacts with the appropriate binding determinants. The compounds may serve as agonists or antagonists of a receptor binding interaction, see, e.g., Goodman, et al . (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed. ) , Pergamon Press. Substantially pure, e.g., in a protein context, typically means that the protein is free from other contaminating proteins, nucleic acids, or other biologicals derived from the original source organism. Purity may be assayed by standard methods, typically by weight, and will ordinarily be at least about 40% pure, generally at least about 50% pure, often at least about 60% pure, typically at least about 80% pure, preferably at least about 90% pure, and in most preferred embodiments, at least about 95% pure. Carriers or excipients will often be added.

Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans and mice, though under certain situations the temperature may be raised or lowered in situ or in vitro. The size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state. The polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents. The solvent and electrolytes will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological aqueous solvent. Usually the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5. On some occasions, one or more detergents will be added, typically a mild non- denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3- [3-cholamidopropyl) dimethylammonio] -1-propane sulfonate) , or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein. In other instances, a harsh detergent may be used to effect significant denaturation. III. Physical Variants

This invention also encompasses proteins or peptides having substantial amino acid sequence identity with the amino acid sequence of the DIL-40 antigen. The variants include species, polymorphic, or allelic variants.

Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. See also Needleham, et al. (1970) J. Mol. Biol. 48:4-43-453;

Sankoff, et al. (1983) Chapter One in Time Warps , String Edits, and Macromolecules : The Theory and Practice of Sequence Comparison, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and the University of Wisconsin Genetics Computer Group, Madison, WI. Sequence identity changes when considering conservative substitutions as matches. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The conservation may apply to biological features, functional features, or structural features. Homologous amino acid sequences are typically intended to include natural polymorphic or allelic and interspecies variations of a protein sequence. Typical homologous proteins or peptides will have from 25-100% identity (if gaps can be introduced) , to 50-100% identity (if conservative substitutions are included) with the amino acid sequence of the DIL-40. Identity measures will be at least about 35%, generally at least about 40%, often at least about 50%, typically at least about 60%, usually at least about 70%, preferably at least about 80%, and more preferably at least about 90%.

The isolated DIL-40 DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of short nucleotide stretches. These modifications result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunogenic, antigenic, or other functional activity. These modified sequences can be used to produce mutant antigens or to enhance expression. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. "Mutant DIL-40" encompasses a polypeptide otherwise falling within the sequence identity definition of the DIL-40 as set forth above, but having an amino acid sequence which differs from that of DIL-40 as normally found in nature, whether by way of deletion, substitution, or insertion. This generally includes proteins having significant identity with a protein having sequence of SEQ ID NO: 2 or 4, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most of the natural full length disclosed sequences. Full length sequences will typically be preferred, though truncated versions will also be useful, likewise, genes or proteins found from natural sources are typically most desired. Similar concepts apply to different DIL-40 proteins, particularly those found in various warm blooded animals, e.g., mammals and birds . These descriptions are generally meant to encompass all DIL-40 proteins, not limited to the particular primate embodiments specifically discussed. DIL-40 mutagenesis can also be conducted by making amino acid insertions or deletions. Substitutions, deletions , insertions , or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions . Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by Ml3 primer mutagenesis or polymerase chain reaction (PCR) techniques. See, e.g., Sambrook, et al . (1989); Ausubel, et al . (1987 and Supplements); and Kunkel, et al . (1987) Methods in Enzymol . 154:367-382. Preferred embodiments include, e.g., 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, etc., preferably conservative substitutions at the nucleotide or amino acid levels. Preferably the substitutions will be away from the conserved cysteines, and often will be in the regions away from the heli'cal structural domains . Such variants may be useful to produce specific antibodies, and often will share many or all biological properties . The present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins . A heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner. A similar concept applies to heterologous nucleic acid sequences .

In addition, new constructs may be made from combining similar functional domains from other proteins . For example, target-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al . (1988) J. Biol. Chem. 263:15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments . A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., PCR techniques. 19491

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IV. Functional Variants

The blocking of physiological response to DIL-40s may result from the competitive inhibition of binding of the ligand to its receptor. In vitro assays of the present invention will often use isolated protein, soluble fragments comprising receptor binding segments of these proteins, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations' and modifications, or cytokine mutations and modifications, e.g., DIL-40 analogs.

This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to the cytokine, or receptor binding fragments compete with a test compound.

"Derivatives" of DIL-40 antigens include amino acid sequence mutants from naturally occurring forms, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in DIL-40 amino acid side chains or at the N- or C- termini, e.g. , by standard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagents for Protein Modification, vols . 1-2, CRC Press, Inc., Boca Raton, FL; Hugli (ed. 1989) Techniques in Protein Chemistry, Academic Press, San Diego, CA; and Wong (1991) Chemistry of Protein Conjugation and Cross Linking . CRC Press, Boca Raton, FL. In particular, glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versions of the peptides with the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine .

Fusion polypeptides between DIL-40s and other homologous or heterologous proteins are also provided. Many cytokine receptors or other surface proteins are multimeric, e.g., homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic cleavage. Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a receptor- binding segment, so that the presence or location of the fused ligand may be easily determined. See, e.g., Dull, et al., U.S. Patent No. 4,859,609. Other gene fusion partners include bacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, yeast alpha mating factor, other cytokines or neurotrophins, and detection or purification tags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, et al. (1988) Science 241:812-816. Fusion peptides will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods . Techniques for nucleic acid manipulation and expression are described generally, e.g., in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed. ) , vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al . (eds. 1993) Current Protocols in Molecular Biology, Greene and Wiley, NY. Techniques for synthesis of polypeptides are described, e.g., in Merrifield (1963) J. Amer. Chem. Soc . 85:2149- 2156; Merrifield (1986) Science 232: 341-347; Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992) Synthetic Peptides: A User's Guide, W.H. Freeman, NY. Refolding methods may be applicable to synthetic proteins. This invention also contemplates the use of derivatives of DIL-40 proteins other than variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical moieties or protein carriers . Covalent or aggregative derivatives will be useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of binding partners, e.g., other antigens. An DIL-40 can be immobilized by covalent bonding to a solid support such as cyanogen bromide- activated SEPHAROSE, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-DIL+-40 antibodies or an alternative binding composition. The DIL-40 proteins can also be labeled with a detectable group, e.g., for use in diagnostic assays. Purification of DIL-40 may be effected by an immobilized antibody or complementary binding partner, e.g., binding portion of a receptor.

A solubilized DIL-40 or fragment of this invention can be used as an immunogen for the production of antisera or antibodies specific for binding. Purified antigen can be used to screen monoclonal antibodies or antigen-binding fragments, encompassing antigen binding fragments of natural antibodies, e.g., Fab, Fab', F(ab)2/ etc. Purified DIL-40 antigens can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of the cytokine, which may be diagnostic of an abnormal or specific physiological or disease condition. This invention contemplates antibodies raised against amino acid sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, or fragments of proteins containing it. In particular, this invention contemplates antibodies having binding affinity to or being raised against specific domains, e.g., helices A, B, C, or D.

The present invention contemplates the isolation of additional closely related species variants. Southern and Northern blot analysis will establish that similar genetic entities exist in other mammals . It is likely that DIL-40s are widespread in species variants, e.g., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigens displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the molecules will be greatly accelerated by the isolation and characterization of additional distinct species or polymorphic variants of them. In particular, the present invention provides useful probes for identifying additional homologous genetic entities in different species .

The isolated genes will allow transformation of cells lacking expression of a DIL-40, e.g., either species types or cells which lack corresponding proteins and exhibit negative background activity. This should allow analysis of the function of DIL-40 in comparison to untransformed control cells. Dissection of critical structural elements which effect the various physiological functions mediated through these antigens is possible using standard techniques of modern molecular biology, particularly in comparing members of the related class. See, e.g., the homolog-scanning mutagenesis technique described in Cunningham, et al . (1989) Science 243:1339-1336; and approaches used in O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al . (1990) EMBO J. 9:4381-4390. Intracellular functions would probably involve receptor signaling. However, protein internalization may occur under certain circumstances, and interaction between intracellular components and cytokine may occur. Specific segments of interaction of DIL-40 with interacting components may be identified by mutagenesis or direct biochemical means, e.g., cross-linking or affinity methods . Structural analysis by crystallographic or other physical methods will also be applicable. Further investigation of the mechanism of signal transduction will include study of associated components which may be isolatable by affinity methods or by genetic means, e.g., complementation analysis of mutants .

Further study of the expression and control of DIL-40 will be pursued. The controlling elements associated with the antigens should exhibit differential physiological, developmental, tissue specific, or other expression patterns. Upstream or' downstream genetic regions, e.g., control elements, are of interest.

Structural studies of the DIL-40 antigens will lead to design of new antigens, particularly analogs exhibiting agonist or antagonist properties on the molecule. This can be combined with previously described screening methods to isolate antigens exhibiting desired spectra of activities.

V. Antibodies

A DIL-40 protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen, such as an immunogen consisting of the amino acid sequence of SEQ ID NO: 2 or 4 is typically determined in an immunoassay. The immunoassay uses a polyclonal antiserum which was raised to a protein of SEQ ID NO: 2 or 4. This antiserum is selected to have low crossreactivity against other similar proteins, e.g., CNTF or other neurotrophic factors, and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay. Alternatively, preparations with defined selectivity can be produced.

In order to produce antisera for use in an immunoassay, the protein, e.g., of SEQ ID NO: 2, is isolated as described herein. For example, recombinant protein may be produced in a mammalian cell line. An 19491

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inbred strain of mice such as Balb/c is immunized with the protein of SEQ ID NO: 2 using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra) . Alternatively, a synthetic peptide, preferably near full length, derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10^ or greater are selected and tested for their cross reactivity against other intracellular proteins, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.

Immunoassays in the competitive binding format can be used for the crossreactivity determinations. For example, a protein of SEQ ID NO: 2 can be immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO: 2. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein (e.g., the DIL-40 protein of SEQ ID NO: 2) . In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the^ immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein, e.g., of SEQ ID NO: 2 that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.

Antibodies can be raised to various epitopes of the DIL-40 proteins, including species, polymorphic, or allelic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to DIL-40s in either their active forms or in their inactive forms, including native or denatured versions. Anti-idiotypic antibodies are also contemplated. Antibodies, including binding fragments and single chain versions, against predetermined fragments of the antigens can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective DIL-40s, or screened for agonistic or antagonistic activity, e.g., mediated through a receptor. Antibodies may be agonistic or antagonistic, e.g., by sterically blocking binding to a receptor. These monoclonal antibodies will usually bind with at least a K-_Q of about 1 mM, more usually at least about 300 μM, typically at least about 100 μM, more typically at least about 30 μM, preferably at least about 10 μM, and more preferably at least about 3 μM or better. The antibodies of this invention can also be useful in diagnostic applications. As capture or non- neutralizing antibodies, they can be screened for ability to bind to the antigens without inhibiting binding to a receptor. As neutralizing antibodies, they can be useful in competitive binding assays. They will also be useful in detecting or quantifying DIL-40 polypeptide or its receptors. See, e.g., Chan (ed. 1987) Immunology: A Practical Guide, Academic Press, Orlando, FLA; Price and Newman (eds. 1991) Principles and Practice of Immunoassay. Stockton Press, N.Y. ; and Ngo (ed. 1988) Nonisotopic Immunoassay, Plenum Press, N.Y. Cross absorptions or other tests will identify antibodies which exhibit various spectra of specificities, e.g., unique or shared species specificities.

Further, the antibodies, including antigen binding fragments, of this invention can be potent antagonists that bind to the antigen and inhibit functional binding, e.g., to a receptor which may elicit a biological response. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides so that when the antibody binds to antigen, a cell expressing it, e.g., on its surface, is killed. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting.

Antigen fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens . An antigen and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions . Dover Publications, New York; Williams, et al . (1967) Methods in Immunology and Immunochemistrv, vol. 1, Academic Press, New York; and Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press, NY, for descriptions of methods of preparing polyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al . (eds.) Basic and Clinical Immunology (4th ed. ) , Lange Medical 9491

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Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies : A Laboratory- Manual . CSH Press; Goding (1986) Monoclonal Antibodies : Principles and Practice (2d ed. ) , Academic Press, New York; and particularly in Kohler and Milstein (1975) in Nature 256:495-497, which discusses one method of generating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of "antibodies in phage or similar vectors. See, Huse, et al . (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda, " Science 246:1275-1281; and Ward, et al . (1989) Nature 341:544- 546. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos . 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567; Moore, et al., U.S. Patent No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.

The antibodies of this invention can also be used for affinity chromatography in isolating the protein. Columns can be prepared where the antibodies are linked to a solid support. See, e.g., Wilchek et al. (1984) Meth. Enzvmol. 104:3-55. Antibodies raised against each DIL-40 will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens .

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful in detecting, isolating, or identifying a DNA clone encoding DIL-40, e.g., from a natural source. Typically, it will be useful in isolating a gene from mammal, and similar procedures will be applied to isolate genes from other species, e.g., warm blooded animals, such as birds and mammals. Cross hybridization will allow isolation of DIL-40 from the same, e.g., polymorphic variants, or other species. A number of different approaches will be available to successfully isolate a suitable nucleic acid clone.

The purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies : A Laboratory Manual , Cold Spring Harbor Press .

For example, the specific binding composition could be used for screening of an expression library made from a cell line which expresses a DIL-40. Screening of intracellular expression can be performed by various staining or immunofluorescence procedures . Binding compositions could be used to affinity purify or sort out cells expressing a surface fusion protein.

The peptide segments can also be used to predict appropriate oligonucleotides to screen a library. The genetic code can be used to select appropriate oligonucleotides useful as probes for screening. See, e.g., SEQ ID NO: 1. In combination with polymerase chain reaction (PCR) techniques, synthetic oligonucleotides will be useful in selecting correct clones from a library. Complementary sequences will also be used as probes, primers, or antisense strands. Various fragments should be particularly useful, e.g., coupled with anchored vector or poly-A complementary PCR techniques or with complementary DNA of other peptides . Antisense nucleic acid constructs may block transcription or translation of message.

This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding DIL-40 polypeptide, particularly lacking the portion coding the untranslated 5 ' portion of the described sequence. In addition, this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide and which is capable of hybridizing under appropriate conditions with the DNA sequences described herein. Said biologically active protein or polypeptide can be an intact antigen, or fragment, and have an amino acid sequence disclosed in, e.g., SEQ ID NO: 2 or 4, particularly a mature, secreted polypeptide. Further, this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which exhibit high identity to a secreted DIL-40. The isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others. Alternatively, expression may be effected by operably linking a coding segment to a heterologous promoter, e.g., by inserting a promoter upstream from an endogenous gene .

An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other components which naturally accompany a native sequence, e.g., ribosomes, polymerases , and/or flanking genomic sequences from the originating species . 19491

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The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule. Generally, the nucleic acid will be in a vector or fragment less than about 50 kb, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.

A "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants. Thus, e.g., products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process.

Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of /19491

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functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. A similar concept is intended for a recombinant, e.g., fusion, polypeptide. Specifically included are synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these "antigens, and fusions of sequences from various^1' different species or polymorphic variants .

A significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 22 nucleotides, ordinarily at least about 29 nucleotides, more often at least about 35 nucleotides, typically at least about 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and in particularly preferred embodiments will be at least about 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, etc.

A DNA which codes for a DIL-40 protein will be particularly useful to identify genes, mRNA, and cDNA species which code for related or similar proteins, as well as DNAs which code for homologous proteins from different species. There will be homologs in other species, including primates, rodents, canines, felines, and birds. Various DIL-40 proteins should be homologous and are encompassed herein. However, even proteins that have a more distant evolutionary relationship to the antigen can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate DIL-40 proteins are of particular interest. Recombinant clones derived from the genomic sequences, e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt (ed. ) Encyclopedia of Immunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al . (1991) Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, IRL Press, Oxford; and Rosenberg (1992) J. Clinical Oncology 10:180-199.

Substantial homology, e.g., identity, in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least about 58%, ordinarily at least about 65%, often at least about 71%, typically at least about 77%, usually at least about 85%, preferably at least about 95 to 98% or more, and in particular embodiments, as high as about 99% or more of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence of DIL-40, e.g., in SEQ ID NO: 1. Typically, selective hybridization will occur when there is at least about 55% identity over a stretch of at least about 30 nucleotides, preferably at least about 75% over a stretch of about 25 nucleotides, and most preferably at least about 90% over about 20 nucleotides. See, Kanehisa (1984) Nuc . Acids Res. 12:203-213. Hybridization under stringent conditions should give a background of at least 2-fold over background, preferably at least 3-5 or more. The length of identity comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 28 nucleotides, typically at least about 40 nucleotides, and preferably at least about 75 to 100 or more nucleotides. Alternatively, the comparisions will involve a plurality of distinct, e.g., nonoverlapping, segments of the specified length. Typically, the plurality will be at least two, more usually at least three, and preferably 5, 7, or even more. While the length minima are provided, longer lengths, of various sizes, may be appropriate, e.g., one of length 7, and two of length 12.

Stringent conditions, in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions. Stringent temperature conditions will usually include temperatures in excess of about 30° C, usually in excess of about 37° C, typically in excess of about 55° C, preferably in excess of about 70° C. Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than about 400 mM, typically less than about 250 mM, preferably less than about 150 mM, including about 100, 50, or even 20 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349- 370.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters .

Optical alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl . Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc . Nat ' 1 Acad. Sci . USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) , or by visual inspection (see generally Ausubel et al . , supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold

(Altschul, et al . , supra). These' initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands .

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5787) . One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

DIL-40 from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species. Homology may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches.

VII. Making DIL-40; Mimetics

DNA which encodes the DIL-40 or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or screening genomic libraries prepared from a wide variety of cell lines or tissue samples. See, e.g.,

Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170;

Gubler and Hoffman (1983) Gene 25:263-269; and Glover

(ed. 1984) DNA Cloning: A Practical Approach, IRL Press, Oxford. Alternatively, the sequences provided herein provide useful PCR primers or allow synthetic or other preparation of suitable genes encoding a DIL-40; including naturally occurring embodiments .

This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length DIL-40 or fragments which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for structure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, "and other vehicles which enable the integration of DNA fragments into the genome of the host. See, e.g., Pouwels, et al . (1985 and Supplements) Cloning Vectors : A Laboratory Manual, Elsevier, N.Y. ; and Rodriguez, et al . (eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, MA.

For purposes of this invention, DNA sequences are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression. See, e.g., Rodriguez, et al., Chapter 10, pp. 205-236; Balbas and Bolivar (1990) Methods in Enzvmology 185:14-37; and Ausubel, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.

Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama, et al . (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199. See also US Pat. 5,593,857 "Production of Homogeneous Truncated CNTF". It will often be desired to express a DIL-40 polypeptide in a system which provides a specific or defined glycosylation pattern. See, e.g. , Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990) Meth. Enzvmol. 185:487-511.

The DIL-40, or a fragment thereof, may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C . This releases the antigen in a biologically active form, and allows purification by standard procedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophvs . Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner, et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the DIL-40 has been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer- Verlag, New York; Bodanszky (1984) The Principles of Peptide Synthesis. Springer-Verlag, New York; and Villafranca (ed. 1991) Techniques in Protein Chemistry II . Academic Press, San Diego, Ca.

VIII . Uses

The present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e.g., in DIL-40 mediated conditions, or below in the description of kits for diagnosis . This invention also provides reagents with significant commercial and/or therapeutic potential. The DIL-40 (naturally occurring or recombinant) , fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to DIL-40, should be useful as reagents for teaching techniques of molecular biology, immunology, or physiology. Appropriate kits may be prepared with the reagents, e.g., in practical laboratory exercises in production or use of proteins, antibodies, cloning methods, histology, etc.

The reagents will also be u≤eful in the treatment of conditions associated with abnormal physiology or development, including inflammatory conditions. They may be useful in vitro tests for presence or absence of interacting components, which may correlate with success of particular treatment strategies. In particular, modulation of physiology of various, e.g., hematopoietic or lymphoid, cells will be achieved by appropriate methods for treatment using the compositions provided herein. CNTF-like reagents have been reported to have various therapeutic effects, see, e.g., US Pat. 5,780,600, 5,648,334, and 5 , 470 , 952 ; in treating impotency (US Pat. 5,691,313); in regulating encapsulated bioartificial organs (US Pat. 5,795,790); and in propagation and differentiation of stem cells from embryonic and adult CNS (US Pat. 5,753,506).

For example, a disease or disorder associated with abnormal expression or abnormal signaling by a DIL-40 should be a likely target for an agonist or antagonist. The new cytokine should play a role in regulation or development of hematopoietic cells, e.g., lymphoid cells, which affect immunological responses, e.g., inflammation and/or autoimmune disorders. Alternatively, it may affect vascular physiology or development, or neuronal effects.

In particular, the cytokine should mediate, in various contexts, cytokine synthesis by the cells, proliferation, etc. Antagonists of DIL-40, such as mutein variants of a naturally occurring form of DIL-40 or blocking antibodies, may provide a selective and powerful way to block immune responses, e.g., in situations as inflammatory or autoimmune responses. See also Samter, et al. (eds.) Immunological Diseases vols. 1 and 2, Little, Brown and Co.

In addition, certain combination compositions would be useful, e.g., with other modulators of inflammation. Such other molecules may include steroids, ^'other versions of IL-6 and/or G-CSF, including species variants, or viral homologs, and their respective antagonists.

Various abnormal conditions are known in each of the cell types shown to produce DIL-40 mRNA by Northern blot analysis. See Berkow (ed. ) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J. ; Thorn, et al. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; and Weatherall, et al . (eds.) Oxford Textbook of Medicine, Oxford University Press, Oxford. Many other medical conditions and diseases involve activation by macrophages or monocytes, and many of these will be responsive to treatment by an agonist or antagonist provided herein. See, e.g., Stites and Terr (eds. 1991) Basic and Clinical Immunology Appleton and Lange, Norwalk, Connecticut; and Samter, et al . (eds.)

Immunological Diseases Little, Brown and Co. These problems should be susceptible to prevention or treatment using compositions provided herein. The pancratic islet localization suggests a possible relevance to diabetes. DIL-40, antagonists, antibodies, etc., can be purified and then administered to a patient, veterinary or human. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers, excipients, or preservatives. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations . This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.

•Drug screening using DIL-40 or fragments thereof can be performed to identify compounds having binding affinity to or other relevant biological effects on DIL-40 functions, including isolation of associated components. Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the cytokine. Likewise, a compound having intrinsic stimulating activity can activate the signal pathway and is thus an agonist in that it simulates the activity of DIL-40. This invention further contemplates the therapeutic use of blocking antibodies to DIL-40 as antagonists and of stimulatory antibodies as agonists . This approach should be particularly useful with other

DIL-40 species variants.

The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al . (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington ' s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn. Methods for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others . Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 μM concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than 'about 1 fM

(femtomolar) , with an appropriate' carrier. Slow release formulations, or a slow release apparatus will often be utilized for continuous or long term administration. See, e.g., Langer (1990) Science 24 : 1527-1533. DIL-40, fragments thereof, and antibodies to it or its fragments, antagonists, and agonists, may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.

Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, topical, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington ' s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, ■Penn.; Avis, et al . (eds. 1993) Pharmaceutical Dosage Forms : Parenteral Medications , Dekker, New York; Lieberman, et al . (eds. 1990) Pharmaceutical Dosage

Forms : Tablets , Dekker, New York; and Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms : Disperse Systems . Dekker, New York. The therapy of this invention may be combined with or used in association with other agents, e.g., other cytokines, or their respective antagonists .

Both naturally occurring and recombinant forms of the DIL-40s of this invention are particularly useful in kits and assay methods which are capable of screening compounds for binding activity to the proteins. Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al . (1991) Science 251:767-773, which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate. The development of suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble DIL-40 as provided by this invention. Other methods can be used to determine the critical residues in DIL-40-DIL-40 receptor interactions. Mutational analysis can be performed, e.g., see Somoza, et al. (1993) J. Exptl . Med. 178:549-558, to determine specific residues critical in the interaction and/or signaling. PHD (Rost and Sander (1994) Proteins 19:55-

72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310) can provide secondary structure predictions of α-helix (H) , β-strand (E) , or coil (L) .

For example, antagonists can normally be found once the antigen has been structurally defined, e.g., by tertiary structure data. Testing of potential interacting analogs is now possible upon the development of highly automated assay methods using a purified DIL-40. In particular, new agonists and antagonists will be discovered by using screening techniques described herein. Of particular importance are compounds found to have a combined binding affinity for a spectrum of DIL-40 molecules, e.g., compounds which can serve as antagonists for species variants of DIL-40.

One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a DIL-40. Cells may be isolated which express a DIL-40 in isolation from other molecules . Such cells , either in viable or fixed form, can be used for standard binding partner binding assays. See also, Parce, et al . (1989) Science 246:243- 247; and Owicki, et al . (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses .

Another technique for drug screening involves an approach which provides high throughput screening for compounds having suitable binding affinity to a DIL-40 and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984. First, large numbers of different small peptide test compounds are synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface, see

Fodor, et al. (1991) . Then all the pins are reacted with solubilized, unpurified or solubilized, purified DIL-40, and washed. The next step involves detecting bound DIL-40. Rational drug design may also be based upon structural studies of the molecular shapes of the DIL-40 and other effectors or analogues. Effectors may be other proteins which mediate other functions in response to binding, or other proteins which normally interact with DIL-40, e.g., a receptor. One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions, as modeled, e.g., against other cytokine-receptor models . For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

IX. Kits This invention also contemplates use of DIL-40 proteins, fragments thereof, pept des, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of another DIL-40 or binding partner. Typically the kit will have a compartment containing either a defined DIL-40 peptide or gene segment or a reagent which recognizes one or the other, e.g., DIL-40 fragments or antibodies.

A kit for determining the binding affinity of a test compound to a DIL-40 would typically comprise a test compound; a labeled compound, for example a binding partner or antibody having known binding affinity for DIL-40; a source of DIL-40 (naturally occurring or recombinant) ; and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the molecule. Once compounds are screened, those having suitable binding affinity to the antigen can be evaluated in suitable biological assays, as are well known in the art, to determine whether they act as agonists or antagonists to the DIL-40 signaling pathway. The availability of recombinant DIL-40 polypeptides also provide well defined standards for calibrating such assays .

A preferred kit for determining the concentration of, e.g., a DIL-40 in a sample would typically comprise a labeled compound, e.g., binding partner or antibody, having known binding affinity for the antigen, a source of cytokine (naturally occurring or recombinant) and a means for separating the bound from free labeled compound, e.g., a solid phase for immobilizing the DIL-40. Compartments containing reagents, and instructions, will normally be provided. Antibodies, including antigen binding fragments, specific for the DIL-40 or fragments are useful in diagnostic applications to detect the presence of elevated levels of DIL-40 and/or its fragments. Such diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, l and further can involve the detection of antigens related to the antigen in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-binding partner complex) or heterogeneous (with a separation step) . Various commercial assays exist, such as radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , enzyme-multiplied immunoassay technique (EMIT) , substrate-labeled fluorescent immunoassay (SLFIA) , and the like. See, e.g., Van

Vunakis, et al . (1980) Meth Enzvmol. 70:1-525; Harlow and Lane (1980) Antibodies : A Laboratory Manual , CSH Press, NY; and Coligan, et al . (eds. 1993) Current Protocols in Immunology, Greene and Wiley, NY. Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a DIL-40, as such may be diagnostic of various abnormal states . For example, overproduction of DIL-40 may result in production of various immunological reactions which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal activation or differentiation. Moreover, the distribution patttern available provides information that the cytokine is expressed in pancreatic islets, suggesting the possibility that the cytokine may be involved in function of that organ, e.g., in a diabetes relevant medical condition. Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody or binding partner, or labeled DIL-40 is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will also contain instructions for proper use and disposal of the contents after use. Typically the kit has compartments for each useful reagent. Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.

Many of the aforementioned constituents of the drug screening and the diagnostic assays may be used without modification or may be modified in a variety of ways. For example, labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In any of these assays, the binding partner, test compound, DIL-40, or antibodies thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups: radiolabels such as 125j_f enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups . There are also numerous methods of separating the bound from the free DIL-40, or alternatively the bound from the free test compound. The DIL-40 can be immobilized on various matrixes followed by washing. Suitable matrixes include plastic such as an ELISA plate, filters, and beads. See, e.g., Coligan, et al. (eds. 1993) Current Protocols in Immunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins' or their fragments to the various labels have been extensively reported in the literature and do not require detailed discussion here. Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like. Fusion proteins will also find use in these applications. Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of a DIL-40. These sequences can be used as probes for detecting levels of the DIL-40 message in samples from patients suspected of having an abnormal condition, e.g., inflammatory or autoimmune. Since the cytokine may be a marker or mediator for activation, it may be useful to determine the numbers of activated cells to determine, e.g., when additional therapy may be called for, e.g., in a preventative fashion before the effects become and progress to significance. The preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature. See, e.g., Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967; and Wilchek et al. (1988) Anal. Biochem. 171:1-32.

Diagnostic kits which also test for the qualitative or quantitative expression of other molecules are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers . Thus, kits may test for combinations of markers. See, e.g., Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97. Other kits may be used to evaluate other cell subsets.

X. Isolating a DIL-40 Receptor

Having isolated a ligand of a specific ligand- receptor interaction, methods exist for isolating the receptor. See, Gearing, et al . (1989) EMBO J. 8:3667- 3676. For example, means to label the DIL-40 cytokine without interfering with the binding to its receptor can be determined. For example, an affinity label can be fused to either the amino- or carboxyl-terminus of the ligand. Such label may be a FLAG epitpe tag, or, e.g., an Ig or Fc domain. An expression library can be screened for specific binding of the cytokine, e.g., by cell sorting, or other screening to detect subpopulations which express such a binding component. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90:11267-11271; and Liu, et al. (1994) J. Immunol. 152:1821-29. Alternatively, a panning method may be used. See, e.g., Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369. Protein cross-linking techniques with label can be applied to isolate binding partners of the DIL-40 cytokine. This would allow identification of proteins which specifically interact with the cytokine, e.g., in a ligand-receptor like manner. Early experiments will be performed to determine whether the known CNTF receptor components are involved in response (s) to DIL-40, or components shared with the IL-6 or G-CSF signal pathways. It is also quite possible that these functional receptor complexes may share many or all components with a DIL-40 receptor complex, either a specific receptor subunit or an accessory receptor subunit.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

EXAMPLES General Methods

Many of the standard methods below are described or referenced, e.g., in Maniatis, et al . (1982) Molecular Cloning, A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSH Press, NY; Ausubel, et al . , Biology Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al. (1987 and Supplements) Current Protocols in Molecular Biology Wiley/Greene, NY; Innis, et al . (eds. 1990) PCR Protocols: A Guide to Methods and Applications Academic Press, NY. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification," Methods in Enzvmology vol. 182, and other volumes in this series; Coligan, et al . (1995 and supplements) Current Protocols in Protein Science John Wiley and Sons, New York, NY; Matsudaira (ed. 1993) A Practical Guide to Protein and Peptide Purification for Microsequencing Academic Press, San Diego, CA; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, NJ, or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments (epitope tags) , e.g., to a FLAG sequence or an equivalent which can be fused, e.g., via a protease-removable sequence. See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed. ) Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, NY; and Crowe, et al . (1992) OIAexpress : The High Level Expression & Protein Purification System QUIAGEN, Inc., Chatsworth, CA.

Standard immunological techniques are described, e.g., in Hertzenberg, et al . (eds. 1996) Weir ' s Handbook of Experimental Immunology vols 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Methods in Enzvmology vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. Assays for vascular biological activities are well known in the art. They will cover angiogenic and angiostatic activities in tumor, or other tissues, e.g., arterial smooth muscle proliferation (see, e.g., Koyoma, et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascular epithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc. See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am. J. Pathol . 147:668-677; Thyberg, et al. (1990) Athersclerosis 10:966-990; and Gumbiner (1996) Cell 84:345-357.

Assays for neural cell biological activities are described, e.g., in Wouterlood (ed. 1995) Neuroscience

Protocols modules 10, Elsevier; Methods in Neurosciences Academic Press; and Neuromethods Humana Press, Totowa, NJ. Methodology of developmental systems is described, e.g., in Meisami (ed.) Handbook of Human Growth and Developmental Biology CRC Press; and Chrispeels (ed.) Molecular Techniques and Approaches in Developmental Biology Interscience. FACS analyses are described in Melamed, et al . (1990) Flow Cvtometrv and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Flow Cvtometrv Liss, New York, NY; and Robinson, et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, NY.

EXAMPLE 1: Cloning of Human DIL-40

The sequence of the gene is provided in Table 1. The sequence is derived from a cDNA library made from human brain frontal cortex. These sequences allow preparation of PCR primers, or probes, to determine cellular distribution of the gene, which is believed to be a rare message, and for which ESTs have not been identified in any public database. The sequences allow isolation of genomic DNA which encode the message.

Using the probe or PCR primers, various tissues or cell types are probed to determine cellular distribution. PCR products are cloned using, e.g., a TA cloning kit (Invitrogen) . The resulting cDNA plasmids are sequenced from both termini on an automated sequencer (Applied Biosysterns) .

EXAMPLE 2: Cellular Expression of Human DIL-40

An appropriate probe or primers specific for cDNA encoding primate DIL-40 are prepared. Typically, the probe is labeled, e.g., by random priming. The expression is probably in brain, but the specific cell subsets have not yet been determined.

EXAMPLE 3: Chromosome mapping of human DIL-40

An isolated cDNA encoding the DIL-40 is used. Chromosome mapping is a standard technique. See, e.g., BIOS Laboratories (New Haven, CT) and methods for using a mouse somatic cell hybrid panel with PCR. EXAMPLE 4: Purification of DIL-40 Protein

Multiple transfected cell lines are screened for one which expresses the cytokine at a high level compared with other cells . Various cell lines are screened and selected for their favorable properties in handling.

Natural DIL-40 can be isolated from natural sources, or by expression from a transformed cell using an appropriate expression vector. Purification of the expressed protein is achieved by standard procedures, or may be combined with engineered means for effective purification at high efficiency from cell lysates or supernatants. FLAG or Hisς segments can be used for such purification features. Alternatively, affinity chromatography may be used with specific antibodies, see below.

Protein is produced in coli, insect cell, or mammalian expression systems, as desired.

EXAMPLE 5: Isolation of Homologous DIL-40 Genes The DIL-40 cDNA can be used as a hybridization probe to screen a library from a desired source, e.g., a primate cell cDNA library. Many different species can be screened both for stringency necessary for easy hybridization, and for presence using a probe. Appropriate hybridization conditions will be used to select for clones exhibiting specificity of cross hybridization.

Screening by hybridization using degenerate probes based upon the peptide sequences will also allow isolation of appropriate clones. Alternatively, use of appropriate primers for PCR screening will yield enrichment of appropriate nucleic acid clones .

Similar methods are applicable to isolate either species, polymorphic, or allelic variants. Species variants are isolated using cross-species hybridization techniques based upon isolation of a full length isolate or fragment from one species as a probe. Alternatively, antibodies raised against human DIL-40 will be used to screen for cells which express cross-reactive proteins from an appropriate, e.g., cDNA library. The 'purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies : A Laboratory Manual Cold

Spring Harbor Press. The resulting antibodies are used for screening, purification, or diagnosis, as described.

EXAMPLE 6: Preparation of antibodies specific for DIL-40 Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press . Polyclonal serum, or hybridomas may be prepared. In appropriate situations, the binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods . Peptides which exhibit greater or lesser measures of identity with other related proteins are selected, as appropriate.

EXAMPLE 7 : Biological Functions

Biological activities of DIL-40 are tested based on the sequence and structural homology between DIL-40 and CNTF, IL-6, and G-CSF.

A. Effects on proliferation of cells

The effect on proliferation of various cell types are evaluated with various concentrations of cytokine. A dose response analysis is performed, in combinations with the related cytokines CNTF, IL-6, G-CSF, etc. Preferably, various cell types found in the brain are used, including neuronal cell lines.

For example, total PBMC are isolated from buffy coats of normal healthy donors by centrifugation through ficoll-hypaque as described by Boyum, et al . (1996)

Scand. J. Immunol. 43:228-235. PBMC are cultured in 200 μl Yssel's medium (Gemini Bioproducts, Calabasas, CA) containing 1% human AB serum in 96 well plates (Falcon, Beeton-Dickinson, NJ) in the absence or presence of DIL-40. Cells are cultured in medium alone or in combination with 100 U/ml IL-2 (R'&D Systems) for 120 hours. 3H-Thymidine (0.1 mCi) is added during the last six hours of culture and 3H-Thymidine incorporation determined by liquid scintillation counting. Similar assays can be performed on other cell types, both brain derived or hematopoietic . B. Effects on the expression of cell surface molecules

Assays on the effects of the cytokine on cell surface molecule expression can be performed on various cell types, both brain derived or hematopoietic.

For example, monocytes are purified, e.g., by negative selection from peripheral blood mononuclear cells of normal healthy donors. Briefly, 3 x 10^ ficoll banded mononuclear cells are incubated on ice with a cocktail of monoclonal antibodies (Becton-Dickinson;

Mountain View, CA) consisting, e.g., of 200 μl of αCD2 (Leu-5A) , 200 μl of αCD3 (Leu-4) , 100 μl of αCD8 (Leu 2a), 100 μl of αCDl9 (Leu 12 ), 100 μl of αCD20 (Leu-16) , 100 μl of αCD56 (Leu-19) , 100 μl of αCD67 (IOM 67; Immunotech,Westbrook, ME ) , and anti-glycophorin antibody (10F7MN, ATCC, Rockville, MD) . Antibody bound cells are washed and then incubated with sheep anti-mouse IgG coupled magnetic beads (Dynal, Oslo, Norway) at a bead to cell ratio of 20:1. Antibody bound cells are separated from monocytes by application of a magnetic field.

Subsequently, human monocytes are cultured in Yssel ' s medium (Gemini Bioproducts, Calabasas, CA) containing 1% human AB serum in the absence or presence of DIL-40, IL-6, G-CSF or combinations.

Analyses of the expression of cell surface molecules can be performed by direct immunoflurescence. For example, 2 x 10^ purified human monocytes are incubated in phosphate buffered saline (PBS) containing 1% human serum on ice for 20 minutes. Cells are pelleted at 200 x g. Cells are resuspended in 20 ml PE or FITC labeled mAb. Following an additional 20 minute incubation on ice, cells are washed in PBS containing 1% human serum followed by two washes in PBS alone. Cells are fixed in PBS containing 1% paraformaldehyde and analyzed on FACScan flow cytometer (Becton Dickinson; Mountain View, CA) . Exemplary mAbs are used, e.g.: CDllb (anti-macl) , CDllc (a gpl50/95), CD14 (Leu-M3), CD54 (Leu 54), CD80 (anti-BBl/B7) , HLA-DR (L243) from Becton-Dickinson and CD86 (FUN 1; Pharmingen), CD64 (32.2; Medarex) , CD40 (mAb89; Schering-Plough France). C . Effects on cytokine production by human monocytes Human monocytes are isolated as described and cultured in Yssel ' s medium (Gemini Bioproducts, Calabasas, CA) containing 1% human AB serum in the absence or presence of DIL-40 (1/100 dilution baculovirus expressed material) . In addition, monocytes are stimulated with LPS (E. coli 0127 :B8 Difco) in the absence or presence of DIL-40 and the concentration of cytokines (IL-lβ, IL-6, TNFα, GM-CSF, and IL-10) in the cell culture supernatant determined by ELISA.

For intracytoplasmic staining for cytokines, monocytes are cultured (1 million/ml) in Yssel ' s medium in the absence or presence of DIL-40 and LPS (E. coli 0127 :B8 Difco) and 10 mg/ml Brefeldin A (Epicentre technologies Madison WI) for 12 hrs. Cells are washed in PBS and incubated in 2% formaldehyde/PBS solution for 20 minutes at RT. Subsequently cells are washed, resuspended in permeabilization buffer (0.5% saponin (Sigma) in PBS/BSA (0.5%) /Azide (1 mM) ) and incubated for 20 minutes at RT. Cells (2 x 10⁵) are centrifuged and resuspended in 20 ml directly conjugated anti- cytokine mAbs diluted 1:10 in permeabilization buffer for 20 minutes at RT. The following antibodies can be used: IL-lα-PE (364-3B3-14) ; IL-6-PE (MQ2-13A5); TNFα-PE

(MAbll); GM-CSF-PE (BVD2-21C11) ; and IL-12-PE (Cll.5.14; Pharmingen San Diego, CA) . Subsequently, cells are washed twice in permeabilization buffer and once in PBS/BSA/Azide and analyzed on FACScan flow cytometer (Becton Dickinson; Mountain View, CA) .

Similar assays can be performed on other cell types, both brain derived or hematopoietic.

D. CNTF-like activities The native, recombinant, and fusion proteins would be tested for agonist and antagonist activity in many other biological assay systems, e.g., on neuronal cells, as have been performed with CNTF. Based upon the structural relationship, assays related to those activities should be analyzed alone or in combination with CNTF, GPA, cardiotropin-1 (CT-1) , leukemia inhibitiory factor (LIF) , onscostatin-M (OSM) , IL-6, or G-CSF. See Shelton (1996) Persp. Developmental Neurobiol . 4:101-107; Ip and Yancopoulos (1996) Ann. Rev. Neurosci . 19:491-515.

DIL-40 is evaluated for agonist or antagonist activity on transfected cells expressing CNTFRα or IL-6 or G-CSF receptor and controls. See, e.g., Ho, et al .

(1993) Proc. Nat'l Acad. Sci. USA 90, 11267-11271; Ho, et al. (1995) Mol. Cell. Biol. 15:5043-5053; and Liu, et al.

(1994). J. Immunol. 152:1821-1829.

DIL-40 is evaluated for effect in macrophage/dendritic cell activation and antigen presentation assays, T cell cytokine production and proliferation in response to antigen or allogeneic stimulus. See, e.g., de Waal Malefyt et al . (1991) . EXP. Med. 174:1209-1220; de Waal Malefyt et al . (1991) J.

EXP. Med. 174:915-924; Fiorentino, et al . (1991) J.

Immunol . 147, 3815-3822; Fiorentino, et al . (1991) J.

Immunol . 146:3444-3451; and Groux, et al . (1996) J. Exp. Med. 184:19-29.

DIL-40 will also be evaluated for effects on NK cell stimulation. Assays may be based, e.g., on Hsu, et al .

(1992) Internat . Immunol . 4:563-569; and Schwarz, et al.

(1994) J. Immunother. 16:95-104. B cell growth and differentiation effects will be analyzed, e.g., by the methodology described, e.g., in

Defrance, et al . (1992). J. EXP. Med. 175:671-682;

Rousset, et al (1992) Proc. Nat'l Acad. Sci. USA 89:1890-

1893 ; including IgG2 and IgA2 switch factor assays . Note that, unlike COS7 supernatants, NIH3T3 and COP supernatants apparently do not interfere with human B cell assays.

All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes .

Claims

WHAT IS CLAIMED IS:

1. An isolated protein comprising an amino acid sequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a conservative or allelic variant thereof.

2. An antigenic peptide fragment of the protein according to Claim 1.

3. A fusion protein comprising a protein or peptide according to either Claim 1 or Claim 2.

4. A composition comprising a protein, peptide or fusion protein according to any one of Claims 1 to 3.

5. An antibody which specifically binds a protein or peptide of either Claim 1 or Claim 2.

6. An isolated nucleic acid encoding a protein, peptide or fusion protein according to any one of Claims

1 to 3.

7. An isolated nucleic acid which: a) hybridizes under wash conditions of 30° C and less than 2M salt to a DNA molecule having the sequence of SEQ ID NO:l or SEQ ID NO: 2; or b) exhibits identity over a stretch of at least about 30 nucleotides to a DNA molecule having the sequence of SEQ ID NO:l or SEQ ID N0:2.

8. A recombinant vector comprising the nucleic acid of Claim 6 or Claim 7.

9. A host cell comprising the recombinant vector of Claim 8.

10. A method for producing a protein, peptide or fusion protein comprising culturing the host cell of Claim 9 under conditions in which the nucleic acid is expressed.

11. A kit comprising: a) a protein, peptide or fusion protein according to any one of Claims 1 to 3 ; b) an antibody which specifically binds to a protein or peptide according to either Claim 1 or Claim 2 ; or c) a nucleic acid according to Claim 6 or Claim 7.

12. A method of modulating physiology or development of a cell or tissue culture cells comprising contacting said cell with an agonist or antagonist of a protein according to Claim 1.